method for preparing a non-human animal, use of the cell or tissue derived from the non-human animal

专利摘要:
method of preparing a non-human animal, use of the cell or tissue derived from the non-human animal, method of preparing an antibody. non-human animals, tissues, cells and genetic material are provided that comprise a modification of an endogenous non-human heavy chain immunoglobulin sequence and comprise a functional activity of adam6 in a mouse, wherein non-human animals express a chain variable domain human immunoglobulin heavy and a cognate human immunoglobulin light chain variable domain (lambda).
公开号:BR112014015238A2
申请号:R112014015238-1
申请日:2012-12-17
公开日:2020-12-29
发明作者:Lynn MacDonald；Cagan Gurer；Karolina A Hosiawa；Sean Stevens；Andrew J Murphy
申请人:Regeneron Pharmaceuticals, Inc.；
IPC主号:

专利说明:

[0001] [0001] Fertile, non-human, genetically modified animals are described that express human immunoglobulin λ light chain variable sequences cognate with human heavy chain variable sequences. Genetically modified mouse cells, embryos and tissues comprising a nucleic acid sequence encoding a functional ADAM6a at a mouse ADAM6 locus are described, wherein the mouse cells, embryos and tissues comprise lambda light chain gene segments from human immunoglobulin that are capable of rearrangement to form a functional immunoglobulin light chain variable domain. Modifications include human and/or humanized immunoglobulin loci. Mice that comprise ADAM6 function are described, including mice that comprise an ectopic nucleic acid sequence encoding an ADAM6 protein. Genetically modified male mice, which comprise a genetic modification of an endogenous mouse immunoglobulin VH region locus, and which further comprise ADAM6 activity are described, including mice which comprise an ectopic nucleic acid sequence that restores fertility in the mouse. male.
[0002] [0002] Fertile, non-human and genetically modified animals are described that comprise a deletion or a modification of an endogenous ADAM6 gene, or homolog or ortholog thereof, and that comprise a genetic modification that restores the function of ADAM6 (or homolog or ortholog thereof) in whole or in part, in which the
[0003] [0003] Pharmaceutical applications for antibodies over the past two decades have substantiated a great deal of research into the preparation of antibodies that are suitable for use as human therapeutics. Early antibody-based therapeutics based on mouse antibodies were not ideal as human therapeutics because repeated administration of mouse antibodies to humans results in immunogenicity problems, which can lead to long-term treatment regimens. Solutions that rely on humanizing mouse antibodies to make them more human-like and less mouse-like have been developed. Methods for expressing human immunoglobulin sequences for later use in antibodies have relied primarily on in vitro expression of human immunoglobulin libraries in phage, bacteria, or yeast. Finally, attempts were made to produce usable human antibodies from human lymphocytes in vitro, in mice engrafted with human hematopoietic cells, and in transchromosomal or transgenic mice with inactivated endogenous immunoglobulin loci. In the transgenic mice, it was necessary to inactivate the endogenous mouse immunoglobulin genes in order to randomly integrate fully human transgenes that can function as the source of immunoglobulin sequences expressed in the mouse. Such mice can produce human antibodies suitable for use as human therapeutics, but these mice exhibit substantial problems with their immune systems. These problems (1) make mice unlikely to generate a sufficiently diverse antibody repertoire, (2) require the
[0004] [0004] Transgenic mice that contain fully human antibody transgenes contain randomly inserted transgenes that contain human immunoglobulin heavy chain unrearranged variable sequences (V, D, and J sequences) linked to human heavy chain constant sequences, and non-rearranged human immunoglobulin light chain variable sequences (V and J) linked to the human light chain constant sequences. Therefore, mice generate antibody rearranged genes from loci that are not mouse endogenous loci, where the antibody rearranged genes are fully human. In general, mice contain both human heavy chain sequences and human κ light chain sequences, although mice with at least some human λ sequences have also been reported. Transgenic mice generally have damaged and nonfunctional immunoglobulin loci, or inactivation of endogenous immunoglobulin loci, such that the mice are unable to rearrange human antibody sequences at an endogenous mouse immunoglobulin locus. The differences of such transgenic mice make them less than ideal for generating a sufficiently diverse human antibody repertoire in mice, likely due at least in part to a sub-ideal clonal selection process, which links fully human antibody molecules into a mouse endogenous selection system.
[0005] [0005] Thus, there remains a necessity in the art to produce
[0006] [0006] Genetically modified non-human animals are described which comprise a modification that reduces or eliminates the activity of an ADAM6 Gene or homolog or ortholog thereof, wherein the modification results in a loss of fertility, and the animals additionally comprise a sequence which encodes an activity that complements or restores the loss or reduced activity of ADAM6 (or homologous or orthologous activity), and non-human animals further comprise modifications that enable them to express human immunoglobulin heavy chain variable regions, which are congnant with human immunoglobulin λ light chain variable regions. In various aspects, the human immunoglobulin λ light chain variable regions are expressed fused to λ constant regions or
[0007] [0007] In several respects, the sequence encoding ADAM6 activity is contiguous with a human immunoglobulin sequence. In several aspects, the sequence encoding ADAM6 activity is contiguous with a non-human immunoglobulin sequence. In several aspects, the sequence is present on the same chromosome as the endogenous non-human immunoglobulin heavy chain locus of the non-human animal. In several respects, the sequence is present on a different chromosome than the immunoglobulin heavy chain locus of the non-human animal.
[0008] [0008] Genetically modified non-human animals are described that comprise a modification that maintains the activity of an ADAM6 gene or homolog or ortholog thereof, wherein the modification includes insertion of one or more human immunoglobulin heavy chain gene segments upstream of a non-human immunoglobulin heavy chain constant region, and non-human animals further comprise modifications that enable them to express human immunoglobulin λ light chain variable regions cognate with human immunoglobulin heavy chain variable regions. In various aspects, human immunoglobulin λ light chain variable regions are expressed fused to λ or κ constant regions.
[0009] [0009] In various aspects, the insertion of one or more human immunoglobulin heavy chain gene segments is performed 3' or downstream of the ADAM6 gene of the non-human animal. In various aspects, the insertion of one or more human immunoglobulin heavy chain gene segments is performed in such a way that the ADAM6 gene(s) from the non-human animal is not disrupted, deleted, and/or functionally silenced, such that the ADAM6 activity of the non-human animal is at the same or comparable level in a
[00010] [00010] In one aspect, nucleic acid constructs, cells, embryos, mice, and methods for producing mice are provided that comprise a modification that results in a non-functional mouse endogenous ADAM6 protein or ADAM6 gene (e.g., an inactivation or a deletion in an endogenous ADAM6 gene), wherein the mice comprise a nucleic acid sequence encoding an ADAM6 protein or ortholog, or homolog, or fragment thereof that is functional in a male mouse.
[00011] [00011] In one aspect, nucleic acid constructs, cells, embryos, mice, and methods for producing mice are provided that comprise a modification of an endogenous mouse immunoglobulin locus, wherein the mice comprise an ADAM6 protein or ortholog, or homolog, or fragment thereof that is functional in a male mouse. In one embodiment, the endogenous mouse immunoglobulin locus is an immunoglobulin heavy chain locus, and the modification reduces or eliminates ADAM6 activity from a male mouse cell or tissue.
[00012] [00012] In one aspect, mice are provided which comprise an ectopic nucleotide sequence encoding a mouse ADAM6 or ortholog, or homolog, or functional fragment thereof; also provided are mice that comprise an endogenous nucleotide sequence encoding a mouse ADAM6 or ortholog, or homolog, or fragment thereof, and at least one genetic modification of an immunoglobulin heavy chain locus.
[00013] [00013] In one aspect, methods are provided for producing mice that comprise a modification of an endogenous mouse immunoglobulin locus, wherein the mice comprise an ADAM6 protein or ortholog, or homolog, or fragment thereof that is functional in a male mouse .
[00014] [00014] In one aspect, methods are provided for producing mice that comprise a genetic modification of an immunoglobulin heavy chain locus, wherein application of the methods results in male mice that comprise a modified immunoglobulin heavy chain locus (or a elimination of it), and male mice are able to generate offspring by mating. In one embodiment, male mice are capable of producing sperm that can transit from a mouse uterus to a mouse oviduct to fertilize a mouse egg.
[00015] [00015] In one aspect, methods are provided for producing mice that comprise a genetic modification of an immunoglobulin heavy chain locus and an immunoglobulin light chain locus, wherein application of the methods to modify the heavy chain locus results in male mice that exhibit a reduction in fertility, and mice comprise a genetic modification that recovers all or part of the reduction in fertility. In various embodiments, reduced fertility is characterized by an inability of male mouse sperm to migrate from a mouse uterus to a mouse oviduct to fertilize a mouse egg. In various embodiments, reduced fertility is characterized by sperm that exhibit a defect in in vivo migration. In various embodiments, the genetic modification that recovers in whole or in part the reduction in fertility is a nucleic acid sequence that encodes a mouse ADAM6 gene or ortholog, or homolog, or fragment.
[00016] [00016] In one embodiment, the genetic modification comprises replacing endogenous immunoglobulin heavy chain variable loci with immunoglobulin heavy chain variable loci from another species (eg, a non-mouse species). In one embodiment, the genetic modification comprises inserting orthologous immunoglobulin heavy chain variable loci into endogenous immunoglobulin heavy chain variable loci. In a specific embodiment, the species is human. In one embodiment, the genetic modification comprises deleting an endogenous variable immunoglobulin heavy chain locus in whole or in part, wherein the deletion results in a loss of endogenous ADAM6 function. In one specific embodiment, loss of endogenous ADAM6 function is associated with a reduction in fertility in male mice.
[00017] [00017] In one embodiment, the genetic modification comprises inactivation of an endogenous non-human immunoglobulin heavy chain variable locus in whole or in part, wherein the inactivation does not result in a 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 is substantially unable to rearrange to encode an antibody heavy chain comprising non-human gene segments. endogenous humans. Inactivation may include other modifications that render the endogenous immunoglobulin heavy chain locus unable to rearrange to encode the heavy chain of an antibody, wherein the modification does not include replacement or deletion of endogenous gene segments. Exemplary modifications include chromosomal inversions and/or translocations mediated by molecular techniques, for example, using exact location of specific recombination sites to
[00018] [00018] In one embodiment, the genetic modification comprises inserting into the genome of the non-human animal a DNA fragment containing one or more human VH gene segments, one or more human DH gene segments, and one or more JH gene segments from a human from another species (e.g., a non-mouse species) operably linked to one or more constant region sequences (e.g., an IgM gene and/or an IgG). In one embodiment, the DNA fragment is capable of undergoing rearrangements in the genome of the non-human animal to form a sequence encoding an antibody heavy chain variable domain. In one embodiment, the species is human. In one embodiment, the genetic modification comprises inserting one or more human immunoglobulin heavy chain gene segments downstream or 3' of an endogenous ADAM6 gene from the non-human animal, such that ADAM6 activity (e.g., expression and/or function of an encoded protein) is the same or comparable to a non-human animal that does not understand the insert.
[00019] [00019] In one aspect, mice are provided that comprise a modification that reduces or eliminates mouse ADAM6 expression from an endogenous allele of ADAM6, such that a male mouse with the modification exhibits reduced fertility (e.g. , a greatly reduced ability to generate offspring by mating), i.e. essentially infertile, by virtue of the reduction or elimination of endogenous ADAM6 function, wherein the mice additionally comprise an ectopic ADAM6 sequence, or homolog, or ortholog or functional fragment of the ADAM6. same. In one aspect, the
[00020] [00020] In one embodiment, the reduction or loss of ADAM6 function comprises an inability or substantial inability of mice to produce sperm that can travel from a mouse uterus to a mouse oviduct to fertilize a mouse egg. In a specific embodiment, at least about 95%, 96%, 97%, 98%, or 99% of the sperm cells produced in a mouse ejaculate volume are unable to cross an oviduct in vivo after copulation and fertilize. a mouse egg.
[00021] [00021] In one embodiment, the reduction or loss of ADAM6 function comprises an inability to form or substantial inability to form a complex of ADAM2, and/or ADAM3, and/or ADAM6 on a surface of a mouse sperm cell. In one embodiment, the loss of ADAM6 function comprises a substantial inability to fertilize a mouse egg by copulation with a female mouse.
[00022] [00022] In one aspect, a mouse is provided that lacks a functional endogenous ADAM6 gene, and comprises a protein (or an ectopic nucleotide sequence encoding a protein) that confers ADAM6 functionality in the mice. In one embodiment, the mouse is a male mouse and the functionality comprises improved fertility compared to a mouse that lacks a functional endogenous ADAM6 gene.
[00023] [00023] In one embodiment, the protein is encoded by a genomic sequence located at an immunoglobulin locus in the germ line of mice. In a specific embodiment, the immunoglobulin locus is a heavy chain locus. in another modality
[00024] [00024] In one embodiment, the mouse comprises a human, or human/mouse chimeric or human/rat chimeric light chain (e.g., human variable, mouse or rat constant) and a chimeric heavy chain variable/rat constant. human, mouse or rat. In a specific embodiment, the mouse comprises a transgene comprising a human/mouse or mouse constant light chain gene chimeric variable operably linked to a transcriptionally active promoter, for example, a ROSA26 promoter. In a further specific embodiment, the human/mouse or rat chimeric light chain transgene comprises a human rearranged light chain variable region sequence in the mouse germ line.
[00025] [00025] In one embodiment, the ectopic nucleotide sequence is localized to an immunoglobulin locus in the germ line of mice. In a specific embodiment, the immunoglobulin locus is a heavy chain locus. In one embodiment, the heavy chain locus comprises at least one human VH gene segment, at least one
[00026] [00026] In one aspect, a mouse is provided that loses a functional endogenous ADAM6 gene, wherein the mouse comprises an ectopic nucleotide sequence that complements the loss of mouse ADAM6 function. In one embodiment, the ectopic nucleotide sequence confers on the mouse an ability to produce offspring that is comparable to a corresponding wild-type mouse that contains a functional endogenous ADAM6 gene. In one embodiment, the sequence confers on the mouse an ability to form a complex of ADAM2, and/or ADAM3, and/or ADAM6 on the sperm cell surface of the mice. In one embodiment, the sequence gives the mouse an ability to migrate from a mouse uterus to a mouse oviduct in a mouse egg, to fertilize the egg.
[00027] [00027] In one embodiment, the mouse that loses the functional endogenous ADAM6 gene that comprises the ectopic nucleotide sequence produces at least about 50%, 60%, 70%, 80%, or 90% of the number of litters of an age-matched wild-type mouse strain and produces within a six-month time period.
[00028] [00028] In one embodiment, the mouse that loses the functional endogenous ADAM6 gene that comprises the ectopic nucleotide sequence produces at least about 1.5-fold, about 2-fold, about
[00029] [00029] In one embodiment, the mouse that loses the functional endogenous ADAM6 gene and that comprises the ectopic nucleotide sequence produces an average of at least about 2-fold, 3-fold, or 4-fold the number of offspring per litter in a gestation period of 4 or 6 months than a mouse that loses the functional endogenous ADAM6 gene, and that loses the ectopic nucleotide sequence, and that is generated for the same period of time.
[00030] [00030] In one embodiment, the mouse that loses the functional endogenous ADAM6 gene that comprises the ectopic nucleotide sequence is a male mouse, and the male mouse produces sperm which, when recovered from the oviducts within about 5-6 hours after copulation reflects a migration in the oviduct that is at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least less 90-fold, 100-fold, 110-fold, or 120-fold or greater than in a mouse that loses the functional endogenous ADAM6 gene and that loses the ectopic nucleotide sequence.
[00031] [00031] In one embodiment, the mouse that loses the functional endogenous ADAM6 gene that comprises the ectopic nucleotide sequence, when copulated with a female mouse, generates sperm that are capable of crossing the uterus and entering and crossing the oviduct in about 6 hours, at an efficiency that is generally equal to that of the
[00032] [00032] In one embodiment, the mouse that loses the functional endogenous ADAM6 gene that comprises the ectopic nucleotide sequence produces about 1.5-fold, about 2-fold, about 3-fold, or about 4-fold or more litters in a period of time comparable to a mouse that loses the functional ADAM6 gene and that loses the ectopic nucleotide sequence.
[00033] [00033] In one aspect, a mouse is provided which comprises in its germ line a non-mouse-associated nucleic acid sequence encoding an immunoglobulin protein, wherein the non-mouse-associated immunoglobulin sequence comprises an insertion of an ADAM6 gene mouse or homolog or ortholog or functional fragment thereof. In one embodiment, the non-mouse-associated immunoglobulin sequence comprises a human immunoglobulin sequence. In one embodiment, the sequence comprises a human immunoglobulin heavy chain sequence. In one embodiment, the sequence comprises a human immunoglobulin light chain sequence. In one embodiment, the sequence comprises one or more V gene segments, one or more D gene segments, and one or more J gene segments; in one embodiment, the sequence comprises one or more V gene segments and one or more J gene segments. In one embodiment, the one or more V, D and J gene segments, or 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 comprises in its genome at least one nucleic acid sequence encoding an ADAM6 gene. mouse, or homolog, or ortholog, or functional fragment thereof. In one embodiment, after rearrangement,
[00034] [00034] In one aspect, mice are provided that express a human immunoglobulin heavy chain variable region or functional fragment thereof from an endogenous mouse immunoglobulin heavy chain locus, wherein the mice comprise an ADAM6 activity that is functional in a male mouse.
[00035] [00035] In one embodiment, male mice comprise a single unmodified endogenous ADAM6 allele, or ortholog, of a homolog or functional fragment thereof at an endogenous ADAM6 locus.
[00036] [00036] In one embodiment, the male mice comprise an ectopic sequence of mouse ADAM6, or homolog or ortholog or functional fragment thereof that encodes a protein that confers ADAM6 function.
[00037] [00037] In one embodiment, male mice comprise an ADAM6 sequence, or homolog, or ortholog, or functional fragment thereof at a location in the mouse genome that approximates the location of the mouse endogenous allele of ADAM6, e.g., 3 ' of a V gene segment sequence and 5' of an initial D gene segment.
[00038] [00038] In one embodiment, male mice comprise an ADAM6 sequence, or homolog, or ortholog, or fragment
[00039] [00039] In one embodiment, male mice comprise an ADAM6 sequence, or homolog, or ortholog, or functional fragment thereof that is located at a position at an endogenous immunoglobulin locus that is the same or substantially the same in a mouse wild type male. In a specific embodiment, the endogenous locus is unable to encode the heavy chain variable region of an antibody, wherein the variable region comprises or is derived from a non-human endogenous gene segment. In a specific embodiment, the endogenous locus is positioned at a location in the male mouse genome that renders it unable to encode the heavy chain variable region of an antibody. In various embodiments, the male mice comprise an ADAM6 sequence located on the same chromosome as the human immunoglobulin gene segments, and the ADAM6 sequence encodes a functional ADAM6 protein.
[00040] [00040] In one aspect, a male mouse is provided that
[00041] [00041] In one aspect, a male mouse is provided which comprises a functional endogenous ADAM6 gene and a modification at an endogenous immunoglobulin heavy chain locus. In one embodiment, the modification is carried out downstream, or 3' of the endogenous ADAM6 gene. In one embodiment, the modification is a replacement of one or more endogenous immunoglobulin heavy chain gene segments with one or more human immunoglobulin heavy chain gene segments. In one embodiment, the modification is an insertion of one or more human immunoglobulin heavy chain gene segments upstream of an endogenous immunoglobulin heavy chain constant region gene.
[00042] [00042] In one aspect, mice are provided that comprise a genetic modification that reduces mouse endogenous ADAM6 function, wherein the mouse comprises at least some ADAM6 functionality provided by either an unmodified endogenous allele that is fully functional or in part (eg, a heterozygote), or by expression of an ectopic sequence encoding an ADAM6, or an ortholog, or homolog or functional fragment thereof, which is functional in a male mouse.
[00043] [00043] In one embodiment, the mice comprise sufficient ADAM6 function to confer in male mice the ability to generate offspring by mating, compared to male mice that lose a functional ADAM6. In one embodiment, ADAM6 function is conferred by the presence of an ectopic nucleotide sequence that encodes a mouse ADAM6, or a homolog, or ortholog, or
[00044] [00044] In one embodiment, male mice that express the human immunoglobulin variable region, or functional fragment of the
[00045] [00045] In one embodiment male mice express enough ADAM6 (or an ortholog, or homolog, or functional fragment thereof) to enable a sperm cell from male mice to cross a female mouse oviduct and fertilize a mouse egg.
[00046] [00046] In one embodiment, ADAM6 functionality is conferred by a nucleic acid sequence that is contiguous with a mouse chromosomal sequence (e.g., the nucleic acid is randomly integrated into a mouse chromosome; or placed at a specific location , e.g., by targeting the nucleic acid at a specific site, e.g., by site-specific recombinase-mediated insertion (e.g., Cre-mediated) or homologous recombination). In one embodiment, the ADAM6 sequence is present in a nucleic acid that is distinct from a mouse chromosome (e.g., the ADAM6 sequence is present in an episome, i.e., extrachromosally, e.g., in an expression construct, a vector, a YAC, a transchromosome, etc.).
[00047] [00047] In one aspect, mice are provided genetically
[00048] [00048] In one aspect, genetically modified mice and cells are provided that comprise a modification of an endogenous immunoglobulin heavy chain locus, wherein the modification reduces or eliminates ADAM6 activity expressed from an ADAM6 locus sequence, and wherein the mice comprise an ADAM6 protein or ortholog, or homolog, or functional fragment thereof. In various embodiments, the ADAM6 protein or fragment thereof is encoded by
[00049] [00049] In various embodiments, the modification is the insertion of one or more human immunoglobulin heavy chain gene segments upstream, or 5', 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.
[00050] [00050] In one embodiment, the second modification is located 3' (with respect to the transcriptional directionality of the mouse V gene segment) of a mouse final V gene segment, and located 5' (with respect to the transcriptional directionality of the sequence constant) of a mouse immunoglobulin heavy chain constant gene (or human/chimeric mouse), or fragment thereof (e.g., a nucleic acid sequence encoding a: CH1 and/or hinge and/or CH2 and/or CH3 human and/or mouse).
[00051] [00051] In one embodiment, the modification is at a first immunoglobulin heavy chain allele, at a first locus encoding a first ADAM6 allele, and the function of ADAM6 results from the expression of an endogenous ADAM6 at a second chain allele heavy immunoglobulin, at a second locus that encodes a functional ADAM6,
[00052] [00052] In one embodiment, the modification is in a first immunoglobulin heavy chain allele at a first locus, and a second immunoglobulin heavy chain allele at a second locus, and ADAM6 function results from the expression of an ectopic ADAM6 at a non-immunoglobulin-associated locus in the germ line of mice. In a specific embodiment, the non-immunoglobulin-associated locus is the ROSA26 locus. In a specific embodiment, the non-immunoglobulin-associated locus is transcriptionally active in reproductive tissue.
[00053] [00053] In one embodiment, the modification is in a first immunoglobulin heavy chain allele at a first locus, and a second immunoglobulin heavy chain allele at a second locus, and ADAM6 function results from an endogenous ADAM6 gene in the mouse germ line. In a specific embodiment, the endogenous ADAM6 gene is juxtaposed by mouse immunoglobulin gene segments.
[00054] [00054] In one embodiment, the modification is in a first immunoglobulin heavy chain allele at a first locus, and a second allele
[00055] [00055] In one aspect, a mouse is provided which comprises a heterozygous inactivation or a homozygote of ADAM6. In one embodiment, the mouse further comprises a modified immunoglobulin sequence, which is a human or humanized immunoglobulin sequence, or a camelid, or camelized human, or mouse immunoglobulin sequence. In one embodiment, the modified immunoglobulin sequence is present at the endogenous immunoglobulin heavy chain locus. In one embodiment, the modified immunoglobulin sequence comprises a human heavy chain variable gene sequence at an endogenous immunoglobulin heavy chain locus. In one embodiment, the human heavy chain variable gene sequence replaces an endogenous heavy chain variable sequence at the endogenous immunoglobulin heavy chain locus.
[00056] [00056] In one aspect, a mouse incapable of expressing a mouse functional endogenous ADAM6 from a functional endogenous ADAM6 locus is provided. In one embodiment, the mouse comprises an ectopic nucleic acid sequence encoding an ADAM6, or functional fragment thereof, that is functional in the mouse. In a specific embodiment, the ectopic nucleic acid sequence encodes a protein that recovers a loss in the ability to generate offspring exhibited by a male mouse that is homozygous for an inactivation.
[00057] [00057] In one aspect, a mouse is provided which lacks a functional endogenous ADAM6 Locus, and which comprises an ectopic nucleic acid sequence that confers mouse ADAM6 function. In one embodiment, the nucleic acid sequence comprises an endogenous mouse ADAM6 sequence or a functional fragment thereof. In one embodiment, the endogenous mouse ADAM6 sequence comprises sequence encoding ADAM6a and ADAM6b located in a wild-type mouse between the mouse immunoglobulin heavy chain V gene segment 3' plus (VH) and the D gene segment of 5' plus mouse immunoglobulin heavy chain (DH).
[00058] [00058] In one embodiment, the nucleic acid sequence comprises a sequence encoding mouse ADAM6a or functional fragment thereof, and/or a sequence encoding mouse ADAM6b or functional fragment thereof, wherein ADAM6a and/or ADAM6b or functional fragment(s) thereof is operably linked to a promoter. In one embodiment, the promoter is a human promoter. In one embodiment, the promoter is the mouse ADAM6 promoter. In a specific embodiment, the ADAM6 promoter comprises sequence located between the first codon of the first ADAM6 gene closest to the mouse 5'-plus DH gene segment and the recombination signal sequence of the 5'-plus DH gene segment, where 5' is indicated with respect to the transcriptional direction of the mouse immunoglobulin genes. In one embodiment, the promoter is a viral promoter. In a specific embodiment, the viral promoter is a cytomegalovirus (CMV) promoter. In one embodiment, the promoter is a ubiquitin promoter.
[00059] [00059] In one embodiment, the promoter is an inducible promoter. In
[00060] [00060] In one embodiment, the mouse ADAM6a and/or ADAM6b are selected from the sequence ADAM6a of SEQ ID NO:1 and/or ADAM6b of SEQ ID NO:2. In one embodiment, the mouse ADAM6 promoter is a promoter of SEQ ID NO:3. In a specific embodiment, the mouse ADAM6 promoter comprises the nucleic acid sequence of SEQ ID NO:3 directly upstream (with respect to the transcriptional direction of ADAM6a) of the first codon of ADAM6a and extending from the end of SEQ ID NO. :3 upstream of the region encoding ADAM6. In another specific embodiment, the ADAM6 promoter is a fragment that extends from about 5 to about 20 nucleotides upstream of the ADAM6a start codon to about 0.5kb, 1kb, 2kb, or 3kb or more upstream of the codon start of ADAM6a.
[00061] [00061] In one embodiment, the nucleic acid sequence comprises SEQ ID NO:3 or a fragment thereof which, when placed in a mouse that is infertile or has poor fertility due to a loss of ADAM6, improves fertility or recovers fertility at about a wild-type fertility. In one embodiment, SEQ ID NO:3 or a fragment thereof confers on a male mouse the ability to produce a sperm cell that is capable of crossing a female mouse oviduct in order to fertilize a mouse egg.
[00062] [00062] In one embodiment, the nucleic acid sequence is any sequence that encodes an ADAM6 gene, or homolog, or ortholog, or functional fragment thereof that, when placed or maintained in a
[00063] [00063] In one aspect, there is provided a mouse comprising a deletion of an endogenous nucleotide sequence encoding an ADAM6 protein, a replacement of an endogenous mouse VH gene segment with a human VH gene segment, and a sequence ectopic nucleotide encoding a mouse ADAM6 protein or ortholog, or homolog, or fragment thereof that is functional in a male mouse.
[00064] [00064] In one embodiment, the mouse comprises an immunoglobulin heavy chain locus that comprises a deletion of an endogenous immunoglobulin nucleotide sequence locus that comprises an endogenous ADAM6 gene, comprises a nucleotide sequence that encodes one or more segments immunoglobulin gene sequence, and wherein the ectopic nucleotide sequence encoding the mouse ADAM6 protein is or is directly adjacent to the nucleotide sequence encoding the one or more human immunoglobulin gene segments.
[00065] [00065] In one embodiment, the mouse comprises a replacement of all or substantially all of the endogenous VH gene segments with a nucleotide sequence encoding one or more human VH gene segments, and the ectopic nucleotide sequence encoding the protein Mouse ADAM6 is, or is directly adjacent to, the nucleotide sequence encoding the one or more human VH gene segments. In one embodiment, the mouse further comprises a substitution of one or more DH gene segments
[00066] [00066] In a specific embodiment, the mouse comprises a replacement of all or substantially all of the endogenous VH gene segments with a nucleotide sequence that encodes one or more human VH gene segments, and the ectopic nucleotide sequence that encodes the The mouse ADAM6 protein is, or is directly adjacent to, the nucleotide sequence that encodes the one or more VH gene segments of
[00067] [00067] In one embodiment, the ectopic nucleotide sequence encoding the mouse ADAM6 protein is present on a transgene in the mouse genome. In one embodiment, the ectopic nucleotide sequence encoding the mouse ADAM6 protein is present extrachromosally in the mouse.
[00068] [00068] In one aspect, there is provided a mouse comprising a modification of an endogenous immunoglobulin heavy chain locus, wherein the mouse expresses a B cell comprising an operably rearranged immunoglobulin sequence operably linked to a constant region gene sequence chain, and the B cell comprises in its genome (for example, on a B cell chromosome) a gene that encodes an ADAM6 or ortholog, or homolog, or fragment thereof, that is functional in a male mouse. In one embodiment, the rearranged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises a human V, D, and/or J heavy chain sequence; a mouse V, D, and/or J heavy chain sequence; a human or mouse V and/or J light chain sequence. In one embodiment, the heavy chain constant region gene sequence comprises a human or mouse heavy chain sequence selected from the group consisting of a CH1, a hinge, a CH2, a CH3, and a combination thereof.
[00069] [00069] In one aspect, there is provided a mouse comprising a functionally silenced endogenous immunoglobulin heavy chain variable gene locus, wherein ADAM6 function is maintained in the mice, and further comprises an insertion of one or more gene segments of human immunoglobulin upstream or 5' of one or more mouse heavy chain constant region. In one embodiment, the one or more human immunoglobulin gene segments
[00070] [00070] In one aspect, there is provided a genetically modified mouse, wherein the mouse comprises a functionally silenced immunoglobulin light chain gene, and further comprises a substitution of one or more endogenous gene segments from a region
[00071] [00071] In one aspect, a mouse is provided which lacks a functional mouse endogenous ADAM6 locus or sequence, and which comprises an ectopic nucleotide sequence encoding a mouse ADAM6 locus or a functional fragment of a mouse ADAM6 locus or sequence. mouse, wherein the mouse is capable of mating with a mouse of the opposite sex to produce progeny that comprise the ectopic ADAM6 locus or sequence. In one embodiment, the mouse is male. In one embodiment, the mouse is female.
[00072] [00072] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a human immunoglobulin heavy chain variable region gene segment at an endogenous mouse immunoglobulin heavy chain variable region gene locus, the mouse loses an endogenous functional ADAM6 sequence at the mouse immunoglobulin heavy chain variable region endogenous gene locus, and wherein the mouse comprises an ectopic nucleotide sequence that expresses a mouse ADAM6 protein, or an ortholog, or homolog or fragment thereof, which is functional in a male mouse.
[00073] [00073] In one embodiment, the ectopic nucleotide sequence expressing the mouse ADAM6 protein is extrachromosomal. In one embodiment, the ectopic nucleotide sequence expressing the mouse ADAM6 protein is integrated into one or more loci in a mouse genome. In a specific embodiment, the one or more loci include a
[00074] [00074] In one aspect, a mouse is provided that expresses an immunoglobulin heavy chain sequence from a modified endogenous mouse immunoglobulin heavy chain locus, wherein the heavy chain is derived from a human V gene segment , a D gene segment, and a J gene segment, wherein the mouse comprises an ADAM6 activity that is functional in the mouse.
[00075] [00075] In one embodiment, the mouse comprises a plurality of human V gene segments, a plurality of D gene segments, and a plurality of J gene segments. In one embodiment, the D gene segments are gene segments D for human. In one embodiment, the J gene segments are human J gene segments. In one embodiment, the mouse further comprises a humanized heavy chain constant region sequence, wherein the humanization comprises substitution of a sequence selected from a CH1, hinge, CH2, CH3, and a combination thereof. In a specific embodiment, the heavy chain is derived from a human V gene segment, a human D gene segment, a human J gene segment, a human CH1 sequence, a human or mouse hinge sequence, a mouse CH2 sequence, and a mouse CH3 sequence. In another specific embodiment, the mouse additionally comprises a human light chain constant sequence.
[00076] [00076] In one embodiment, the mouse comprises an ADAM6 gene that is flanked 5' and 3' by endogenous immunoglobulin heavy chain gene segments. In a specific embodiment, the endogenous immunoglobulin heavy chain gene segments are incapable of encoding an antibody heavy chain. In a specific embodiment, the mouse ADAM6 gene is in a
[00077] [00077] In one embodiment, the V gene segment is flanked 5' (with respect to the transcriptional direction of the V gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse.
[00078] [00078] In one embodiment, the V gene segment is flanked 3' (with respect to the transcriptional direction of the V gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse.
[00079] [00079] In one embodiment, the D gene segment is flanked 5' (with respect to the transcriptional direction of the D gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse.
[00080] [00080] In one embodiment, ADAM6 activity that is functional in the mouse results from the expression of a nucleotide sequence located 5' of the 5'-plus D gene segment and 3' of the 3'-plus V gene segment (with relative to the direction of transcription of the V) gene segment of the modified endogenous mouse immunoglobulin heavy chain locus.
[00081] [00081] In one embodiment, ADAM6 activity that is functional in the mouse results from the expression of a nucleotide sequence located between two human V gene segments at the modified endogenous mouse immunoglobulin heavy chain locus. In one embodiment, the two human V gene segments are a human VH1-2 gene segment and a human VH6-1 gene segment.
[00082] [00082] In one embodiment, the nucleotide sequence comprises a sequence selected from a mouse ADAM6b sequence or functional fragment thereof, an ADAM6a sequence from
[00083] [00083] In one embodiment, the nucleotide sequence between the two human V gene segments is placed opposite the transcriptional orientation with respect to the human V gene segments. In a specific embodiment, the nucleotide sequence encodes, 5' to 3' with respect to the direction of transcription of ADAM6 genes, and sequence of ADAM6a followed by a sequence of ADAM6b.
[00084] [00084] In one embodiment, the mouse comprises a substitution of a human ADAM6 pseudogene sequence between human VH1-2 and VH6-1 V gene segments with a mouse ADAM6 sequence or a functional fragment thereof .
[00085] [00085] In one embodiment, the sequence encoding ADAM6 activity that is functional in the mouse is a mouse ADAM6 sequence or functional fragment thereof.
[00086] [00086] In one embodiment, the mouse comprises an endogenous mouse DFL16.1 gene segment (e.g., in a mouse heterozygous for the modified endogenous mouse immunoglobulin heavy chain locus), or a DH1-1 gene segment of human. In one embodiment, the immunoglobulin heavy chain D gene segment expressed by the mouse is derived from an endogenous mouse DFL16.1 gene segment or a human DH1-1 gene segment.
[00087] [00087] In one aspect, a mouse is provided that comprises a nucleic acid sequence that encodes a mouse ADAM6 (or homolog or ortholog or functional fragment thereof) in a cell that carries the DNA of the unrearranged, but does not comprise the nucleic acid sequence encoding mouse ADAM6 (or homolog or ortholog or functional fragment thereof) in a B cell comprising rearranged immunoglobulin loci, wherein the
[00088] [00088] In one aspect, a mouse is provided that comprises a nucleic acid sequence that encodes a mouse ADAM6 (or homolog or ortholog or functional fragment thereof) in all or substantially all cells that carry DNA, including B cells that comprise rearranged immunoglobulin loci, in which the nucleic acid sequence encoding mouse ADAM6 (or homolog or ortholog or functional fragment thereof) occurs in the genome at a position that is different from a position in which a mouse ADAM6 gene appears in a wild type mouse. In one embodiment, the nucleic acid sequence encoding mouse ADAM6 (or homolog or ortholog or functional fragment thereof) is in a nucleic acid that is contiguous with the rearranged immunoglobulin locus. In one embodiment, the nucleic acid that is contiguous with the rearranged immunoglobulin locus is a chromosome. In one embodiment, the chromosome is a chromosome that is found in a wild-type mouse, and the chromosome comprises a modification of a mouse immunoglobulin locus.
[00089] [00089] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a B cell that
[00090] [00090] In one embodiment, 90% or more of the mouse B cells comprise a gene encoding an ADAM6 protein, or an ortholog thereof or a homologue thereof, or a fragment thereof that is functional in the mouse. In a specific embodiment, the mouse is a male mouse.
[00091] [00091] In one embodiment, the B cell genome comprises a first allele and a second allele comprising the sequence of ADAM6 or ortholog or homolog thereof. In one embodiment, the B cell genome comprises a first allele, but not a second allele, which comprises the sequence of ADAM6 or ortholog or homolog thereof.
[00092] [00092] In one aspect, there is provided a mouse which comprises a modification in one or more endogenous immunoglobulin heavy chain alleles, wherein the modification maintains one or more endogenous alleles of ADAM6, and the mouse further comprises an insertion of one or more more human Vλ gene segments and one or more human Jλ gene segments upstream of a mouse light chain constant region. In various embodiments, the mouse light chain constant region is either a mouse Cκ or a mouse Cλ.
[00093] [00093] In one embodiment, the modification makes the mouse
[00094] [00094] In one embodiment, the mice are unable to express a functional heavy chain that comprises rearranged endogenous heavy chain gene segments from at least one of the endogenous immunoglobulin heavy chain alleles, and the mice express and ADAM6 protein from one allele endogenous to ADAM6. In a specific embodiment, mice are unable to express a functional heavy chain that comprises rearranged endogenous heavy chain gene segments from two endogenous immunoglobulin heavy chain alleles, and mice express an ADAM6 protein from one or more endogenous ADAM6 alleles. .
[00095] [00095] In one embodiment, mice are unable to express a functional heavy chain from each of the endogenous heavy chain alleles, and mice comprise a functional allele of ADAM6 located at 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 or more Mbp upstream (with respect to the transcriptional direction of the mouse heavy chain locus) of a chain constant region sequence heavy mouse immunoglobulin. In a specific embodiment, the functional ADAM6 allele is at the endogenous immunoglobulin heavy chain locus (e.g., in a VD intergenic region, between two V gene segments, between a V and a D gene segment, between a gene D and a J, etc.). In a specific embodiment, the functional ADAM6 allele is located in an intergenic sequence 90 to 100 kb between the mouse final V gene segment and the mouse first D gene segment.
[00096] [00096] In one aspect, a mouse is provided that comprises
[00097] [00097] In one embodiment, the modification renders the mouse incapable of expressing a functional ADAM6 protein from at least one of the one or more endogenous ADAM6 alleles. In a specific embodiment, the mouse is unable to express a functional ADAM6 protein from each of the endogenous ADAM6 alleles.
[00098] [00098] In one embodiment, the mice are unable to express a functional ADAM6 protein from each endogenous allele of ADAM6, and the mice comprise an ectopic sequence of ADAM6.
[00099] [00099] In one embodiment, mice are unable to express a functional ADAM6 protein from each endogenous allele of ADAM6, and mice comprise an ectopic sequence of ADAM6 located at 1, 2, 3, 4, 5, 10, 20, 30 , 40, 50, 60, 70, 80, 90, 100, 110, or 120 or more kb upstream (with respect to the transcriptional direction of the mouse heavy chain locus) of an immunoglobulin heavy chain constant region sequence of mouse. In a specific embodiment, the ectopic sequence of ADAM6 is at the endogenous heavy chain locus (e.g., in a VD intergenic region, between two V gene segments, between a V and a D gene segment, between a D gene segment and a J, etc.). In a specific embodiment, the ectopic sequence of ADAM6 is located in a 90 to 100 kb intergenic sequence between the final mouse V gene segment and the first mouse D gene segment. In another specific embodiment, the 90 to 100 kb endogenous V-D intergenic sequence is removed, and the ectopic ADAM6 sequence is placed between the final V gene segment and the first D.
[000100] [000100] In one aspect, a non-fertile male mouse is provided, wherein the mouse comprises a deletion of two or more alleles
[000101] [000101] In one aspect, there is provided a mouse comprising an endogenous immunoglobulin heavy chain V, D, and/or J gene segment that is unable to rearrange to encode an antibody heavy chain, wherein most of the mouse B cells comprise a functional ADAM6 gene. In various embodiments, most mouse B cells additionally comprise one or more human Vλ gene segments and one or more human Jλ gene segments upstream of a mouse immunoglobulin light chain constant region. In one embodiment, the mouse immunoglobulin light chain constant region is selected from a mouse Cκ and a mouse Cλ.
[000102] [000102] In one embodiment, the mouse comprises intact endogenous immunoglobulin heavy chain V, D and J gene segments that are unable to rearrange to encode a functional heavy chain of an antibody. In one embodiment, the mouse comprises at least one and up to 89 V gene segments, at least one and up to 13 D gene segments, at least one and up to four J gene segments, and a combination thereof; wherein the at least one and up to 89 V gene segments, at least one and up to 13 D gene segments, at least one and up to four J gene segments are unable to rearrange to encode a heavy chain variable region of an antibody . In a specific embodiment, the mouse comprises a functional ADAM6 gene located in the intact endogenous immunoglobulin heavy chain V, D and J gene segments. In one embodiment, the mouse comprises an endogenous heavy chain locus that includes an endogenous ADAM6 locus, wherein the endogenous locus of
[000103] [000103] In one aspect, a mouse is provided that loses an endogenous immunoglobulin heavy chain V, D, and J gene segment, wherein a majority of the mice's B cells comprise an ADAM6 sequence or ortholog or homolog thereof. In one embodiment, the majority of mouse B cells express an immunoglobulin light chain comprising a human lambda variable domain and an endogenous immunoglobulin light chain constant region.
[000104] [000104] In one embodiment, the mouse loses endogenous immunoglobulin heavy chain gene segments selected from two or more V gene segments, two or more D gene segments, two or more J gene segments, and a combination thereof. In one embodiment, the mouse loses immunoglobulin heavy chain gene segments selected from at least one and up to 89 V gene segments, at least one and up to 13 D gene segments, at least one and up to four J gene segments, and a combination of this. In one embodiment, the mouse loses a fragment of chromosome 12 genomic DNA comprising about three megabases from the endogenous immunoglobulin heavy chain locus. In a specific embodiment, the mouse loses all functional endogenous heavy chain V, D, and J gene segments. In a specific embodiment, the mouse loses 89 VH gene segments, 13 DH gene segments, and four JH gene segments.
[000105] [000105] In one aspect, a mouse is provided, wherein the mouse has a germline genome comprising a modification of an immunoglobulin heavy chain locus, wherein the
[000106] [000106] In one aspect, there is provided a mouse that expresses an antibody comprising at least one human variable domain polypeptide/non-human immunoglobulin constant, wherein the mouse expresses a mouse ADAM6 protein or ortholog or homolog
[000107] [000107] In one embodiment, the ADAM6 protein or ortholog or homolog thereof is expressed in a mouse B cell, wherein the B cell comprises a rearranged immunoglobulin sequence comprising a human variable sequence and a non-human constant sequence.
[000108] [000108] In one embodiment, the non-human constant sequence is a rodent sequence. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster.
[000109] [000109] In one aspect, a method is provided for producing a non-fertile male mouse, comprising making an endogenous allele of ADAM6 from a non-functional donor ES cell (or inactivating said allele), introducing the donor ES cell into a host embryo , generate the host embryo in a surrogate mother, and allow the surrogate mother to give rise to progeny derived in whole or in part from the donor ES cell. In one embodiment, the method further comprises generating progeny to obtain a non-fertile male mouse.
[000110] [000110] In one aspect, there is provided a method for producing a mouse with a genetic modification of interest, wherein the mouse is infertile, the method comprising the steps of (a) masking a genetic modification of interest in a genome; (b) modifying the genome to inactivate an endogenous allele of ADAM6, or render an endogenous allele of ADAM6 non-functional; and (c) employing the genome to mask a mouse. In various embodiments, the genome is from an ES cell or used in a nuclear transfer experiment.
[000111] [000111] In one aspect, there is provided a mouse produced using a targeting vector, nucleotide construct, or cell in the manner described herein.
[000112] [000112] In one aspect, a progeny of a cross is provided
[000113] [000113] In one aspect, a method for maintaining a mouse strain is provided, wherein the mouse strain comprises a replacement of a mouse immunoglobulin heavy chain sequence with one or more heterologous immunoglobulin heavy chain sequences. In one embodiment, the one or more heterologous immunoglobulin heavy chain sequences are the human immunoglobulin heavy chain sequences.
[000114] [000114] In one embodiment, the mouse strain comprises a deletion of one or more mouse VH, DH, and/or JH gene segments. In one embodiment, the mouse further comprises one or more human VH gene segments, one or more human DH gene segments, and/or one or more human JH gene segments. In one embodiment, the mouse comprises at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 human VH segments, at least 27 human DH gene segments, and at least six JH gene segments. In a specific embodiment, the mouse comprises at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 human VH segments, the at least 27 human DH gene segments, and the at least six JH gene segments are operably linked to a constant region gene. In one embodiment, the constant region gene is a mouse constant region gene. In one embodiment, the constant region gene comprises a mouse constant region gene sequence selected from a CH1, a hinge, a CH2, a CH3, and/or a CH4 or a combination thereof.
[000115] [000115] In one embodiment, the method comprises generating a heterozygous male mouse to replace the mice
[000116] [000116] In one embodiment, the method comprises obtaining cells from male or female mice homozygous or heterozygous for the human heavy chain sequence, and employing those cells as donor cells or nuclei thereof as donor nuclei, and using the cells or nuclei to producing genetically modified animals using host cells, and/or generating the cells and/or nuclei in surrogate mother.
[000117] [000117] In one embodiment, only male mice that are heterozygous for the substitution at the heavy chain locus are mated to female mice. In a specific embodiment, the female mice are homozygous, heterozygous, or wild-type with respect to a substituted heavy chain locus.
[000118] [000118] In one embodiment, the mouse further comprises a substitution of light chain λ and/or κ variable sequences, at an endogenous immunoglobulin light chain locus, with heterologous immunoglobulin light chain sequences. In one embodiment, the heterologous immunoglobulin light chain sequences are human immunoglobulin light chain λ and/or κ variable sequences.
[000119] [000119] In one embodiment, the mouse further comprises a transgene at a locus other than an endogenous immunoglobulin locus, wherein the transgene comprises a sequence encoding a heterologous λ or κ sequence of rearranged or unrearranged light chain (e.g., Unrearranged VL and unrearranged JL, or VJ
[000120] [000120] In one aspect, there is provided a nucleic acid construct, comprising an upstream homology arm and a downstream homology arm, wherein the upstream homology arm comprises a sequence that is identical or substantially identical to a sequence immunoglobulin heavy chain variable region sequence, the downstream homology arm comprises a sequence that is identical or substantially identical to a human or mouse immunoglobulin variable region sequence, and disposed between the upstream and downstream homology arms. downstream is a sequence comprising a nucleotide sequence encoding a mouse ADAM6 protein. In a specific embodiment, the sequence encoding the mouse ADAM6 genes is operably linked with a mouse promoter to which the mouse ADAM6 is linked in a wild-type mouse.
[000121] [000121] In one aspect, a targeting vector is provided, comprising (a) a nucleotide sequence that is identical or substantially identical to a nucleotide human variable region gene segment sequence; and, (b) a nucleotide sequence that
[000122] [000122] In one embodiment, the targeting vector further comprises a promoter operably linked to the mouse ADAM6 encoding sequence. In a specific embodiment, the promoter is a mouse ADAM6 promoter.
[000123] [000123] In one aspect, there is provided a nucleotide construct for modifying a mouse immunoglobulin heavy chain variable locus, wherein the construct comprises at least a site-specific recombinase recognition site and a sequence encoding an ADAM6 protein or ortholog, or homolog, or fragment thereof, which is functional in a mouse.
[000124] [000124] In one aspect, mouse cells and mouse embryos are provided including, but not limited to, ES cells, pluripotent cells, and induced pluripotent cells comprising genetic modifications in the manner described herein. Cells that are XX and cells that are XY are given. Cells comprising a nucleus containing a modification in the manner described herein are also provided, for example, a modification introduced into a cell by pronuclear injection. Cells, embryos, and mice that comprise a virus-introduced ADAM6 gene are also provided, for example, cells, embryos and mice comprising a transduction construct comprising an ADAM6 gene, which is functional in the mouse.
[000125] [000125] In one aspect, a genetically modified mouse cell is provided, wherein the cell lacks a functional mouse endogenous ADAM6 locus, and the cell comprises an ectopic nucleotide sequence encoding a mouse ADAM6 protein or functional fragment thereof . In one embodiment, the cell further comprises a modification of a variable gene sequence
[000126] [000126] In one embodiment, the cell is a totipotent cell, a pluripotent cell, or an induced pluripotent cell. In a specific embodiment, the cell is a mouse ES cell.
[000127] [000127] In one aspect, a mouse B cell is provided, wherein the mouse B cell comprises a rearranged immunoglobulin heavy chain gene, wherein the B cell comprises on a chromosome of the B cell a nucleic acid sequence that encodes an ADAM6 protein or ortholog, or homolog, or fragment thereof, that is functional in a male mouse. In one embodiment, the mouse B cell comprises two alleles of the nucleic acid sequence.
[000128] [000128] In one embodiment, the nucleic acid sequence is on a nucleic acid molecule (eg, a B cell chromosome) that is contiguous with the rearranged mouse immunoglobulin heavy chain locus.
[000129] [000129] In one embodiment, the nucleic acid sequence is on a nucleic acid molecule (e.g., a B cell chromosome), which is distinct from the nucleic acid molecule comprising the locus
[000130] [000130] In one embodiment, the mouse B cell comprises a non-mouse-associated immunoglobulin rearranged gene variable sequence operably linked to a mouse or human immunoglobulin constant region gene, wherein the B cell comprises an acid sequence nucleic acid encoding an ADAM6 protein or ortholog, or homolog, or fragment thereof, that is functional in a male mouse.
[000131] [000131] In one aspect, a mouse somatic cell is provided, comprising a chromosome comprising a modified immunoglobulin heavy chain locus, and a nucleic acid sequence encoding a mouse ADAM6 or ortholog, or homolog, or fragment thereof. , which is functional in a male mouse. In one embodiment, the nucleic acid sequence is on the same chromosome at the modified immunoglobulin heavy chain locus. In one embodiment, the nucleic acid is on a different chromosome than the modified immunoglobulin heavy chain locus. In one embodiment, the somatic cell comprises a single copy of the nucleic acid sequence. In one embodiment, the somatic cell comprises at least two copies of the nucleic acid sequence. In a specific embodiment, the somatic cell is a B cell. In a specific embodiment, the cell is a germ cell. In a specific embodiment, the cell is an embryonic cell.
[000132] [000132] In one aspect, a mouse germ cell is provided, comprising a nucleic acid sequence encoding a mouse ADAM6 (or homolog or ortholog or functional fragment thereof) on a chromosome of the germ cell, wherein the acid sequence nucleic acid encoding mouse ADAM6 (or homolog or ortholog or functional fragment thereof) is at a position on the chromosome
[000133] [000133] In one aspect, a mouse-derived pluripotent, induced pluripotent or totipotent cell, in the manner described herein, is provided. In a specific embodiment, the cell is a mouse embryonic stem (ES) cell.
[000134] [000134] In one aspect, a cell or tissue derived from a mouse, in the manner described herein, is provided. In one embodiment, the cell or tissue is derived from a mouse spleen, lymph node, or bone marrow in the manner described herein. In one embodiment, the cell is a B cell. In one embodiment the cell is an embryonic stem cell. In one embodiment, the cell is a germ cell.
[000135] [000135] In one embodiment, the tissue is selected from connective, muscular, nervous, and epithelial tissue. In a specific embodiment, the tissue is reproductive tissue.
[000136] [000136] In one embodiment, the cell and/or tissue derived from a mouse in the manner described herein is isolated for use in one or more ex vivo assays. In various embodiments, one or more ex vivo assays
[000137] [000137] In aspect, there is provided the use of cell or tissue derived from a mouse in the manner described herein to produce an antibody. In one aspect, there is provided the use of a cell or tissue derived from a mouse in the manner described herein to produce a hybridoma or quadroma.
[000138] [000138] In one aspect, a non-human cell comprises a chromosome, or fragment thereof, from a non-human animal, in the manner described herein. In one embodiment, the non-human cell comprises a nucleus from a non-human animal in the manner described herein. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of nuclear transfer.
[000139] [000139] In one aspect, a nucleus derived from a mouse in the manner described herein is provided. In one embodiment, the nucleus is from a diploid cell that is not a B cell.
[000140] [000140] In one aspect, there is provided a nucleotide sequence encoding an immunoglobulin variable region produced in a mouse in the manner described herein.
[000141] [000141] In one aspect, an immunoglobulin heavy chain or immunoglobulin light chain variable region amino acid sequence of an antibody produced in a mouse in the manner described herein is provided.
[000142] [000142] In one aspect, there is provided an immunoglobulin heavy chain or nucleotide immunoglobulin light chain variable region sequence encoding a variable region of an antibody produced in a mouse in the manner described herein.
[000143] [000143] In one aspect, there is provided an antibody or antigen-binding fragment thereof (e.g. Fab, F(ab)2, scFv) produced in a mouse in the manner described herein.
[000144] [000144] In one aspect, there is provided a method for producing a genetically modified mouse, comprising replacing one or more immunoglobulin heavy chain gene segments upstream (with respect to transcription of the immunoglobulin heavy chain gene segments) of a endogenous ADAM6 locus of mice with one or more human immunoglobulin heavy chain gene segments, and replace one or more immunoglobulin gene segments downstream (with respect to transcription of the immunoglobulin heavy chain gene segments) of ADAM6 locus of mice by one or more human immunoglobulin light chain or heavy chain gene segments. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments upstream of an endogenous ADAM6 locus of mice include V gene segments. In one embodiment, the gene segments human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments upstream of an endogenous ADAM6 locus of the mice 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 a mouse endogenous ADAM6 locus include J gene segments. In one embodiment, the one or more human immunoglobulin gene segments that replaces one or more endogenous gene segments of immunoglobulin downstream of an endogenous ADAM6 locus of mice includes both D and J gene segments. In one embodiment, the one or more immunoglobulin gene segments human line that replaces one or more endogenous gene segments of
[000145] [000145] In one embodiment, the one or more immunoglobulin heavy chain gene segments upstream and/or downstream of the ADAM6 gene are substituted in a pluripotent, induced pluripotent or totipotent cell to form a genetically modified progenitor cell; the genetically modified progenitor cell is introduced into a host; and the host comprising the genetically modified progenitor cell is generated to form a mouse which comprises a genome derived from the genetically modified progenitor cell. In one embodiment, the host is an embryo. In a specific embodiment, the host is selected from a mouse premorula (eg, 8- or 4-cell stage), a tetraploid embryo, an embryonic cell aggregate, or a blastocyst.
[000146] [000146] In one aspect, there is provided a method for producing a genetically modified mouse, comprising substituting a mouse nucleotide sequence that comprises a mouse immunoglobulin gene segment and a mouse ADAM6 nucleotide sequence (or ortholog, or homolog, or fragment thereof functional in a male mouse) by a sequence comprising a human immunoglobulin gene segment to form a first chimeric locus, and then inserting a sequence comprising a mouse ADAM6-encoding sequence (or a sequence encoding mouse ADAM6 an ortholog or homolog or functional fragment thereof) in the sequence comprising the human immunoglobulin gene segment to form a second chimeric locus.
[000147] [000147] In one embodiment, the second chimeric locus comprises a human immunoglobulin (VH) heavy chain variable gene segment. In one embodiment, the second chimeric locus comprises
[000148] [000148] In one aspect, there is provided the use of a mouse comprising an ectopic nucleotide sequence comprising a mouse ADAM6 locus or sequence to produce a male fertile mouse, wherein the use comprises mating the mouse comprising the ectopic sequence of nucleotide, comprising the mouse ADAM6 locus or sequence, with a mouse that loses a functional mouse endogenous ADAM6 locus or sequence, and obtain progeny that is a female capable of producing progeny with the ectopic ADAM6 locus or sequence , or that it is a male that comprises the ectopic ADAM6 locus or sequence, and the male exhibits a fertility that is approximately the same as a fertility exhibited by a male wild-type mouse.
[000149] [000149] In one aspect, the use of a mouse in the manner described herein to produce an immunoglobulin variable region nucleotide sequence is provided.
[000150] [000150] In one aspect, there is provided the use of a mouse in the manner described herein to produce a fully human Fab or a fully human F(ab)2 fully human.
[000151] [000151] In one aspect, the use of a mouse in the manner described herein to produce an immortalized cell line is provided.
[000152] [000152] In one aspect, the use of a mouse in the manner described herein to produce a hybridoma or quadroma is provided.
[000153] [000153] In one aspect, there is provided the use of a mouse in the manner described herein to produce a phage library containing human heavy chain variable regions and human light chain variable regions.
[000154] [000154] In one aspect, there is provided the use of a mouse in the manner described herein to generate a variable region sequence for producing a human antibody, comprising (a) immunizing a mouse in the manner described herein with an antigen of interest, ( b) isolating a lymphocyte from the immunized mouse from (a), (c) exposing the lymphocyte to one or more labeled antibodies, (d) identifying a lymphocyte that is capable of binding to the antigen of interest, and (e) amplifying one or more further nucleic acid variable region sequences from the lymphocyte, thereby generating a variable region sequence.
[000155] [000155] In one embodiment, the lymphocyte is derived from the spleen of mice. In one embodiment, the lymphocyte is derived from a mouse lymph node. In one embodiment, the lymphocyte is derived from the bone marrow of mice.
[000156] [000156] In one embodiment, the labeled antibody is a fluorophore-conjugated antibody. In one embodiment, the one or more fluorophore-conjugated antibodies are selected from an IgM, an IgG, and/or a combination thereof.
[000157] [000157] In one embodiment, the lymphocyte is a B cell.
[000158] [000158] In one embodiment, the one or more nucleic acid variable region sequences comprises a heavy chain variable region sequence. In one embodiment, the one or more nucleic acid variable region sequences comprise a light chain variable region sequence. In a specific embodiment, the variable region sequence of
[000159] [000159] In one embodiment, there is provided the use of a mouse in the manner described herein to generate a κ heavy and light chain variable region sequence to produce a human antibody, comprising (a) immunizing a mouse in the manner described herein with an antigen of interest, (b) isolating the spleen of the immunized mouse from (a), (c) exposing spleen B lymphocytes to one or more labeled antibodies, (d) identifying a B lymphocyte from (c) that is capable of bind to the antigen of interest, and (e) amplify a nucleic acid heavy chain variable region sequence and a B lymphocyte nucleic acid κ light chain variable region sequence, thereby generating the chain variable region sequences heavy and light chain κ.
[000160] [000160] In one embodiment, there is provided the use of a mouse in the manner described herein to generate a κ heavy and light chain variable region sequence to produce a human antibody, comprising (a) immunizing a mouse in the manner described herein with an antigen of interest, (b) isolating one or more lymph nodes from the immunized mouse from (a), (c) exposing B lymphocytes from the one or more lymph nodes to one or more labeled antibodies, (d) identifying a B lymphocyte from (c) that is capable of binding to the antigen of interest, and (e) amplifying a nucleic acid heavy chain variable region sequence and a B lymphocyte nucleic acid κ light chain variable region sequence, thereby generating the sequences heavy chain and κ light chain variable region.
[000161] [000161] In one embodiment, there is provided the use of a mouse in the manner described herein to generate a κ heavy and light chain variable region sequence to produce a human antibody, comprising (a)
[000162] [000162] In various embodiments, there is provided the use of a mouse in the manner described herein to generate a κ light and heavy chain variable region sequence to produce a human antibody, further comprising fusing the amplified light chain variable region sequences and heavy to human light and heavy chain constant region sequences, expressing the fused light and heavy chain sequences in a cell, and recovering the expressed light and heavy chain sequences, thereby generating a human antibody.
[000163] [000163] In various embodiments, the human heavy chain constant regions are selected from IgM, IgD, IgA, IgE and IgG. In various specific embodiments, the IgG is selected from an IgG1, an IgG2, an IgG3 and an IgG4. In various embodiments, the human heavy chain constant region comprises a CH1, a hinge, a CH2, a CH3, a CH4, or a combination thereof. In various embodiments, the light chain constant region is an immunoglobulin κ constant region. In various embodiments, the cell is selected from a HeLa cell, a DU145 cell, an Lncap cell, an MCF-7 cell, an MDA-MB-438 cell, a PC3 cell, a T47D cell, a THP-1 cell, a U87, a SHSY5Y cell (human neuroblastoma), a Saos-2 cell, a Vera cell,
[000164] [000164] In one aspect, there is provided a method for generating a specific rodent-human reverse-chimeric antibody against an antigen of interest, comprising the steps of immunizing a mouse in the manner described herein with the antigen, isolating at least one cell from the mouse that produces a mouse-human reverse chimeric antibody specific against the antigen, culture at least one cell that produces the mouse-human reverse chimeric antibody specific against the antigen, and obtain said antibody.
[000165] [000165] In one embodiment, the mouse-human reverse-chimeric antibody comprises a human heavy chain variable domain fused to a mouse or rat heavy chain constant gene, and a human light chain variable domain fused to a mouse, rat, or human light chain constant gene.
[000166] [000166] In one embodiment, the cultivation of at least one cell that produces the specific rodent-human reverse-chimeric antibody against the antigen is performed on at least one hybridoma cell generated from the at least one cell isolated from the mice.
[000167] [000167] In one aspect, there is provided a method for generating a fully human antibody specific to an antigen of interest, comprising the steps of immunizing a mouse in the manner described herein with the antigen, isolating at least one cell from the mouse that produces an antibody antigen-specific rodent-human reverse-chimeric antibody, generating at least one cell that produces a fully human antibody derived from the antigen-specific rodent-human reverse-chimeric antibody, and cultivating at least one cell that produces the fully human antibody , and obtain said antibody
[000168] [000168] In various embodiments, the at least one cell isolated from the mouse that produces a specific rodent-human reverse-chimeric antibody against the antigen is a splenocyte or a B cell.
[000169] [000169] In various embodiments, the antibody is a monoclonal antibody.
[000170] [000170] In various embodiments, immunization with the antigen of interest is performed with protein, DNA, a combination of DNA and protein, or cells expressing the antigen.
[000171] [000171] In one aspect, there is provided the use of a mouse in the manner described herein to produce a nucleic acid sequence encoding an immunoglobulin variable region or fragment thereof. In one embodiment, the nucleic acid sequence is used to produce a human antibody or antigen-binding fragment thereof. In one embodiment, the mouse is used to produce an antigen-binding protein selected from an antibody, a multispecific antibody (e.g., a bispecific antibody), a Fvsc, a bispecific Fvsc, a diabody, a triabody, a tetrabody, a V-NAR, a VHH, a VL, an F(ab), an F(ab)2, a DVD (i.e. dual variable domain antigen binding protein), an SVD (i.e. single variable domain antigen), or a bispecific T cell (BiTE).
[000172] [000172] In one aspect, the use of a mouse in the manner described herein to introduce an ectopic ADAM6 sequence into a mouse that loses a functional mouse endogenous ADAM6 sequence is provided, wherein the use comprises mating a mouse in the manner herein described with the mouse that loses the functional mouse endogenous ADAM6 sequence.
[000173] [000173] In one aspect, the use of genetic material from a mouse in the manner described herein to produce a mouse with
[000174] [000174] In one aspect, there is provided a method of producing a fertile male mouse comprising a modified immunoglobulin heavy chain locus, comprising fertilizing a first mouse germ cell which comprises a modification of an endogenous immunoglobulin heavy chain locus with a second mouse germ cell comprising an ADAM6 gene or ortholog, or homolog, or fragment thereof, which is functional in a male mouse; form a fertilized cell; allowing the fertilized cell to develop into an embryo; and, generating the embryo into a surrogate to obtain a mouse.
[000175] [000175] In one embodiment, fertilization is achieved by mating a male mouse and a female mouse. In one embodiment, the female mouse comprises the ADAM6 gene or ortholog, or homolog, or fragment thereof. In one embodiment, the male mouse comprises the ADAM6 gene or ortholog, or homolog, or fragment thereof.
[000176] [000176] In one aspect, there is provided the use of a nucleic acid sequence encoding a mouse ADAM6 protein or an ortholog or homolog thereof, or a functional fragment of the corresponding ADAM6 protein to restore or enhance the fertility of a mouse with a genome comprising a modification of a chain locus
[000177] [000177] In one embodiment, the nucleic acid sequence is integrated into the mouse genome at an ectopic position. In one embodiment, the nucleic acid sequence is integrated into the mouse genome at an endogenous immunoglobulin locus. In a specific embodiment, the endogenous immunoglobulin locus is a heavy chain locus. In one embodiment, the nucleic acid sequence is integrated into the mouse genome at a position that is not an endogenous immunoglobulin locus.
[000178] [000178] In one aspect, the use of mice in the manner described herein is provided for the manufacture of a drug (e.g., an antigen-binding protein), or for the manufacture of a sequence encoding a variable sequence of a drug. (e.g., an antigen-binding protein), for the treatment of a human disease or disorder.
[000179] [000179] In one aspect, a genetically modified mouse cell is provided, wherein the cell is incapable of expressing a heavy chain comprising endogenous rearranged immunoglobulin heavy chain gene segments, and the cell comprises a functional gene of ADAM6 encoding a mouse ADAM6 protein or functional fragment thereof. In one embodiment, the cell further comprises an insert of human immunoglobulin gene segments. In a specific embodiment, the human immunoglobulin gene segments are heavy chain gene segments that are operably linked to mouse heavy chain constant regions such that the rearrangement encodes a functional heavy chain of an antibody that comprises a human variable region.
[000180] [000180] Non-human animals are provided genetically
[000181] [000181] Chimeric and human antigen binding proteins (e.g. antibodies), and nucleic acids encoding them, which comprise somatically mutated variable regions, including antibodies that display light chains comprising a variable domain derived from a human Vλ gene segment and a human Jλ fused to a mouse light chain constant domain.
[000182] [000182] In one aspect, a mouse is provided that expresses a human λ variable region sequence in a light chain that
[000183] [000183] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a human light chain λ variable unrearranged gene segment (hVλ) and a human λ splice gene segment (hJλ). In one embodiment, the unrearranged hVλ and hJλ are at a mouse light chain locus. In one embodiment, the non-rearranged hVλ and non-rearranged hJλ are in a transgene and operably linked to a human or mouse constant region sequence. In one embodiment, unrearranged hVλ and unrearranged hJλ are in an episome. In one embodiment, the mouse is capable of producing an immunoglobulin that comprises a light chain that is derived from an unrearranged hVλ sequence and an hJλ sequence, and a mouse light chain constant region (CL) nucleic acid sequence. Methods and compositions for producing and using genetically modified mice are also provided. Antibodies are provided that comprise (a) a human heavy chain variable domain (hVH) fused to a mouse heavy chain constant region, and (b) a human Vα domain fused to a mouse CL; including what one or more of the variable domains are
[000184] [000184] In one aspect, a mouse is provided which comprises in its germ line, at an endogenous mouse light chain locus, a light chain human λ variable region sequence, wherein the human lambda variable region sequence is expressed in a light chain comprising a mouse immunoglobulin constant region sequence gene.
[000185] [000185] In one embodiment, the endogenous mouse light chain locus is a λ locus. In one embodiment, the endogenous mouse light chain locus is a κ locus.
[000186] [000186] In one embodiment, the mouse loses an endogenous light chain variable sequence at the mouse endogenous light chain locus.
[000187] [000187] In one embodiment, all or substantially all of the endogenous mouse light chain variable region gene segments are replaced by one or more human λ variable region gene segments.
[000188] [000188] In one embodiment, the light chain human λ variable region sequence comprises a human Jλ sequence. In one embodiment, the human sequence Jλ is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and a combination thereof.
[000189] [000189] In one embodiment, the human light chain λ variable region sequence comprises a cluster A fragment of the human light chain locus. In a specific embodiment, the fragment
[000190] [000190] In one embodiment, the human light chain λ variable region sequence comprises a fragment of cluster B from the human light chain locus. In a specific embodiment, the human light chain λ locus cluster B fragment spans from hVλ5-52 to hVλ1-40.
[000191] [000191] In one embodiment, the light chain human λ variable region sequence comprises a cluster A genomic fragment and a cluster B genomic fragment. In one embodiment, the human light chain λ variable region sequence comprises at least one cluster A gene segment and at least one cluster B gene segment.
[000192] [000192] In one embodiment, more than 10% of the mouse naive light chain repertoire is derived from at least two hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9- 49. In one embodiment, more than 20% of the mouse's naive light chain repertoire is derived from at least three hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49. In one embodiment, more than 30% of the mouse's naive light chain repertoire is derived from at least four hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49.
[000193] [000193] In one aspect, there is provided a mouse that expresses an immunoglobulin light chain comprising a human λ variable sequence fused to a mouse constant region, wherein the mouse exhibits a κ use ratio for λ use. about 1:1.
[000194] [000194] In one embodiment, the immunoglobulin light chain is expressed from an endogenous mouse light chain locus.
[000195] [000195] In one aspect, a mouse is provided that comprises a light chain variable region λ (Vλ) sequence and at least one
[000196] [000196] In one embodiment, the mouse loses a functional mouse Vκ and/or mouse Jκ gene segment.
[000197] [000197] In one embodiment, the Vλ is a human Vλ (hVλ), and the J is a human Jλ (hJλ). In one embodiment, the hVλ and hJλ are unrearranged gene segments.
[000198] [000198] In one embodiment, the mouse comprises a plurality of unrearranged hVλ gene segments and at least one hJλ gene segment. In a specific embodiment, the plurality of unrearranged hVλ gene segments is at least 12 gene segments, at least 28 gene segments, or at least 40 gene segments.
[000199] [000199] In one embodiment, the at least one hJλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and a combination thereof.
[000200] [000200] In one embodiment, an endogenous mouse λ light chain locus is eliminated in whole or in part.
[000201] [000201] In one embodiment, the mouse κ light chain constant region sequence is at an endogenous mouse κ light chain locus.
[000202] [000202] In one embodiment, about 10% to about 45% of mouse B cells express an antibody that comprises a light chain comprising a human λ light chain variable domain (Vλ) and a κ light chain constant domain mouse (Cκ).
[000203] [000203] In one embodiment, the human λ variable domain is derived from a rearranged hVλ/hJλ sequence selected from the group consisting of 3-1/1, 3-1/7, 4-3/1, 4-3/ 7, 2-8/1, 3-9/1, 3-10/1, 3-10/3, 3-10/7, 2-14/1, 3-19/1, 2-23/1, 3-25/1, 1-40/1, 1-40/2, 1-40/3, 1-40/7, 7-43/1, 7-43/3, 1-
[000204] [000204] In one embodiment, the mouse further comprises a human Vκ-Jκ intergenic region from a human light chain κ locus, wherein the human Vκ-Jκ intergenic region is contiguous with the Vλ sequence and the J. In a specific embodiment, the human Vκ-Jκ intergenic region is placed between the Vλ sequence and the J sequence.
[000205] [000205] In one aspect, a mouse is provided that comprises (a) at least 12 to at least 40 unrearranged human light chain λ variable region gene segments and at least one human Jλ gene segment at a locus endogenous mouse light chain; (b) a human Vκ-Jκ intergenic sequence located between the at least 12 to at least 40 human light chain variable region gene segments and the at least one human Jλ sequence; wherein the mouse expresses an antibody comprising a light chain comprising a human Vλ domain and a mouse Cκ domain.
[000206] [000206] In one aspect, there is provided a mouse that expresses an antibody comprising a light chain comprising a variable sequence λ and a constant sequence κ.
[000207] [000207] In one embodiment, the mouse exhibits a ratio of κ usage to λ usage of about 1:1.
[000208] [000208] In one embodiment, a population of immature B cells obtained from the bone marrow of mice exhibits a ratio of κ use to λ use of about 1:1.
[000209] [000209] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a Vλ gene segment and a non-rearranged immunoglobulin Jλ operably linked to a locus of
[000210] [000210] In one embodiment, the Vλ and/or Jλ gene segments are human gene segments. In one embodiment, the Vλ and/or Jλ gene segments are mouse gene segments, and the CL is a mouse Cκ.
[000211] [000211] In one embodiment, the endogenous mouse light chain locus is a light chain κ locus. In one embodiment, the endogenous mouse light chain locus is a light chain λ locus.
[000212] [000212] In one embodiment, the unrearranged Vλ and Jλ gene segments are at an endogenous mouse light chain locus.
[000213] [000213] In one embodiment, the non-rearranged immunoglobulin Vλ and Jλ gene segments are in a transgene.
[000214] [000214] In one embodiment, the mouse further comprises a replacement of one or more heavy chain V, D and/or J gene segments with one or more human V, D and/or J gene segments at an endogenous locus of mouse immunoglobulin heavy chain.
[000215] [000215] In one embodiment, the mouse comprises a Vλ gene segment and an immunoglobulin non-rearranged Jλ at an endogenous mouse κ light chain locus, which comprises a mouse Cκ gene.
[000216] [000216] In one embodiment, the mouse comprises a non-rearranged human immunoglobulin λ light chain variable gene (Vλ) segment, and a λ-binding gene segment (Jλ) at an endogenous mouse λ light chain locus which comprises a mouse Cλ gene.
[000217] [000217] In one embodiment, the light chain variable gene locus (the "VL locus") comprises at least one human Vλ (hVλ) gene segment. In one embodiment, the VL locus comprises at least one human Jλ (hJλ) gene segment. In another embodiment, the VL locus
[000218] [000218] In one embodiment, the κ light chain variable gene locus (the "κ locus") comprises at least one human Vλ (hVλ) gene segment. In one embodiment, the κ locus comprises at least one human Jλ (hJλ) gene segment. In one embodiment, the κ locus comprises up to four hJλ gene segments. In one embodiment, the κ locus comprises at least one hVλ and at least one hJλ, and loses or substantially loses a functional Vκ gene segment region and loses or substantially loses a functional Jκ gene segment region. In one embodiment, the mouse does not comprise any functional Vκ gene segment regions. In one embodiment, the mouse does not comprise any functional Jκ gene segment regions.
[000219] [000219] In one embodiment, the λ light chain variable gene locus (the "λ locus") comprises at least one hVλ gene segment. In one embodiment, the λ locus comprises at least one human Jλ (hJλ) gene segment. In another embodiment, the λ locus comprises up to four hJλ gene segments.
[000220] [000220] In one embodiment, the VL locus comprises a plurality of hVλs. In one embodiment, the plurality of hVλs is selected so as to result in expression of a λ light chain variable region repertoire, which reflects about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of the observed Vλ usage in a human. In one embodiment, the VL locus comprises hVλ gene segments 1-40, 1-44, 2-8, 2-14, 3-21, and a combination thereof.
[000221] [000221] In one embodiment, the hVλs include 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, and 3-12. In a specific embodiment, the VL locus comprises a
[000222] [000222] In one embodiment, the VL locus comprises 13 to 28 or more hVλs. In a specific embodiment, the hVλs include 2-14, 3-16, 2-18, 3-19, 3-21, 3-22, 2-23, 3-25, and 3-27. In a specific embodiment, the κ locus comprises a contiguous sequence of the human λ locus that spans from Vλ3-27 to Vλ3-1. In one embodiment, the VL locus is at the endogenous κ locus. In a specific embodiment, the VL locus is at the endogenous κ locus and the endogenous light chain λ locus is eliminated in part or completely. In another embodiment, the VL locus is at the endogenous λ locus. In a specific embodiment, the VL locus is at the endogenous λ locus and the endogenous κ locus is eliminated in part or completely.
[000223] [000223] In one embodiment, the VL locus comprises 29 to 40 hVλs. In a specific embodiment, the κ locus comprises a contiguous sequence from the human λ locus that spans from Vλ3-29 to Vλ3-1, and a contiguous sequence from the human λ locus that spans from Vλ5-52 to Vλ1-40. In a specific embodiment, all or substantially all of the sequences between hVλ1-40 and hVλ3-29 in the genetically modified mouse essentially consist of an approximately 959 bp human λ sequence found in nature (e.g., in the
[000224] [000224] In one embodiment, the VL locus comprises at least one hJλ. In one embodiment, the VL locus comprises a plurality of hJλs. In one embodiment, the VL locus comprises at least 2, 3, 4, 5, 6, or 7 hJλ. In a specific embodiment, the VL locus comprises four hJλ. In a specific embodiment, the four hJλs are hJλ1, hJλ2, hJλ3, and hJλ7. In one embodiment, the VL locus is a κ locus. In a specific embodiment, the VL locus is at the endogenous κ locus and the endogenous light chain λ locus is eliminated in part or completely. In one embodiment, the VL locus comprises an hJλ. In a specific embodiment, the one hJλ is hJλ1. In one embodiment, the VL locus is at the endogenous κ locus. In a specific embodiment, the VL locus is at the endogenous κ locus and the endogenous light chain λ locus is eliminated in part or completely. In another embodiment, the VL locus is at the endogenous λ locus. In a specific embodiment, the VL locus is at the endogenous λ locus and the endogenous κ locus is eliminated in part or completely.
[000225] [000225] In one embodiment, the VL locus comprises at least one hVλ, at least one hJλ, and a mouse Cκ gene. In one embodiment, the VL locus comprises at least one hVλ, at least one hJλ,
[000226] [000226] In one embodiment, the mouse comprises a substitution at the mouse endogenous κ locus of endogenous mouse Vκ gene segments with one or more hVλ gene segments, wherein the hVλ gene segments are operably linked to an endogenous gene of the mouse. mouse Cκ region, such that the mouse rearranges the human Vλ gene segments and expresses an immunoglobulin reverse chimeric light chain comprising a human Vλ domain and a mouse Cκ. In one embodiment, 90-100% of unrearranged mouse Vκ gene segments are replaced by at least one unrearranged hVλ gene segment. In a specific embodiment, all or substantially all of the endogenous mouse Vκ gene segments are replaced by at least one unrearranged hVλ gene segment. In one embodiment, the replacement is for at least 12, at least 28, or at least 40 unrearranged hVλ gene segments. In one embodiment, the replacement is for at least 7 non-rearranged functional hVλ gene segments, at least 16 non-rearranged functional hVλ gene segments, or at least 27 non-rearranged functional hVλ gene segments. In one embodiment, the mouse comprises a replacement of all mouse Jκ gene segments with at least one unrearranged hJλ gene segment. In one embodiment, the at least one unrearranged hJλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ4, Jλ5, Jλ6, Jλ7, and a combination thereof. In a specific embodiment, the one or more hVλ gene segment is selected from an hVλ gene segment 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, 3-12,
[000227] [000227] In one embodiment, the mouse comprises a replacement of mouse endogenous Vλ gene segments at the mouse endogenous λ locus with one or more human Vλ gene segments at the mouse endogenous λ locus, wherein the hVλ gene segments are operably linked to a mouse Cλ region gene such that the mouse rearranges the hVλ gene segments and expresses a reverse immunoglobulin chimeric light chain comprising an hVλ domain and a mouse Cλ. In a specific embodiment, the mouse Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to mouse Cλ2. In one embodiment, 90-100% of unrearranged mouse Vλ gene segments are replaced by at least one unrearranged hVλ gene segment. In a specific embodiment, all or substantially all of the mouse endogenous Vλ gene segments are replaced by at least one unrearranged hVλ gene segment. In one embodiment, the replacement is for at least 12, at least 28, or at least 40 unrearranged hVλ gene segments. In one embodiment, the replacement is for at least 7 non-rearranged functional hVλ gene segments, at least 16 non-rearranged functional hVλ gene segments, or at least 27 non-rearranged functional hVλ gene segments. In one embodiment, the mouse comprises a replacement of all mouse Jλ gene segments with at least one unrearranged hJλ gene segment. In one embodiment, the at least one unrearranged hJλ gene segment is selected from Jλ1,
[000228] [000228] In one aspect, a genetically modified mouse is provided that comprises a human Vκ-Jκ intergenic region sequence located at an endogenous mouse κ light chain locus.
[000229] [000229] In one embodiment, the human Vκ-Jκ intergenic region sequence is at an endogenous mouse κ light chain locus comprising a hVλ and hJλ gene segment, and the human Vκ-Jκ intergenic region sequence is arranged between the hVλ and hJλ gene segments. In a specific embodiment, the hVλ and hJλ gene segments are capable of recombining to form a functional human λ light chain variable domain in the mouse.
[000230] [000230] In one embodiment, a mouse is provided that comprises a plurality of hVλ and one or more hJλ, and the human Vκ-Jκ intergenic region sequence is arranged, with respect to transcription, downstream of the proximal hVλ sequence or 3 ' plus, and upstream or 5' of the first sequence hJλ.
[000231] [000231] In one embodiment, the human Vκ-Jκ intergenic region is a region located about 130 bp downstream or 3' of a human Vκ4-1 gene segment, about 130 bp downstream of the 3' non translated from the human Vκ4-1 gene segment, and spaced about 600 bp upstream or 5' of a human Jκ1 gene segment. In a specific embodiment, the human Vκ-Jκ intergenic region is about 22.8 kb in size. In one embodiment, the Vκ-Jκ intergenic region is about 90% or
[000232] [000232] In one aspect, a non-human animal, a non-human cell (e.g., an ES cell or a pluripotent cell), a non-human embryo, or a non-human tissue comprising the intergenic region sequence Vκ- cited human Jκ, wherein the intergenic region sequence is ectopic. In a specific embodiment, the ectopic sequence is placed at an endogenous non-human humanized immunoglobulin locus. In one embodiment, the non-human animal is selected from a mouse, a rat, a hamster, a goat, a cow, a sheep and a non-human primate.
[000233] [000233] In one aspect, there is provided an isolated nucleic acid construct comprising the recited human Vκ-Jκ intergenic region sequence. In one embodiment, the nucleic acid construct comprises targeting arms to target the human Vκ-Jκ intergenic region sequence at a mouse light chain locus. In a specific embodiment, the mouse light chain locus is a κ locus. In a specific embodiment, the targeting arms target the human Vκ-Jκ intergenic region at a modified mouse endogenous κ locus, where the target is at a position between an hVλ sequence and an hJλ sequence.
[000234] [000234] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises no more than two alleles of
[000235] [000235] In one embodiment, the endogenous mouse light chain locus is a κ locus. In another embodiment, the endogenous mouse light chain locus is a λ locus.
[000236] [000236] In one embodiment, no more than two light chain alleles are selected from one κ allele and one λ allele, two κ alleles, and two λ alleles. In a specific embodiment, one of the two light chain alleles is a λ allele that comprises a Cλ2 gene.
[000237] [000237] In one embodiment, the mouse comprises a functional immunoglobulin light chain locus and a nonfunctional light chain locus, wherein the functional light chain locus comprises an unrearranged human immunoglobulin Vλ gene segment and a Jλ at an endogenous mouse κ light chain locus, which comprises a mouse Cκ gene.
[000238] [000238] In one embodiment, the mouse comprises a functional immunoglobulin light chain locus and a nonfunctional light chain locus, wherein the functional light chain locus comprises an unrearranged gene segment Vλ and a human immunoglobulin Jλ at an endogenous mouse λ light chain locus, which comprises a mouse Cλ gene. In one embodiment, the Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to mouse Cλ2.
[000239] [000239] In one embodiment, the mouse comprises
[000240] [000240] In one embodiment, the mouse comprises a first light chain allele comprising a non-rearranged hVκ and a non-rearranged hJκ, at an endogenous mouse κ locus comprising an endogenous Cκ gene; and a second light chain allele comprising a non-rearranged hVλ and a non-rearranged hJλ, at an endogenous mouse κ locus comprising an endogenous Cκ gene. In a specific embodiment, the first and second light chain alleles are the only functional light chain alleles of the genetically modified mouse. In a specific embodiment, the mouse comprises a nonfunctional λ locus. In one embodiment, the genetically modified mouse does not express a light chain comprising a λ constant region.
[000241] [000241] In one embodiment, the mouse comprises a first light chain allele comprising a non-rearranged hVκ and a non-rearranged hJκ, at an endogenous mouse κ locus comprising an endogenous Cκ gene; and a second light chain allele comprising a non-rearranged hVλ and a non-rearranged hJλ, at an endogenous mouse λ locus comprising an endogenous Cλ gene. In a specific embodiment, the first and second light chain alleles are the only functional light chain alleles of the genetically modified mouse. In one embodiment, the endogenous Cλ gene is Cλ2. In a specific mode,
[000242] [000242] In one embodiment, the mouse comprises six immunoglobulin alleles, wherein the first allele comprises a segment of unrearranged immunoglobulin Vλ and Jλ gene at an endogenous mouse κ light chain locus, which comprises a mouse Cκ gene , the second comprises an immunoglobulin Vκ and Jκ unrearranged gene segment at an endogenous mouse κ light chain locus comprising a mouse Cκ gene, the third comprises an immunoglobulin Vλ and Jλ unrearranged gene segment at a locus endogenous mouse λ light chain comprising a mouse Cλ gene, the fourth and fifth comprise independently a segment of unrearranged VH and DH and JH gene at an endogenous mouse heavy chain locus comprising a heavy chain gene of mouse, and the sixth comprises both (a) a non-segment of rearranged immunoglobulin Vλ and Jλ gene at an endogenous mouse λ light chain locus comprising a mouse Cλ gene, (b) a λ locus that is nonfunctional, or (c) a complete or partial deletion of the λ locus.
[000243] [000243] In one embodiment, the first allele comprises an unrearranged hVλ and hJλ. In one embodiment, the second allele comprises an unrearranged hVκ and hJκ. In one embodiment, the third allele comprises an unrearranged hVλ and hJλ. In one embodiment, the fourth and fifth each independently comprise an hVH and hDH and unrearranged hJH. In one embodiment, the sixth allele comprises an endogenous mouse λ locus that is eliminated in whole or in part.
[000244] [000244] In one embodiment, the mouse comprises six immunoglobulin alleles, wherein the first allele comprises a segment of
[000245] [000245] In one embodiment, the first allele comprises an unrearranged hVλ and hJλ gene segment. In one embodiment, the second allele comprises an unrearranged hVλ and hJλ gene segment. In one embodiment, the third allele comprises an unrearranged hVκ and hJκ gene segment. In one embodiment, the fourth and fifth each independently comprise a hVH and hDH and unrearranged hJH gene segment. In one embodiment, the sixth allele comprises an endogenous mouse κ locus that is functionally silenced.
[000246] [000246] In one embodiment, the genetically modified mouse comprises a B cell comprising a rearranged antibody gene comprising a rearranged hVλ domain operably linked to a mouse CL domain. In one embodiment, the mouse CL domain is selected from a mouse Cκ domain and a mouse Cλ domain. In a specific embodiment, the mouse Cλ domain
[000247] [000247] In one aspect, a genetically modified mouse is provided that expresses a Vλ region in a CL which is a Cκ. In one aspect, a genetically modified mouse is provided that expresses an hVλ region in a CL selected from a human Cκ, a human Cλ, or a mouse Cκ. In one aspect, a genetically modified mouse is provided that expresses an hVλ region in a mouse Cκ.
[000248] [000248] In one embodiment, about 10-50% of the mouse splenocytes are B cells (i.e., CD19-positive), or that about 9-28% express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.
[000249] [000249] In a specific embodiment, about 23-34% of mouse splenocytes are B cells (i.e., CD19-positive), or wherein about 9-11% express an immunoglobulin light chain comprising a fused hVλ domain to a mouse Cκ domain.
[000250] [000250] In a specific embodiment, about 19-31% of mouse splenocytes are B cells (i.e., CD19-positive), or about 9-17% express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.
[000251] [000251] In a specific embodiment, about 21-38% of mouse splenocytes are B cells (i.e., CD19-positive), or about 24-27% express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.
[000252] [000252] In a specific embodiment, about 10-14% of mouse splenocytes are B cells (i.e. CD19-positive), or about
[000253] [000253] In a specific embodiment, about 31-48% of mouse splenocytes are B cells (i.e., CD19-positive), or about 15-21% express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain. In a specific embodiment, about 30-38% of mouse splenocytes are B cells (i.e., CD19-positive), of which about 33-48% express an immunoglobulin light chain comprising an hVλ domain fused to a Cκ domain. of mouse.
[000254] [000254] In one embodiment, about 52-70% of the mouse bone marrow is B cells (i.e. CD19-positive), or about 31-47% of immature B cells (i.e., CD19-positive/B220 -intermediate positive/IgM-positive) express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.
[000255] [000255] In one embodiment, about 60% of the mouse bone marrow is B cells (i.e., CD19-positive), or about 38.3% of immature B cells (i.e., CD19-positive/B220-intermediate). positive/IgM-positive) express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.
[000256] [000256] In one embodiment, the mouse expresses an antibody comprising a light chain comprising a variable domain derived from a human V and a human J gene segment, and a constant domain derived from a mouse constant region gene. In one embodiment, the mouse constant region gene is a Cκ gene. In another embodiment, the mouse constant region gene is a Cλ gene. In a specific embodiment, the Cλ region is Cλ2. In a specific embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90
[000257] [000257] In one embodiment, the mouse expresses an antibody comprising a light chain comprising a human Vλ-Jλ rearranged sequence and a mouse Cκ sequence. In one embodiment, the human Vλ-Jλ rearranged sequence is derived from a rearrangement of hVλ gene segments selected from a 3-1, 4-3, 2-8, 3-9, 3-10, 2- 14, 3-19, 2-23, 3-25, 1-40, 7-43, 1-44, 5-45, 7-46, 1-47, 9-49, and 1-
[000258] [000258] In one embodiment, the mouse expresses an antibody comprising a light chain comprising an immunoglobulin rearranged λ variable region light chain comprising a human Vλ/Jλ sequence selected from 3-1/1, 3-1/7, 4 -3/1, 4-3/7, 2-8/1, 3-9/1, 3-10/1, 3-10/3, 3-10/7, 2-14/1, 3-19 /1, 2-23/1, 3-25/1, 1-40/1, 1-40/2, 1-40/3, 1-
[000259] [000259] In one aspect, there is provided a mouse that expresses an antibody comprising (a) a heavy chain comprising a heavy chain variable domain derived from a human heavy chain variable region unrearranged gene segment, wherein the domain heavy chain variable is fused to a mouse heavy chain constant region (CH); and, (b) a light chain comprising a light chain variable domain derived from an hVλ and an unrearranged hJλ, wherein the light chain variable domain is fused to a mouse CL region.
[000260] [000260] In one embodiment, the mouse comprises (i) a heavy chain locus comprising a substitution of all or substantially all of the mouse functional endogenous V, D and J gene segments with all or substantially all of the V gene segments, functional human D and J, a mouse CH gene, (ii) a first light chain κ locus comprising a replacement of all or substantially all functional mouse endogenous Vκ and Jκ gene segments pot all, substantially all, or a plurality of functional hVλ and hJλ gene segments, and a mouse C gene, (iii) a second light chain κ locus comprising a replacement of all or substantially all functional endogenous mouse Vκ and Jκ gene segments by all, substantially all, or a plurality of functional hVκ and hJκ gene segments, and a mouse Cκ gene. In one embodiment, the mouse does not express an antibody that comprises
[000261] [000261] In one embodiment, the mouse comprises (i) a heavy chain locus comprising a replacement of all or substantially all functional mouse endogenous V, D and J gene segments with all or substantially all of the V gene segments, functional human D and J, a mouse CH gene, (ii) a first light chain λ locus comprising a substitution of all or substantially all functional mouse endogenous Vλ gene segments and Jλ by all, substantially all, or a plurality of functional hVλ and hJλ gene segments, and a mouse Cλ gene, (iii) a second light chain λ locus comprising a substitution of all or substantially all functional mouse endogenous Vλ gene segments and Jλ by all, substantially all , or a plurality of functional hVλ and hJλ gene segments, and a mouse Cλ gene. In a specific embodiment, the mouse Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to mouse Cλ2.
[000262] [000262] In one embodiment, the mouse comprises a deletion of a Cκ gene, and/or a Vκ gene segment and/or a Jκ. In one embodiment, the mouse comprises a non-functional light chain κ locus.
[000263] [000263] In one aspect, a genetically modified mouse that expresses an antibody is provided, wherein greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, more than 40%,
[000264] [000264] In one embodiment, the variable domain derived from λ is derived from an hVλ and an hJλ. In one embodiment, the λ-derived variable domain is on a light chain comprising a mouse Cκ region. In a specific embodiment, the λ-derived variable region is in a light chain comprising a mouse Cλ region. In another specific embodiment, the Cλ region is a Cλ2 region. In one embodiment, the κ-derived variable domain is derived from an hVκ and an hJκ, and in a specific embodiment it is on a light chain comprising a mouse Cκ region.
[000265] [000265] In one aspect, an isolated DNA construct is provided comprising an upstream homology arm and a downstream homology arm, wherein the upstream and downstream homology arm target the construct at a mouse κ locus, and the construct comprises a functional non-rearranged segment hVλ and a functional segment non-rearranged hJλ, and a selection or marker sequence.
[000266] [000266] In one aspect, an isolated DNA construct is provided, comprising, 5' to 3' to the direction of transcription, a targeting arm for targeting a mouse λ sequence upstream of mouse Vλ2, a cassette flanked 5' and 3' with recombinase recognition sites, and a targeting arm to target a mouse λ sequence 3' of mouse Jλ2. In one embodiment, the selection cassette is a Frt'ed Hyg-TK cassette. In one mode, the
[000267] [000267] In one aspect, an isolated DNA construct is provided, comprising, 5' to 3' to the direction of transcription, a targeting arm for targeting the mouse λ locus 5' to Vλ1, a cassette flanked 5' and 3' with recombinase recognition sites, and a 3' targeting arm to target a mouse λ sequence 3' to mouse Cλ1. In one embodiment, the selection cassette is a neomycin cassette. In one embodiment, the 3' targeting arm comprises mouse λ 3' enhancer and mouse λ 3'3.1 enhancer.
[000268] [000268] In one aspect, an isolated DNA construct is provided, comprising 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse λ locus 5' with respect to Vλ2, a cassette of 5' and 3' flanked selection with recombinase recognition sites, and a 3' targeting arm to target a mouse λ sequence 3' to mouse Jλ2 and 5' to mouse Cλ2. In one embodiment, the selection cassette is a Frt'ed Hygromycin-TK cassette. In one embodiment, the 3' targeting arm comprises the mouse Cλ2 -Jλ4-Cλ4 and mouse λ 2,4 enhancer gene segments.
[000269] [000269] In one aspect, an isolated DNA construct is provided, comprising, 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse λ locus 5' with respect to Vλ2, a cassette flanked 5' and 3' with recombinase recognition sites, a human genomic fragment comprising a contiguous region of the human light chain λ locus of hVλ3-12 downstream at the end of hJλ1, and a 3' targeting arm to target a mouse λ sequence 3' to mouse Jλ2. In a
[000270] [000270] In one aspect, an isolated DNA construct is provided, comprising a contiguous region of the human light chain λ locus of hVλ3-12 downstream at the end of hJλ1.
[000271] [000271] In one aspect, an isolated DNA construct is provided, comprising from 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse λ 5' locus with respect to Vλ2, a cassette flanked 5' and 3' with recombinase recognition sites, and a human genomic fragment comprising a contiguous region of the human light chain λ locus of hVλ3-27 downstream at the end of hVλ2-8. In one embodiment, the selection cassette is a Frt'ed Hygromycin cassette. In one embodiment, the human genomic fragment comprises a 3' targeting arm. In a specific embodiment, the 3' targeting arm comprises about 53 kb of the human light chain λ locus of hVλ3-12 downstream at the end of hVλ2-8.
[000272] [000272] In one aspect, an isolated DNA construct is provided, comprising a contiguous region of the human light chain λ locus of hVλ3-27 downstream at the end of hVλ3-12.
[000273] [000273] In one aspect, an isolated DNA construct is provided, comprising, 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse λ locus 5' with respect to Vλ2, a cassette 5' and 3' flanked with recombinase recognition sites, a first human genomic fragment comprising a contiguous region of the human light chain λ locus of hVλ5-52 downstream at the end of hVλ1-40, an enzyme site restriction, and a second human genomic fragment comprising a contiguous region of the human light chain λ locus of hVλ3-29 downstream in the
[000274] [000274] In one aspect, an isolated DNA construct is provided, comprising a contiguous region of the human light chain λ locus of hVλ5-52 downstream at the end of hVλ1-40.
[000275] [000275] In one aspect, an isolated DNA construct is provided, comprising, 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse κ locus 5' with respect to Vκ gene segments endogenous, two juxtaposed recombinase recognition sites, a 3' selection cassette in the juxtaposed recombinase recognition sites, and a 3' targeting arm to target a mouse 5' κ sequence to variable light chain gene segments κ. In one embodiment, the juxtaposed recombinase recognition sites are in opposite orientation with respect to each other. In a specific embodiment, the recombinase recognition sites are different. In another specific embodiment, the recombinase recognition sites are a loxP site and a lox511 site. In one embodiment, the selection cassette is a neomycin cassette.
[000276] [000276] In one aspect, an isolated DNA construct is provided, comprising, 5' to 3' to the direction of transcription, a targeting arm for targeting the mouse κ locus 5' to the Jκ gene segments mouse, a selection cassette, a 3' recombinase recognition site in the selection cassette, and a
[000277] [000277] In one aspect, an isolated DNA construct is provided, comprising, 5' to 3' with respect to the direction of transcription, a first mouse genomic fragment comprising 5' sequence of the mouse endogenous Vκ gene segments, a first recombinase recognition site, a second recombinase recognition site, and a second mouse genomic fragment comprising sequence 3' of the mouse endogenous Jκ gene segments and 5' of the mouse κ intronic enhancer.
[000278] [000278] In one aspect, a genetically modified mouse is provided, wherein the genetic modification comprises a modification with one or more of the DNA constructs described above or herein.
[000279] [000279] In one aspect, the use of an isolated DNA construct to produce a mouse in the manner described herein is provided. In one aspect, there is provided the use of a DNA construct isolated in the manner described herein, in a method for producing an antigen-binding protein.
[000280] [000280] In one aspect, there is provided a non-human embryonic cell comprising a targeting vector comprising a DNA construct in the manner described above and herein. In one aspect, a non-human embryonic cell is provided, wherein the non-human embryonic cell is derived from a mouse described herein.
[000281] [000281] In one embodiment, the non-human embryonic cell is an embryonic stem (ES) cell. In a specific embodiment, the cell
[000282] [000282] In one aspect, there is provided the use of a non-human embryonic cell, in the manner described herein, to produce a mouse in the manner described herein. In one aspect, the use of a non-human embryonic cell in the manner described herein to produce an antigen-binding protein is provided.
[000283] [000283] In one aspect, a mouse embryo is provided, wherein the mouse embryo comprises a genetic modification in the manner provided herein. In one embodiment, a mouse embryo host comprising a donor ES cell is provided, wherein the donor ES cell comprises a genetic modification in the manner described herein. In one embodiment, the mouse embryo is a premorula stage embryo. In one specific embodiment, the premorula stage embryo is either a 4-cell stage embryo or an 8-cell stage embryo. In another specific embodiment, the mouse embryo is a blastocyst.
[000284] [000284] In one aspect, there is provided the use of a mouse embryo in the manner described herein, to produce a mouse in the manner described herein. In one aspect, the use of a mouse embryo in the manner described herein to produce an antigen-binding protein is provided.
[000285] [000285] In one aspect, a non-human cell is provided, wherein the non-human cell comprises a rearranged immunoglobulin light chain gene sequence derived from a genetically modified mouse, in the manner described herein. In one embodiment, the cell is a B cell. In one embodiment, the cell is a hybridoma. In one embodiment, the cell encodes an immunoglobulin light chain variable domain and/or an immunoglobulin heavy chain variable domain that are somatic mutated.
[000286] [000286] In one aspect, a non-human cell is provided, wherein the non-human cell comprises a rearranged immunoglobulin light chain gene sequence derived from a genetically modified mouse, in the manner described herein. In one embodiment, the cell is a B cell. In one embodiment, the cell is a hybridoma. In one embodiment, the cell encodes an immunoglobulin light chain variable domain and/or an immunoglobulin heavy chain variable domain, which are somatic mutated.
[000287] [000287] In one aspect, the use of a non-human cell in the manner described herein to produce a non-human animal in the manner described herein is provided. In one aspect, the use of a non-human cell in the manner described herein to produce an antigen-binding protein is provided. In one embodiment, the non-human animal is selected from a mouse, a rat, a hamster, a sheep, a goat, a cow and a non-human primate.
[000288] [000288] In one aspect, there is provided a mouse B cell that expresses an immunoglobulin light chain comprising: (a) a variable region derived from an hVλ gene segment and an hJλ gene segment; and, (b) a mouse CL gene. In one embodiment, the mouse CL gene is selected from a Cκ and a Cλ gene. In a specific embodiment, the Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to mouse Cλ2. In one embodiment, the mouse B cell further expresses a heavy chain cognate comprising (c) a variable region derived from an hVH segment, an hDH, and (d) an hJH. In one embodiment, the B cell does not comprise a rearranged λ gene. In another embodiment, the B cell does not comprise a rearranged κ gene.
[000289] [000289] In one aspect, there is provided a method for producing an antibody in a non-human genetically modified animal, comprising: (a) exposing a non-human genetically modified animal to an antigen, wherein the animal has a genome comprising at least one hVλ and at least one hJλ at an endogenous light chain locus, wherein the endogenous light chain locus comprises a non-human CL gene; (b) allowing the genetically modified animal to develop an immune response to the antigen; and (c) isolating from the animal of (b) an antibody that specifically recognizes the antigen, or isolating from the animal of (b) a cell comprising an immunoglobulin domain that specifically recognizes the antigen, wherein the antibody comprises a light chain derived from an hVλ, an hJλ and an animal CL gene. In a specific embodiment, the non-human CL gene is a mouse Cκ gene. In one embodiment, the non-human animal is selected from a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a cow and a non-human primate.
[000290] [000290] In one embodiment, there is provided a method for producing an antibody in a non-human genetically modified animal, comprising: (a) exposing a genetically modified animal to an antigen, wherein the animal has a genome comprising at least one hVλ in an endogenous κ locus and at least one hJλ at the κ locus, wherein the κ locus comprises a non-human Cκ gene; (b) allowing the genetically modified animal to develop an immune response to the antigen; and, (c) isolating from the animal of (b) an antibody that specifically recognizes the antigen, or isolating from the mouse of (b) a cell comprising an immunoglobulin domain that specifically recognizes the antigen, wherein the antibody comprises a derived light chain of a Cκ hVλ, a hJλ and a non-human gene.
[000291] [000291] In one embodiment, the constant light chain κ gene is
[000292] [000292] In one embodiment, a method is provided for producing an antibody in a genetically modified non-human animal, comprising: (a) exposing a genetically modified non-human animal to an antigen, wherein the animal has a genome comprising at least one hVλ at a light chain λ locus and at least one Jλ at the light chain λ locus, wherein the light chain λ locus comprises a non-human Cλ gene; (b) allowing the genetically modified animal to develop an immune response to the antigen; and, (c) isolating from the animal of (b) an antibody that specifically recognizes the antigen, or isolating from the animal of (b) a cell comprising an immunoglobulin domain that specifically recognizes the antigen, or identifying in the animal from B is a nucleic acid sequence encoding an antigen-binding heavy and/or light chain variable domain, wherein the antibody comprises a light chain derived from an hVλ gene, an hJλ and a non-human Cλ. In one embodiment, the non-human animal is selected from a mouse, a rat, a hamster, a sheep, a goat, a cow and a non-human primate.
[000293] [000293] In one embodiment, the constant light chain λ gene is selected from a human Cλ gene and a non-human Cλ gene. In one embodiment, the constant light chain λ gene is a human Cλ gene. In a specific embodiment, the human Cλ gene is selected from Cλ1, Cλ2, Cλ3 and Cλ7. In one embodiment, the constant light chain λ gene is a mouse or rat Cλ gene. In a specific embodiment, the mouse Cλ gene is selected from Cλ1, Cλ2 and Cλ3. In a more specific embodiment, the mouse Cλ gene is Cλ2. In another specific embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to mouse Cλ2.
[000294] [000294] In one aspect, there is provided a method for producing a rearranged antibody gene in a genetically modified non-human animal, comprising: (a) exposing a genetically modified non-human animal to an antigen, wherein the genetic modification comprises an hVλ and an hJλ at an endogenous light chain locus, wherein the endogenous light chain locus comprises a non-human CL gene or functional fragment thereof; and, (b) identifying a rearranged immunoglobulin gene in said non-human animal, wherein the rearranged immunoglobulin gene comprises a light chain λ variable region gene segment, and a CL gene or functional fragment thereof.
[000295] [000295] In one embodiment, the method further comprises cloning a nucleic acid sequence encoding a heavy and/or light chain variable region of the animal, wherein the heavy and/or light chain variable region is from an antibody comprising a human Vλ and a mouse CL.
[000296] [000296] In one embodiment, the mouse CL gene or functional fragment thereof is selected from a human CL gene and a mouse CL gene, or functional fragment thereof.
[000297] [000297] In one embodiment, a method is provided for producing a rearranged antibody gene in a genetically modified non-human animal, comprising: (a) exposing a genetically modified non-human animal to an antigen, wherein the genetic modification comprises an hVλ and an hJλ at a light chain κ locus, wherein the light chain κ locus comprises a non-human Cκ gene or functional fragment thereof; and, (b) identifying a rearranged immunoglobulin gene in said animal, wherein the rearranged immunoglobulin gene comprises a λ light chain variable region gene segment and a Cκ gene or functional fragment thereof.
[000298] [000298] In one embodiment, the constant light chain κ gene or functional fragment thereof is selected from a human Cκ gene and a
[000299] [000299] In one embodiment, the method further comprises cloning a nucleic acid sequence encoding a heavy and/or light chain variable region of the animal, wherein the heavy and/or light chain variable region is from an antibody comprising a human Vλ and a non-human Cκ (eg mouse or rat).
[000300] [000300] In one embodiment, a method for producing a rearranged antibody gene in a genetically modified non-human animal is provided, comprising: (a) exposing a genetically modified non-human animal to an antigen, wherein the genetic modification comprises a hVλ and an hJλ at a non-human light chain λ locus, wherein the light chain λ locus comprises a non-human Cλ gene or a functional fragment thereof; and, (b) identifying a rearranged immunoglobulin gene in said animal, wherein the rearranged immunoglobulin gene comprises a λ light chain variable region gene segment and a Cλ gene or functional fragment thereof.
[000301] [000301] In one embodiment, the constant light chain λ gene or functional fragment thereof is selected from a human Cλ gene and a mouse or rat Cλ gene, or a functional fragment thereof. In a specific embodiment, the constant light chain λ gene is a mouse or rat Cλ gene, or a functional fragment thereof.
[000302] [000302] In one embodiment, the method further comprises cloning a nucleic acid sequence encoding a heavy and/or light chain variable region of the animal, wherein the heavy and/or light chain variable region is from an antibody comprising a human Vλ and a non-human Cλ (eg mouse or rat).
[000303] [000303] In one aspect, there is provided a method of producing an antibody, comprising exposing a non-human animal in the manner herein
[000304] [000304] In one embodiment, a method for producing an antibody is provided, comprising exposing a non-human animal in the manner described herein to an antigen, allowing the animal to mount an immune response comprising producing an antibody that specifically binds to the antigen, identify a rearranged nucleic acid sequence in the mouse that encodes a heavy chain and a rearranged nucleic acid sequence in the animal that encodes a cognate sequence of an antibody light chain variable domain, in which the antibody specifically binds to the antigen, and employ the heavy and light chain variable domain nucleic acid sequences fused to human nucleic acid constant domain sequences to produce a desired antibody, wherein the desired antibody comprises a light chain comprising a Vλ domain fused to a Cκ domain.
[000305] [000305] In one embodiment, a method is provided for producing an antibody, comprising exposing a non-human animal in the manner herein
[000306] [000306] In one embodiment, the Cλ region is mouse, and in one embodiment it is selected from Cλ1, Cλ2 and Cλ3. In a specific embodiment, the mouse Cλ region is Cλ2.
[000307] [000307] In one aspect, there is provided a method for producing an antibody light chain variable region rearranged gene sequence, comprising (a) exposing a non-human animal in the manner described herein to an antigen; (b) allowing the animal to mount an immune response; (c) identifying a cell in the animal that comprises a nucleic acid sequence encoding a rearranged human Vλ domain sequence fused to a non-human CL domain, wherein the cell also encodes a cognate heavy chain comprising a human VH domain, and a non-human CH domain, and wherein the cell expresses an antibody that binds to the antigen; (d) cloning from the cell a nucleic acid sequence encoding the human Vλ domain and a nucleic acid sequence encoding the cognate human VH domain; and, (e) use the cloned nucleic acid sequence encoding the human Vλ domain and the cloned sequence
[000308] [000308] In one embodiment, a method is provided for producing an antibody light chain variable region rearranged gene sequence, comprising (a) exposing a non-human animal in the manner described in that disclosure to an antigen; (b) allowing the animal to mount an immune response; (c) identifying a cell in the animal that comprises a nucleic acid sequence encoding a contiguous human Vλ-domain rearranged sequence on the same nucleic acid molecule with a nucleic acid sequence encoding a non-human animal Cκ domain, in that the cell also encodes a cognate heavy chain comprising a human VH domain and a non-human animal CH domain, and wherein the cell expresses an antibody that binds the antigen; (d) cloning from the cell a nucleic acid sequence encoding the human Vλ domain and a nucleic acid sequence encoding the cognate human VH domain; and, (e) using the cloned nucleic acid sequence encoding the human Vλ domain and the cloned nucleic acid sequence encoding the cognate human VH domain to produce a fully human antibody.
[000309] [000309] In one embodiment, a method is provided for producing an antibody light chain variable region rearranged gene sequence, comprising (a) exposing a non-human animal in the manner described herein to an antigen; (b) allowing the animal to mount an immune response to the antigen; (c) identifying a cell in the animal that comprises DNA encoding a rearranged human Vλ domain sequence fused to a non-human Cλ domain of the animal, wherein the cell also encodes a cognate heavy chain comprising a human VH domain and a
[000310] [000310] In one aspect, a genetically modified non-human animal is provided that expresses a human λ-derived light chain fused to an endogenous light chain (CL) constant region, wherein the animal, upon immunization with antigen, produces a antibody comprising a human Vλ domain fused to a non-human CL domain of the animal. In one embodiment, the non-human CL domain is selected from a Cκ domain and a Cλ domain. In one embodiment, the CL domain is a Cκ domain. In one embodiment, the animal is a mouse. In one embodiment, the mouse CL domain is a Cλ domain. In a specific embodiment, the Cλ domain is Cλ2. In a specific embodiment, the mouse Cλ domain is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to mouse Cλ2.
[000311] [000311] In one aspect, there is provided a non-human genetically modified animal comprising an endogenously modified κ or λ light chain locus, in the manner described herein, which expresses a plurality
[000312] [000312] In one aspect, there is provided a non-human animal suitable for producing antibodies having a human-derived λ light chain, wherein all or substantially all of the antibodies produced in the non-human animal are expressed with a human-derived λ light chain . In one embodiment, the human-derived λ light chain is expressed from an endogenous light chain locus. In one embodiment, the endogenous light chain locus is a light chain κ locus. In a specific embodiment, the animal is a mouse and the light chain κ locus is a mouse light chain κ locus.
[000313] [000313] In one aspect, a method is provided for producing a
[000314] [000314] In one aspect, there is provided a method for producing an antigen-binding protein, comprising exposing a non-human animal in the manner described herein to an antigen; allowing the non-human animal to mount an immune response; and obtaining from the non-human animal an antigen-binding protein that binds the antigen, or obtaining from the non-human animal a sequence to be used in the production of an antigen-binding protein that binds the antigen.
[000315] [000315] In one aspect, a cell derived from a non-human animal (eg, a mouse or rat) is provided in the manner described herein. In one embodiment, the cell is selected from an embryonic stem cell, a pluripotent cell, an induced pluripotent cell, a B cell, and a hybridoma.
[000316] [000316] In one aspect, there is provided a cell comprising a genetic modification in the manner described herein. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is selected from a hybridoma and a quadroma. In one embodiment, the cell expresses an immunoglobulin light chain comprising a human λ variable sequence fused to a mouse constant sequence. In a specific embodiment, the mouse constant sequence is a mouse constant κ sequence.
[000317] [000317] In one aspect, tissue derived from a non-human animal is provided in the manner described herein.
[000318] [000318] In one aspect, there is provided the use of a non-human animal or a cell in the manner described herein to produce a binding protein
[000319] [000319] In one aspect, there is provided an antigen-binding protein produced by a non-human animal, cell, tissue, or method in the manner described herein. In one embodiment, the antigen-binding protein is a human protein. In one embodiment, the human protein is a human antibody.
[000320] [000320] Any of the modalities and aspects described herein may be used together with each other unless otherwise indicated or evident from the context. Other embodiments will become apparent to those skilled in the art from a review of the following description. BRIEF DESCRIPTION OF THE FIGURES
[000321] [000321] FIG. 1A shows a general illustration, not used for classification, of direct genomic replacement of about three megabases (Mb) of a mouse immunoglobulin heavy chain variable gene locus (closed symbols) for about one megabase (Mb) of the locus of mouse immunoglobulin heavy chain variable gene (open symbols).
[000322] [000322] FIG. 1B shows a general illustration, not used for classification, of direct genomic replacement of about three megabases (Mb) of a mouse immunoglobulin κ light chain variable gene locus (closed symbols) by about 0.5 megabases (Mb ) of the first, or near, two nearly identical repeats of the human immunoglobulin κ light chain variable gene locus (open symbols).
[000323] [000323] FIG. 2A shows a detailed illustration, not used for classification, of three initial steps (A-C) for the direct genomic replacement of a mouse immunoglobulin heavy chain variable gene locus, which results in elimination of all VH gene segments,
[000324] [000324] FIG. 2B shows a detailed illustration, not used for classification, of six additional steps (DI) for the direct genomic replacement of a mouse immunoglobulin heavy chain variable gene locus, which results in the insertion of 77 additional human VH gene segments and removing a final selection cassette. A targeting vector for inserting additional human VH gene segments (18hVH BACvec) into the initial insertion of human D and heavy chain gene segments (3hVH-CRE Hybrid Allele) is shown with a 20 kb mouse homology arm 5 ', a selection cassette (open rectangle), a 196 kb human genomic fragment, and a 62 kb human homology arm that overlaps with the 5' end of the initial insertion of human D gene and heavy chain segments , which is shown with a site-specific recombination site (open triangle) located 5' of the human gene segments. Human (open symbols) and mouse (closed symbols) immunoglobulin gene segments and additional selection cassettes (open rectangle) inserted by subsequent targeting vectors are shown.
[000325] [000325] FIG. 2C shows a detailed illustration, not used for classification, of three initial steps (AC) for the direct genomic replacement of a mouse immunoglobulin κ light chain variable gene locus, which results in elimination of all Vκ gene segments and mouse Jκ (Igκ-CRE Hybrid Allele). Selection cassettes (open rectangle) and site-specific recombination sites (open triangle) inserted from the targeting vectors are shown.
[000326] [000326] FIG. 2D shows a detailed illustration, not used for classification, of five additional steps (DH) for the direct genomic replacement of a mouse immunoglobulin κ light chain variable gene locus, which results in the insertion of all Vκ gene segments and Human Jκ in the proximal repeat and elimination of the final selection cassette (40hVκdHyg Hybrid Allele). Human (open symbols) and mouse (closed symbols) immunoglobulin gene segments, and additional selection cassettes (open rectangle), inserted by subsequent targeting vectors are shown.
[000327] [000327] FIG. 3A shows a general illustration, not used for sorting, of a selection strategy including quantitative PCR (qPCR) primer/probe oligonucleotide sites to detect insertion of human heavy chain gene sequences and loss of gene sequences of mouse heavy chain in targeted embryonic stem (ES) cells. The selection strategy in ES cells and mice for a first human heavy chain gene insertion is shown with qPCR primer/probe oligonucleotide sets for the deleted region ("loss" of probes C and D), the inserted region ( “hIgH” probes G and H) and flanking regions (“retention” of probes A, B, E and F) on an unmodified mouse chromosome (top) and a correctly targeted chromosome (bottom).
[000328] [000328] FIG. 3B shows a representative calculation of the number of
[000329] [000329] FIG. 3C shows a representative calculation of copy numbers for four mice of each genotype, calculated using D and H probes only. Wild-type mice: WT mice; Mice heterozygous for a first insertion of human immunoglobulin gene segments: HET mice; Mice homozygous for a first insertion of human immunoglobulin gene segments: Homo mice.
[000330] [000330] FIG. 4A shows a detailed illustration, not used for classification, of the three steps employed to construct a 3hVH BACvec by bacterial homologous recombination (BHR). Human (open symbols) and mouse (closed symbols) immunoglobulin gene segments, selection cassettes (open rectangle) and site-specific recombination sites (open triangle) inserted from targeting vectors are shown.
[000331] [000331] FIG. 4B shows pulsed field gel electrophoresis (PFGE) of three BAC clones (B1, B2 and B3) after NotI digestion. Markers M1, M2 and M3 are low variance, medium variance markers
[000332] [000332] FIG. 5A shows a schematic illustration, not used for classification, of sequential modifications of the immunoglobulin heavy chain locus of mice with increased amounts of human immunoglobulin heavy chain gene segments. Homozygous mice were produced from each of three different stages of heavy chain humanization. Open symbols indicate human sequence; closed symbols indicate mouse sequence.
[000333] [000333] FIG. 5B shows a schematic illustration, not used for classification, of sequential modifications of the immunoglobulin light chain κ locus of mice with increased amounts of human immunoglobulin κ light chain gene segments. Homozygous mice were produced from each of three different stages of κ light chain humanization. Open symbols indicate human sequence; closed symbols indicate mouse sequence.
[000334] [000334] FIG. 6 shows FACS dot plots of B cell populations in humanized wild-type and VELOCIMMUNE® mice. Spleen cells (top row, third row from top and bottom row) or inguinal lymph node (second row from top) from wild type (wt) mice, VELOCIMMUNE® 1 (V1), VELOCIMMUNE® 2 (V2) or VELOCIMMUNE® 3 (V3) were stained for surface IgM expressing B cells (top row and second row from top), surface immunoglobulin containing both κ and λ light chains (third row from top), or IgM from surface of specific haplotypes (bottom row), and populations separated by FACS.
[000335] [000335] FIG. 7A shows representative CDR3 heavy chain sequences from randomly selected VELOCIMMUNE® antibodies around the VH-DH-JH junction (CDR3), demonstrating junctional diversity and
[000336] [000336] FIG. 7B shows representative CDR3 sequences of light chain from VELOCIMMUNE® antibodies randomly selected around the Vκ-Jκ junction (CDR3), demonstrating junctional diversity and nucleotide additions. The Vκ gene segments for each light chain CDR3 sequence are noted in parentheses at the 5' end of each sequence (e.g., 1-6 is human Vκ1-6). The Jκ gene segments for each light chain CDR3 are noted in parentheses at the 3' end of each sequence (e.g., 1 is human Jκ1). SEQ ID NOs for each sequence shown are as follows, preceded from top to bottom: SEQ ID NO:40; SEQ ID NO:41; SEQ ID NO:42; SEQ ID NO:43; SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58.
[000337] [000337] FIG. 8 shows somatic hypermutation frequencies of heavy and light chains of selected VELOCIMMUNE® antibodies (after
[000338] [000338] FIG. 9A shows serum immunoglobulin levels for IgM and IgG isotypes in wild type (open bars) or VELOCIMMUNE® (closed bars) mice.
[000339] [000339] FIG. 9B shows serum immunoglobulin levels for IgA isotype in wild type (open bars) or VELOCIMMUNE® (closed bars) mice.
[000340] [000340] FIG. 9C shows serum immunoglobulin levels for IgE isotype in wild type (open bars) or VELOCIMMUNE® (closed bars) mice.
[000341] [000341] FIG. 10A shows IgG antigen-specific titers against interleukin-6 receptor (IL-6R) from the serum of seven VELOCIMMUNE® (VI) and five wild-type (WT) mice after two (bleeding 1) or three (bleeding 2) cycles of immunization with IL-6R ectodomain.
[000342] [000342] FIG. 10B shows isotype-specific IgG titers of IL-6R from seven VELOCIMMUNE® (VI) and five wild-type (WT) mice.
[000343] [000343] FIG. 11A shows affinity distribution of anti-interleukin-6 receptor monoclonal antibody generated in VELOCIMMUNE® mice.
[000344] [000344] FIG. 11B shows antigen-specific blocking of anti-interleukin-6 receptor monoclonal antibodies generated in VELOCIMMUNE® (VI) and wild type (WT) mice.
[000345] [000345] FIG. 12 shows a schematic illustration, not used for
[000346] [000346] FIG. 13 shows a schematic illustration, not used for classification, of a human ADAM6 pseudogene (hADAM6Ψ) located between human heavy chain 1-2 (VH1-2) and 6-1 (VH6-1) variable gene segments. A targeting vector for bacterial homologous recombination (hADAM6Ψ Targeting Vector) to eliminate a human ADAM6 pseudogene, and insert unique restriction sites at a human heavy chain locus, is shown with a selection cassette (NEO: neomycin) flanked by site-specific recombination (loxP) sites, including genetically modified restriction sites at the 5' and 3' ends. An illustration, not used for classification, of the resulting targeted heavy chain locus, containing a genomic fragment encoding the mouse ADAM6a ADAM6b genes and including a selection cassette flanked by site-specific recombination sites, is shown.
[000347] [000347] FIG. 14A shows FACS contour plots of singlet-spliced lymphocytes for surface expression of IgM and B220 in the bone marrow of mice homozygous for human heavy chain and κ light chain variable gene loci (H+/+κ+/+), and mice homozygous for human heavy chain and light chain κ variable gene loci with an inserted mouse genomic fragment comprising the mouse ADAM6 gene (H+/+A6resκ+/+). The percentage of immature (B220intIgM+) and mature (B220highIgM+) B cells is noted on each graph of
[000348] [000348] FIG. 14B shows the total number of immature (B220intIgM+) and mature (B220highIgM+) B cells in bone marrow isolated from femurs of mice homozygous for human heavy chain and light chain κ (H+/+κ+/+) variable gene loci, and mice homozygous for human heavy chain and light chain κ variable gene loci with a mouse ectopic genomic fragment encoding the mouse ADAM6 gene (H+/+A6resκ+/+).
[000349] [000349] FIG. 15A shows FACS contour plots of CD19+ bound B cells for surface expression of c-kit and CD43 in bone marrow in mice homozygous for human heavy chain and κ light chain variable gene loci (H+/+κ+/ +) and mice homozygous for human heavy chain and light chain κ variable gene loci with a mouse ectopic genomic fragment encoding the mouse ADAM6 gene (H+/+A6resκ+/+). The percentage of pro-B (CD19+CD43+ckit+) and pre-B (CD19+CD43-ckit-) cells is noted in the upper right and lower left quadrants, respectively, of each contour plot.
[000350] [000350] FIG. 15B shows the total number of pro-B cells (CD19+CD43+ckit+) and pre-B cells (CD19+CD43-ckit-) in bone marrow, isolated from femurs of mice homozygous for heavy chain and heavy chain variable gene loci. human κ light chain (H+/+κ+/+), and mice homozygous for human heavy chain and light chain κ variable gene loci with a mouse ectopic genomic fragment comprising the mouse ADAM6 gene (H+/+A6resκ+ /+).
[000351] [000351] FIG. 16A shows FACS contour plots of singlet-linked lymphocytes for surface expression of CD19 and CD43 in bone marrow in mice homozygous for human heavy chain and κ light chain variable gene loci (H+/+κ+/+), and mice
[000352] [000352] FIG. 16B shows histograms of immature (CD19+CD43-) and pre-B (CD19+CD43int) B cells in the bone marrow of mice homozygous for human heavy chain and κ light chain variable gene loci (H+/+κ+/+ ), and mice homozygous for human heavy chain and light chain κ variable gene loci with a mouse ectopic genomic fragment encoding the mouse ADAM6 gene (H+/+A6resκ+/+).
[000353] [000353] FIG. 17A shows FACS contour plots of singlet-linked lymphocytes for surface expression of CD19 and CD3 on splenocytes in mice homozygous for human heavy chain and κ light chain variable gene loci (H+/+κ+/+), and mice homozygous for human heavy chain and light chain κ variable gene loci with a mouse ectopic genomic fragment encoding the mouse ADAM6 gene (H+/+A6resκ+/+). The percentage of B (CD19+CD3-) and T (CD19-CD3+) cells is noted on each contour plot.
[000354] [000354] FIG. 17B shows FAC contour plots for CD19+ bound B cells for surface expression of Igλ and Igκ light chain in the spleen of mice homozygous for human heavy chain and κ light chain variable gene loci (H+/+κ+/ +), and mice homozygous for human heavy chain and light chain κ variable gene loci with a mouse ectopic genomic fragment encoding the mouse ADAM6 gene (H+/+A6resκ+/+). The percentage of B cells with Igλ+ (upper left quadrant) and with Igκ+ (lower right quadrant) is noted in each contour plot.
[000355] [000355] FIG. 17C shows the total number of CD19+ B cells in the spleen of mice homozygous for human heavy chain and κ light chain variable gene loci (H+/+κ+/+), and mice homozygous for heavy chain variable gene loci and human κ light chain with an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+/+A6resκ+/+).
[000356] [000356] FIG. 18A shows contour plots of B cell FACs bound to CD19+ for surface expression of IgD and IgM in the spleen of mice homozygous for human heavy chain and κ light chain variable gene loci (H+/+κ+/+), and mice homozygous for human heavy chain and light chain κ variable gene loci with a mouse ectopic genomic fragment encoding the mouse ADAM6 gene (H+/+A6resκ+/+). The percentage of mature B cells (CD19+IgDhighIgMint) is noted for each contour plot. The arrow in the contour plot on the right illustrates the maturation process for B cells in relation to surface expression of IgM and IgD.
[000357] [000357] FIG. 18B shows the total number of B cells in the spleen of mice homozygous for human heavy chain and light chain κ variable gene loci (H+/+κ+/+), and mice homozygous for heavy chain and light chain variable gene loci light human κ with an ectopic mouse genomic fragment encoding the mouse ADAM6 gene (H+/+A6resκ+/+), during the maturation of CD19+IgMhighIgDint to CD19+IgMintIgDhigh.
[000358] [000358] FIG. 19 shows a detailed illustration, not used for classification, of the human light chain λ locus, including the paired clusters of the Vλ gene segment region (A, B, and C) and the Jλ and Cλ (J-C pairs)
[000359] [000359] FIG. 20 shows a general illustration, not used for classification, of a targeting strategy used to inactivate the locus
[000360] [000360] FIG. 21 shows a general illustration, not used for classification, of a targeting strategy used to inactivate the endogenous mouse κ light chain locus.
[000361] [000361] FIG. 22A shows a general illustration, not used for classification of an initial targeting vector to target the mouse endogenous λ light chain locus with human λ light chain sequences, including 12 hVλ gene segments and 1 hJλ gene segment (Vector of 12/1-λ targeting).
[000362] [000362] FIG. 22B shows a general illustration, not used for classification, of four initial targeting vectors to target the endogenous mouse κ light chain locus with human λ light chain sequences, including 12 hVλ gene segments and 1 hJλ gene segment ( 12/1-κ targeting vector), 12 hVλ gene segments and 1, 2, 3 and 7 hJλ gene segments (12/4-κ targeting vector), 12 hVλ gene segments, a Vκ-Jκ genomic sequence and 1 hJλ gene segment (Targeting vector 12(κ)1-κ) and 12 hVλ gene segments, a human Vκ-Jκ genomic sequence and 1, 2, 3 and 7 hJλ gene segments (Vector of targeting 12(κ)4-κ).
[000363] [000363] FIG. 23A shows a general illustration, not used for classification, of a targeting strategy for the progressive insertion of 40 hVλ gene segments and a single hJλ gene segment into the mouse light chain λ locus.
[000364] [000364] FIG. 23B shows a general illustration, not used for classification, of a targeting strategy for the progressive insertion of 40 hVλ gene segments and a single hJλ gene segment into the mouse κ locus.
[000365] [000365] FIG. 24 shows a general illustration, not used for classification, of the genetic and molecular targeting and modification steps
[000366] [000366] FIG. 25A shows a general illustration, not used for classification, of the locus structure for a modified mouse λ light chain locus containing 40 hVλ gene segments, and a single hJλ gene segment operably linked to the endogenous Cλ2 gene.
[000367] [000367] FIG. 25B shows a general illustration, not used for classification, of the locus structure for four independent, modified mouse κ light chain loci containing 40 hVλ gene segments, and either one or four hJλ gene segments with or without a contiguous genomic sequence. Human Vκ-Jκ operably linked to the endogenous Cκ gene.
[000368] [000368] FIG. 26A shows contour plot of CD19+-linked Igλ+ and Igκ+ splenocytes from a wild-type (WT) mouse, a mouse homozygous for 12 hVλ and four hJλ gene segments, including a human Vκ-Jκ genomic sequence (12hVλ- VκJκ-4hJλ), and a mouse homozygous for 40 hVλ gene segment and one hJλ gene segment (40hVλ-1hJλ).
[000369] [000369] FIG. 26B shows the total number of CD19+ B cells in flasks collected from wild-type (WT) mice, homozygous for 12 hVλ and four hJλ gene segments, including a human Vκ-Jκ genomic sequence (12hVλ-VκJκ-4hJλ) and homozygous mice. for 40 hVλ gene segments and one hJλ (40hVλ-1hJλ).
[000370] [000370] FIG. 27A, top panel, shows contour plot of splenocytes bound in singlets and stained for B and T cells (CD19+ and CD3+, respectively), from a wild-type mouse (WT) and a mouse homozygous for 40 hVλ gene segments and Jλ, including
[000371] [000371] FIG. 27B shows the total number of CD19+, CD19+Igκ+ and CD19+Igλ+ B cells in spleens collected from wild-type (WT) mice and mice homozygous for 40 hVλ and four Jλ gene segments, including a Vκ- genomic sequence. Human Jκ (40hVλ- VκJκ-4hJλ).
[000372] [000372] FIG. 27C shows contour plots of CD19+ bound splenocytes and stained for immunoglobulin D (IgD) and immunoglobulin M (IgM) from a wild-type mouse (WT) and a mouse homozygous for 40 hVλ gene segments and four Jλ, including a genomic sequence Human Vκ-Jκ (40hVλ-VκJκ-4hJλ). Mature (72 for WT, 51 for 40hVλ-VκJκ-4hJλ) and transitional (13 for WT, 22 for 40hVλ-VκJκ-4hJλ) B cells are noted in each of the contour plots.
[000373] [000373] FIG. 27D shows the total number of CD19+ B cells, transitional B cells (CD19+IgMhiIgDlo) and mature B cells (CD19+IgMloIgDhi) in spleens collected from wild-type (WT) mice and mice homozygous for 40 hVλ and four Jλ gene segments, including a human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ).
[000374] [000374] FIG. 28A, in the top panel, shows bone marrow contour plots stained for B and T cells (CD19+ and CD3+, respectively) from a wild-type (WT) mouse and a mouse homozygous for 40 hVλ gene segments and four Jλ, including a human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ). The panel at the bottom shows
[000375] [000375] FIG. 28B shows the number of Pro (CD19+CD43+ckit+) and Pre (CD19+CD43-ckit-) B cells in bone marrow, collected from the femurs of wild-type (WT) mice and mice homozygous for 40 hVλ gene segments and four Jλ, including a human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ).
[000376] [000376] FIG. 28C shows contour plots of bone marrow ligated in singlets stained for immunoglobulin M (IgM) and B220 from a wild-type (WT) mouse and a mouse homozygous for 40 hVλ gene segments and four Jλ, including a Vκ-Jκ genomic sequence from human (40hVλ-VκJκ-4hJλ). Immature, mature, and pro/pre B cells are annotated on each of the contour plots.
[000377] [000377] FIG. 28D shows the total number of immature (B220intIgM+) and mature (B220hiIgM+) B cells in bone marrow isolated from the femurs of wild-type (WT) mice and mice homozygous for 40 hVλ gene segments and four Jλ, including a Vκ- genomic sequence. Human Jκ (40hVλ-VκJκ-4hJλ).
[000378] [000378] FIG. 28E shows contour plots of bone marrow bound in immature (B220intIgM+) and mature (B220hiIgM+) B cells stained for Igλ and Igκ expression isolated from the femurs of a wild-type (WT) mouse and a mouse homozygous for 40 hVλ gene segments and four Jλ, including a human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ).
[000379] [000379] FIG. 29 shows a Vλ-Jλ-Cκ junction nucleotide alignment sequence of eighteen independent RT-PCR clones,
[000380] [000380] FIG. 30 shows a Vλ-Jλ-Cκ junction nucleotide alignment sequence of twelve independent RT-PCR clones, amplified from mouse splenocyte RNA carrying human λ light chain gene sequences, including a Vκ- Contiguous human Jκ at an endogenous mouse κ light chain locus. 5-2 = SEQ ID NO:145; 2-5 = SEQ ID NO:146; 1-3 = SEQ ID NO:147; 4B-1 = SEQ ID NO:148; 3B-5 = SEQ ID NO:149; 7A-1 = SEQ ID NO:150; 5-1 = SEQ ID NO:151; 4A-1 = SEQ ID NO:152; 11A-1 = SEQ ID NO:153; 5-7 = SEQ ID NO:154; 5-4 = SEQ ID NO:155; 2-3 = SEQ ID NO:156. Lowercase bases indicate non-germ lineage bases, resulting from either mutation or addition of N during recombination. The consensus amino acids in framework region 4 (FWR4) encoded by the nucleotide sequence of each human Jλ and mouse Cκ are noted at the bottom of the sequence alignment.
[000381] [000381] FIG. 31 shows a Vλ-Jλ-Cλ junction nucleotide alignment sequence of three independent RT-PCR clones,
[000382] [000382] This invention is not limited to the particular methods, and experimental conditions described, as such methods and conditions may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention is defined by the claims.
[000383] [000383] Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have reached in the technology, unless otherwise clearly stated or clearly evidenced in the context in which the term or phrase is used. used. While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, particular methods and materials are now described. All patent applications mentioned are incorporated herein by reference.
[000384] [000384] The phrase "substantially" or "substantially" when used to refer to a number of gene segments (e.g., "substantially all" V gene segments) includes both functional and non-functional gene segments and includes, in various modalities, e.g. 80 % or more, 85 % or more, 90 % or more, 95 % or more 96 % or more, 97 % or more, 98 % or more, or 99 % or more of all segments
[000385] [000385] The term "replacement" includes where a DNA sequence is placed into a genome of a cell in such a way as to replace a sequence in the genome with a heterologous sequence (e.g., a sequence from human in a mouse), at the locus of the genomic sequence. The so-placed DNA sequence may include one or more regulatory sequences that are part of the source DNA used to obtain the so-placed sequence (e.g., promoters, enhancers, 5'- or 3' untranslated regions, recombination signal sequences). appropriate, etc.). For example, in various embodiments, substitution is a substitution of an endogenous sequence for a heterologous sequence that results in no production of a gene product from the DNA sequence so placed (comprising the heterologous sequence), but no expression of the endogenous sequence; the replacement is of an endogenous genomic sequence with a DNA sequence that encodes a protein that has a similar function to a protein encoded by the endogenous genomic sequence (for example, the endogenous genomic sequence encodes an immunoglobulin gene or domain, and the fragment of DNA encodes one or more human immunoglobulin genes or domains). In various embodiments, an endogenous gene or fragment thereof is replaced by a corresponding human gene or fragment thereof. A corresponding human gene or fragment thereof is a human gene or fragment that is an ortholog of, a homolog of, or is substantially identical to, or the same in structure and/or function, as the endogenous gene or fragment thereof that is replaced.
[000386] [000386] The term "contiguous" includes reference to occurrence in the same nucleic acid molecule, for example, two nucleic acid sequences are "contiguous" if they occur in the same nucleic acid molecule, but are
[000387] [000387] The phrase "derived from" when used with respect to a variable region "derived from" a cited gene or gene segment includes the ability to sequence backward to a particular unrearranged gene segment or gene segments that have been rearranged to form a gene that expresses the variable domain (responsible, where applicable, for minor differences and somatic mutations).
[000388] [000388] The phrase "functional" when used with respect to a variable region gene segment or splice gene segment refers to use in an antibody expressed repertoire; for example, in human gene segments Vλ 3-1, 4-3, 2-8, etc. are functional, while Vλ 3-2, 3-4, 2-5, etc. are not functional.
[000389] [000389] A “heavy chain locus” includes a location on a chromosome, for example a mouse chromosome, where in a mouse wild-type variable heavy chain (VH) region, heavy chain diversity (DH), joining heavy chain (JH), and constant heavy chain (CH) DNA sequences are found.
[000390] [000390] A “κ locus” includes a location on a chromosome, for
[000391] [000391] A “λ locus” includes a location on a chromosome, for example a mouse chromosome, where in the λ variable region (Vλ), λ splice (Jλ), and λ constant region (Cλ) DNA sequences ) wild-type mice are found.
[000392] [000392] The term "cell," when used in conjunction with an expression sequence includes any cell that is suitable for expressing a recombinant nucleic acid sequence. Cells include those of prokaryotes and eukaryotes (single cell or multiple cells), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (eg. 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, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (eg, CHO K1, DXB-11 CHO, Veggie-CHO), COS (eg, COS-7), retinal cell, Vero, CV1, kidney (e.g. HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g. BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3 cell, L, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell and a derived cell line of a cell mentioned above. In some embodiments, the cell comprises one or more viral genes, for example a retinal cell that expresses a viral gene (for example, a cell
[000393] [000393] The phrase "complementarity determining region," or the term "CDR," includes an amino acid sequence encoded by a nucleic acid sequence of an immunoglobulin gene of the organism that normally (i.e., in a wild-type animal) appears between two framework regions in a variable region of a light or heavy chain of an immunoglobulin molecule (eg, an antibody or a T cell receptor). A CDR may 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 a T cell. In some cases (e.g. for a CDR3 ), CDRs 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 sequence of nucleic acid, for example, as the result of spacing or linking to sequences (e.g. VDJ recombination to form a CDR3 heavy chain).
[000394] [000394] The phrase "gene segment," or "segment" includes reference to an immunoglobulin V (light or heavy) or D or J (light or heavy) gene segment, which includes sequences not rearranged at the immunoglobulin loci ( for example in humans and mice) that can participate in a rearrangement (mediated by, for example, endogenous recombinases) to form a V/J or V/D/J rearranged sequence. Unless otherwise noted, the V, D, and J segments comprise recombination signal sequences (RSS) that allow for V/J recombination or V/D/J recombination in accordance with the 12/23 rule. Unless otherwise indicated, the segments further comprise sequences with which they are associated in nature or functional equivalents thereof (e.g., for V promoter(s) and leader(s) segments).
[000395] [000395] The term "unrearranged" includes the state of an immunoglobulin locus, in which the V and J gene segments (for heavy chains, D gene segments alike) are maintained separately but are capable of being joined to form a rearranged V(D)J gene comprising a single V,(D),J repertoire of V(D)J.
[000396] [000396] The phrase “micromolar range” shall mean 1-999 micromolar; the phrase “nanomolar range” must mean 1-999 nanomolar; the phrase “picomolar range” should mean 1-999 picomolar.
[000397] [000397] The term "non-human animals" shall include any non-human animals, such as cyclotomes, spiny fish, cartilaginous fish, such as sharks and rays, amphibians, reptiles, mammals, and birds. Suitable non-human animals include mammals. Suitable mammals include non-human primates, goats, sheep, pigs, dogs, cows and rodents. Suitable non-human animals are selected from the rodent family including rat and mouse. In one embodiment, the non-human animals are mice.
[000398] [000398] The mouse as a genetic model was greatly improved by transgenic and inactivation technologies, which made it possible to study the effects of direct over-expression or elimination of specific genes. For all its advantages, the mouse still has genetic obstacles that make it an imperfect model for human disease and an imperfect platform for testing human therapeutics or making them. First, although about 99% of human genes have a mouse homolog (Waterston et al. (2002), early sequencing and comparative analysis of the mouse genome, Nature 420, 520-562), potential therapeutics often fail to cross-react, or inappropriately cross-react with orthologous mice actually destined for human targets. To solve this problem, selected target genes can be “humanized,” that is, the mouse gene can be
[000399] [000399] Human immunoglobulin transgenes exogenously
[000400] [000400] Genetically modified animals that comprise a substitution at the endogenous immunoglobulin heavy chain locus with heterologous (e.g. from another species) immunoglobulin sequences can be produced together with substitutions at endogenous immunoglobulin light chain loci or together with transgenes immunoglobulin light chain transgenes (e.g. chimeric immunoglobulin light chain or fully human mouse transgenes, etc.). The species from which the heterologous immunoglobulin heavy chain sequences are derived can vary widely; as well as immunoglobulin light chain sequences employed in immunoglobulin light chain sequence substitutions or light chain transgenes from
[000401] [000401] Immunoglobulin variable region nucleic acid sequences, for example, V, D, and/or J segments, are in various embodiments obtained from a human or non-human animal. Non-human animals suitable for providing V, D, and/or J segments include, for example, spiny fish, cartilaginous fish, such as sharks and rays, amphibians, reptiles, mammals, birds (e.g., chickens). Non-human animals include, for example, mammals. Mammals include, for example, non-human primates, goats, sheep, pigs, dogs, cattle (e.g. cow, buffalo), deer, camels, ferrets and rodents, and non-human primates (e.g. chimpanzees, orangutans, gorillas, marmosets , rhesus monkeys). Suitable non-human animals are selected from the rodent family including rats, mice, and hamsters. In one embodiment, the non-human animals are mice. As is evident from the context, various non-human animals can be used as sources of gene segments from variable domains or variable regions (eg sharks, rays, mammals, eg camels, rodents such as mice and rats).
[000402] [000402] Depending on the context, non-human animals are also used as sources of constant region sequences to be used in conjunction with variable sequences or segments, e.g. rodent constant sequences can be used in transgenes operably linked to variable sequences human or non-human (e.g. human or non-human primate variable sequences operably linked to constant sequences, e.g. rodent, e.g. mouse or rat or hamster,). Thus, in various embodiments, human V, D, and/or J segments are operably linked to rodent (e.g., mouse or rat or hamster) constant region gene sequences. In some embodiments, the human V, D, and/or J segments (or one or
[000403] [000403] In a specific embodiment, a mouse is provided that comprises a substitution of VH, DH, and JH gene segments at an endogenous immunoglobulin heavy chain locus with one or more human VH, DH, and JH segments, wherein the one or more human VH, DH, and JH segments are operably linked to an endogenous immunoglobulin heavy chain constant gene; wherein the mouse comprises a transgene at a locus other than an endogenous immunoglobulin locus, wherein the transgene comprises an unrearranged or rearranged human VL and human JL segment operably linked to a mouse or rat or human constant region.
[000404] [000404] In a specific embodiment, a mouse is provided which comprises an insertion of one or more human VH, DH and JH gene segments into an endogenous immunoglobulin heavy chain locus. In one embodiment, the insertion is upstream of an endogenous immunoglobulin heavy chain constant gene; in one embodiment, the insertion is downstream of an endogenous variable gene segment (V); in one embodiment, the insertion is downstream of an endogenous diversity gene segment (D); in one embodiment, the insertion is downstream of an endogenous splicing gene segment (J). In various embodiments, the insertion is such that the one or more human VH, DH, and JH gene segments are positioned in operable linkage with one or more endogenous heavy chain constant genes.
[000405] [000405] A method is described for a large in situ genetic replacement of the mouse germline immunoglobulin variable gene loci with the lineage immunoglobulin variable gene loci
[000406] [000406] Humanized VELOCIMMUNE® mice exhibit a fully functional humoral immune system that is essentially indistinguishable from that of wild-type mice. These exhibit normal cell populations at all stages of B cell development. They exhibit normal lymphoid organ morphology. VELOCIMMUNE® mouse antibody sequences exhibit normal V(D)J rearrangement frequencies and normal somatic hypermutation. Antibody populations in these mice reflect isotype distributions that result from normal class switching (eg, normal cis-swap isotype). Immunization of VELOCIMMUNE® mice results in
[000407] [000407] It is the exact replacement of mouse immunoglobulin variable sequences with human immunoglobulin variable sequences that allows the production of VELOCIMMUNE® mice. Yet exact replacement of endogenous mouse immunoglobulin sequences at heavy and light chain loci with equivalent human immunoglobulin sequences, sequentially recombining the very large spaces of human immunoglobulin sequences, can present certain challenges because of the divergent evolution of the loci. of immunoglobulin between mouse and man. For example, intergenic sequences interspersed at immunoglobulin loci are not identical between mice and humans and, in some cases, may be nonfunctionally equivalent. Differences between mice and humans at their immunoglobulin loci may still result in abnormalities in humanized mice, particularly in the humanization or manipulation of certain heavy chain portions of endogenous mouse immunoglobulin loci. Some modifications at the mouse immunoglobulin loci heavy chain are harmful. Harmful modifications can include, for example, loss of the modified mice's ability to mate and produce offspring. In various embodiments, genetic engineering of human immunoglobulin sequences in a mouse genome includes methods that maintain endogenous sequences that, when absent in modified mouse strains, are harmful. Exemplary harmful effects may include inability to propagate modified strains, loss of gene function
[000408] [000408] A large-scale, in situ exact replacement of six megabases of the variable regions of the mouse immunoglobulin heavy and light chain loci (VH-DH-JH and Vκ-Jκ) with the corresponding 1.4 megabase genomic sequences was performed, while allowing for the flanking of intact and functional mouse sequences at the hybrid loci, including all mouse constant chain genes and transcriptional control region loci (FIG. 1A and FIG. 1B). Specifically, the human VH, DH, JH, Vκ and Jκ gene sequences were introduced stepwise until the insertion of 13 chimeric BAC targeting vectors that carry the overlapping fragments of the human germline variable loci into ES cells of mouse, using VELOCIGENE® genetic modification technology (see, for example, US patent 6,586,251 and Valenzuela, DM et al. (2003). High-throughput engineering of the mice genome coupled with high-resolution expression analysis. Nat Biotechnol 21 , 652-659).
[000409] [000409] The humanization of mouse immunoglobulin genes represents the largest genetic modification in the mouse genome to date. While previous efforts with randomly integrated human immunoglobulin transgenes have had some success (discussed earlier), the direct replacement of mouse immunoglobulin genes with their human counterparts greatly increases the efficiency of which fully human antibodies can be efficiently generated, unlike normal mice. Additionally, such mice exhibit a markedly greater diversity of fully human antibodies that can be obtained after immunization with virtually any antigen, compared to mice that
[000410] [000410] Notwithstanding the neighboring wild-type humoral immune function seen in mice with immunoglobulin-substituted loci (i.e., VELOCIMMUNE® mice), there are other challenges encountered employing a direct immunoglobulin replacement that is not found in some approaches that employ integrated transgenes. . Differences in the genetic makeup of immunoglobulin loci between mice and humans led to the discovery of sequences beneficial for the propagation of mice with substituted immunoglobulin gene segments. Specifically, mouse ADAM genes located at the endogenous immunoglobulin heavy chain locus are ideally present in mice with substituted immunoglobulin loci, because of their role in fertility. Genomic location and function of mouse ADAM6
[000411] [000411] Male mice that lack the ability to express any functional ADAM6 protein surprisingly show a defect in the ability of mice to mate and produce offspring. Mice lack the ability to express a functional ADAM6 protein due to a replacement of all or substantially all of the mouse immunoglobulin variable region gene segments with
[000412] [000412] In several respects, male mice that comprise a damaged (ie, non-functional or marginally functional) ADAM6 gene show a reduction or elimination of fertility. Because the ADAM6 gene in mice (and other rodents) is located at the immunoglobulin heavy chain locus, the inventors determined that in order to propagate mice, or create and maintain a strain of mice, which comprise modifications at an endogenous chain locus heavy immunoglobulin, various modified breeding or propagation schemes are employed. The low fertility, or infertility, of male mice homozygous for a substitution of the immunoglobulin heavy chain endogenous variable gene locus makes maintaining such a modification in a mouse strain
[000413] [000413] In one aspect, a method for maintaining a mouse strain, in the manner described herein, is provided. The mouse strain need not comprise an ectopic sequence of ADAM6, and in various embodiments the mouse strain is homozygous or heterozygous for an inactivation (eg, a functional inactivation) of ADAM6.
[000414] [000414] The mouse strain comprises a modification of an endogenous immunoglobulin heavy chain locus that results in a reduction or loss of fertility in a male mouse. In one embodiment, the modification comprises a deletion of a regulatory region and/or a coding region of an ADAM6 gene. In a specific embodiment, the modification comprises a modification of an endogenous ADAM6 gene (regulatory and/or coding region) that reduces or eliminates fertility of a male mouse that comprises the modification; in a specific embodiment, the modification reduces or eliminates fertility in a male mouse that is homozygous for the modification.
[000415] [000415] In one embodiment, the mouse strain is homozygous or heterozygous for an inactivation (eg, a functional inactivation) or a deletion of an ADAM6 gene.
[000416] [000416] In one embodiment, the mouse strain is maintained by isolating from a mouse that is homozygous or heterozygous for the modification a cell, and employing the donor cell in the host embryo, and gestating the host embryo and donor cell in a surrogate mother, and obtaining from the surrogate mother progeny that comprise genetic modification. In one embodiment, the donor cell is an ES cell. In one embodiment, the donor cell is a pluripotent cell, for example, an induced pluripotent cell.
[000417] [000417] In one embodiment, the mouse strain is maintained by isolating from a mouse that is homozygous or heterozygous for the modification a nucleic acid sequence comprising the modification, and introducing the nucleic acid sequence into a host nucleus, and gestating a cell comprising the nucleic acid sequence and the core host in a suitable animal. In one embodiment, the nucleic acid sequence is introduced into an embryo oocyte of the host.
[000418] [000418] In one embodiment, the mouse strain is maintained by isolating from a mouse that is homozygous or heterozygous for the modification a nucleus, and introducing the nucleus into a host cell, and gestating the nucleus and host cell in a suitable animal to obtain a progeny that is homozygous or heterozygous for the modification.
[000419] [000419] In one embodiment, the mouse strain is maintained by employing in vitro fertilization (IVF) of a female mouse (wild type, homozygous for the modification, or heterozygous for the modification) employing a sperm from a male mouse comprising the genetic modification . In one embodiment, the male mouse is heterozygous for the genetic modification. In one embodiment, the male mouse is homozygous for the genetic modification.
[000420] [000420] In one embodiment, the mouse strain is maintained by breeding a male mouse that is heterozygous for the genetic modification with a female mouse to obtain progeny comprising the genetic modification, identifying a male and female progeny comprising the genetic modification, and employing a male who is heterozygous for the genetic modification in breeding with a female who is wild-type, homozygous, or heterozygous for the genetic modification to obtain progeny comprising the genetic modification. In one embodiment, the step of breeding a male heterozygous for the genetic modification with a wild-type female, a female heterozygous for the
[000421] [000421] In one aspect, there is provided a method for maintaining a mouse strain which comprises a replacement of an endogenous immunoglobulin heavy chain variable gene locus with one or more human immunoglobulin heavy chain sequences, comprising crossing the strain of mice in order to generate heterozygous male mice, in which heterozygous male mice are bred to maintain the genetic modification in the strain. In a specific embodiment, the strain is not maintained by any cross between a homozygous male and a wild-type female, or a homozygous or heterozygous female for the genetic modification.
[000422] [000422] The ADAM6 protein is an element of the ADAM family of proteins, where ADAM is an acronym for a Disintegrin E Metalloprotease. The ADAM family of proteins is large and diverse, with diverse functions, including cell adhesion. Some elements of the ADAM family are implicated in spermatogenesis and fertilization. For example, ADAM2 encodes a subunit of the protein fertilin, which is implicated in sperm-egg interactions. ADAM3, or cyritestin, and appears necessary for sperm binding in the zona pellucida. The absence of both ADAM2 and ADAM3 results in infertility. ADAM2, ADAM3, and ADAM6 are postulated to form a complex on the surface of mouse sperm cells.
[000423] [000423] The human ADAM6 gene, generally found between human VH gene segments VH1-2 and VH6-1, appears to be a pseudogene (FIG. 12). In mice, there are two ADAM6 genes, ADAM6a and ADAM6b, which are found in an intergenic region between the mouse VH and DH gene segments, and in the mouse the genes
[000424] [000424] The position of the intergenic sequence in mice encoding ADAM6a and ADAM6b makes the intergenic sequence susceptible to modification by modifying an endogenous heavy chain. When VH gene segments are deleted or replaced, or when DH gene segments are deleted or replaced, there is a high probability that a mouse will experience a severe deficit in fertility. In order to compensate for the deficit, the mouse is modified to include a nucleotide sequence that encodes a protein that will complement the loss of ADAM6 activity due to a modification of the endogenous ADAM6 locus. In various embodiments, the complementation nucleotide sequence is one that encodes a mouse ADAM6a, a mouse ADAM6b, or a homologous or orthologous or functional fragment thereof that rescues the fertility deficit. Alternatively, suitable methods to conserve the endogenous ADAM6 locus can be employed, while rendering the endogenous immunoglobulin heavy chain sequences flanking the mouse ADAM6 locus unable to rearrange to encode a functional endogenous heavy chain variable region. Exemplary alternative methods include manipulating portions of the mouse chromosome larger than the position of the endogenous loci of immunoglobulin heavy chain variable regions in such a way that they are unable to rearrange to encode a heavy chain variable region.
[000425] [000425] The nucleotide sequence that rescues fertility can be placed in any suitable position. It can be placed in an intergenic region, or at any suitable position in the genome (ie, ectopic). In one embodiment, the nucleotide sequence can be introduced into a transgene that randomly integrates into the mouse genome. In one embodiment, the sequence may be maintained episomally, that is, on a separate nucleic acid rather than on a mouse chromosome. Suitable positions include positions that are transcriptionally permissive or active, for example, a ROSA26 locus (Zambrowicz et al., 1997, PNAS USA 94:3789-3794), a BT-5 locus (Michael et al., 1999, Mech. Dev 85:35-47), or an Oct4 locus ( Wallace et al., 2000, Nucleic Acids Res. 28:1455-1464 ). Nucleotide sequences that target transcriptionally active loci are described, for example, in US
[000426] [000426] Alternatively, the fertility-rescuing nucleotide sequence can be coupled with an inducible promoter in order to facilitate optimal expression in the appropriate cells and/or tissues, eg, reproductive tissues. Exemplary inducible promoters include promoters activated by physical (e.g. heat shock promoter) and/or chemical (e.g. IPTG or Tetracycline) means.
[000427] [000427] Additionally, expression of the nucleotide sequence can be linked to other genes in order to achieve expression at specific stages of development or in specific tissues. Such expression can be achieved by placing the nucleotide sequence in operable linkage with the promoter of a gene expressed at a specific stage of
[000428] [000428] Yet another method to achieve robust expression of an inserted nucleotide sequence is to employ a constitutive promoter. Exemplary constitutive promoters include SV40, CMV, UBC, EF1A, PGK and CAGG. In a similar manner, the desired nucleotide sequence is placed in operable linkage with a selected constitutive promoter, which provides high level expression of the protein(s) encoded by the nucleotide sequence.
[000429] [000429] The term “ectopic” shall include a displacement, or a placement in a position not normally found in nature (e.g., placement of a nucleic acid sequence in a position that is not in the same position as the sequence of nucleic acid is found in a wild-type mouse). The term in various modalities is used in the sense of its aim being out of its normal, or proper, position. For example, the phrase "an ectopic nucleotide sequence encoding ..." refers to a nucleotide sequence that appears in a position where it is not normally found in the mouse. For example, in the case of an ectopic nucleotide sequence encoding a mouse ADAM6 protein (or an ortholog or homolog or fragment thereof that provides the same fertility benefit or similar benefit in male mice), the sequence can be placed in a position different in the mouse genome from what is normally found in a wild-type mouse. In such cases, novel mouse sequence junctions will be created.
[000430] [000430] The ectopic position can be anywhere (e.g., as with random insertion of a transgene containing a mouse ADAM6 sequence), or it can be, for example, at a position that approximates (but is not precisely the same) its location in a wild-type mouse (e.g. at a modified endogenous immunoglobulin locus but either upstream or downstream from its natural position, e.g. at a modified immunoglobulin locus but between different gene segments, or at a different position in a mouse VD intergenic sequence). An example of an ectopic placement is the maintenance of the position normally found in wild-type mice at the endogenous immunoglobulin heavy chain locus while making the endogenous heavy chain gene segments able to rearrange to encode a functional heavy chain containing a constant region. endogenous heavy chain. In this example, this can be accomplished by inverting the chromosomal fragment containing the endogenous immunoglobulin heavy chain variable loci, for example using site-specific engineered recombination sites placed
[000431] [000431] Another example of an ectopic substitution is the substitution at a humanized immunoglobulin heavy chain locus. For example, a mouse that comprises a replacement of one or more endogenous VH gene segments with human VH gene segments, where the substitution removes an endogenous ADAM6 sequence, can be genetically modified to have a localized mouse ADAM6 sequence in the sequence containing the human VH gene segments. The resulting modification can generate a (ectopic) mouse ADAM6 sequence in a human gene sequence, and the (ectopic) placement of the mouse ADAM6 sequence in the human gene sequence can approximate the position of the human ADAM6 pseudogene (i.e., between two V segments) or may approximate the position of the mouse ADAM6 sequence (ie, in the VD intergenic region). The resulting sequence junctions created by joining a mouse (ectopic) ADAM6 sequence to or adjacent to a human gene sequence (e.g., an immunoglobulin light chain gene sequence) in the
[000432] [000432] In various embodiments, non-human animals are provided that do not have an ADAM6 or ortholog or homolog thereof, the lack of which renders the non-human animal infertile, or substantially reduces the fertility of the non-human animal. In various embodiments, the lack of ADAM6 or ortholog or homolog thereof is due to a modification of an endogenous immunoglobulin heavy chain locus. A substantial reduction in fertility is, for example, a reduction in fertility (e.g. breeding frequency, pups per litter, litters per year, etc.) of about 50%, 60%, 70%, 80%, 90% , or 95% or more. In various embodiments, the non-human animals are supplemented with a mouse ADAM6 gene or ortholog, or homolog, or a functional fragment thereof that is functional in a male of the non-human animal, wherein the ADAM6 gene or ortholog, or homolog is supplemented, or functional fragment of this rescues the reduction in fertility in whole or in substantial part. A substantial fertility rescue is, for example, a restoration of fertility such that the non-human animal exhibits a fertility that is at least 70%, 80%, or 90% or more compared to an unmodified locus of modification. (i.e., an animal lacking an ADAM6 gene or ortholog or homolog thereof) heavy chain.
[000433] [000433] The sequence conferred by the genetically modified animal (that is, the animal that loses a functional ADAM6 or ortholog or homolog thereof, due to, for example, a modification of an immunoglobulin heavy chain locus) is, in various embodiments, selected from an ADAM6 gene or ortholog or homolog thereof. For example, in a mouse, loss of ADAM6 function is rescued by adding, in one embodiment, a mouse ADAM6 gene. In one embodiment, the loss
[000434] [000434] Thus, in various embodiments, genetically modified animals that exhibit no fertility or a reduction in fertility due to modification of a nucleic acid sequence encoding an ADAM6 protein (or ortholog or homolog thereof) or a regulatory region operably linked with the nucleic acid sequence, comprise a nucleic acid sequence that complements, or restores, the loss of fertility where the nucleic acid sequence that complements or restores the loss of fertility is from a different strain of the same species or from a
[000435] [000435] In one embodiment, the genetically modified animal is from the superfamily Dipodoidea, and the ADAM6 ortholog or homolog or functional fragment thereof is from the superfamily Muroidea. In one embodiment, the genetically modified animal is from the superfamily Muroidea, and the orthologous or homologous ADAM6 or functional fragment thereof is from the superfamily Dipodoidea.
[000436] [000436] 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 in the
[000437] [000437] In various embodiments, one or more ADAM6 orthologous or homologous rodents or functional fragments thereof from a rodent in a family restores fertility to a genetically modified rodent of the same family that loses an ADAM6 ortholog or homolog (e.g., Cricetidae (e.g. , hamsters, New World rats and mice, voles); Muridae (e.g. true mice and rats, gerbils, spinner mice, crown rat)).
[000438] [000438] In various embodiments, ADAM6 orthologs, homologs, and fragments thereof are estimated for functionality by verifying whether the ortholog, homolog, or fragment restores fertility to a genetically modified male non-human animal that loses ADAM6 activity (e.g.
[000439] [000439] In various aspects, mice that comprise deletions or substitutions of the endogenous heavy chain variable region locus or portions thereof can be prepared that contain an ectopic nucleotide sequence that encodes a conference protein similar to the fertility benefits of mouse ADAM6 (eg, an ortholog or a homolog or a fragment thereof that is functional in a male mouse). The ectopic nucleotide sequence may include a nucleotide sequence that encodes a protein that is a homolog or ortholog of ADAM6 (or fragment thereof) from a different strain of mouse or from a different species, e.g., a different species of rodent, and that confer a benefit on fertility, e.g., greater number of litters over a specified period of time, and/or greater number of offspring per litter, and/or the ability of a sperm cell from a male mouse to cross through a mouse oviduct to fertilize a mouse egg.
[000440] [000440] In one embodiment, ADAM6 is a homolog or ortholog that is at least 89% to 99% identical to a mouse ADAM6 protein (e.g., at least 89% to 99% identical to mouse ADAM6a or mouse ADAM6b ). In one embodiment, the ectopic nucleotide sequence encodes one or more independently selected proteins of a protein at least 89% identical to mouse ADAM6a, a protein at least 89% identical to mouse ADAM6b, and a combination thereof. In one mode, the
[000441] [000441] In one aspect, non-human animals are provided, wherein the non-human animals comprise (a) an insertion of one or more human Vλ and Jλ gene segments upstream of a non-human immunoglobulin light chain constant region , (b) an insertion of one or more human VH gene segments, one or more DH and one or more human JH upstream of a non-human immunoglobulin heavy chain constant region, and (c) a nucleotide sequence that encodes an ADAM6 protein or a functional fragment thereof. In one embodiment, the heavy chain and/or non-human chain constant regions are rodent constant regions (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 specific embodiment, the light chain constant region is a mouse Cκ or rat Cκ region. In a specific embodiment, the light chain constant region is a mouse Cλ or rat Cκ region. Suitable non-human animals include rodents, for example, mice, rats and hamsters. In one embodiment, the rodent is a mouse or rat.
[000442] [000442] In one embodiment, the non-human animal comprises at least 12 to at least 40 human Vλ gene segments and at least one human Jλ gene segment. In a specific embodiment, the non-human animal comprises 12 human Vλ gene segments and at least one human Jλ gene segment. In a specific mode, the
[000443] [000443] In one embodiment, the nucleotide sequence encoding an ADAM6 protein or functional fragment thereof is ectopic in the non-human animal. In one embodiment, the nucleotide sequence encoding an ADAM6 protein or functional fragment thereof (which is functional in the non-human animal) is present at the same location compared to a wild-type non-human ADAM6 locus. In one embodiment, the non-human animal is a mouse and the nucleotide sequence encodes a mouse ADAM6 protein or functional fragment thereof and is present at an ectopic site in the genome of the non-human animal. In one embodiment the non-human animal is a mouse and the nucleotide sequence encodes a mouse ADAM6 protein or functional fragment thereof and is present in immunoglobulin gene segments. In a specific embodiment, the immunoglobulin gene segments are heavy chain gene segments. In one embodiment, the heavy chain gene segments are human. In one embodiment, the heavy chain gene segments are endogenous heavy chain gene segments from the non-human animal. In one embodiment, the mouse comprises a contiguous ectopic sequence comprising one or more endogenous unrearranged heavy chain gene segments, and the ADAM6 sequence is in the contiguous ectopic sequence.
[000444] [000444] In one embodiment, the non-human animal loses an endogenous VL gene segment and/or an immunoglobulin JL at an endogenous immunoglobulin light chain locus. In one embodiment, the non-human animal comprises endogenous immunoglobulin VL and/or JL gene segments that are unable to rearrange to form an immunoglobulin VL domain in the non-human animal. In one embodiment, all or substantially all of the immunoglobulin endogenous Vκ and Jκ gene segments are replaced by one or more Vλ and Jλ gene segments. In one embodiment, all or substantially all of the endogenous immunoglobulin Vλ and Jλ gene segments are replaced by one or more human Vλ and Jλ gene segments. In one embodiment, all or substantially all of the immunoglobulin endogenous VL and JL gene segments are intact in the non-human animal, and the non-human animal comprises one or more human Vλ gene segments and one or more human Jλ gene segments inserted between the immunoglobulin endogenous VL and/or JL gene segments and an endogenous immunoglobulin light chain constant region. In a specific embodiment, intact endogenous immunoglobulin VL and JL gene segments are rendered unable to rearrange to form a VL domain of an antibody in the non-human animal. In various embodiments, the endogenous immunoglobulin light chain locus of the non-human animal is an immunoglobulin light chain κ locus. In various embodiments, the endogenous immunoglobulin light chain locus of the non-human animal is an immunoglobulin light chain λ locus. In various embodiments, the immunoglobulin endogenous VL and JL gene segments are Vκ and Jκ gene segments. In various embodiments, the immunoglobulin endogenous VL and JL gene segments are Vλ and Jλ gene segments.
[000445] [000445] In one embodiment, the non-human animal additionally comprises a human Vκ-Jκ intergenic region from a κ locus of
[000446] [000446] In one aspect, cells and/or tissues derived from non-human animals in the manner described herein are provided, wherein the cells and/or tissues comprise (a) an insertion of one or more Vκ and Jκ gene segments upstream of a non-human immunoglobulin light chain constant region, (b) an insertion of one or more VH gene segments, one or more DH and one or more human JH upstream of a heavy chain constant region of non-human immunoglobulin, and (c) a nucleotide sequence encoding an ADAM6 protein or a functional fragment thereof. In one embodiment, the non-human heavy and/or light chain constant regions are mouse constant regions. In one embodiment, the non-human heavy and/or light chain constant regions are mouse constant regions. In one embodiment, the non-human heavy and/or light chain constant regions are hamster constant regions.
[000447] [000447] In one embodiment, the nucleotide sequence encoding an ADAM6 protein or functional fragment thereof is ectopic in the cell and/or tissue. In one embodiment, the nucleotide sequence encoding an ADAM6 protein or functional fragment thereof is present at the same location compared to a wild-type non-human ADAM6 locus. In one embodiment the non-human cell and/or tissue is derived from a mouse and the nucleotide sequence encodes a mouse ADAM6 protein or functional fragment thereof and is present at an ectopic site. In one embodiment, the non-human cell and/or tissue is derived from a mouse and the nucleotide sequence encodes a mouse ADAM6 protein or functional fragment thereof and is present in gene segments of
[000448] [000448] In one aspect, there is provided use of a non-human animal, in the manner described herein, to produce an antigen-binding protein, wherein the non-human animal expresses (a) an antibody comprising (i) a light chain immunoglobulin comprising a human Vλ domain and a non-human light chain constant region and (ii) an immunoglobulin heavy chain comprising a human VH domain and a non-human constant region; and (b) an ADAM6 protein or 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 regions are rodent constant regions. In one particular embodiment, the rodent is a mouse.
[000449] [000449] In one aspect, a non-human cell or tissue derived from a non-human animal, in the manner described herein, is provided. In one embodiment, the non-human cell or tissue comprises one or more Vλ gene segments, and at least one human immunoglobulin Jλ gene segments, contiguous with a non-human immunoglobulin light chain constant region gene and one or more human VH gene segments, one or more human DH and one or more human JH contiguous with a non-human immunoglobulin heavy chain constant region gene, wherein the cell or tissue expresses an ADAM6 protein or functional fragment thereof . In one embodiment, the non-human light chain constant region gene is a mouse Cκ or Cλ
[000450] [000450] In one embodiment, the nucleotide sequence encoding the ADAM6 protein or functional fragment thereof is ectopic. In one embodiment, the nucleotide sequence encoding the ADAM6 protein or functional fragment thereof is located at a position that is the same as a wild-type non-human cell. In various embodiments, the non-human cell is a mouse B cell. In various embodiments, the non-human cell is an embryonic stem cell.
[000451] [000451] In one embodiment, the tissue is derived from the spleen, bone marrow, or lymph node of the non-human animal.
[000452] [000452] In one aspect, use of a cell or tissue derived from a non-human animal, in the manner described herein, to produce a hybridoma or quadroma is provided.
[000453] [000453] In one aspect, a non-human cell comprising a modified genome, in the manner described herein, is provided, wherein the non-human cell is an oocyte, a host embryo, or a fusion of a cell from a non-human animal, in the manner described herein, and a cell from a different non-human animal.
[000454] [000454] In one aspect, use of a cell or tissue derived from a non-human animal in the manner described herein to produce a fully human antibody is provided. In one embodiment, the fully human antibody comprises a human VH domain and a human Vλ domain isolated from a non-human animal in the manner described herein.
[000455] [000455] In one aspect, a method for producing an antibody that binds to an antigen of interest is provided, wherein the method comprises (a) exposing a non-human animal in the manner described herein to an antigen of interest, (b) isolating one or more B lymphocytes from the non-human animal, wherein the one or more B lymphocytes express an antibody that binds to the antigen of interest, and (c) identifying a nucleic acid sequence that encodes a
[000456] [000456] In one embodiment, the non-human constant light chain domain is a mouse Cκ. In one embodiment, the non-human light decay constant domain is a mouse Cλ. In one embodiment, the non-human animal is a mouse.
[000457] [000457] In one aspect, a male fertile mouse comprising a modification in an immunoglobulin heavy chain locus is provided, wherein the male fertile mouse comprises an ectopic sequence of ADAM6 that is functional in the male mouse. Ectopic ADAM6 in mice with humanized heavy chain
[000458] [000458] Development in genes that target, for example, the development of bacterial artificial chromosomes (BACs), now capable of recombination of relatively large genomic fragments. Genetically engineered BACs have enabled the ability to produce large deletions and large insertions in mouse ES cells.
[000459] [000459] Mice that make human antibodies (ie, human variable regions) are currently available. Although they represent an important advance in the development of human antibody therapeutics, these mice exhibit numerous significant abnormalities that limit their usefulness. For example, they exhibit compromised B-cell development. The compromised development may be due to a variety of differences between transgenic mice and wild-type mice.
[000460] [000460] Human antibodies should ideally not interact with
[000461] [000461] Mice that make whole human antibodies generally comprise endogenous immunoglobulin loci that are deficient in some way, and human transgenes that comprise immunoglobulin variable and constant gene segments are introduced at a random location in the mouse genome. Provided the endogenous locus is sufficiently deficient that it does not rearrange gene segments to form a functional immunoglobulin gene, the goal of preparing whole human antibodies in a mouse like this can be achieved—albeit with compromised B-cell development.
[000462] [000462] Although forced to produce full human transgene locus antibodies, the generation of human antibodies in a mouse is apparently an unfavorable process. In some mice, the process is so unfavorable that it results in the formation of mouse human chimeric variable/constant heavy chains (but not light chains) up to the trans-exchange mechanism. By this mechanism, transcripts encoding whole human antibodies undergo isotype switching in trans from the human isotype to a mouse isotype. The process is in trans, as the fully human transgene is located far from the endogenous locus that retains an undamaged copy of
[000463] [000463] A major concern in preparing human antibody-based therapeutics is to prepare a sufficiently large diversity of human immunoglobulin variable region sequences to identify useful variable domains that specifically recognize particular epitopes and bind them with desirable affinity, usually-but not always-with high affinity. Prior to the development of VELOCIMMUNE® mice (described herein), there was no indication that mice that express human variable regions with mouse constant regions might show any significant difference from mice that make human antibodies to a transgene. This assumption, however, was incorrect.
[000464] [000464] VELOCIMMUNE® mice, which contain a precise substitution of mouse immunoglobulin variable regions with human immunoglobulin variable regions at the endogenous loci, show a striking and striking similarity to wild-type mice with respect to B-cell development.
[000465] [000465] VELOCIMMUNE® mice contain a precise, large-scale substitution of mouse germline immunoglobulin heavy chain (IgH) and immunoglobulin light chain (e.g. light chain κ, Igκ) variable regions with corresponding variable regions of human immunoglobulin at the endogenous loci. In total, about six megabases of mouse loci are replaced by about 1.5 megabases of human genomic sequence. This precise substitution results in a mouse with hybrid immunoglobulin loci that make heavy and light chains that have human variable regions and a mouse constant region. Precise replacement of mouse VH-DH-JH and Vκ-Jκ segments left intact and functional mouse sequences flanking at the immunoglobulin hybrid loci. The mouse humoral immune system functions like that of a wild-type mouse. B cell development is not impeded with respect to any significance and a rich diversity of human variable regions is generated in the mouse upon antigen challenge.
[000466] [000466] VELOCIMMUNE® mice are possible because immunoglobulin gene segments for κ heavy and light chains similarly rearranged in humans and mice, which is not the same as saying that their loci are the same or nearly the same - clearly they are not. they are. However, the loci are similar enough that humanization of the variable heavy chain gene locus can be performed by replacing about three million base pairs of the contiguous mouse sequence that contains all the VH, DH, and JH gene segments with about of a million
[000467] [000467] In some embodiments, additional substitution of certain mouse constant region gene sequences for human gene sequence (e.g., substitution of mouse CH1 sequence for human CH1 sequence, and substitution of mouse CL sequence for sequence human CL) results in mice with hybrid immunoglobulin loci that make antibodies that have human variable regions and human partially constant regions, suitable for, for example, making fully human antibody fragments, for example, fully human Fab. Mice with hybrid immunoglobulin loci exhibit normal variable gene segment rearrangement, normal somatic hypermutation, and normal class switching. These mice exhibit a humoral immune system that cannot be distinguished from wild-type mice, and have normal cell populations at all stages of B-cell development and normal lymphoid organ structures—even where mice do not have a complete repertoire of segments. gene of human variable regions. Immunization of these mice results in robust humoral responses that show a wide diversity of use of the variable gene segment.
[000468] [000468] Precise replacement of gene segments from variable regions of the mouse germline allows for the production of mice that have partially human immunoglobulin loci. Because the partially human immunoglobulin loci normally rearrange, hypermutate, and switch classes, the partially human immunoglobulin loci generate antibodies in a mouse that comprises human variable regions. Nucleotide sequences encoding the variable regions can be identified and cloned, then
[000469] [000469] Large-scale humanization by recombination methods was used to modify mouse embryonic stem (ES) cells to exactly replace up to three megabases of mouse immunoglobulin heavy chain loci, which included essentially all VH gene segments , DH, and JH of mice per human-equivalent gene segments with up to a human megabase genomic sequence containing some or essentially all of the VH, DH, and JH gene segments. Up to half a megabase of the human genome segment, comprising one of two repeats that encode essentially all human Vκ and Jκ gene segments, was used to replace a three-megabase segment of the immunoglobulin light chain κ locus. mice containing essentially all mouse Vκ and Jκ gene segments.
[000470] [000470] Mice with such substituted immunoglobulin loci may comprise an interruption or deletion of the functional endogenous ADAM6 locus, which is generally found between the 3'-plus VH gene segment and the 5'-plus DH gene segment at the locus of mouse immunoglobulin heavy chain. Disruption in this region can lead to reduced or eliminated functionality of the functional endogenous ADAM6 locus. If the repertoire of human 3'-plus heavy chain VH gene segments is used in a substitution, an intergenic region containing a pseudogene that appears to be a human ADAM6 pseudogene is present between these VH gene segments, that is, between VH1 -2 and human VH1-6. However, male mice that comprise this human intergenic sequence exhibit a reduction in fertility.
[000471] [000471] Mice are described which comprise the loci substituted in the manner described above, and which also comprise an ectopic nucleic acid sequence encoding a mouse ADAM6, where the mice exhibit essentially normal fertility. In one embodiment, the ectopic nucleic acid sequence comprises a mouse ADAM6a and/or a mouse ADAM6b sequence, or functional fragments thereof, placed between a human VH1-2 gene segment and a human VH6-1 gene segment. human at a modified endogenous heavy chain locus. In one embodiment, the ectopic nucleic acid sequence is SEQ ID NO:3, placed between human VH1-2 and VH1-6 at the modified endogenous heavy chain locus. The transcriptional direction of the ADAM6 genes of SEQ ID NO:3 are opposite with respect to the transcriptional direction of the VH gene segments around human. Although the examples here show fertility recovery by placing the ectopic sequence between the indicated human VH gene segments, those skilled in the art will recognize that this substitution of the ectopic sequence at any transcriptionally permissive locus in the mouse genome (or even extrachromosally) would be expected. as similar fertility recovery in a male mouse.
[000472] [000472] The phenomenon of complementation of a mouse that loses a functional ADAM6 locus with an ectopic sequence comprising a mouse ADAM6 gene or ortholog, or homolog, or functional fragment thereof is a general method that is applicable to rescue any mice with loci Non-functional or minimally functional endogenous ADAM6. Thus, many mice that comprise a modification of the immunoglobulin heavy chain locus that disrupts ADAM6 can be showered with the compositions and methods of the invention. Thus, the invention comprises mice with a wide variety of immunoglobulin heavy chain loci modifications that
[000473] [000473] In one aspect, there is provided a mouse comprising an ectopic sequence of ADAM6 encoding a functional ADAM6 protein (or ortholog, or homolog, or functional fragment thereof), a replacement of all or substantially all of the VH gene segments of mice with one or more human VH gene segments, a replacement of all or substantially all of the DH gene segments, and mice JH gene segments with human DH and human JH gene segments; wherein the mouse misses a CH1 and/or hinge region. In one embodiment, the mouse produces a single variable domain binding protein that is a dimer of immunoglobulin chains selected from: (a) human VH -mouse CH1 -mouse CH2 -mouse CH3; (b) human VH -mouse hinge -mouse CH2 -mouse CH3; and (c) human VH -mouse CH2 -mouse CH3.
[000474] [000474] In one aspect, the nucleotide sequence that restores fertility is placed in a human immunoglobulin heavy chain variable region sequence (e.g., between human VH1-2 and VH1-6 gene segments) in a mouse which features a substitution of one or more mouse immunoglobulin heavy chain variable gene segments (mVH's, mDH's, and/or mJH's) with one or more human immunoglobulin heavy chain variable gene segments (hVH's, hDH's, and /or hJH's), and the mouse further comprises a substitution of one or more variable light chain immunoglobulin κ gene segments
[000475] [000475] In one embodiment, the one or more mouse immunoglobulin heavy chain variable gene segments comprise about three megabases of the mouse immunoglobulin heavy chain locus. In one embodiment, the one or more mouse immunoglobulin heavy chain variable gene segments comprises at least 89 VH gene segments, at least 13 DH gene segments, at least four JH gene segments, or a combination thereof from the locus of mouse immunoglobulin heavy chain. In one embodiment, the one or more human immunoglobulin heavy chain variable gene segments comprise about one megabase of a human immunoglobulin heavy chain locus. In one embodiment, the one or more human immunoglobulin heavy chain variable gene segments comprise at least 80 VH gene segments, at least 27 DH gene segments, at least six JH gene segments, or a combination thereof from a locus of human immunoglobulin heavy chain.
[000476] [000476] In one embodiment, the one or more mouse variable light chain immunoglobulin κ gene segments comprises about
[000477] [000477] In one embodiment, the nucleotide sequence is placed between two human immunoglobulin gene segments. In a specific embodiment, the two human immunoglobulin gene segments are heavy chain gene segments.
[000478] [000478] In one aspect, a functional mouse ADAM6 locus (or ortholog, or homolog, or functional fragment thereof) is present in half of the mouse gene segments that are present in the mouse heavy chain variable region endogenous locus , said locus unable to rearrange to encode a functional heavy chain containing an endogenous heavy chain constant region. In one embodiment, the endogenous mouse heavy chain locus comprises at least one and up to 89 VH gene segments, at least one and up to 13 DH gene segments, at least one and up to four gene segments
[000479] [000479] In one aspect, a functional mouse ADAM6 locus (or ortholog, or homolog, or functional fragment thereof) is present in half of the human VH gene segments that replace the mouse endogenous VH gene segments. In one embodiment, at least 89 mouse VH gene segments are removed and placed with one or more human VH gene segments, and the mouse ADAM6 locus is present immediately adjacent to the 3' end of the human VH gene segments, or between two human VH gene segments. In a specific embodiment, the mouse ADAM6 locus is present between two VH gene segments at about 20 kilo bases (kb) to about 40 kilo bases (kb) from the 3' end of the inserted human VH gene segments. In a specific embodiment, the mouse ADAM6 locus is present between two VH gene segments at about 29 kb to about 31 kb from the 3' end of the inserted human VH gene segments. In a specific embodiment, the mouse ADAM6 locus is present within about 30 kb of the 3' end of the inserted human VH gene segments. In a specific embodiment, the mouse ADAM6 locus is present at about 30,184 bp from the 3' end of the inserted human VH gene segments. In a specific embodiment, the substitution includes human VH, VH1-2, and VH6-1 gene segments, and the mouse ADAM6 locus is present downstream of the VH1-2 gene segment and upstream of the VH6-1 gene segment. . In a specific embodiment, the mouse ADAM6 locus is present between a human VH1-2 gene segment and a human VH6-1 gene segment, where
[000480] [000480] Similarly, a mouse modified with one or more human VL gene segments (e.g., Vκ or Vλ segments) that replaces all or substantially all of the endogenous VH gene segments can be modified in such a way as to maintain the endogenous ADAM6 locus as described above, for example employing a targeting vector with a downstream homology arm that includes a mouse ADAM6 locus or functional fragment thereof, or replacing a damaged mouse ADAM6 locus with a positioned ectopic sequence between two human VL gene segments or between human VL gene segments and a DH gene segment (either human or mouse, e.g. Vλ + m/hDH), or a J gene segment
[000481] [000481] In one aspect, a mouse is provided with a replacement of one or more mouse endogenous VH gene segments, and which comprises at least one mouse endogenous DH gene segment. In such a mouse, modification of the mouse endogenous VH gene segments may comprise a modification of one or more of the 3'-plus VH gene segments, but not the 5'-plus DH gene segment, where care if modification of one or more 3'-plus VH gene segments does not interrupt or render the functional endogenous ADAM6 locus non-functional. For example, in one embodiment the mouse comprises a replacement of all or substantially all of the mouse endogenous VH gene segments with one or more human VH gene segments, and the mouse comprises one or more mouse endogenous DH gene and a functional endogenous mouse ADAM6 locus.
[000482] [000482] In another embodiment, the mouse comprises the
[000483] [000483] In various aspects, employment of mice that contain an ectopic sequence encoding a mouse ADAM6 protein or an ortholog or homolog or functional homolog thereof is used where modifications disrupt the function of endogenous mouse ADAM6. The probability of disruption of the endogenous function of ADAM6 in the mouse is high by making modifications to the mouse immunoglobulin loci, in particular the modification of the mouse immunoglobulin heavy chain variable regions and surrounding sequences. In this way, such mice provide particular benefit by making mouse immunoglobulin heavy chain loci that are deleted in whole or in part, are fully or in part humanized, or are replaced (e.g., with Vκ or Vλ sequences) in full. or in part. Methods for producing the genetic modifications described for the mice described below are known to those skilled in the art.
[000484] [000484] Mice containing an ectopic sequence encoding
[000485] [000485] In some aspects, genetically modified mice that are provided comprise an ectopic mouse, rodent, or other ADAM6 gene (or ortholog or homolog or fragment) functional in a mouse, and one or more variable region gene segments and/or human immunoglobulin constant. In various embodiments, other orthologous or homologous ADAM6 genes or functional fragments in a mouse may include sequences from bovine, canine, primate, rabbit, or other non-human sequences.
[000486] [000486] In one aspect, there is provided a mouse comprising an ectopic ADAM6 sequence encoding a functional ADAM6 protein, a replacement of all or substantially all of the mouse VH gene segments with one or more human VH gene segments; a replacement of all or substantially all of the segments
[000487] [000487] In one embodiment, the mouse further comprises a replacement of a mouse CH1 nucleotide sequence with a human CH1 nucleotide sequence. In one embodiment, the mouse further comprises a replacement of a mouse hinge nucleotide sequence with a human hinge nucleotide sequence. In one embodiment, the mouse further comprises a replacement of a variable immunoglobulin light chain locus (VL and JL) with a human immunoglobulin variable light chain locus. In one embodiment, the mouse further comprises a substitution of a mouse immunoglobulin constant region light chain nucleotide sequence with a human immunoglobulin constant region light chain nucleotide sequence. In a specific embodiment, the VL, JL, and CL are immunoglobulin κ light chain sequences. In a specific embodiment, the mouse comprises a mouse CH2 and mouse CH3 immunoglobulin constant region sequence fused to a human hinge and a human CH1 sequence such that the mouse immunoglobulin loci rearrange to form a gene. which encodes a binding protein comprising (a) a heavy chain having a human variable region, a human CH1 region, a human hinge region, and a mouse CH2 and a mouse CH3 region; and (b) a gene encoding an immunoglobulin light chain comprising a human variable domain and a human constant region.
[000488] [000488] In one aspect, a mouse is provided that comprises
[000489] [000489] In one embodiment, the mouse comprises a replacement of all or substantially all of the mouse VH, DH, and JH gene segments with one or more VL gene segments, one or more DH, and one or more J (e.g. , Jκ, or Jλ), wherein the gene segments are operatively linked to a mouse hinge region, wherein the mouse forms a rearranged immunoglobulin chain gene that contains, 5' to 3' in the transcriptional direction, VL from human -DH from human or mouse -J from human or mouse -hinge from mouse - CH2 from mouse -CH3 from mouse. In one embodiment, the J region is a human Jκ region. In one embodiment, the J region is a human JH region. In one embodiment, the J region is a human Jλ region. In one embodiment, the human VL region is selected from a human Vλ region and a human Vκ region.
[000490] [000490] In specific embodiments, the mouse expresses a single variable domain antibody with a mouse or human constant region and a variable region derived from a human Vκ, a human DH, and a human Jκ; a human Vκ, a human DH, and a human JH; a human Vλ, a human DH, and a human Jλ; a human Vλ, a human DH, and a human JH; a human Vκ, a human DH, and a human Jλ; a Vλ of
[000491] [000491] In one aspect, there is provided a mouse comprising an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or ortholog or homolog or functional fragment thereof), a replacement of all or substantially all mouse VH gene segments with a or more human VL gene segments, a replacement of all or substantially all of the mouse DH gene segments and JH gene segments with human JL gene segments; in which the mouse loses a CH1 and/or hinge region.
[000492] [000492] In one embodiment, the mouse loses a sequence encoding a CH1 domain. In one embodiment, the mouse misses a sequence encoding a hinge region. In one embodiment, the mouse loses a sequence encoding a CH1 domain and a hinge region.
[000493] [000493] In a specific embodiment, the mouse expresses a binding protein comprising a human immunoglobulin light chain variable domain (λ or κ) fused to a mouse CH2 domain that is attached to a mouse CH3 domain.
[000494] [000494] In one aspect, a mouse is provided which comprises an ectopic sequence of ADAM6 encoding a functional ADAM6 protein (or ortholog or homolog or functional fragment thereof), a replacement of all or substantially all mouse VH gene segments with a or more human VL gene segments, a replacement of all or substantially all of the mouse DH and JH gene segments with human JL gene segments.
[000495] [000495] In one embodiment, the mouse comprises a
[000496] [000496] In one embodiment, the mouse makes a single variable domain binding protein comprising a homodimer selected from the following: (a) human VL -mouse CH1 -mouse CH2 -mouse CH3; (b) human VL -mouse hinge -mouse CH2 -mouse CH3; (c) Human VL - Mouse CH2 - Mouse CH3.
[000497] [000497] In one aspect, there is provided a mouse with an inactivated endogenous immunoglobulin heavy chain locus, comprising an inactivated or deleted locus of functional endogenous ADAM6, wherein the mouse comprises a nucleic acid sequence that expresses a human or mouse, or human/mouse, or other chimerical. In one embodiment, the nucleic acid sequence is present in an integrated transgene, which is randomly integrated into the mouse genome. In one embodiment, the nucleic acid sequence is in an episome (eg, a chromosome) not found in a wild-type mouse.
[000498] [000498] In one embodiment, the mouse further comprises an endogenous inactivated immunoglobulin light chain locus. In a specific embodiment, the endogenous immunoglobulin light chain locus is selected from a kappa (κ) and a lambda (λ) light chain locus. In a specific embodiment, the mouse comprises an endogenous inactivated light chain κ locus and an inactivated light chain λ locus, wherein the mouse expresses an antibody comprising a human immunoglobulin heavy chain variable domain and a light chain domain. of human immunoglobulin. In one mode,
[000499] [000499] In one aspect, there is provided a genetically modified animal which expresses a chimeric antibody and expresses an ADAM6 protein or ortholog or homolog thereof which is functional in the genetically modified animal.
[000500] [000500] In one embodiment, the genetically modified animal is selected from a mouse and a rat. In one embodiment, the genetically modified animal is a mouse, and the ADAM6 protein or ortholog or homologue thereof is from a strain of mouse that is a different strain of the genetically modified animal. In one embodiment, the genetically modified animal is a rodent of the family Cricetidae (e.g., a hamster, a new world mouse or mouse, a mole rat), and the ortholog or homolog of the ADAM6 protein is from a rodent of the family Muridae ( e.g. mouse or true mouse, gerbil, spiny mouse, crested mouse). In one embodiment, the genetically modified animal is a rodent of the family Muridae, and the ortholog or homolog of the ADAM6 protein is from a rodent of the family Cricetidae.
[000501] [000501] In one embodiment, the chimeric antibody comprises a
[000502] [000502] In one embodiment, the chimeric antibody is expressed from an immunoglobulin locus. In one embodiment, the chimeric antibody heavy chain variable domain is expressed from an endogenous rearranged immunoglobulin heavy chain locus. In one embodiment, the chimeric antibody light chain variable domain is expressed from an endogenous rearranged immunoglobulin light chain locus. In one embodiment, the chimeric antibody heavy chain variable domain and/or the chimeric antibody light chain variable domain is expressed from a rearranged transgene (e.g., a rearranged nucleic acid sequence derived from a nonspecific sequence).
[000503] [000503] In a specific embodiment, the transgene is expressed from a transcriptionally active locus, eg, a ROSA26 locus, eg, a murine (eg, mouse) ROSA26 locus.
[000504] [000504] In one aspect, there is provided a non-human animal comprising a humanized immunoglobulin heavy chain locus, wherein the humanized immunoglobulin heavy chain locus comprises a non-human ADAM6 sequence or ortholog or homolog thereof.
[000505] [000505] In one embodiment, the non-human animal is a rodent selected from a mouse, a rat, and a hamster.
[000506] [000506] In one embodiment, the non-human ADAM6 ortholog or homolog is a sequence that is orthologous and/or homologous to a mouse ADAM6 sequence, wherein the ortholog or homolog is functional in the non-human animal.
[000507] [000507] In one embodiment, the non-human animal is selected from a mouse, a rat, and a hamster and the ADAM6 ortholog or homolog is from a non-human animal selected from a mouse, a rat, and a hamster. In a specific embodiment, the non-human animal is a mouse and the ADAM6 ortholog or homolog is from an animal that is selected from a different species of mouse, a rat, and a hamster. In a specific embodiment, the non-human animal is a mouse, and the ADAM6 ortholog or homolog is from a rodent that is selected from a different species of mouse, a mouse, and a hamster. in a modality
[000508] [000508] In a specific embodiment, the non-human animal is of the suborder Myomorpha, and the ADAM6 sequence is from an animal selected from a rodent of the subfamily Dipodoidea and a rodent of the subfamily Muroidea. In a specific embodiment, the rodent is a mouse of the subfamily Muroidea, and the ortholog or homolog of ADAM6 is a mouse or a rat or a hamster of the subfamily Muroidea.
[000509] [000509] In one embodiment, the humanized heavy chain locus comprises one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments. In a specific embodiment, the one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments are operably linked to one or more human chimeric constant region genes. and/or rodent (eg mouse or rat). In one embodiment, the constant region genes are from mice. In one embodiment, the constant region genes are from mouse. In one embodiment, the constant region genes are from hamsters. In one embodiment, the constant region genes comprise a selected sequence of a hinge, a CH2, a CH3, and a combination thereof. In a specific embodiment, the constant region genes comprise a hinge, a CH2, and a CH3 sequence.
[000510] [000510] In one embodiment, the non-human ADAM6 sequence is contiguous with a human immunoglobulin heavy chain sequence. In one embodiment, the non-human ADAM6 sequence is positioned in a human immunoglobulin heavy chain sequence. In a specific embodiment, the human immunoglobulin heavy chain sequence comprises a V, D and/or J gene segment.
[000511] [000511] In one embodiment, the non-human ADAM6 sequence is juxtaposed with a V gene segment. In one embodiment, the non-human ADAM6 sequence is positioned between two V gene segments. In one embodiment, the ADAM6 sequence is not is juxtaposed between a V and a D gene segment. In one embodiment, the mouse ADAM6 sequence is positioned between a V and a J gene segment. In one embodiment, the mouse ADAM6 sequence is juxtaposed between a segment of gene D and a J.
[000512] [000512] In one aspect, a genetically modified non-human animal is provided, comprising a B cell that expresses a human VH domain cognate with a human VL domain from an immunoglobulin locus, wherein the non-human animal expresses a protein non-human non-immunoglobulin from the immunoglobulin locus. In one embodiment, the non-human non-immunoglobulin protein is an ADAM protein. In a specific embodiment, the ADAM protein is an ADAM6 protein, or homolog, or ortholog, or functional fragment thereof.
[000513] [000513] In one embodiment, the non-human animal is a rodent (eg, mouse or rat). In one embodiment, the rodent is of the Muridae family. In one embodiment, the rodent is of the Murinae subfamily. In a specific embodiment, the rodent of the subfamily Murinae is selected from a mouse and a rat.
[000514] [000514] In one embodiment, the non-human non-immunoglobulin protein is a rodent protein. In one embodiment, the rodent is of the Muridae family. In one embodiment, the rodent is of the Murinae subfamily. In a specific embodiment, the rodent is selected from a mouse, a rat, and a hamster.
[000515] [000515] In one embodiment, the human VH and VL domains are directly attached or even linked in a domain sequence
[000516] [000516] In one aspect, a genetically modified non-human animal is provided, comprising in its germ line a human immunoglobulin sequence, wherein the sperm of a male non-human animal is characterized by a defect in in vivo migration. In one embodiment, the in vivo migration defect comprises an inability of the sperm of the male non-human animal to migrate from a uterus to an oviduct of a female non-human animal of the same species. In one embodiment, the non-human animal loses a nucleotide sequence encoding the ADAM6 protein or functional fragment thereof. In a specific embodiment, the ADAM6 protein or functional fragment thereof includes an ADAM6a protein and/or an ADAM6b or functional fragments thereof. In one embodiment, the non-human animal is a rodent. In a specific embodiment, the rodent is selected from a mouse, a rat, and a hamster.
[000517] [000517] In one aspect, there is provided a non-human animal, comprising a human immunoglobulin sequence contiguous with a non-human sequence encoding an ADAM6 protein or ortholog, or homolog, or functional fragment thereof. In one embodiment, the non-human animal is a rodent. In a specific embodiment, the rodent is selected from a mouse, a rat, and a hamster.
[000518] [000518] In one embodiment, the human immunoglobulin sequence is an immunoglobulin heavy chain sequence. In one embodiment, the immunoglobulin sequence comprises one or more VH gene segments. In one embodiment, the human immunoglobulin sequence comprises one or more DH gene segments. In a
[000519] [000519] In one embodiment, the immunoglobulin sequence comprises one or more VH gene segments exhibiting a high frequency in natural human repertoires. In a specific embodiment, the one or more VH gene segments comprise not more than two VH gene segments, not more than three VH gene segments, not more than four VH gene segments, not more than five VH gene segments, not more than six VH gene segments, not more than seven VH gene segments, not more than eight VH gene segments, not more than nine VH gene segments, not more than 10 VH gene segments, not more than 11 segments of VH gene, not more than 12 VH gene segments, not more than 13 VH gene segments, not more than 14 VH gene segments, not more than 15 VH gene segments, not more than 16, VH gene segments, not more than 17 VH gene segments, not more than 18 VH gene segments, not more than 19 VH gene segments, not more than 20 VH gene segments, not more than 21 VH gene segments, not more than 22 segments of VH gene or not more than 23 VH gene segments.
[000520] [000520] In a specific embodiment, the one or more VH gene segments comprise five VH gene segments. In a specific embodiment, the one or more VH gene segments comprise 10 VH gene segments. In a specific embodiment, the one or more VH gene segments comprise 15 VH gene segments. In a specific embodiment, the one or more VH gene segments comprise 20 VH gene segments.
[000521] [000521] In various embodiments, the VH gene segments are selected from VH6-1, VH1-2, VH1-3, VH2-5, VH3-7, VH1-8, VH3-9, VH3-11, VH3-13 , VH3-15, VH3-16, VH1-18, VH3-20, VH3-21, VH3-23, VH1-24, VH2-
[000522] [000522] In a specific embodiment, human immunoglobulin sequence comprises at least 18 VH gene segments, 27 DH gene segments, and six JH gene segments. In a specific embodiment, the human immunoglobulin sequence comprises at least 39 VH gene segments, 27 DH gene segments, and six JH gene segments. In a specific embodiment, the human immunoglobulin sequence comprises at least 80 VH gene segments, 27 DH gene segments, and six JH gene segments.
[000523] [000523] In one embodiment, the non-human animal is a mouse, and the mouse comprises a replacement gene segments
[000524] [000524] In various embodiments, the human immunoglobulin sequence is operably linked to a constant region in the germ line of the non-human animal (e.g., the rodent, e.g., the mouse, rat, or hamster). In one embodiment, the constant region is a human, human/chimeric mouse or human/chimeric mouse or human/chimeric hamster constant region, a mouse, a rat, or a hamster. In one embodiment, the constant region is a rodent constant region (eg, mouse or rat or hamster). In a specific embodiment, the rodent is a mouse or rat. In various embodiments, the constant region comprises at least a CH2 domain and a CH3 domain.
[000525] [000525] In one embodiment, the human immunoglobulin heavy chain sequence is located at an immunoglobulin heavy chain locus in the germ line of the non-human animal (e.g., the rodent, e.g., the mouse or rat or hamster) . In one embodiment, the human immunoglobulin heavy chain sequence is located at an immunoglobulin heavy chain locus in the germline of the non-human animal, wherein the non-heavy chain locus is a transcriptionally active locus. In a specific embodiment, the non-heavy chain locus is a ROSA26 locus.
[000526] [000526] In various aspects, the non-human animal further comprises a human immunoglobulin light chain sequence (e.g., one or more unrearranged V and J light chain sequences, or one or more rearranged VJ sequences) in the germ line of the non-human animal. In a specific embodiment, the immunoglobulin light chain sequence is an immunoglobulin λ light chain sequence. In one embodiment, the human immunoglobulin light chain sequence comprises one or more Vλ gene segments. In one embodiment, the human immunoglobulin light chain sequence
[000527] [000527] In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 12 Vλ gene segments and one Jλ gene segment. In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 12 Vλ gene segments and four Jλ gene segments.
[000528] [000528] In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 28 Vλ gene segments and one Jλ gene segments. In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 28 Vλ gene segments and four Jλ gene segments.
[000529] [000529] In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 40 Vλ gene segments and one Jλ gene segment. In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 40 Vλ gene segments and four Jλ gene segments.
[000530] [000530] In various embodiments, the human immunoglobulin light chain sequence is operably linked to a constant region in the germ line of the non-human animal (e.g. rodent, e.g. mouse or rat or hamster). In one embodiment, the constant region is a human, human/chimeric rodent, mouse, rat, or hamster constant region. In a specific embodiment, the constant region is a mouse or rat constant region. In a specific embodiment, the constant region is a mouse constant κ region (mCκ) or a rat κ constant region (rCκ). In a specific embodiment, the constant region is either a mouse λ constant region (mCl) or a rat λ constant region (rCλ). In a
[000531] [000531] In one embodiment, the human immunoglobulin light chain sequence is located at an immunoglobulin light chain locus in the germ line of the non-human animal. In a specific embodiment, the immunoglobulin light chain locus in the germ line of the non-human animal is an immunoglobulin light chain κ locus. In a specific embodiment, the immunoglobulin light chain locus in the germ line of the non-human animal is an immunoglobulin light chain λ locus. In one embodiment, the human immunoglobulin light chain sequence is located at a non-light chain immunoglobulin locus in the germ line of the non-human animal, which is transcriptionally active. In a specific embodiment, the non-immunoglobulin-associated locus is a ROSA26 locus.
[000532] [000532] In one aspect, there is provided a method of producing a human antibody, wherein the human antibody comprises variable domains derived from one or more encoded variable region nucleic acid sequences in a cell of a non-human animal in the manner described here.
[000533] [000533] In one aspect, there is provided a pharmaceutical composition, comprising a polypeptide comprising antibody or antibody fragment that is derived from one or more variable region nucleic acid sequences isolated from a non-human animal in the manner described herein. In one embodiment, the polypeptide is an antibody. In one embodiment, the polypeptide is a heavy chain-only antibody. In one embodiment, the polypeptide is a single-chain variable fragment (e.g., an Fvsc).
[000534] [000534] In one aspect, there is provided the use of a non-human animal in the manner described herein to produce an antibody. In various embodiments, the antibody comprises one or more variable domains that are derived from
[000535] [000535] Genetically modified non-human animals (eg, mice, rats, etc.) comprising a modification that reduces fertility, by virtue of the loss of an ADAM protein activity (eg, ADAM6-dependent), can be crossed with non-human animals. humans in the manner described herein, which comprise human λ variable sequences in non-human, endogenous, or (eg, transgenic) light constant genes. For example, non-human animals such as mice or rats that comprise a damaged ADAM6 gene (or a deleted ADAM6 gene), e.g., animals with a humanized immunoglobulin heavy chain loci, are matched with mice that comprise a locus of light chain (endogenous or transgenic) comprising human λ segments and JL segments linked to human or non-human (e.g., endogenous mouse or rat) light chain constant region genes, wherein non-human animals comprise an activity which restores ADAM-dependent fertility. The genetic modification that restores ADAM-dependent fertility can be either in the non-human animal, for example, in a mouse with a humanized heavy chain, or in a mouse with humanized λ variable segments. The progeny comprise genes that form a humanized heavy-chain locus (i.e., results in expression of a human heavy-chain variable domain) and a humanized light-chain (i.e., results in expression of a human heavy-chain variable domain).
[000536] [000536] VELOCIMMUNE® genetically modified mice comprise a replacement of unrearranged V(D)J gene segments at endogenous mouse loci with human V(D)J gene segments. VELOCIMMUNE® mice express chimeric antibodies with human variable domains and mouse constant domains (see, for example, U.S. Patent 7,605,237). Still other lelates pertain to mice that express fully human antibodies from fully human transgenes in mice that have inactivated endogenous immunoglobulin loci.
[000537] [000537] Antibody light chains are encoded by one of two separate loci: kappa (κ) and lambda (λ). Mouse antibody light chains are primarily of the κ type. Mice that produce mouse antibodies, and modified mice that produce fully human or human-mouse chimeric antibodies, exhibit a tendency towards light chain use. Humans also exhibit a light chain tendency, but not as pronouncedly as in mice; the ratio of κ light chains to λ light chains in mice is about 95:5, whereas in humans the ratio is about 60:40. The most evident trend in mice is not knowing how to seriously affect antibody diversity, as the variable λ locus in mice is not as diverse as in the first example. This is not just in humans. The human light chain λ locus is very diverse.
[000538] [000538] The human light chain λ locus spans 1000 kb and contains more than 80 genes encoding variable (V) or splice (J) segments (FIG. 19). At the human light chain λ locus, nearly half of all
[000539] [000539] The λ light chain locus in humans is similar in structure to the λ locus of both mice and humans, in that the human light chain λ locus has several variable region gene segments that are capable of recombining to form a functional light chain protein. The human light chain λ locus contains approximately 70 V gene segments and 7 Jλ-Cλ gene segment pairs. Only four of these Jλ-Cλ gene segment pairs appear to be functional. In some alleles, a fifth Jλ-Cλ gene segment pair is reported as a pseudo gene (Cλ6). The 70 Vλ gene segments appear to contain 38 functional gene segments. The 70 Vλ sequences are arranged in three clusters, all of which contain different elements from distinct V family gene clusters (clusters A, B and C; FIG. 19). This is a potentially rich source of relatively untapped diversity to generate antibodies with human V regions in non-human animals.
[000540] [000540] In sharp contrast, the mouse light chain λ locus contains only two or three (depending on the strain) mouse Vλ region gene segments (FIG. 20). For this reason at least, the large κ bias in mice is not known to be particularly detrimental to overall antibody diversity.
[000541] [000541] According to published maps of the mouse light chain λ locus, the locus essentially consists of two clusters of gene segments in a space of approximately 200 kb (FIG. 20). The two clusters contain two sets of V, J, and C genes that are capable of independent rearrangement: Vλ2-Jλ2-Cλ2-Jλ4-Cλ4 and Vλ1-Jλ3-Cλ3-Jλ1-Cλ1.
[000542] [000542] The mouse light chain κ locus is very different. The structure and number of gene segments that participate in recombination events lead to a locus in the functional mouse κ light chain protein that is much more complex (FIG. 21). Thus, mouse λ light chains do not contribute much to the diversity of an antibody population in a typical mouse.
[000543] [000543] Exploring the rich diversity of the human λ light chain locus in mice is likely to result in, among other things, a source of a more complete human repertoire of light chain V domains. Previous attempts to exploit this diversity have used human transgenes containing fragments of the human light chain λ locus randomly incorporated into the mouse genome (see, for example, U.S. 6,998,514 and U.S. 7,435,871). Mice containing these randomly integrated transgenes are reported to express fully human λ light chains, however, in some cases, one or both of the endogenous light chain loci remain intact. This situation is undesirable for the human λ light chain sequence, which deals with the mouse (κ or λ) light chain in the expressed antibody repertoire of mice.
[000544] [000544] Rather, the inventors describe genetically modified mice that are capable of expressing one or more nucleic acid λ light chain sequences directly from a mouse light chain locus, including by substitution at an endogenous light chain locus of mouse. Genetically modified mice capable of expressing human λ light chain sequence from an endogenous locus can be additionally crossed with the
[000545] [000545] Many advantages can be realized for various modalities of expressing binding proteins derived from human Vλ and Jλ gene segments in mice. The advantages can be realized by placing the human λ sequences at an endogenous light chain locus, for example the mouse κ or λ locus. Antibodies produced from such mice may have light chains comprising human Vλ domains fused to a mouse CL region, specifically a mouse Cκ or Cλ region. Mice will also express human Vλ domains that are suitable for identification and cloning for use with human CL regions, specifically Cκ and/or Cλ regions. Because B cell development in such mice is different from normal, it is possible to generate compatible Vλ domains (including somatically mutated Vλ domains) in the context of both the Cλ and Cκ regions.
[000546] [000546] Genetically modified mice comprising an unrearranged Vλ gene segment at an immunoglobulin light chain κ or λ locus are described. Mice expressing antibodies comprising a light chain with a human Vλ domain fused to a Cκ and/or Cλ region are described.
[000547] [000547] In one aspect, a genetically modified non-human animal is described that comprises (1) one or more segments of
[000548] [000548] In one embodiment, the non-human light chain constant domain is a Cκ domain or a Cλ domain. In one embodiment, the ADAM6 protein or functional fragment thereof is encoded by an ectopic sequence in the germ line of mice. In one embodiment, the ADAM6 protein or functional fragment thereof is encoded by a sequence endogenous to the non-human animal.
[000549] [000549] In one embodiment, the endogenous light chain locus of the non-human animal is an immunoglobulin light chain λ locus. In one embodiment, the endogenous light chain locus of the non-human animal is an immunoglobulin light chain κ locus.
[000550] [000550] In one embodiment, the non-human animal loses an endogenous VL and/or JL gene segment at the endogenous light chain locus. In a specific embodiment, the VL and/or JL gene segment is a segment of
[000551] [000551] In one embodiment, the VL and JL gene segments of the non-human animal are replaced by one or more human Vλ gene segments and one or more human Jλ. In a specific embodiment, the VL and JL gene segments of the non-human animal are κ gene segments. In a specific embodiment, the VL and JL gene segments of the non-human animal are λ gene segments.
[000552] [000552] In one embodiment, the one or more human Vλ gene segments are from a cluster A fragment of the human immunoglobulin light chain λ locus. In a specific embodiment, the cluster A fragment extends from human Vλ3-27 to human Vλ3-1. In a specific embodiment, the cluster A fragment extends from human Vλ3-12 to human Jλ1. In one embodiment, the one or more human Vλ gene segments are from a cluster B fragment of the human immunoglobulin light chain λ locus. In a specific embodiment, the cluster B fragment extends from human Vλ5-52 to human Vλ1-40. In a specific embodiment, the one or more human Vλ gene segments are from a cluster A fragment and a cluster B fragment of the human immunoglobulin light chain λ locus in the manner described herein.
[000553] [000553] In one embodiment, the non-human animal comprises at least 12 human Vλ gene segments. In one embodiment, the non-human animal comprises at least 28 human Vλ gene segments. In one embodiment, the non-human animal comprises at least 40 human Vλ gene segments.
[000554] [000554] In one embodiment, the at least one human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and a combination thereof.
[000555] [000555] In one aspect, a male, fertile non-human animal is provided, wherein the fertile non-human animal expresses (1) an immunoglobulin light chain comprising a human Vλ domain or a human Vκ domain, and (2) an immunoglobulin heavy chain comprising a human VH domain, wherein the male non-human animal comprises a heavy chain modified variable region locus and an ectopic ADAM6 gene that is functional in the male non-human animal. In one embodiment, the male non-human animal is a mouse.
[000556] [000556] In one aspect, the use of a non-human animal in the manner described herein to produce an antigen-binding protein is provided. In one embodiment, the antigen-binding protein is human. In one embodiment, the antigen-binding protein is an antibody. In one embodiment, the antigen-binding protein comprises a human VH domain and/or a human Vλ domain derived from a non-human animal in the manner described herein.
[000557] [000557] In one aspect, a cell or tissue derived from a non-human animal in the manner described herein is provided. In one embodiment, the tissue is derived from a spleen, bone marrow, or a lymph node. In one embodiment, the cell is a B cell. In one embodiment, the cell is an embryonic stem (ES) cell. In one embodiment, the cell is a germ cell.
[000558] [000558] In one aspect, there is provided an oocyte comprising a diploid genome from a non-human animal genetically modified in the manner described herein. Sterile immunoglobulin light chain κ locus transcripts
[000559] [000559] Variations in the theme of expressing human immunoglobulin λ sequences in mice are reflected in various modalities of genetically modified mice capable of such expression. Thus, in some embodiments, mice genetically
[000560] [000560] The human and mouse κ light chain loci contain sequences encoding sterile transcripts that miss both a start codon and an open reading frame, and which are listed as elements that regulate transcription of the κ light chain loci. These sterile transcripts arise from an intergenic sequence located downstream or 3' of the most proximal Vκ gene segment, and upstream or 5' of the κ light chain intronic enhancer (Eκi), which is upstream of the κ constant region gene of light chain (Cκ). Sterile transcripts arise from the rearrangement of the intergenic sequence to form a VκJκ1 segment fused to a Cκ.
[000561] [000561] A substitution of the light chain κ locus upstream of the Cκ gene can remove the intergenic region encoding sterile transcripts. Therefore, in various embodiments, a substitution of a mouse κ light chain sequence upstream of the mouse Cκ gene with human λ light chain gene segments can result in a humanized mouse light chain κ locus, which contains segments of human Vλ and Jλ gene, but not the κ light chain intergenic region that encodes the sterile transcripts.
[000562] [000562] In the manner described herein, humanization of the endogenous mouse κ light chain locus with human λ light chain gene segments, where humanization removes the intergenic region, results in a
[000563] [000563] Humanization of the endogenous mouse κ light chain locus with human Vλ and Jλ gene segments coupled to an insert of a human κ intergenic region to create a Vλ locus containing, with respect to transcription, is also described. between the final human Vλ gene segment and the first human Jλ gene segment, a κ intergenic region; which exhibits a B cell population with an increased expression of a locus that misses the κ intergenic region. This observation is consistent with a hypothesis that the intergenic region, either directly to a sterile transcript, or indirectly, suppresses the use of the endogenous light chain λ locus. A hypothesis like this, including the intergenic region, could result in a decrease in the use of the endogenous light chain λ locus, leaving the mouse with a restricted choice but to employ the modified locus (λ in κ) to generate antibodies.
[000564] [000564] In various embodiments, a substitution of mouse κ light chain sequence upstream of the mouse Cκ gene with human λ light chain sequence further comprises a human light chain κ intergenic region arranged, with respect to transcription, between the 3' untranslated region of the 3' plus and 5' Vλ gene segment in the first human Jλ gene segment. Alternatively, an intergenic region such as this can be omitted from an endogenous light chain κ locus (upstream of the mouse Cκ genes) substituted by crossing a deletion at the endogenous light chain λ locus. Likewise, in this embodiment, the mouse generates antibodies from an endogenous light chain κ locus containing human λ light chain sequences.
[000565] [000565] Various approaches are described for producing genetically modified mice that produce antibodies, which contain a light chain that has a human Vλ domain fused to an endogenous CL region (eg, Cκ or Cλ). Genetic modifications are described which, in various embodiments, comprise a deletion of one or both of the endogenous light chain loci. For example, to eliminate mouse λ light chains from the endogenous antibody repertoire, a deletion of a first Vλ-Jλ-Cλ gene cluster and replacement, in whole or in part, of the Vλ-Jλ gene segments of a second Gene clustering with human Vλ-Jλ gene segments can be produced. Embryos, cells, and mouse targeting constructs genetically modified to produce the mice, mouse embryos, and cells are also provided.
[000566] [000566] Deletion of an endogenous Vλ-Jλ-Cλ gene cluster and replacement of the Vλ-Jλ gene segments from another endogenous Vλ-Jλ-Cλ gene cluster employs relatively minimal disruption in antibody constant region association and function in the animal, in various modalities, as a result of endogenous Cλ genes being left intact and therefore maintaining normal functionality and ability to associate with the constant region of an endogenous heavy chain. Thus, in such embodiments, the modification does not affect other endogenous heavy chain constant regions dependent on functional light chain constant regions for assembly of a functional antibody molecule containing two heavy chains and two light chains. Additionally, in several embodiments, the modification does not affect the assembly of a functional membrane-bound antibody molecule involving an endogenous heavy chain and light chain, e.g., an hVλ domain linked to a Cλ region.
[000567] [000567] A schematic illustration (not used for classification) of a deleted mouse endogenous Vλ-Jλ-Cλ gene cluster is provided in FIG. 20. As illustrated, the mouse light chain λ locus is organized into two gene clusters, both of which contain gene segment function capable of recombining to form a mouse λ light chain function. The endogenous mouse Vλ1-Jλ3-Cλ3-Jλ1-Cλ1 gene cluster is eliminated by a targeting construct (Targeting Vector 1) with a neomycin cassette flanked by recombination sites. The other endogenous gene cluster (Vλ2-Vλ3-Jλ2-Cλ2-Jλ4-Cλ4) is eliminated in part by a targeting construct (Targeting Vector 2) with a hygromycin-thymidine kinase cassette flanked by recombination sites. In this second targeting event, the endogenous Cλ2-Jλ4-Cλ4 gene segments are maintained. The second targeting construct (Targeting Vector 2) is constructed using recombination sites that are different from those in the first targeting construct (Targeting Vector 1), thereby allowing the selective elimination of the selection cassette after a satisfactory targeting has been hit. The resulting double-targeted locus is functionally silenced, whereby no endogenous λ light chain can be produced. This modified locus can be used for the insertion of human Vλ and Jλ gene segments to create an endogenous mouse λ locus comprising Vλ and Jλ gene segments from
[000568] [000568] Genetically modifying a mouse to render endogenous λ gene segments non-functional, in various modalities, results in a mouse that exclusively displays κ light chains in its antibody repertoire, producing the mouse used to assess the role of λ light chains in immune response, and used to produce an antibody repertoire comprising Vκ domains, but not Vλ domains.
[000569] [000569] A genetically modified mouse that expresses an hVλ linked to a mouse Cλ gene, which is recombined at the endogenous mouse λ light chain locus, can be produced by any method recognized in the art. A schematic illustration (not used for classification) of the replacement of endogenous mouse Vλ2-Vλ3-Jλ2 gene segments with human Vλ and Jλ gene segments is provided in FIG. 22A. As illustrated, an endogenous mouse λ light chain locus that has been rendered non-functional is replaced by a targeting construct (Targeting Vector 12/1-λ) that includes a neomycin cassette flanked by recombination sites. The Vλ2-Vλ3-Jλ2 gene segments are replaced by a genomic fragment containing human λ sequence, which includes 12 hVλ gene segments and a single hJλ gene segment.
[000570] [000570] Thus, this first approach positions one or more hVλ gene segments at the endogenous light chain λ locus contiguous with a single hJλ gene segment (FIG. 22A).
[000571] [000571] Additional modifications at the modified endogenous light chain λ locus can be achieved using similar techniques to insert more hVλ gene segments. For example, schematic illustrations of two additional targeting constructs (targeting vectors +16-λ and +12-λ)
[000572] [000572] Previous approaches to inserting human λ light chain gene segments into the mouse λ locus maintain the enhancers positioned downstream of the Cλ2-Jλ4-Cλ4 gene segments (determined Enh 2.4, Enh and Enh 3.1 FIG. 22A FIG. 22A and Fig. 23A). This approach results in a single modified allele at the endogenous mouse λ light chain locus (FIG. 25A).
[000573] [000573] Compositions and methods for producing a mouse that expresses a light chain comprising hVλ and Jλ gene segments operably linked to a mouse Cλ gene segment are provided, including compositions and method for producing a mouse that expresses such genes from from an endogenous mouse λ light chain locus. Methods include selectively ternating a non-functional mouse endogenous Vλ-Jλ-Cλ gene cluster (e.g., by a targeted deletion), and employing a segment of the hVλ and Jλ gene at the mouse endogenous λ light chain locus to express a hVλ domain in a mouse.
[000574] [000574] Alternatively, in a second approach, human λ light chain gene segments can be positioned at the κ locus
[000575] [000575] For reasons stated above, deletion of the Vκ and Jκ gene segments from mice employs relatively minimal disruption. A schematic illustration (not used for classification) of deleted mouse Vκ and Jκ gene segments is provided in FIG. 21. The mouse endogenous Vκ and Jκ gene segments are deleted via positional recombinase-mediated deletion of mouse sequences between two precisely positioned targeting vectors, each employing site-specific recombination sites. A first targeting vector (Jκ targeting vector) is employed in a first targeting event to eliminate mouse Jκ gene segments. A second targeting vector (Vκ targeting vector) is employed in a second sequential targeting event to eliminate a sequence located 5' of the most distal mouse Vκ gene segment. Both targeting vectors contain site-specific recombination sites, thereby allowing selective elimination of both the selection cassettes and the entire κ light chain intervening sequence of mice after satisfactory targeting is achieved. The resulting deleted locus is functionally silenced, whereby no endogenous κ light chain can be produced. This modified locus can be used for the insertion of hVλ and Jλ gene segments to create an endogenous mouse κ locus comprising hVλ and Jλ gene segments, where, upon recombination
[000576] [000576] Thus, in a second approach, one or more human Vλ gene segments are positioned at the mouse light chain κ locus contiguous with a single human Jλ gene segment (Targeting vector 12/1-κ, FIG 22B).
[000577] [000577] In various embodiments, modifications to this approach can be made to add gene segments and/or regulatory sequences to optimize the use of the mouse κ human λ light chain sequences in the mouse antibody repertoire.
[000578] [000578] In a third approach, one or more hVλ gene segments are positioned at the light chain mouse κ locus contiguous with four hJλ gene sequences (Targeting Vector 12/4-κ, FIG. 22B).
[000579] [000579] In a third approach, one or more hVλ gene segments are positioned at the light chain mouse κ locus contiguous with a human κ intergenic sequence and a single hJλ gene sequence (Targeting Vector 12(κ)1- κ, Fig. 22B).
[000580] [000580] In a fourth approach, one or more hVλ gene segments are positioned at the light chain mouse κ locus contiguous with a human κ intergenic sequence and four hJλ gene sequences (12(κ)4-κ Targeting vector Fig. 22B).
[000581] [000581] All previous approaches to inserting human λ light chain gene segments into the mouse κ locus maintain the κ intronic enhancer element upstream of the Cκ gene (determined Eκi, FIG. 22B and
[000582] [000582] In various embodiments, genetically modified mice comprise an inactivation of the endogenous mouse λ light chain locus. In one embodiment, the light chain λ locus is inactivated by a strategy that eliminates the region spaced from Vλ2 to Jλ2, and the region spaced from Vλ1 to Cλ1 (FIG. 20). Any strategy that reduces or eliminates the ability of the endogenous light chain λ locus to express the endogenous λ domain is suitable for use with modalities in this disclosure. Lambda domain antibodies from genetically modified mice
[000583] [000583] Mice comprising human λ sequences in both the mouse κ and λ light chain locus will express a light chain comprising an hVλ region fused to a mouse CL region (Cκ or Cλ). These are advantageously crossed with mice that (a) comprise a functionally silenced light chain locus (eg, an inactivation of the endogenous mouse light chain κ or λ locus); (b) comprise an endogenous mouse λ light chain locus comprising hV and hJ gene segments operably linked to an endogenous mouse Cλ gene; (c) comprise an endogenous mouse κ light chain locus comprising hVκ and hJκ gene segments operably linked to an endogenous mouse Cκ gene; and, (d) a mouse in which a κ allele comprises hVκs and hJκs; the other κ allele comprising hVλs and hJλs; a λ allele comprising hVλs and hJλs and a silenced or inactivated λ allele, or both λ alleles comprising hVλs and hJλs; and, two heavy chain alleles each comprising hVHs, hDHs, and hJHs.
[000584] [000584] Antibodies that comprise the expressed hVλ domains
[000585] [000585] The following examples are provided in order to describe how to make and use methods and compositions of the invention, and are not intended to be limiting of the scope within which the inventors report their invention. Unless otherwise indicated, temperature is indicated in Celsius, and pressure is at or near atmospheric. Example 1. Humanization of mouse immunoglobulin genes
[000586] [000586] Human and mouse bacterial artificial chromosomes (BACs) were used to genetically modify 13 different BAC targeting vectors (BACvecs) for humanization of the mouse immunoglobulin heavy chain and light chain κ loci. Tables 1 and 2 present detailed descriptions of the steps performed for the construction of all BACvecs used in the humanization of the heavy chain and light chain loci κ of mouse immunoglobulin, respectively.
[000587] [000587] Identification of human and mouse BACs. Mouse BACs that space the 5' and 3' ends of the immunoglobulin heavy chain and κ light chain loci were identified by hybridization of stained filters with BAC library or by PCR selection of mouse library of BAC DNA clusters. Filters were hybridized under standard conditions using probes corresponding to regions of interest. Library clusters were selected by PCR using unique oligonucleotide primer pairs flanking the
[000588] [000588] Construction of BACvecs by bacterial recombination and homologous ligation. Bacterial homologous recombination (BHR) was performed as described (Valenzuela et al., 2003; Zhang, Y., Buchholz, F., Muyrers, JP, and Stewart, AF (1998).A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20, 123-128). In many cases, linear fragments were generated by ligating PCR-derived homology boxes to the cloned cassettes, followed by gel isolation of ligation products and electroporation into BHR-compliant bacteria that carry the target BAC. After selection in Petri dishes with suitable antibiotics, correctly recombined BACs were identified by PCR at both novel junctions followed by restriction analysis on pulsed field gels (Schwartz, DC, and Cantor, CR (1984). Separation of yeast chromosome- sized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67-75) and PCR blot verification using oligonucleotide primers distributed over the human sequences.
[000589] [000589] A BACvec 3hVH was constructed using three sequential steps of BHR for the initial step of humanization of the chain locus
[000590] [000590] In a similar manner, 12 additional BACvecs were genetically modified by humanization of the κ heavy chain and light chain loci. In some examples, BAC binding was performed in place of BHR to co-link two large BACs until the introduction of rare restriction sites in both parental BACvecs by BHR, along with careful replacement of selectable markers. This allowed survival of the desired ligation product upon selection with
[000591] [000591] Modification of embryonic stem cell (ES)s and generation of mice. ES cell targeting (F1H4) was performed using the VELOCIGENE® genetic alteration method, as described (Valenzuela et al., 2003). The derivation of mice from ES cells modified either by blastocyst (Valenzuela et al., 2003) or by 8-cell injection (Poueymirou et al., 2007) was as described. ES cells and targeted mice were confirmed by selecting DNA from ES cells or mice with unique sets of probes and primers in a PCR-based assay (eg, FIG. 3A, 3B, and 3C). All mouse studies were observed and approved by Regeneron's Institutional Animal Care and Use Committee (IACUC).
[000592] [000592] Karyotype analysis and fluorescent in situ hybridization (FISH). Karyotype analysis was performed by Coriell Cell Repositories (Coriell Institute for Medical Research, Camden, NJ). FISH was performed in
[000593] [000593] Immunoglobulin heavy chain variable gene locus. Humanization of the variable region of the heavy chain locus was achieved in nine sequential steps by the direct replacement of about three million base pairs (Mb) of contiguous mouse genomic sequence containing all the VH, DH, and JH gene segments with about an Mb of contiguous human genomic sequence containing the human equivalent gene segments (FIG. 1A and Table 1), using VELOCIGENE® gene alteration technology (see, for example, US patent
[000594] [000594] The intron between JH gene segments and constant region genes (the JC intron) contains a transcriptional enhancer (Neuberger, MS (1983). Expression and regulation of heavy chain immunoglobulin gene transfected into lymphoid cells. Embo J 2, 1373 -1378), followed by a region of single repeats required for recombination during isotype switching (Kataoka, T., Kawakami, T., Takahashi, N., and Honjo, T. (1980). Rearrange of immunoglobulin gamma 1- chain gene and mechanism for heavy-chain class switch (Proc Natl Acad Sci USA 77, 919-923). The junction between the human VH-DH-JH region and the mouse CH region (the proximal junction) was chosen to retain the mouse heavy chain intronic enhancer and switch the domain in order to conserve both efficient expression and class switching. the humanized heavy chain locus in mice. The exact nucleotide position of these and subsequent junctions in all substitutions was made possible by the use of the VELOCIGENE® GENETIC ALTERATION method (supra), which employed recombination
[000595] [000595] A first mouse human immunoglobulin DNA sequence insertion was achieved using 144 kb from the proximal end of the human heavy chain locus containing 3 VH gene segments, all 27 DH and 9 human JH inserted at the end proximal to the mouse IgH locus, with a concomitant deletion of 16.6 kb of mouse genomic sequence, using about 75 kb of mouse homology arms (Step A, FIG. 2A; Tables 1 and 3, 3hVH). This large insertion of 144kb and accompanying the deletion of 16.6kb was performed in a single step (Step A), which occurred with a frequency of 0.2% (Table 3). Correctly targeted ES cells were selected by a natural allele loss (LONA) assay (Valenzuela et al., 2003) using probes and flanking the mouse deleted sequence, and the inserted human sequence, and the integrity of the large insert of human was verified using multiple probes that fully space the insert (FIG. 3A, 3B and 3C). Because many cycles of sequential ES cell targeting were anticipated, the ES cell clones targeted at this and all subsequent steps were
[000596] [000596] The targeted ES cells from step A were re-targeted with a BACvec that produced a 19 kb deletion at the distal end of the heavy chain locus (Step B, FIG. 2A). BACvec from step B contained a hygromycin resistance gene (hyg), in contrast to the neomycin resistance gene (neo) contained in BACvec from step A. The resistance genes of the two BACvecs were determined in such a way that, upon satisfactory targeting on the same chromosome, approximately three Mb heavy chain variable gene loci from mice containing all mice VH gene segments, other than VH1-86, and all DH gene segments, other than DQ52, as well as the two resistance gene , were flanked by loxP sites; DQ52 and all mouse JH chain gene segments were deleted in step A. Double-targeted ES cell clones on the same chromosome were identified by targeting the 3hVH proximal cassette to high G418 homozygosity (Mortensen, RM et al. (1992) Production of homozygot mutant ES cells with a single targeting construct. Mol Cell Biol 12, 2391-2395), and after the fate of the distal hyg cassette. Segments up to four Mb in mouse size, modified in such a way as to be flanked by loxP sites, were satisfactorily eliminated in ES cells by transient expression of CRE recombinase with high efficiencies (up to ≈11%) even in the absence of drug selection (Zheng, B. et al. (2000) Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications. Mol Cell Biol 20, 648-655). In a similar manner, the inventors achieved a three Mb deletion in 8% of the ES cell clones following transient CRE expression (Step C, FIG. 2A; Table 3). Elimination was selected by the LONA assay using probes both in the
[000597] [000597] The remainder of the human heavy chain variable region was added to the 3hVH allele in a series of 5 steps using the VELOCIGENE® genetic alteration method (Steps EH, FIG. 2B), with each step involving the exact insertion of up to 210 kb of human gene sequences. For each step, the proximal end of each new BACvec was determined to overlap the most distal human sequence of the previous step, and the distal end of each new BACvec contained in the same distal region of mouse homology as used in step A. BACvecs from steps D, F and H contained neo selection cassettes, whereas those from steps E and G contained hyg selection cassettes, thus selections were alternated between G418 and hygromycin. Targeting in step D was assayed by the loss of the unique PCR product through the distal loxP site of the 3hVH hybrid allele. The targeting of steps E through I was tested by the loss of the pre-selection cassette. In the final step (Step I, FIG. 2B), the neo selection cassette flanked by the Frt sites (McLeod, M. et al. (1986) Identification of the crossover site during FLP-mediated recombination on the Saccharomyces cerevisiae plasmide 2 microns circle Mol Cell Biol 6, 3357-3367), was removed by transient expression of FLPe (Buchholz, F. et al. (1998) Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 16, 657-662). The human sequences of the BACvecs for steps D, E and G were derived from two human parental BACs each, whereas those of steps F and H were from a single BACs. Retention of human sequences was confirmed at each step using multiple probes spacing the inserted human sequence (in the manner
[000598] [000598] Immunoglobulin light chain variable gene κ locus. The κ light chain variable region was humanized in eight sequential steps by the direct replacement of about three Mb of mouse sequence containing all the Vκ and Jκ gene segments, with about 0.5 Mb of human sequence containing the κ gene segments. human proximal Vκ and Jκ gene in a manner similar to that of the heavy chain (FIG. 1B; Tables 2 and 4).
[000599] [000599] The human light chain κ locus variable region contains two nearly identical 400 kb repeats separated by an 800 kb spacer (Weichhold, GM et al. (1993) The human immunoglobulin kappa locus consists in two copies that are organized in opposite polarity. Genomics 16, 503-511). Because the repeats are so similar, almost the entire diversity of the locus can be reproduced in mice using the proximal repeat. Additionally, a naturally occurring human allele of the light chain κ locus that misses the distal repeat has been reported (Schaible, G. et al. (1993) The immunoglobulin kappa locus: polymorphism and haplotypes of
[000600] [000600] A human genomic fragment of about 480 kb in size, containing the entire immunoglobulin κ light chain variable region, was inserted in four sequential steps (FIG. 2D; Tables 2 and 4), with up to 150 kb of human immunoglobulin κ light chain sequence inserted in a single step, using methods similar to those employed for the heavy chain (see example 1). The final hygromycin resistance gene was removed by transient expression of FLPe. As with the heavy chain, the targeted ES cell clones were evaluated for complete human insert integrity, normal karyotype, and germline potential after each step. Mice homozygous for each of the κ light chain alleles were generated and found to be healthy and normal in appearance.
[000601] [000601] At various points, ES cells carrying a portion of the human immunoglobulin heavy chain or κ light chain variable repertoire, in the manner described in example 1, were microinjected and the resulting mice were bred to create multiple versions of mice. VELOCIMMUNE® with progressively larger fractions of the human germline immunoglobulin repertoires (Table 5; FIG. 5A and 5B). VELOCIMMUNE® 1 (V1) mice have 18 human VH gene segments and all human DH and JH gene segments combined with 16 human Vκ gene segments and all human Jκ gene segments. VELOCIMMUNE® 2 (V2) and VELOCIMMUNE® (V3) mice had larger variable repertoires carrying a total of 39 VH and 30 Vκ, and 80 VH and 40 Vκ, respectively. Once the genomic regions encoding mouse VH, DH, and JH gene segments, and Vκ and Jκ gene segments have been completely replaced, antibodies produced by any mouse version of VELOCIMMUNE® contain human variable regions linked to constant regions. of mouse. The mouse λ light chain loci remain intact in all versions of the VELOCIMMUNE® mice, and serve as a comparator for the expression efficiency of the various VELOCIMMUNE® κ light chain loci.
[000602] [000602] Mice doubly homozygous for both humanization of immunoglobulin heavy chain and light chain κ were generated from a subset of the alleles described in example 1. All genotypes observed during the course of mating to generate the doubly homozygous mice occurred in approximately Mendelian proportions. Progeny from males homozygous for each of the human heavy chain alleles showed reduced fertility. Reduced fertility resulted from loss of mouse ADAM6 activity. The mouse heavy chain variable gene locus contains two functional inserted ADAM6 genes (ADAM6a and ADAM6b). During humanization of the mouse heavy chain variable gene locus, the inserted human genomic sequence contained an ADAM6 pseudogene. Mouse ADAM6 may be required for fertility, and thus loss of the mouse ADAM6 gene at humanized heavy chain variable gene loci may lead to reduced fertility in these mice despite the presence of the human pseudogene. Examples 7-9 describe the exact replacement of the mouse-deleted ADAM6 gene placed at a humanized heavy chain variable gene locus, and the recovery of a wild-type fertility level in mice with a humanized immunoglobulin heavy chain locus. TABLE 5 Heavy Chain Light Chain κ Mouse Version VH of Gene 5' Vκ of Gene 5' VELOCIMMUNE® Allele Human Allele Human VH Vκ V1 18 18hVH VH1-18 16 16hVκ Vκ1-16 V2 39 39hVH VH4-39 30 30hVκ Vκ2-29 V3 80 80hVH VH3-74 40 40hVκ Vκ2-40 Example 3. Lymphocyte populations in mice with humanized immunoglobulin genes
[000603] [000603] Mature B cell populations in the three different versions of VELOCIMMUNE® mice were evaluated by cytometry of
[000604] [000604] Briefly, bone marrow, spleen and thymus cell suspensions were produced using standard methods. Cells were resuspended at 5x10 5 cells/mL in BD Pharmingen FACS staining buffer, blocked with anti-mouse CD16/32 (BD Pharmingen), stained with the appropriate cocktail of antibodies and fixed with BD Cytofix™, all according to instructions. from the manufacturer. Final cell pellets were resuspended in 0.5 mL of staining buffer and analyzed using BD FACSCALIBUR™ and BD CELLQUEST PRO™ software. All antibodies (BD Pharmingen) were prepared in a dilution/mass cocktail and added to a final concentration of 0.5 mg/10 5 cells. Bone marrow staining antibody cocktails (A-D) were as follows: A: anti-mouse IgMb-FITC, anti-mouse IgMa-PE, anti-mouse CD45R(B220)-APC; B: anti-mouse CD43(S7)-PE, anti-mouse CD45R(B220)-APC; C: anti-mouse CD24(HSA)-PE; anti-mouse CD45R(B220)-APC; D: anti-mouse BP-1-PE, anti-mouse CD45R(B220)-APC. Antibody cocktails for staining spleen and inguinal lymph node (E-H) were as follows: E: anti-mouse IgMb-FITC, anti-mouse IgMa-PE, anti-mouse CD45R(B220)-APC; F: anti-mouse Ig, 1, 2, 3 Light chain-FITC, anti-mouse Igκ-PE light chain, anti-mouse CD45R(B220)-APC; G: anti-mouse Ly6G/C-FITC, anti-mouse CD49b(DX5)-PE, anti-mouse CD11b-APC; H: anti-mouse CD4(L3T4)-FITC, anti-mouse CD45R(B220)-PE, anti-mouse CD8a-APC. The results are shown in FIG. 6.
[000605] [000605] Lymphocytes isolated from the spleen or lymph node of homozygous VELOCIMMUNE® mice were stained for surface expression of markers B220 and IgM and analyzed using flow cytometry (FIG. 6). The sizes of mature B220+ IgM+ B cell populations
[000606] [000606] Allelic exclusion and locus selection. The ability to maintain allelic exclusion was examined in mice heterozygous for
[000607] [000607] The humanization of the immunoglobulin loci was performed in an F1 ES strain (F1H4 (Valenzuela et al., 2003)), derived from heterozygous 129S6/SvEvTac and C57BL/6NTac embryos. The human germline heavy chain variable gene sequences are targeted to the 129S6 allele, which carries the IgMa halotype, whereas the unmodified mouse C576BL/6N allele carries the IgMb halotype. These allelic forms of IgM can be distinguished by flow cytometry using antibodies specific to the polymorphisms found in the IgMa or IgMb alleles. In the manner shown in FIG. 6 (bottom row), B cells identified in mice heterozygous for each version of the humanized heavy chain locus express only a single allele, both IgMa (the humanized allele) and IgMb (the wild-type allele). This demonstrates that the mechanisms involved in allelic exclusion are intact in VELOCIMMUNE® mice. Furthermore, the relative number of B cells positive for the humanized allele (IgMa) is likely proportional to the number of VH gene segments present. The humanized immunoglobulin locus is expressed in approximately 30% of B cells in heterozygous VELOCIMMUNE® 1 mice, have 18 human VH gene segments, and in 50% of B cells in heterozygous VELOCIMMUNE® 2 and 3 mice (not shown), with 39 and 80 human VH gene segments, respectively. Notably, the ratio of cells expressing the humanized versus wild type mouse allele (0.5 for VELOCIMMUNE® 1 mice and 0.9 for VELOCIMMUNE® 2 mice) is more than the ratio of the number of variable gene segments contained in the humanized versus wild type loci (0.2 for VELOCIMMUNE® 1 mice and 0.4 for VELOCIMMUNE® 2 mice). This may indicate that the probability of choosing
[000608] [000608] Polymorphisms of the Cκ regions are not available in 129S6 or C57BL/6N to examine allelic exclusion of humanized versus non-humanized κ light chain loci. However, VELOCIMMUNE® mice all have wild-type mouse λ light chain loci, so it is possible to observe whether rearrangement and expression of humanized κ light chain loci can prevent mouse λ light chain expression. The ratio of the number of cells expressing humanized κ light chain to the number of cells expressing mouse λ light chain was relatively unchanged in mice.
[000609] [000609] B cell development. Because the mature B cell populations in VELOCIMMUNE® mice resemble those of wild-type mice (described above), it is possible that defects in early B cell differentiation are compensated for by expanding populations of mature B cell. The various stages of B cell differentiation were examined by analyzing B cell populations using flow cytometry. Table 6 presents the ratio of cell fractions in each B cell lineage defined by FACs, using specific cell surface markers, in VELOCIMMUNE® mice compared to wild type pups.
[000610] [000610] Early B cell development occurs in the bone marrow, and different stages of B cell differentiation are characterized by changes in the types and amounts of cell surface marker expression. These differences in surface expression are related to the
[000611] [000611] No major defects were observed in B cell differentiation in any of the VELOCIMMUNE® mice. The introduction of human heavy chain D gene segments does not appear to affect the pro-B to pre-B transition, and the introduction of human κ light chain gene segments does not affect the pre-B to B transition in VELOCIMMUNE® mice. This demonstrates that “reverse chimeric” immunoglobulin molecules, which have human variable and mouse constant regions, function normally in the context of B cell signaling and co-receptor molecules, leading to proper B cell differentiation in a mouse environment. . In contrast, the balance between different populations during B cell differentiation is disturbed in different variations in mice that contain randomly integrated immunoglobulin transgenes and inactivated endogenous heavy chain or κ light chain loci (Green and Jakobovits, 1998). Example 4. Variable gene repertoire in mice with humanized immunoglobulin
[000612] [000612] The use of human variable gene segments in
[000613] [000613] Briefly, total RNA was extracted from 1 x 107-2 x 107 splenocytes or about 104-105 hybridoma cells using TRIZOL™ (Invitrogen) or Qiagen RNEASY™ Mini Kit (Qiagen), and prepared with specific oligonucleotide primers constant region test using the SUPERSCRIPT™ III One-Step RT-PCR system (Invitrogen). Reactions were performed with 2-5 µL of RNA from each sample using the aforementioned constant-specific 3' oligonucleotide primers, paired with grouped master oligonucleotide primers for each family of human variable regions, for both the heavy chain and the light chain. κ, separately. Reagent and oligonucleotide primer volumes and RT-PCR/PCR conditions were performed according to the manufacturer's instructions. Oligonucleotide primer sequences were based on multiple sources (Wang, X. and Stollar, BD (2000) Human immunoglobulin variable region gene analysis by single cell RT-PCR. J Immunol Methods 244:217-225; Ig-primer sets, Novagen). Where appropriate, nested and secondary PCR reactions were performed with pooled family structure-specific oligonucleotide primers and the same mouse 3' constant immunoglobulin-specific oligonucleotide primer used in the main reaction. Aliquots (5 µL) of each reaction were analyzed by agarose electrophoresis and the reaction products were purified from the agarose using a MONTAGE™ gel extraction kit (Millipore). Purified products were cloned using the TOPO™ TA Cloning System (Invitrogen) and
[000614] [000614] Use of immunoglobulin variable gene. Plasmid DNA from both heavy chain and κ light chain clones were sequenced with both T7 and M13 reverse oligonucleotide primers on the ABI 3100 genetic analyzer (Applied Biosystems). The raw sequence data was imported into SEQUENCHER™ (v4.5, Gene Codes). each sequence was assembled into contiguous sequences and aligned to human immunoglobulin sequences using IMGT V-Quest function search (Brochet, X., Lefranc, MP, and Giudicelli, V. (2008). IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized VJ and VD-J sequence analysis. Nucleic acids Res 36, W503-508) to identify the use of human VH, DH, JH and Vκ and Jκ segments. Sequences were compared with germline sequences by somatic hypermutation junction analysis and junction recombination.
[000615] [000615] Mice were generated from ES cells containing the initial heavy chain modification (3hVH-CRE Hybrid Allele, lower part of FIG. 2A) by RAG complementation (Chen, J. et al. (1993) RAG-2 - deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proc Natl Acad Sci USA 90, 4528-4532), and cDNA was prepared from splenocyte RNA. The cDNA was amplified using oligonucleotide primer sets (described above) specific for the predicted chimeric heavy chain mRNA, which can originate by V(D)J recombination in the inserted human gene segments and subsequent splicing of each of the mouse IgM or IgG constant domains. The sequences derived from these cDNA clones (not
[000616] [000616] In a similar experiment, B cells from unimmunized and VELOCIMMUNE® wild-type mice were separated by flow cytometry, based on surface expression of B220 and IgM or IgG. B220+ IgM+ or IgG+ (sIgG+) surface cells were pooled and VH and Vκ sequences were obtained after RT-PCR amplification and cloning (described above). Representative gene usage in a set of RT-PCR amplified cDNAs from unimmunized VELOCIMMUNE® 1 mice (Table 7) and VELOCIMMUNE® 3 mice (Table 8) was recorded (*RSS defective; †nonsense or pseudogene). TABLE 7 Observed VH Observed DH Observed Vκ Observed 1-18 3 1-1 1 1-16 2 1-17P 0 2-2 2 3-15 1 3-16* 0 3-3 4 1-12 5 3-15 13 4 -4 0 3-11 1 3-13 9 5-5 0 1-9 5 3-11 6 5-18 4 1-8 2 3-9 8 6-6 5 3-7* 0 1-8 6 1- 7 7 1-6 5 3-7 2 2-8 0 1-5 8 2-5 2 3-9 4 5-2 6 1-3 0 3-10 2 4-1 8 1-2 11 4-11 1 6-1 5 5-12 1 Jκ Observed 6-13 3 1 12 JH Observed 1-14 0 2 10 1 2 2-15 0 3 5 2 1 3-16 1 4 10 3 8 4-17 0 5 0 4 33 6-19 2 5 5 1-20 2 6 16 2-21 1 3-22 0 4-23 2
[000617] [000617] As shown in Tables 7 and 8, almost all functional human VH, DH, JH, Vκ and Jκ gene segments are utilized. Of the functional variable gene segments described but not detected
[000618] [000618] The heavy and light chain sequences of antibodies are known to show exceptional variability, especially in short segments of polypeptides in the rearranged variable domain. These regions, known as hypervariable regions or complementary determining regions (CDRs) create the antigen-binding site in the structure of the antibody molecule. Intervening polypeptide sequences are called framework regions (FRs). There are three CDRs (CDR1, CDR2, CDR3) and 4 FRs (FR1, FR2, FR3, FR4) in both heavy and light chains. one CDR, CDR3, is unique, in that this CDR is created by recombination of both the VH, DH and JH and Vκ and Jκ gene segments, and generates a significant amount of repertoire diversity before the antigen is encountered. This union is imprecise because of both nucleotide deletions by
[000619] [000619] In the manner shown in FIG. 7A, template-free encoded nucleotide additions (N additions) are seen at both the VH-DH and DH-JH junctions in VELOCIMMUNE® mouse antibodies, indicating optimal TdT function with the human segments. The endpoints of the VH, DH, and JH segments relative to their germline counterparts indicate that exonuclease activity also occurred. Unlike the heavy chain locus, human κ light chain rearrangements exhibit little or no TdT addition to CDR3, which is formed by recombination of the Vκ and Jκ segments (FIG. 7B). This is expected because of the loss of TdT expression in mice during light chain rearrangements in the pre-B to B cell transition. of recombination.
[000620] [000620] Somatic hypermutation. Additional diversity is added to the variable regions of rearranged immunoglobulin genes during germinal center reaction by a process called somatic hypermutation. B cells that express somatic mutated variable regions compete with other B cells to access the
[000621] [000621] Sequences of random VH or Vκ clones of sIgM+ or sIgG+ B cells from unimmunized VELOCIMMUNE® mice or sIgG+ B cells from immunized mice were compared with their germline variable gene segments, and exchanged with respect to the germline sequence annotated. The resulting nucleotide sequences were translated in silico and the mutations that led to the amino acid changes were also noted. Data were collected from all variable regions and the percentage shift at a given position was calculated (FIG. 8).
[000622] [000622] In the manner shown in FIG. 8 , human heavy chain variable regions derived from sIgG+ B cells from unimmunized VELOCIMMUNE® mice exhibit much more nucleotides than sIgM+ B cells from the same splenocyte clusters, and heavy chain variable regions derived from immunized mice exhibit even more changes. The number of alterations is greater in the complementarity determining regions (CDRs) than in the framework regions, indicating antigen selection. The corresponding amino acid sequences of the human heavy chain variable regions also exhibit significantly greater numbers of mutations in IgG vs IgM, and even more in immunized IgG. These mutations again appear to be more frequent in CDRs compared to framework sequences, suggesting
[000623] [000623] Gene use and somatic hypermutation observed in VELOCIMMUNE® mice demonstrates that essentially all gene segments present are able to rearrange to form fully functional reverse chimeric antibodies in these mice. Additionally, VELOCIMMUNE® antibodies fully participate in the mouse immune system to undergo selection and affinity maturation to create fully mature human antibodies that can efficiently neutralize their target antigen. VELOCIMMUNE® mice are able to mount robust immune responses to multiply classes of antigens that result in the use of a wide range of human antibodies that are both high-finity and suitable for therapeutic use (data not shown). Example 5. Analysis of lymphoid structure and serum isotypes
[000624] [000624] The thick structures of spleen, inguinal lymph nodes, Peyer's patches and thymus of tissue samples from wild type or VELOCIMMUNE® mice, stained with H&E, were examined by light microscopy. Immunoglobulin isotype levels in serum collected from wild-type and VELOCIMMUNE® mice were analyzed using LUMINEX™ technology.
[000625] [000625] Structure of the lymphoid organ. The structure and function of lymphoid tissues are in part dependent on the proper development of hematopoietic cells. A defect in B cell development or function can be displayed as a change in the structure of lymphoid tissues. Upon analysis of stained tissue sections, no significant differences in the appearance of secondary lymphoid organs between wild-type and VELOCIMMUNE® mice were identified (data not shown).
[000626] [000626] Serum immunoglobulin levels. The expression level of each isotype is similar in wild type and VELOCIMMUNE® mice (FIG. 9A, 9B and 9C). This demonstrates that humanization of the variable gene segments does not appear to have an adverse effect upon class switching or immunoglobulin expression and secretion and therefore apparently maintains all of the mouse endogenous sequences necessary for these functions. Example 6. Immunization and antibody production in mice with humanized immunoglobulin
[000627] [000627] Different versions of VELOCIMMUNE® mice were immunized with antigen to examine the humoral response to challenge the foreign antigen.
[000628] [000628] Immunization and hybridoma development. VELOCIMMUNE® and wild-type mice can be immunized with an antigen in the form of protein, DNA, a combination of DNA and protein, or cells that express the antigen. Animals are typically revaccinated every three weeks for a total of two to three times. After each antigen boost, serum samples from each animal are collected and analyzed for specific antigen-antibody responses by determining the serum titer. Prior to fusion, mice received a final pre-fusion boost of 5 µg of protein or DNA, as desired, via intraperitoneal and/or intravenous injections. Splenocytes are collected and fused into Ag8.653 myeloma cells in an electrofusion chamber, according to the manufacturer's suggested protocol (Cyto Pulse Sciences Inc., Glen Burnie, MD). Ten days after culture, hybridomas are selected for antigen specificity using an ELISA assay (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Press, New York). Alternatively, specific B cell antigens are isolated directly from immunized VELOCIMMUNE® mice and selected using standard techniques, including those here
[000629] [000629] Determination of serum titration. To monitor the animal's anti-antigen serum responses, serum samples are collected approximately 10 days after each boost and titers are determined using antigen-specific ELISA. Briefly, 96-well Nunc MAXISORP™ plates are coated with 2 #g/mL antigen overnight at 4°C and blocked with bovine serum albumin (Sigma, St. Louis, MO). Serum samples at a 3-fold serial dilution are allowed to bind to the plates for one hour at room temperature. Plates are then washed with PBS containing 0.05% Tween-20 and bound IgG is detected using HRP-conjugated goat anti-mouse Fc (Jackson Immuno Research Laboratories, Inc., West Grove, PA) for total titration of IgG, or isotype-specific biotin-labeled or specific light chain polyclonal antibodies (SouthernBiotech Inc.) for isotype-specific titers, respectively. For biotin-labeled antibodies, after washing the plate, HRP-conjugated streptavidin (Pierce, Rockford, IL) is added. All plates are developed using colorimetric substrates such as BD OPTEIA™ (BD Biosciences Pharmingen, San Diego, CA). After the reaction is stopped with 1M phosphoric acid, optical absorptions at 450 nm are recorded and the data analyzed using Graph Pad's PRISM™ software. Dilutions required to obtain two background signal times are defined as titration.
[000630] [000630] In one experiment, VELOCIMMUNE® mice were immunized with human interleukin-6 receptor (hIL-6R). A representative set of serum titers for VELOCIMMUNE® and wild-type mice immunized with hIL-6R is shown in FIG. 10A and 10B.
[000631] [000631] VELOCIMMUNE® and wild type mice
[000632] [000632] Determination of binding affinity of antibody with antigen in solution. An ELISA-based collation competition assay is typically designed to determine the binding affinity of the antibody with the antigen.
[000633] [000633] Briefly, antibodies in conditioned medium are premixed with serial dilutions of antigen protein ranging from 0 to 10 mg/mL. The antibody and antigen mixture solutions are then incubated for two to four hours at room temperature to reach binding equilibrium. The amounts of free antibody in the mixtures are then measured using a quantitative sandwich ELISA. Ninety-six-well MAXISORB™ plates (VWR, West Chester, PA) are coated with 1 µg/mL antigen protein in PBS solution overnight at 4°C, followed by non-specific blocking of BSA. The mixture of antibody-antigen solutions is then transferred to these plates, followed by an hour of incubation. The plates are then washed with wash buffer and the antibodies bound on the plate were detected with an HRP-conjugated goat anti-mouse IgG polyclonal antibody reagent (Jackson Immuno Research Lab), and developed using colorimetric substrates such as BD
[000634] [000634] In one experiment, VELOCIMMUNE® mice were immunized with hIL-6R (in the manner described above). FIGS. 11A and 11B show a representative set of affinity measurements for anti-hIL6R antibodies from VELOCIMMUNE® and wild-type mice.
[000635] [000635] After immunized mice receive a third antigen boost, serum titers are determined by ELISA. Splenocytes are isolated from selected cohorts of wild-type and VELOCIMMUNE® mice and fused to Ag8.653 myeloma cells to form hybridomas and grown in selection (in the manner described above). Of a total of 671 anti-IL-6R hybridomas produced, 236 were observed to express antigen-specific antibodies. Media collected from the antigen-positive wells was used to determine the antigen-binding affinity of antibody using a competitive ELISA solution. Antibodies derived from VELOCIMMUNE® mice exhibit a wide range of affinity in binding antigen in solution (FIG. 11A). In addition, 49 of 236 anti-IL-6R hybridomas were observed to block IL-6 from binding to the receptor in an in vitro bioassay (data not shown). Additionally, these anti-IL-6R blocking antibodies exhibited a high-affinity solution range, similar to those blocking antibodies derived from parallel immunization of wild-type mice (FIG. 11B).
[000636] [000636] A targeting vector for the insertion of mouse ADAM6a and ADAM6b genes into a humanized heavy chain locus was constructed using VELOCIGENE® gene alteration technology (supra) to modify a bacterial artificial chromosome (BAC) 929d24, obtained from Dr. .Fred Alt (Havard University). The BAC 929d24 DNA was genetically modified to contain genomic fragments containing the mouse ADAM6a and ADAM6b genes and a hygromycin cassette for targeted elimination of a human ADAM6 pseudogene (hADAM6Ψ), located between the VH1-2 and VH6- gene segments. 1 human from a humanized heavy chain locus (FIG. 12).
[000637] [000637] First, a genomic fragment containing the mouse ADAM6b gene, ~800 bp of the upstream (5') sequence and ~4800 bp of the downstream (3') sequence, was subcloned from the BAC clone 929d24. A second genomic fragment containing the mouse ADAM6a gene, ~300 bp of the upstream (5') sequence and ~3400 bp of the downstream (3') sequence, was subcloned separately from clone BAC 929d24. The two genomic fragments containing the mouse ADAM6b and ADAM6 genes were ligated into a hygromycin cassette flanked by the Frt recombination sites to create the targeting vector (mouse ADAM6 targeting vector, FIG. 20; SEQ ID NO:3). Different restriction enzyme sites were genetically modified at the 5' end of the targeting vector after the mouse ADAM6b gene, and at the 3' end after the mouse ADAM6a gene (bottom of FIG. 12) for ligation at the humanized chain locus. heavy.
[000638] [000638] A separate modification was produced in a BAC clone containing a replacement of the mouse heavy chain locus with the human heavy chain locus, including the ADAM6 pseudogene from
[000639] [000639] Briefly, a neomycin cassette flanked by the loxP recombination sites was genetically modified to contain homology arms containing the human genomic sequence at positions 3' of the human VH1-2 gene segment (5' to hADAM6Ψ) and 5' of human VH6-1 gene segment (3' to hADAM6Ψ; see middle part of FIG. 13). The insertion site location of this targeting construct was about 1.3 kb 5' and ~350 bp 3' of the human ADAM6 pseudogene. The targeting construct also included the same restriction sites as the mouse ADAM6 targeting vector to allow for subsequent BAC binding between the BAC clone containing the human ADAM6 pseudogene deletion and the mouse ADAM6 targeting vector.
[000640] [000640] Following digestion of BAC DNA derived from both constructs, the genomic fragments were ligated together to construct a genetically modified BAC clone containing a humanized heavy chain locus that contains an ectopically placed genomic sequence comprising ADAM6a and mouse ADAM6b. The final targeting construct for deleting a human ADAM6 gene at a humanized heavy chain locus, and inserting mouse ADAM6a and ADAM6b sequences into ES cells contained, from 5' to 3', a 5' genomic fragment containing ~13 kb of human genomic sequence 3' of the human VH1-2 gene segment, ~800 bp of mouse genomic sequence downstream of the mouse ADAM6b gene, the mouse ADAM6b gene, ~4800 bp of upstream genomic sequence from the mouse ADAM6b gene, a 5' Frt site, a hygromycin cassette, a 3' Frt site, ~300 bp of genomic sequence from
[000641] [000641] The genetically modified BAC clone (described above) was used to electroporate mouse ES cells that contained a humanized heavy chain locus to create modified ES cells comprising an ectopically placed mouse genomic sequence comprising sequences from ADAM6a and ADAM6b of mice at a humanized heavy chain locus. Positive ES cells containing the mouse ectopic genomic fragment at the humanized heavy chain locus were identified by a quantitative PCR assay using TAQMAN™ probes (Lie, YS and Petropoulos, CJ (1998) Advances in quantitative PCR technology: 5'nuclease assays Curr Opin Biotechnol 9(1):43-48). The upstream and downstream regions outside the modified portion of the humanized heavy chain locus were confirmed by PCR using oligonucleotide primers and probes located in the modified region, to confirm the presence of the mouse ectopic genomic sequence at the humanized heavy chain locus, as well as in the hygromycin cassette. The nucleotide sequence through the upstream insertion point included the following, which indicates the human heavy chain genomic sequence upstream of the insertion point and an I-Ceu I restriction site (contained in parentheses below) contiguously linked to the mouse genomic sequence present at the insertion point: (CCAGCTTCAT TAGTAATCGT TCATCTGTGG TAAAAAGGCA GGATTTGAAG CGATGGAAGA TGGGAGTACG GGGCGTTGGA AGACAAAGTG CCACACAGCG CAGCCTTCGT CTAGACCCCC GGGCTAACTA TAACGGTCCT AAGGTAGCGA G)
[000642] [000642] The previously described targeted ES cells were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® mouse genetic modification method (see, for example, US patents 7,6598,442, 7,576 .259,
[000643] [000643] Mice carrying a humanized heavy chain locus that contain mouse ADAM6a and ADAM6b genes are crossed with a mouse FLPe deleter strain (see, eg, Rodríguez, CI et al. (2000) ) High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nature Genetics 25:139-140) in order to remove
[000644] [000644] The offspring are genotyped and a offspring heterozygous for a humanized heavy chain locus containing a mouse ectopic genomic fragment, which comprises the mouse ADAM6a and ADAM6b sequences, is selected to characterize the mouse ADAM6 gene expression and fertility . Example 8. Characterization of mice with recovered ADAM6
[000645] [000645] Flow cytometry. Three mice at age 25 weeks, homozygous for human heavy chain and human κ light chain (H/κ) variable gene loci, and three mice at age 18-20 weeks, homozygous for human heavy chain and human κ light, with the mouse ectopic genomic fragment encoding the mouse ADAM6a and ADAM6b genes in both alleles of the human heavy chain locus (H/κ-A6) were sacrificed for identification and analysis of human cell populations. lymphocyte by FACs in the BD LSR II system (BD Bioscience). Lymphocytes were linked by specific cell lines and analyzed for progression to various stages of B cell development. Tissues collected from the animals included blood, spleen, and bone marrow. Blood was collected in BD microtainer tubes with EDTA (BD Biosciences). Bone marrow was collected from the femurs flushed with complete RPMI medium and supplemented with fetal bovine serum, sodium pyruvate, HEPES, 2-mercaptoethanol, essential amino acids and gentamicin. Red blood cells from the blood, spleen and bone marrow preparations were lysed with an ammonium chloride-based lysis buffer (eg, ACK lysis buffer), followed by washing with RPMI complete medium.
[000646] [000646] For staining of cell populations, 1 x 106 cells from
[000647] [000647] Bone marrow: anti-mouse FITC-CD43 (1B11, BioLegend), PE-ckit (2B8, BioLegend), PeCy7-IgM (II/41, eBioscience), PerCP-Cy5.5-IgD (11-26c. 2a, BioLegend), APC-eFluor780-B220 (RA3-6B2, eBioscience), A700-CD19 (1D3, BD Biosciences).
[000648] [000648] Peripheral blood and spleen: anti-mouse FITC-κ (187.1, BD Biosciences), PE-λ (RML-42, BioLegend), PeCy7-IgM (II/41, eBioscience), PerCP-Cy5.5-IgD (11-26c.2a, BioLegend), APC-CD3 (145-2C11, BD), A700-CD19 (1D3, BD), APC-eFluor780-B220 (RA3-6B2, eBioscience). After incubation with labeled antibodies, cells were washed and fixed in 2% formaldehyde. Acquisition data were performed on an LSRII flow cytometer and analyzed with FlowJo. Representative results from an H/κ and H/κ-A6 mouse are shown in FIGS. 14 - 18.
[000649] [000649] The results demonstrate that B cells from H/κ-A6 mice progress to the stages of B cell development, in a similar manner to H/κ mice in bone marrow and peripheral compartments, and show normal patterns of maturation, once they enter the periphery. H/κ-A6 mice demonstrated a higher CD43intCD19+ cell population compared to H/κ mice (FIG. 16B). This may indicate an accelerated IgM expression from the humanized heavy chain locus, containing an ectopic mouse genomic fragment comprising the mouse ADAM6a and ADAM6b sequences in H/κ-A6 mice. In the periphery, populations and B and T cells of H/κ-A6 mice appear normal and similar to H/κ mice.
[000650] [000650] Testicle morphology and characterization of
[000651] [000651] Briefly, the testes of two groups of five mice per group (Group 1: mice homozygous for human heavy chain and κ light chain variable gene loci, mADAM6-/-; Group 2: mice heterozygous for gene loci human heavy chain variable and homozygous for κ light chain variable gene loci, mADAM6+/-) were dissected with the epididymis intact and weighed. The specimens were then fixed, embedded in paraffin, sectioned and stained with hematoxylin and eosin (HE) dye. Testis sections (2 tests per mouse, for a total of 20) were examined for defects in morphology and evidence of sperm production, while epididymal sections were examined for the presence of sperm.
[000652] [000652] In this experiment, no difference in testis weight or morphology was observed between mADAM6-/- mice and mADAM6+/- mice. Sperm were observed in all genotypes, both in the testis and in the epididymis. These results establish that the absence of mouse ADAM6a and ADAM6b genes does not lead to detectable alterations in testis morphology, and that sperm are produced in mice in the presence and absence of these two genes. Deficiencies in fertility in ADAM6-/- male mice are therefore not likely due to poor sperm production.
[000653] [000653] Sperm motility and migration. Mice that lose other elements of the ADAM gene family are infertile due to defects in sperm motility or migration. Sperm migration is defined as the ability of sperm to pass from the uterus into the oviduct, and is generally required for fertilization in mice.
[000654] [000654] Briefly, sperm was obtained from testing (1) mice heterozygous for human heavy chain variable gene loci and homozygous for human κ light chain variable gene loci (ADAM6+/-); (2) mice homozygous for human heavy chain variable gene loci and homozygous for human κ light chain variable gene loci (ADAM6-/-); (3) mice homozygous for human heavy chain variable gene loci and homozygous for wild-type κ light chain (ADAM6-/-mκ); and, (4) wild type C57 BL/6 (WT) mice. No significant abnormalities were observed in sperm count or overall sperm motility by inspection. For all mice, cumulus dispersion was observed, indicating that each sperm sample was able to penetrate cumulus cells and bind to the zona pellucida in vitro. These results establish that ADAM6-/- mice have sperm that are able to penetrate the cumulus and bind to the zona pellucida.
[000655] [000655] Mouse in vitro fertilization (IVF) was performed using mouse sperm in the manner described above. A small number of cleaved embryos were present for ADAM6 -/- the day after IVF, as well as a reduced number of sperm bound to the eggs. These results establish that sperm from ADAM6-/- mice, once exposed to an egg, are able to penetrate the cumulus and bind to the zona pellucida.
[000656] [000656] In another experiment, the ability of sperm from ADAM6 -/- mice to migrate from the uterus to the oviduct was determined in a sperm migration assay.
[000657] [000657] Briefly, a first group of five superovulated female mice were mated with five ADAM6-/- males. A second group of five superovulated female mice was mated with five ADAM6+/- males. Mating pairs were observed for mating, and five to six hours after mating the uterus and attached oviduct of all females were removed and stained for analysis. Staining solutions were checked by eggs to check for ovulation and obtain a sperm count. Sperm migration was assessed in two different ways. First, both oviducts were removed from the uterus, washed with saline, and any identified sperm were counted. The presence of eggs has also been observed as evidence of ovulation. Second, the oviducts were left attached to the uterus and both tissues were fixed, embedded in paraffin, sectioned and stained (in the manner described above). The sections were examined for the presence of sperm, both in the uterus and in the oviducts.
[000658] [000658] For the five females mated with the five ADAM6-/- males, very little sperm was found in the stained solution of the oviduct. The stained solutions from the oviducts of the five females mated to the five ADAM6+/- males exhibited a sperm level about 25- to 30 times higher (mean, n = 10 oviducts) than that present in the stained solutions from the oviducts of the five females. mated with the five ADAM6-/- males.
[000659] [000659] Histological sections of uterus and oviduct were prepared. The sections were examined for the presence of sperm in the uterus and in the oviduct (the tubal colliculus). Inspection of histological sections of oviduct and uterus revealed that for female mice mated with ADAM6-/- mice, sperm was found in the uterus but not in the oviduct. Additionally, sections of females mated with ADAM6-/- mice revealed that sperm was not found.
[000660] [000660] These results establish that mice that lose the ADAM6a and ADAM6b genes produce sperm that exhibit a defect in migration in vivo. In all cases, sperm was observed in the uterus, indicating that copulation and sperm release apparently proceed in a normal manner, but little or no sperm were observed in the oviducts after copulation, as measured by both sperm count and observation. histological These results establish that mice that lose the ADAM6a and ADAM6b genes produce sperm that exhibit an inability to migrate from the uterus to the oviduct. This defect apparently leads to infertility, as sperm are unable to cross the uterine-tubule junction into the oviduct, where eggs are fertilized. Taken together, all these results converge to support the hypothesis that the ADAM6 gene from mice directly assists normally motile sperm to migrate out of the uterus, up to the uterotubal junction and into the oviduct, and thus approach an egg to reach the fertilization event. The mechanism by which ADAM6 achieves this can be directly through the action of ADAM6 proteins, or even coordinated expression with other proteins, eg other ADAM proteins, in the sperm cell, as described below.
[000661] [000661] ADAM family gene expression. A complex of ADAM proteins are known to be present as a complex on the surface of maturing sperm. Mice that lose other ADAM family gene elements lose this complex as mature sperm, and exhibit a reduction of multiple ADAM proteins in mature sperm. To determine whether a loss of genes
[000662] [000662] In this experiment, protein extracts were analyzed from four ADAM6-/- and four ADAM6+/- mice. The results show that the expression of ADAM2 and ADAM3 was not affected in testis extracts. However, both ADAM2 and ADAM3 were greatly reduced in epididymal extracts. This demonstrates that the absence of ADAM6a and ADAM6b in sperm from ADAM6-/- mice may have a direct effect on the expression and perhaps the function of other ADAM proteins such as mature sperm (eg ADAM2 and ADAM3). This suggests that ADAM6a and ADAM6b are part of an ADAM protein complex on the sperm surface, which may be important for proper sperm migration. Example 9. Use of human heavy chain variable gene in mice that rescue ADAM6
[000663] [000663] Use of selected human heavy chain variable gene was determined for mice homozygous for human heavy and light chain variable gene loci κ, either missing the mouse ADAM6a and ADAM6b genes (mADAM6-/-) or containing a ectopic genomic fragment encoding mouse ADAM6a and ADAM6b genes (ADAM6+/+; see example 1) by a quantitative PCR assay using TAQMAN™ probes (in the manner described above).
[000664] [000664] Briefly, CD19+ B cells were purified from the spleens of mADAM6-/- and ADAM6+/+ mice using CD19 mouse microspheres (Miltenyi Biotec), and total RNA was purified using the RNEASY™ Mini Kit (Qiagen). The genomic RNA was removed using
[000665] [000665] In this experiment, the expression of all four human VH genes was observed in the analyzed samples. Additionally, expression levels were comparable between mADAM6-/- and ADAM6+/+ mice. These results demonstrate that human VH genes that were both distal at the modification site (VH3-23 and VH1-69) and proximal at the modification site (VH1-2 and VH6-1) were also able to recombine to form a chain. functionally expressed human weight. These results demonstrate that the ectopic genomic fragment comprising mouse ADAM6a and ADAM6b sequences inserted into a genomic human heavy chain sequence did not affect the recombination of V(D)J gene segments and human heavy chain at the locus, and these mice are able to recombine D gene segments
[000666] [000666] Several targeting constructs have been produced using VELOCIGENE® technology (see, for example, US patent 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech 21(6):652-659) to modify mouse bacterial artificial chromosome (BAC) genomic libraries to inactivate the κ and λ light chain loci.
[000667] [000667] Elimination of mouse light chain λ locus. DNA from mouse BAC clone RP23-135k15 (Invitrogen) was modified by homologous recombination to inactivate the endogenous mouse λ light chain locus until targeted elimination of the Vλ-Jλ-Cλs gene cluster (FIG. 20).
[000668] [000668] Briefly, the entire proximal cluster, comprising Vλ1-Jλ3-Cλ3-Jλ1-Cλ1 gene segments, was eliminated in a single targeting event using a targeting vector comprising a neomycin cassette flanked by loxP sites, with a mouse 5' homology arm containing the 5' sequence of the Vλ1 gene segment and a mouse 3' homology arm containing the 3' sequence of the Cλ1 gene segment (FIG. 20, Targeting Vector 1).
[000669] [000669] A second targeting construct was prepared to exactly eliminate the mouse endogenous distal λ gene cluster containing Vλ2-Jλ2-Cλ2-Jλ4-Cλ4, with the exception that the targeting construct contained a 5' homology arm of mouse that contained the 5' sequence of the Vλ2 gene segment, and a mouse 3' homology arm that contained the 5' sequence of the endogenous Cλ2 gene segment (FIG. 20, Targeting Vector 2). So the second construct
[000670] [000670] Elimination of mouse light chain κ locus. Several targeting constructs were produced using similar methods described above to modify DNA from mouse BAC clones RP23-302g12 and RP23-254m04 (Invitrogen) by homologous recombination to inactivate the mouse light chain κ locus in a two-step process. (FIG. 21).
[000671] [000671] Briefly, the Jκ gene segments (1-5) from the endogenous mouse κ light chain locus were eliminated in a single targeting event using a targeting vector comprising a hygromycin-thymidine kinase (hyg-TK) cassette, containing a single 3' loxP site of the hyg-TK cassette (FIG. 21, Jκ targeting vector). The homology arms used to produce this targeting vector contained mouse 5' and 3' genomic sequence of endogenous mouse Jκ gene segments. In a second targeting event, a second targeting vector was prepared to eliminate a portion of mouse genomic sequence upstream (5') of the most distal mouse endogenous Vκ gene segment (FIG. 21, Vκ targeting vector). This targeting vector contained an inverted lox511 site, a loxP site and a neomycin cassette. The homology arms used to produce this targeting vector contained mouse genomic sequence at
[000672] [000672] Thus, two endogenous modified light chain (κ and λ) loci containing intact enhancers and constant regions were created to progressively insert the unrearranged λ gene segments from the human germline, in an exact manner using vectors of targeting described below. Example 11. Replacement of mouse light chain loci with a human λ light chain mini locus
[000673] [000673] Multiple targeting vectors were genetically engineered for the progressive insertion of human λ gene segments into endogenous mouse κ and λ light chain loci, using similar methods as described above. Multiple and independent initial modifications were produced at the endogenous light chain loci, each of which produced a chimeric light chain locus containing hVλ and Jλ gene segments operably linked to the light chain constant genes and mouse enhancers.
[000674] [000674] A human λ mini-locus containing 12 human Vλ gene segments and one human Jλ gene segment. A series of
[000675] [000675] For a first set of initial targeting vectors, a 124,125 bp DNA fragment from BAC clone 729g4 containing 12 hVλ gene segments and one hJλ1 gene segment was genetically modified to contain a 996 bp PI-SceI site downstream (3') of the hJλ1 gene segment, for ligation of a mouse 3' homology arm. Two different sets of homology arms were used to link to this human fragment; one set of homology arms contained endogenous λ sequence from mice of clone BAC 135k15 (FIG. 22A), and another set contained endogenous κ sequence 5' and 3' of the mouse Vκ and Jκ gene segments of BAC clones RP23-302g12 and mouse RP23-254m04, respectively (FIG. 22B).
[000676] [000676] For the 12/1-λ targeting vector (FIG. 22A), a PI-SceI site was genetically modified at the 5' end of a 27,847 bp DNA fragment containing the mouse Cλ2-Jλ4-Cλ4 and enhancer 2,4 of the modified mouse λ locus described in example 10. The ~28 kb mouse fragment was used as a 3' homology arm by ligation to the ~124 kb human λ fragment, which created a 3' junction containing, from 5' to 3', a hJλ1 gene segment, 996 bp of human λ 3' sequence from the hJλ gene segment, 1229 bp of mouse 5' λ sequence in the mouse Cλ2 gene, in the mouse Cλ2 gene and in the remaining portion of the ~28 kb mouse fragment. Upstream (5') of the human Vλ3-12 gene segment was an additional λ sequence of
[000677] [000677] Thus, the 12/1-λ targeting vector included, from 5' to 3', a 5' homology arm containing ~24 kb of mouse genomic λ sequence, 5' to the endogenous λ locus, a Frt site 5', a neomycin cassette, a 3' Frt site, ~123 kb of human genomic λ sequence containing the first 12 consecutive hVλ gene segments and a hJλ1 gene segment, a PI-SceI site, and a homology arm 3 ' containing ~28 kb of mouse genomic sequence, including the endogenous Cλ2-Jλ4-Cλ4 gene segments, the mouse 2.4 enhancer sequence, and additional mouse genomic sequence downstream (3') of the 2.4 enhancer (FIG. 22A) .
[000678] [000678] In a similar manner, the 12/1-κ targeting vector (FIG. 22B) employed the same human λ ~124 fragment, with the exception that mouse homology arms containing mouse κ sequence were used. , such that targeting of the endogenous κ locus could be achieved by homologous recombination. Thus, the 12/1-κ targeting vector included, from 5' to 3', a 5' homology arm containing ~23 kb of mouse genomic sequence, 5' from the endogenous κ locus, an I-CeuI site, a 5' Frt, a neomycin cassette, a 3' Frt site, ~124 kb of human λ genomic sequence containing the first 12 consecutive hVλ gene segments and a hJλ1 gene segment, a PI-SceI site, and a 3' homology containing ~28 kb of mouse genomic sequence, including the endogenous mouse Cκ, Eκi and Eκ3' gene and additional mouse genomic sequence downstream (3') of Eκ3' (FIG. 22B, Targeting vector 12 /1-κ).
[000679] [000679] Homologous recombination with each of these two initial targeting vectors to create a modified mouse light chain (κ or λ) locus containing 12 hVλ gene segments and one hJλ1 gene segment operably linked to the constant light chain gene mouse endogenous and enhancer genes (Cκ or Cλ2 and Eκi/Eκ3' or Enh 2.4/Enh
[000680] [000680] A human λ mini-locus with 12 human Vλ gene segments and four human Jλ. In another approach to adding diversity to a chimeric λ light chain locus, a third initial targeting vector was genetically modified to insert the first 12 consecutive human Vλ gene segments from cluster A and hJλ1, 2, 3 and 7 in the mouse light chain κ locus (FIG. 22B, 12/4-κ Targeting vector). A DNA segment containing hJλ1, Jλ2, Jλ3 and Jλ7 gene segments was produced by de novo DNA synthesis (Integrated DNA Technologies), including each Jλ gene segment and ~100 bp human genomic sequence from both intermediate regions 5 ' and 3' of each Jλ gene segment. A PI-SceI site was genetically modified at the 3' end of this ~1 kb DNA fragment and ligated into a chloramphenicol cassette. Homology arms were amplified by PCR from the human λ sequence at positions 5' and 3', with respect to the hJλ1 gene segment of human BAC clone 729g4. Homologous recombination with this intermediate targeting vector was performed in a modified BAC 729g4 clone that was previously targeted upstream (5') of the human Vλ32 gene segment with a neomycin cassette flanked by Frt sites, which also contained a 5' I-CeuI site at the 5' Frt site. The double-targeted BAC clone 729g4 included from 5' to 3' an I-CeuI site, a 5' Frt site, a neomycin cassette, a 3' Frt site, a ~123 kb fragment containing the first
[000681] [000681] This ligation resulted in a third targeting vector for insertion of human λ sequences into the endogenous light chain κ locus, which included, from 5' to 3', a mouse 5' homology arm containing ~23 kb 5' genomic sequence of the mouse endogenous κ locus, an I-CeuI site, a 5' Frt site, a neomycin cassette, a 3' Frt site, a ~123 kb fragment containing the first 12 hVλ gene segments, a ~1 kb fragment containing hJλ1, 2, 3 and 7 gene segments, a PI-SceI site and a 3' homology arm containing ~28 kb of mouse genomic sequence, including the endogenous mouse gene Cκ, Eκi and Eκ3', and additional mouse genomic sequence downstream (3') of Eκ3' (FIG. 22B, 12/4-κ Targeting vector). Homologous recombination with this third targeting vector created a modified mouse light chain κ locus containing 12 hVλ gene segments and four hJλ gene segments operably linked to the endogenous mouse Cκ gene which, upon recombination, leads to the formation of a chimeric human λ light/mouse κ light chain.
[000682] [000682] A human λ mini-locus with an integrated human κ light chain sequence. In a similar manner, two additional targeting vectors similar to those genetically engineered to produce an initial insertion of human λ gene segments into the endogenous light chain κ locus (FIG. 22B, 12/1-κ and 12/4-κ Vectors targeting), were genetically modified to progressively insert human λ light chain gene segments, using exclusively targeting vectors.
[000683] [000683] Both targeting vectors containing the human genomic sequence κ were produced using the modified BAC clone RP11-729g4 described above (FIG. 24). This modified BAC clone was targeted with a spectinomycin selection cassette flanked by NotI and AsiSI restriction sites (FIG. 24, upper left). Homologous recombination with the spectinomycin cassette resulted in a double-targeted BAC clone 729g4 that included, from 5' to 3', an I-CeuI site, a 5' Frt site, a neomycin cassette, a 3' Frt site, a ~123 kb fragment containing the first 12 hVλ gene segments, a NotI site about 200 bp downstream (3') of the nonamer sequence of the hVλ3-1 gene segment, a spectinomycin cassette, and an AsiSI site. A separate human BAC clone containing human κ sequence (CTD-2366j12) was targeted twice at genetically modified restriction sites, at sites between the hVκ4-1 and hJκ1 gene segments, to allow subsequent cloning of a ~ 23 kb for ligation with the hVλ gene segments contained in the double-targeted modified BAC clone 729g4 (FIG. 24, upper right).
[000684] [000684] Briefly, clone BAC 2366j12 is about 132 kb in size and contains hVκ gene segments 1-6, 1-5, 2-4, 7-3, 5-2, 4-1, genomic sequence κ of downstream of the Vκ gene segments, hJκ gene segments 1-5, the hCκ and about 20 kb of additional human locus κ locus genomic sequence. This clone was first targeted with
[000685] [000685] Double-targeted clones 729g4 and 2366j12 were digested with NotI and AsiSI, yielding a fragment containing the neomycin cassette and hVλ gene segments, and another fragment containing the ~23 kb genomic fragment from the human κ locus containing the region
[000686] [000686] Additional hVλ gene segments have been added
[000687] [000687] Introduction of 16 additional human Vλ gene segments. Upstream, homology arms (5') used in the construction of targeting vectors to add 16 additional hVλ gene segments to the modified light chain loci described in example 11 contained 5' mouse genomic sequence from each of the endogenous loci light chain λ κ or. The 3' homology arms were the same for all targeting vectors and contained human genomic sequence that overlaps with the 5' end of the human λ sequence of the modifications in the manner described in example 11.
[000688] [000688] Briefly, two targeting vectors were genetically modified by introducing 16 additional hVλ gene segments into the modified mouse light chain loci described in example 11 (FIG. 23A and 5B, +16-λ or +16-κ targeting vector). A ~172 kb DNA fragment from human BAC clone RP11-761l13 (Invitrogen), containing 21 consecutive hVλ gene segments from cluster A, was genetically modified with a 5' homology arm containing mouse 5' genomic sequence in both endogenous κ and λ light chain loci, and a 3' homology arm containing human λ genomic sequence. The mouse 5' κ or λ homology arms used in these targeting constructs were the same as the 5' homology arm described in example 11 (FIG. 23A and 23B). The 3' homology arm included a 53,057 bp overlap of human λ genomic sequence, which corresponds to the 5'-equivalent end of the ~123 kb human λ genomic fragment sequence described in Example 11. These two targeting vectors included , 5' to 3', a mouse 5' homology arm containing both ~23 kb of 5' genomic sequence from the locus
[000689] [000689] In a similar manner, the +16-κ targeting vector was also used to introduce the additional 16 hVλ gene segments in the other early modifications described in example 11, which incorporated multiple hJλ gene segments with and without a κ sequence integrated human (FIG. 22B). Homologous recombination with this targeting vector at the mouse endogenous κ locus containing the other initial modifications created mouse κ light chain loci containing 28 hVλ gene segments and 1, 2, 3 and 7 hJλ gene segments with and without a sequence human Vκ-Jκ genome operably linked to the endogenous mouse Cκ gene which, upon recombination, leads to the formation of a chimeric λ and κ light chain.
[000690] [000690] Introduction of 12 additional human Vλ gene segments. Additional hVλ gene segments were added independently of each of the modifications described above, using targeting vectors and similar methods. The structure of the final locus resulting from homologous recombination with targeting vectors containing
[000691] [000691] Briefly, a targeting vector was genetically modified by introducing 12 additional hVλ gene segments into the previously described mouse modified κ and λ light chain loci (FIG. 23A and 23B, Targeting Vectors +12-λ or 12 -κ). A 93,674 bp DNA fragment from human BAC clone RP11-22I18 (Invitrogen), containing 12 consecutive hVλ gene segments from cluster B, was genetically modified with a 5' homology arm containing mouse 5' genomic sequence at both loci endogenous light chain κ and mouse λ, and a 3' homology arm containing the human λ genomic sequence. The 5' homology arms used in this targeting construct were the same 5' homology arms used for the addition of 16 hVλ gene segments described previously (FIG. 23A and 23B). The 3' homology arm was produced by genetic modification of a ~3431 bp 5' PI-SceI site in the human Vλ3-29P gene segment, contained in a human 27,468 bp genomic fragment λ sequence of the BAC clone RP11- 761I13. This PI-SceI site functions as a binding point for joining the λ sequence of the additional ~94 kb human fragment to the λ sequence of the ~27 kb human fragment that overlaps the 5' end of the human λ sequence in the modification. preview, using the +16-λ or +16-κ targeting vectors (FIG. 23A and 23B). These two targeting vectors included, from 5' to 3', a 5' homology arm containing both a ~23 kb mouse genomic sequence 5' of the endogenous light chain κ locus and a ~24 kb mouse genomic sequence 5' of the endogenous light chain λ locus, a 5' Frt site, a neomycin cassette, a 3' Frt site and 121,188 bp of human genomic λ sequence containing 16 hVλ gene segments and a PI-SceI site, ~27 kb that overlap the 5' end of the human λ sequence of the insertion of 16 additional hVλ gene segments, and functions as the homology arm
[000692] [000692] In a similar manner, the +12-κ targeting vector was also used to introduce the additional 12 hVλ gene segments in the other early modifications, which incorporated multiple hJλ gene segments with and without an integrated human κ sequence ( Fig. 22B). Homologous recombination with this targeting vector at the mouse endogenous κ locus containing the other modifications created a mouse κ light chain locus containing 40 hVλ gene segments and 1, 2, 3 and 7 hJλ gene segments, with and without a human Vκ-Jκ genomic sequence operably linked to the endogenous mouse Cκ gene which, upon recombination, leads to the forcing of a chimeric κ and λ light chain. Example 13. Identification of targeted ES cells that carry human λ light chain gene segments
[000693] [000693] The BAC DNA targeted and produced according to the preceding examples was used to electroporate mouse ES cells to create modified ES cells to generate chimeric mice expressing human λ light chain gene segments. ES cells containing an insert of unrearranged human λ light chain gene segments were identified by a TAQMAN™ quantitative assay. Specific oligonucleotide primers and probe sets were determined by inserting human λ sequences and associated selection cassettes (allele gain, GOA), loss of endogenous mouse sequences, and any of the selection cassettes.
[000694] [000694] ES cells carrying the human λ light chain gene segments are optionally transfected with a construct expressing FLP in order to remove the Frt'ed neomycin cassette introduced by the insertion of the targeting construct containing Vλ5 gene segments -52 - Human Vλ1-40 (FIG. 23A and 23B). The neomycin cassette can optionally be removed by mating mice expressing FLP recombinase (e.g., U.S. 6,774,279). Optionally, the neomycin cassette is maintained in the mice. Table 10 oligonucleotide initiator SEQ ID NO: SAME SEQ ID NO: HL2F 60 HL2P 82 HL2R 61 HL3F 62 HL3P 83 HL3R 63 NEOF 64 NEOP 84 NEOR 65 67HT1F 68 67HT1P 86 67HT1R 69 67HT3F 70 67HT3P 87 67HT3R 71 HYGF 72 HygP 88 HygR 73 MKD2F 74 MKD2P 89 MKD2R 75 MKP8F 76 MKP8P 90 MKP8R 77 MKP15F 78 MKP15P 91 MKP15R 79 MK20F 80  
[000695] [000695] The previously described targeted ES cells were used as the donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, for example, US patent 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyzes Nature Biotech. Human λ gene segments were identified by genotyping using an allele modification assay (Valenzuela et al., supra) that detected the presence of unique human λ gene segments (supra).
[000696] [000696] Use of κ:λ light chain from mice that carry human λ light chain gene segments. Mice homozygous for each of three successive insertions of hVλ gene segments with a single hJλ gene segment (FIG. 23B), and mice homozygous for a first insertion of hVλ gene segments with either a single hJλ gene segment or with four human Jλ gene segments, including a human Vκ-Jκ genomic sequence (FIG. 22B), were analyzed for κ and λ light chain expression in splenocytes using flow cytometry.
[000697] [000697] Briefly, spleens were collected from groups of mice (ranging from three to seven animals per group) and ground using glass slides. After lysing red blood cells (RBCs) with ACK lysis buffer (Lonza Walkersville), splenocytes were stained
[000698] [000698] In a similar experiment, the B cell contents of the splenic compartment of mice homozygous for a first insertion of 12 hVλ and four hJλ gene segments, including a human Vκ-Jκ genomic sequence operably linked to the mouse Cκ gene ( bottom of FIG. 22B), and mice homozygous for 40 hVλ gene segments and one hJλ (bottom of FIG. 23B or top of FIG. 25B) were analyzed for Igκ and Igλ expression using flow cytometry (from FIG. manner described above). FIG. 26A shows the expression of Igλ and Igκ on CD19+ B cells for a representative mouse from each group. The number of CD19+ B cells per spleen was also recorded for each mouse (FIG. 26B).
[000699] [000699] In another experiment, the B cell contents of the spleen and bone marrow compartments of mice homozygous for 40 hVλ and four hJλ gene segments, including a human Vκ-Jκ genomic sequence operably linked to the mouse Cκ gene ( bottom of Fig. 26B) were analyzed for progression to B cell development using flow cytometry of various cell surface markers.
[000700] [000700] Briefly, two groups (N=3 each, 9-12 weeks, male and female) of wild type mice homozygous for 40 segments of
[000701] [000701] Bone marrow panel: anti-mouse FITC-CD43 (1B11, BioLegend), PE-ckit (2B8, BioLegend), PeCy7-IgM (II/41, eBioscience), PerCP-Cy5.5-IgD (11- 26c.2a, BioLegend), APC-B220 (RA3-6B2, eBioscience), APC-H7-CD19 (ID3, BD) and Pacific Blue-CD3 (17A2, BioLegend).
[000702] [000702] Bone marrow and spleen panel: anti-mouse FITC-Igκ (187.1, BD), PE-Igλ (RML-42, BioLegend), PeCy7-IgM (II/41, ebioscience), PerCP-Cy5.5- IgD (11-26c.2a, BioLegend), Pacific Blue-CD3 (17A2, BioLegend), APC-B220 (RA3-6B2, eBioscience), APC-H7-CD19 (ID3, BD).
[000703] [000703] After staining, cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on a FACSCANTOII™ flow cytometer (BD Biosciences) and analyzed with FLOWJO™ software (Tree Star, Inc.). FIGS. 27A - 27D show the result for the splenic compartment of a representative mouse from each group. FIGS. 28A - 28E show the result for the bone marrow compartment of a representative mouse from each group. Table 13 presents the mean percentage values for the expression of B cells (CD19+), chain
[000704] [000704] Use of human Vλ gene in mice carrying human λ light chain gene segments. Mice heterozygous for a first insertion of human λ sequences (hVλ3-12 - hVλ3-1 and hJλ1, FIG. 23B) and homozygous for a third insertion of human λ sequences (hVλ5-52 - hVλ3-1 and hJλ1, FIG. 23B) were analyzed for use of the human λ light chain gene by reverse transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from splenocytes.
[000705] [000705] Briefly, spleens were collected and perfused in 10 mL of RPMI-1640 (Sigma) with 5% HI-FBS in sterile disposable bags. Each bag containing a single spleen was then placed in a STOMACHER™ (Seward) and homogenized on a medium setting for 30 seconds. Homogenized spleens were filtered using a 0.7µm cell sieve and then precipitated with a centrifuge (1000 rpm per minute), and RBCs were isolated in BD PHARM LYSE™ (BD Biosciences) for three minutes. Splenocytes were diluted with RPMI-1640 and centrifuged again, followed by resuspension in 1 mL PBS (Irvine Scientific). RNA was isolated from the precipitated splenocytes using standard techniques known in the art.
[000706] [000706] RT-PCR was performed on splenocyte RNA using oligonucleotide primers specific for segments of the human hVλ gene and the mouse Cκ gene (Table 15). PCR products were gel purified and cloned into pCR2.1-TOPO TA vector (Invitrogen), and sequenced with M13 sense (GTAAAACGAC GGCCAG; SEQ ID NO:113) and reverse M13 (CAGGAAACAG CTATGAC; SEQ ID NO:) primers. 114) located in the vector at sites that flank the cloning site. A total of eighty-four clones derived from the first and third insertions of human λ sequences were sequenced to determine the use of the hVλ gene (Table 16). The nucleotide sequence of the hVλ-hJλ1-mCκ junction for clones selected by RT-PCR is shown in FIG. 29.
[000707] [000707] In a similar manner, mice homozygous for a third insertion of human λ light chain gene sequences (i.e., 40 hVλ gene segments and four hJλ gene segments, including a human Vκ-Jκ genomic sequence, 25B) operably linked to the endogenous mouse Cκ gene were analyzed for the use of human λ light chain gene by RT-PCR,
[000708] [000708] In a similar manner, mice homozygous for a first insertion of human λ light chain gene segments (12 hVλ and hJλ1 gene segments, FIG. 22A & FIG. 23A), operably linked to the endogenous Cλ2 gene of mouse were analyzed for the use of human λ light chain gene by RT-PCR using RNA isolated from splenocytes (in the manner described above). The primers specific for segments of the hVλ gene (Table 15) were paired with one of two primers specific for the mouse Cλ2 gene; Cλ2-1 (SEQ ID NO:162) or Cλ2-2 (SEQ ID NO:163).
[000709] [000709] Multiple hVλ gene segments rearranged into hλ1 were observed from clones by RT-PCR of mice that carry human λ light chain gene segments at the endogenous mouse λ light chain locus. The nucleotide sequence of the hVλ-hJλ-mCλ2 junction for clones selected by RT-PCR is shown in FIG. 31. TABLE 15 Oligonucleotide primer hVλ 5' Sequence (5'-3') SEQ ID NO: VLL-1 CCTCTCCTCC TCACCCTCCT 98 VLL-1n ATGRCCDGST YYYCTCTCCT 99 VLL-2 CTCCTCACTC AGGGCACA 100 VLL-2n ATGGCCTGGG CTCTGCTSCT 101 VTCGCCTG Y ACSTGCC 102 VII-4 TCACCATGGC YTGGRYCYCM YTC 103 VII-4.3 TCACCATGGC CTGGGTCTCC TT 104 VLL-5 TCACCATGGC CTGGAMTCYT CT 105 VII-6 TCACCATGGC CTGGGCTCCA CTACTT 106 VII-7 TCACCATGGC CTGGACTCCT 107 VII-8 TCACCATGGC CTGGATGATG CTT 108 VII-9 TAAATATGGC CTGGGCTCCT CT 109 VLL -10 TCACCATGCC CTGGGCTCTG CT 110 VLL-11 TCACCATGGC CCTGACTCCT CT 111 Sequence (5'-3') Cκ Oligonucleotide Primer SEQ ID NO: mouse 3' mIgKC3'-1 CCCAAGCTTA CTGGATGGTG GGAAGATGGA 112
[000710] [000710] FIG. 29 shows the hVλ-hJλ1-mCκ junction sequence for RT-PCR clones from mice that carry a first and third insertion of hVλ gene segments with a single hJλ gene segment. The sequence shown in FIG. 29 illustrates unique rearrangements involving different segments of the hVλ gene with hJλ1 recombined in the Cκ gene of v. Heterozygous mice carrying a single endogenous κ-modified locus containing 12 hVλ and hJλ1 gene segments, and homozygous mice carrying two endogenous κ-modified loci containing 40 hVλ and hJλ1 gene segments were both capable of producing human λ gene segments, operably linked to the mouse Cκ gene and produce B cells that express human λ light chains. These rearrangements demonstrate that the chimeric loci were able to independently rearrange human λ gene segments into multiple independent B cells in these mice. Additionally, these changes to the
[000711] [000711] FIG. 30 shows the hVλ-hJλ-mCκ junction sequence for clones selected by RT-PCR from mice homozygous for 40 hVλ and four hJλ gene segments, including a human Vκ-Jκ genomic sequence. The sequences shown in FIG. 30 illustrate additional unique rearrangements involving multiple different hVλ gene segments, which space the entire chimeric locus, with multiple different hJλ gene segments rearranged and operably linked to the mouse Cκ gene. Homozygous mice carrying modified endogenous κ loci containing 40 hVλ and four hJλ gene segments were also able to produce human λ gene segments operably linked to the mouse Cκ gene and produce B cells that express human λ light chains. These rearrangements further demonstrate that all stages of chimeric loci were able to independently rearrange human λ gene segments into multiple independent B cells in these mice. Additionally, these additional modifications to the endogenous light chain κ locus demonstrate that each insertion of human λ gene segments did not render any of the hVλ and/or Jλ gene segments inoperative or prevent the chimeric locus from recombining with the hVλ and Jλ gene segments. Jλ during B cell development, as evidenced by 12 hVλ gene segments different from those that were
[000712] [000712] FIG. 31 shows the hVλ-hJλ-mCλ junction sequence for three individual clones by RT-PCR from mice homozygous for 12 hVλ and hJλ1 gene segments. The sequences shown in FIG. 31 illustrate additional unique rearrangements involving different hVλ gene segments, which space the length of the first insertion, with hJλ1 rearranged and operably linked to the mouse Cλ2 gene (2D1 = Vλ2-8Jλ1; 2D9 = Vλ3-10Jλ1; 3E15 = Vλ3-1Jλ1 ). One clone demonstrated a non-productive rearrangement by virtue of N additions at the hVλ-hJλ junction (2D1, FIG. 31). This is not uncommon in V(D)J recombination, as the joining of gene segments during recombination has been shown to be imprecise. Although this clone represents a non-productive recombinant present in the light chain repertoire of these mice, it demonstrates that the genetic mechanism that contributes to the junctional diversity between antibody genes is functionally normal in these mice, and leads to an antibody repertoire containing light chains. with greater diversity.
[000713] [000713] Homozygous mice carrying modified endogenous λ loci, containing 12 hVλ and hJλ1 gene segments, were also able to produce λ human gene segments operably linked to an endogenous mouse Cλ gene and produce B cells that express λ light chains chimeric reverses containing hVλ regions linked to mouse Cλ regions. These rearrangements further demonstrate that human λ light chain gene segments placed at the other light chain locus (i.e., the λ locus) were able to rearrange independently of the
[000714] [000714] As shown in this example, mice that carry human λ light chain gene segments at the endogenous κ and λ light chain loci are able to rearrange human λ light chain gene segments, and express them in context of a mouse Cκ and/or Cλ region as part of the normal mouse antibody repertoire, as a functional light chain is required at various checkpoints in B cell development in both the spleen and bone marrow. Additionally, early subsets of B cells (eg, pre-, pro- and transitional B cells) demonstrate a normal phenotype in these mice compared to wild-type pups (FIGS. 27D, 28A and 28B). A small deficit in bone marrow and peripheral B cell populations was observed, which can be attributed to a deletion of a subset of immature, autoreactive B cells and/or a sub-optimal association of human λ light chain with human λ heavy chain. mouse. However, the use of Igκ/Igλ observed in these mice demonstrates a more likely situation of human light chain expression than observed in mice. Example 15. Crossing mice expressing human λ light chains from an endogenous light chain locus
[000715] [000715] To optimize the use of human λ gene segments in a
[000716] [000716] Mice that carry an unrearranged human λ light chain locus are also crossed with mice that contain a replacement of the mouse endogenous variable heavy chain gene locus with the human heavy chain variable gene locus (see US 6,596,541, Regeneron Pharmaceuticals, the mouse genetically modified by VELOCIMMUNE®). The VELOCIMMUNE® mouse includes, in part, a genome comprising human heavy chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces antibodies comprising a human heavy chain variable region and a mouse heavy chain constant in response to antigenic stimulus. DNA encoding the variable regions of the antibody heavy chain can be isolated and
[000717] [000717] After crossing mice that contain the unrearranged human light chain λ locus into several desired strains containing modifications and deletions of other endogenous Ig loci (in the manner described above), the selected mice are immunized with an antigen of interest .
[000718] [000718] In general, a VELOCIMMUNE® mouse containing one of the only rearranged regions of the human germline light chain is challenged with an antigen, and lymph cells (such as B cells) are recovered from the animals' serum. Lymph cells can be fused to a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are selected and classified to identify hybridoma cell lines that produce antibodies containing human heavy chain and light human λ, which are specific for the antigen used for immunization. DNA encoding the heavy chain and λ light chain variable regions can be isolated and ligated into the desired heavy chain and light chain isotypic constant regions. Due to the presence of gene segments
[000719] [000719] Initially, high-affinity chimeric antibodies are isolated with a human variable region and a mouse constant region. In the manner described above, antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody, containing a somatically mutated human heavy chain and a human λ light chain derived from an unrearranged human λ light chain locus. of the invention. Suitable human constant regions include, for example, wild-type or modified IgG1, IgG2, IgG3, or IgG4. Example 17. Crossing ADAM6 mice and human λ variable mice
[000720] [000720] Any of the mice described herein that comprise a modification of an endogenous ADAM6 gene or ortholog or homolog thereof, and further comprise a gene that confers ADAM6 function in mice, is crossed with a mouse that comprises a modification that comprises a segment human variable λ (eg, a V and a J segment) operably linked to a human or mouse constant λ or κ gene. The mouse that comprises the human variable segment λ may have the variable segment present
[000721] [000721] A mouse that comprises a humanized heavy chain variable locus (human V, D, and J segments that replace all or substantially all of mouse V, D, and J segments) that further comprises an ectopic sequence of ADAM6 ( or a sequence from an ortholog or homolog of ADAM6 that confers ADAM6 function in mice) is crossed with a mouse comprising a replacement of all or substantially all of the light chain V and J segments with a λ V and J light chain segment of human at the mouse λ locus and/or the mouse κ locus. Progeny are then further outcrossed if necessary, and mice that express an antibody comprising a human VH fused to a heavy chain constant sequence, and a human λ VL cognate fused to either a λ constant sequence or a κ light chain are identified.
[000722] [000722] Mice are exposed to an antigen of interest and allowed to generate an immune response. Antibodies specific to the antigen of interest are identified, and human VH sequences and human λ variable sequences (including human λ variable sequences linked to mouse κ constant regions) are identified and employed to produce a human antibody by genetically modifying the sequences. domain in combination with human constant region genes.
[000723] [000723] In one example, a mouse that is bred by cross-breeding comprises a replacement of all or substantially all
[000724] [000724] Mice as described in this example comprise one or more of the Vκ-Jκ intergenic regions described in the text and in FIGS. here.

权利要求:
Claims (76)
[1]
1. Method for preparing a non-human animal, characterized in that it comprises genetically modifying a non-human animal so that its henoma includes: (a) an insertion of one or more human Vλ gene segments and one or more human Vλ gene segments human Jλ gene upstream of a non-human immunoglobulin light chain constant region, (b) an insertion of one or more human VH gene segments, one or more human DH gene segments, and one or more gene segments human JH upstream of a non-human immunoglobulin heavy chain constant region, and (c) an ectopic nucleotide sequence encoding an ADAM6 protein or a functional fragment thereof, wherein the ADAM6 protein or functional fragment thereof is expressed from the ectopic nucleic acid sequence.
[2]
2. Method according to claim 1, characterized in that the non-human animal is a rodent.
[3]
Method according to claim 1 or 2, characterized in that the non-human immunoglobulin heavy chain constant region is a rodent immunoglobulin heavy chain constant region and/or the immunoglobulin light chain constant region non-human is a rodent immunoglobulin light chain constant region.
[4]
4. Method according to claim 1 or 2, characterized in that the rodent light chain constant region is a mouse Cκ region, a rat Cκ region, a mouse Cλ region, or a rat Cλ region .
[5]
5. Method according to any one of claims 1 to 4, characterized in that the non-human animal comprises 12 to 40 human Vλ gene segments.
[6]
6. Method according to any one of claims 1 to 5, characterized in that the non-human animal comprises at least four human Jλ gene segments.
[7]
7. Method according to any one of claims 1 to 6, characterized in that the ectopic nucleotide sequence encodes an ADAM6 protein or a functional fragment thereof.
[8]
A method according to any one of claims 1 to 7, characterized in that the ectopic nucleotide sequence is present at the same location compared to a wild type non-human ADAM6 locus.
[9]
Method according to any one of claims 1 to 8, characterized in that the ectopic nucleotide sequence is present between immunoglobulin gene segments.
[10]
10. Method according to claim 9, characterized in that the immunoglobulin gene segments are immunoglobulin heavy chain gene segments.
[11]
11. Method according to any one of claims 1 to 10, characterized in that the non-human animal additionally comprises an intergenic human Vκ-Jκ region from a human immunoglobulin κ light chain locus, wherein the human region Intergenic Vκ-Jκ is continuous with one or more human Vλ gene segments and/or one or more human Jλ gene segments.
[12]
12. Method according to any one of claims 1 to 11, characterized in that the ectopic nucleic acid sequence is ectopic because it is inserted or extrachromosomal.
[13]
13. Method according to any one of claims 1 to 12, characterized in that the ectopic nucleotide sequence encodes ADAM6a protein and/or ADAM6b protein.
[14]
14. Use of the cell or tissue derived from the non-human animal as defined in any one of claims 1 to 13, characterized in that it produces an antigen-binding protein.
[15]
15. Use of a cell or tissue derived from a non-human animal as defined in any one of claims 1 to 13, characterized in that it is to produce hybridoma or quadroma.
[16]
16. Use according to claim 14 or 15, characterized in that the cell is a B cell.
[17]
17. Use of the cell or tissue derived from a non-human animal according to any one of claims 1 to 13, characterized in that it is to prepare a fully human antibody.
[18]
18. Use of a cell or tissue derived from a non-human animal as defined in any one of claims 1 to 13, characterized in that it is for preparing a human immunoglobulin λ variable domain and/or an immunoglobulin heavy chain variable domain human.
[19]
19. Method for preparing an antibody that binds to a provided antigen of interest, characterized in that the method comprises: (a) exposing a non-human animal prepared as defined in any one of claims 1 to 13 to an antigen of interest , (b) isolating one or more B lymphocytes from the non-human animal, wherein the one or more B lymphocytes express an antibody that binds to the antigen of interest, (c) identifying a nucleic acid sequence that encodes an immunoglobulin light chain of the antibody that binds the antigen of interest, wherein the immunoglobulin light chain comprises a human immunoglobulin λ light chain variable domain and a non-human immunoglobulin light chain constant domain, and (d) employing the nucleic acid sequence of (c) with a human immunoglobulin light chain constant region in the nucleic acid sequence to prepare a human antibody that binds to the antigen of interest.
1 / 11
1. Non-human animal, characterized in that it comprises: (a) an insertion of one or more human Vλ and Jλ gene segments upstream of a non-human immunoglobulin light chain constant region, (b) an insertion of one or more human VH gene segments, one or more human DH, and one or more human JH upstream of a non-human immunoglobulin heavy chain constant region, and (c) a nucleotide sequence that encodes an ADAM6 protein or a functional fragment thereof, wherein the ADAM6 protein is expressed from an ectopic ADAM6 nucleic acid sequence.
2. Non-human animal according to claim 1, characterized in that the non-human heavy and/or light chain constant regions are selected from mouse, rat or hamster constant regions.
3. Non-human animal according to claim 1, characterized in that the non-human light chain constant region is a rodent constant region.
4. Non-human animal according to claim 1 or 2, characterized in that the rodent light chain constant region is a mouse Cκ region.
5. Non-human animal according to claim 1 or 2, characterized in that the rodent light chain constant region is a rat Cκ region.
6. Non-human animal according to claim 1 or 2, characterized in that the rodent light chain constant region is a mouse Cλ region.
A non-human animal according to claim 1 or 2,
2 / 11 characterized by the fact that the rodent light chain constant region is a mouse Cλ region.
8. Non-human animal according to any one of the preceding claims, characterized in that the non-human animal comprises at least 12 to at least 40 human Vλ gene segments and at least one human Jλ gene segment.
9. Non-human animal according to claim 8, characterized in that the non-human animal comprises 12 human Vλ gene segments and at least one human Jλ gene segment.
10. Non-human animal according to claim 8, characterized in that the non-human animal comprises 28 human Vλ gene segments and at least one human Jλ gene segment.
11. Non-human animal according to claim 8, characterized in that the non-human animal comprises 40 human Vλ gene segments and at least one human Jλ gene segment.
12. Non-human animal according to any one of the preceding claims, characterized in that the at least one human Jλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ7 and a combination thereof.
13. Non-human animal according to any one of the preceding claims, characterized in that the non-human comprises at least four human Jλ gene segments.
14. Non-human animal according to claim 13, characterized in that the at least four human Jλ gene segments comprise at least Jλ1, Jλ2, Jλ3 and Jλ7.
15. Non-human animal according to any of the
3 / 11 preceding claims, characterized in that the nucleotide sequence that encodes an ADAM6 protein or a functional fragment thereof is ectopic in the non-human animal.
16. Non-human animal according to any one of the preceding claims, characterized in that the nucleotide sequence that encodes an ADAM6 protein or a functional fragment thereof is present at the same location compared to a wild-type non-human ADAM6 locus.
17. Non-human animal according to any one of the preceding claims, characterized in that the nucleotide sequence that encodes an ADAM6 protein or a functional fragment thereof is present in an ectopic location in the genome of the non-human animal.
18. Non-human animal according to any one of the preceding claims, characterized in that the nucleotide sequence that encodes an ADAM6 protein or a functional fragment thereof is present in immunoglobulin gene segments.
19. Non-human animal according to claim 18, characterized in that the immunoglobulin gene segments are heavy chain gene segments.
[20]
20. Non-human animal according to claim 19, characterized in that the heavy chain gene segments are from humans.
[21]
21. Non-human animal according to claim 19, characterized in that the heavy chain gene segments are endogenous heavy chain gene segments of the non-human animal.
[22]
22. Non-human animal according to any of the preceding claims, characterized in that the non-human animal does not have a segment of endogenous immunoglobulin VL gene
4 / 11 and/or a JL at an endogenous immunoglobulin heavy chain locus.
[23]
23. Non-human animal according to any one of the preceding claims, characterized in that the non-human animal comprises endogenous VL and/or JL immunoglobulin gene segments that are incapable of rearrangement to form a VL immunoglobulin domain in the animal not human.
[24]
24. Non-human animal according to any one of the preceding claims, characterized in that all or substantially all of the endogenous immunoglobulin Vκ and Jκ gene segments are replaced by one or more human Vλ and Jλ gene segments.
[25]
25. Non-human animal according to any one of the claims, characterized in that all or substantially all of the endogenous immunoglobulin Vλ and Jλ gene segments are replaced by one or more human Vλ and Jλ gene segments.
[26]
26. A non-human animal according to any one of the preceding claims, characterized in that all or substantially all of the VL and JL endogenous immunoglobulin gene segments are intact in the non-human animal and the non-human animal comprises one or more segments of human Vλ gene and one or more human Jλ gene segments inserted between the VL and/or JL endogenous immunoglobulin gene segments, and an endogenous immunoglobulin light chain constant region.
[27]
27. Non-human animal according to claim 26, characterized in that the intact VL and JL endogenous immunoglobulin gene segments have become unable to rearrange themselves to form a VL domain of an antibody in the non-human animal.
[28]
28. Non-human animal according to any one of the preceding claims, characterized in that the animal is not
The human Vκ-Jκ region additionally comprises an intergenic human Vκ-Jκ region from a human κ light chain locus, wherein the human intergenic Vκ-Jκ region is continuous with the one or more human Vλ and Jλ gene segments.
[29]
29. Non-human animal according to claim 28, characterized in that the intergenic human Vκ-Jκ region is placed between a human Vλ gene segment and a human Jλ gene segment.
[30]
30. Cell or tissue, characterized in that it is derived from the non-human animal according to any one of the preceding claims.
[31]
31. Use of a cell or tissue as defined in claim 30, characterized in that it produces an antigen-binding protein.
[32]
32. Use of a cell or tissue as defined in claim 30, characterized in that it is to produce hybridoma or quadroma.
[33]
33. Use according to claim 31 or 32, characterized in that the cell is a B cell.
33. Use according to claim 31 or 32, characterized in that the tissue is derived from the spleen, bone marrow or lymph node of the non-human animal.
[34]
34. Use of a cell or tissue as defined in claim 30, characterized in that it is to prepare a fully human antibody.
[35]
35. Use of a cell or tissue, as defined in claim 30, characterized in that it is to prepare a human Vλ domain and/or a human VH domain.
[36]
36. Use according to any one of claims 31 to
6 / 11 35, characterized by the fact that the cell or tissue is a derivative of a mouse.
[37]
37. Method for preparing an antibody that binds to a provided antigen of interest, characterized in that the method comprises: (a) exposing a non-human animal as defined in any one of claims 1 to 29 to an antigen of interest, (b) isolating one or more B lymphocytes from the non-human animal, wherein the one or more B lymphocytes express an antibody that binds to the antigen of interest, and (c) identifying a nucleic acid sequence that encodes an immunoglobulin light chain of the antibody that binds the antigen of interest, wherein the immunoglobulin light chain comprises a human Vλ domain and a non-human light chain constant domain, and (d) employing the nucleic acid sequence of (c) with a region human immunoglobulin light chain constant in the nucleic acid sequence to prepare a human antibody that binds to the antigen of interest.
[38]
38. Method according to claim 37, characterized in that the non-human light chain constant region is a mouse Cκ.
[39]
39. Method according to claim 37, characterized in that the non-human light chain constant region is a mouse Cλ.
[40]
Method according to claim 37, characterized in that the antibody comprises an immunoglobulin heavy chain comprising a human VH domain and a non-human CH domain.
[41]
41. Method according to any one of the claims
7 / 11 37 to 40, characterized by the fact that the non-human animal is a mouse.
[42]
42. A genetically modified non-human animal, characterized in that it comprises: (a) one or more unarranged human Vλ gene segments and one or more unrearranged human Jλ gene segments at an endogenous immunoglobulin heavy chain locus from the non-human animal, (b) one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments at an endogenous immunoglobulin heavy chain locus of the non-human animal human, wherein the non-human animal is capable of expressing an ADAM6 protein or functional fragment thereof.
[43]
43. Non-human animal according to claim 42, characterized in that the non-human animal expresses antibodies containing heavy chains that comprise human VH domains, and non-human heavy chain and light chain constant regions that comprise human Vλ domains and non-human light chain constant regions.
[44]
44. Non-human animal according to claim 42 or 41, characterized in that the non-human light chain constant domain is a Cκ domain or a Cλ domain.
[45]
45. Non-human animal according to any one of claims 42 to 44, characterized in that the ADAM6 protein or a functional fragment thereof is encoded by an ectopic sequence in the mouse germ line.
[46]
46. Non-human animal according to any one of claims 42 to 45, characterized in that the ADAM6 protein or
8 / 11 a functional fragment thereof is encoded by an endogenous sequence of the non-human animal.
[47]
47. Non-human animal according to any one of claims 42 to 46, characterized in that the endogenous light chain locus of the non-human animal is an immunoglobulin λ light chain locus.
[48]
48. Non-human animal according to any one of claims 42 to 46, characterized in that the endogenous light chain locus of the non-human animal is an immunoglobulin κ light chain locus.
[49]
49. Non-human animal according to any one of claims 42 to 48, characterized in that the non-human animal does not have an endogenous VL and/or JL gene segment in the endogenous light chain locus.
[50]
50. Non-human animal according to claim 49, characterized in that the VL and/or JL gene segment is a Vκ and/or Jκ gene segment.
[51]
51. Non-human animal according to claim 49, characterized in that the VL and/or JL gene segment is a Vλ and/or Jλ gene segment.
[52]
52. Non-human animal, according to any one of claims 42 to 51, characterized in that the VL and JL gene segments of the non-human animal are replaced by one or more human Vλ and one or more gene segments Jλ of human.
[53]
53. Non-human animal according to claim 52, characterized in that the VL and JL gene segments are κ gene segments.
[54]
54. Non-human animal according to claim 52, characterized in that the VL and JL gene segments are segments of
9 / 11 λ gene.
[55]
55. Non-human animal according to any one of claims 42 to 54, characterized in that the one or more human Vλ gene segments are from a fragment of the A cluster of the human immunoglobulin λ light chain locus.
[56]
56. Non-human animal according to claim 55, characterized in that the fragment of cluster A extends from human Vλ3-27 to human Vλ3-1.
[57]
57. Non-human animal according to claim 55, characterized in that the cluster fragment A extends from human Vλ3-12 to human Jλ1.
[58]
58. Non-human animal according to any one of claims 42 to 54, characterized in that the one or more human Vλ gene segments are from a fragment of cluster B of the human immunoglobulin λ light chain locus.
[59]
59. Non-human animal according to claim 58, characterized in that the cluster B fragment extends from human Vλ5-52 to human Vλ1-40.
[60]
60. Non-human animal according to any one of claims 42 to 54, characterized in that the one or more human Vλ gene segments are from a cluster A fragment and one from a cluster B fragment of the chain locus light λ of human immunoglobulin.
[61]
61. Non-human animal according to any one of claims 42 to 60, characterized in that the non-human animal comprises at least 12 human Vλ gene segments.
[62]
62. Non-human animal according to any one of claims 42 to 60, characterized in that the non-human animal comprises at least 28 human Vλ gene segments.
10 / 11
[63]
63. Non-human animal according to any one of claims 42 to 60, characterized in that the non-human animal comprises at least 40 human Vλ gene segments.
[64]
64. Non-human animal according to any one of claims 42 to 60, characterized in that the at least one human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and a combination of these.
[65]
65. Non-human animal according to any one of claims 42 to 64, characterized in that the non-human animal is a rodent.
[66]
66. Non-human animal according to claim 65, characterized in that the rodent is a mouse or rat.
[67]
67. Use of a non-human animal, as defined in any one of claims 42 to 66, characterized in that it is to prepare an antigen-binding protein.
[68]
68. Use according to claim 67, characterized in that the antigen-binding protein is human.
[69]
69. Use according to claim 67, characterized in that the antigen-binding protein is an antibody.
[70]
70. Cell or tissue, characterized in that it is derived from a non-human animal as defined in any one of claims 42 to 66.
[71]
71. Cell or tissue according to claim 70, characterized in that the tissue is derived from a spleen, bone marrow or a lymph node.
[72]
72. Cell or tissue according to claim 70, characterized in that the cell is a B cell.
[73]
73. Cell or tissue according to claim 70, characterized in that the cell is an umbilical cord cell
11 / 11 (ES).
[74]
74. Cell or tissue as defined in claim 68, characterized in that the cell is a germ cell.
[75]
75. Fertile male non-human animal, characterized in that it expresses an immunoglobulin light chain comprising a human Vλ domain or a human Vκ domain, and an immunoglobulin heavy chain comprising a human VH domain, wherein the animal does not male human comprises a modified heavy chain variable region locus, and an ectopic ADAM6 gene that is functional in the male non-human animal.
[76]
76. Male and fertile non-human animal according to claim 75, characterized in that the non-human animal is a mouse.

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法律状态:
2021-01-12| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-01-19| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-12-14| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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US201161578097P| true| 2011-12-20|2011-12-20|

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