Humanized rodents expressing heavy chains containing the VL domain

Abstract

To provide humanized rodents that express heavy chains containing VL domains.SOLUTION: Non-human animals, tissues, cells, and genetic material are provided that comprise a modification of an endogenous non-human heavy chain immunoglobulin sequence and that comprise an ADAM6 activity functional in a rodent (e.g., a mouse). The non-human animals rearrange human immunoglobulin light chain gene segments in the context of heavy chain constant regions and express immunoglobulin-like molecules comprising human immunoglobulin light chain variable domains fused to heavy chain constant domains that are cognate with human immunoglobulin light chain variable domains fused to light chain constant domains.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Field of Invention Provided are genetically modified non-human fertility animals expressing human immunoglobulin-like binding proteins containing an immunoglobulin heavy chain constant region fused to an immunoglobulin light chain variable domain, as well as binding proteins having an immunoglobulin light chain variable domain fused to a light chain constant domain and an immunoglobulin light chain variable domain fused to a heavy chain constant domain. Genetically modified mice, cells, embryos, and tissues containing a nucleic acid sequence encoding a functional ADAM6 protein are described, and these mice, cells, embryos, and tissues contain a human immunoglobulin light chain gene segment operably linked to one or more non-human immunoglobulin heavy chain constant genes. The modification includes a human and / or humanized immunoglobulin locus. Mice containing ADAM6 function are described as containing a mouse containing an ectopic nucleic acid sequence encoding an ADAM6 protein. Endogenous mouse immunoglobulin V H A genetically modified male mouse, which includes a regional locus gene modification and further includes ADAM6 activity, is described as a mouse containing an ectopic nucleic acid sequence that restores or maintains fertility in the male mouse. Exemplary fertility is comparable to that of a wild-type mouse.

[0002] A genetically modified non-human fertility animal is provided, comprising a gene modification that includes the deletion or alteration of the endogenous ADAM6 gene or its homolog or ortholog, thereby restoring all or part of the ADAM6 (or its homolog or ortholog) function, wherein the non-human animal expresses a human immunoglobulin light chain variable sequence in the heavy chain constant sequence. Cells expressing such binding proteins, rodents (e.g., mice) producing them, and related methods and compositions are also provided.

[0003] A genetically modified animal expressing an antibody containing a light chain variable region fused to a heavy chain constant region is described, wherein the non-human animal lacks the functional endogenous ADAM6 gene but retains ADAM6 function, and the animal is a rodent (e.g., a mouse) that is prevented from producing the functional ADAM6 protein, resulting in loss of fertility, is described, which contains endogenous immunoglobulin heavy chain variable (V H Examples include rodents (e.g., mice) that have undergone modification of the ) region gene locus. Multiple human V L , J L , and D as needed H Immunoglobulin V characterized by gene segments, or combinations thereof H Genetically modified mice containing a gene locus and further incorporating ADAM6 function have been described, including mice containing an ectopic nucleic acid sequence that restores fertility in male mice. These genetically modified mice lack a heavy chain variable domain and instead express an antibody containing a variable domain that includes a rearranged light chain gene segment.

[0004] Genetically modified rodents (e.g., mice), cells, embryos, and tissues containing nucleic acid sequences encoding a functional ADAM6 locus are described, and these mice, cells, embryos, and tissues express immunoglobulin heavy chains containing human light chain variable domains. Furthermore, these mice, cells, embryos, and tissues lack the functional endogenous ADAM6 gene but retain ADAM6 function characterized by the presence of ectopic nucleic acid sequences encoding the ADAM6 protein. Methods for constructing antibody sequences in pregnant non-human animals useful for antigen binding are provided. [Background technology]

[0005] background Over the past two decades, the pharmaceutical applications of antibodies have spurred extensive research into the creation of antibodies suitable for use as human therapeutics. Initial antibody therapies based on mouse antibodies were not ideal for human use because repeated administration of mouse antibodies to humans resulted in immunogenicity problems that could disrupt long-term treatment regimens. Solutions were developed based on the humanization of mouse antibodies, making them more human-like and less mouse-like. This was followed by methods for expressing human immunoglobulin sequences for use as antibodies, primarily based on the in vitro expression of human immunoglobulin libraries in phages, bacteria, or yeast. Finally, attempts were made to produce useful human antibodies from human lymphocytes in vitro in mice engrafted with human hematopoietic cells, and in transgenic mice or chromosome-transfected mice in which the endogenous immunoglobulin locus was rendered inactive.

[0006] To create these mice, it was necessary to inactivate endogenous mouse immunoglobulin genes so that randomly incorporated fully human transgenes could function as the immunoglobulin repertoire expressed in the mice. While such mice can produce human antibodies suitable for use as human therapeutics, they present significant problems related to the mouse's immune system. These problems present several experimental hurdles. For example, the mice cannot produce a sufficiently diverse antibody repertoire, requiring the use of extensive re-engineering fixes, potentially leading to suboptimal clonal selection processes due to incompatibility between human and mouse elements, and resulting in an unreliable source of a large and diverse population of human variable sequences necessary for truly useful human therapeutics.

[0007] Transgenic mice containing a complete human antibody transgene contain randomly inserted transgenes containing unrearranged human immunoglobulin heavy chain variable sequences (V, D, and J sequences) that bind to the human heavy chain constant sequence and unrearranged human immunoglobulin light chain variable sequences (V and J) that bind to the human light chain constant sequence. Therefore, these mice produce antibody genes that are completely human, rearranged from loci other than the endogenous locus. Generally, mice with at least some human λ sequences have also been reported, but these mice contain human heavy chain sequences and human κ light chain sequences. Transgenic mice generally have damaged and non-functional endogenous immunoglobulin loci, or knockouts of endogenous immunoglobulin loci, and therefore these mice cannot rearrange human antibody sequences at the endogenous immunoglobulin locus. The changes in such transgenic mice are not optimal for producing a sufficiently diverse human antibody repertoire in mice, likely due to a suboptimal clonal selection process that binds complete human antibody molecules within the endogenous selection system, and due to the detrimental effects of the alteration of the endogenous genetic makeup of such mice.

[0008] There remains a need in the art to create improved genetically modified non-human animals that are useful for producing immunoglobulin sequences containing human antibody sequences and for producing a diverse repertoire of immunoglobulin-like molecules that exhibit diversity apart from conventional antibody molecules, while simultaneously reducing or eliminating potentially harmful changes resulting from such genetic modification. There also remains a need for non-human animals that can rearrange immunoglobulin gene segments to form useful rearranged immunoglobulin genes, including human immunoglobulin light chain variable domains in relation to the heavy chain constant domain, which are cognitive with the human immunoglobulin light chain variable domain in relation to the light chain constant domain, or that can produce proteins from altered immunoglobulin loci containing a sufficiently diverse collection of human light chain variable gene segments. There remains a need for non-human animals that can produce immunoglobulin-like binding proteins, which include human immunoglobulin light chain variable domains linked to the heavy chain constant domain. [Overview of the Initiative] [Means for solving the problem]

[0009] A genetically modified non-human animal having an immunoglobulin locus is provided, wherein the immunoglobulin locus has multiple human light chain variable (V) operably linked to one or more non-human constant regions. L The gene segment comprises, for example, human Vκ and Jκ, or human Vλ and Jλ, and in various embodiments, the locus lacks a sequence encoding the endogenous functional ADAM6 protein. The non-human animals mentioned above include rodents, such as mice and rats.

[0010] Genes encoding light chain variable domains derived from rearrangements involving human light chain Vκ or Vλ gene segments and human Jκ or Jλ gene segments, and in various embodiments, additionally, D HLoci are provided that can rearrange and form gene segments, and in various embodiments, the locus lacks an endogenous functional ADAM6 gene or a functional fragment thereof.

[0011] The modified immunoglobulin locus lacks an endogenous functional ADAM6 gene and comprises human V segments operably linked to a human immunoglobulin sequence, e.g., a human (or human / non-human chimeric) or non-human immunoglobulin constant sequence (and, e.g., operably linked to a V segment and / or a J segment). L Loci that include a plurality of V gene segments are provided. L Modified loci are provided that include a plurality of V gene segments and a heterologous nucleotide sequence encoding an ADAM6 protein or a fragment thereof that is functional in a non-human animal. One or more D segments and / or one or more J or J segments operably linked to a non-human immunoglobulin constant sequence and a plurality of V gene segments, e.g., a rodent (e.g., mouse or rat) sequence or a human sequence, are provided. H Segments and / or one or more J L Or J H Modified loci are provided that include a plurality of V gene segments operably linked to human D and / or human J gene segments, e.g., a rodent (e.g., mouse or rat) sequence or a human sequence. Non-human animals that include such humanized loci are also provided, and the non-human animals exhibit wild-type fertility. L Non-human animals are provided that include an immunoglobulin heavy chain variable gene locus that includes a plurality of human V gene segments operably linked to human D and / or human J gene segments (either on a transgene or as an insertion or replacement in an endogenous non-human animal heavy chain variable locus). In various embodiments, the plurality of human V

[0012] gene segments. L gene segments. LThe gene segment is operably linked to one or more human D and / or one or more human J gene segments at the endogenous immunoglobulin heavy chain variable gene locus of the non-human animal. In various embodiments, the non-human animal further comprises an ectopic nucleotide sequence encoding the ADAM6 protein or its homolog or ortholog, which is functional in the male non-human animal containing the modified heavy chain locus. In various embodiments, the ectopic nucleotide sequence comprises at least one human V L Segment, D H gene segment, or J L It is in close proximity to a gene segment. In various embodiments, the ectopic nucleotide sequence is in close proximity to a non-immunoglobulin sequence in the genome of the non-human animal. In one embodiment, the ectopic nucleotide sequence is on the same chromosome as the modified heavy chain locus. In one embodiment, the ectopic nucleotide sequence is on a different chromosome than the modified heavy chain locus.

[0013] Non-human animals, all or substantially all endogenous immunoglobulin V H The immunoglobulin heavy chain variable region locus is modified to delete a segment (e.g., all functional segments, or almost all functional segments), and the endogenous immunoglobulin heavy chain variable region locus of the non-human animal is D H and J segment or J L Multiple human V cells operably linked to a gene segment L Non-human animals containing a gene segment are provided. Non-human animals containing such a gene locus and lacking the endogenous ADAM6 gene are also provided.

[0014] A method is provided for generating human immunoglobulin sequences in non-human animals. In various embodiments, the human immunoglobulin sequence is rearranged and operably linked to a constant region of the immunoglobulin heavy chain. L A repertoire of immunoglobulin heavy chain sequences containing gene segments, e.g., VL , and one or more D H , and J segments, or one or more J L The method is derived from a segment. A method is provided for generating human immunoglobulin sequences in non-human animals, tissues, and cells, the human immunoglobulin sequences which bind to a target antigen.

[0015] In one embodiment, nucleic acid constructs, cells, embryos, rodents (e.g., mice), and methods are provided for constructing a rodent (e.g., mouse) comprising a modification (e.g., knockout or deletion of the endogenous ADAM6 gene) that produces a non-functional endogenous rodent (e.g., mouse) ADAM6 protein or ADAM6 gene, wherein the rodent (e.g., mouse) comprises a nucleic acid sequence encoding the ADAM6 protein or its orthologue, homolog, or fragment, which is functional in male rodents (e.g., mice) of the same species. In one embodiment, the mouse comprises an ectopic nucleotide sequence encoding the rodent ADAM6 protein or its orthologue, homolog, or functional fragment; in a particular embodiment, the rodent ADAM6 protein is the mouse ADAM6 protein. In one embodiment, the mouse comprises an ectopic nucleotide sequence encoding one or more rodent ADAM6 proteins, wherein the one or more proteins comprise Sequence ID No. 1 or Sequence ID No. 2, or a fragment thereof, which is functional in the mouse.

[0016] In various embodiments, the sequence encoding ADAM6 activity is adjacent to a human immunoglobulin sequence. In various embodiments, the sequence encoding ADAM6 activity is adjacent to a non-human immunoglobulin sequence. In various embodiments, the sequence is located on the same chromosome as the endogenous non-human immunoglobulin heavy chain locus of the non-human animal. In various embodiments, the sequence is located on a different chromosome from the immunoglobulin heavy chain locus of the non-human animal.

[0017] The genetically modified non-human animal includes modifications that maintain the activity of the ADAM6 gene or its homolog or ortholog, the modifications including the insertion of one or more human immunoglobulin light chain gene segments upstream of the non-human immunoglobulin heavy chain constant region, and the non-human animal further includes modifications that enable the expression of a human immunoglobulin light chain variable region of the same origin as the human immunoglobulin light chain variable region. In various embodiments, the human immunoglobulin λ light chain variable region is expressed in relation to the constant regions of the light chain and heavy chain.

[0018] In various embodiments, the insertion of one or more human immunoglobulin light chain gene segments is performed at the 3' or downstream of the ADAM6 gene of the non-human animal. In various embodiments, the insertion of one or more human immunoglobulin light chain gene segments is performed such that the ADAM6 gene(s) of the non-human animal are not disrupted, deleted, and / or functionally silenced, so that the ADAM6 activity of the non-human animal is at the same or equivalent level as that of a non-human animal not containing such insertion. Exemplary disruption, deletion, and / or functionally silenced modifications include any modifications that result in a reduction, erasure, and / or loss of the activity of the ADAM6 protein(s) encoded by the ADAM6 gene(s) of the non-human animal.

[0019] In one embodiment, nucleic acid constructs, cells, embryos, mice, and methods are provided for producing mice comprising a modification of an endogenous immunoglobulin locus, wherein the mice contain the ADAM6 protein or its orthologue, homolog, or fragment, which is functional in male mice. In one embodiment, the endogenous immunoglobulin locus is an immunoglobulin heavy chain locus, and the modification reduces or eliminates the ADAM6 activity of cells or tissues of male mice.

[0020] In one embodiment, a mouse is provided comprising an ectopic nucleotide sequence encoding mouse ADAM6 or its ortholog, homolog, or functional fragment; also provided a mouse comprising an endogenous nucleotide sequence encoding mouse ADAM6 or its ortholog, homolog, or functional fragment, and comprising at least one genetic modification of the heavy chain immunoglobulin locus. In one embodiment, the endogenous nucleotide sequence encoding mouse ADAM6 or its ortholog, homolog, or functional fragment is located in an ectopic position compared to the endogenous ADAM6 gene in wild-type mice.

[0021] In one embodiment, a method is provided for producing mice comprising a modification of the endogenous immunoglobulin locus, wherein the mice contain the ADAM6 protein or its ortholog, homolog, or fragment, which is functional in male mice. In various embodiments, the modification comprises one or more human V proteins at the endogenous immunoglobulin locus. L Includes insertion of gene segments.

[0022] In one embodiment, a method is provided for producing a mouse containing a genetically modified heavy chain immunoglobulin locus, and by applying these methods, a male mouse containing the modified heavy chain immunoglobulin locus (or deletion thereof) is obtained, which can produce offspring by mating. In one embodiment, the male mouse can produce sperm that can travel from the mouse uterus through the mouse fallopian tube to fertilize a mouse egg.

[0023] In one embodiment, a method is provided for producing mice comprising genetic modifications of immunoglobulin heavy chain loci and immunoglobulin light chain loci, wherein the application of these methods to modify the heavy chain loci results in male mice exhibiting reduced fertility, and the mice comprising genetic modifications that restore all or part of the reduced fertility. In various embodiments, the reduced fertility is characterized by the inability of the male mouse's sperm to migrate from the mouse uterus through the mouse fallopian tubes to fertilize mouse eggs. In various embodiments, the reduced fertility is characterized by sperm exhibiting an in vivo migration defect. In various embodiments, the genetic modification that restores all or part of the reduced fertility is a nucleic acid sequence encoding the mouse ADAM6 gene or its ortholog, homolog, or fragment, which is functional in male mice.

[0024] In one embodiment, the gene modification includes replacing the endogenous immunoglobulin heavy chain variable locus with the immunoglobulin light chain variable locus of another species (e.g., a non-mouse species). In one embodiment, the gene modification includes inserting the immunoglobulin light chain variable locus of another species (e.g., a non-mouse species) into the endogenous immunoglobulin heavy chain variable locus. In a particular embodiment, the species is human. In one embodiment, the gene modification includes the deletion of all or part of the endogenous immunoglobulin heavy chain variable locus, which 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.

[0025] In one embodiment, the gene modification includes inactivation of all or part of an endogenous non-human immunoglobulin heavy chain variable locus, which does not result in loss of endogenous ADAM6 function. Inactivation may include replacement or deletion of one or more endogenous non-human gene segments resulting in an endogenous non-human immunoglobulin heavy chain locus that cannot be substantially rearranged to encode the antibody heavy chain containing the endogenous non-human gene segment. Inactivation may include other modifications that prevent the endogenous immunoglobulin heavy chain locus from being rearranged to encode the antibody heavy chain, which do not involve replacement or deletion of the endogenous gene segment. Exemplary modifications include chromosomal inversions and / or translocations using molecular techniques, for example, precise placement of site-directed recombination sites (e.g., Cre-lox technique). Other exemplary modifications include disabling the operable linkage between the non-human immunoglobulin variable gene segment and the non-human immunoglobulin constant region.

[0026] In one embodiment, the gene modification operably links one or more constant region sequences (e.g., IgM and / or IgG genes) to one or more human V genes of another species (e.g., a non-mouse species) in the genome of the non-human animal. L Gene segment, and one or more human J L Gene segments, and, if necessary, 1 or more human D H The method includes inserting a DNA fragment containing a gene segment. In one embodiment, the DNA fragment can be rearranged in the genome of the non-human animal to form a sequence encoding a light chain variable domain operably linked to a heavy chain constant region. In one embodiment, the species is human. In one embodiment, the gene modification includes inserting one or more human immunoglobulin light chain gene segments downstream or into the 3' of the endogenous ADAM6 gene of the non-human animal such that the ADAM6 activity (e.g., expression and / or function of the encoded protein) is the same as or equivalent to that of a non-human animal without the insertion.

[0027] In one embodiment, a method is provided for producing a mouse comprising a genetic modification of an immunoglobulin heavy chain locus, wherein the application of these methods yields a male mouse comprising the modified heavy chain immunoglobulin locus (or deletion thereof), the male mouse exhibiting reduced fertility, and the mouse comprising a genetic modification that restores all or part of the reduced fertility. In various embodiments, the reduced fertility is characterized by the inability of the male mouse's sperm to migrate from the mouse uterus through the mouse fallopian tube to fertilize a mouse egg. In various embodiments, the reduced fertility is characterized by sperm exhibiting an in vivo migration defect. In various embodiments, the genetic modification that restores all or part of the reduced fertility is a nucleic acid sequence encoding the mouse ADAM6 gene or its ortholog, homolog, or fragment, which is functional in the male mouse.

[0028] In one embodiment, the gene modification involves converting an endogenous immunoglobulin heavy chain variable locus to an immunoglobulin light chain variable locus, for example, one or more light chain variable (V) loci of another species (e.g., a non-mouse species). L ) Gene segment, one or more heavy chain variability (D H ) gene segment and one or more linked (J) gene segments, or one or more light chain linked (J) L ) Includes replacing with a gene segment. In one embodiment, the gene modification is a single orthologous immunoglobulin light chain variable locus V L gene segment, at least one D H A gene segment and at least one J gene segment, or at least one J LThe modification includes the insertion of a gene segment into the endogenous immunoglobulin heavy chain variable locus. In certain embodiments, the species is human. In one embodiment, the gene modification includes the deletion of all or part of the endogenous immunoglobulin heavy chain variable locus, the deletion resulting in a loss of endogenous ADAM6 function. In certain embodiments, the loss of endogenous ADAM6 function is associated with reduced fertility in male mice. In one embodiment, the gene modification includes the inactivation of all or part of the endogenous immunoglobulin heavy chain variable locus, the deletion of which does not result in a loss of endogenous ADAM6 function. Inactivation may include the replacement or deletion of one or more endogenous gene segments that result in an endogenous immunoglobulin heavy chain locus that is substantially incapable of rearranging to encode an antibody heavy chain containing the endogenous gene segment. Inactivation may include other modifications that prevent the endogenous immunoglobulin heavy chain locus from being rearranged to encode the antibody heavy chain, and such modifications do not involve replacement or deletion of an endogenous gene segment. Exemplary modifications include chromosomal inversions and / or alterations that result in a heavy chain locus that is not operably linked to one or more endogenous constant regions.

[0029] In one embodiment, the gene modification is operably linked to one or more constant region sequences (e.g., IgM gene and / or IgG gene) of one or more human V genes of another species (e.g., non-mouse species). L The process involves inserting a DNA fragment containing a gene segment, one or more J gene segments, and optionally one or more D gene segments into the genome of the mouse. In various embodiments, the J gene segment contains J H Gene segment or J LExamples include gene segments. In one embodiment, the DNA fragment can be rearranged to form a sequence encoding an antibody heavy chain, the heavy chain comprising a rearranged human light chain variable gene segment fused to the heavy chain constant region. In one embodiment, the gene modification comprises at least 6, at least 16, at least 30, or at least 40, or more human V L A gene segment, and at least one or at least five human J L This includes the insertion of a gene segment into the genome of the mouse described above. In a particular embodiment, the species is human, and the gene segment is the human κ light chain gene segment. In one embodiment, the gene modification includes the deletion of all or part of the endogenous immunoglobulin heavy chain variable locus, such that the endogenous immunoglobulin heavy chain locus becomes non-functional, and the deletion further 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.

[0030] In one embodiment, a mouse is provided having a modification that reduces or eliminates mouse ADAM6 expression from an endogenous ADAM6 allele, such that a male mouse exhibits reduced fertility (e.g., a significant reduction in the ability to produce offspring through mating) or is essentially infertile as a result of the reduction or elimination of endogenous ADAM6 function, and further comprising an ectopic ADAM6 sequence or its homolog, orthologue, or functional fragment. In one embodiment, the modification that reduces or eliminates mouse ADAM6 expression is a modification of a mouse immunoglobulin locus (e.g., insertion, deletion, substitution, etc.). In one embodiment, the immunoglobulin locus is an immunoglobulin heavy chain locus.

[0031] In one embodiment, reduction or loss of ADAM6 function includes the inability, or substantial inability, for a mouse to produce sperm capable of traveling from the mouse uterus through the mouse fallopian tubes to fertilize mouse eggs. In certain embodiments, at least about 95%, 96%, 97%, 98%, or 99% of the spermatids produced in the ejaculatory volume of the mouse are unable to travel through the fallopian tubes in vivo after mating and are unable to fertilize mouse eggs.

[0032] In one embodiment, reduction or loss of ADAM6 function includes the inability or substantial inability to form ADAM2 and / or ADAM3 and / or ADAM6 complexes on the surface of the mouse spermatogonial cells. In one embodiment, loss of ADAM6 function includes the substantial inability to fertilize mouse oocytes by mating with female mice.

[0033] In one embodiment, the present invention provides a mouse lacking the functionally endogenous ADAM6 gene, the mouse comprising 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 includes enhanced fertility compared to a mouse lacking the functionally endogenous ADAM6 gene.

[0034] In one embodiment, the protein is encoded by a genomic sequence located within an immunoglobulin locus in the mouse germline. In a particular embodiment, the immunoglobulin locus is a heavy chain locus. In another particular embodiment, the heavy chain locus is at least one human V H , at least one human D H and at least one human J H Includes a gene segment. In another specific embodiment, the heavy chain locus is at least one human V L and at least one human J LIncludes a gene segment. In another specific embodiment, the heavy chain locus is at least one human V L , at least one human D H , and at least one human J L This includes. In another specific embodiment, the heavy chain locus is at least one human V L , at least one human D H , and at least one human J H Includes a gene segment. In another specific embodiment, the heavy chain locus is at least one human V L and at least one human J L Includes a gene segment. In another specific embodiment, the heavy chain locus is at least one human V L and at least one human J H The heavy chain locus includes a gene segment. In another specific embodiment, the heavy chain locus includes six human Vκ and five human Jκ gene segments. In another specific embodiment, the heavy chain locus includes sixteen human Vκ and five human Jκ gene segments. In another specific embodiment, the heavy chain locus includes thirty human Vκ and five human Jκ gene segments. In another specific embodiment, the heavy chain locus includes forty human Vκ and five human Jκ gene segments.

[0035] In one embodiment, the ectopic protein is encoded by a genomic sequence located within a non-immunoglobulin locus in the mouse germline. In one embodiment, the non-immunoglobulin locus is a transcriptionally active locus. In a particular embodiment, the transcriptionally active locus is a ROSA 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 germ tissue. In one embodiment, the protein is encoded by a genomic sequence randomly inserted into the mouse germline.

[0036] 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 particular embodiment, the mouse comprises a transgene containing a chimeric human variable / rat or mouse constant light chain gene operably linked to a transcriptional activity promoter, e.g., the ROSA26 promoter. In a further particular embodiment, the chimeric human / mouse or rat light chain transgene comprises a rearranged human light chain variable region sequence within the germline of the mouse.

[0037] In one embodiment, the ectopic nucleotide sequence is located within an immunoglobulin locus in the mouse germline. In a particular embodiment, the immunoglobulin locus is a heavy chain locus. In one embodiment, the heavy chain locus is located within at least one human V L and at least one human J L The gene segment is included. In a particular embodiment, the heavy chain locus comprises at least six, up to 40, human Vκ gene segments and five human Jκ gene segments. In one embodiment, the ectopic nucleotide sequence is located within a non-immunoglobulin locus in the mouse germline. 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 mouse germline.

[0038] In one embodiment, a mouse lacking the functionally endogenous ADAM6 gene is provided, comprising an ectopic nucleotide sequence that compensates for the loss of mouse ADAM6 function. In one embodiment, the ectopic nucleotide sequence confers to the mouse the ability to produce offspring comparable to that of a corresponding wild-type mouse containing the functionally endogenous ADAM6 gene. In one embodiment, the sequence confers to the mouse the ability to form a complex of ADAM2 and / or ADAM3 and / or ADAM6 on the surface of the mouse's spermatogonial cells. In one embodiment, the sequence confers to the mouse the ability to travel from the mouse uterus through the mouse fallopian tube to a mouse oocyte and fertilize the oocyte.

[0039] In one embodiment, a mouse lacking the functional endogenous ADAM6 gene and containing the ectopic nucleotide sequence produces at least about 50%, 60%, 70%, 80%, or 90% of the number of littermates produced by a wild-type mouse of the same age and strain over a period of six months.

[0040] In one embodiment, mice lacking the functional endogenous ADAM6 gene and containing the ectopic nucleotide sequence, when mated over a period of 6 months, produce 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 mice of the same age and same or similar strain lacking the functional endogenous ADAM6 gene and the ectopic nucleotide sequence, when mated under substantially the same conditions over substantially the same period.

[0041] In one embodiment, mice lacking the functional endogenous ADAM6 gene and containing the ectopic nucleotide sequence produce, on average, at least two, three, or four times more offspring per litter during a mating period of four or six months compared to mice lacking the functional endogenous ADAM6 gene and the ectopic nucleotide sequence that are mated for the same period.

[0042] In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and containing the ectopic nucleotide sequence is a male mouse that, when retrieved from the oviduct about 5-6 hours after mating, produces sperm exhibiting tubal migration 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 or more compared to a mouse lacking the functional endogenous ADAM6 gene and the ectopic nucleotide sequence.

[0043] In one embodiment, a mouse lacking the functional endogenous ADAM6 gene and containing the ectopic nucleotide sequence produces sperm that, when mated with a female mouse, can pass through the uterus and enter and pass through the fallopian tubes within approximately 6 hours with nearly the same efficiency as sperm from wild-type mice.

[0044] In one embodiment, mice lacking the functional endogenous ADAM6 gene and containing the ectopic nucleotide sequence produce approximately 1.5 times, 2 times, 3 times, or 4 times more littermates within a comparable period compared to mice lacking both the functional ADAM6 gene and the ectopic nucleotide sequence.

[0045] In one embodiment, a mouse is provided having a non-mouse nucleic acid sequence encoding an immunoglobulin protein within its germline, wherein the non-mouse immunoglobulin sequence includes an insertion of the mouse ADAM6 gene or its homolog, orthologue, or functional fragment. In one embodiment, the non-mouse immunoglobulin sequence includes a human immunoglobulin sequence. In one embodiment, the sequence includes a human immunoglobulin heavy chain sequence. In one embodiment, the sequence includes a human immunoglobulin light chain sequence. In one embodiment, the sequence includes a human heavy chain sequence adjacent to the human light chain sequence. In one embodiment, the sequence includes one or more V gene segments, one or more D gene segments, and one or more J gene segments; in one embodiment, the sequence includes 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 one or more V and J gene segments, are unrearranged. In one embodiment, the one or more V, D, and J gene segments, or one or more V and J gene segments, are rearranged. In one embodiment, after rearrangement of one or more V, D, and J gene segments, or one or more V and J gene segments, the mouse contains in its genome at least one nucleic acid sequence encoding the mouse ADAM6 gene or its homolog, ortholog, or functional fragment. In one embodiment, after rearrangement, the mouse contains in its genome at least two nucleic acid sequences encoding the mouse ADAM6 gene or its homolog, ortholog, or functional fragment. In one embodiment, after rearrangement, the mouse contains in its genome at least one nucleic acid sequence encoding the mouse ADAM6 gene or its homolog, ortholog, or functional fragment. In one embodiment, the mouse contains the ADAM6 gene or its homolog, ortholog, or functional fragment in its B cells.In one embodiment, the mouse contains the ADAM6 gene or its homolog, ortholog, or functional fragment within non-B cells.

[0046] In one embodiment, a mouse is provided that expresses a human immunoglobulin heavy chain variable region or a functional fragment thereof from an endogenous immunoglobulin heavy chain locus, and which contains ADAM6 activity that is functional in male mice. In one embodiment, the heavy chain locus is one or more human V L Gene segment and one or more J L Gene segment, and, if necessary, 1 or more D H The heavy chain locus includes gene segments. In one embodiment, the heavy chain locus includes at least 6 human Vκ gene segments and 5 human Jκ gene segments. In one embodiment, the heavy chain locus includes at least 16 human Vκ gene segments and 5 human Jκ gene segments. In one embodiment, the heavy chain locus includes at least 30 human Vκ gene segments and 5 human Jκ gene segments. In one embodiment, the heavy chain locus includes at least 40 human Vκ gene segments and 5 human Jκ gene segments.

[0047] In one embodiment, the present invention provides a mouse expressing a human immunoglobulin light chain variable region or a functional fragment thereof from an endogenous immunoglobulin heavy chain locus, the mouse comprising ADAM6 activity that is functional in male mice.

[0048] In one embodiment, the male mouse contains a single unmodified endogenous ADAM6 allele or its ortholog, homolog, or functional fragment at the endogenous ADAM6 locus.

[0049] In one embodiment, the male mouse comprises an ectopic mouse ADAM6 sequence or its homolog, ortholog, or functional fragment that encodes a protein conferring ADAM6 function.

[0050] In one embodiment, the male mouse contains an ADAM6 sequence or its homolog, orthologue, or functional fragment at a location in the mouse genome close to the location of the endogenous ADAM6 allele (e.g., 3' of the last V gene segment sequence and 5' of the first J gene segment).

[0051] In one embodiment, the male mouse contains an ADAM6 sequence or its homolog, orthologue, or functional fragment at a location in the mouse genome different from the location of the endogenous ADAM6 allele (e.g., 5' of the 5' end of the V gene segment sequence of the V gene locus).

[0052] In one embodiment, the male mouse contains an ADAM6 sequence or its homolog, orthologue, or functional fragment adjacent to (with respect to the transcription direction of the ADAM6 sequence) the nucleic acid sequences encoding the immunoglobulin V gene segment and / or the immunoglobulin J gene segment. In a particular embodiment, the immunoglobulin V and J gene segments are human gene segments. In one embodiment, the immunoglobulin V gene segment and J gene segment are human gene segments, and the sequence encoding functional mouse ADAM6 or its orthologue, homolog, or fragment in a mouse is located between the human V gene segment and the J gene segment; in one embodiment, the mouse comprises two or more human V gene segments, and the sequence is located at the 5' position of the furthest 5' human V gene segment; in one embodiment, the mouse comprises two or more human V gene segments, and the sequence is located between the last V gene segment and the second to last V gene segment; in one embodiment, the mouse comprises a plurality of human V gene segments, and the sequence is located upstream of the furthest 5' human V gene segment; in one embodiment, the mouse further comprises a D gene segment, and the sequence is located following the furthest 3' V gene segment and the furthest 5' D gene segment, and in one embodiment, the sequence is located between the V gene segment and the J gene segment.

[0053] In one embodiment, the human V gene segment is a light chain V gene segment. In a particular embodiment, the light chain V gene segment is a Vκ gene segment. In another particular embodiment, the light chain V gene segment is a Vλ gene segment. In one embodiment, the J gene segment is a J H Gene segment and J L Selected from gene segments. In a particular embodiment, the above J LThe gene segment is the Jκ gene segment. In another specific embodiment, the above J L The gene segment is the Jλ gene segment.

[0054] In one embodiment, the male mouse includes an ADAM6 sequence or its homolog, orthologue, or functional fragment located at the same or substantially the same location within an endogenous immunoglobulin locus as that of a wild-type male mouse. In a particular embodiment, the endogenous locus is the heavy chain variable region of the antibody and does not include or encode a variable region derived from an endogenous non-human gene segment. In a particular embodiment, the endogenous locus is located at a genomic position in the male mouse such that the heavy chain gene segment of the locus cannot encode the heavy chain variable region of the antibody in the male mouse. In various embodiments, the male mouse includes an ADAM6 sequence located on the same chromosome as a human immunoglobulin gene segment, and the ADAM6 sequence encodes a functional ADAM6 protein.

[0055] In one embodiment, a male mouse is provided whose germline contains a non-functional endogenous ADAM6 gene or a deletion of the endogenous ADAM6 gene, the spermatids of which can pass through the fallopian tubes of female mice and fertilize their eggs. In one embodiment, the mouse contains an extrachromosomal copy of the functional mouse ADAM6 gene or its ortholog, homolog, or functional fragment in the male mouse. In one embodiment, the mouse contains an ectopic mouse ADAM6 gene or its ortholog, homolog, or functional fragment that is functional in the male mouse.

[0056] In one embodiment, a male mouse is provided comprising modifications to the functional endogenous ADAM6 gene and the endogenous immunoglobulin heavy chain gene locus. In one embodiment, the modification is made downstream or 3' of the endogenous ADAM6 gene or locus. In one embodiment, the modification is the replacement of one or more endogenous immunoglobulin heavy chain gene segments with one or more human immunoglobulin light chain gene segments. In one embodiment, the modification is the insertion of one or more human immunoglobulin light chain gene segments upstream of the endogenous immunoglobulin heavy chain constant region gene.

[0057] In one embodiment, the present invention provides a mouse comprising a genetic modification that reduces endogenous ADAM6 function, wherein the mouse possesses at least some ADAM6 functionality, which is brought about by expression of an endogenous unmodified allele that is fully or partially functional (e.g., a heterozygote) or by expression from an ectopic sequence encoding ADAM6 or its ortholog, homolog, or functional fragment that is functional in male mice. In various embodiments, the ADAM6 or its ortholog, homolog, or functional fragment comprises a nucleic acid sequence encoding the ADAM6 protein as shown in SEQ ID NO: 1, SEQ ID NO: 2, or a combination thereof.

[0058] In one embodiment, the mouse contains sufficient ADAM6 function to confer the ability to produce offspring through mating to a male mouse compared to a male mouse lacking functional ADAM6. In one embodiment, the ADAM6 function is conferred by the presence of an ectopic nucleotide sequence encoding mouse ADAM6 or its homolog, orthologue, or functional fragment. In one embodiment, the ADAM6 function is conferred by an endogenous ADAM6 gene located at an endogenous immunoglobulin locus, where the mouse is unable to express an antibody containing the endogenous immunoglobulin heavy chain locus segment. Functional ADAM6 homologs, orthologues, or fragments in male mice include those that fully or partially restore the loss of offspring production ability observed in male mice lacking sufficient endogenous ADAM6 activity, for example, the loss of ability observed in ADAM6 knockout mice. In this sense, ADAM6 knockout mice include mice that contain the endogenous locus or a fragment thereof but are non-functional, i.e., mice that do not express ADAM6 (ADAM6a and / or ADAM6b) at all, or mice that express ADAM6 (ADAM6a and / or ADAM6b) at levels insufficient to support the essentially normal reproductive capacity of wild-type male mice. Loss of function may result, for example, from alteration of the structural gene (i.e., the ADAM6a or ADAM6b coding region) of the locus, or from alteration of the regulatory region of the locus (e.g., the 5' sequence of the ADAM6a gene or the 3' sequence of the ADAM6a or ADAM6b coding region, which controls all or part of the transcription of the ADAM6 gene, the expression of ADAM6 RNA, or the expression of ADAM6 protein). In various embodiments, an orthologue or homolog or fragment thereof that is functional in male mice allows the sperm (or the majority of spermatids in the ejaculate) of male mice to pass through the mouse oviduct and fertilize mouse eggs.

[0059] In one embodiment, a male mouse expressing the human immunoglobulin variable region or a functional fragment thereof contains sufficient ADAM6 activity to confer to the male mouse the ability to produce offspring by mating with a female mouse, and in one embodiment, when mated with a female mouse, the male mouse exhibits a procreative capacity that is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and in one embodiment approximately the same as the procreative capacity of a mouse having one or two endogenous unmodified ADAM6 alleles.

[0060] In one embodiment, a male mouse expresses sufficient ADAM6 (or its ortholog, homolog, or functional fragment) to enable sperm cells from the male mouse to pass through the ovary of a female mouse and fertilize the mouse egg.

[0061] In one embodiment, the ADAM6 functionality is conferred by a nucleic acid sequence adjacent to the mouse chromosome sequence (for example, the nucleic acid is randomly incorporated into the mouse chromosome; or, for example, the nucleic acid is targeted to a specific location, for example, by site-directed recombinase-mediated (e.g., Cre-mediated) insertion or homologous recombination). In one embodiment, the ADAM6 sequence resides on a nucleic acid different from the mouse chromosome (for example, the ADAM6 sequence resides on an episome, i.e., outside the chromosome, for example, in an expression construct, vector, YAC, transchromosome, etc.).

[0062] In one embodiment, genetically modified mice and cells are provided, comprising a modification of an endogenous heavy chain immunoglobulin locus, wherein the mice express at least a portion of an immunoglobulin light chain sequence, for example, at least a portion of a human sequence, and the mice contain ADAM6 activity that is functional in male mice. In one embodiment, the modification reduces or eliminates the ADAM6 activity of the mice. In one embodiment, the mice are modified so that both alleles encoding ADAM6 activity are absent or express ADAM6 that is substantially non-functional to support normal mating in male mice. In one embodiment, the mice further comprise an ectopic nucleic acid sequence encoding mouse ADAM6 or its ortholog, homolog, or functional fragment. In one embodiment, the modification maintains the ADAM6 activity of the mice and prevents the endogenous immunoglobulin heavy chain locus from encoding the heavy chain of an antibody. In certain embodiments, the modifications include chromosomal inversions and / or translocations that prevent the endogenous immunoglobulin heavy chain locus from rearranging to encode the variable region of the antibody heavy chain.

[0063] In one embodiment, genetically modified mice and cells are provided, comprising a modification of an endogenous immunoglobulin heavy chain locus, the modification reducing or eliminating ADAM6 activity expressed from the ADAM6 sequence of the locus, and the mouse comprising the ADAM6 protein or its orthologue, homolog, or functional fragment. In various embodiments, the ADAM6 protein or fragment is encoded by an ectopic ADAM6 sequence. In various embodiments, the ADAM6 protein or fragment is expressed from an endogenous ADAM6 allele. In various embodiments, the mouse comprises a first heavy chain allele comprising a first modification that reduces or eliminates the expression of functional ADAM6 from a first heavy chain allele, and a second heavy chain allele comprising a second modification that does not substantially reduce or eliminate the expression of functional ADAM6 from a second heavy chain allele.

[0064] In various embodiments, the modification involves inserting one or more human immunoglobulin light chain gene segments upstream of or into the 5' position of an endogenous immunoglobulin heavy chain constant region gene. In various embodiments, the modification maintains the endogenous ADAM6 gene located at the endogenous immunoglobulin heavy chain locus.

[0065] In one embodiment, the second modification described above is located at 3' of the last mouse V gene segment (relative to the transcriptional orientation of the mouse V gene segment), and the mouse (or chimeric human / mouse) immunoglobulin heavy chain constant gene or a fragment thereof (e.g., human and / or mouse:C H 1 and / or hinge and / or C H 2 and / or C H It is located at 5' (relative to the transcriptional directionality of the constant sequence) of the nucleic acid sequence that codes for 3.

[0066] 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 the expression of endogenous ADAM6 in a second immunoglobulin heavy chain allele at a second locus encoding functional ADAM6, the second immunoglobulin heavy chain allele comprising at least one modification of the 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 may be a deletion, a substitution in the human V, D, and / or J gene segment, a substitution in the camelid V, D, and / or J gene segment, a substitution in the humanized or camelidized V, D, and / or J gene segment, a substitution of the light chain sequence in the heavy chain sequence, and a combination thereof. In one embodiment, the at least one modification is the deletion of one or more heavy chain V, D, and / or J gene segments at the heavy chain locus and their replacement with one or more light chain V and / or J gene segments (e.g., human light chain V and / or J gene segments).

[0067] In one embodiment, the modification is in the first immunoglobulin heavy chain allele at the first locus and the second immunoglobulin heavy chain allele at the second locus, and the ADAM6 function results from ectopic ADAM6 expression at a non-immunoglobulin locus in the mouse germline. In a particular embodiment, the non-immunoglobulin locus is the ROSA26 locus. In a particular embodiment, the non-immunoglobulin locus is transcriptional activity in germ tissue.

[0068] In one embodiment, the modifications are in the first immunoglobulin heavy chain allele at the first locus and the second immunoglobulin heavy chain allele at the second locus, and the ADAM6 function arises as a result of the endogenous ADAM6 gene in the mouse germline. In a particular embodiment, the endogenous ADAM6 gene is placed in parallel with the mouse immunoglobulin heavy chain gene segment.

[0069] In one embodiment, the modification is in the first immunoglobulin heavy chain allele at the first locus and the second immunoglobulin heavy chain allele at the second locus, and the ADAM6 function results from the expression of the ectopic ADAM6 sequence in the first immunoglobulin heavy chain allele. In one embodiment, the modification is in the first immunoglobulin heavy chain allele at the first locus and the second immunoglobulin heavy chain allele at the second locus, and the ADAM6 function or activity results from the expression of the ectopic ADAM6 in the second immunoglobulin heavy chain allele.

[0070] In one embodiment, a mouse comprising a heterozygous or homozygous knockout of ADAM6 is provided. In one embodiment, the mouse further comprises a modified immunoglobulin sequence which is a human or humanized immunoglobulin sequence or a camelid or camelid human or mouse immunoglobulin sequence. In one embodiment, the modified immunoglobulin sequence is located at the endogenous immunoglobulin heavy chain locus. In one embodiment, the modified immunoglobulin sequence comprises a human light chain variable region sequence at the endogenous immunoglobulin heavy chain locus. In one embodiment, the human light chain variable region sequence replaces the endogenous heavy chain variable sequence at the endogenous immunoglobulin heavy chain locus. In one embodiment, the modified immunoglobulin sequence comprises a human κ light chain variable region sequence at the endogenous immunoglobulin heavy chain locus. In one embodiment, the modified immunoglobulin sequence comprises a human λ light chain variable region sequence at the endogenous immunoglobulin heavy chain locus.

[0071] In one embodiment, a mouse is provided that is unable to express functional endogenous ADAM6 from the endogenous ADAM6 locus. In one embodiment, the mouse comprises an ectopic nucleic acid sequence encoding 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 reproductive capacity exhibited by male mice that are homozygous for ADAM6 knockout. In a particular embodiment, the ectopic nucleic acid sequence encodes a mouse ADAM6 protein.

[0072] In one embodiment, a mouse is provided that lacks a functional endogenous ADAM6 locus and comprises an ectopic nucleic acid sequence that confers ADAM6 function to the mouse. In one embodiment, the nucleic acid sequence comprises an endogenous ADAM6 sequence or a functional fragment thereof. In one embodiment, the endogenous ADAM6 sequence comprises the 3'-most mouse immunoglobulin heavy chain V gene segment (V) in wild-type mice. H ) and the 5'-most mouse immunoglobulin heavy chain D gene segment (DH Includes ADAM6a and ADAM6b code sequences between ).

[0073] In one embodiment, the nucleic acid sequence includes a sequence encoding mouse ADAM6a or a functional fragment thereof, and / or a sequence encoding mouse ADAM6b or a functional fragment thereof, wherein ADAM6a and / or ADAM6b or its functional fragment (one or more) 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 located at the 5' end of mouse D H The first codon of the first ADAM6 gene closest to the gene segment, and the D at the 5' end. H The sequence includes a sequence between the gene segment and the recombinant signal sequence, where 5' is indicated with respect to the transcription direction of the mouse immunoglobulin gene. 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 a ubiquitin promoter.

[0074] In one embodiment, the promoter is an inductive promoter. In one embodiment, the inductive promoter regulates expression in non-germ tissues. In one embodiment, the inductive promoter regulates expression in germ tissues. In a particular embodiment, the expression of the mouse ADAM6a and / or ADAM6b sequence or its functional fragment(s) is developmentally regulated in germ tissues by the inductive promoter.

[0075] In one embodiment, the mouse ADAM6a and / or ADAM6b described above are selected from ADAM6a of sequence SEQ ID NO: 1 and / or ADAM6b of sequence SEQ ID NO: 2.

[0076] In one embodiment, the mouse ADAM6 promoter is the promoter of SEQ ID NO: 3. In a particular embodiment, the ADAM6 promoter is the nucleic acid sequence of SEQ ID NO: 3 immediately upstream of the first codon of ADAM6a (with respect to the transcription direction of ADAM6a) and includes an extending nucleic acid sequence of SEQ ID NO: 3 upstream of the ADAM6 coding region. In another particular embodiment, the ADAM6 promoter is a fragment extending from 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.

[0077] In one embodiment, the nucleic acid sequence includes Sequence ID No. 3 or a fragment thereof, which, when placed in the body of a mouse that is infertile or has low fertility due to ADAM6 deficiency, improves fertility or restores fertility to near wild-type fertility. In one embodiment, Sequence ID No. 3 or a fragment thereof confers to a male mouse the ability to produce spermatids that can pass through the oviduct of a female mouse and fertilize a mouse egg.

[0078] In one embodiment, the mouse ADAM6 promoter is the promoter of SEQ ID NO: 4. In a particular embodiment, the ADAM6 promoter is the nucleic acid sequence of SEQ ID NO: 4 immediately upstream of the first codon of ADAM6a (with respect to the transcription direction of ADAM6a) and includes an extending nucleic acid sequence of SEQ ID NO: 4 upstream of the ADAM6 coding region. In another particular embodiment, the ADAM6 promoter is a fragment extending from 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.

[0079] In one embodiment, the nucleic acid sequence includes Sequence ID No. 4 or a fragment thereof, which, when placed in the body of a mouse that is infertile or has low fertility due to ADAM6 deficiency, improves fertility or restores fertility to near wild-type fertility. In one embodiment, Sequence ID No. 4 or a fragment thereof confers to a male mouse the ability to produce spermatids that can pass through the oviduct of a female mouse and fertilize a mouse egg.

[0080] In one embodiment, the mouse ADAM6 promoter is the promoter of SEQ ID NO: 5. In a particular embodiment, the mouse ADAM6 promoter includes the nucleic acid sequence of SEQ ID NO: 5 immediately upstream of the first codon of ADAM6a (with respect to the transcription direction of ADAM6a) and extends to the end of SEQ ID NO: 5 upstream of the ADAM6 coding region. In another particular embodiment, the ADAM6 promoter is a fragment extending 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.

[0081] In one embodiment, the nucleic acid sequence includes Sequence ID No. 5 or a fragment thereof, which, when placed in the body of a mouse that is infertile or has low fertility due to ADAM6 deficiency, improves fertility or restores fertility to near wild-type fertility. In one embodiment, Sequence ID No. 5 or a fragment thereof confers to a male mouse the ability to produce spermatids that can pass through the oviduct of a female mouse and fertilize a mouse egg.

[0082] In various embodiments, the ectopic nucleic acid sequence conferring ADAM6 function to the mouse encodes one or more ADAM6 proteins, the one or more ADAM6 proteins including SEQ ID NO: 1, SEQ ID NO: 2, or a combination thereof.

[0083] In various embodiments, the ectopic nucleic acid sequence comprises a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and the ectopic nucleic acid sequence confers ADAM6 function to the mouse by one or more ADAM6 proteins encoded by the ectopic nucleic acid sequence.

[0084] In one embodiment, the nucleic acid sequence is any sequence encoding the ADAM6 gene or its homolog, orthologue, or functional fragment, which, when placed in or maintained in a mouse, produces the same or equivalent level of fertility as that of a wild-type mouse. Exemplary levels of fertility can be demonstrated by the ability of male mice to produce spermatids that can pass through the oviducts of female mice and fertilize mouse eggs.

[0085] In one embodiment, a deletion of the endogenous nucleotide sequence encoding the ADAM6 protein and endogenous V H Human V gene segment H The present invention provides a mouse comprising a gene segment replacement and an ectopic nucleotide sequence encoding the mouse ADAM6 protein or its ortholog, homolog, or fragment, which is functional in male mice.

[0086] In one embodiment, a deletion of the endogenous nucleotide sequence encoding the ADAM6 protein and endogenous V H Human V gene segment L The present invention provides a mouse comprising a gene segment replacement and an ectopic nucleotide sequence encoding the mouse ADAM6 protein or its ortholog, homolog, or fragment, which is functional in male mice. In one embodiment, the above human V L The gene segment is the Vκ gene segment. In one embodiment, the above V L The gene segment is the Vλ gene segment.

[0087] In one embodiment, the mouse is further subjected to human J LA heterologous nucleotide sequence encoding a mouse ADAM6 protein, or an ortholog, homolog, or fragment thereof, that contains a gene segment and is functional in male mice is between the human V L gene segment and the human J L gene segment. In one embodiment, the mouse contains one or more human V L gene segments, and one or more V L gene segments, and the heterologous nucleotide sequence encoding a mouse ADAM6 protein, or an ortholog, homolog, or fragment thereof, that is functional in male mice is upstream (or 5' side) of the one or more human V L gene segments. In certain embodiments, the human V L and J L gene segments are Vκ and Jκ gene segments.

[0088] In one embodiment, the mouse contains an immunoglobulin heavy chain locus that contains a deletion of an endogenous immunoglobulin locus nucleotide sequence containing an endogenous ADAM6 gene, contains a nucleotide sequence encoding one or more human immunoglobulin gene segments, and the heterologous nucleotide sequence encoding the mouse ADAM6 protein is within or immediately adjacent to the nucleotide sequence encoding the one or more human immunoglobulin gene segments.

[0089] In one embodiment, the mouse contains replacement of all or substantially all endogenous V L gene segments with a nucleotide sequence encoding one or more human V H gene segments, and the heterologous nucleotide sequence encoding the mouse ADAM6 protein is within the nucleotide sequence encoding the one or more human V L gene segments or the one or more human V LIs directly adjacent to the nucleotide sequence encoding the gene segment. In one embodiment, the mouse has one or more endogenous D H gene segments at the endogenous D L gene locus replaced by one or more human V L and / or human J L gene segments. In one embodiment, the mouse further comprises replacement of one or more endogenous J H gene segments at the endogenous J H gene locus with one or more human J L gene segments. In one embodiment, the mouse comprises replacement of all or substantially all of the endogenous V H , D H and J H gene segments, as well as replacement of the endogenous V H , D H and J H gene segments at the endogenous V L ]>and J L gene locus with human V L and J L gene segments, wherein the mouse comprises a heterologous sequence encoding the mouse ADAM6 protein. In one embodiment, the mouse comprises insertion of one or more human V L and J L gene segments into the endogenous immunoglobulin heavy chain locus, and the mouse comprises an ADAM6 gene that is functional in the mouse. In certain embodiments, the human V L and J L gene segments are Vκ and Jκ gene segments. In certain embodiments, the heterologous sequence encoding the mouse ADAM6 protein is located between the second V L gene segment from the most 3'-terminal end of the existing human V L gene segment and the ultimate most 5'-terminal JL The V at the 5' end of the gene segment L It is located upstream (or 5' side) of the gene segment. In certain embodiments, the above mouse is all or substantially all mouse V H Deletion of gene segment, and at least 40 human V L Including substitutions in the gene segment, the ectopic nucleotide sequence encoding the above mouse ADAM6 protein is human V κ 4-1 is located upstream of the human Jκ1 gene segment. In certain embodiments, the above mouse is all or substantially all mouse V H Deletion of gene segment, and at least 40 human V L The ectopic nucleotide sequence encoding the mouse ADAM6 protein, including substitutions in the gene segment, is located upstream of the human Vκ2-40 gene segment.

[0090] In a particular embodiment, the mouse is one or more human V L In the nucleotide sequence encoding the gene segment, all or substantially all endogenous V H The ectopic nucleotide sequences encoding the mouse ADAM6 protein, including the substitution of gene segments, are one or more human V L The nucleotides encoding the gene segment are located within one or more human V L It is directly adjacent to the nucleotide that codes for the gene segment.

[0091] In one embodiment, the above V L The gene segment is the Vκ gene segment. In one embodiment, the above V L The gene segment is the Vλ gene segment.

[0092] In one embodiment, the ectopic nucleotide sequence encoding the mouse ADAM6 protein is located on a transgene within the mouse genome. In one embodiment, the ectopic nucleotide sequence encoding the mouse ADAM6 protein is located outside the mouse chromosome.

[0093] In one embodiment, a mouse is provided comprising a modification of the endogenous heavy chain immunoglobulin locus, expressing B cells comprising a rearranged immunoglobulin sequence operably linked to a heavy chain constant region gene sequence, wherein the B cells contain in their genome (e.g., on the B cell chromosome) a gene encoding ADAM6 or its orthologue, homolog, or fragment that is functional in male mice. In one embodiment, the rearranged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises human light chain V, J, and optionally D gene sequences; mouse heavy chain V, D, and / or J sequences; and human or mouse light chain V and / or J sequences. In one embodiment, the heavy chain constant region gene sequence is C H 1. Hinge, C H 2, C H It includes a human or mouse heavy chain sequence selected from the group consisting of 3 and combinations thereof.

[0094] In one embodiment, the human light chain V and / or J sequence is selected from human Vκ, Vλ, Jκ, and Jλ sequences.

[0095] In one embodiment, a mouse is provided that contains a functionally silenced endogenous immunoglobulin heavy chain locus, wherein ADAM6 function is maintained within the mouse, and further comprises the insertion of one or more human immunoglobulin gene segments, wherein the one or more human immunoglobulin gene segments are human V L and the JL gene segment, and human D as needed. H The gene segment is included. In one embodiment, the one or more human immunoglobulin gene segments include human Vκ, Vλ, Jκ, and Jλ gene sequences.

[0096] In one embodiment, the present invention provides a genetically modified mouse comprising a functionally silenced immunoglobulin light chain gene, further comprising the replacement of one or more endogenous immunoglobulin heavy chain variable region gene segments with one or more human immunoglobulin light chain variable region gene segments; lacking a functional endogenous ADAM6 locus; and comprising an ectopic nucleotide sequence expressing a mouse ADAM6 protein or its ortholog, homolog, or fragment that is functional in male mice.

[0097] In one embodiment, the present invention provides a genetically modified mouse comprising a functionally silenced immunoglobulin light chain gene locus, further comprising the replacement of one or more endogenous immunoglobulin light chain variable gene segments with one or more human immunoglobulin light chain variable gene segments; lacking a functional endogenous ADAM6 locus; and comprising an ectopic nucleotide sequence encoding a mouse ADAM6 protein or its ortholog, homolog, or fragment that is functional in male mice.

[0098] In one embodiment, one or more of the human immunoglobulin light chain variable gene segments are in close proximity to the ectopic nucleotide sequence.

[0099] In one embodiment, a mouse is provided that lacks a functional endogenous mouse ADAM6 locus or sequence and contains an ectopic nucleotide sequence encoding a functional fragment of the mouse ADAM6 locus or sequence, and which can be crossed with a mouse of the opposite sex to produce offspring containing the ectopic ADAM6 locus or sequence. In one embodiment, the mouse is male. In one embodiment, the mouse is female.

[0100] In one embodiment, the present invention provides a genetically modified mouse comprising: a human immunoglobulin light chain variable region gene segment at the endogenous immunoglobulin heavy chain variable region gene locus; a lack of an endogenous functional ADAM6 sequence at the endogenous immunoglobulin heavy chain variable region gene locus; and an ectopic nucleotide sequence expressing a functional mouse ADAM6 protein or its orthologue, homolog, or fragment in male mice.

[0101] In one embodiment, the present invention provides a genetically modified mouse comprising: a human immunoglobulin light chain variable gene segment at the endogenous immunoglobulin heavy chain variable region gene locus; a lack of an endogenous functional ADAM6 sequence at the endogenous immunoglobulin heavy chain variable gene locus; and an ectopic nucleotide sequence expressing a functional mouse ADAM6 protein or its ortholog, homolog, or fragment in male mice.

[0102] 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 within the mouse genome. In certain embodiments, the one or more loci include an immunoglobulin locus.

[0103] In one aspect, human V L Gene segment and J gene segment, and D as needed H The present invention provides a mouse that expresses an immunoglobulin light chain sequence derived from a gene segment from a modified endogenous immunoglobulin heavy chain locus, and that contains functional ADAM6 activity in the mouse. In one embodiment, the above human V L The gene segment is selected from the human Vκ and Vλ gene segments. In various embodiments, the J gene segment is J H This refers to the Jκ or Jλ gene segment or a combination thereof.

[0104] In one embodiment, the mouse is a multiple human V L It includes a gene segment and multiple J gene segments. In certain embodiments, the J gene segment is J L It is a gene segment.

[0105] In one embodiment, a mouse expressing an immunoglobulin light chain sequence from a modified endogenous immunoglobulin heavy chain locus, wherein the heavy chain is human V L Gene segment and J L The present invention provides a mouse that contains ADAM6 activity, which is functional in the mouse, derived from a gene segment.

[0106] In one embodiment, the mouse comprises a plurality of human V gene segments, a plurality of J gene segments, and optionally a plurality of D 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, wherein the humanization is C H 1. Hinge, C H 2, C H This includes the substitution of sequences selected from 3 and combinations thereof. In a particular embodiment, the heavy chain is a human V gene segment, a human J gene segment, and a human C H 1 sequence, human or mouse hinge sequence, mouse C H 2 arrays, and mouse C H3. Derived from sequences. In another specific embodiment, the mouse further comprises a human light chain constant sequence. In one embodiment, the mouse comprises an ADAM6 gene whose 5' and 3' positions are adjacent to an endogenous immunoglobulin heavy chain gene segment. In a specific embodiment, the endogenous immunoglobulin heavy chain variable gene segment is not capable of encoding the antibody heavy chain variable region. In a specific embodiment, the ADAM6 gene in the mouse is in the same position as that of a wild-type mouse, and the endogenous immunoglobulin heavy chain variable gene locus in the mouse cannot be rearranged to encode the antibody heavy chain.

[0107] In one embodiment, the plurality of human V gene segments are adjacent to a sequence encoding ADAM6 activity that is functional in the mouse, with respect to the transcription direction of the V gene segment, at 5'. In a particular embodiment, the plurality of human V gene segments are human Vκ gene segments Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, Vκ1-6, Vκ3-7, Vκ1-8, Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15, Vκ1-16, Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3 The human Vκ2-40 gene segment includes Vκ2-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39 and Vκ2-40, and the human Vκ2-40 gene segment is adjacent to the sequence encoding ADAM6 activity that is functional in the mouse (with respect to the transcription direction of the human Vκ2-40 gene segment) at 5'. In certain embodiments, the sequence encoding ADAM6 activity that is functional in the mouse is located in the same transcription direction with respect to the human Vκ gene segment. In certain embodiments, the sequence encoding ADAM6 activity that is functional in the mouse is located in the opposite transcription direction with respect to the human Vκ gene segment.

[0108] In one embodiment, the V gene segment is adjacent to a sequence encoding ADAM6 activity that is functional in the mouse, with respect to the transcriptional direction of the V gene segment, at 3'.

[0109] In one embodiment, the D gene segment is adjacent to a sequence encoding ADAM6 activity that is functional in the mouse, with respect to the transcriptional direction of the D gene segment, at its 5' position.

[0110] In one embodiment, the J gene segment is adjacent to a sequence encoding ADAM6 activity that is functional in the mouse, with respect to the transcription direction of the J gene segment, at its 5' position.

[0111] In one embodiment, the ADAM6 activity that is functional in the mouse results from the expression of nucleotide sequences located at the 5' end of the D gene segment and the 3' end of the V gene segment (relative to the transcription direction of the V gene segment) of the modified endogenous heavy chain immunoglobulin locus.

[0112] In one embodiment, the ADAM6 activity that is functional in the mouse is located at the 5' of the J gene segment furthest 5' and the 3' of the V gene segment furthest 3' (relative to the transcription direction of the V gene segment) of the modified endogenous immunoglobulin heavy chain locus. It results from the expression of a specific nucleotide sequence.

[0113] In one embodiment, the ADAM6 activity that is functional in the mouse results from the expression of a nucleotide sequence between two human V gene segments at the modified endogenous immunoglobulin heavy chain locus. In one embodiment, the two human V gene segments are the human Vκ5-2 gene segment and the Vκ4-1 gene segment.

[0114] In one embodiment, the functional ADAM6 activity in the mouse results from the expression of a nucleotide sequence between the human V gene segment and the human J gene segment at the modified endogenous immunoglobulin heavy chain locus. In one embodiment, the human V gene segment is the human Vκ4-1 gene segment, and the human J segment is the Jκ1 gene segment.

[0115] In one embodiment, the nucleotide sequence includes 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.

[0116] In various embodiments, the sequence encoding the functional ADAM6 activity in the mouse encodes the ADAM6b protein shown in SEQ ID NO: 2 and / or the ADAM6a protein shown in SEQ ID NO: 1.

[0117] In one embodiment, the nucleotide sequences between the two human V gene segments are arranged in opposite transcription directions with respect to the human V gene segments. In a particular embodiment, the nucleotide sequences encode the ADAM6a sequence, followed by the ADAM6b sequence, from 5' to 3' with respect to the transcription direction of the ADAM6 gene. In one embodiment.

[0118] In one embodiment, the nucleotide sequence between the human V gene segment and the human J gene segment is arranged in opposite transcription directions with respect to the human V and human J gene segments. In a particular embodiment, the nucleotide sequence encodes the ADAM6a sequence, followed by the ADAM6b sequence, from 5' to 3' with respect to the transcription direction of the ADAM6 gene.

[0119] In one embodiment, the mouse comprises a hybrid immunoglobulin sequence, the hybrid immunoglobulin sequence comprising a human immunoglobulin κ light chain sequence adjacent to a non-human ADAM6 sequence.

[0120] In one embodiment, the mouse contains a human sequence adjacent to the mouse sequence at the endogenous immunoglobulin heavy chain locus, the adjacent sequence being at least one human V L Gene segments, mouse ADAM6 sequences or their orthologs, homologs, or functional fragments, and human J L Includes a gene segment. In certain embodiments, the mouse ADAM6 sequence or its ortholog, homolog, or functional fragment is the same as at least one human V L It is located directly adjacent to the gene segment. In one embodiment, the above human V L The gene segment is the human Vκ gene segment. In one embodiment, the mouse ADAM6 sequence or its ortholog, homolog, or functional fragment is the same as at least one human Vκ sequence. L Directly adjacent to the 3' side of the gene segment, and human J L It is located directly adjacent to the 5' side of the gene segment. In certain embodiments, the above human V L The gene segment is the human Vκ gene segment, and the above human J L The gene segment is the human Jκ gene segment.

[0121] In one embodiment, the sequence encoding the ADAM6 activity that is functional in the mouse is a mouse ADAM6 sequence or a functional fragment thereof.

[0122] In one embodiment, the mouse is either an endogenous mouse DFL16.1 gene segment (for example, in the case of a mouse heterozygous with respect to the modified endogenous mouse immunoglobulin heavy chain locus) or human D H Includes the 1-1 gene segment. In one embodiment, the D gene segment of the immunoglobulin heavy chain expressed by the above mouse is either the endogenous mouse DFL16.1 gene segment or the human D H It originates from the 1-1 gene segment.

[0123] In one embodiment, a mouse is provided in which DNA-holding cells of a non-rearranged B cell lineage contain a nucleic acid sequence encoding mouse ADAM6 (or its homolog, ortholog, or functional fragment), but B cells containing a rearranged immunoglobulin locus do not contain the nucleic acid sequence encoding mouse ADAM6 (or its homolog, ortholog, or functional fragment), and the nucleic acid sequence encoding mouse ADAM6 (or its homolog, ortholog, or functional fragment) is located at a genomic location different from the location where the mouse ADAM6 gene appears in wild-type mice. In one embodiment, the nucleic acid sequence encoding mouse ADAM6 (or its homolog, ortholog, or functional fragment) is present in all or substantially all DNA-holding cells that are not of a rearranged B cell lineage; in one embodiment, the nucleic acid sequence is present in germline cells of the mouse but not in the chromosomes of rearranged B cells.

[0124] In one embodiment, a mouse is provided in which all or substantially all DNA-holding cells, including B cells containing a rearranged immunoglobulin locus, contain a nucleic acid sequence encoding mouse ADAM6 (or its homolog, orthologue, or functional fragment), wherein the nucleic acid sequence encoding mouse ADAM6 (or its homolog, orthologue, or functional fragment) is located in a genomic location different from the location where the mouse ADAM6 gene appears in wild-type mice. In one embodiment, the nucleic acid sequence encoding mouse ADAM6 (or its homolog, orthologue, or functional fragment) is located 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 wild-type mice, and the chromosome includes a modification of the mouse immunoglobulin locus.

[0125] In one embodiment, a genetically modified mouse is provided, comprising B cells whose genome contains an ADAM6 sequence or its ortholog or homolog. In one embodiment, the ADAM6 sequence or its ortholog or homolog is located at an immunoglobulin heavy chain locus. In a particular embodiment, the heavy chain locus includes an endogenous immunoglobulin heavy chain gene segment that cannot be rearranged to encode an antibody heavy chain. In one embodiment, the ADAM6 sequence or its ortholog or homolog is located at a locus that is not an immunoglobulin locus. In one embodiment, the ADAM6 sequence is located on a transgene driven by a heterologous promoter. In a particular embodiment, the heterologous promoter is a non-immunoglobulin promoter. In a particular embodiment, the B cells express the ADAM6 protein or its ortholog or homolog.

[0126] In one embodiment, more than 90% of the mouse B cells contain a gene encoding the ADAM6 protein or its ortholog or homolog or a fragment thereof, which is functional in the mouse. In a particular embodiment, the mouse is a male mouse.

[0127] In one embodiment, the B cell genome includes a first allele and a second allele, each containing the ADAM6 sequence or its ortholog or homolog. In another embodiment, the B cell genome includes a first allele, each containing the ADAM6 sequence or its ortholog or homolog, but does not include a second allele.

[0128] In one embodiment, a mouse is provided having a modification in one or more endogenous immunoglobulin heavy chain alleles, wherein the modification maintains one or more endogenous ADAM6 alleles.

[0129] In one embodiment, the modification prevents the mouse from expressing a functional heavy chain that includes an endogenous heavy chain gene segment rearranged from at least one heavy chain allele, while maintaining the endogenous ADAM6 allele located within the at least one endogenous immunoglobulin heavy chain allele.

[0130] In one embodiment, the mouse is unable to express a functional heavy chain containing an endogenous heavy chain gene segment rearranged from at least one of the endogenous immunoglobulin heavy chain alleles, and the mouse expresses the ADAM6 protein from an endogenous ADAM6 allele. In a specific embodiment, the mouse is unable to express a functional heavy chain containing an endogenous heavy chain gene segment rearranged from two endogenous immunoglobulin heavy chain alleles, and the mouse expresses the ADAM6 protein from one or more endogenous ADAM6 alleles.

[0131] In one embodiment, the mouse is unable to express a functional heavy chain from each endogenous heavy chain allele, and the mouse contains a functional ADAM6 allele located upstream of the mouse immunoglobulin heavy chain constant region sequence (with respect to the transcription direction of the mouse immunoglobulin heavy chain locus) within 1 Mbp, 2 Mbp, 3 Mbp, 4 Mbp, 5 Mbp, 10 Mbp, 20 Mbp, 30 Mbp, 40 Mbp, 50 Mbp, 60 Mbp, 70 Mbp, 80 Mbp, 90 Mbp, 100 Mbp, 110 Mbp, 120 Mbp, or more than 120 Mbp. In a particular embodiment, the functional ADAM6 allele is located at the endogenous heavy chain locus (for example, within the intergeneric 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 particular embodiment, the functional ADAM6 allele is located within a 90-100kb intergenetic sequence between the last mouse V gene segment and the first mouse D gene segment. In another particular embodiment, the endogenous 90-100kb intergenetic VD sequence is removed, and the ectopic ADAM6 sequence is positioned between the last V gene segment and the first D gene segment.

[0132] One embodiment provides a mouse comprising modifications in one or more endogenous ADAM6 alleles.

[0133] In one embodiment, the modification prevents the mouse from expressing a functional ADAM6 protein from at least one of the one or more endogenous ADAM6 alleles. In a particular embodiment, the mouse is unable to express a functional ADAM6 protein from each of the endogenous ADAM6 alleles.

[0134] In one embodiment, the mouse is unable to express a functional ADAM6 protein from each endogenous ADAM6 allele, and the mouse contains an ectopic ADAM6 sequence.

[0135] In one embodiment, the mouse is unable to express a functional ADAM6 protein from each endogenous ADAM6 allele, and the mouse contains an ectopic ADAM6 sequence located upstream of the mouse immunoglobulin heavy chain constant region sequence (with respect to the transcription direction of the mouse heavy chain locus) within 1kb, 2kb, 3kb, 4kb, 5kb, 10kb, 20kb, 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb, 100kb, 110kb, or 120kb, or larger kb. In a particular embodiment, the ectopic ADAM6 sequence is located at the endogenous immunoglobulin heavy chain locus (e.g., within the intergeneric VD region, between two V gene segments, between a V gene segment and a J gene segment, between a D gene segment and a J gene segment, etc.). In a particular embodiment, the ectopic ADAM6 sequence is located within a 90-100kb intergenetic sequence between the last mouse V gene segment and the first mouse D gene segment. In another particular embodiment, the endogenous 90-100kb intergenetic VD sequence is removed, and the ectopic ADAM6 sequence is positioned between the last human Vκ gene segment and the first human Jκ gene segment. In yet another particular embodiment, the endogenous 90-100kb intergenetic VD sequence is removed, and the ectopic ADAM6 sequence is positioned at the 5' end of the human Vκ gene segment or upstream of the segment, and the human Vκ gene segment is selected from the human Vκ4-1 or Vκ2-40 gene segment.

[0136] In one embodiment, the mouse can express a functional ADAM6 protein from one or more endogenous ADAM6 alleles, and the modification includes the insertion of a human sequence encoding an immunoglobulin variable domain. In one embodiment, the human sequence includes an unrearranged immunoglobulin gene segment. In a particular embodiment, the human sequence includes a V gene segment and a J gene segment. In another particular embodiment, the human sequence includes a V gene segment, a J gene segment, and a D gene segment.

[0137] One embodiment provides a sterile male mouse containing the deletion of two or more endogenous ADAM6 alleles. Another embodiment provides a female mouse that carries the male infertility trait and contains a non-functional ADAM6 allele or an endogenous ADAM6 allele knockout in its germline.

[0138] In one embodiment, a mouse is provided comprising endogenous immunoglobulin heavy chain V, D, and / or J gene segments that cannot be rearranged to encode an antibody heavy chain, wherein the majority of the mouse's B cells contain a functional ADAM6 gene. In various embodiments, the majority of the B cells further comprises one or more human V genes upstream of the mouse immunoglobulin heavy chain constant region. L Gene segment and J L Includes a gene segment. In one embodiment, human V L Gene segment and J L The gene segments are the Vκ gene segment and the Jκ gene segment.

[0139] In one embodiment, the mouse includes intact endogenous immunoglobulin heavy chain V, D, and J gene segments that cannot be rearranged to encode the functional heavy chain of an antibody. In one embodiment, the mouse includes at least one and 89 or fewer V gene segments, at least one and 13 or fewer D gene segments, at least one and 4 or fewer J gene segments, and combinations thereof, wherein the at least one and 89 or fewer V gene segments, at least one and 13 or fewer D gene segments, and at least one and 4 or fewer J gene segments cannot be rearranged to encode the variable region of the antibody heavy chain. In a particular embodiment, the mouse includes a functional ADAM6 gene located within intact endogenous immunoglobulin heavy chain V, D, and J gene segments. In one embodiment, the mouse comprises an endogenous heavy chain locus including an endogenous ADAM6 locus, the endogenous heavy chain locus comprising 89 V gene segments, 13 D gene segments, and 4 J gene segments, the endogenous heavy chain gene segments being unable to be rearranged to encode the heavy chain variable region of an antibody, and the ADAM6 locus encoding the ADAM6 protein which is functional in the mouse.

[0140] In one embodiment, a method is provided for producing a sterile male mouse, the method comprising rendering the endogenous ADAM6 allele of a donor ES cell nonfunctional (or knocking out the allele), introducing the donor ES cell into a host embryo, impregnating a surrogate mother with the host embryo, and having the surrogate mother give birth to a progeny that is all or partly derived from the donor ES cell. In one embodiment, the method further comprises mating the progeny to obtain a sterile male mouse.

[0141] In one embodiment, a method is provided for producing a mouse having a target gene modification and being infertile, the method comprising: (a) performing the target gene modification within the genome; (b) modifying the genome to knock out or render the endogenous ADAM6 allele nonfunctional; and (c) using the genome to produce a mouse. In various embodiments, the genome is derived from ES cells or used in nuclear transplantation experiments.

[0142] In one embodiment, the present invention provides a mouse lacking the endogenous immunoglobulin heavy chain V, D, and J gene segments, wherein the majority of the mouse's B cells contain the ADAM6 sequence or its ortholog or homolog.

[0143] In one embodiment, the mouse lacks an endogenous immunoglobulin heavy chain gene segment 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 at least one V gene segment with 89 or fewer segments, at least one D gene segment with 13 or fewer segments, at least one J gene segment with 4 or fewer segments, and combinations thereof. In one embodiment, the mouse lacks a genomic DNA fragment from chromosome 12 containing the endogenous immunoglobulin heavy chain locus of approximately 3 megabases. 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 89 V H Gene segment, 13 D H Gene segment and 4 J H It lacks a gene segment.

[0144] In one embodiment, a mouse is provided having a genome in the germline that includes a modification of an immunoglobulin heavy chain locus, wherein the modification of the immunoglobulin heavy chain locus includes the replacement of one or more mouse immunoglobulin variable region sequences with one or more non-mouse immunoglobulin variable region sequences, and the mouse includes a nucleic acid sequence encoding the mouse ADAM6 protein. In one embodiment, the D of the endogenous immunoglobulin heavy chain locus H and J H array and at least 3, at least 10, at least 20, at least 40, at least 60 or at least 80 V H The sequence is replaced by a non-mouse immunoglobulin light chain sequence. In one embodiment, the D of the endogenous immunoglobulin heavy chain locus is replaced. H , J H and all V H The sequence consists of multiple non-mouse immunoglobulin V L Gene segment, 1 or more J L The gene segment and, optionally, one or more D gene segment sequences are replaced. The above non-mouse immunoglobulin sequence may be unrearranged. In one embodiment, the above non-mouse immunoglobulin sequence is a complete unrearranged V of a non-mouse species. L and J L Includes a region. In one embodiment, the non-mouse immunoglobulin sequence is operably linked to one or more endogenous constant regions, the fully variable regions of the non-mouse species, i.e., sequences that bind together to encode a light chain variable region, V L Gene segment and J L A rearranged variable region containing a gene segment can be formed. The non-mouse species may be Homo sapiens, and the non-mouse immunoglobulin sequence may be a human sequence.

[0145] In one embodiment, a genetically modified mouse is provided that includes a nucleotide sequence encoding the ADAM6 protein or a functional fragment thereof in close proximity to a human immunoglobulin light chain variable gene segment.

[0146] In one embodiment, the mouse lacks the unmodified endogenous ADAM6 gene sequence. In one embodiment, the mouse lacks the functional endogenous ADAM6 gene sequence.

[0147] In one embodiment, the human immunoglobulin light chain variable gene segment is an immunoglobulin κ light chain variable gene segment. In one embodiment, the human immunoglobulin light chain variable gene segment is an immunoglobulin λ light chain variable gene segment. In one embodiment, the human immunoglobulin light chain variable gene segment is operably linked to a constant immunoglobulin heavy chain gene sequence.

[0148] In one embodiment, the immunoglobulin heavy chain constant gene sequence is a mouse, rat, or human heavy chain gene sequence. In one embodiment, the heavy chain constant gene sequence is C H Includes 1 and / or hinge area.

[0149] In one embodiment, the mouse comprises a deletion or substitution of one or more endogenous immunoglobulin heavy chain gene sequences.

[0150] In one embodiment, the mouse further comprises an unrearranged human Vκ or unrearranged human Vλ gene segment operably ligated to a human, mouse, or rat light chain constant region sequence. In one embodiment, the mouse comprises multiple unrearranged human Vκ gene segments (e.g., two or more human Vκ segments and one or more human Jκ segments) or multiple unrearranged human Vλ gene segments (e.g., two or more human Vλ segments and one or more human Jλ segments). In one embodiment, the unrearranged human Vκ or unrearranged human Vλ gene segment is operably ligated to a constant region sequence at an endogenous immunoglobulin light chain locus.

[0151] In one embodiment, the mouse further includes modifications that render the endogenous κ light chain locus and / or the endogenous λ light chain locus nonfunctional. In one embodiment, the mouse includes knockout or deletion of the endogenous mouse κ and / or endogenous mouse λ light chain locus.

[0152] In one embodiment, a method for maintaining a mouse strain is provided, comprising replacing a mouse immunoglobulin heavy chain sequence with one or more human immunoglobulin light chain sequences. In one embodiment, the one or more human immunoglobulin light chain sequences are human immunoglobulin Vκ and / or Jκ gene segments.

[0153] In one embodiment, the mouse strain is one or more mice V H , D H and / or J H This includes deletion of a gene segment. In one embodiment, the mouse is one or more human V L Gene segment, and one or more human J L The gene segments further include. In one embodiment, the mouse comprises at least 6, at least 16, at least 30, or at least 40 human Vκ gene segments and at least 5 Jκ gene segments. In a particular embodiment, the human light chain gene segments are operably linked to a constant region gene. In one embodiment, the constant region gene is a mouse constant region gene. In one embodiment, the constant region gene is C H 1. Hinge, C H 2, C H 3 and / or C H It includes a mouse constant region gene sequence selected from 4 or a combination thereof.

[0154] In one embodiment, the method includes the steps of producing a male mouse that is heterozygous with respect to the replacement of the mouse immunoglobulin heavy chain sequence, and mating the heterozygous male mouse with a wild-type female mouse or a female mouse that is homozygous or heterozygous with respect to the human heavy chain sequence. In one embodiment, the method includes the steps of maintaining the strain by repeatedly mating the heterozygous male with a wild-type female or a female that is homozygous or heterozygous with respect to the human heavy chain sequence.

[0155] In one embodiment, the method comprises the steps of: obtaining cells from homozygous or heterozygous male or female mice with respect to human heavy chain sequences; utilizing these cells as donor cells or nuclei from these cells as donor nuclei; and using the cells or nuclei to produce genetically modified animals by using host cells and / or by impregnating a surrogate mother with the cells and / or nuclei.

[0156] In one embodiment, only male mice that are heterozygous with respect to the replacement at the heavy chain locus are mated with female mice. In a particular embodiment, the female mice are homozygous, heterozygous, or wild-type with respect to the heavy chain locus being replaced.

[0157] In one embodiment, the mouse further comprises replacing the λ and / or κ light chain variable sequence at the 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 λ and / or κ light chain variable sequence.

[0158] In one embodiment, the mouse further comprises a transgene at a locus other than the endogenous immunoglobulin locus, wherein the transgene is operably linked to (for non-rearranged regions) or fused to (for rearranged sequences) a rearranged or non-rearranged heterologous λ or κ light chain sequence (e.g., non-rearranged V L and non-rearranged JL It includes a sequence encoding a (or rearranged VJ) light chain. In one embodiment, the heterologous λ or κ light chain sequence is human. In one embodiment, the constant region sequence is selected from rodents, humans, and non-human primates. In one embodiment, the constant region sequence is selected from mice, rats, and hamsters. In one embodiment, the transgene includes a non-immunoglobulin promoter that drives the expression of the light chain sequence. In certain embodiments, the promoter is a transcriptional activity promoter. In certain embodiments, the promoter is a ROSA26 promoter.

[0159] In one embodiment, a pregnant mouse is provided comprising a modification of the endogenous ADAM6 gene, the mouse comprising an ectopic sequence that confers ADAM6 function to the mouse, and the mouse comprising in its germline an unrearranged human immunoglobulin light chain gene segment operably linked to a nucleic acid sequence encoding an immunoglobulin heavy chain sequence.

[0160] In one embodiment, a pregnant mouse is provided having a modification of the endogenous ADAM6 locus, the modification rendering the ADAM6 locus nonfunctional, and the mouse expresses an immunoglobulin-binding protein containing a human immunoglobulin light chain variable domain adjacent to the heavy chain constant sequence.

[0161] In one embodiment, the immunoglobulin-binding protein further comprises a human immunoglobulin light chain modification domain of the cognitive fused with a light chain constant sequence.

[0162] In one embodiment, the heavy chain constant sequence and the light chain constant sequence are non-human.

[0163] In one embodiment, one or more immunoglobulin heavy chain variable regions (V) in the endogenous immunoglobulin heavy chain locus H ) Gene segment, heavy chain diversity (D H ) gene segment, and heavy chain linkage (J H) One or more light chain variable regions (V) of the gene segment L ) Gene segment and one or more light chain linkage regions (J L A mouse is provided that contains an immunoglobulin heavy chain locus, including a substitution in a gene segment, and is capable of expressing the ADAM6 protein.

[0164] In one embodiment, the endogenous heavy chain locus (VL) H V at the gene locus L All or substantially all V for forming the gene segment sequence H Gene segment, D H gene segment, and J H One or more V of the gene segment L Gene segment and one or more J L Replacement by gene segment, said VL H The gene locus is endogenous C H Recombined with genes, V L Gene segment, J L Gene segment, and endogenous C H A mouse is provided that contains an immunoglobulin heavy chain locus, which includes a substitution capable of forming a rearranged gene derived from the gene.

[0165] In one embodiment, the above V L The segment is Human V L It is a segment. In one embodiment, the above J L The segment is human J L It is a segment. In a particular embodiment, the above V L Segments and J L The segment is Human V L Segments and human J L It is a segment.

[0166] In one embodiment, all or substantially all V H Gene segment, D H gene segment, and J HReplace the gene segment with at least six human Vκ gene segments and at least one Jκ gene segment. In one embodiment, all or substantially all V H Gene segment, D H gene segment, and J H The gene segment is 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 V H Gene segment, D H gene segment, and J H Replace the gene segment with at least 30 human Vκ gene segments and at least one Jκ gene segment. In one embodiment, all or substantially all V H Gene segment, D H gene segment, and J H The gene segment is 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 two, three, four, or five human Jκ gene segments.

[0167] In one embodiment, the above V L The segment is a human Vκ segment. In one embodiment, the human Vκ segment includes 4-1, 5-2, 7-3, 2-4, 1-5, and 1-6. In one embodiment, the human Vκ segment includes 3-7, 1-8, 1-9, 2-10, 3-11, 1-12, 1-13, 2-14, 3-15, and 1-16. In one embodiment, the human Vκ segment includes 1-17, 2-18, 2-19, 3-20, 6-21, 1-22, 1-23, 2-24, 3-25, 2-26, 1-27, 2-28, 2-29, and 2-30. In one embodiment, the human Vκ segment includes 3-31, 1-32, 1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and 2-40.

[0168] In one embodiment, the above V LThe segment is a human Vκ segment and includes 4-1, 5-2, 7-3, 2-4, 1-5, 1-6, 3-7, 1-8, 1-9, 2-10, 3-11, 1-12, 1-13, 2-14, 3-15, and 1-16. In one embodiment, the human Vκ segment further includes 1-17, 2-18, 2-19, 3-20, 6-21, 1-22, 1-23, 2-24, 3-25, 2-26, 1-27, 2-28, 2-29, and 2-30. In one embodiment, the human Vκ segment further includes 3-31, 1-32, 1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and 2-40.

[0169] In one embodiment, the above V L The segment is a human Vλ segment and contains a fragment of cluster A of the human λ light chain locus. In certain embodiments, the fragment of cluster A of the human λ light chain locus extends from hVλ3-27 to hVλ3-1.

[0170] In one embodiment, the above V L The segment includes a fragment of cluster B of the human λ light chain locus. In a particular embodiment, the fragment of cluster B of the human λ light chain locus extends from hVλ5-52 to hVλ1-40.

[0171] In one embodiment, the above V L The segment includes a human λ light chain variable region sequence containing genomic fragments from cluster A and genomic fragments from cluster B. In one embodiment, the human λ light chain variable region sequence includes at least one gene segment from cluster A and at least one gene segment from cluster B.

[0172] In one embodiment, the above V L The segment includes at least one gene segment from cluster B and at least one gene segment from cluster C.

[0173] In one embodiment, the above V LThe segment includes hVλ3-1, hVλ4-3, hVλ2-8, hVλ3-9, hVλ3-10, hVλ2-11, and hVλ3-12. In a particular embodiment, the above V L The segment includes adjacent sequences of the human λ light chain locus extending from Vλ3-12 to Vλ3-1. In one embodiment, the adjacent sequences include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hVλ. In a particular embodiment, the hVλ includes 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, and 3-12. In a particular embodiment, the hVλ includes adjacent sequences of the human λ locus extending from Vλ3-12 to Vλ3-1.

[0174] In one embodiment, the hVλ comprises 13 to 28 or more hVλ. In a particular embodiment, the hVλ comprises 2-14, 3-16, 2-18, 3-19, 3-21, 3-22, 2-23, 3-25, and 3-27. In a particular embodiment, the hVλ comprises adjacent sequences of the human λ locus extending from Vλ3-27 to Vλ3-1.

[0175] In one embodiment, the above V L The segment includes hVλ of 29-40. In a particular embodiment, the above V L The segment includes adjacent sequences of the human λ locus extending from Vλ3-29 to Vλ3-1, and adjacent sequences of the human λ locus extending from Vλ5-52 to Vλ1-40. In certain embodiments, all or substantially all sequences in hVλ1-40 to hVλ3-29 in the genetically modified mouse consist of a human λ sequence of approximately 959 bp found downstream of the hVλ1-40 gene segment (downstream of the 3' untranslated portion) in nature (e.g., in human populations), a restriction enzyme recognition site (e.g., PI-SceI site), and a subsequent human λ sequence of approximately 3,431 bp found upstream of the hVλ3-29 gene segment in nature.

[0176] In one embodiment, Jκ is human Jκ, selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and combinations thereof. In a particular embodiment, Jκ includes Jκ1 to Jκ5.

[0177] In one embodiment, the above V L The segment is a human Vλ segment, and the Jκ gene segment is an RSS having 12-mer spacers, and includes an RSS that is juxtaposed at the upstream end of the Jκ gene segment. In one embodiment, the V L The gene segment is human Vλ, and the above VL H The locus comprises two or more Jκ gene segments, each Jκ gene segment having an RSS with 12-mer spacers, and each Jκ gene segment comprises adjacent RSSs at its upstream end.

[0178] In a particular embodiment, the above V L The segment includes adjacent human κ gene segments spanning the above human κ locus Vκ4-1 to Vκ2-40, and the above J L The segment includes adjacent κ gene segments spanning the human κ locus Jκ1 to Jκ5.

[0179] The above V L The segment is a Vλ segment, and the V L Between segment and segment J, D H In one embodiment where no segment exists, the V L If the segment is adjacent to a 23-mar RSS downstream (i.e., close on its downstream side), and the Jκ segment (if present) or the Jλ segment (if present) is adjacent to a 12-mar RSS upstream (i.e., close on its upstream side).

[0180] The above V gene segment is the Vκ gene segment, and between the V gene segment and the J gene segment, D HIn one embodiment where no gene segments exist, each of the Vκ gene segments is adjacent to a 12-mer RSS downstream, and each of the present Jκ segments or present Jλ segments is adjacent to a 23-mer RSS upstream.

[0181] In one embodiment, the mouse is V L Gene segment, J L Gene segment, and endogenous C H It includes a rearranged gene derived from a gene. In one embodiment, the rearranged gene is somatically mutated. In one embodiment, the rearranged gene includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N additions. In one embodiment, the above V L Segment and the above J L The above N addition and / or somatic mutation observed in rearranged genes derived from a segment rearranges the rearranged light chain variable domain (same V) at the endogenous light chain locus. L Gene segment and the same J L The number of N additions and / or somatic mutations observed in (derived from gene segments) is 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or at least 5 times greater. In one embodiment, the rearranged gene is a B cell that specifically binds to the target antigen, and the target antigen contains K at a low nanomolar concentration range or less. D (For example, K at concentrations of 10 nanomoles or less) D It is present in B cells that bind via ( ). In certain embodiments, the above V L Segment, above J L The segment, or both, is a human gene segment. In a particular embodiment, the above V L Segments and J L The segment is the human κ gene segment. In one embodiment, the above mouse C HThe gene is selected from IgM, IgD, IgG, IgA, and IgE. In a particular embodiment, the IgG of the mouse is selected from IgG1, IgG2A, IgG2B, IgG2C, and IgG3. In another particular embodiment, the IgG of the mouse is IgG1.

[0182] In one embodiment, the mouse comprises B cells, wherein the B cells express a binding protein consisting of four polypeptide chains from a gene locus on the B cell chromosome, wherein the four polypeptide chains are (a)V L Endogenous C fused with the region H (b) two identical polypeptides including the region, and the mouse C H It is fused with the region, and in one embodiment, human (e.g., human κ)V L The region is V L V is cognitive in terms of domain L Endogenous C fused with the region L It essentially consists of two identical polypeptides, including the region. In one embodiment, the endogenous C H V is fused with the domain. L The region is Human V L This is the region. In a specific embodiment, the above mouse C H Human V fused with the domain L The region is the Vκ region. In a specific embodiment, the above mouse C H Human V fused with the domain L The region is identical to the V region encoded by a rearranged human germline light chain nucleotide sequence. In a particular embodiment, the above mouse C H Human V fused into the domain L The region contains two, three, four, five, six or more somatic hypermutations. In one embodiment, the above mouse C H Human V fused into the domain L The region is encoded by a rearranged gene that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[0183] In one embodiment, at least 50% of all IgG molecules expressed by the above mouse are IgG isotype C H Region and V L The polypeptide comprises a region, wherein the length of the polypeptide is 535, 530, 525, 520, or 515 amino acids or less. In one embodiment, at least 75% of all IgG molecules contain the polypeptides listed in this paragraph. In one embodiment, at least 80%, 85%, 90%, or 95% of all IgG molecules contain the polypeptides listed in this paragraph. In a particular embodiment, all IgG molecules expressed by the mouse contain polypeptides whose length is less than or equal to the polypeptides listed in this paragraph.

[0184] In one embodiment, the above mouse encodes the rearranged human V gene segment and J gene segment, but D H In some gene segments, endogenous C is fused with a variable domain that is not encoded. H It is encoded by a first polypeptide containing the region, as well as rearranged human V gene segment and J gene segment, but D H In some gene segments, endogenous C is fused with a V domain that is not encoded. L A binding protein containing a second polypeptide including the region is expressed, and this binding protein specifically binds to the antigen with affinity in the range of micromolar, nanomolar, or picomolar concentrations. In one embodiment, the J segment is a human J segment (e.g., a human κ gene segment). In one embodiment, the human V segment is a human Vκ segment. In one embodiment, the endogenous C H The variable domain fused with the region is the endogenous C mentioned above. L The variable region fused with the region contains numerous somatic hypermutations, and in certain embodiments, the endogenous C H The variable region fused with the region is the endogenous C L The somatic hypermutation includes approximately 1.5, 2, 3, 4, or 5 times or greater multipliers of the V region fused to the region, and in certain embodiments, the above mouse CH The V region, which is fused with the region, is the mouse C L The region contains at least 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more somatic hypermutations than the V region fused with the region. In one embodiment, the above mouse C H The V region fused with the region is encoded by a rearranged gene containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[0185] In one embodiment, the mouse is fused with a first light chain variable domain (V) which is a constant region of the immunoglobulin heavy chain. L 1) and the second light chain variable domain (V) fused with the constant region of the immunoglobulin light chain L 2) expresses a binding protein including V L 1 is V L It contains approximately 1.5 to 5 times or more the number of somatic hypermutations present in 2. In one embodiment, V L The number of somatic hypermutations in 1 is V L The number is approximately 2 to 4 times greater than the number in 2. In one embodiment, V L The number of somatic hypermutations in 1 is V L The number is about 2 to 3 times greater than the number in 2. In one embodiment, V L 1 is encoded by an array containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[0186] In one embodiment, the following polypeptides: each is V L Gene segment and J L A variable domain derived from a gene segment is fused with a gene segment that is essentially a gene segment. H Two identical polypeptides essentially derived from the region, and each of them is V L Segments and J L C is fused with a variable domain derived from a gene segment that is essentially a segment. L A genetically modified mouse is provided that expresses an immunoglobulin essentially derived from two identical polypeptides, which are essentially derived from a region.

[0187] In a particular embodiment, C H Two identical polypeptides having a region were found in mouse C H It has a domain.

[0188] In a particular embodiment, C L Two identical polypeptides having a region were found in mouse C L It has a domain.

[0189] In one embodiment, the above C L The variable domain that is fused with the region is the above C H These are variable domains and cognito variable domains that are fused into the domain.

[0190] In one embodiment, the endogenous C H The variable domain fused with the region is the endogenous C mentioned above. L The region contains numerous somatic hypermutations from the variable domain fused with the region, and in certain embodiments, the endogenous C H The variable domain fused with the region is the endogenous C L The region includes somatic hypermutations of approximately 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times or greater multiples of the variable domain fused with the region. In one embodiment, the above endogenous C L The variable domain fused with the region is encoded by a gene containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N additions.

[0191] In one embodiment, one or more of the V segment and the J segment are human gene segments. In a particular embodiment, both the V segment and the J segment are human κ gene segments. In another particular embodiment, both the V segment and the J segment are human λ gene segments. In one embodiment, the V segment and the J segment are selected independently from human κ gene segments and human λ gene segments. In a particular embodiment, the V segment is a Vκ segment and the J segment is a Jλ segment. In another particular embodiment, the V segment is a Vλ segment and the J segment is a Jκ segment.

[0192] In one embodiment, the above C L Variable domains fused with the region and the above C H One or more of the variable domains fused with the region are human variable domains. In certain embodiments, the human variable domain is a human Vκ domain. In other specific embodiments, the human variable domain is a Vλ domain. In one embodiment, the human domain is selected independently from the human Vκ domain and the human Vλ domain. In certain embodiments, the above C L The human variable domain fused with the region is the human Vλ domain, and the above C H The human variable domain fused with the region is the human Vκ domain. In another embodiment, C L The human variable domain fused with the region is the human Vκ domain, and the above C H The human variable domain that is fused with the region is the human Vλ domain.

[0193] In one embodiment, V of the two identical first polypeptides L The gene segment is selected from the human Vλ segment and the human Vκ segment. In one embodiment, V is selected from the two identical polypeptides described above. LThe segment is selected from the human Vλ segment and the human Vκ segment. In a particular embodiment, V is selected from the two identical first polypeptides. L The segment is a human Vκ segment, and V is one of the two identical polypeptides mentioned above. L The segment is selected from the human Vκ segment and the human Vλ segment. In a particular embodiment, V is selected from the two identical first polypeptides. L The segment is a human Vλ segment, and V is one of the two identical polypeptides mentioned above. L The segment is selected from the human Vλ segment and the human Vκ segment. In a particular embodiment, the human V of the two identical first polypeptides is selected. L The segment is a human Vκ segment, and of the two identical polypeptides mentioned above, human V L The segment is the human Vκ segment.

[0194] In one embodiment, the mouse IgG described above includes a binding protein produced by a reaction with an antigen, wherein the binding protein has a variable domain and C H The region comprises a polypeptide which is essentially a variable domain, where the variable domain is rearranged V L Encoded by a nucleotide sequence essentially consisting of a segment and a rearranged J segment, where the binding protein contains K in the range of micromolar, nanomolar, or picomolar concentrations at the epitope of the antigen. D It binds specifically to it.

[0195] In one embodiment, a mouse is provided in which all or substantially all of the IgG produced by the mouse in response to an antigen comprises a heavy chain containing a variable domain, wherein the variable domain is encoded by a rearranged gene, the rearranged gene comprising a gene segment essentially consisting of a V gene segment and a J gene segment. In one embodiment, the rearranged gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[0196] In one embodiment, the V segment is a V segment of a light chain. In one embodiment, the light chain is selected from a κ light chain and a λ light chain. In a particular embodiment, the light chain is a κ light chain. In a particular embodiment, the V segment is a human V segment. In a particular embodiment, the V segment is a human Vκ segment and the J segment is a human Jκ segment.

[0197] In one embodiment, the J segment is a light chain J segment. In one embodiment, the light chain is selected from a κ light chain and a λ light chain. In a particular embodiment, the light chain is a κ light chain. In a particular embodiment, the J segment is a human J segment. In another embodiment, the J segment is a heavy chain J segment (i.e., J H (segment). In certain embodiments, the heavy chain is of mouse origin. In other specific embodiments, the heavy chain is of human origin.

[0198] In one embodiment, the variable domain of the heavy chain, which is made up solely from the V and J segments, is a variable domain that has undergone somatic mutation.

[0199] In one embodiment, a variable heavy chain domain made from only the V segment and J segment is used in mouse C H It is merging into the domain.

[0200] In certain embodiments, all or substantially all of the IgG produced by the mouse in response to the antigen comprises a variable domain derived from one or fewer human V segments and one or fewer human J segments, wherein the variable domain is fused to the constant region of mouse IgG, and the IgG is mouse C L Human V fused with the domain L Further comprising a light chain containing a domain. In a particular embodiment, the above mouse C L V is fused with the domain. L The domains are derived from the human Vκ segment and the human Jκ segment. In certain embodiments, the above mouse CL V is fused with the domain. L The domains are derived from the human Vλ segment and the human Jλ segment.

[0201] In one embodiment, C H First CDR3 and C in polypeptides containing the region L A mouse that produces IgG containing a second CDR3 in a polypeptide containing a region, wherein both the first CDR3 and the second CDR3 are independently derived from two or fewer gene segments, wherein the two gene segments are essentially V L Gene segment and J L A mouse comprising a gene segment is provided. In one embodiment, the above C H The CDR3 in the polypeptide containing the region contains a sequence derived from a nucleotide sequence of CDR3 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[0202] In one embodiment, the above V L Segment and the above J L The segment is a human gene segment. In one embodiment, the above V L Segment and the above J L The segment is the κ gene segment. In one embodiment, the above V L Segment and the above J L The segment is the λ gene segment.

[0203] In one embodiment, C H First CDR3 and C in the first polypeptide containing the region L A mouse is provided that produces IgG containing a second CDR3 in a second polypeptide containing a region, wherein both the first CDR3 and the second CDR3 each contain an amino acid sequence, wherein more than 75% of the amino acids are derived from the V gene segment. In one embodiment, the above C HThe CDR3 in the polypeptide containing the region contains a sequence derived from a nucleotide sequence of CDR3 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[0204] In one embodiment, more than 80%, more than 90%, or more than 95% of the amino acids of the first CDR3, and more than 80%, more than 90%, or more than 95% of the amino acids of the second CDR3, are derived from the light chain V segment.

[0205] In one embodiment, two or fewer amino acids in the first CDR3 are derived from a gene segment other than the light chain V segment. In one embodiment, two or fewer amino acids in the second CDR3 are derived from a gene segment other than the light chain V segment. In a specific embodiment, two or fewer amino acids in the first CDR3 and two or fewer amino acids in the second CDR3 are derived from a gene segment other than the light chain V segment. In one embodiment, the CDR3 of the IgG does not contain an amino acid sequence derived from the D gene segment. In one embodiment, the CDR3 of the first polypeptide does not contain a sequence derived from the D segment.

[0206] In one embodiment, the V segment is the human V gene segment. In a specific embodiment, the V segment is the human Vκ gene segment.

[0207] In one embodiment, the first and / or second CDR3 has at least one, two, three, four, five, or six somatic hypermutations. In one embodiment, the first CDR3 is encoded by a nucleic acid sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[0208] In one embodiment, the first CDR3 essentially consists of amino acids derived from the human light chain V gene segment and the human light chain J gene segment, and the second CDR3 essentially consists of amino acids derived from the human light chain V gene segment and the human light chain J gene segment. In one embodiment, the first CDR3 is derived from a nucleic acid sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions. In one embodiment, the first CDR3 is derived from two or fewer gene segments, where the two or fewer gene segments are the human Vκ gene segment and the human Jκ gene segment, and the second CDR3 is derived from two or fewer gene segments, where the two or fewer gene segments are the human Vκ gene segment and the human Jκ segment, the human Jλ segment, and the human J H This is a J gene segment selected from the segments. In one embodiment, the first CDR3 is derived from two or fewer gene segments, where these two or fewer gene segments are the human Vλ segment, the human Jκ segment, the human Jλ segment, and the human J H This is the J segment selected from the segments.

[0209] In one embodiment, D H IgG that does not contain an amino acid sequence derived from a gene segment, where mouse C L The first V is fused with the domain. L A first polypeptide having a domain, and mouse C H The second V is fused with the domain. L It comprises a second polypeptide having a domain, wherein the first V L Domain and the second V L A mouse is provided that produces IgG with non-identical domains. In one embodiment, the first and second V L The domains originate from different V segments. In another embodiment, the first and second V segments described above L The domains originate from different J segments. In one embodiment, the first and second V LThe domain originates from the same V segment and J segment, where the second V L The domain is the first V L It contains a larger number of somatic hypermutations compared to domains.

[0210] In one embodiment, the first and second V L The domain is Human V L Domain and Mouse V L Selected independently of the domain. In one embodiment, the first and second V L The domain is selected independently of the Vκ domain and the Vλ domain. In a particular embodiment, the first V L The domain is selected from the Vκ domain and the Vλ domain, and the second V L The domain is the Vκ domain. In another specific embodiment, the Vκ domain is the human Vκ domain.

[0211] In one embodiment, a mouse wherein all or substantially all of the IgG produced by the mouse is mouse C L The first human V fused with the domain L Light chain having a domain, and mouse C H Second Human V fused with the domain L A mouse is provided that essentially consists of a heavy chain having a domain.

[0212] In one embodiment, the above mouse C H Human V merging with the domain L The domain is the human Vκ domain.

[0213] In one embodiment, the first and second human V L The domains are not the same.

[0214] In one embodiment, a mouse wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% of the immunoglobulin G produced by the mouse is (a) immunoglobulin V LDomains and immunoglobulin C L (b) a first polypeptide which is essentially derived from the region, and (b) a second polypeptide which is 535 amino acids or less in length, where C H Region and D H A mouse is provided that essentially consists of a dimer with a second polypeptide which is essentially derived from a V domain lacking a sequence derived from a gene segment.

[0215] In one embodiment, the second polypeptide is approximately 435 to 535 amino acids long. In a specific embodiment, the second polypeptide is approximately 435 to 530 amino acids long. In a specific embodiment, the second polypeptide is approximately 435 to 525 amino acids long. In a specific embodiment, the second polypeptide is approximately 435 to 520 amino acids long. In a specific embodiment, the second polypeptide is approximately 435 to 515 amino acids long.

[0216] In one embodiment, in over 90% of the IgG produced by the above-mentioned mice, the second polypeptide has a length of approximately 535 amino acids or less.

[0217] In one embodiment, in about 50% or more of the IgG produced by the above mouse, the second polypeptide is about 535 amino acids or less in length. In one embodiment, in about 50% or more of the immunoglobulin G produced by the above mouse, the second polypeptide is about 530 amino acids or less, 525 amino acids or less, 520 amino acids or less, 515 amino acids or less, 510 amino acids or less, 505 amino acids or less, 500 amino acids or less, 495 amino acids or less, 490 amino acids or less, 485 amino acids or less, 480 amino acids or less, 475 amino acids or less, 470 amino acids or less, 465 amino acids or less, 460 amino acids or less, 455 amino acids or less, or 450 amino acids or less in length. In one embodiment, about 60%, 70%, 80%, 90%, or 95% or more of the IgG produced by the above mouse is IgG of the lengths listed above. In a particular embodiment, all or substantially all of the IgG produced by the above mouse is IgG of the lengths listed above.

[0218] In one embodiment, the V domain of the second polypeptide is V L This is a domain. In certain embodiments, the V domain of the second polypeptide is selected from a Vκ domain and a Vλ domain. In certain embodiments, the V domain of the second polypeptide is a human Vκ domain or a human Vλ domain.

[0219] In one embodiment, a mouse is used, and from the nucleotide sequence in its germ cell line, a light chain variable sequence (e.g., V sequence and / or J sequence), D H A mouse is provided that expresses a polypeptide containing a sequence and a heavy chain constant region.

[0220] In one embodiment, the mouse expresses a polypeptide derived from the endogenous heavy chain locus, which includes the replacement of all or substantially all functional endogenous heavy chain variable locus gene segments with multiple human gene segments at the endogenous heavy chain locus.

[0221] In one embodiment, the polypeptide is derived from the Vλ gene segment or the Vκ gene segment. L The sequence includes, and the polypeptide is D H The polypeptide contains CDR3 derived from a gene segment, and the polypeptide is J H It contains sequences derived from a gene segment, or from the Jλ gene segment, or the Jκ gene segment.

[0222] In one embodiment, the mouse contains an endogenous heavy chain immunoglobulin locus, and this locus contains all functional V H The gene segment is replaced by one or more human light chain Vλ gene segments, each of which has a 23-mer spaced recombinant signal sequence (RSS) downstream thereof, and the Vλ segment has 12-mer spaced RSSs upstream and downstream of it, in human D H Segment or Mouse D H The segment is operably connected, and the D H The gene segment is the D H A J segment adjacent upstream is operably linked to an RSS with a 23-mer space, suitable for recombining with the 12-mer spaced RSS that brings the gene segments into close proximity, where the V segment, D H The segments and J segments are operably ligated to the nucleic acid sequence encoding the heavy chain constant region.

[0223] In one embodiment, the mouse contains an endogenous heavy chain immunoglobulin locus, and this locus contains all functional V H The gene segment comprises a recombinant signal sequence (RSS) spaced 12 mars apart and replacement with one or more human Vκ gene segments adjacent downstream thereto, where the V segment is adjacent both upstream and downstream to the RSS spaced 23 mars apart. HSegment or Mouse D H The segment is operably connected, and the D H The segment is the D H Suitable for rearranging the segment in the RSS with a 23-mer space, the RSS with a 12-mer space is operably linked to the upstream adjacent J segment, where the V gene segment, D H A gene segment, and the gene segment, are operably ligated to a nucleic acid sequence encoding a heavy chain constant region.

[0224] In one embodiment, the heavy chain constant region is an endogenous heavy chain constant region. In one embodiment, its nucleic acid sequence is C H 1-row, hinged row, C H 2 arrays, C H It codes for 3 sequences and sequences selected from combinations thereof. In one embodiment, the above C H 1-row, hinged row, C H 2 arrays, and C H One or more of the three sequences are human sequences.

[0225] In one embodiment, the mouse contains an endogenous heavy chain immunoglobulin locus, and this locus contains all functional V H Multiple human Vλ gene segments or human Vκ gene segments that are adjacent downstream to each other by an RSS with 23 mar spaces between the gene segments, and multiple human D13 gene segments that are adjacent both upstream and downstream by an RSS with 12 mar spaces between them. H Segment, RSS with 23 mer spaces and multiple human J segments (J) in close proximity both upstream and downstream. H This includes substitution by a segment (or Jλ segment or Jκ segment), where the locus is C H 1-row, hinged row, C H 2 arrays, C HThe mouse comprises three sequences and an endogenous constant region sequence selected from combinations thereof. In certain embodiments, the mouse comprises all or substantially all functional human Vλ segments or human Vκ segments, and all or substantially all functional human D H Segments, and all or substantially all J H Includes segments, Jλ segments, or Jκ segments.

[0226] In one embodiment, the mouse expresses an antigen-binding protein comprising (a) a polypeptide comprising a human light chain sequence linked to a heavy chain constant sequence comprising a mouse sequence, and (b) a polypeptide comprising a human light chain variable region linked to a human light chain constant sequence or a mouse light chain constant sequence. In a particular embodiment, the light chain sequence is a human light chain sequence, and when exposed to a protease capable of cleaving the antibody into Fc and Fab, a complete human Fab is formed comprising at least four light chain CDRs, where the at least four light chain CDRs are selected from λ sequences, κ sequences, and combinations thereof. In one embodiment, the Fab comprises at least five light chain CDRs. In one embodiment, the Fab comprises six light chain CDRs. In one embodiment, at least one CDR of the Fab comprises a sequence derived from a Vλ segment or a Vκ segment, and the at least one CDR further comprises a sequence derived from a D segment. In one embodiment, the at least one CDR is CDR3, and the CDR is derived from a human Vκ segment, a human D segment, and a human Jκ segment.

[0227] In one embodiment, the polypeptide is a human Vλ gene segment or a human Vκ gene segment, human D H Gene segment, and human J H It includes a variable region derived from a gene segment or a human Jλ gene segment or a human Jκ gene segment. In certain embodiments, the heavy chain constant sequence is human C H 1 array and mouse C H 2 arrays and mouse C H It originates from sequence 3.

[0228] In one embodiment, a mouse having in its germline an unrearranged human Vκ gene segment or human Vλ gene segment operably linked to a human J gene segment and a heavy chain constant region sequence, wherein the human Vκ domain fused to the heavy chain constant region and the human Vλ domain fused to the light chain constant domain L V including domain L A mouse expressing the binding protein is provided. In one embodiment, the above human V L The domain is a rearranged human V selected from the human Vκ gene segment and the human Vλ gene segment. L Includes a gene segment. In a particular embodiment, the above human V L The domain is further a rearranged human J selected from the human Jκ gene segment and the human Jλ gene segment. L Includes gene segments.

[0229] In one embodiment, a mouse is provided that expresses an immunoglobulin protein derived from a modified endogenous heavy chain locus in its germline, wherein the modified endogenous heavy chain locus lacks a functional mouse heavy chain V gene segment and comprises an unrearranged light chain V gene segment and an unrearranged J gene segment, wherein the unrearranged light chain V gene segment and the unrearranged J gene segment are operably linked to a heavy chain constant region sequence, wherein the immunoglobulin protein essentially consists of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an immunoglobulin light chain sequence and an immunoglobulin heavy chain constant sequence, and the second polypeptide comprises an immunoglobulin light chain variable domain and a light chain constant region.

[0230] In one embodiment, a mouse is provided that expresses an immunoglobulin protein, wherein the immunoglobulin protein lacks a heavy chain immunoglobulin variable domain, and comprises a first variable domain derived from a light chain gene and a second variable domain derived from a light chain gene, wherein the first variable domain and the second variable domain are cognitive with respect to each other, wherein the first and second light chain variable domains are not identical, and wherein the first and second light chain variable domains associate and, upon association, specifically bind to a target antigen.

[0231] In one embodiment, a mouse expresses an immunoglobulin protein that contains a variable region entirely derived from an unrearranged human gene segment, wherein the immunoglobulin protein contains a constant immunoglobulin light chain sequence and C H 1-row, hinged row, C H 2 arrays, C H A mouse is provided containing an immunoglobulin heavy chain constant sequence selected from a group consisting of three sequences and combinations thereof.

[0232] In one embodiment, a mouse is provided that expresses an immunoglobulin protein containing a variable region from an unrearranged gene segment in its germline, wherein all CDR3s in all variable regions are generated exclusively from the light chain V gene segment and the light chain J gene segment (optionally having one or more somatic hypermutations (e.g., one or more N additions)).

[0233] In one embodiment, a mouse is provided that expresses a somatically mutated immunoglobulin protein in its germline, derived from an unrearranged human immunoglobulin light chain variable region gene segment, wherein the immunoglobulin protein lacks a CDR containing a sequence derived from the D gene segment, and wherein the immunoglobulin protein contains a first CDR3 in a light chain variable domain fused to the light chain constant region, and a second CDR3 in a light chain variable domain fused to the heavy chain constant region, wherein the second CDR3 is derived from a rearranged light chain variable region sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[0234] In one embodiment, a mouse described herein is provided which includes a functionally silenced light chain locus selected from a λ locus, a κ locus, and a combination thereof. In one embodiment, the mouse includes deletions of the λ locus and / or κ locus, in whole or in part, such that the λ locus and / or κ locus are non-functional.

[0235] In one embodiment, a mouse embryo is provided comprising cells containing a modified immunoglobulin locus as described herein. In one embodiment, the mouse is a chimera, and at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the cells of the embryo contain the modified immunoglobulin locus as described herein. In one embodiment, at least 96%, 97%, 98%, 99%, or 99.8% of the cells of the embryo contain the modified immunoglobulin locus as described herein. In one embodiment, the embryo comprises cells derived from host cells and donor ES cells, wherein the cells derived from the donor ES cells contain the modified immunoglobulin locus as described herein. In one embodiment, the embryo is a host embryo or blastocyst at the 2-cell, 4-cell, 8-cell, 16-cell, 32-cell, or 64-cell stage, and further comprises donor ES cells containing a modified immunoglobulin locus as described herein.

[0236] In one embodiment, a mouse or cells prepared using a nucleic acid construct described herein are provided.

[0237] In one embodiment, a mouse produced using the cells described herein is provided. In one embodiment, the cells are mouse ES cells.

[0238] In one embodiment, mouse cells and mouse embryos, including but not limited to ES cells, pluripotent cells and induced pluripotent cells, containing the genetic modifications described herein. Cells that are XX and cells that are XY are provided. Cells containing nuclei containing the modifications described herein, for example, modifications introduced into cells by pronuclear injection are also provided. Cells, embryos and mice containing the virally introduced ADAM6 gene, for example, cells, embryos and mice containing transduction constructs containing the ADAM6 gene that is functional in the mouse are also provided.

[0239] In one embodiment, cells or tissue derived from a mouse described herein are provided. In one embodiment, the cells or tissue are derived from the spleen, lymph node, or bone marrow of a mouse described herein. In one embodiment, the cells are B cells. In one embodiment, the cells are embryonic stem cells. In one embodiment, the cells are germ cells.

[0240] In one embodiment, the tissue is selected from connective tissue, muscle tissue, nerve tissue, and epithelial tissue. In a particular embodiment, the tissue is reproductive tissue.

[0241] In one embodiment, mouse-derived cells and / or tissues described herein are isolated for use in one or more ex vivo assays. In various embodiments, the one or more ex vivo assays include measuring physical, thermal, electrical, mechanical, or optical properties, surgical techniques, interactions between different types of tissues, developing imaging techniques, or a combination thereof.

[0242] In one embodiment, the use of mouse-derived cells or tissues as described herein for producing antibodies is provided. In one embodiment, the use of mouse-derived cells or tissues as described herein for producing hybridomas or quadromas is provided.

[0243] In one embodiment, the non-human cell comprises a chromosome or fragment thereof from a non-human animal described herein. In one embodiment, the non-human cell comprises a nucleus from a non-human animal described herein. In one embodiment, the non-human cell comprises a chromosome or fragment thereof as a result of nuclear transfer.

[0244] In one embodiment, a nucleus derived from a mouse described herein is provided. In one embodiment, the nucleus is from a diploid cell that is not a B cell.

[0245] In one embodiment, genetically modified mouse cells are provided that are unable to express a heavy chain comprising a rearranged endogenous immunoglobulin heavy chain gene segment and that contain a functional ADAM6 gene encoding the mouse ADAM6 protein or a functional fragment thereof. In one embodiment, the cells further include the insertion of a human immunoglobulin light chain gene segment. In a particular embodiment, the human immunoglobulin light chain gene segment is a Vκ and / or Jκ gene segment operably linked to the mouse heavy chain constant region such that, upon rearrangement, it encodes a functional light chain variable domain fused to the mouse heavy chain constant domain.

[0246] In one embodiment, genetically modified mouse cells are provided that lack a functional endogenous ADAM6 locus and contain an ectopic nucleotide sequence encoding mouse ADAM6. In one embodiment, the cells further include modification of the endogenous immunoglobulin heavy chain variable region sequence. In a particular embodiment, the modification of the endogenous immunoglobulin heavy chain variable region sequence is mouse V H Deletion of gene segment, mouse D H Deletion of gene segment, mouse J H This includes deletions of gene segments and deletions selected from combinations thereof. In a particular embodiment, the mouse is one or more mice immunoglobulin V H , D H , and / or J H This includes replacing the sequence with a human immunoglobulin sequence. In certain embodiments, the human immunoglobulin sequence is human V H Human V L Human D H Human J H Human J L , and combinations thereof are selected.

[0247] In one embodiment, the cells are totipotent, pluripotent, or induced pluripotent cells. In a particular embodiment, the cells are mouse ES cells.

[0248] In one embodiment, mouse B cells are provided, comprising a rearranged immunoglobulin gene, wherein the chromosome of the B cell contains a nucleic acid sequence encoding the ADAM6 protein or its orthologue, homolog, or fragment, which is functional in male mice. In one embodiment, the mouse B cell comprises two alleles of the nucleic acid sequence. In one embodiment, the rearranged immunoglobulin gene comprises a rearranged immunoglobulin light chain sequence adjacent to a heavy chain constant sequence. In one embodiment, the light chain sequence is a κ sequence, and in one embodiment, the light chain sequence is a λ sequence.

[0249] In one embodiment, the nucleic acid sequence is located on a nucleic acid molecule (e.g., a B cell chromosome) adjacent to a rearranged mouse heavy chain immunoglobulin locus.

[0250] In one embodiment, the nucleic acid sequence is located on a nucleic acid molecule different from the nucleic acid molecule containing the rearranged mouse heavy chain immunoglobulin locus (e.g., a B cell chromosome).

[0251] In one embodiment, the mouse B cells comprise a rearranged non-mouse light chain immunoglobulin variable region sequence operably linked to a mouse or human heavy chain immunoglobulin constant region gene, wherein the B cells comprise a nucleic acid sequence encoding the ADAM6 protein or its orthologue, homolog, or fragment, which is functional in male mice.

[0252] In one embodiment, a mouse somatic cell is provided, comprising a chromosome containing a modified immunoglobulin heavy chain locus and a nucleic acid sequence encoding mouse ADAM6 or its ortholog, homolog, or fragment, which is functional in male mice. In one embodiment, the nucleic acid sequence is located on the same chromosome as the modified immunoglobulin heavy chain locus. In one embodiment, the nucleic acid is located on a different chromosome from the modified immunoglobulin heavy chain locus. In one embodiment, the somatic cell contains a single copy of the nucleic acid sequence. In one embodiment, the somatic cell contains at least two copies of the nucleic acid sequence. In a particular embodiment, the somatic cell is a B cell. In one embodiment, the modified immunoglobulin heavy chain locus comprises an unrearranged immunoglobulin light chain gene segment operably linked to a heavy chain constant region sequence.

[0253] In one embodiment, a mouse germ cell is provided, the germ cell having a nucleic acid sequence on its chromosome encoding mouse ADAM6 (or its homolog, ortholog, or functional fragment), wherein the nucleic acid sequence encoding mouse ADAM6 (or its homolog, ortholog, or functional fragment) is located at a different chromosomal location than that of the wild-type mouse germ cell, and the mouse further includes a modification comprising an unrearranged light chain immunoglobulin gene segment (Vκ, and / or Vλ, and / or Vκ and Jκ, and / or Vλ and Jλ) operably linked to a heavy chain constant region sequence. In one embodiment, the nucleic acid sequence is located at a mouse immunoglobulin locus. In one embodiment, the nucleic acid sequence is located on the same germ cell chromosome as the mouse immunoglobulin locus. In one embodiment, the nucleic acid sequence is located on a different germ cell chromosome than the mouse immunoglobulin locus. In one embodiment, the mouse immunoglobulin locus includes the replacement of at least one mouse immunoglobulin sequence with at least one non-mouse immunoglobulin sequence. In certain embodiments, the at least one non-mouse immunoglobulin sequence is a human immunoglobulin sequence.

[0254] In one embodiment, pluripotent, induced pluripotent, or totipotent cells derived from mouse are provided herein. In certain embodiments, the cells are mouse embryonic stem (ES) cells.

[0255] In one embodiment, cells containing a modified immunoglobulin locus as described herein are provided. In one embodiment, the cells are selected from totipotent cells, pluripotent cells, induced pluripotent stem cells (iPS cells), and ES cells. In a particular embodiment, the cells are mouse cells, for example, mouse ES cells. In one embodiment, the cells are homozygous for the modified immunoglobulin locus.

[0256] In one embodiment, cells are provided that contain a nucleic acid sequence encoding a first polypeptide comprising a somatically mutated first human Vκ sequence or a first human Vλ sequence fused to a human heavy chain constant region sequence.

[0257] In one embodiment, the cells further comprise a second polypeptide chain containing a somatically mutated second human Vκ sequence or a second human Vλ sequence fused to a human light chain constant region sequence.

[0258] In one embodiment, the human Vκ sequence or the human Vλ sequence of the first polypeptide is cognitive with the human Vκ sequence or the human Vλ sequence of the second polypeptide.

[0259] In one embodiment, when the Vκ or Vλ of the first polypeptide and the human Vκ or human Vλ of the second polypeptide associate, they specifically bind to the target antigen. In a particular embodiment, the first polypeptide comprises a variable domain essentially derived from human Vκ, the second polypeptide comprises a variable domain consisting of human Vκ of the first polypeptide and a cognitive human Vκ, and the human constant region sequence is an IgG sequence.

[0260] In one embodiment, the cells are selected from CHO cells, COS cells, 293 cells, HeLa cells, and human retinal cells expressing a viral nucleic acid sequence (e.g., PERC.6® cells).

[0261] In one embodiment, rodent (e.g., mouse) somatic cells are provided that include chromosomes with the genetic modifications described herein.

[0262] In one embodiment, rodent (e.g., mouse) germ cells are provided that contain nucleic acid sequences including the genetic modifications described herein.

[0263] In one embodiment, pluripotent, induced pluripotent, or totipotent cells derived from a rodent (e.g., mouse) as described herein are provided. In certain embodiments, the cells are mouse embryonic stem (ES) cells.

[0264] In one embodiment, the use of cells described herein for producing rodents (e.g., mice), cells, or therapeutic proteins (e.g., antibodies or other antigen-binding proteins) is provided. In one embodiment, the use of cells described herein for producing a therapeutic protein is provided, the therapeutic protein comprising a human variable domain. In a particular embodiment, the human variable domain comprises rearranged Vκ and Jκ gene segments.

[0265] In one embodiment, a rodent (e.g., a mouse) is provided that is constructed using the targeting vector, nucleotide construct, or cells described herein.

[0266] In one embodiment, the use of the targeting vector described herein for the production of rodents (e.g., mice) or cells (e.g., mouse ES cells, mouse fibroblasts, etc.) is provided. In one embodiment, the targeting vector comprises a human genome fragment containing an unrearranged immunoglobulin gene segment. In a particular embodiment, the unrearranged immunoglobulin gene segment may include a V gene segment and a J gene segment. In a particular embodiment, the unrearranged immunoglobulin gene segment may include a V gene segment, a D gene segment, and a J gene segment.

[0267] In one embodiment, a nucleic acid construct is provided comprising an upstream homology arm and a downstream homology arm, wherein the upstream homology arm comprises a sequence identical or substantially identical to a human immunoglobulin heavy chain variable region sequence, and the downstream homology arm comprises a sequence identical or substantially identical to a human or mouse immunoglobulin variable region sequence, and a sequence comprising a nucleotide sequence encoding the mouse ADAM6 protein is positioned between the upstream and downstream homology arms. In a particular embodiment, the sequence encoding the mouse ADAM6 gene is operably ligated to a mouse promoter to which mouse ADAM6 is ligated in the case of a wild-type mouse.

[0268] In one embodiment, 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 mouse ADAM6 or its ortholog, homolog, or fragment, which is functional in mouse.

[0269] In one embodiment, the targeting vector further includes a promoter operably linked to a sequence encoding mouse ADAM6. In a particular embodiment, the promoter is a mouse ADAM6 promoter.

[0270] In one embodiment, a nucleotide construct for modifying a mouse immunoglobulin heavy chain variable locus is provided, comprising at least one site-specific recombinase recognition site and a sequence encoding the ADAM6 protein or its orthologue, homolog, or fragment, which is functional in mouse.

[0271] In one embodiment, RSS with a space of 23 meters is placed and Human D is in close proximity upstream and downstream. HA nucleic acid construct containing a gene segment is provided. In certain embodiments, the nucleic acid construct includes homology arms homologous to a human genome sequence containing a human Vκ gene segment. In one embodiment, the targeting construct includes all or substantially all human D23s adjacent to the RSS upstream and downstream, respectively, with a 23-mar space between them. H Includes gene segments.

[0272] In one embodiment, a nucleic acid construct is provided that includes a human Jκ gene segment adjacent upstream to an RSS spaced 12 marks apart. In a particular embodiment, the nucleic acid construct includes a human genome D29 adjacent upstream and downstream to an RSS spaced 23 marks apart. H The nucleic acid construct includes a first homology arm containing homology to a gene sequence. In one embodiment, the nucleic acid construct includes a second homology arm containing homology to the human genome J gene sequence, or homology to the mouse heavy chain constant region sequence, or homology to the upstream JC gene sequence of the mouse constant region heavy chain sequence.

[0273] In one embodiment, a human Vλ segment is adjacent downstream to an RSS with a 23-mar space, and a human D is adjacent upstream and downstream to an RSS with a 12-mar space. H The segment, as well as the Jκ segment adjacent upstream to the RSS with a 23-mar space, the human Jλ segment adjacent upstream to the RSS with a 23-mar space, and the human J H A nucleic acid construct is provided that includes a human J segment selected from the segments. In one embodiment, the construct includes a homology arm that contains homology to a mouse constant region sequence, a mouse JC intergene sequence, and / or a human Vλ sequence.

[0274] In one embodiment, the nucleic acid construct includes a human λ light chain variable region sequence containing a fragment of cluster A of the human λ light chain locus. In a particular embodiment, the fragment of cluster A of the human λ light chain locus extends from hVλ3-27 to hVλ3-1.

[0275] In one embodiment, the nucleic acid construct includes a human λ light chain variable region sequence containing a fragment of cluster B of the human λ light chain locus. In a particular embodiment, the fragment of cluster B of the human λ light chain locus extends from hVλ5-52 to hVλ1-40.

[0276] In one embodiment, the nucleic acid construct includes a human λ light chain variable region sequence comprising a genomic fragment of cluster A and a genomic fragment of cluster B. In one embodiment, the human λ light chain variable region sequence includes at least one gene segment of cluster A and at least one gene segment of cluster B.

[0277] In one embodiment, the human λ light chain variable region sequence includes at least one gene segment of cluster B and at least one gene segment of cluster C.

[0278] In one embodiment, the Jκ segment is normally found in nature, J H Segment, Vλ segment, or V H Human D, located adjacent to any of the segments, is found with a 23-mar space between it and the RSS, and is adjacent to it both upstream and downstream. H A nucleic acid construct containing a segment is provided. In one embodiment, the nucleic acid construct includes a first homology arm which is homologous to the human VJ intergene region or to a human genome sequence containing the human V gene segment. In one embodiment, the nucleic acid construct includes a second homology arm which is homologous to a human heavy chain constant region sequence or a mouse heavy chain constant region sequence. In a particular embodiment, the human heavy chain constant region sequence or the mouse heavy chain constant region sequence is C H 1-row, hinged row, C H 2 arrays, C H3 sequences and combinations thereof are selected. In one embodiment, the nucleic acid construct includes a human J gene segment adjacent to a 12-mer RSS upstream. In one embodiment, the nucleic acid construct includes a second homology arm containing homology to the 12-mer RSS and the J gene segment adjacent upstream. In one embodiment, the J gene segment includes a human Jκ segment, a human Jλ segment, and a human J H Selected from a segment.

[0279] In one embodiment, RSS with a space of 23 meters is placed and Human D is in close proximity upstream and downstream. H A nucleic acid construct is provided that includes segments and site-specific recombinase recognition sequences, such as sequences recognized by site-specific recombinases, including Cre protein, Flp protein, or Dre protein.

[0280] In one embodiment, a human Vλ segment or human Vκ segment, with a 12-mar or 23-mar spaced RSS, is adjacent to a D upstream and downstream. H A nucleic acid construct is provided comprising a human J segment with a 12-mer or 23-mer spaced RSS, where the 12-mer or 23-mer spaced RSS is located immediately 5' to the human J segment (i.e., with respect to the transcription direction). In one embodiment, the construct comprises a human Vλ segment adjacent to the 23-mer spaced RSS on the 3' side, and a human D segment adjacent to the 12-mer spaced RSS upstream and downstream. H The structure includes a human Jκ segment adjacent to the RSS with a 23-mar space on the 5' side, and a human Dκ segment adjacent to the RSS with a 12-mar space on the 3' side, and a human Dκ segment adjacent to the RSS with a 23-mar space on the upstream and downstream sides. H The segment includes the RSS and a human Jλ segment adjacent to it on the 5' side, spaced 12 mars apart.

[0281] In one embodiment, a targeting vector comprising (a) a first targeting arm and a second targeting arm, independently selected from a human targeting arm and a mouse targeting arm, which direct the vector to the locus of an endogenous immunoglobulin V region gene or a modified immunoglobulin V region gene, and (b) (i) hVκ4-1 to hVκ1-6 and Jκ1, (ii) hVκ4-1 (iii) hVκ1-6 and Jκ1-Jκ2, (iv) hVκ4-1-hVκ1-6 and Jκ1-Jκ3, (v) hVκ4-1-hVκ1-6 and Jκ1-Jκ4, (vi) hVκ3-7-hVκ1-16, (vii) hVκ1-17-hVκ2-30, (viii) hVκ3-31-hVκ2-40, and (ix) selected from the group consisting of combinations thereof, human V L Adjacent sequences of a gene segment, or human V L A targeting vector is provided, comprising a gene segment and adjacent sequences of at least one human Jκ gene segment.

[0282] In one embodiment, the targeting arm that directs the vector to an endogenous immunoglobulin locus or a modified immunoglobulin locus is identical or substantially identical to the sequence at the endogenous immunoglobulin locus or the modified immunoglobulin locus.

[0283] In one embodiment, a method is provided for producing a genetically modified mouse, the method comprising the steps of replacing one or more immunoglobulin heavy chain gene segments upstream of the endogenous ADAM6 locus of the mouse (based on transcription of immunoglobulin heavy chain gene segments) with one or more human immunoglobulin light chain and / or heavy chain gene segments, and replacing one or more immunoglobulin gene segments downstream of the ADAM6 locus of the mouse (based on transcription of immunoglobulin heavy chain gene segments) with one or more human immunoglobulin heavy chain or light chain gene segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments upstream of the endogenous ADAM6 locus of the mouse include a V gene segment. In one embodiment, the human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments upstream of the endogenous ADAM6 locus of the mouse include 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 endogenous ADAM6 locus include the J gene segment. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments downstream of the mouse endogenous ADAM6 locus include the 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 endogenous ADAM6 locus include the V, D and J gene segments.

[0284] In one embodiment, a method is provided for producing a genetically modified mouse, the method comprising the steps of replacing one or more immunoglobulin heavy chain gene segments upstream of the endogenous ADAM6 locus of the mouse (based on transcription of immunoglobulin heavy chain gene segments) with one or more human immunoglobulin light chain gene segments, and replacing one or more immunoglobulin gene segments downstream of the ADAM6 locus of the mouse (based on transcription of immunoglobulin heavy chain gene segments) with one or more human immunoglobulin light chain 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 include a V gene segment. In one embodiment, the human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments upstream of the endogenous ADAM6 locus of the mouse include V 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 endogenous ADAM6 locus include the J gene segment. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments downstream of the mouse endogenous ADAM6 locus include the V 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 endogenous ADAM6 locus include the V gene segment.

[0285] In a particular embodiment, the V gene segment is V L It is a gene segment. In another specific embodiment, the above J gene segment is J L It is a gene segment.

[0286] In one embodiment, one or more immunoglobulin heavy chain gene segments upstream and / or downstream of the ADAM6 gene are replaced in pluripotent cells, induced pluripotent cells, or totipotent cells to form genetically modified progenitor cells; these genetically modified progenitor cells are introduced into a host; and the host containing the genetically modified progenitor cells is made pregnant to form a mouse containing a genome derived from the genetically modified progenitor cells. 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.

[0287] In one embodiment, a method is provided for producing a genetically modified mouse, the method comprising the steps of: forming a first chimeric locus by replacing a mouse nucleotide sequence containing a mouse immunoglobulin gene segment and a mouse ADAM6 (or a functional ortholog or homolog or fragment thereof in male mice) nucleotide sequence with a sequence containing a human immunoglobulin gene segment; and subsequently, forming a second chimeric locus by inserting a mouse ADAM6 coding sequence (or a sequence coding its ortholog or homolog or functional fragment) into the sequence containing the human immunoglobulin gene segment.

[0288] In one embodiment, the second chimeric locus is the human immunoglobulin heavy chain variable gene segment (V H ) includes. In one embodiment, the second chimeric locus is the human immunoglobulin light chain variable gene segment (V L ) includes. In certain embodiments, the second chimeric locus is human D H Gene segments and human J H Human V operably linked to gene segments H Gene segment or human V L Includes a gene segment. In certain embodiments, the second chimeric locus is human J H Gene segment or human J LHuman V operably linked to gene segments L Includes a gene segment. In a further specific embodiment, the second chimeric locus is mouse C H 2+C H Human C fusion with 3 sequences H 1 sequence, or human C H It is operably linked to a third chimeric locus containing 1 and a human hinge sequence.

[0289] One embodiment provides a method for modifying a mouse heavy chain immunoglobulin locus, comprising the steps of (a) making a first modification of the mouse heavy chain immunoglobulin locus that results in a reduction or elimination of endogenous mouse ADAM6 activity in male mice; and (b) making a second modification to add a nucleic acid sequence that confers functional ADAM6 activity to the mouse in male mice.

[0290] In one embodiment, the first modification includes the addition of a human immunoglobulin sequence or the replacement of a mouse immunoglobulin sequence with a human immunoglobulin sequence.

[0291] In one embodiment, the human immunoglobulin sequence is a heavy chain sequence. In one embodiment, the human immunoglobulin sequence is a light chain sequence.

[0292] In one embodiment, the first and second modifications described above are performed in a single ES cell, which is then introduced into a host embryo to produce the mouse.

[0293] In one embodiment, the offspring of a cross between a mouse described herein and a second mouse that is either a wild-type mouse or a genetically modified mouse are provided.

[0294] In one embodiment, the use of a mouse containing an ectopic nucleotide sequence including the mouse ADAM6 locus or sequence is provided for producing fertile male mice, the use of which includes crossing the mouse containing the ectopic nucleotide sequence including the mouse ADAM6 locus or sequence with a mouse lacking the functional endogenous ADAM6 locus or sequence, and obtaining a female offspring capable of producing offspring having the ectopic ADAM6 locus or sequence, or obtaining a male offspring containing the ectopic ADAM6 locus or sequence that exhibits fertility substantially the same as that shown by wild-type male mice.

[0295] In one embodiment, the use of the mouse described herein is provided for introducing an ectopic ADAM6 sequence into a mouse lacking a functional endogenous ADAM6 sequence, the use comprising crossing the mouse described herein with the mouse lacking the functional endogenous ADAM6 sequence.

[0296] In one embodiment, the use of genetic material from the mouse described herein for producing a mouse having an ectopic ADAM6 sequence is provided. In one embodiment, the use includes nuclear transfer using the nucleus of the mouse cells described herein. In one embodiment, the use includes cloning the mouse cells described herein to produce an animal derived from those cells. In one embodiment, the use includes the use of the mouse sperm or oocyte described herein in a process for producing a mouse containing the ectopic ADAM6 sequence.

[0297] In one embodiment, a method is provided for producing a fertilizing male mouse containing a modified immunoglobulin heavy chain locus, comprising the steps of: fertilizing a first mouse germ cell containing a modification of the endogenous heavy chain immunoglobulin locus with a second mouse germ cell containing the ADAM6 gene or its ortholog, homolog, or fragment, which is functional in male mice; forming fertilized cells; developing the fertilized cells into embryos; and obtaining a mouse by having a surrogate mother become pregnant with the embryos.

[0298] In one embodiment, fertilization is achieved by mating a male mouse with a female mouse. In one embodiment, the female mouse contains the ADAM6 gene or its ortholog, homolog, or fragment. In one embodiment, the male mouse contains the ADAM6 gene or its ortholog, homolog, or fragment.

[0299] In one embodiment, the use of nucleic acid sequences encoding the mouse ADAM6 protein or its ortholog or homolog or a corresponding functional fragment of the ADAM6 protein is provided for restoring or enhancing fertility in mice having a genome that includes a modification of the immunoglobulin heavy chain locus, wherein the modification reduces or eliminates endogenous ADAM6 function.

[0300] In one embodiment, the nucleic acid sequence is incorporated into an ectopic location in the mouse genome. In one embodiment, the nucleic acid sequence is incorporated into an endogenous immunoglobulin locus in the mouse genome. In a particular embodiment, the endogenous immunoglobulin locus is a heavy chain locus. In one embodiment, the nucleic acid sequence is incorporated into a location other than an endogenous immunoglobulin locus in the mouse genome.

[0301] In one embodiment, a method is provided for producing a genetically modified mouse, comprising the step of replacing one or more mouse immunoglobulin heavy chain gene segments with one or more human immunoglobulin light chain gene segments at an endogenous heavy chain locus. In one embodiment, the replacement replaces all or substantially all functional mouse immunoglobulin heavy chain segments (i.e., V H , D H , and J H One or more functional human light chain segments (i.e., V) L and J L This is a replacement in the segment. In one embodiment, the above replacement is all or substantially all of the functional mouse heavy chain V H , DH , and J H This involves replacing all or substantially all human Vλ or Vκ segments and at least one Jλ or Jκ segment of a segment. In certain embodiments, the above replacement encompasses all or substantially all functional human Jλ or Jκ segments.

[0302] In one embodiment, endogenous heavy chain immunoglobulin variable segment (V H , D H , and J H A method is provided for producing mice expressing polypeptides containing sequences derived from human immunoglobulin Vλ or Vκ and / or Jλ or Jκ segments fused to a mouse heavy chain constant region, comprising the step of replacing the mouse heavy chain constant region with at least one human Vλ or Vκ segment and at least one human Jλ or Jκ segment, wherein the replacement is present in pluripotent, induced pluripotent, or totipotent mouse cells to form genetically modified mouse progenitor cells, which are introduced into a mouse host, and the mouse host containing the genetically modified progenitor cells is made pregnant to form a mouse containing a genome derived from the genetically modified mouse progenitor cells. In one embodiment, the host is an embryo. In a particular embodiment, the host is selected from a mouse premorula (e.g., 8 or 4 cell stage), tetraploid embryo, aggregate of embryonic cells, or blastocyst.

[0303] In one embodiment, a method is provided for producing a genetically modified mouse as described herein, comprising the steps of introducing nucleic acids containing the modification described herein into cells by nuclear transfer, and maintaining the cells under appropriate conditions to develop them into a mouse as described herein (including, for example, the steps of culturing the cells and having an embryo containing the cells conceive in a surrogate mother).

[0304] In one embodiment, a method for producing a modified mouse is provided, comprising the steps of: modifying mouse ES cells or mouse pluripotent cells or mouse totipotent cells or mouse induced pluripotent cells to include one or more unrearranged immunoglobulin light chain variable gene segments operably linked to a constant immunoglobulin heavy chain sequence as described herein; culturing the ES cells; introducing the cultured ES cells into a host embryo to form a chimeric embryo; and introducing the chimeric embryo into a suitable host mouse to develop into a modified mouse. In one embodiment, the one or more unrearranged immunoglobulin light chain variable region gene segments are human λ gene segments or human κ gene segments. In one embodiment, the one or more unrearranged immunoglobulin light chain variable region gene segments are human Vλ segments or human Vκ segments and one or more Jλ segments, Jκ segments, or J H Includes a segment. In one embodiment, the heavy chain constant gene sequence is C H 1-row, hinged row, C H 2 arrays, C H 3. Human sequences selected from combinations thereof. In one embodiment, one or more unrearranged immunoglobulin light chain variable gene segments replace all or substantially all functional endogenous heavy chain variable region gene segments at the endogenous heavy chain locus, and the heavy chain constant sequence is C H 1-row, hinged row, C H 2 arrays, and C H This is a mouse sequence containing 3 sequences.

[0305] In one embodiment, nucleic acid constructs, cells, embryos, mice, and methods are provided for producing proteins comprising one or more κ light chain and / or λ light chain variable domain immunoglobulin sequences and immunoglobulin heavy chain constant domain sequences, including proteins comprising human λ or κ light chain variable domains and human or mouse heavy chain constant domain sequences.

[0306] In one embodiment, a nucleotide sequence encoding an immunoglobulin variable region, produced in the mouse described herein, is provided.

[0307] In one embodiment, an amino acid sequence of the heavy chain or light chain variable region of an antibody produced in a mouse as described herein is provided.

[0308] In one embodiment, a heavy chain or light chain variable region nucleotide sequence encoding a variable region of an antibody produced in a mouse described herein is provided.

[0309] In one embodiment, an antibody or its antigen-binding fragment (e.g., Fab, F(ab)2, scFv) produced in a mouse described herein is provided.

[0310] In one embodiment, a binding protein is described that contains an immunoglobulin variable domain derived from a light chain (i.e., kappa (κ) and / or lambda (λ)) immunoglobulin variable domain, but not from a full-length heavy chain immunoglobulin variable domain. Methods and compositions for producing the binding protein, including genetically modified mice, are also provided.

[0311] In one embodiment, a method is provided for producing an antigen-binding protein that does not contain an immunoglobulin heavy chain variable domain, comprising the steps of immunizing a mouse described herein with a target antigen and maintaining the mouse under conditions that enable the mouse to produce an antigen-binding protein that specifically binds to the target antigen.

[0312] In one embodiment, a method is provided for producing an antigen-binding protein comprising a first immunoglobulin light chain variable domain adjacent to a heavy chain constant region sequence and a second immunoglobulin light chain variable domain adjacent to a light chain constant region sequence, comprising the steps of immunizing a mouse described herein with a target antigen and maintaining the mouse under conditions that enable the mouse to produce an antigen-binding protein that specifically binds to the target antigen.

[0313] In one embodiment, a method is provided for producing an antigen-binding protein lacking an immunoglobulin light chain, comprising an immunoglobulin light chain variable domain adjacent to a heavy chain constant region sequence, the method comprising the steps of immunizing a mouse described herein, which includes knockout of endogenous κ and λ loci (or non-functional endogenous κ and / or λ loci), with a target antigen, and maintaining the mouse under conditions that enable the mouse to produce an antigen-binding protein that specifically binds to the target antigen.

[0314] In one embodiment, the use of the mouse described herein for producing an immunoglobulin variable region nucleotide sequence is provided. In one embodiment, the variable region nucleotide sequence is a rearranged V L , D H , and J L Includes a gene segment. In one embodiment, the variable region nucleotide sequence is rearranged V L , D H , and J H It includes gene segments. In one embodiment, the variable region nucleotide sequence includes rearranged Vκ and Jκ gene segments. In one embodiment, the variable region nucleotide sequence includes rearranged Vλ and Jλ gene segments.

[0315] In one embodiment, the use of the mouse described herein for producing a fully human Fab or fully human F(ab)2 is provided. In one embodiment, the fully human Fab or fully human F(ab)2 is a rearranged V L , D H , and J L Includes a gene segment. In one embodiment, the above-mentioned whole human Fab or whole human F(ab)2 is rearranged V L , D H , and J HIncludes gene segments. In one embodiment, the whole human Fab or whole human F(ab)2 includes rearranged Vκ and Jκ gene segments. In one embodiment, the whole human Fab or whole human F(ab)2 includes rearranged Vλ and Jλ gene segments.

[0316] In one embodiment, a complete human Fab (first human V fused with a human light chain constant region) L , and a second human V fused with a human heavy chain constant region sequence L The use of the mice described herein for producing ) or complete human F(ab)2 is provided.

[0317] In one embodiment, the use of the mouse described herein for producing an immortalized cell line is provided. In one embodiment, the immortalized cell line comprises a nucleic acid sequence encoding a human Vλ or Vκ domain, operably ligated to a nucleic acid sequence comprising a mouse constant region nucleic acid sequence. In one embodiment, the immortalized cell line expresses an antibody comprising a human variable domain. In a particular embodiment, the human variable domain is a light chain variable domain.

[0318] In one embodiment, the use of the mouse described herein for producing a hybridoma or quadroma is provided. In one embodiment, the hybridoma or quadroma expresses a polypeptide comprising a human variable domain that binds to a target antigen.

[0319] In one embodiment, the use of the mouse described herein is provided for preparing a phage library containing a human light chain variable region. In one embodiment, the light chain variable region is a human Vκ region.

[0320] In one embodiment, the use of the mice described herein is provided for generating variable region sequences for the production of human antigen-binding proteins, the use comprising (a) immunizing the mice described herein with a target antigen, (b) isolating lymphocytes from the immunized mice in (a), (c) exposing the lymphocytes to one or more labeled antibodies, (d) identifying lymphocytes capable of binding to the target antigen, and (e) amplifying one or more human light chain variable region nucleic acid sequences from the lymphocytes to generate variable region sequences.

[0321] 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.

[0322] In one embodiment, the labeled antibody is a fluorophore-conjugated antibody. In one embodiment, one or more of the fluorophore-conjugated antibodies are selected from IgM, IgG, and / or a combination thereof.

[0323] In one embodiment, the lymphocytes are B cells.

[0324] In one embodiment, the one or more variable region nucleic acid sequences include a light chain variable region sequence. In a particular embodiment, the light chain variable region sequence is an immunoglobulin κ light chain variable region sequence. In one embodiment, the one or more variable region nucleic acid sequences are a λ light chain variable region sequence.

[0325] In one embodiment, the use of the mouse described herein is provided for generating one or more κ light chain variable region sequences for the production of a human antigen-binding protein, the use comprising (a) immunizing the mouse described herein with a target antigen, (b) isolating the spleen from the immunized mouse of (a), (c) exposing B lymphocytes from the spleen to one or more labeled antibodies, (d) identifying the B lymphocytes of (c) that can bind to the target antigen, and (e) amplifying the κ light chain variable region nucleic acid sequences from the B lymphocytes to generate the κ light chain variable region sequences.

[0326] In one embodiment, the use of the mouse described herein is provided for generating a κ light chain variable region sequence for producing a human antigen-binding protein, the use comprising: (a) immunizing the mouse described herein with a target antigen; (b) isolating one or more lymph nodes from the immunized mouse in (a); (c) exposing B lymphocytes from the one or more lymph nodes to one or more labeled antibodies; (d) identifying the B lymphocytes in (c) that can bind to the target antigen; and (e) amplifying the κ light chain variable region nucleic acid sequence from the B lymphocytes to generate the κ light chain variable region sequence.

[0327] In one embodiment, the use of the mouse described herein is provided for generating a κ light chain variable region sequence for producing a human antigen-binding protein, the use comprising (a) immunizing the mouse described herein with a target antigen, (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) that can bind to the target antigen, and (e) amplifying the κ light chain variable region nucleic acid sequence from the B lymphocytes to generate the κ light chain variable region sequence. In various embodiments, the one or more labeled antibodies are selected from IgM, IgG, and / or a combination thereof.

[0328] In various embodiments, the use of the mouse described herein is provided for generating κ light chain variable region sequences for producing human antigen-binding proteins, the use further comprising fusing the amplified κ light chain variable region sequence to a human heavy or light chain constant region sequence or, optionally, a human heavy chain variable region sequence, expressing the fused sequence in cells, and recovering the expressed sequence to thereby produce human antigen-binding proteins.

[0329] 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 C H 1. Hinge, C H 2, C H 3, C H This includes 4 or a combination thereof. In various embodiments, the light chain constant region is an immunoglobulin κ constant region. In various embodiments, the cells are selected from 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, CHO cells, GH3 cells, PC12 cells, human retinal cells (e.g., PER.C6® cells), and MC3T3 cells. In a particular embodiment, the cells are CHO cells.

[0330] In one embodiment, a method is provided for producing a human light chain variable region specific to a target antigen, the method comprising the steps of: immunizing a mouse described herein with the antigen; isolating at least one cell from a mouse producing a human light chain variable region specific to the antigen; producing at least one cell that produces a human antigen-binding protein containing the light chain variable region specific to the antigen; culturing at least one cell that produces the human antigen-binding protein; and obtaining the human antigen-binding protein. In one embodiment, the human light chain variable region is a human Vκ region.

[0331] In various embodiments, at least one cell isolated from a mouse producing a human light chain variable region specific to the above antigen is a splenocyte or a B cell.

[0332] In various embodiments, the antigen-binding protein is an antibody.

[0333] In various embodiments, immunization with the target antigen is carried out using a protein, DNA, a combination of DNA and protein, or cells expressing the antigen.

[0334] In one embodiment, the use of the mouse described herein is provided for producing nucleic acid sequences encoding immunoglobulin variable regions or fragments thereof. In one embodiment, the above nucleic acid sequence is used to produce human antibodies or antigen-binding fragments thereof. In one embodiment, the above mouse is used to produce antibodies, multispecific antibodies (e.g., bispecific antibodies), scFv, bis-scFv, diabody, triabody, tetrabody, V-NAR, V HH , V L An antigen-binding protein is created by selecting from F(ab), F(ab)2, DVD (i.e., a bivariable domain antigen-binding protein), SVD (i.e., a single variable domain antigen-binding protein), or a bispecific T-cell engager (BiTE).

[0335] In one embodiment, the use of the mouse described herein is provided for producing a drug (e.g., an antigen-binding protein) or a sequence encoding a variable sequence of a drug (e.g., an antigen-binding protein) for the treatment of a human disease or disorder.

[0336] In one embodiment, a second human light chain immunoglobulin variable sequence (V L 2) The first human light chain immunoglobulin variable sequence (V) is a cognitive sequence. LUse of a mouse as described herein for producing a nucleic acid sequence encoding 1), wherein the V is fused with the constant region of a human immunoglobulin light chain (polypeptide 1). L V is fused with the constant region of the human immunoglobulin heavy chain (polypeptide 2). L Along with 2, it is expressed as a polypeptide 1 / polypeptide 2 dimer and V L 1-V L It forms two antibodies.

[0337] In one embodiment, the use of a mouse described herein for producing a nucleic acid sequence encoding a human immunoglobulin light chain variable sequence fused with a human immunoglobulin heavy chain sequence, wherein the nucleic acid sequence is human V L -C H Encodes a polypeptide, here the human V L -C H The polypeptide is expressed as a dimer, and here the dimer is expressed in the absence of immunoglobulin light chains (for example, in the absence of human λ light chains or human κ light chains). In one embodiment, its V L -C H The dimer specifically binds to the target antigen in the absence of the λ light chain and in the absence of the κ light chain.

[0338] In one embodiment, the use of a mouse described herein for producing a nucleic acid sequence encoding all or part of an immunoglobulin variable domain. In one embodiment, the immunoglobulin variable domain is a human Vλ domain or a human Vκ domain.

[0339] In one embodiment, the use of nucleic acid constructs described herein for producing mice, cells, or therapeutic proteins (e.g., antibodies or other antigen-binding proteins) is provided.

[0340] In one embodiment, the use of mouse-derived nucleic acid sequences described herein for creating a cell line for producing a therapeutic agent for human use is provided. In one embodiment, the therapeutic agent for human use is a binding protein comprising a human light chain variable sequence (e.g., a human light chain variable sequence derived from a human Vλ segment or a human Vκ segment) fused to a human heavy chain constant sequence. In one embodiment, the therapeutic agent for human use comprises a first polypeptide which is a human λ immunoglobulin light chain or a human κ immunoglobulin light chain, and a second polypeptide which comprises a human Vλ variable sequence or a human Vκ variable sequence fused to a human heavy chain constant sequence.

[0341] In one embodiment, human C H Human V, fused with a domain, somatically mutated. L An expression system is provided, including mammalian cells transfected with a DNA construct encoding a polypeptide containing a domain.

[0342] In one embodiment, the expression system is human C L Immunoglobulin V fused to the domain L Further comprising a nucleotide sequence encoding the domain, wherein the human C L The V is fused with the domain. L The domain is the above human C H V is fused with the domain. L It is a cognitive light chain with respect to the domain.

[0343] In one embodiment, the mammalian cells are selected from CHO cells, COS cells, Vero cells, 293 cells, and retinal cells expressing viral genes (e.g., PER.C6® cells).

[0344] In one embodiment, a method for producing a binding protein, V L The nucleotide sequence encoding the domain is derived from the mouse cells described herein. H V fused into the domain L The process of obtaining it from the gene encoding the region, and human C HThe V L A method is provided which includes the steps of cloning a nucleotide sequence encoding a region sequence to form a human-binding protein sequence, and expressing the human-binding protein sequence in appropriate cells.

[0345] In one embodiment, the above mouse is immunized with the target antigen, and the above C H V fused into the domain L The region specifically binds to the target antigen epitope (for example, K in the range of micromolar, nanomolar, or picomolar concentrations). D (Specifically binds to C) In one embodiment, the above C H V fused into the domain L The nucleotide sequence encoding the region is subjected to somatic mutation in the above-mentioned mice.

[0346] In one embodiment, the appropriate cells are selected from B cells, hybridomas, quadromas, CHO cells, COS cells, 293 cells, HeLa cells, and human retinal cells expressing a viral nucleic acid sequence (e.g., PERC.6® cells).

[0347] In one embodiment, the above C H The region contains a human IgG isotype. In certain embodiments, the human IgG is selected from IgG1, IgG2, and IgG4. In another particular embodiment, the human IgG is IgG1. In another particular embodiment, the human IgG is IgG4. In another particular embodiment, the human IgG is a modified IgG4. In one embodiment, the modified IgG4 includes a substitution in the hinge region. In certain embodiments, the modified IgG4 includes a substitution at amino acid residue 228, numbered according to the Kabat EU numbering index, compared to wild-type human IgG4. In certain embodiments, the substitution at amino acid residue 228 is an S228P substitution, numbered according to the Kabat EU numbering index.

[0348] In one embodiment, the above cell is CH V fused into the domain L V originates from the domain and the light chain, which is the cognitive component. L The cognitive V further comprises a nucleotide sequence encoding a domain, wherein the method is fused to a human Cκ domain or a human Cλ domain. L The process further includes expressing a nucleotide sequence that codes for the domain.

[0349] In one embodiment, a method for producing a bispecific antigen-binding protein, comprising exposing a first mouse described herein to a first target antigen and producing a first human V that specifically binds to the first target antigen. L Steps to identify the domain sequence; Expose a second mouse described herein to a second target antigen and a second human V that specifically binds to the second target antigen. L A step of identifying the domain sequence, wherein the first human V L The domain does not bind to the second target antigen, and the second human V L The domain does not bind the antigen of the first target; and the first human V L The domain sequence is fused to the first heavy chain constant sequence to form the first antigen-binding polypeptide, and the second human V L A method is provided which includes the steps of: fusing the domain sequence to a second heavy chain constant sequence to form a second antigen-binding polypeptide; and utilizing the first antigen-binding polypeptide and the second antigen-binding polypeptide in a bispecificity binding protein.

[0350] In one embodiment, the antigen-binding protein further comprises the first human V L A first immunoglobulin light chain comprising a human κ or λ variable domain which is a domain and a cognitive domain, and the second human V L It contains a second immunoglobulin light chain, which includes a domain and a human κ or λ variable domain, which is a cognitive domain.

[0351] In one embodiment, the first heavy chain constant sequence is identical to the second heavy chain constant sequence. In one embodiment, the first heavy chain constant sequence includes modifications that reduce or eliminate the binding affinity of its first heavy chain constant region to protein A, and the second heavy chain constant sequence binds protein A.

[0352] In one embodiment, the first and second human V L The domain contains sequences derived from human Vκ and human Jκ gene segments. In one embodiment, the first and second human V L The domain contains sequences derived from human Vλ and human Jλ gene segments. In one embodiment, the first and second human V L The domain is Human D H Includes sequences derived from gene segments. In one embodiment, the first and second human V L The domain is Human J H Includes sequences derived from gene segments. In certain embodiments, the first and second human V L The domain is Human D H and human J H Includes sequences derived from gene segments. In certain embodiments, the first and second human V L The domain is Human D H and includes sequences derived from the human Jκ gene segment.

[0353] In one embodiment, the first human V L The domain contains sequences derived from the human Vκ and human Jκ gene segments, and the second human V L The domain contains sequences derived from human Vλ and human Jλ gene segments. In one embodiment, the first human V L The domain contains sequences derived from the human Vλ and human Jλ gene segments, and the second human V L The domain contains sequences derived from the human Vκ and human Jκ gene segments.

[0354] In one embodiment, an immunoglobulin variable region (VR) (e.g., human J) is produced in a mouse as described herein. L Sequence, or human J H Sequence, or human D H Sequence and Human J H Sequence, or human D H Sequence and Human J L Human V fused with the sequence L A sequence (including) is provided. In a particular embodiment, the immunoglobulin VR is derived from a germline human gene segment selected from the Vκ segment and the Vλ segment, where the VR is encoded by a rearranged sequence derived from the mouse, where the rearranged sequence is somatically hypermutated. In one embodiment, the rearranged sequence contains 1 to 5 somatic hypermutations. In one embodiment, the rearranged sequence contains at least 6, 7, 8, 9, or 10 somatic hypermutations. In one embodiment, the rearranged sequence contains more than 10 somatic hypermutations. In one embodiment, the rearranged sequence contains one or more human heavy chain constant region sequences or mouse heavy chain constant region sequences (e.g., human or mouse C H 1-row, hinged row, C H 2 arrays, C H It is fused with 3 sequences (and combinations thereof).

[0355] In one embodiment, the amino acid sequence of the immunoglobulin variable domain of a binding protein produced in a mouse as described herein is provided. In one embodiment, the VR is made up of one or more human heavy chain constant region sequences or mouse heavy chain constant region sequences (e.g., human or mouse C H 1-row, hinged row, C H 2 arrays, C H It is fused with 3 sequences (and combinations thereof).

[0356] In one embodiment, a light chain variable domain encoded by a nucleic acid sequence derived from a mouse as described herein is provided.

[0357] In one embodiment, an antibody or its antigen-binding fragment (e.g., Fab, F(ab)2, scFv) is provided, which is produced in the mice described herein or derived from a sequence produced in the mice described herein. In certain embodiments, for example, the following are provided: (Item 1) Non-human animals, and the following: (a) 1 or more human V L Gene segments and one or more human J L Insertion of a gene segment upstream of the non-human immunoglobulin light chain constant region, (b) Human V that can contain 1 L Gene segments and one or more human J L Insertion of a gene segment upstream of the non-human immunoglobulin heavy chain constant region, and (c) A nucleotide sequence encoding the ADAM6 protein or a functional fragment thereof, comprising a nucleotide sequence expressed from an ectopic ADAM6 nucleic acid sequence, Non-human animals. (Item 2) The non-human animal described in item 1, wherein the non-human heavy chain and / or light chain constant regions are rodent constant regions. (Item 3) A non-human animal as described in item 1 or 2, wherein the constant light chain region is mouse Cκ. (Item 4) Upstream of the non-human immunoglobulin light chain constant region of the human V L Gene segments and human J L A non-human animal as described in either of the preceding items, wherein the gene segment is the human Vκ gene segment and the human Jκ gene segment. (Item 5) Upstream of the non-human immunoglobulin heavy chain constant region of the human V L Gene segments and human J L A non-human animal as described in either of the preceding items, wherein the gene segment is the human Vκ gene segment and the human Jκ gene segment. (Item 6) A non-human animal according to any one of the preceding items, wherein the nucleotide sequence encoding the ADAM6 protein or a functional fragment thereof is located at the same location as the wild-type non-human ADAM6 locus. (Item 7) A non-human animal according to any one of the preceding items, wherein the nucleotide sequence encoding the ADAM6 protein or a functional fragment thereof is located within an immunoglobulin gene segment. (Item 8) The non-human animal described in item 7, wherein the immunoglobulin gene segment is a human κ-light chain gene segment. (Item 9) The non-human animal according to item 7 or 8, wherein the immunoglobulin gene segment is the endogenous heavy chain gene segment of the non-human animal. (Item 10) The aforementioned non-human animal has immunoglobulin V L Endogenous immunoglobulin V that cannot be rearranged to form a domain. L and / or J L A non-human animal, including a gene segment, as described in any one of the preceding items. (Item 11) (a) One or more unrearranged human Vκ gene segments and one or more unrearranged human Jκ gene segments at the endogenous immunoglobulin heavy chain gene locus of the non-human animal, (b) One or more unrearranged human Vκ gene segments and one or more unrearranged human Jκ gene segments at the endogenous immunoglobulin light chain locus of the non-human animal. A genetically modified non-human animal that contains the ADAM6 protein or a functional fragment thereof and is capable of expressing the ADAM6 protein or a functional fragment thereof. (Item 12) Cells derived from non-human animals as described in item 1 or 11. (Item 13) The cell described in item 12, wherein the cell is a B cell. (Item 14) A hybridoma generated from B cells as described in item 13. (Item 15) The cells described in item 12, wherein the non-human animal is a rodent. (Item 16) The cells described in item 15, wherein the rodent is selected from mice and rats. (Item 17) (a) The step of exposing a non-human animal described in item 1 or 11 to the target antigen; (b) A step of isolating one or more B lymphocytes of the non-human animal, wherein the one or more B lymphocytes bind to the target antigen. L The process of expressing the binding protein; (c) The V to which the target antigen is bound L Binding protein V L A step of identifying the nucleic acid sequence encoding the domain, the V L The binding protein comprises a human Vκ domain and a non-human light chain constant domain, as well as a human Vκ domain and a non-human heavy chain constant domain; A method is provided for producing an antigen-binding protein that binds to a target antigen, comprising the steps of (d) and (c) using the nucleic acid sequence of a human immunoglobulin constant region together with a human immunoglobulin constant region nucleic acid sequence to produce a human antigen-binding protein that binds to the target antigen. (Item 18) The aforementioned V L The method according to item 17, wherein the non-human light chain constant region of the binding protein is mouse Cκ, and the non-human heavy chain constant region is the mouse heavy chain constant region. (Item 19) The method according to item 17 or 18, wherein the non-human animal is a mouse. (Item 20) A rodent comprising one or more human immunoglobulin light chain gene segments operably linked to a non-human immunoglobulin heavy chain constant region gene, and expressing one or more ADAM6 proteins. (Item 21) The rodent according to item 20, wherein the one or more human immunoglobulin light chain gene segments are a human Vκ gene segment and a human Jκ gene segment. (Item 22) A rodent as described in item 20 or 21, wherein the non-human immunoglobulin heavy chain constant region gene is a mouse or rat heavy chain constant region gene. (Item 23) The immunoglobulin heavy chain constant region gene is C H A rodent as described in any one of items 20 to 22, including one region and / or a hinge region. (Item 24) A rodent as described in any one of items 20 to 23, comprising the deletion or substitution of one or more endogenous immunoglobulin heavy chain gene sequences. (Item 25) The aforementioned endogenous V H Gene segment, D H gene segment, and J H A rodent as described in any one of items 20-24, whose gene segment cannot be rearranged to form a rearranged V / D / J sequence. (Item 26) A rodent as described in any one of items 20 to 25, comprising a deletion of the endogenous ADAM6 gene and further comprising an ectopic mouse ADAM6 gene. (Item 27) A rodent selected from mice or rats, as described in any one of items 20-26. (Item 28) (a) A step of making a first modification of a rodent heavy chain immunoglobulin locus that results in a reduction or elimination of endogenous ADAM6 activity in male rodents, wherein the first modification is one or more human V L Gene segment, 1 or more human J L A process comprising the insertion of gene segments and gene segments selected from combinations thereof; and (b) A second modification step of adding a nucleic acid sequence to the rodent that confers ADAM6 activity, which is functional in male rodents, to the rodent. A method for modifying the heavy chain immunoglobulin gene locus in rodents, including [specific example]. (Item 29) The first modification described above is one or more V L Gene segment and one or more J L One or more human D2 genes that can be rearranged together with the gene segment. H The method described in item 28, further comprising the insertion of a gene segment. (Item 30) The one or more V L Gene segments and one or more human J L The method according to item 28, wherein the gene segment is a Vκ gene segment and a Jκ gene segment, or a Vλ gene segment and a Jλ gene segment. (Item 31) The nucleic acid sequence that confers functional ADAM6 activity to the rodent in male rodents is one or more human V L Gene segment and / or the one or more human J L The method described in any one of items 28-30, which is adjacent to the gene segment. (Item 32) The method according to any one of items 28 to 31, wherein the first and second modifications are performed in a single ES cell, and the single ES cell is introduced into a host embryo to produce the rodent. [Brief explanation of the drawing]

[0358] [Figure 1]Figure 1 illustrates an overview (not in a fixed proportion) of the mouse heavy chain locus (top) and the human κ light chain locus (bottom). The mouse heavy chain locus is approximately 3 Mb long and contains approximately 200 heavy chain variable (VH) gene segments, 13 heavy chain diversity (DH) gene segments, and 4 heavy chain ligation (JH) gene segments, as well as enhancer (Enh) and heavy chain constant (CH) regions. The human κ light chain locus overlaps into distal and proximal contigs, each approximately 440 kb to 600 kb in length, with opposite polarity. Between these two contigs, there is approximately 800 kb of DNA that is not thought to contain Vκ gene segments. The human κ light chain locus contains approximately 76 Vκ gene segments, 5 Jκ gene segments, an intron enhancer (Enh), and a single constant region (Cκ).

[0359] [Figure 2] Figure 2 shows an exemplary targeting strategy for sequentially inserting 40 human Vκ gene segments and 5 human Jκ gene segments into a mouse immunoglobulin heavy chain locus, resulting in a modified mouse immunoglobulin heavy chain locus containing human Vκ and Jκ gene segments operably linked to the constant region of the mouse immunoglobulin heavy chain. Hygromycin (hyg) and neomycin (neo) selective cassettes are shown along with their recombinase recognition sites (R1, R2, etc.).

[0360] [Figure 3] Figure 3 shows an exemplary targeting strategy for sequentially inserting multiple human Vλ gene segments and a single human Jλ gene segment into mouse heavy chain loci. Hygromycin (hyg) and neomycin (neo) selector cassettes are shown along with their recombinase recognition sites (R1, R2, etc.).

[0361] [Figure 4]Figure 4 shows an exemplary targeting strategy for sequentially inserting multiple human Vλ gene segments and four human Jλ gene segments into mouse heavy chain loci. Hygromycin (hyg) and neomycin (neo) selector cassettes are shown along with their recombinase recognition sites (R1, R2, etc.).

[0362] [Figure 5] Figure 5 shows an exemplary targeting strategy for sequentially inserting the human Vλ gene segment, human DH gene segment, and human JH gene segment into the mouse heavy chain locus. Hygromycin (hyg) and neomycin (neo) selector cassettes are shown along with their recombinase recognition sites (R1, R2, etc.).

[0363] [Figure 6] Figure 6 shows an exemplary targeting strategy for sequentially inserting the human Vλ gene segment, human DH gene segment, and human Jκ gene segment into the mouse heavy chain locus. Hygromycin (hyg) and neomycin (neo) selector cassettes are shown along with their recombinase recognition sites (R1, R2, etc.).

[0364] [Figure 7] Figure 7 shows the steps for cloning a genomic fragment encoding the mouse ADAM6 gene from the intergenic region of the mouse immunoglobulin heavy chain (VD), and the engineering steps for modifying the genomic fragment for insertion into a modified immunoglobulin heavy chain locus.

[0365] [Figure 8] Figure 8 shows a targeting strategy for inserting a genomic fragment encoding the mouse ADAM6 gene into the Vκ-Jκ intergenic region of a modified mouse immunoglobulin heavy chain locus containing human Vκ and Jκ gene segments operably linked to the constant region of the mouse immunoglobulin heavy chain.

[0366] [Figure 9] Figure 9 shows a targeting strategy for inserting a genomic fragment encoding the mouse ADAM6 gene upstream (5' side) of the human Vκ gene segment (i.e., hVκ2-40) of a modified mouse immunoglobulin heavy chain locus containing human Vκ and Jκ gene segments operably linked to the constant region of the mouse immunoglobulin heavy chain. [Modes for carrying out the invention]

[0367] Detailed explanation The present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may change. The scope of the present invention is defined by the claims, and it should be understood that the technical terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0368] Unless otherwise defined, all terms and phrases used herein include the meanings they have acquired in the art, unless it is clearly pointed out or evident from the context in which they are used. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but specific methods and materials are described here. All publications mentioned are incorporated herein by reference.

[0369] When used to refer to the quantity of gene segments, the phrase “substantial” or “substantially” (e.g., “substantially all” V gene segments) includes both functional and non-functional gene segments and, in various embodiments, includes, for example, 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 include, for example, at least 95%, 96%, 97%, 98% or 99% of functional (i.e., non-pseudogene) gene segments.

[0370] The term “placement” includes cases where a DNA sequence is placed in the cellular genome in such a way that it replaces a sequence within the genome with a heterologous sequence (e.g., a human sequence in a mouse) at a locus of the genome sequence. The DNA thus 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 thus placed sequence. For example, in various embodiments, the substitution is the replacement of an endogenous sequence in place of a heterologous sequence, resulting in the production of a gene product from the placed DNA sequence (including the heterologous sequence), and not the expression of the endogenous sequence; the substitution is the replacement of an endogenous genome sequence with a DNA sequence that encodes a protein having a similar function to the protein encoded by the endogenous genome sequence (e.g., the endogenous genome 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 is an orthologue of the endogenous gene or fragment being replaced, a homolog of the endogenous gene or fragment being replaced, or a human gene or fragment that is substantially identical or the same in terms of structure and / or function as the endogenous gene or fragment being replaced.

[0371] The term “contiguous” encompasses references to being located on the same nucleic acid molecule. For example, two nucleic acid sequences are “contiguous” if they are located on the same nucleic acid molecule but are interrupted by another nucleic acid sequence. For instance, the last codon of a rearranged V(D)J sequence does not directly follow the first codon of a constant region sequence, but the V(D)J sequence is “contiguous” with the constant region gene sequence. In another example, two V gene segment sequences located on the same genomic fragment are “contiguous,” but they may be separated by sequences that do not code for codons in the V region, for example, they may be separated by regulatory sequences, such as a promoter or other non-coding sequences. In one embodiment, a contiguous sequence comprises a genomic fragment containing a genomic sequence arranged to be found in a wild-type genome.

[0372] When the phrase "derived from" is used in reference to a variable region "derived from" a gene or gene segment being referred to, it includes the ability to trace its sequence back to a specific unrearranged gene segment or a gene segment that has been rearranged to form a gene expressing that variable domain (including, where applicable, splice differences and somatic mutations).

[0373] The term "functional," when used in relation to variable region gene segments or linked gene segments, refers to the use of the antibody repertoire expressed; for example, in humans, Vλ gene segments 3-1, 4-3, and 2-8 are functional, while Vλ gene segments 3-2, 3-4, and 2-5 are non-functional.

[0374] The "heavy chain locus" is heavy chain variable in wild-type mice (V H ), heavy chain diversity (D H ), heavy chain linkage (J H ) and heavy chain steady state (C HThis includes locations on chromosomes where the DNA sequence of the region is found, for example, on mouse chromosomes.

[0375] The term "bispecific binding protein" encompasses binding proteins that are capable of selectively binding to two or more epitopes. A bispecific binding protein is a first C H The first light chain variable domain (V) is fused with the region. L 1) and the second C H The second light chain variable domain (V) is fused with the region. L 2) comprises two different polypeptides including the first and second C above. H The regions are either identical or differ only by one or more amino acid substitutions (e.g., amino acid substitutions described herein). L 1 and V L 2 specifically binds to different epitopes (to two different molecules (e.g., antigens) or to the same molecule (e.g., the same antigen)). When a bispecific binding protein selectively binds to two different epitopes (a first epitope and a second epitope), V for the first epitope L Affinity 1 is generally V for the second epitope mentioned above. L Affinity of 1 is at least 1-2 or 3 or 4 orders of magnitude smaller, V L The opposite is true for point 2. Epitopes recognized by the bispecific binding proteins mentioned above may be present on the same target or on different targets (for example, they may be present on the same antigen or on different antigens). Bispecific binding proteins recognize, for example, different epitopes of the same antigen. L 1 and V L It can be produced by combining with 2. For example, V that recognizes different epitopes of the same antigen as above. L The nucleic acid sequence that codes for the sequence is different C HThese can be fused to nucleic acid sequences that encode a region, and such sequences can be expressed in cells that express immunoglobulin light chains, as well as in cells that do not express immunoglobulin light chains. Typical bispecific binding proteins each consist of three light chain CDRs followed by (N-terminus to C-terminus) C H 1 domain, hinge, C H 2 domains, and C H Two heavy chains having 3 domains, and an immunoglobulin light chain that does not confer antigen-binding specificity but can associate with each heavy chain, or is capable of associating with each heavy chain, V L 1 and / or V L The molecule comprises an immunoglobulin light chain capable of binding to one or more of the epitopes to which 2 binds, or an immunoglobulin light chain capable of associating with each heavy chain, enabling or assisting one or both of the heavy chains to bind to one or both of the epitopes.

[0376] Therefore, the two common types of bispecific binding proteins are (1) V L 1-C H (Dimer), and (2)V L 1-C H : Light chain + V L 2-C H : Light chain (whether the light chain is the same or different). In either case, the above C H The heavy chain constant region (i.e., the heavy chain constant region described herein) may also be differentially modified as described herein (for example, modified to differentially bind to protein A, modified to extend the serum half-life, etc.), or it may remain the same.

[0377] The term “cell” when used in relation to sequence expression includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include those of prokaryotes and eukaryotes (single or multiple cells), bacterial cells (e.g., strains such as Escherichia coli, Bacillus spp., Streptomyces spp.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, B cells, or cell fusions, such as hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells are eukaryotic cells and are selected from the following: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo205, HB8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT060562, Sertoli cells, BRL3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cells include one or more viral genes, for example, retinal cells expressing the viral genes (e.g., PER.C6® cells).

[0378] The meaning of "~and cognitive," for example, the second V L The first V is in the domain "~and Cognate". L When used in the sense of a domain, the term "cognite" refers to two Vs derived from the same binding protein produced by mice according to the present invention. LIt is intended to include references to relationships between domains. For example, a mouse genetically modified according to an embodiment of the present invention, e.g., V H area, D H Region, and J H The region is V L Region and J L The first human V was produced by mice with a heavy chain locus replaced in the region. L The same mouse C that is fused with the domain H Two identical polypeptide chains were created from a region (e.g., an IgG isotype), as well as a second human V L The same mouse C that is fused with the domain L Antibody-like binding proteins are produced from the region, each having two identical polypeptide chains. During clonal selection in the above mice, the first and second human V proteins appear together in relation to a single antibody-like binding protein. L The domains were selected by a clone selection process. Therefore, as a result of the above clone selection process, the first and second Vs appear together in a single antibody-like molecule. L The domain is referred to as a "cognite." In contrast, unless the first and second antibody-like molecules have the same heavy chain (i.e., the V fused to the first human heavy chain region), L V is fused to the domain and the second human heavy chain region mentioned above. L (Unless it is identical to the domain) V appearing in the first antibody-like molecule L The V that appears in the domain and the second antibody-like molecule L A domain is not a cognitive entity.

[0379] The term “complementarity-determining region,” or “CDR,” refers to an amino acid sequence encoded by a nucleic acid sequence of an immunoglobulin gene in a living organism that typically appears (i.e., in wild-type animals) between two framework regions of the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor). A CDR can be encoded, for example, by a germline sequence or a rearranged or unrearranged sequence, and, for example, by a naive or mature B cell or T cell. In some circumstances (e.g., in CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, for example, as a result of splicing or joining the above sequences (e.g., VDJ recombination to form a heavy chain CDR3).

[0380] The terms “gene segment” or “segment” include references to V (light or heavy chain) or D or J (light or heavy chain) immunoglobulin gene segments, including unrearranged sequences at immunoglobulin loci (e.g., human and mouse) that can be involved in rearrangements (e.g., mediated by endogenous recombinases) that form rearranged V / J or V / D / J sequences. Unless otherwise indicated, the above V, D, and J segments include recombinant signaling sequences (RSS) that enable V / J or V / D / J recombination according to the 12 / 23 rule. Unless otherwise indicated, the above segments further include sequences associated with native or functional equivalents thereof (e.g., V segment promoters and leaders).

[0381] The terms "heavy chain" or "immunoglobulin heavy chain" encompass the constant region sequence of an immunoglobulin heavy chain derived from any organism, and unless otherwise specified, include the heavy chain variable domain (V H ) includes. Unless otherwise specified, V HThe domain contains three heavy chain CDRs and four framework (FR) regions. The heavy chain fragments include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has the following variable domain (from N-terminus to C-terminus): C H 1 domain, hinge, C H 2 domains, C H 3 domains, and C as needed. HIt essentially consists of four domains (e.g., in the case of IgM or IgE) and a transmembrane (M) domain (e.g., in the case of membrane-bound immunoglobulins in lymphocytes). The heavy chain constant region is the region of the heavy chain that extends from the outside of FR4 to the C-terminus of the heavy chain (from the N-terminus to the C-terminus). In addition to heavy chain constant regions with slight deviations, e.g., cleavage of one, two, three or several amino acids from the C-terminus, heavy chain constant regions with sequence modifications, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, are also included by the term "heavy chain constant region". The amino acid substitutions are, for example (referring to the EU numbering for the immunoglobulin constant region, e.g., the human IgG constant region), at positions 228, 233, 234, 235, 236, 237, 238, 239, 241, 248, 249, 250, 252, 254, 255, 256, 258, and 265. 267th, 268th, 269th, 270th, 272nd, 276th, 278th, 280th, 283rd, 285th, 286th, 289th, 290th, 292nd , 293rd, 294th, 295th, 296th, 297th, 298th, 301st, 303rd, 305th, 307th, 308th, 309th, 311th, 312th, 315th, 318th, 320th, 322nd, 324th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 3 35th, 337th, 338th, 339th, 340th, 342nd, 344th, 356th, 358th, 359th, 360th, 361st, 362nd, 373rd, 37th It can be manufactured at one or more positions selected from 5th, 376th, 378th, 380th, 382nd, 383rd, 384th, 386th, 388th, 389th, 398th, 414th, 416th, 419th, 428th, 430th, 433rd, 434th, 435th, 437th, 438th, and 439th.

[0382] Without intending to limit the possibilities, for example, the heavy chain constant region may be modified to exhibit an extension of the serum half-life (compared to the same heavy chain constant region without the listed modifications(s)), and may have modifications at position 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at position 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F) and 434. In another example, the above modifications may include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, modifications of 259I (e.g., V259I), and modifications of 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), and modifications of 307 and / or 308 (e.g., 308F or 308P).

[0383] The term "light chain" refers to the constant state of immunoglobulin light chains (C) derived from any organism. L ) Includes the region sequence and, unless otherwise specified, includes the human κ light chain and human λ light chain. Unless otherwise specified, includes the light chain variable (V L The ) domain typically contains three light chain CDRs and four framework (FR) regions. Generally, the full-length light chain (V L +C L ) contains V, which has FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus. L Domain, and C L Includes the region. Light chain (V) that can be used in this invention L +C L) is, for example, a first or second (in the case of a bispecific binding protein) epitope to which the above-mentioned binding protein selectively binds (for example, the above-mentioned C H V is fused with the domain. L It includes light chains that do not selectively bind to the epitopes (or multiple epitopes) to which the domain selectively binds. H V is fused with the domain. L V does not selectively bind to the epitope(s) to which the domain binds. L The domain can be identified by screening for light chains that are most commonly used in existing antibody libraries (wet libraries or in silico) and that do not substantially interfere with the affinity and / or selectivity of the epitope-binding domain of the binding protein. L It includes the domain. The appropriate light chain is C above. H V fused into the domain L The region can bind to an epitope to which it specifically binds (by itself or the C H The cognition that is fused with the domain ItoV L (In combination with) it includes a light chain.

[0384] The term "micromolar range" is intended to mean a concentration range of 1 to 999 micromoles, the term "nanomolar range" is intended to mean a concentration range of 1 to 999 nanomoles, and the term "picomolar range" is intended to mean a concentration range of 1 to 999 picomoles.

[0385] The term “non-human animal” is intended to include any non-human animal, e.g., cyclostomes, bony fish, cartilaginous fish, e.g., sharks and rays, amphibians, reptiles, mammals, and birds. Suitable non-human animals include mammals. Suitable mammals include non-human primates, goats, sheep, pigs, dogs, cattle, and rodents. Suitable non-human animals are selected from the family of rodents, including rats and mice. In one embodiment, the non-human animal is a mouse.

[0386] Mice as genetic models have been greatly improved by transgenic and knockout techniques, which have enabled the study of the effects of targeted overexpression or deletion of specific genes. Despite all its advantages, the aforementioned mice still present genetic limitations that make them imperfect models for human diseases and imperfect platforms for testing or developing human therapeutics. Firstly, while approximately 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 cannot cross-react, or cross-react improperly, with mouse orthologues of their intended human targets. To prevent this problem, the selected target gene can be "humanized," that is, the mouse gene can be erased and replaced with the corresponding orthologous human gene sequence (see, for example, U.S. Patents 6,586,251, 6,596,541 and 7,105,348; these are incorporated herein by reference).Initially, efforts to humanize mouse genes using the "knockout-plus-transgenic humanization" strategy required crossing mice carrying the aforementioned endogenous gene deletion (i.e., knockout) with mice carrying randomly incorporated human transgenes (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 models of classic and intermediate maple syrup urine disease, BMC Med Genet 7:33; Jamsai et al. (2006), A humanized BAC transgenic / knockout mouse model for HbE / beta-thalassemia, Genomics). 88(3):309-15; Pan et al. (2006), Different role for mouse and human 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; conventional knockout techniques were not sufficient to directly replace large mouse genes with their larger human genome counterparts.A direct homologous substitution approach, in which an endogenous mouse gene is directly replaced by a human counterpart gene at the exact same location (i.e., the endogenous mouse locus) in the mouse gene, is rarely attempted due to technical difficulties. To date, efforts toward direct substitution 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.

[0387] Exogenously introduced human immunoglobulin transgenes rearrange in mouse precursor B cells (Alt et al. (1985), Immunoglobulin genes in transgenic mice, Trends Genet 1:231-236). This discovery was utilized in the engineering of 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, N. (2005), Human antibodies from transgenic animals. Nat Biotechnol 23:1117-1125;Lonberg, N. et al. (1994)Antigen-specific human antibodies from mice comprising four distinct genetic modifications, Nature 368:856-859; Jakobovits et al. (2007) From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice. Nat Biotechnol 25:1134-1143). In these mice, the endogenous mouse immunoglobulin heavy chain and kappa light chain gene loci were inactivated by targeted deletion of small but significant portions of each endogenous locus, followed by the introduction of human immunoglobulin gene loci as randomly incorporated large transgenes or as minichromosomes, as described above (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 symbolized a significant advance in genetic engineering; the fully human monoclonal antibodies isolated from them showed promising therapeutic potential for treating various 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ら、2007;Kimら(2007)Clinical efficacy of zanolimumab(HuMax-CD4):two Phase II studies in refractory cutaneous T-cell lymphoma.Blood 109(11):4655-62;Lonberg、2005;Makerら(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ら(2006)、Denosumab in postmenopausal women with low bone Mineral density (N 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 mice's ability to maintain active humoral responses and therefore produce fully human antibodies against certain antigens. The above failures are due to (1) insufficient functionality resulting from the random introduction of human immunoglobulin transgenes, and inaccurate expression resulting from the lack 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 176I6839-685 1); (2) Inefficient interspecies interactions between the human constant domain and the mouse elements of the B-cell receptor signaling complex on the cell surface that can impair the signaling processes required for the 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 serum immunoglobulin concentration (Brambell et al. (1964), A Theoretical Model of Gamma-Globulin Catabolism, Nature 203: 1352-1354; Junghans and Anderson (1996), The protection receptor for IgG catabolism is the 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; Nimmerjahn and Ravetch (2007), Fc-receptors as regulators of immunity. Adv Immunol 96, 179-204) may be reduced due to inefficient interspecies interactions between soluble human immunoglobulins and mouse Fc receptors. These deficiencies can be corrected by in situ humanization of only the variable region of the mouse immunoglobulin locus in its native location at the endogenous heavy and light chain loci.This would effectively result in mice producing "reverse chimeric" (i.e., human V:mouse C) antibodies that, based on the preservation of the mouse constant region, would be capable of normal interaction and selection in the mouse environment. Furthermore, such reverse chimeric antibodies can be readily rearranged into fully human antibodies for therapeutic purposes.

[0388] Genetically modified animals can be created that include insertions or replacements of heterologous (e.g., from another species) immunoglobulin sequences at the endogenous immunoglobulin heavy chain locus, either with insertions or replacements at the endogenous immunoglobulin light chain locus, or with immunoglobulin light chain transgenes (e.g., chimeric immunoglobulin light chain transgenes or completely human-to-complete mice). The species from which the heterologous immunoglobulin sequences originate can be diverse. Exemplary heterologous immunoglobulin sequences include human sequences.

[0389] In various embodiments, immunoglobulin variable region nucleic acid sequences, e.g., V, D, and / or J segments, are obtained from human or non-human animals. Suitable non-human animals for supplying V, D, and / or J segments include, for example, bony fish, cartilaginous fish, e.g., sharks and rays, amphibians, reptiles, mammals, and birds (e.g., chickens). Examples of non-human animals include mammals. Examples of mammals include non-human primates, goats, sheep, pigs, dogs, cattle (e.g., cows, cattle, 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 is evident from this context, various non-human animals (e.g., sharks, rays, mammals (e.g., camels, rodents, e.g., mice and rats)) can be used as sources for variable domains or variable region gene segments.

[0390] In the context described above, non-human animals are also used as sources of constant region sequences used in conjunction with variable sequences or segments. For example, rodent constant sequences can be used in transgenes that are operably linked to human or non-human variable sequences (e.g., rodents, e.g., mice, rats, or hamsters, or human or non-human primate variable sequences operably linked to constant sequences). Thus, in various embodiments, human V, D, and / or J segments are operably linked to rodent (e.g., mouse, rat, or hamster) constant region gene sequences. In some embodiments, the above 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 in transgenes that are incorporated, for example, into a locus other than an endogenous immunoglobulin locus.

[0391] In certain embodiments, V at the endogenous immunoglobulin heavy chain locus H , D H and J H One or more human V segments L Gene segments and one or more human J L Including replacement in a gene segment, one or more human V L Gene segment and one or more J L We provide a mouse in which a segment is operably linked to an endogenous immunoglobulin heavy chain gene, the mouse in which the transgene is located at a locus other than the endogenous immunoglobulin locus, and the transgene is operably linked to a non-rearranged or rearranged human V locus in a mouse, rat, or human constant region. L and human J L Includes a gene segment. In various embodiments, one or more of the above human V L Examples of gene segments include the human Vκ gene segment or the human Vλ gene segment. In one embodiment, one or more of the above human J L Gene segments include the human Jκ gene segment or the human Jλ gene segment.

[0392] A method for large in situ genetic replacement of the mouse germline immunoglobulin heavy chain variable gene with that of the human germline immunoglobulin light chain variable gene is described, while maintaining the ability of the mouse to produce offspring. Specifically, the precise replacement of the mouse heavy chain variable gene locus with that of the human light chain variable gene locus is described, while keeping the mouse constant region intact. As a result, mice expressing immunoglobulin-like binding proteins in relation to the endogenous constant region are created. The human light chain variable region is linked to and rearranged with the mouse heavy chain constant region to form a chimeric human-mouse immunoglobulin locus that expresses a unique immunoglobulin-like molecule. The expressed immunoglobulin-like molecule is a "reverse chimera," meaning it contains both the human variable region sequence and the mouse constant region sequence.

[0393] Even at precise locations, such as the endogenous mouse immunoglobulin locus described above, engineering human immunoglobulin sequences in the mouse genome can present some challenges due to the diverse evolution of immunoglobulin loci between mice and humans. For example, inter-gene sequences scattered within immunoglobulin loci are not identical between mice and humans, and in some situations, may not be functionally equivalent. These differences in immunoglobulin loci between mice and humans can also result in abnormalities in humanized mice, particularly when humanizing or manipulating certain portions of the endogenous mouse immunoglobulin heavy chain locus. Some modifications to the mouse immunoglobulin heavy chain locus are harmful. Harmful modifications include, for example, the loss of mating and offspring-producing ability in modified mice. In various embodiments, the engineering of human immunoglobulin sequences in the mouse genome described above involves methods for maintaining endogenous sequences that would be harmful if absent in the modified mouse strain. Exemplary adverse effects may include the inability of the modified strain to grow, loss of function of essential genes, and inability to express polypeptides. Such adverse effects may be directly or indirectly related to the engineered modifications to the mouse genome described above.

[0394] Despite the near-wild-type humoral immune function observed in mice with replaced immunoglobulin loci, there are other challenges encountered when using direct immunoglobulin substitution that are not encountered with some approaches that utilize randomly incorporated transgenes. Differences in the genetic composition of immunoglobulin loci between mice and humans have led to the discovery of sequences beneficial for breeding mice with replaced immunoglobulin gene segments. Specifically, the mouse ADAM gene, located within the aforementioned endogenous immunoglobulin heavy chain locus, is optimally present in mice with replaced immunoglobulin loci due to its role in fertility.

[0395] Including all mouse constant-chain genes and locus transcriptional regulatory regions, the hybrid locus leaves adjacent mouse sequences intact and functional, while the 6 megabase variable region (V) of the mouse heavy-chain immunoglobulin locus is preserved. H -D H -J H ) the human immunoglobulin light chain variable gene locus (V L -J L Precise in-situ substitution is performed at (Figures 2-6). Manipulation steps are carried out to maintain the mouse sequence that confers the ability to mate and produce offspring in a manner comparable to wild-type mice (Figures 7-9). Specifically, a human immunoglobulin κ light chain locus of approximately 0.5 megabases containing the proximal arm (i.e., 40 functional human Vκ gene segments and 5 human Jκ gene segments) and the mouse ADAM6 gene were introduced into mouse ES cells using VELOCIGENE® gene engineering technology via a chimeric BAC targeting vector (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al., 2003, High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nat Biotechnol 21:652-659).

[0396] Location and function of the mouse ADAM6 genome Male mice lacking the ability to express any functional ADAM6 protein exhibit a severe defect in their ability to mate and produce offspring. These mice lack the ability to express functional ADAM6 protein by replacing all or substantially all mouse immunoglobulin heavy chain variable gene segments with human light chain variable gene segments. The ADAM6 locus is D H V located upstream of the gene segment HBecause the ADAM6 function is located within the region of the endogenous immunoglobulin heavy chain variable gene locus, proximal to the 3' end of the gene segment locus, this results in a loss of ADAM6 function. Breeding mice that are homozygous for the substitution of all or substantially all endogenous heavy chain variable gene segments with human light chain variable gene segments is generally a cumbersome approach, as it requires supplying homozygous males and females awaiting productive mating. Well-formed littermates are relatively rare, and the average litter size is very small. Instead, heterozygous males with respect to the substitution were used to breed homozygous females to produce heterozygous progeny, from which homozygous mice were subsequently bred. The inventors determined that a possible cause of the loss of fertility in these male mice is the absence of functional ADAM6 protein in homozygous male mice.

[0397] In various embodiments, male mice containing a damaged (i.e., non-functional or slightly functional) ADAM6 gene exhibit reduced or extinct fertility. Because the ADAM6 gene is located at the immunoglobulin heavy chain locus in mice (and other rodents), the inventors have decided to utilize various modification breeding or breeding schemes to breed mice containing modifications to the endogenous immunoglobulin heavy chain locus, or to create and maintain strains of such mice. The low fertility or infertility of homozygous male mice with respect to the replacement of the endogenous immunoglobulin heavy chain variable locus makes it difficult to maintain such modified mouse strains. In various embodiments, maintaining the strains involves avoiding the infertility problem exhibited by homozygous male mice with respect to the replacement.

[0398] In one embodiment, a method for maintaining the mouse strain described herein is provided. The mouse strain does not need to contain an ectopic ADAM6 sequence, and in various embodiments, the mouse strain is homozygous or heterozygous with respect to ADAM6 knockout (e.g., functional knockout).

[0399] The mouse strain described above includes modifications to the endogenous immunoglobulin heavy chain locus that result in reduced or lost fertility in male mice. In one embodiment, the modification includes deletion of the regulatory and / or coding regions of the ADAM6 gene. In a particular embodiment, the modification includes modifications to the endogenous ADAM6 gene (regulatory and / or coding regions) that reduce or eliminate fertility in male mice containing the modification; in a particular embodiment, the modification reduces or eliminates fertility in male mice that are homozygous for the modification.

[0400] In one embodiment, the mouse strain is homozygous or heterozygous with respect to ADAM6 gene knockout (e.g., functional knockout) or deletion.

[0401] In one embodiment, the mouse strain is maintained by isolating cells from a mouse that is homozygous or heterozygous with respect to the modification, using the donor cells in a host embryo, impregnating a surrogate mother with the host embryo and donor cells, and obtaining offspring containing the genetic modification from the surrogate mother. In one embodiment, the donor cells are ES cells. In one embodiment, the donor cells are pluripotent cells, such as introduced pluripotent cells.

[0402] In one embodiment, the mouse strain is maintained by isolating a nucleic acid sequence containing the modification from a mouse that is homozygous or heterozygous with respect to the modification, introducing the nucleic acid sequence into a host nucleus, and impregnating a suitable animal with cells containing the nucleic acid sequence and the host nucleus. In one embodiment, the nucleic acid sequence is introduced into the embryo of a host oocyte.

[0403] In one embodiment, the mouse strain is maintained by isolating a nucleus from a mouse that is homozygous or heterozygous with respect to the modification, introducing the nucleus into a host cell, and impregnating a suitable animal with the nucleus and host cell to obtain a progeny that is homozygous or heterozygous with respect to the modification.

[0404] In one embodiment, the mouse strain is maintained by in vitro fertilization (IVF) of female mice (wild-type, homozygous with respect to the modification, or heterozygous with respect to the modification) using semen from male mice containing the gene modification. In one embodiment, the male mouse is heterozygous with respect to the gene modification. In one embodiment, the male mouse is homozygous with respect to the gene modification.

[0405] In one embodiment, the mouse strain is maintained by mating a male mouse that is heterozygous for the gene modification with a female mouse to obtain offspring containing the gene modification, identifying the male and female offspring containing the gene modification, and using the heterozygous male to mat with a wild-type female, a homozygous or heterozygous female to obtain offspring containing the gene modification. In another embodiment, the gene modification in the mouse strain is maintained by repeating the process of mating a heterozygous male with the gene modification with a wild-type female, a heterozygous female to the gene modification, or a homozygous female to the gene modification.

[0406] In one embodiment, one or more human immunoglobulin light chain sequences of an endogenous immunoglobulin heavy chain variable gene locus, and optionally one or more human D HA method for maintaining a mouse strain that includes a replacement in a gene segment, comprising the step of mating the mouse strain to produce a heterozygous male mouse, and a method for maintaining the gene modification in the strain by mating the heterozygous male mouse. In a particular embodiment, the strain is not maintained by mating a homozygous male with a wild-type female, or with a female that is homozygous or heterozygous with respect to the gene modification.

[0407] The ADAM6 protein is a member of the A Disintegrin And Metalloprotease (ADAM) family of proteins, a large family 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-oocyte interaction. ADAM3, or silitestin, appears to be required for sperm to bind to the zona pellucida. The absence of either ADAM2 or ADAM3 results in infertility. ADAM2, ADAM3, and ADAM6 are hypothesized to form a complex on the surface of mouse spermatogonial cells.

[0408] In humans, the DAM6 gene is reported to be a pseudogene, and in human V H Gene segment V H 1-2 and V H It is located between 6-1. Mice have two ADAM6 genes - ADAM6a and ADAM6b - and these are mouse V H Gene segment and D HIt is located in the intergenetic region between gene segments and is oriented to a transcriptional orientation opposite to that of the surrounding immunoglobulin gene segments. In mice, a functional ADAM6 locus is clearly required for normal fertilization. Therefore, a functional ADAM6 locus or sequence refers to an ADAM6 locus or sequence that can compensate for or rescue the thorough reduction in fertilization shown in male mice with a deficient or damaged endogenous ADAM6 locus.

[0409] The position of the intergene sequence in mice encoding ADAM6a and ADAM6b makes it more susceptible to modification when the endogenous heavy chain is altered. H When deleting or replacing a gene segment, or D HWhen a gene segment is deleted or replaced, the resulting mice are highly likely to exhibit severe fertility defects. To compensate for this defect, the mice are modified to include a nucleotide sequence encoding a protein that compensates for the loss of ADAM6 activity resulting from the modification of the endogenous ADAM6 locus. In various embodiments, the compensatory nucleotide sequence encodes mouse ADAM6a, mouse ADAM6b, or their homologs, orthologues, or functional fragments that rescue the fertility defect. In various embodiments, the compensatory nucleotide sequence encodes the mouse ADAM6a protein as shown in SEQ ID NO: 1 and / or the mouse ADAM6b protein as shown in SEQ ID NO: 2. Alternatively, a suitable method for preserving the endogenous ADAM6 locus can be used, while ensuring that the endogenous immunoglobulin heavy chain sequence adjacent to the mouse ADAM6 locus cannot be rearranged to encode a functional endogenous heavy chain variable region. An exemplary alternative method involves manipulating a large portion of a mouse chromosome to locate the endogenous immunoglobulin heavy chain variable region gene locus in such a way that it cannot be rearranged to encode a functional heavy chain variable region operably linked to an endogenous heavy chain constant gene. In various embodiments, the method involves inversion and / or translocation of a mouse chromosome fragment containing the endogenous immunoglobulin heavy chain gene segment.

[0410] The nucleotide sequence that rescues fertility can be placed at any suitable location. The sequence can be placed in an intergene region (e.g., between the V gene segment and the J gene segment or upstream of the V gene segment), or at any suitable location within the genome (i.e., ectopically). In one embodiment, the nucleotide sequence can be introduced into a transgene that is randomly incorporated into the mouse genome. In one embodiment, the sequence can be maintained in an episome, i.e., on a separate nucleic acid rather than on a mouse chromosome. Suitable locations include transcriptionally permissible 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 nucleotide sequences to transcriptional activity loci is described, for example, in U.S. Patent No. 7,473,557, which is incorporated herein by reference.

[0411] Alternatively, the nucleotide sequence that rescues fertility can be coupled with an inducible promoter to promote optimal expression in appropriate cells and / or tissues, such as reproductive tissue. Exemplary inducible promoters include promoters activated by physical means (e.g., heat shock promoters) and / or promoters activated by chemical means (e.g., IPTG or tetracycline).

[0412] Furthermore, the expression of the above nucleotide sequence can be linked to other genes to achieve expression at a specific developmental stage or within a specific tissue. Such expression can be achieved by operably linking the above nucleotide sequence to the promoter of a gene expressed at a specific developmental stage. For example, an immunoglobulin sequence from a single species, engineered into the genome of a host species, can be operably linked to the promoter sequence of the CD19 gene (a B cell-specific gene) from that host species. This enables B cell-specific expression at the precise developmental stage in which the immunoglobulin is expressed.

[0413] Another method for achieving robust expression of an inserted nucleotide sequence is the use of a constitutive promoter. Exemplary constitutive promoters include SV40, CMV, UBC, EF1A, PGK, and CAGG. Similarly, a desired nucleotide sequence can be operably linked to a selected constitutive promoter, thereby achieving a high expression level of the protein(s) encoded by that nucleotide sequence.

[0414] The term “ectopic” is intended to include transposition or arrangement to a location not normally encountered in nature (e.g., the arrangement of a nucleic acid sequence to a location other than the one in which it is found in wild-type mice). In various embodiments, the term is used to mean that the object is outside its normal or correct location. For example, the phrase “ectopic nucleotide sequence encoding…” refers to a nucleotide sequence that appears at a location not normally encountered in mice. For example, in the case of an ectopic nucleotide sequence encoding the mouse ADAM6 protein (or its orthologue, homolog, or fragment that provides the same or similar fertility benefits to male mice), the sequence may be arranged at a location in the mouse genome different from the location normally found in wild-type mice. In such cases, arranging the sequence at a location in the mouse genome different from its location in wild-type mice creates a novel sequence junction of the mouse sequence. A functional homolog or orthologue of mouse ADAM6 is ADAM6- / - This sequence provides rescue from fertility loss observed in mice (e.g., loss of the ability of male mice to produce offspring through mating). Functional homologs or orthologues include proteins having at least approximately 89% identity, e.g., no more than 99% identity, to the amino acid sequence of ADAM6a and / or ADAM6b, as well as proteins that can compensate for or rescue the ability of mice having genotypes including ADAM6a and / or ADAM6b deletion or knockout to successfully mate.

[0415] The above ectopic location may be anywhere (for example, in the case of random insertion of a transgene containing the mouse ADAM6 sequence), or it may be a location close to (but not exactly the same as) that location in wild-type mice (for example, within the modified endogenous immunoglobulin locus but either upstream or downstream of its native location, for example, within the modified immunoglobulin locus but between different gene segments, or at different locations within the mouse VD inter-gene sequence). One example of ectopic arrangement is to maintain the location normally found in wild-type mice within the endogenous immunoglobulin heavy chain locus, while preventing the surrounding endogenous heavy chain gene segments from rearranging to encode a functional heavy chain containing the endogenous heavy chain constant region. In this example, this can be achieved, for example, by inversion of the chromosomal fragment containing the endogenous immunoglobulin heavy chain variable locus, using an engineered site-specific recombination site located adjacent to the variable region locus. Therefore, during recombination, the endogenous heavy chain variable region locus is positioned at a considerable distance from the endogenous heavy chain constant region gene, thereby preventing rearrangement that encodes a functional heavy chain containing the endogenous heavy chain constant region. Other exemplary methods for achieving functional silencing of the endogenous immunoglobulin heavy chain variable gene locus while maintaining the functional ADAM6 locus will be apparent to those skilled in the art when read in conjunction with the present disclosure and / or methods known in the art. With such arrangement of the endogenous heavy chain locus, the endogenous ADAM6 gene is maintained and the endogenous immunoglobulin heavy chain locus is functionally silenced.

[0416] Another example of ectopic placement is placement within a modified immunoglobulin heavy chain locus. For example, one or more endogenous V H Human V gene segment L A mouse whose ADAM6 sequence is replaced in a gene segment, and which includes a replacement that removes the endogenous ADAM6 sequence, is considered to have a human V LIt can be engineered to be positioned within a sequence containing a gene segment. The resulting modification creates an (ectopic) mouse ADAM6 sequence within a human gene sequence, and this (ectopic) positioning of the mouse ADAM6 sequence within the human gene sequence may be close to the location of the human ADAM6 pseudogene (i.e., between two V segments) or close to the location of the mouse ADAM6 sequence (i.e., within the VD intergene region). The resulting sequence ligation region, created by the ligation of an (ectopic) mouse ADAM6 sequence within or adjacent to a human gene sequence (e.g., an immunoglobulin light chain gene sequence) in the mouse germline, will be novel compared to the same or similar location in the wild-type mouse genome.

[0417] In various embodiments, non-human animals lacking ADAM6 or its ortholog or homolog are provided, where this lack makes the non-human animals infertile or substantially reduces their fertility. In various embodiments, the lack of ADAM6 or its ortholog or homolog is due to an alteration of the endogenous immunoglobulin heavy chain locus. Substantial reduction in fertility is, for example, a reduction of about 50%, 60%, 70%, 80%, 90%, or 95% or more in fertility (e.g., mating frequency, offspring per litter, littermates per year, etc.). In various embodiments, the non-human animals are supplemented with a mouse ADAM6 gene or its ortholog, homolog or functional fragment that is functional in male non-human animals, and the supplemented ADAM6 gene or its ortholog, homolog or functional fragment rescues all or substantial of the reduction in fertility. Rescuing a substantial portion of fertility would be, for example, a restoration of fertility such that the non-human animals described above exhibit at least 70%, 80%, or 90% or more fertility compared to unmodified (i.e., animals without modifications to the ADAM6 gene or its orthologs or homologs) heavy chain loci.

[0418] In various embodiments, the sequence conferred to the genetically modified animal (i.e., an animal lacking functional ADAM6 or its ortholog or homolog due to, for example, modification of the immunoglobulin heavy chain locus) is selected from the ADAM6 gene or its ortholog or homolog. For example, in one embodiment, the loss of ADAM6 function in a mouse is rescued by the addition of the mouse ADAM6 gene. In one embodiment, the loss of ADAM6 function in a mouse is rescued by the addition of an ortholog or homolog of a closely related species to the mouse, for example, a rodent, for example, a different strain or species of mouse, any species of rat, or rodent, in which case the addition of the mouse ortholog or homolog rescues the loss of fertility resulting from the loss of ADAM6 function or the loss of the ADAM6 gene. Orthologues and homologs from other species are selected from phylogenetically related species in various embodiments and exhibit identity percentages with endogenous ADAM6 (or orthologues) of approximately 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, or 97% or more in various embodiments, and rescue fertility loss associated with ADAM6 or (in the case of non-mouse) ADAM6 orthologues. For example, in genetically modified male rats lacking ADAM6 function (e.g., rats with an endogenous immunoglobulin heavy chain variable region replaced by a human immunoglobulin heavy chain variable region, or rats with a knockout of the rat immunoglobulin heavy chain region), fertility loss in those rats is rescued by addition of rat ADAM6, or in some embodiments by addition of an orthologue of rat ADAM6 (e.g., an ADAM6 orthologue from another rat strain or species, or in one embodiment from mouse).

[0419] Thus, in various embodiments, genetically modified animals that are fertile or exhibit reduced fertility due to modifications of the nucleic acid sequence encoding the ADAM6 protein (or its ortholog or homolog), or a regulatory region operably linked to said nucleic acid sequence, include a nucleic acid sequence that compensates for or restores the loss of fertility, in which case the nucleic acid sequence that compensates for or restores the loss of fertility is from a different strain of the same species or from a phylogenetically related species. In various embodiments, the supplementary nucleic acid sequence is an ADAM6 ortholog or homolog or a functional fragment thereof. In various embodiments, the supplementary ADAM6 ortholog or homolog or a functional fragment thereof is from a non-human animal closely related to the genetically modified animal with the fertility defect. For example, if the genetically modified animal is a particular strain of mouse, the ADAM6 ortholog or homolog or a functional fragment thereof can be obtained from a different strain of mouse or from a related species of mouse. In one embodiment, if the genetically modified animal having the fertility defect is of the order Rodentia, the ADAM6 ortholog or homolog or functional fragment is from another animal of the order Rodentia. In one embodiment, the genetically modified animal having the fertility defect is of the suborder Myomoropha (e.g., jerboas, jumping mice) The ADAM6 orthologues or homologs or functional fragments described above are selected from rodents or murexes.

[0420] In one embodiment, the genetically modified animal is from the superfamily Dipodoidea, and the ADAM6 ortholog or homolog or functional fragment is from the superfamily Muroidea.

[0421] In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from the superfamily Muroidea, and the ADAM6 ortholog or homolog is from a different species within the superfamily Muroidea. In one embodiment, the genetically modified animal is from a family selected from Calomyscidae (e.g., kangaroo hamster), Cricetidae (e.g., hamster, New World rat and mouse, field vole), Muridae (purebred mouse and rat, gerbil, spiny mouse, maned mouse), Nesomyidae (tree mouse, rock mouse, white-tailed porgy, Madagascar rat and mouse), Platacanthomyidae (e.g., spiny dormouse), and Moroccanidae (e.g., mole rat, bamboo rat and burrowing mouse); and the ADAM6 ortholog or homolog is selected from different species of the same family. In certain embodiments, the genetically modified rodent is selected from purebred mice or rats (Muridae), and the ADAM6 orthologue or homolog is from a species selected from gerbils, spiny mice, or maned mice. In one embodiment, the genetically modified mouse is from a member of the Muridae family, and the ADAM6 orthologue or homolog is from a different species of the Muridae family. In certain embodiments, the genetically modified rodent is a mouse of the Muridae family, and the ADAM6 orthologue or homolog is from a rat, gerbil, spiny mouse, or maned mouse of the Muridae family.

[0422] In various embodiments, one or more rodent ADAM6 orthologues or homologs or functional fragments of rodents within a family restore fertility to genetically modified rodents of the same family (e.g., Cricetidae (e.g., hamsters, New World rats and mice, voles); Muridae (e.g., purebred mice and rats, gerbils, spiny mice, maned mice)) that lack the ADAM6 orthologue or homolog.

[0423] In various embodiments, ADAM6 orthologues, homologs, and fragments are evaluated for their functionality by determining whether the orthologue, homolog, or fragment restores fertility in genetically modified male non-human animals lacking ADAM6 activity (e.g., rodents, such as mice or rats, including knockouts of ADAM6 or its orthologues). In various embodiments, functionality is defined as the ability of sperm from genetically modified animals lacking endogenous ADAM6 or its orthologue or homolog to travel through the fallopian tubes of the same species of genetically modified animal to fertilize eggs.

[0424] In various embodiments, mice can be constructed that include a deletion or substitution of an endogenous heavy chain variable region locus or a portion thereof, containing an ectopic nucleotide sequence encoding a protein that provides similar fertility benefits to mouse ADAM6 (e.g., its ortholog, homolog, or fragment that is functional in male mice). Examples of such ectopic nucleotide sequences include nucleotide sequences encoding a protein that is an ADAM6 homolog or ortholog (or fragment thereof) of a different mouse strain or a different species, e.g., a different rodent species, which confers fertility benefits, e.g., an increase in litter size over a specified period, and / or an increase in the number of offspring per litter, and / or the ability of male mouse spermatids to pass through the mouse oviduct and fertilize mouse eggs.

[0425] In one embodiment, the ADAM6 is a homolog or ortholog that is at least 89% to 99% identical to the 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 at least 89% identical to mouse ADAM6a, a protein 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 modified to be at least 89% 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to mouse ADAM6a and / or mouse ADAM6b. In a particular embodiment, mouse ADAM6a comprises SEQ ID NO: 1 or a functional fragment thereof, and mouse ADAM6b comprises SEQ ID NO: 2 or a functional fragment thereof.

[0426] In one embodiment, (a) one or more human V L and J L (b) Insertion of a gene segment upstream of the non-human immunoglobulin heavy chain constant region, (b) one or more human V L , and J LThe present invention provides a non-human animal comprising (c) an insertion of a gene segment upstream of a non-human immunoglobulin light chain constant region, and a nucleotide sequence encoding the ADAM6 protein or a functional fragment thereof. In one embodiment, the non-human heavy chain and / or light chain constant region is a rodent constant region (e.g., selected from mouse, rat, or hamster constant regions). In one embodiment, the non-human light chain constant region is a rodent constant region. In a particular embodiment, the light chain constant region is a mouse Cκ or rat Cκ region. In a particular embodiment, the light chain constant region is a mouse Cλ or rat Cκ region. In one embodiment, the human V L and J L The gene segments are the Vκ and Jκ gene segments. In one embodiment, the above human V L and J L The gene segments are the Vλ and Jλ gene segments. In one embodiment, the non-human animal is human V L and J L One or more human D2 present between gene segments H The gene segment is further included. Suitable non-human animals include rodents, such as mice, rats, and hamsters. In one embodiment, the rodent is a mouse or a rat.

[0427] In one embodiment, the non-human animal comprises at least 6 to at least 40 human Vκ gene segments and at least 1 to at least 5 human Jκ gene segments. In a particular embodiment, the non-human animal comprises 6 human Vκ gene segments and 5 human Jκ gene segments. In a particular embodiment, the non-human animal comprises 16 human Vκ gene segments and 5 human Jκ gene segments. In a particular embodiment, the non-human animal comprises 30 human Vκ gene segments and 5 human Jκ gene segments. In a particular embodiment, the non-human animal comprises 40 human Vκ gene segments and 5 human Jκ gene segments. In various embodiments, the human Jκ gene segments are selected from Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and combinations thereof.

[0428] In one embodiment, the nucleotide sequence encoding the ADAM6 protein or a functional fragment thereof is ectopic in the non-human animal. In one embodiment, the nucleotide sequence encoding the ADAM6 protein or a functional fragment thereof (which is functional in the non-human animal) is located at the same location as the wild-type non-human ADAM6 locus. In one embodiment, the non-human animal is a mouse, and the nucleotide sequence encoding the mouse ADAM6 protein or a functional fragment thereof is located at an ectopic location in the genome of the non-human animal. In one embodiment, the non-human animal is a mouse, and the nucleotide sequence encoding the mouse ADAM6 protein or a functional fragment thereof is located within an immunoglobulin gene segment. In a particular embodiment, the immunoglobulin gene segment is a heavy chain gene segment of the non-human animal. In a particular embodiment, the immunoglobulin gene segment is a light chain gene segment of another species. In one embodiment, the light chain gene segment is a human κ light chain gene segment. In one embodiment, the mouse comprises an ectopic sequence containing one or more endogenous, unrearranged heavy chain gene segments, wherein the ADAM6 sequence is located within the ectopic sequence.

[0429] In one embodiment, the non-human animal has endogenous immunoglobulin V at the endogenous immunoglobulin light chain locus. L and / or J L Lacking a gene segment. In one embodiment, the non-human animal has immunoglobulin V L Endogenous immunoglobulin V that cannot be rearranged to form a domain L and / or J LIncludes gene segments. In one embodiment, all or substantially all endogenous immunoglobulin Vκ and Jκ gene segments are replaced with one or more human Vκ and Jκ gene segments. In one embodiment, all or substantially all endogenous immunoglobulin Vλ and Jλ gene segments are deleted in whole or in part. In one embodiment, all or substantially all endogenous immunoglobulin V L and J L The gene segment is intact in the non-human animals mentioned above, and these non-human animals contain endogenous immunoglobulin V. L and / or J L The present invention comprises one or more human Vκ gene segments and one or more human Jκ gene segments inserted between the gene segment and the constant region of the endogenous immunoglobulin light chain. In certain embodiments, the present invention comprises the intact endogenous immunoglobulin V L and J L The gene segment is the antibody V in the non-human animals mentioned above. L To prevent rearrangement to form a domain. In various embodiments, the endogenous immunoglobulin light chain locus of the non-human animal is the immunoglobulin κ light chain locus. In one embodiment, the endogenous immunoglobulin V L and J L The gene segments are the Vκ and Jκ gene segments.

[0430] In one embodiment, the Specified Non-Human Animal-Derived Cells and / or Tissues are provided, the cells and / or tissues comprising (a) one or more human Vκ and Jκ gene segments inserted upstream of a non-human immunoglobulin light chain constant region, (b) one or more human Vκ and Jκ gene segments inserted upstream of a non-human immunoglobulin heavy chain constant region, and (c) a nucleotide sequence encoding the ADAM6 protein or a functional fragment thereof. In one embodiment, the non-human heavy chain and / or light chain constant region is a mouse constant region. In one embodiment, the non-human heavy chain and / or light chain constant region is a rat constant region. In one embodiment, the non-human heavy chain and / or light chain constant region is a hamster constant region.

[0431] In one embodiment, the nucleotide sequence encoding the ADAM6 protein or a functional fragment is ectopic in the cells and / or tissues. In one embodiment, the nucleotide sequence encoding the ADAM6 protein or a functional fragment is located at the same location as the wild-type non-human ADAM6 locus. In one embodiment, the non-human cells and / or tissues are mouse-derived, and the nucleotide sequence encodes the mouse ADAM6 protein or a functional fragment and is located at an ectopic location. In one embodiment, the non-human cells and / or tissues are mouse-derived, and the nucleotide sequence encodes the mouse ADAM6 protein or a functional fragment and is located within an immunoglobulin gene segment. In a particular embodiment, the immunoglobulin gene segment is a heavy chain gene segment. In a particular embodiment, the immunoglobulin gene segment is a light chain gene segment. In one embodiment, a sequence of endogenous heavy chain gene segments is ectopically arranged in the non-human animal, and the sequence of ectopically arranged endogenous heavy chain gene segments includes the ADAM6 gene, which is functional in the mouse (for example, in male mice).

[0432] In one embodiment, the use of a non-human animal as described herein is provided for producing an antigen-binding protein, the non-human animal expressing (a)(i) an immunoglobulin light chain comprising a human Vκ domain and a non-human light chain constant region, and (ii) an antibody comprising an immunoglobulin heavy chain comprising a human Vκ domain and a non-human light chain constant region, and (b) an ADAM6 protein or a functional fragment thereof. In one embodiment, the antigen-binding protein is human. In one embodiment, the non-human animal is a rodent, and the non-human constant region is a rodent constant region. In a particular embodiment, the rodent is a mouse.

[0433] In one embodiment, a non-human cell or tissue derived from a non-human animal is provided herein. In one embodiment, the non-human cell or tissue comprises one or more human immunoglobulin Vκ gene segments and at least one human immunoglobulin Jκ gene segment adjacent to a non-human immunoglobulin light chain constant region gene, and one or more human Vκ and one or more human Jκ gene segments adjacent to a non-human immunoglobulin heavy chain constant region gene, wherein the cell or tissue expresses the ADAM6 protein or a functional fragment thereof. In one embodiment, the non-human light chain constant region gene is mouse Cκ.

[0434] In one embodiment, the nucleotide sequence encoding the ADAM6 protein or its functional fragment is ectopic. In one embodiment, the nucleotide sequence encoding the ADAM6 protein or its functional fragment is at the same position as in wild-type non-human cells. In various embodiments, the non-human cells are mouse B cells. In various embodiments, the non-human cells are embryonic stem cells.

[0435] In one embodiment, the tissue is derived from the spleen, bone marrow, or lymph nodes of the non-human animal.

[0436] In one embodiment, the use of non-human animal-derived cells or tissues described herein for producing hybridomas or quadromas is provided.

[0437] In one embodiment, a non-human cell comprising a modified genome as described herein is provided, which is a fusion of an oocyte, a host embryo, or a cell from a non-human animal as described herein with a cell from a different non-human animal.

[0438] In one embodiment, the use of cells or tissues derived from non-human animals as described herein for producing a human antigen-binding protein is provided. In one embodiment, the human antigen-binding protein comprises a human Vκ domain isolated from a non-human animal as described herein.

[0439] One embodiment provides a method for producing an antigen-binding protein that binds to a target antigen, the method comprising: (a) exposing a non-human animal described herein to a target antigen; and (b) isolating one or more B lymphocytes from the non-human animal (the one or more B lymphocytes binding to the target antigen). L (c) expresses a binding protein, and (c) binds to the target antigen. L Binding protein V L Steps to identify the nucleic acid sequence encoding the domain (where, V above) L (d) The binding protein comprises a human Vκ domain and a non-human light chain constant domain and a human Vκ domain and a non-human heavy chain constant domain, and (d) the step of using the nucleic acid sequence of (c) and the human immunoglobulin constant region nucleic acid sequence to produce a human antigen-binding protein that binds to the target antigen.

[0440] In one embodiment, the above V L The non-human light chain constant domain of the binding protein is mouse Cκ. In one embodiment, the above V L The non-human heavy chain constant domain of the binding protein is mouse Cγ. In one embodiment, the non-human animal is a mouse.

[0441] In one embodiment, a fertile male mouse is provided that includes a modification at an immunoglobulin heavy chain locus, the fertile male mouse containing an ectopic ADAM6 sequence that is functional in the male mouse.

[0442] Ectopic ADAM6 at modified immunoglobulin heavy chain locus Advances in gene targeting, such as the development of bacterial artificial chromosomes (BACs), now make it possible to recombinate relatively large genomic fragments. BAC engineering has enabled the introduction of large deletions and insertions into mouse ES cells.

[0443] Mice that produce human antibodies (i.e., human variable regions) have become available in recent years. While these mice represent a significant advance in the development of human therapeutic antibodies, they exhibit numerous critical abnormalities that limit their usefulness. For example, these mice exhibit impaired B-cell development, which can be attributed to various differences between transgenic and wild-type mice.

[0444] Human antibodies may not optimally interact with mouse pre-B cell receptors or B cell receptors on the surface of mouse cells that transmit signals about survival during maturation, proliferation, or clonal selection. Fully human antibodies may not optimally interact with the mouse Fc receptor system, and mice express Fc receptors that do not present a one-to-one correspondence with human Fc receptors. Ultimately, various mice that produce fully human antibodies do not contain all the true mouse sequences that may be required for wild-type B cell development, such as downstream enhancer elements and other locus regulatory elements.

[0445] Mice that produce fully human antibodies generally contain an endogenous immunoglobulin locus that is rendered inactive in some way, and human transgenes containing variable and constant immunoglobulin gene segments are introduced at random locations within the mouse genome. As long as the endogenous locus is rendered inactive to the extent that it cannot rearrange its gene segments to form functional immunoglobulin genes, the objective of producing fully human antibodies can be achieved in such mice, even if B cell development is impaired.

[0446] While it is necessary to produce fully human antibodies from the human transgene locus, the production of human antibodies in mice appears to be an undesirable process. In some mice, this process is undesirable because it results in the formation of chimeric human variable / mouse constant heavy chains (not light chains) via a transswitching mechanism. This mechanism causes the transcript encoding the fully human antibody to undergo trans isotype switching from the human isotype to the mouse isotype. Since the fully human transgene is located far from the endogenous locus that holds an undamaged copy of the mouse heavy chain constant region gene, this process occurs in trans. Transswitching is readily apparent in such mice, but this phenomenon is still insufficient to rescue B cell development, and B cell development remains clearly impaired. In any case, since the transswitching phenomenon does not occur with respect to the light chain, the transswitched antibodies in such mice retain the fully human light chain; presumably, the transswitching depends on the switch sequence at the endogenous locus used (if different) for normal cis isotype switching. Therefore, even if mice engineered to produce fully human antibodies select a transswitch mechanism to produce antibodies with a mouse constant region, this strategy is still insufficient for rescuing normal B cell development.

[0447] A primary concern in the development of antibody-based human therapeutics is to create sufficiently diverse human immunoglobulin variable region sequences to identify useful variable domains that specifically recognize a particular epitope and bind that epitope with desirable affinity, usually—but not always—high affinity. Prior to the development of the VELOCIMMUNE® mouse (as described herein), there was no indication that mice expressing human variable regions along with the mouse constant region would show any significant difference compared to mice from which human antibodies were produced from the transgene. However, that assumption was incorrect.

[0448] VELOCIMMUNE® mice, which contain a precise substitution of the mouse immunoglobulin variable region in the human immunoglobulin variable region at the endogenous locus, exhibit remarkable and noteworthy similarities to wild-type mice in terms of B cell development. Remarkably and truly impressively, VELOCIMMUNE® mice present an essentially normal wild-type immune response, differing from wild-type mice only in one crucial aspect: the variable region produced in response to immunity is entirely human.

[0449] VELOCIMMUNE® mice contain precise, large-scale substitutions of the germline variable regions of mouse immunoglobulin heavy chains (IgH) and immunoglobulin light chains (e.g., κ light chain, Igκ) at endogenous loci with the corresponding human immunoglobulin variable regions. In total, approximately 6 megabases of mouse loci are replaced with approximately 1.5 megabases of human genome sequence. This precise substitution results in mice with hybrid immunoglobulin loci that produce heavy and light chains containing both human variable and mouse constant regions. H -D H -J H and V κ -J κPrecise segmental replacement leaves adjacent mouse sequences intact and functional at the hybrid immunoglobulin locus. The humoral immune system of the mice described above functions similarly to that of wild-type mice. B cell development is not disrupted in any significant way, and antigen challenge produces highly diverse human variable regions in the mice described above.

[0450] VELOCIMMUNE® mice are feasible because the immunoglobulin gene segments for heavy and κ light chains are rearranged similarly in humans and mice, but the loci are not the same or nearly the same—clearly they are not. However, the loci are all V H , D H and J H The mouse sequence containing the gene segment, consisting of approximately 3 million base pairs, is so similar to the human genome sequence of approximately 1 million base pairs that essentially covers the equivalent sequence from the human immunoglobulin locus, that humanization of the heavy chain variable gene locus can be achieved.

[0451] In some embodiments, a certain mouse constant region gene sequence is further replaced with a human gene sequence (e.g., mouse C H Human C, 1 sequence H Replacement in 1, and mouse C L The sequence, Human C LSequence substitution results in mice possessing a hybrid immunoglobulin locus that produces antibodies with human variable regions and partial human constant regions, suitable for, for example, the production of fully human antibody fragments, such as fully human Fabs. Mice with the hybrid immunoglobulin locus exhibit normal variable gene segment rearrangements, normal somatic hypermutation, and normal class switching. These mice exhibit a humoral immune system indistinguishable from wild-type mice and present normal cell populations at all B-cell developmental stages and normal lymphoid organ structure, even when the mice lack a complete repertoire of human variable region gene segments. Immune responses in these mice result in robust humoral responses that exhibit a wide diversity of variable gene segment usage frequencies.

[0452] Precise replacement of the gene segment of the mouse germline variable region makes it possible to create mice with a partial human immunoglobulin locus. Because the partial human immunoglobulin locus rearranges, hypermutates, and successfully class-switches, antibodies are produced by this locus in mice containing the human variable region. The nucleotide sequence encoding this variable region can be identified, cloned, and fused (e.g., in vitro) with any selected sequence, such as any immunoglobulin isotype suitable for a specific application, to obtain antibodies or antigen-binding proteins entirely derived from human sequences.

[0453] The above mouse V H , D H , and J HMouse embryonic stem (ES) cells were modified using a large-scale recombinant humanization method to create a unique immunoglobulin heavy chain locus by precisely replacing the above mouse heavy chain immunoglobulin locus up to 3 megabases, which essentially contains all of the gene segments, with a segment of the human genome up to 0.5 megabases containing one of the two repeats that essentially encode all of the human Vκ and Jκ gene segments. Furthermore, the 3 megabase segment of the mouse immunoglobulin κ light chain locus, which essentially contains all of the mouse Vκ and Jκ gene segments, was replaced with a segment of the human genome of less than half a megabase, which contains one of the two repeats that essentially encode all of the human Vκ and Jκ gene segments. Mice with such a substituted immunoglobulin locus have the most 3' V of the mouse immunoglobulin heavy chain locus. H Gene segment and the 5' end D H This may involve disruption or deletion of the mouse ADAM6 locus, which is normally found between gene segments. Disruption of this region may result in reduced or eliminated functionality of the mouse ADAM6 locus.

[0454] We describe a mouse that contains the above-mentioned substituted gene locus and also contains an ectopic nucleic acid sequence encoding mouse ADAM6, and that exhibits essentially normal fertility. In one embodiment, the above-mentioned ectopic nucleic acid sequence is the human V of the above-mentioned modified endogenous heavy chain locus. L Genetic segments and human J L Between gene segments, or at the 5' end of human V L It is located upstream of the gene segment. The transcription direction of the ADAM6 gene is the same as that of the surrounding human V L The direction of transcription of the gene segment may be reversed (Figure 7) or the same (Figure 8). An example in this specification is shown in human V L and J L Between gene segments, or at the 5' end of human V LWhile this demonstrates fertility rescue by placing an ectopic sequence upstream of a gene segment, those skilled in the art will acknowledge that the placement of an ectopic sequence to any suitable transcription-permissible locus within the mouse genome (or even outside the chromosome) is expected to similarly rescue fertility in male mice. In various embodiments, the ectopic nucleic acid sequence is selected from SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and the ectopic nucleic acid sequence encodes one or more ADAM6 proteins, the one or more ADAM6 proteins comprising SEQ ID NO: 1, SEQ ID NO: 2, or a combination thereof.

[0455] The phenomenon of rescuing mice lacking a functional ADAM6 locus having an ectopic sequence containing the mouse ADAM6 gene or its ortholog, homolog, or functional fragment is a common method applicable to the rescue of any mouse having a non-functional or minimally functional endogenous ADAM6 locus. Therefore, a very large number of mice, including those with ADAM6 disruption modifications of the immunoglobulin heavy chain locus, can be rescued with the compositions and methods of the present invention. Accordingly, the present invention includes mice having a wide variety of modifications of the immunoglobulin heavy chain locus, including endogenous ADAM6 function. Some (non-limiting) examples are provided herein. In addition to the described mice, the compositions and methods relating to ADAM6 can be used in a very wide range of applications, for example, when modifying heavy chain loci in a wide variety of ways.

[0456] In one embodiment, an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or its ortholog, homolog, or functional fragment) and all or substantially all mouse V H One or more human V gene segments L Replacement in gene segments and all or substantially all mouse D H Gene segment and J H Human J gene segment L A mouse comprising a replacement in a gene segment, C HA mouse is provided that lacks one region and / or a hinge region. In one embodiment, the mouse is (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; and (c) Human V L -Mouse C H 2-Mouse C H A single variable domain-binding protein, which is a dimer of an immunoglobulin chain selected from 3, is constructed.

[0457] In one embodiment, all or substantially all mouse immunoglobulin light chain variable gene segments (mV H , mD H and mJ H A mouse having substitutions in one or more human immunoglobulin κ light chain variable gene segments (hVκ and hJκ), further comprising substitutions of all or substantially all mouse immunoglobulin κ light chain variable gene segments (mVκ, mJκ) in one or more human immunoglobulin κ light chain variable gene segments (hVκ and hJκ), wherein a nucleotide sequence for rescuing fertility is located within the human immunoglobulin heavy chain variable region sequence (e.g., between the human Vκ4-1 gene segment and the Jκ1 gene segment).

[0458] In one embodiment, the functional mouse ADAM6 locus (or its ortholog, homolog, or functional fragment) is human V L Within the gene segment, or the most 5' end of human V L It can be positioned upstream of the gene segment, and the human V L The gene segment is endogenous V H Replace the gene segment. In one embodiment, all or substantially all mouse V H The gene segment is removed, and one or more human V LReplaced by the gene segment, the above mouse ADAM6 locus is the most 5'-side human V L Either located directly adjacent to the 5' end of the gene segment, or two human V L It is positioned between gene segments. In a particular embodiment, the mouse ADAM6 locus is inserted into the human V L Two Vs near the 3' end of the gene segment L It is placed between gene segments. In a particular embodiment, the next is human V L The sequence of the gene segment is as follows (Human V above) L With respect to the transcription direction of the gene segment (upstream to downstream): human Vκ5-2 - mouse ADAM6 locus - human Vκ4-1. In certain embodiments, then human V L The sequence of the gene segment is as follows (Human V above) L Regarding the transcription direction of the gene segment, from upstream to downstream): mouse ADAM6 locus - human Vκ2-40, where human Vκ2-40 is the most 5'-side human V in the modified immunoglobulin heavy chain locus. L This is a gene segment. In one embodiment, the orientation of one or more of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is the same as that of human V L In comparison to the orientation of the gene segment, it is opposite with respect to the transcription direction. In one embodiment, the orientation of one or more of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is the same as that of human V L This is the same with respect to transcription direction, compared to the orientation of gene segments.

[0459] In one embodiment, the functional mouse ADAM6 locus (or its ortholog, homolog, or functional fragment) is human V L Genetic segments and human J L Between gene segments (i.e., the 3' end of human V L Gene segment and the 5' end of human J L It can be placed (within the intergenetic region between gene segments), and the human VL and J L The gene segment is endogenous V H It replaces the gene segment. In one embodiment, all or substantially all mouse V H The gene segment is removed, and one or more human V L Gene segments and one or more human J L Replaced by the gene segment, the above mouse ADAM6 locus is the most 3'-side human V L The human J gene segment directly adjacent to the 3' end and furthest to the 5' end. L It is positioned directly adjacent to the 5' end of the gene segment. In certain embodiments, one or more of the above human V L Gene segments and one or more human J L The gene segments are the Vκ gene segment and the Jκ gene segment. In a particular embodiment, the following is human V L The sequence of the gene segment is as follows (Human V above) L With respect to the transcription direction of the gene segment, from upstream to downstream): Human Vκ4-1 - Mouse ADAM6 locus - Human Jκ1. In one embodiment, the orientation of one or more of the mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is the same as that of the human V L In comparison to the orientation of the gene segment, it is opposite with respect to the transcription direction. In one embodiment, the orientation of one or more of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is the same as that of human V L This is the same with respect to transcription direction, compared to the orientation of gene segments.

[0460] All or substantially all endogenous V H Replace the gene segment with one or more of the above human V LMice modified in a gene segment (e.g., Vκ or Vλ segment) can be modified to maintain the endogenous ADAM6 locus by using a targeting vector having a downstream homology arm containing, for example, the mouse ADAM6 locus or a functional fragment thereof, as described above, or by using two human V L Between gene segments, or human V L Gene segment and D H Between gene segments (whether human or mouse, for example, Vλ+m / hD) H ) or between the J gene segment (whether human or mouse, for example, Vκ+J) H The damaged mouse ADAM6 gene locus can be modified to replace the ectopic sequence located at ). In one embodiment, the above replacement can be performed on two or more human V L The gene segment includes, and the mouse ADAM6 locus or its functional fragment is located at the two most 3' ends of the V L It is positioned between gene segments. Therefore, in a particular embodiment, human V L The gene segment is structured as follows (from upstream to downstream with respect to the transcriptional direction of the human gene segment): Human V L 3'-1-Mouse ADAM6 locus-Human V L 3'. In one embodiment, with respect to transcription direction, the orientation of one or more of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is human V L It is the opposite of the orientation of the gene segment. Alternatively, the mouse ADAM6 locus is the most 3'-side human V L Gene segment and the 5' end J L They can be located in intergenetic regions between gene segments.

[0461] In one embodiment, one or more endogenous V H Having a gene segment replacement and at least one endogenous D H A mouse containing a gene segment is provided. In such a mouse, the endogenous V HModi...

Claims

1. A method for producing a human immunoglobulin chain, (a) A step of immunizing a genetically modified mouse with an antigen, wherein the genetically modified mouse (i) Below: (A) One or more human V activatably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus Ｌ Genetic segment and one or more human J Ｌ Gene segment and, (B) One or more human V operably connected to the light chain steady region Ｌ Genetic segment and one or more human J Ｌ Gene segment and, (C) Nucleic acid encoding mouse ADAM6 protein and The genetically modified mouse has a germline genome that includes the mouse ADAM6 protein, and the mouse ADAM6 protein is expressed in the genetically modified mouse; (ii) When immunized with the antigen, antibodies are produced, and each antibody comprises a human light chain variable domain operably linked to a mouse heavy chain constant domain and a human light chain variable domain operably linked to a light chain constant domain; and (iii) Fertile process; (b) A step of determining the human light chain variable domain sequence of an antibody that specifically binds to the antigen and is produced by the genetically modified mouse; and (c) The step of forming the human immunoglobulin chain by operably linking the human light chain variable domain sequence to a human light chain constant domain sequence or a human heavy chain constant domain sequence. Methods that include...

2. The method according to claim 1, wherein the step of determining the human light chain variable domain sequence includes determining the nucleic acid encoding the human light chain variable domain sequence.

3. The method according to claim 2, wherein the step of operably linking the human light chain variable domain sequence to the human light chain constant domain sequence or the human heavy chain constant domain sequence includes operably linking the nucleic acid encoding the human light chain variable domain sequence to the human light chain constant domain sequence or the nucleic acid encoding the human heavy chain constant domain sequence.

4. The one or more human V operably connected to the light chain steady-state region Ｌ Genetic segment and one or more human J Ｌ The method according to any one of claims 1 to 3, wherein a gene segment is operably linked to the constant region of the mouse light chain at an endogenous mouse light chain locus.

5. The method according to claim 4, wherein the mouse light chain constant region is the mouse Cκ gene.

6. The method according to claim 4, wherein the mouse light chain constant region is a mouse Cλ gene.

7. The one or more human V operably connected to the light chain steady-state region Ｌ Genetic segment and one or more human J Ｌ The method according to any one of claims 1 to 6, wherein the gene segment is one or more human Vκ gene segments and one or more human Jκ gene segments.

8. In the endogenous mouse immunoglobulin heavy chain locus, the one or more human V Ｌ gene segments and one or more human J Ｌ gene segments are one or more human Vκ gene segments and one or more human Jκ gene segments. The method according to any one of claims 1 to 7.

9. The one or more human V operably connected to the light chain steady-state region Ｌ Genetic segment and one or more human J Ｌ The method according to any one of claims 1 to 6, wherein the gene segment is one or more human Vλ gene segments and one or more human Jλ gene segments.

10. One or more human V genes are operably linked to the constant region of the mouse heavy chain at the endogenous mouse immunoglobulin heavy chain gene locus. Ｌ Genetic segment and one or more human J Ｌ The method according to any one of claims 1 to 6 and 9, wherein the gene segment is one or more human Vλ gene segments and one or more human Jλ gene segments.

11. The method according to any one of claims 1 to 10, wherein the mouse ADAM6 protein is mouse ADAM6a protein and / or mouse ADAM6b protein.

12. (i) The nucleic acid encoding the mouse ADAM6 protein is operably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, one or more human V Ｌ One of the gene segments and the one or more of the aforementioned human J Ｌ It exists between one of the gene segments; (ii) The germline genome of the genetically modified mouse is operably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and contains two or more human V Ｌ The nucleic acid comprising the gene segment and encoding the mouse ADAM6 protein is operably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and the two or more human V Ｌ It exists between two of the gene segments; or (iii) The germline genome of the genetically modified mouse is operably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and contains two or more human J Ｌ The nucleic acid comprising the gene segment and encoding the mouse ADAM6 protein is operably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and the two or more human J Ｌ Located between two of the gene segments, The method according to any one of claims 1 to 11.

13. One or more endogenous mice V in which the genetically modified mouse cannot be rearranged to form an immunoglobulin light chain variable region in the genetically modified mouse. Ｌ Genetic segment and / or one or more endogenous mouse J Ｌ The method according to any one of claims 1 to 12, comprising a gene segment.

14. The method according to any one of claims 1 to 12, wherein the genetically modified mouse comprises the deletion or substitution of one or more endogenous mouse immunoglobulin heavy chain gene sequences.

15. A method for producing nucleic acids encoding human immunoglobulin chains, (a) A step of immunizing a genetically modified mouse with an antigen, wherein the genetically modified mouse (i) Below: (A) One or more human V activatably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus Ｌ Genetic segment and one or more human J Ｌ Gene segment and, (B) One or more human V operably connected to the light chain steady region Ｌ Genetic segment and one or more human J Ｌ Gene segment and, (C) Nucleic acid encoding mouse ADAM6 protein and The genetically modified mouse has a germline genome that includes the mouse ADAM6 protein, and the mouse ADAM6 protein is expressed in the genetically modified mouse; (ii) When immunized with the antigen, antibodies are produced, and each antibody comprises a human light chain variable domain operably linked to a mouse heavy chain constant domain and a human light chain variable domain operably linked to a light chain constant domain; and (iii) Fertile process; (b) A step of inducing an immune response to the antigen in the genetically modified mouse; (c) A step of determining a human light chain variable region sequence that specifically binds to the antigen and encodes the human light chain variable domain of the antibody produced by the genetically modified mouse; (d) A step of generating the human light chain variable region sequence that encodes the human light chain variable domain; and (e) A step of forming the nucleic acid encoding the human immunoglobulin chain by operably linking the human light chain variable region sequence to a human heavy chain constant region sequence or a human light chain constant region sequence. Methods that include...

16. The step of determining the human light chain variable region sequence that encodes the human light chain variable domain is, (i) Antibodies isolated from the serum of the genetically modified mouse, which are produced by the genetically modified mouse in response to antigen stimulation; or (ii) B cells isolated from the genetically modified mouse The method according to claim 15, comprising determining the sequence of the human light chain variable region from the above.

17. The method according to claim 16, wherein determining the human light chain variable region sequence from the B cells isolated from the genetically modified mouse is a method that includes determining the human light chain variable region sequence that encodes a human light chain variable domain expressed on the surface of the B cells.

18. The method according to claim 16, wherein determining the human light chain variable region sequence from the B cells isolated from the genetically modified mouse includes determining the human light chain variable region sequence in the genome of the B cells.

19. The one or more human V operably connected to the light chain steady-state region Ｌ Genetic segment and one or more human J Ｌ The method according to any one of claims 15 to 18, wherein the gene segment is operably linked to the constant region of the mouse light chain at an endogenous mouse light chain locus.

20. The method according to claim 19, wherein the mouse light chain constant region is the mouse Cκ gene.

21. The method according to claim 19, wherein the mouse light chain constant region is a mouse Cλ gene.

22. The one or more human V operably connected to the light chain steady-state region Ｌ Genetic segment and one or more human J Ｌ The method according to any one of claims 15 to 21, wherein the gene segment is one or more human Vκ gene segments and one or more human Jκ gene segments.

23. One or more human V genes are operably linked to the constant region of the mouse heavy chain at the endogenous mouse immunoglobulin heavy chain gene locus. Ｌ Genetic segment and one or more human J Ｌ The method according to any one of claims 15 to 22, wherein the gene segment is one or more human Vκ gene segments and one or more human Jκ gene segments.

24. The one or more human V operably connected to the light chain steady-state region Ｌ Genetic segment and one or more human J Ｌ The method according to any one of claims 15 to 21, wherein the gene segment is one or more human Vλ gene segments and one or more human Jλ gene segments.

25. One or more human V genes are operably linked to the constant region of the mouse heavy chain at the endogenous mouse immunoglobulin heavy chain gene locus. Ｌ Genetic segment and one or more human J Ｌ The method according to any one of claims 15 to 21 and 24, wherein the gene segment is one or more human Vλ gene segments and one or more human Jλ gene segments.

26. The method according to any one of claims 15 to 25, wherein the mouse ADAM6 protein is mouse ADAM6a protein and / or mouse ADAM6b protein.

27. (i) The nucleic acid encoding the mouse ADAM6 protein is operably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, one or more human V Ｌ One of the gene segments and the one or more of the aforementioned human J Ｌ It exists between one of the gene segments; (ii) The germline genome of the genetically modified mouse is operably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and contains two or more human V Ｌ The nucleic acid comprising the gene segment and encoding the mouse ADAM6 protein is operably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and the two or more human V Ｌ It exists between two of the gene segments; or (iii) The germline genome of the genetically modified mouse is operably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and contains two or more human J Ｌ The nucleic acid comprising the gene segment and encoding the mouse ADAM6 protein is operably linked to the mouse heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and the two or more human J Ｌ Located between two of the gene segments, The method according to any one of claims 15 to 26.

28. One or more endogenous mice V in which the genetically modified mouse cannot be rearranged to form an immunoglobulin light chain variable region in the genetically modified mouse. Ｌ Genetic segment and / or one or more endogenous mouse J Ｌ The method according to any one of claims 15 to 27, comprising a gene segment.

29. The method according to any one of claims 15 to 27, wherein the genetically modified mouse comprises the deletion or substitution of one or more endogenous mouse immunoglobulin heavy chain gene sequences.

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