MX2014009300A - Humanized rodents that express heavy chains containing vl domains. - Google Patents

Abstract

Non-human animals, tissues, cells, and genetic material are provided that comprise a modification of an endogenous non-human heavy chain immunoglobulin sequence and that comprise an ADAM6 activity functional in a rodent (e.g., a mouse), wherein the non-human animals rearrange human immunoglobulin light chain gene segments in the context of heavy chain constant regions and express immunoglobulin-like molecules comprising human immunoglobulin light chain variable domains fused to heavy chain constant domains that are cognate with human immunoglobulin light chain variable domains fused to light chain constant domains.

Description

HUMANIZED RODENTS THAT EXPRESS HEAVY CHAINS WHICH CONTAINS DOMAINS V.

FIELD OF THE INVENTION 5 Genetically modified non-human fertile animals expressing human immunoglobulin-like binding proteins comprising a constant region of the immunoglobulin heavy chain fused to a variable domain of the light chain of immunoglobulin, as well as binding proteins having a domain are provided. variable of the immunoglobulin light chain fused to a constant domain of the light chain and a variable domain of the immunoglobulin light chain fused to a constant domain of the heavy chain. Described are mice, cells, embryos, and genetically modified tissues comprising a nucleic acid sequence encoding a functional ADAM6 protein, in which the mice, cells, embryos, and tissues comprise segments of the immunoglobulin light chain gene of human bound operably to one or more non-human immunoglobulin heavy chain constant genes. Modifications include human and / or humanized immunoglobulin loci. Mice comprising the function of ADAM6 are described, including mice comprising an ectopic nucleic acid sequence encoding an ADAM6 protein. Mice of genetic modification of a locus of the VH region of endogenous mouse immunoglobulin, and also comprising ADAM6 activity, including mice comprising an ectopic nucleic acid sequence that restores or maintains fertility for the male mouse. The fertility example is fertility that is comparable with wild-type mice.

Genetically modified non-human fertile animals are provided which comprise a deletion or modification of an endogenous ADAM6 gene or homologous or orthologous thereof, and which comprises a genetic modification that restores the function of ADAM6 (or homologue or ortholog thereof) in whole or in part, in which the non-human animals express a variable sequence of the human immunoglobulin light chain in the context of a constant sequence of the heavy chain. Also provided are cells expressing said binding proteins, rodents (e.g. mice) that make them, and related methods and compositions.

Genetically engineered animals are described which express antibodies comprising light chain variable regions fused to heavy chain constant regions, in which non-human animals lack a functional endogenous ADAM6 gene but retain the function of ADAM6, including rodents ( for example, mice) comprising a modification of a locus of the variable region of the endogenous immunoglobulin heavy chain (VH) which renders the mouse incapable of develop a functional ADAM6 protein and result in loss of fertility. The genetically modified mice comprise an immunoglobulin VH locus characterized by a plurality of segments of VL gene, JL and optionally DH of human or a combination thereof, and which also comprise the function of ADAM6, including mice comprising a ectopic nucleic acid sequence that restores fertility to a male mouse. Genetically modified mice express antibodies lacking the variable domains of the heavy chain and instead comprise variable domains comprising rearranged light chain gene segments.

Rodents (e.g., mice), cells, embryos, and genetically modified tissues comprising a nucleic acid sequence encoding a functional ADAM6 locus are described, in which mice, cells, embryos, and tissues express a heavy chain of immunoglobulin comprising a variable domain of the human light chain. In addition, mice, cells, embryos, and tissues lack a functional endogenous ADAM6 gene but retain the function of ADAM6 characterized by the presence of an ectopic nucleic acid sequence encoding an ADAM6 protein. Methods are provided for making antibody sequences in fertile non-human animals that are useful for binding antigens.

BACKGROUND OF THE INVENTION Over the past two decades, pharmaceutical applications for antibodies have caused a great deal of research in the development of antibodies that are suitable for use as therapeutic agents for humans. The first antibody therapeutic agents, based on mouse antibody, were not ideal as therapeutic agents for humans because the repeated administration of mouse antibodies to humans results in immunogenicity problems that may confuse long-term treatment regimens. Solutions based on humanizing mouse antibodies were developed to make them look more human-like and less mouse-like. Then followed methods for expressing human immunoglobulin sequences for use in antibodies, mostly based on in vitro expression of human immunoglobulin libraries in phage, bacteria, or yeast. Finally, attempts were made to make useful human antibodies from human lymphocytes in vitro, in mice grafted with human hematopoietic cells, and in transchromosomal or transgenic mice with uninhabited endogenous immunoglobulin loci.

For the creation of these mice, it was necessary to disable the endogenous mouse immunoglobulin genes so that the fully human transgenes integrated into random could work as the expressed repertoire of immunoglobulins in the mouse. Such mice can make human antibodies suitable for use as therapeutic agents for humans, but these mice display substantial problems with their immune systems. These problems led to several experimental obstacles, for example, the mice are impractical to generate antibody repertoires sufficiently diverse, require the use of extensive re-engineering corrections, provide a suboptimal selection procedure for clones probably due to incompatibility between the elements of human and mouse, and an unreliable source of large and diverse populations of variable human sequences needs to be really useful in developing therapeutic agents for humans.

Transgenic mice containing completely human antibody transgenes contain randomly inserted transgenes containing variable sequences of the human immunoglobulin heavy chain that are not rearranged (sequences V, D and J) linked to constant sequences of the human heavy chain, and variable sequences of the non-rearranged human immunoglobulin light chain (V and J) linked to constant sequences of the human light chain. The mice therefore generate rearranged antibody genes from loci different from the endogenous loci, in which the rearranged antibody genes are completely human. In general, mice contain sequences of the heavy chain of human and sequences of the light chain k of human, although they have also been reported mice with at least some human A sequences. Transgenic mice generally have damaged and non-functional endogenous immunoglobulin loci, or blocked expression of endogenous immunoglobulin loci, such that mice are unable to rearrange human antibody sequences at an endogenous immunoglobulin locus. The ramblings of said transgenic mice make them less than optimal to generate a repertoire of human antibody sufficiently diverse in mice, probably due at least in part to a sub-optimal clone selection procedure that relates antibody molecules to one another. completely human within an endogenous selection system and the detrimental effects of changes to the endogenous genetic constitution of said mice.

A need to develop improved genetically modified non-human animals that are useful for generating immunoglobulin sequences still exists in the art., including human antibody sequences, and which are useful for generating a diverse repertoire of immunoglobulin-like molecules that exhibit diversity in addition to traditional antibody molecules, while at the same time reducing or eliminating the deleterious changes that could result from genetic modifications . There also remains a need for non-human animals that are able to rearrange segments of the immunoglobulin gene to form immunoglobulin genes useful rearrangements, including variable domains of the human immunoglobulin light chain in the context of constant domains of the heavy chain that are cognate with variable domains of the human immunoglobulin light chain in the context of constant domains of the light chain, or that they are capable of making proteins from altered immunoglobulin loci, including loci that contain a sufficiently diverse collection of variable gene segments of the human light chain. There remains a need for non-human animals that can generate immunoglobulin-like binding proteins, in which the binding proteins comprise human immunoglobulin light chain variable domains linked to constant domains of the heavy chain.

BRIEF DESCRIPTION OF THE INVENTION Genetic modified non-human animals having immunoglobulin loci are provided, in which the immunoglobulin loci comprise a plurality of human light chain (VL) variable gene segments operably linked to one or more non-human constant regions, for example, VK and JK of human or VA and human JA, and in various embodiments the loci lack a sequence encoding an endogenous functional ADAM6 protein. Non-human animals include rodents, for example, mice and rats.

Loci are provided that are capable of rearrangement and form a gene coding for a variable domain of the light chain that is derived from a rearrangement involving a VK or VA gene segment of the human light chain and a gene segment. JK O JA of human and in various modalities, addition a segment of DH gene, in which in various embodiments the loci lack an endogenous functional ADAM6 gene or functional fragment thereof.

Modified immunoglobulin loci are provided including loci that lack an endogenous functional ADAM6 gene and comprise human immunoglobulin sequences, eg, a VL segment of human operably linked to a constant sequence of human or non-human (or chimeric) immunoglobulin human / non-human) (and in operable linkage with, for example, a segment V and / or a segment J). Modified loci are provided comprising multiple VL gene segments and an ectopic nucleotide sequence encoding an ADAM6 protein or fragment thereof that is functional in the non-human animal. Modified loci are provided which comprise multiple VL gene segments, operably linked with one or more DH segments and / or one or more JL or JH segments, operably linked to a constant sequence of non-human immunoglobulin, eg, a Rodent sequence (for example, mouse or rat) or human. Also provided are non-human animals comprising said humanized loci, in which non-human animals exhibit wild-type fertility.

Non-human animals comprising a locus are provided variable of the immunoglobulin heavy chain (eg, in a transgene or as an insertion or replacement at a variable locus of the endogenous non-human animal heavy chain) comprising a plurality of human VL gene segments linked in operable form to a segment of human D gene and / or human J gene. In various embodiments, the plurality of human VL gene segments are operably linked to one or more human D gene segments and / or one or more human J gene segments at the variable heavy chain gene locus of endogenous immunoglobulin of the non-human animal. In various embodiments, non-human animals further comprise an ectopic nucleotide sequence that encodes an ADAM6 protein or homologue or ortholog thereof thereof that is functional in the non-human male animal comprising the modified heavy chain locus. In various embodiments, the ectopic nucleotide sequence is contiguous with at least one human VL segment, one DH gene segment, or one JL gene segment. In various embodiments, the ectopic nucleotide sequence is contiguous with a non-human immunoglobulin sequence in the non-human animal. In one embodiment, the ectopic nucleotide sequence is on the same chromosome as the locus of the modified heavy chain. In one embodiment, the ectopic nucleotide sequence is on a chromosome different from the locus of the modified heavy chain.

Non-human animals are provided that are modified at their loci in the variable region of the immunoglobulin heavy chain to eliminate all or substantially all (for example, all functional segments, or almost all functional segments) the endogenous immunoglobulin VH segments and comprising a plurality of human VL gene segments operably linked to a DH and J segment or a JL gene segment at the locus of the variable region of the endogenous immunoglobulin heavy chain of the non-human animal. Also provided are non-human animals comprising said loci and lacking an endogenous ADAM6 gene.

Methods for making human immunoglobulin sequences in non-human animals are provided. In various embodiments, human immunoglobulin sequences are derived from a repertoire of immunoglobulin heavy chain sequences comprising human Vu gene sequences rearranged and operably linked to constant regions of the immunoglobulin heavy chain, e.g. , VL, and one or more segments DH and J or one or more JL segments. Methods for making human immunoglobulin sequences in animals, tissues, and non-human cells are provided, in which human immunoglobulin sequences bind an antigen of interest.

In one aspect, constructs of nucleic acid, cells, embryos, rodents (e.g., mice), and methods for making rodents (e.g., mice) are provided which comprise a modification that results in an ADAM6 gene or ADAM6 protein. endogenous non-functional rodent (e.g., mouse) (e.g., a blocked expression of or a deletion in an endogenous ADAM6 gene), in which rodents (e.g., mice) comprise a sequence of nucleic acid encoding an ADAM6 protein or ortholog or homologue or fragment thereof that is functional in a male genus rodent of the same class (eg, mouse). In one embodiment, the mice comprise an ectopic nucleotide sequence that encodes a rodent ADAM6 protein or ortholog or homologue or functional fragment thereof; in a specific embodiment, the rodent ADAM6 protein is a mouse ADAM6 protein. In one embodiment, the mice comprise an ectopic nucleotide sequence that codes for one or more rodent ADAM6 proteins, wherein said one or more proteins comprise SEQ ID NO: 1 or SEQ ID NO: 2 or a fragment thereof. It is functional in mice.

In various aspects, the sequence encoding the activity of ADAM6 is contiguous with a human immunoglobulin sequence. In various aspects, the sequence encoding the activity of ADAM6 is contiguous with a non-human immunoglobulin sequence. In various aspects, the sequence is present on the same chromosome as that of the endogenous non-human immunoglobulin heavy chain locus of the non-human animal. In various aspects, the sequence is present on a chromosome different from the locus of the immunoglobulin heavy chain of the non-human animal.

Genetically modified non-human animals are described that comprise a modification that maintains the activity of an ADAM6 gene or homologue or ortholog thereof, in which the modification includes the insertion of one or more gene segments of the human immunoglobulin light chain upstream of a constant region of the non-human immunoglobulin heavy chain, and the non-human animals further comprise modifications allowing them to express regions human immunoglobulin light chain variables cognate with variable regions of the human immunoglobulin light chain. In various aspects, the variable regions of the human immunoglobulin light chain are expressed in the context of constant regions of the heavy chain and the light chain.

In various aspects, the insertion of one or more gene segments of the human immunoglobulin light chain takes place 3 'or downstream of the ADAM6 gene of the non-human animal. In various aspects, the insertion of one or more human immunoglobulin light chain gene segments is effected in such a way that the ADAM6 gene or genes of the non-human animal are not altered, deleted (n) and / or functionally silencing (n) such that the ADAM6 activity of the non-human animal is at the same or comparable level as in a non-human animal that does not contain said insertion. Examples of functionally silencing alterations, deletions and / or modifications include any modification that results in a reduction, deletion and / or loss of activity of the ADAM6 protein (s) encoded by the ADAM6 gene or genes of the animal not human.

In one aspect, nucleic acid constructs are provided, cells, embryos, mice, and methods to make mice that they comprise a modification of an endogenous immunoglobulin locus, in which 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 immunoglobulin locus is a locus of the immunoglobulin heavy chain, and the modification reduces or eliminates the ADAM6 activity of a cell or tissue of a male mouse.

In one aspect, mice are provided comprising an ectopic nucleotide sequence encoding a mouse ADAM6 or ortholog or homologue or functional fragment thereof; also provided are mice comprising an endogenous nucleotide sequence encoding a mouse ADAM6 or ortholog or homologue or fragment thereof, and at least one genetic modification of an immunoglobulin locus of the heavy chain. In one embodiment, the endogenous nucleotide sequence encoding a mouse ADAM6 or ortholog or homologue or functional fragment thereof is located at an ectopic position compared to an endogenous ADAM6 gene from a wild-type mouse.

In one aspect, methods are provided for making mice comprising a modification of an endogenous immunoglobulin locus, wherein the mice comprise an ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse. In various embodiments, the modification comprises an insertion of one or more segments of the human VL gene in the endogenous immunoglobulin locus.

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

In one aspect, methods are provided for making mice comprising a genetic modification of an immunoglobulin heavy chain locus and a locus of the immunoglobulin light chain, in which the application of methods to modify the locus of the heavy chain it results in male mice exhibiting a reduction in fertility, and the mice comprise a genetic modification that restores in whole or in part the reduction in fertility. In various modalities, the reduction in fertility is characterized by an inability of the sperm of male mice to migrate from a mouse uterus through a mouse oviduct to fertilize a mouse ovule. In various modalities, the reduction in fertility is characterized by sperm exhibiting a defect of live migration. In various modalities, the genetic modification that restores in whole or in part the Reduction in fertility is a nucleic acid sequence that encodes a mouse ADAM6 or orthologous gene or homologue or fragment thereof that is functional in a male-gender mouse.

In one embodiment, the genetic modification comprises replacing endogenous immunoglobulin heavy chain variable loci with variable loci of the immunoglobulin light chain of another species (eg, a non-mouse species). In one embodiment, the genetic modification comprises the insertion of variable loci of the immunoglobulin light chain of another species (e.g., a non-mouse species) at variable loci of the endogenous immunoglobulin heavy chain. In a specific modality, the species is human. In one embodiment, the genetic modification comprises the deletion of a variable locus of the endogenous immunoglobulin heavy chain in whole or in part, in which the deletion results in a loss of the function of endogenous ADAM6. In a specific embodiment, the loss of endogenous ADAM6 function is associated with a reduction in fertility in male mice.

In one embodiment, the genetic modification comprises the inactivation of a variable locus of the endogenous non-human immunoglobulin heavy chain in whole or in part, in which inactivation does not result in a loss of the function of endogenous ADAM6. Inactivation may include replacement or deletion of one or more segments of endogenous non-human gene what gives as resulting in a locus of the endogenous non-human immunoglobulin heavy chain that is substantially unable to rearrange to code for a heavy chain of an antibody comprising endogenous non-human gene segments. The inactivation may include other modifications that make the locus of the endogenous immunoglobulin heavy chain incapable of rearrangement to code for the heavy chain of an antibody, wherein the modification does not include the replacement or deletion of endogenous gene segments. Examples of modifications include inversions and / or chromosomal translocations mediated by molecular techniques, for example, using precise placement of site-specific recombination sites (eg, Cre-lox technology). Other examples of modifications include disabling operable linkage between the non-human immunoglobulin variable gene segments and the non-human immunoglobulin constant regions.

In one embodiment, the genetic modification comprises inserting into the non-human animal genome a DNA fragment containing one or more human VL gene segments and one or more human JL gene segments, and optionally one or more gene segments. DH of human, of another species (for example, a non-mouse species) operably linked to one or more sequences of the constant region (eg, an IgM gene and / or an IgG gene). In one embodiment, the DNA fragment is capable of undergoing rearrangement in the genome of the non-human animal to form a sequence that codes for a variable domain of the light chain bound in operable to a constant region of the heavy chain. In one modality, the species is human. In one embodiment, the genetic modification comprises the insertion of one or more gene segments of the human immunoglobulin light chain downstream or 3 'of an endogenous ADAM6 gene of the non-human animal such that the activity of ADAM6 (per example, expression and / or function of an encoded protein) is the same or comparable to a non-human animal that does not comprise the insertion.

In one aspect, methods are provided for making mice comprising a genetic modification of a locus of the immunoglobulin heavy chain, in which the application of the methods results in male mice comprising a locus of the immunoglobulin heavy chain modified (or a deletion thereof), and male mice exhibit a reduction in fertility, and mice comprise a genetic modification that restores in whole or in part the reduction in fertility. In various modalities, the reduction in fertility is characterized by an inability of the sperm of male mice to migrate from a mouse uterus through a mouse oviduct to fertilize a mouse ovule. In various modalities, the reduction in fertility is characterized by sperm exhibiting an in vivo migration defect. In various embodiments, the genetic modification that restores in whole or in part the reduction in fertility is a nucleic acid sequence that encodes a mouse ADAM6 or orthologous gene or homologue or fragment thereof that is functional in a mouse from male gender.

In one embodiment, the genetic modification comprises replacing variable loci of the endogenous immunoglobulin heavy chain with variable loci of the immunoglobulin light chain, eg, one or more segments of the variable gene of the light chain (VL), one or more segments of heavy chain diversity gene (DH) and one or more segments of the binding gene (J), or one or more segments of the light chain (JL) binding gene of another species (for example, a non-mouse species). In one embodiment, the genetic modification comprises the insertion of a single segment of the VL gene of the variable loci of the orthologous immunoglobulin light chain, at least one segment of the DH gene and at least one segment of the J gene, or minus one segment of the Ju gene in variable loci of the endogenous immunoglobulin heavy chain. In a specific modality, the species is human. In one embodiment, the genetic modification comprises a deletion of a variable locus of the endogenous Immunoglobulin heavy chain in whole or in part, in which the deletion results in a loss of endogenous ADAM6 function. In a specific embodiment, the loss of endogenous ADAM6 function is associated with a reduction in fertility in male mice. In one embodiment, the genetic modification comprises the inactivation of a variable locus of the endogenous immunoglobulin heavy chain in whole or in part, in which the deletion does not result in a loss of ADAM6 function more segments of the endogenous gene which results in a locus of the endogenous immunoglobulin heavy chain that is substantially unable to rearrange to code for a heavy chain of an antibody comprising endogenous gene segments. The inactivation may include other modifications that make the locus of the endogenous immunoglobulin heavy chain unable to rearrange to code for the heavy chain of an antibody, wherein the modification does not include replacement or deletion of endogenous gene segments. Examples of modifications include inversions and / or chromosomal alterations that result in a locus of the heavy chain that is not operably linked to one or more endogenous constant regions.

In one embodiment, the genetic modification comprises inserting into the mouse genome a DNA fragment containing one or more segments of the human VL gene, one or more segments of the J gene, and optionally one or more segments of the D gene of another species. (eg, a non-mouse species) operably linked to one or more sequences of the constant region (eg, an IgM gene and / or an IgG gene). In various embodiments, the J gene segments include the JH or JL gene segments. In one embodiment, the DNA fragment is capable of undergoing rearrangement to form a sequence encoding an antibody heavy chain, in which the heavy chain comprises a variable gene segment of the rearranged human light chain fused to a region constant of the heavy chain. In one embodiment, genetic modification comprises the insertion of at least six, at least 16, at least 30, or at least 40 or more human VL gene segments, and at least one or at least 5 human JL gene segments in the mouse genome. In a specific embodiment, the species is human and the gene segments are gene segments of the human light chain k. In one embodiment, the genetic modification comprises the deletion of a variable locus of the endogenous immunoglobulin heavy chain in whole or in part to render non-functional the locus of the endogenous immunoglobulin heavy chain, in which the deletion also results in a loss of the function of endogenous ADAM6. In a specific embodiment, the loss of endogenous ADAM6 function is associated with a reduction in fertility in male mice.

In one aspect, mice are provided comprising a modification that reduces or eliminates expression of mouse ADAM6 from an endogenous ADAM6 allele such that a male mouse having the modification exhibits reduced fertility (e.g. a highly reduced ability to generate offspring by mating), ie essentially infertile, due to the reduction or elimination of the endogenous ADAM6 function, in which the mice further comprise an ectopic or homologous or orthologous ADAM6 sequence or functional fragment of the same In one aspect, the modification that reduces or eliminates the expression of mouse ADAM6 is a modification (eg, an insertion, a deletion, a replacement, etc.) at a mouse immunoglobulin locus. In a modality, the immunoglobulin locus is a locus of the immunoglobulin heavy chain.

In one embodiment, the reduction or loss of ADAM6 function comprises a substantial inability or inability of the mouse to produce sperm that can travel from a mouse uterus through a mouse oviduct to fertilize a mouse ovule. In a specific embodiment, at least about 95%, 96%, 97%, 98%, or 99% of the sperm produced in a cyaculatory volume of the mouse are unable to cross through an oviduct in vivo after copulation and fertilize a mouse ovule.

In one embodiment, the reduction or loss of the function of AQAM6 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. In one embodiment, the loss of the APAM6 function comprises a substantial inability to fertilize a mouse ovule by copulation with a female mouse.

In one aspect, there is provided a mouse lacking a functional endogenous ADAM6 gene, and comprising a protein (or an ectopic nucleotide sequence encoding a protein) that confers ADAM6 functionality in the mouse. In one embodiment, the mouse is a male mouse and the functionality comprises increased fertility compared to a mouse lacking a functional endogenous ADAM6 gene.

In one embodiment, the protein is encoded by a genomic sequence located within a locus of immunoglobulin in the mouse germline. In a specific embodiment, the immunoglobulin locus is a locus of the heavy chain. In another specific embodiment, the heavy chain locus comprises at least one human VH gene segment, at least one human DH gene segment and at least one human JH gene segment. In another specific embodiment, the heavy chain locus comprises at least one segment of human VL gene and at least one segment of human JL gene. In another specific embodiment, the heavy chain locus comprises at least one human VL, at least one human DH, and at least one human JL. In another specific embodiment, the heavy chain locus comprises at least one segment of human VL gene, at least one segment of human DH gene, and at least one segment of human JH gene. In another specific embodiment, the heavy chain locus comprises at least one segment of human VL gene and at least one segment of human JL gene. In another specific embodiment, the heavy chain locus comprises at least one segment of human VL gene and at least one segment of human JH gene. In another specific embodiment, the heavy chain locus comprises six segments of the human VK gene and five segments of the human JK gene. In another specific embodiment, the heavy chain locus comprises 16 segments of the human VK gene and five segments of the human JK gene. In another specific embodiment, the heavy chain locus comprises 30 segments of the human VK gene and five JK gene segments of human. In another specific embodiment, the heavy chain locus comprises 40 human VK gene segments and five human JK gene segments.

In one embodiment, the ectopic protein is encoded by a genomic sequence located within a non-immunoglobulin locus in the mouse germline. In one embodiment, the non-immunoglobulin locus is an active locus from the transcription point of view. In a specific modality, the active locus from the transcription point of view is the ROSA locus. In a specific embodiment, the active locus from the transcription point of view is associated with tissue-specific expression. In one embodiment, tissue-specific expression is present in the reproductive tissues. In one embodiment, the protein is encoded by a genomic sequence randomly inserted into the germline of the mouse.

In one embodiment, the mouse comprises a light chain of human or human chimeric light chain / mouse or human chimeric / rat (eg, human variable, mouse constant or rat) and a chimeric heavy chain of human variable / constant of mouse or rat. In a specific embodiment, the mouse comprises a transgene comprising a chimeric light chain gene of human variable / rat constant or mouse operably linked to a transcriptionally active promoter, eg, a promoter. ROSA26 In a further specific embodiment, the transgene of the chimeric human / mouse or rat light chain comprises a variable region sequence of the human light chain rearranged in the germ line of the mouse.

In one embodiment, the ectopic nucleotide sequence is located within an immunoglobulin locus in the mouse germline. In a specific modality, the immunoglobulin locus is a locus of the heavy chain. In one embodiment, the heavy chain locus comprises at least one segment of human VL gene and at least one segment of human JL gene. In a specific embodiment, the heavy chain locus comprises at least six and up to 40 human Vk gene segments, and five human JK gene segments. In one embodiment, the ectopic nucleotide sequence is located within a non-immunoglobulin locus in the mouse germline. In one embodiment, the non-immunoglobulin locus is an active locus from the transcription point of view. In a specific modality, the active locus from the transcription point of view is the ROSA26 locus. In one embodiment, the ectopic nucleotide sequence is positioned randomly inserted into the germline of the mouse.

In one aspect, a mouse lacking a functional endogenous ADAM6 gene is provided, in which the mouse comprises an ectopic nucleotide sequence that complements the loss of mouse ADAM6 function. In one embodiment, the ectopic nucleotide sequence gives the mouse an ability to produce offspring that is comparable to that of a corresponding wild-type mouse that contains a functional endogenous ADAM6 gene. In one embodiment, the sequence gives the mouse an ability to form a complex of ADAM2 and / or ADAM3 and / or ADAM6 on the surface of the sperm of the mouse. In one embodiment, the sequence gives the mouse an ability to travel from a mouse uterus through a mouse oviduct to a mouse egg to fertilize the ovule.

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

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces at least about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 4 times, about 6 times, approximately 7 times, approximately 8 times, or approximately 10 times or more progeny when reared over a period of time of six months than a mouse of the same age and of the same strain or similar strain lacking the ADAM6 gene functional endogenous and lacking the ectopic nucleotide sequence that is reared over substantially the same period of time and under substantially the same conditions.

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces an average of at least about 2 times, 3 times, or 4 times more the number of offspring per litter in a breeding period of four to six months than a mouse that lacks the functional endogenous ADAM6 gene that lacks the ectopic nucleotide sequence, and that is reared during the same period of time.

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence is a male-gender mouse, and the male-type mouse produces sperm which when recovered from the oviducts at approximately 5-6 hours after copulation reflects an oviduct migration that is at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, so at least 70 times, at least 80 times, at least 90 times, 100 times, 110 times, or 120 times or more than that of a mouse lacking the functional endogenous ADAM6 gene and lacking the ectopic nucleotide sequence.

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence when copulated with a female mouse generates sperm that is capable of crossing the uterus and entering and traversing the oviduct within approximately six hours to a deficiency that is approximately equal to that of sperm from a wild-type mouse.

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces about 1.5 times, about 2 times, about 3 times, or about 4 times or more litters in a comparable period of time as the mice. of a mouse that lacks the functional ADAM6 gene and lacks the ectopic nucleotide sequence.

In one aspect, a mouse is provided comprising in its germ line a non-mouse nucleic acid sequence encoding an immunoglobulin protein, wherein the non-mouse immunoglobulin sequence comprises an insertion of a mouse ADAM6 or homologous or orthologous gene or functional fragment thereof. In one embodiment, the non-mouse immunoglobulin sequence comprises a human immunoglobulin sequence. In one embodiment, the sequence comprises a human immunoglobulin heavy chain sequence. In one embodiment, the sequence comprises a human immunoglobulin light chain sequence. In one embodiment, the sequence comprises a sequence of the human heavy chain contiguous with a human light chain sequence. In one embodiment, the sequence comprises one or more segments of gene V, one or more segments of gene D, and one or more segments of gene J; in one embodiment, the sequence comprises one or more segments of gene V and one or more segments of gene J. In one embodiment, said one or more segments of the gene V, D, and J, or one or more segments of gene V and J, they are not rearranged. In one embodiment, said one or more segments of the V, D, and J gene, or one or more segments of gene V and J, are rearranged. In one embodiment, after rearrangement of said one or more segments of the V, D, and J gene, or one or more segments of V and J gene, the mouse comprises in its genome at minus a nucleic acid sequence that codes for a gene from Mouse ADAM6 or homologous or ortholog or functional fragment thereof. In one embodiment, after rearrangement the mouse comprises in its genome at least two nucleic acid sequences encoding a mouse ADAM6 or homologous or orthologous gene or functional fragment thereof. In one embodiment, after rearrangement the mouse comprises in its genome at least one nucleic acid sequence encoding a mouse ADAM6 or homologous or orthologous gene or functional fragment thereof. In one embodiment, the mouse comprises the ADAM6 gene or homologous or orthologous or functional fragment thereof in a B cell. In one embodiment, the mouse comprises the ADAM6 gene or homologous or orthologous or functional fragment thereof in a cell which is not a B cell In one aspect, mice are provided that express a variable region of the human immunoglobulin heavy chain or functional fragment thereof from a locus of the endogenous immunoglobulin heavy chain, in which the mice comprise an ADA 6 activity. which is functional in a male mouse. In one embodiment, the heavy chain locus comprises one or more human VL gene segments and one or more JL gene segments, and optionally one or more DH gene segments. In one embodiment, the heavy chain locus comprises at least six human VK gene segments and five human JK gene segments. In one embodiment, the heavy chain locus comprises at least 16 human VK gene segments and five gene segments JK of human. In one embodiment, the locus of the heavy chain it comprises at least 30 human VK gene segments and five human JK gene segments. In one embodiment, the heavy chain locus comprises at least 40 human VK gene segments and five human JK gene segments.

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

In one embodiment, the male mice comprise an allele of individual unmodified endogenous ADAM6 or ortholog or homologue or functional fragment thereof at a locus of endogenous ADAM6.

In one embodiment, male mice comprise a mouse ectopic or homologous or orthologous ADAM6 sequence or functional fragment thereof encoding a protein conferring ADAM6 function.

In one embodiment, the male mice comprise a sequence of ADAM6 or homologous or orthologous or functional fragment thereof at a location in the mouse genome that approaches the location of the endogenous ADAM6 allele, eg, 3 'of a sequence of the final and 5 'V gene segments of an initial J gene segment.

In one embodiment, male mice comprise a sequence of ADAM6 or homologous or orthologous or functional fragment thereof at a location in the mouse genome that is different from that of the endogenous ADAM6 allele, eg, 5 'of the V gene segment sequence further towards the 5' end of a locus of the V gene.

In one embodiment, the male-gender mice comprise a sequence of ADAM6 or homologous or orthologous or functional fragment thereof flanked towards the 5 'end, towards the 3' end, or towards the 5 'end and towards the 3' end ( with respect to the transcription direction of the ADA sequence 6) of a nucleic acid sequence encoding an immunoglobulin V gene segment and / or an immunoglobulin J gene segment. In a specific embodiment, the V and J immunoglobulin gene segments are human gene segments. In one embodiment, the V and J gene segments of Immunoglobulin are human gene segments, and the sequence encoding the mouse ADAM6 or ortholog or homologous or functional fragment in a mouse is between the V gene segments and J of human; in one embodiment, the mouse comprises two or more segments of human V gene, and the sequence is in a 5 'position of the human V gene segment further towards the 5' end; in one embodiment, the mouse comprises two or more human V gene segments, and the sequence is at a position between the final V gene segment and the penultimate V gene segment; in one embodiment, the mouse comprises a plurality of human V gene segments, and the sequence is in a position towards the 5 'end of the human V gene segment further towards the 5' end; in one modality, the mouse it further comprises a segment of gene D, and the sequence is in a position after the V gene segment more towards the 3 'end and a segment of gene D more towards the 5' end; in one embodiment, the sequence is in a position between a segment of gene V and a segment of gene J.

In one embodiment, the human V-gene segments are V-gene segments of the light chain. In a specific embodiment, the V-gene segments of the light chain are VK gene segments. In another specific embodiment, the V-gene segments of the light chain are segments of the VA gene. In one embodiment, the J gene segment is selected from a JH gene segment and a JL gene segment. In a specific embodiment, the JL gene segment is a segment of the JK gene. In another specific embodiment, the JL gene segment is a segment of the JA gene.

In one embodiment, the male mice comprise a sequence of ADAM6 or homologous or orthologous or functional fragment thereof that is located at a position in an endogenous immunoglobulin locus that is the same or substantially the same as in a genus mouse male of wild type. In a specific embodiment, the endogenous locus is unable to code for the variable region of the heavy chain of an antibody, in which the variable region comprises or is derived from an endogenous non-human gene segment. In a specific embodiment, the endogenous locus is positioned at a location in the genome of the male gender mouse that makes the gene segments of the Heavy chain loci are unable to code for a variable region of the heavy chain of an antibody. In various embodiments, the male-gender mice comprise an ADAM6 sequence located on the same chromosome as that of the human immunoglobulin gene segments and the ADAM6 sequence codes for a functional ADAM6 protein.

In one aspect, a male mouse comprising an endogenous non-functional ADAM6 gene, or a deletion of an endogenous ADAM6 gene, is provided in its germline; in which mouse sperm are able to transit an oviduct of a female mouse and fertilize an egg. In one embodiment, the mice comprise an extrachromosomal copy of a mouse ADAM6 gene or ortholog or homologue or functional fragment thereof that is functional in a male mouse. In one embodiment, the mice comprise an ectopic mouse ADAM6 or orthologous gene or homologue or functional fragment thereof that is functional in a male mouse.

In one aspect, a male mouse comprising a functional endogenous ADAM6 gene and a modification to a locus of the endogenous immunoglobulin heavy chain is provided. In one embodiment, the modification is made downstream, or 3 ', of a gene or locus of endogenous ADAM6. In one embodiment, the modification is a replacement of one or more segments of the endogenous immunoglobulin heavy chain gene with one or more human immunoglobulin light chain gene segments. In one modality, the modification is an insertion of one or more gene segments of the human immunoglobulin light chain towards the 5 'end of a gene of the constant region of the endogenous immunoglobulin heavy chain.

In one aspect, mice are provided comprising a genetic modification that reduces the function of endogenous ADAM6, in which the mouse comprises at least some functionality of ADAM6 provided either by an endogenous non-modified allele that is functional in its entirety or in part (eg, a heterozygote), or by expression from an ectopic sequence encoding an ADAM6 or an ortholog or homologue or functional fragment thereof that is functional in a male mouse. In various embodiments, the ADAM6 or ortholog or homologue or functional fragment thereof comprises a nucleic acid sequence encoding an ADAM6 protein indicated in SEQ ID NO: 1, SEQ ID NO: 2, or a combination thereof.

In one embodiment, the mice comprise function of ADA 6 sufficient to give male mice the ability to generate offspring by mating, compared to male mice lacking a functional ADAM6. In one embodiment, the function of ADAM6 is conferred by the presence of an ectopic nucleotide sequence encoding a mouse ADAM6 or a homologue or ortholog or functional fragment thereof. In one modality, the function of ADAM6 is Endogenous immunoglobulin, in which the mouse is unable to express an antibody comprising an endogenous immunoglobulin heavy chain gene segment. ADAM6 homologs or orthologs or fragments thereof that are functional in a male-gender mouse include those that restore, in whole or in part, the loss of the ability to generate offspring observed in a male mouse that lacks sufficient activity of endogenous ADAM6, for example, the loss in capacity observed in a mouse with blocked expression of ADAM6. In this sense, the ADAM6 blocked expression mice include mice comprising an endogenous locus or fragment thereof, but which is non-functional, i.e., not expressing ADAM6 (ADAM6a and / or ADAM6b) at all, or expressing ADAM6 (ADAM6a and / or ADAM6b) at a level that is insufficient to support an essentially normal capacity to generate offspring of a male wild type mouse. The loss of function may be due to, for example, a modification in a structural gene of the locus (i.e., in a region encoding ADAM6a or ADAM6b) or in a regulatory region of the locus (eg, in a 5 'sequence). to the ADAM6a gene, or 3 'of the coding region of ADAM6a or ADAM6b, in which the sequence controls, in whole or in part, the transcription of an ADAM6 gene, the expression of an ADAM6 RNA, or the expression of an ADAM6 protein). In various embodiments, orthologs or homologs or fragments thereof that are functional in a male mouse are those that allow a sperm of a male mouse (or a most sperm in the cyaculate of a male-genus mouse) transits a mouse oviduct and fecundates a mouse ovule.

In one embodiment, male mice expressing the human immunoglobulin variable region or functional fragment thereof comprise sufficient ADAM6 activity to confer on male mice the ability to generate offspring by mating female and female mice. , in one embodiment, male mice exhibit an ability to generate offspring when they mate with female mice that is in a modality at least 25%, in one modality, at least 30%, in a modality so less 40%, in a modality of at least 50%, in a modality of at least 60%, in a modality of at least 70%, in a modality of at least 80%, in a modality of at least 90%, and in a mode approximately the same as that of mice with one or two alleles of unmodified endogenous ADAM6.

In one embodiment, the male mice express sufficient ADAM6 (or an ortholog or homologous or functional fragment thereof) to allow a sperm from the male mice to cross the oviduct of a female mouse and fecundate a mouse ovule .

In one embodiment, the functionality of ADAM6 is conferred by a nucleic acid sequence that is contiguous with a mouse chromosomal sequence (eg, the nucleic acid is randomly integrated into a mouse chromosome, or it is placed in a specific location, for example, by directing the nucleic acid to a specific location, for example, by site-specific recombinase-mediated insertion (eg, mediated by Cre) 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., extra-chromosomally, e.g. expression, a vector, a YAC, a trans-chromosome, etc.).

In one aspect, there are provided genetically modified mice and cells comprising a modification of an immunoglobulin locus of the endogenous heavy chain, in which the mice express at least a portion of an immunoglobulin light chain sequence, for example, at least a portion of a human sequence, in which the mice comprise an ADAM6 activity that is functional in a male mouse. In one embodiment, the modification reduces or eradicates a mouse ADAM6 activity. In one embodiment, the mouse is modified such that both alleles encoding the activity of ADAM6 are either absent or express an ADAM6 that does not substantially function to support normal pairing in a male mouse. In one embodiment, the mouse further comprises an ectopic nucleic acid sequence encoding a mouse ADAM6 or ortholog or homologue or functional fragment thereof. In one mode, the modification maintains the ADAM6 activity of the mouse and causes a locus of the endogenous immunoglobulin heavy chain is unable to code for a heavy chain of an antibody. In a specific embodiment, the modification includes inversions and or chromosomal translocations that render the locus of the endogenous immunoglobulin heavy chain incapable of rearrangement to code for a variable region of the heavy chain of an antibody.

In one aspect, there are provided genetically modified mice and cells comprising a modification of a locus of the endogenous immunoglobulin heavy chain, wherein the modification reduces or eliminates ADAM6 activity expressed from an ADAM6 sequence of the locus, and wherein the mice comprise an ADAM6 protein or ortholog or homologue or functional fragment thereof. In various embodiments, the ADAM6 protein or fragment thereof is encoded by an ectopic ADAM6 sequence. In various embodiments, the ADAM6 protein or fragment thereof is expressed from an endogenous ADAM6 allele. In various embodiments, the mouse comprises a first allele of the heavy chain comprising a first modification that reduces or eliminates the expression of a functional ADAM6 from the first allele of the heavy chain, and the mouse comprises a second allele of the heavy chain comprising a second modification that does not substantially reduce or eliminate the expression of a functional ADAM6 from the second allele of the heavy chain.

In various embodiments, the modification is the insertion of one or more gene segments of the immunoglobulin light chain of human upstream, or 5 ', of a gene of the endogenous immunoglobulin heavy chain constant region. In various embodiments, the modification maintains the endogenous ADAM6 gene located at the locus of the endogenous immunoglobulin heavy chain.

In one embodiment, the second modification is located 3 '(with respect to the transcriptional directionality of the mouse V gene segment) of a final and localized 5' mouse V gene segment (with respect to the directionality of the transcript of the constant sequence) of a mouse immunoglobulin heavy chain (or human / mouse chimeric) constant gene or fragment thereof (eg, a nucleic acid sequence encoding a human and / or mouse: CH1 and / or hinge and / or CH2 and / or CH3).

In one embodiment, the modification is in a first allele of the immunoglobulin heavy chain at a first locus coding for a first allele of ADAM6, and the function of ADAM6 results from the expression of an endogenous ADAM6 in a second allele of the chain heavy immunoglobulin at a second locus encoding a functional ADAM6, wherein the second allele of the immunoglobulin heavy chain comprises at least one modification of a V, D, and / or J gene segment. In a specific embodiment, said at least one modification of the gene segment V, D, I J is a deletion, a replacement with a segment of V, D, and / or human J gene, a replacement with a segment of gene V, D, and / or J of camelid, a replacement with a humanized or camelized V, D, and / or J gene segment, a replacement of a heavy chain sequence with a sequence of the light chain, and a combination thereof. In one embodiment, said at least one modification is the deletion of one or more segments of gene V, D, and / or J of the heavy chain and a replacement with one or more segments of gene V and / or J of the chain light (eg, a segment of V gene and / or J of the human light chain) at the locus of the heavy chain.

In one embodiment, the modification is in a first allele of the immunoglobulin heavy chain at a first locus and a second allele of the immunoglobulin heavy chain at a second locus, and the function of ADAM6 results from the expression of an ectopic ADAM6 in a non-immunoglobulin locus in the germ line of the mouse. In a specific modality, the non-immunoglobulin locus is the ROSA26 locus. In a specific embodiment, the non-immunoglobulin locus is active from the point of transcription in reproductive tissue.

In one embodiment, the modification is in a first allele of the immunoglobulin heavy chain at a first locus and a second allele of the immunoglobulin heavy chain at a second locus, and the function of ADAM6 results from an endogenous ADAM6 gene in the germ line of the mouse. In a specific embodiment, the endogenous ADAM6 gene is juxtaposed with mouse immunoglobulin heavy chain gene segments.

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

In one aspect, a mouse comprising a blockade of the heterozygous or homozygous expression of ADAM6 is provided. In one embodiment, the mouse further comprises a modified immunoglobulin sequence that is a human immunoglobulin sequence or a humanized immunoglobulin sequence, or a camelid or camelized human or mouse immunoglobulin sequence. In one embodiment, the modified immunoglobulin sequence is present at the locus of the endogenous immunoglobulin heavy chain. In one embodiment, the modified immunoglobulin sequence comprises a variable region sequence of the human light chain at a locus of the endogenous immunoglobulin heavy chain. In one embodiment, the variable region sequence of the human light chain replaces a variable sequence of the endogenous heavy chain at the locus of the endogenous immunoglobulin heavy chain. In one embodiment, the modified immunoglobulin sequence comprises a variable region sequence of the human light chain k at a locus of the immunoglobulin heavy chain endogenous. In one embodiment, the immunoglobulin sequence modified comprises a variable region sequence of the human light chain A at a locus of the endogenous immunoglobulin heavy chain.

In one aspect, a mouse incapable of expressing a functional endogenous ADAM6 from a locus of endogenous ADAM6 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 rescues a loss in the ability to generate offspring exhibited by a male mouse that is homozygous for a blocked expression of ADAM6. In a specific embodiment, the ectopic nucleic acid sequence codes for a mouse ADAM6 protein.

In one aspect, a mouse lacking a functional endogenous ADAM6 locus is provided, and comprises an ectopic nucleic acid sequence that confers the function of ADAM6 to the mouse. In one embodiment, the nucleic acid sequence comprises an endogenous ADAM6 sequence or functional fragment thereof. In one embodiment, the endogenous ADAM6 sequence comprises the sequence coding for ADAM6a and for ADAM6b located in a wild-type mouse between the V-gene segment of the mouse immunoglobulin heavy chain (VH) more towards the 3 'end and the D-gene segment of the mouse immunoglobulin heavy chain (DH) more towards the 5 'end.

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, in which ADAM6a and / or ADAM6b or functional fragment (s) (is ) of the same (s) are linked in operable form 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 the sequence located between the first codon of the first ADAM6 gene closest to the mouse DH gene segment most toward the 5 'end and the recombination signal sequence of the DH gene segment plus towards the 5 'end, in which 5' is indicated with respect to the transcription 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.

In one embodiment, the promoter is an inducible promoter. In one embodiment, the inducible promoter regulates expression in non-reproductive tissues. In one embodiment, the inducible promoter regulates expression in reproductive tissues. In a specific embodiment, the expression of mouse ADAM6a and / or ADAM6b sequences or functional fragment (s) thereof is regulated from the developmental point of view by the promoter inducible in reproductive tissues.

In one embodiment, the mouse ADAM6a and / or ADAM6b are selected from the ADAM6a of SEQ ID NO: 1 and / or ADAM6b of the sequence 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 to the 5 'end ( with respect to the transcription direction of ADAM6a) of the first codon of ADAM6a and extending towards the end of SEQ ID NO: 3 towards the 5 'end of the coding region of ADAM6. In another specific embodiment, the ADAM6 promoter is a fragment extending from within about 5 to about 20 nucleotides to the 5 'end of the start codon of ADAM6a to about 0.5 kb, 1 kb, 2 kb, or 3 kb or more towards the 5 'end of the start codon of ADAM6a.

In one embodiment, the nucleic acid sequence comprises SEQ ID NO: 3 or a fragment thereof that when placed inside a mouse that is not fertile or that has low fertility due to a lack of ADAM6 improves fertility or restores Fertility up to 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 that is capable of traversing the oviduct of a female mouse in order to fertilize a mouse ovule .

In one embodiment, the mouse ADAM6 promoter is a promoter of SEQ ID NO: 4. In a specific embodiment, the mouse ADAM6 promoter comprises the nucleic acid sequence of SEQ ID NO: 4.

NO: 4 directly towards the 5 'end (with respect to the transcription direction of ADAM6a) of the first codon of ADAM6a and extending towards the end of SEQ ID NO: 4 towards the 5' end of the coding region of ADAM6. In another specific embodiment, the ADAM6 promoter is a fragment extending from within about 5 to about 20 nucleotides to the 5 'end of the start codon of ADAM6a to about 0.5 kb, 1 kb, 2 kb, or 3 kb or more towards the 5 'end of the start codon of ADAM6a.

In one embodiment, the nucleic acid sequence comprises SEQ ID NO: 4 or a fragment thereof that when placed inside a mouse that is not fertile or that has low fertility due to a lack of ADAM6 improves fertility or restores fertility up to approximately a wild type fertility. In one embodiment, SEQ ID NO: 4 or a fragment thereof confers on a male mouse the ability to produce a sperm that is capable of traversing the oviduct of a female mouse in order to fertilize a mouse ovule .

In one embodiment, the mouse ADAM6 promoter is a promoter of SEQ ID NO: 5. In a specific embodiment, the mouse ADAM6 promoter comprises the nucleic acid sequence of SEQ ID NO: 5 directly to the 5 'end ( with respect to the transcription direction of ADAM6a) of the first codon of ADAM6a and extending towards the end of SEQ ID NO: 5 towards the 5 'end of the region encoder of ADAM6. In another specific modality, the promoter of ADAM6 is a fragment extending from about 5 to about 20 nucleotides to the 5 'end of the start codon of ADAM6a to about 0.5 kb, 1 kb, 2 kb, or 3 kb or more towards the 5' end of the codon of start of ADAM6a.

In one embodiment, the nucleic acid sequence comprises SEQ ID NO: 5 or a fragment thereof that when placed inside a mouse that is not fertile or that has low fertility due to a lack of ADAM6 improves fertility or restores fertility up to approximately a wild type fertility. In one embodiment, SEQ ID NO: 5 or a fragment thereof confers to a male mouse the ability to produce a sperm that is capable of traversing the oviduct of a female mouse in order to fertilize a mouse ovule .

In various embodiments, the ectopic nucleic acid sequence which confers to the mouse the function of ADAM6 codes for one or more ADAM6 proteins, wherein said one or more ADAM6 proteins comprise SEQ ID NO: 1, SEQ ID NO: 2 or a combination from the same.

In various embodiments, the ectopic nucleic acid sequence comprises a sequence that is selected from SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, in which the ectopic nucleic acid sequence confers the mouse the function of ADAM6 through one or more ADAM6 proteins encoded by the acid sequence ectopic nucleus.

In one embodiment, the nucleic acid sequence is any sequence that encodes an ADAM6 gene or homologous or orthologous or functional fragment thereof that when placed within or maintained in a mouse produces a level of fertility that is the same or comparable to that of a wild-type mouse. An example of fertility level can be demonstrated by the ability of a male mouse to produce a sperm that is capable of traversing the oviduct of a female mouse in order to fertilize a mouse ovule.

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 VH gene segment with a human VH gene segment, and an ectopic nucleotide sequence that encodes a mouse ADAM6 protein or ortholog or homologue or fragment thereof that is functional in a male mouse.

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 VH gene segment with a human VL gene segment, and an ectopic nucleotide sequence that encodes a mouse ADAM6 protein or ortholog or homologue or fragment thereof that is functional in a male mouse. In one embodiment, the human VL gene segment is a VK gene segment. In one embodiment, the VL gene segment is a segment of the VA gene.

In one embodiment, the mouse further comprises a human JL gene segment, and the ectopic nucleotide sequence encoding a mouse ADAM6 protein or ortholog or homologue or fragment thereof that is functional in a male mouse is positioned between a human VL gene segment and the human JL gene segment. In one embodiment, the mouse comprises one or more human VL gene segments and one or more human VL gene segments and the ectopic nucleotide sequence encoding a mouse ADAM6 or ortholog protein or homologue or fragment thereof which is functional in a male mouse is positioned towards the 5 'end (or 5') of said one or more human VL gene segments. In a specific embodiment, the human VL and JL gene segments are the VK and JK gene segments.

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

In one embodiment, the mouse comprises a replacement of all or substantially all of the endogenous VH gene segments with a nucleotide sequence that codes for one or more segments of VL gene of human, and the ectopic nucleotide sequence encoding the mouse ADAM6 protein is within, or directly adjacent to, the nucleotide sequence encoding said one or more human VL gene segments. In one embodiment, the mouse further comprises a replacement of one or more endogenous DL gene segments with one or more human VL gene segments and / or human JL gene segments at the locus of the endogenous DH gene. In one embodiment, the mouse further comprises a replacement of one or more segments of endogenous JH gene with one or more segments of human JL gene at the locus of the endogenous JH gene. In one embodiment, the mouse comprises a replacement of all or substantially all of the endogenous VH, DH, and JH gene segments and a replacement at the endogenous VH, DH, and JH gene loci with human VL and JL gene segments, wherein the mouse comprises an ectopic sequence encoding a mouse ADAM6 protein. In one embodiment, the mouse comprises an insertion of one or more human VL and JL gene segments at a locus of the endogenous immunoglobulin heavy chain, in which the mouse comprises an ADAM6 gene that is functional in the mouse. In a specific embodiment, the human VL and JL gene segments are the VK and JK gene segments. In a specific embodiment, the ectopic sequence encoding the mouse ADAM6 protein is placed between the penultimate VL gene segment more towards the 3 'end of the human VL gene segments present, and the last of the JL gene segments more towards the 5 'end of the JL gene segments of human present In a specific modality, the ectopic sequence encoding the mouse ADAM6 protein is positioned towards the 5 '(or 5') end of the VL gene segment further towards the 5 'end of the human VL gene segments present. In a specific embodiment, the mouse comprises a deletion of all or substantially all mouse VH gene segments, and a replacement with at least 40 segments of human VL gene, and the ectopic nucleotide sequence encoding the ADAM6 protein of mouse is positioned towards the 3 'end of the human VK4-1 gene segment and towards the 5' end of the human JK1 gene segment. In a specific embodiment, the mouse comprises a deletion of all or substantially all mouse VH gene segments, and a replacement with at least 40 segments of human VL gene, and the ectopic nucleotide sequence encoding the ADAM6 protein of mouse is positioned towards the 5 'end of a human VK2-40 gene segment.

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 codes for one or more segments of the human VL gene, and the ectopic nucleotide sequence encoding the protein Mouse ADAM6 is within, or directly adjacent to, the nucleotide sequence encoding said one or more human VL gene segments.

In one embodiment, the VL gene segments are VK gene segments. In one embodiment, the VL gene segments are segments of the VA gene.

In one embodiment, the ectopic nucleotide sequence that coding for the mouse ADAM6 protein is present in a transgene in the mouse genome. In one embodiment, the ectopic nucleotide sequence encoding the mouse ADAM6 protein is extra-chromosomally present in the mouse.

In one aspect, there is provided a mouse comprising a modification of a locus of immunoglobulin of the endogenous heavy chain, in which the mouse expresses a B cell comprising a sequence of rearranged immunoglobulin operably linked to a gene sequence. of the constant region of the heavy chain, and the B cell comprises in its genome (e.g., on a B cell chromosome) a gene encoding an ADAM6 or ortholog or homologue or fragment thereof that is functional in a mouse of masculine gender. In one embodiment, the rearranged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises a V gene sequence, J, and optionally a D gene sequence of the human light chain; a sequence of V, D, and / or J of the mouse heavy chain; a sequence of V and / or J of the human or mouse light chain. In one embodiment, the sequence of the heavy chain constant region gene comprises a human heavy chain sequence or a mouse heavy chain sequence that is selected from the group consisting of a CH1, a hinge, a CH2, a CH3, and a combination thereof.

In one embodiment, the V and / or J sequence of the human light chain is selected from a sequence VK, VA, JK and JA of human.

In one aspect, there is provided a mouse comprising a locus of the functionally silenced endogenous immunoglobulin heavy chain, in which the function of ADAM6 is maintained in the mouse, and further comprises an insertion of one or more gene segments of the same gene. human immunoglobulin, in which said one or more human immunoglobulin gene segments include human VL and JL gene segments, and optionally human DH gene segments. In one embodiment, said one or more human immunoglobulin gene segments include human VK, VA, JK and JA gene segments.

In one aspect, a genetically modified mouse is provided, in which the mouse comprises a functionally silenced immunoglobulin light chain gene, and further comprises a replacement of one or more segments of the variable region of the heavy chain Endogenous immunoglobulin with one or more segments of the variable region of the human immunoglobulin light chain, in which the mouse lacks a locus of functional endogenous ADAM6, and in which the mouse comprises an ectopic nucleotide sequence which expresses a mouse ADAM6 protein or an ortholog or homologue or fragment thereof that is functional in a male mouse.

In one aspect, a genetically modified mouse is provided, in which the mouse comprises a locus of the functionally silenced immunoglobulin light chain gene, and further comprises a replacement of one or more segments of the variable gene of the light chain of endogenous immunoglobulin with one or more segments of the variable gene of the human immunoglobulin light chain, in which the mouse lacks a functional endogenous ADAM6 locus, and in which the mouse comprises an ectopic nucleotide sequence encoding for a mouse ADAM6 protein or an ortholog or homologue or fragment thereof that is functional in a male-gender mouse.

In one embodiment, said one or more segments of the variable gene of the human immunoglobulin light chain are contiguous with the ectopic nucleotide sequence.

In one aspect, there is provided a mouse lacking a locus or functional endogenous ADAM6 sequence and comprising an ectopic nucleotide sequence encoding a mouse ADAM6 locus or functional fragment from a locus or mouse ADAM6 sequence, which the mouse is capable of mating with a mouse of the opposite sex to produce a progeny comprising the locus or sequence of ectopic ADAM6. In one modality, the mouse is male. In one modality, the mouse is female.

In one aspect, there is provided a genetically modified mouse, in which the mouse comprises a segment of the variable region of the human immunoglobulin light chain at a locus of the variable region gene of the endogenous immunoglobulin heavy chain, the mouse lacks an endogenous functional ADAM6 sequence at the locus of the variable region gene of the it comprises an ectopic nucleotide sequence expressing a mouse ADAM6 protein or an ortholog or homologue or fragment thereof that is functional in a male-gender mouse.

In one aspect, a genetically modified mouse is provided, in which the mouse comprises a segment of the variable gene of the human immunoglobulin light chain at a locus of the variable region gene of the endogenous immunoglobulin heavy chain, the mouse lacks of a functional endogenous ADAM6 sequence in the locus of the variable gene of the endogenous immunoglobulin heavy chain, and in which the mouse comprises an ectopic nucleotide sequence expressing a mouse ADAM6 protein or an ortholog or homologue or fragment thereof which is functional in a male mouse.

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, said one or more loci include an immunoglobulin locus.

In one aspect, a mouse is provided which expresses a sequence of the immunoglobulin light chain from a locus of the modified endogenous immunoglobulin heavy chain, in which the heavy chain is derived from a VL gene segment, a J gene segment, and optionally a DH gene segment, of human, in which the mouse comprises an ADAM6 activity that is functional in the mouse. In one embodiment, the human VL gene segment is selected from a human VK gene segment and a human VA gene segment. In various embodiments, the J gene segment is a JH gene segment, a JK segment OR a JA segment or a combination thereof.

In one embodiment, the mouse comprises a plurality of human VL gene segments and a plurality of gene segments J. In a specific embodiment, the J gene segments are JL gene segments.

In one aspect, a mouse is provided which expresses a sequence of the immunoglobulin light chain from a locus of the modified endogenous immunoglobulin heavy chain, in which the heavy chain is derived from a segment of the human VL gene. and a JL gene segment, in which the mouse comprises an ADAM6 activity that is functional in the mouse.

In one embodiment, the mouse comprises a plurality of human V gene segments, a plurality of J gene segments, and optionally a plurality of D gene segments. In one embodiment, the D gene segments are D gene segments of human, in one embodiment, the J gene segments are human J gene segments. In one embodiment, the mouse further comprises a sequence of the humanized heavy chain constant region, wherein the humanization comprises the replacement of a sequence that is selected from a CH1 hinge, CH2, CH3, and a combination of Ijj ?? IRR? 6P yfl? Specificity, the heavy chain is derived from a human V gene segment, a human J gene segment, a human CH1 sequence, a human or mouse hinge sequence, a mouse CH2 sequence, and a mouse CH3 sequence. In another specific embodiment, the mouse further comprises a constant sequence of the human light chain. In one embodiment, the mouse comprises an ADAM6 gene that is flanked 5 'and 3' by segments of the endogenous immunoglobulin heavy chain gene. In a specific embodiment, the variable gene segments of the endogenous immunoglobulin heavy chain are unable to code for a variable region of the heavy chain of an antibody. In a specific embodiment, the mouse ADAM6 gene is in a position that is the same as in a wild type mouse and the variable gene loci of the endogenous immunoglobulin heavy chain of the mouse are unable to rearrange to code for a chain heavy of an antibody.

In one embodiment, the plurality of human V gene segments are flanked 5 '(with respect to the transcription direction of the V gene segments) by a sequence encoding an ADAM6 activity that is functional in the mouse. In a specific embodiment, the plurality of human V gene segments include the human VK gene segments VK4-1, VK5-2, VK7-3, VK2-4, VK1 -5, VK1 -6, VK3-7, VK1 -8, VK1 -9, VK2-10, VK3-1 1, VK1 -12, VK1 -13, VK2-14, VK3-15, VK1 -16, VK1 -17, V 2-18, V 2-19 , VK3-20, VK6-21, VK1-22, VK1-23, VK2-24, VK3-25, VK2-26, VK1-27, VK2-28, VK2-29, VK2-30, VK3-31, VKI-32, VKI-33, VK3-34, VKI-35, VK2-36, V I-37, VK2-38, V1-39, and VK2-40 and the human VK2-40 gene segment is flanked 5 '(with respect to the transcription direction of the human VK2-40 gene segment) by a sequence coding for an activity of ADAM6 that is functional in the mouse. In a specific embodiment, the sequence encoding an ADAM6 activity that is functional in the mouse is placed in the same transcription orientation with respect to the human VK gene segments. In a specific embodiment, the sequence encoding an ADAM6 activity that is functional in the mouse is placed in the opposite transcription orientation with respect to the human VK gene segments.

In one embodiment, the V gene segment is flanked 3 '(with respect to the transcription direction of the V gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse.

In one embodiment, the D gene segment is flanked 5 '(with respect to the transcription direction of the D gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse.

In one embodiment, the J gene segment is flanked 5 '(with respect to the transcription direction of the J gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse.

In one embodiment, the activity of ADAM6 that is functional in the mouse results from the expression of a nucleotide sequence located 5 'of the D gene segment more towards the 5' and 3 'end of the V gene segment more towards the 3' end (with respect to the transcription direction of the V gene segment) of the heavy chain immunoglobulin locus modified endogenous In one embodiment, the activity of ADAM6 that is functional in the mouse results from the expression of a nucleotide sequence located 5 'of the J gene segment further towards the 5' and 3 'end of the V gene segment more towards the 3 rd end. '(with respect to the transcription direction of the V gene segment) of the modified endogenous immunoglobulin heavy chain locus.

In one embodiment, the 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 locus of the modified endogenous immunoglobulin heavy chain. In one embodiment, the two human V gene segments are a VK5-2 gene segment and a human VK4-1 gene segment.

In one embodiment, the activity of ADAM6 that is functional in the mouse results from the expression of a nucleotide sequence located between a human V gene segment and a human J gene segment at the locus of the endogenous immunoglobulin heavy chain modified. In one embodiment, the human V gene segment is a human VK4-1 gene segment and the human J segment is a JK 1 gene segment.

In one embodiment, the nucleotide sequence comprises a sequence that is selected from a sequence of ADAM6b of mouse or functional fragment thereof, a mouse ADAM6a sequence or functional fragment thereof, and a combination thereof.

In various embodiments, the sequence encoding an ADAM6 activity that is functional in the mouse encoding an ADAM6b protein indicated in SEQ ID NO: 2 and / or encoding an ADAM6a protein indicated in SEQ ID NO: 1.

In one embodiment, the nucleotide sequence between the two human V gene segments is placed in opposite transcription orientation with respect to human V gene segments. In a specific embodiment, the nucleotide sequence encodes, from 5 'to 3' with respect to the transcription direction of ADAM6 genes, a sequence of ADAM6a followed by a sequence of ADAM6b. In one modality, In one embodiment, the nucleotide sequence between the human V gene segment and the human J gene segment is placed in opposite transcription orientation with respect to the human V and J gene segments. In a specific embodiment, the nucleotide sequence encodes, from 5 'to 3' with respect to the transcription direction of ADAM6 genes, a sequence of ADAM6a followed by a sequence of ADAM6b.

In one embodiment, the mouse comprises a hybrid immunoglobulin sequence, in which the hybrid immunoglobulin sequence comprises a sequence of the human immunoglobulin light chain k contiguous with a non-ADAM6 sequence. human In one embodiment, the mouse comprises a contiguous human sequence with a mouse sequence in a locus of the endogenous immunoglobulin heavy chain, in which the contiguous sequence comprises at least one segment of human VL gene, a sequence of ADAM6 of mouse or ortholog or homologue or functional fragment thereof, and a segment of human JL gene. In a specific embodiment, the ADAM6 sequence of mouse or orthologous or homologue or functional fragment thereof is positioned immediately adjacent to said at least one segment of human VL gene. In one embodiment, the human VL gene segment is a human VK gene segment. In one embodiment, the ADAM6 sequence of mouse or orthologous or homologue or functional fragment thereof is positioned immediately adjacent and 3 'to said at least one segment of human VL gene and immediately adjacent and 5' to the JL gene segment of human. In a specific embodiment, the human VL gene segment is a human VK gene segment and the human JL gene segment is a human JK gene segment.

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

In one embodiment, the mouse comprises an endogenous mouse DFL16.1 gene segment (eg, in a mouse heterozygous for the modified endogenous mouse immunoglobulin heavy chain locus), or a human DH1 -1 gene segment from human . In one modality, the D-gene segment of the immunoglobulin heavy chain expressed by the mouse is derived from an endogenous mouse DFL16.1 gene segment or a human DH1-1 gene segment.

In one aspect, there is provided a mouse comprising a nucleic acid sequence encoding a mouse ADAM6 (or homologous or orthologous or functional fragment thereof) in a cell carrying non-rearranged B cell lineage DNA., but does not comprise the nucleic acid sequence encoding mouse ADAM6 (or homologue or ortholog or functional fragment thereof) in a B cell comprising rearranged immunoglobulin loci, in which the nucleic acid sequence encoding mouse ADAM6 (or homologous or orthologous or functional fragment thereof) is presented in the genome in 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 homologous or orthologous or functional fragment thereof) is present in all or substantially all cells bearing DNA that are not of rearranged B cell lineage.; in one embodiment, the nucleic acid sequence is present in germline cells of the mouse, but not in a chromosome of a rearranged B cell.

In one aspect, there is provided a mouse comprising a nucleic acid sequence encoding a mouse ADAM6 (or homologue or ortholog or functional fragment thereof) in all or substantially all cells carrying DNA, including B cells comprising locus of immunoglobulin rearranged, in which the nucleic acid sequence encoding mouse ADAM6 (or homologous or orthologous or functional fragment thereof) is presented in the genome in a position that is different from a position in which a mouse ADAM6 gene appears in a mouse of wild type. In one embodiment, the nucleic acid sequence encoding mouse ADAM6 (or homologue or ortholog or functional fragment thereof) is in a nucleic acid that is contiguous with the locus of rearranged immunoglobulin. 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 found in a wild-type mouse and the chromosome comprises a modification of a mouse immunoglobulin locus.

In one aspect, a genetically modified mouse is provided, in which the mouse comprises a B cell comprising in its genome a sequence of ADAM6 or ortholog or homologue thereof. In one embodiment, the ADAM6 sequence or ortholog or homologue thereof is at a locus of the immunoglobulin heavy chain. In a specific embodiment, the heavy chain locus comprises endogenous immunoglobulin heavy chain gene segments that are unable to rearrange to code for the heavy chain of an antibody. In one embodiment, the ADAM6 sequence or orthologue or homolog thereof is in a locus that is not an immunoglobulin locus. In one embodiment, the sequence of ADAM6 is in a transgene controlled by a promoter heterologous, In a specific modality, the heterologous promoter is a non-immunoglobulin type promoter. In a specific embodiment, the B cell expresses an ADAM6 protein or ortholog or homologue thereof.

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

In one embodiment, the B cell genome comprises a first allele and a second allele comprising the ADAM6 sequence or ortholog or homologue thereof. In one embodiment, the B cell genome comprises a first allele but not a second allele comprising the sequence of ADAM6 or ortholog or homologue thereof.

In one aspect, a mouse is provided comprising a modification in one or more alleles of the endogenous immunoglobulin heavy chain, in which the modification maintains one or more endogenous ADAM6 alleles.

In one embodiment, the modification causes the mouse to be unable to express a functional heavy chain comprising endogenous heavy chain gene segments rearranged from at least one allele of the heavy chain and maintains an endogenous ADAM6 allele located within said at least one allele of the endogenous immunoglobulin heavy chain.

In one embodiment, the mice are incapable of expressing a functional heavy chain comprising endogenous heavy chain gene segments rearranged from at least one of the endogenous immunoglobulin heavy chain alleles, and the mice express an ADAM6 protein from an endogenous ADAM6 allele. In a specific embodiment, the mice are unable to express a functional heavy chain comprising endogenous heavy chain gene segments rearranged from two alleles of the endogenous immunoglobulin heavy chain, and the mice express an ADAM6 protein from one or more endogenous ADAM6 alleles.

In one embodiment, the mice are unable to express a functional heavy chain from each allele of the endogenous heavy chain, and the mice comprise a functional ADAM6 allele located within 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 or more Mpb towards the 5 'end (with respect to the transcription direction of the mouse immunoglobulin heavy chain locus) of a constant region sequence of the mouse immunoglobulin heavy chain. In a specific embodiment, the functional ADAM6 allele is at a locus of the endogenous heavy chain (e.g., in an endogenous VD region, between the V gene segments, between a V gene segment and a D gene segment, between a segment of gene D and a segment of gene J, etc.). In a specific embodiment, the functional ADAM6 allele is located within 90 to 100 kb of an intergenic sequence between the final V gene segment of the mouse and the first mouse D gene segment. In another specific embodiment, the endogenous 90 to 100 kb endogenous V-D sequence is removed, and the ectopic ADAM6 sequence is placed between the final V gene segment and the first D gene segment.

In one aspect, a mouse comprising a modification in one or more alleles of endogenous ADAM6 is provided.

In one embodiment, the modification renders the mouse incapable of expressing a functional ADAM6 protein from at least one of said 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.

In one embodiment, the mouse is unable to express a functional ADAM6 protein from each allele of endogenous ADAM6, and the mice comprise an ectopic ADAM6 sequence.

In one embodiment, the mouse is unable to express a functional ADAM6 protein from each allele of endogenous ADAM6, and the mouse comprises an ectopic ADAM6 sequence located within 1, 2, 3, 4, 5, 10, 20, 30 , 40, 50, 60, 70, 80, 90, 100, 110, or 120 or more kb towards the 5 'end (with respect to the transcription direction of the mouse heavy chain locus) of a sequence from the region constant of the mouse immunoglobulin heavy chain. In a specific embodiment, the ectopic ADAM6 sequence is at the locus of the endogenous immunoglobulin heavy chain (eg, in an intergenic VD region, between two segments of the V gene, between a segment of the V gene and a segment of the J gene). , between a segment of the D gene and a segment of the J gene, etc.). In a specific embodiment, the ectopic ADAM6 sequence is located within an intergenic sequence of 90 to 100 kb between the final segment of the mouse V gene and the first segment of the mouse D gene. In another specific modality, the endogenous intergenic V-D sequence of 90 to 100 kb is deleted, and the ectopic ADAM6 sequence is positioned between a human VK gene segment and a first human JK gene segment. In another specific embodiment, the endogenous intergenic VD sequence of 90 to 100 kb is deleted, and the ectopic ADAM6 sequence is placed 5 'or upstream of a human VK gene segment, in which the human VK gene segment it is selected from a human VK4-1 or VK2-40 gene segment.

In one embodiment, the mouse is capable of expressing a functional ADAM6 protein from one or more endogenous ADAM6 alleles and the modification includes an insertion of a human sequence encoding an immunoglobulin variable domain. In one embodiment, the human sequence comprises segments of the non-rearranged immunoglobulin gene. In a specific embodiment, the human sequence comprises a V gene segment and a J gene segment. In another specific embodiment, the human sequence comprises a V gene segment, a J gene segment, and a D gene segment .

In one aspect, an infertile male gender mouse is provided, in which the mouse comprises a deletion of two or more endogenous ADAM6 alleles. In one aspect, a female mouse carrying a male infertility trait is provided, in which the female mouse comprises in its germline a non-functional ADAM6 allele or a blocked expression of an allele of Endogenous ADAM6.

In one aspect, a mouse is provided comprising an endogenous V, D, I, and J gene segment of the immunoglobulin heavy chain that is unable to rearrange to code for an antibody heavy chain, in which the majority of the cells B of the mouse comprises a functional ADAM6 gene. In various embodiments, the majority of B cells further comprise one or more human VL and JL gene segments toward the 5 'end of a constant region of the mouse immunoglobulin heavy chain. In one embodiment, the human VL and JL gene segments are the VK and Jk gene segments.

In one embodiment, the mouse comprises intact intact V, D, and J gene segments of the immunoglobulin heavy chain that are unable to rearrange to code for 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 said 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 code for a variable region of the heavy chain of an antibody. In a specific embodiment, the mouse comprises a functional ADAM6 gene located within the intact endogenous V, D, and J gene segments of the immunoglobulin heavy chain. In one embodiment, the mouse comprises a locus of the wherein the locus of the endogenous heavy chain comprises 89 V gene segments, 13 D gene segments, and four J gene segments, in which the endogenous heavy chain gene segments are unable to rearrange to code for a region variable of the heavy chain of an antibody and the ADAM6 locus encodes an ADAM6 protein that is functional in the mouse.

In one aspect, a method for making a male infertile mouse is provided, which comprises making an allele of endogenous ADAM6 from a donor embryonic stem cell (IS cell) non-functional (or blocking the expression of said allele), introducing the donor ES cell in a host embryo, carrying out the gestation of the host embryo in a surrogate mother, and allowing the surrogate mother to give birth to the progeny derived in whole or in part from the donor ES cell. In one embodiment, the method further comprises reproducing the progeny to obtain a male infertile mouse.

In one aspect, a method is provided for making a mouse with a genetic modification of interest, in which the mouse is infertile, the method comprising the steps of (a) making a genetic modification of interest in a genome; (b) modifying the genome to block the expression of an endogenous ADAM6 allele, or making an endogenous ADAM6 allele non-functional; and, (c) employing the genome in the making of a mouse. In various embodiments, the genome comes from an ES cell or is used in a nuclear transfer experiment.

In one aspect, a mouse is provided that lacks a V, D, and endogenous J gene segment of the immunoglobulin heavy chain, in which a majority of the B cells of the mouse comprises a sequence of ADAM6 or ortholog or homologue thereof.

In one embodiment, the mouse lacks endogenous immunoglobulin heavy chain gene segments that are selected from two or more V gene segments, two or more D gene segments, two or more J gene segments, and one combination of them. In one embodiment, the mouse lacks immunoglobulin heavy chain gene segments that are selected from at least one and up to 89 V gene segments, at least one and up to 13 D gene segments, at least one and up to four J gene segments, and a combination thereof. In one embodiment, the mouse lacks a genomic DNA fragment of chromosome 12 comprising approximately three megabases of the endogenous Immunoglobulin heavy chain locus. In a specific embodiment, the mouse lacks all the functional endogenous heavy chain V, D, and J segments of the gene. In a specific embodiment, the mouse lacks 89 VH gene segments, 13 DH gene segments and four JH gene segments.

In one aspect, a mouse is provided, in which the mouse has a germline genome comprising a modification of a locus of the immunoglobulin heavy chain, wherein the modification to the locus of the immunoglobulin heavy chain comprises the replacement of one or more sequences of the mouse immunoglobulin variable region with one or more immunoglobulin variable region sequences of the non-mouse type, and in which the mouse comprises a nucleic acid sequence encoding a mouse ADAM6 protein. In one embodiment, the DH and JH sequences and at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 VH sequences of the heavy chain locus of endogenous immunoglobulin are replaced by non-mouse immunoglobulin light chain sequences. In one embodiment, the DH, JH, and all VH sequences of the endogenous immunoglobulin heavy chain locus are replaced by a plurality of VL gene segment sequences, one or more JL gene segment sequences, and optionally one or more immunoglobulin D gene segment sequences that are not mouse. Non-mouse immunoglobulin sequences may not be rearranged. In a modality, the non-mouse immunoglobulin sequences comprise whole VL and non-rearranged regions of the non-mouse type species. In one embodiment, the non-mouse immunoglobulin sequences are capable of forming a complete variable region, i.e., a rearranged variable region containing the VL and JL gene segments linked together to form a sequence encoding a region variable of the light chain, of the non-mouse type species, operably linked to one or more endogenous constant regions. The non-mouse type species can be Homo sapiens and the non-mouse immunoglobulin sequences can be human sequences.

In one aspect, a genetically modified mouse is provided comprising a nucleotide sequence encoding an ADAM6 protein or functional fragment thereof which is contiguous with a variable gene segment of the human immunoglobulin light chain.

In one embodiment, the mouse lacks an endogenous unmodified ADAM6 gene sequence. In one embodiment, the mouse lacks a functional endogenous ADAM6 gene sequence.

In one embodiment, the variable gene segment of the human immunoglobulin light chain is a variable gene segment of the immunoglobulin light chain k. In one embodiment, the variable gene segment of the human immunoglobulin light chain is a variable gene segment of the immunoglobulin light chain A. In one embodiment, the variable gene segment of the human immunoglobulin light chain is operably linked to a constant gene sequence of the immunoglobulin heavy chain.

In one embodiment, the constant gene sequence of the immunoglobulin heavy chain is a mouse or rat or human heavy chain gene sequence. In one embodiment, the constant gene sequence of the heavy chain comprises a CH1 region and / or a hinge region.

In one embodiment, the mouse comprises deleting, or replacing, one or more endogenous immunoglobulin heavy chain gene sequences.

In one embodiment, the mouse further comprises a non-rearranged human VK gene segment or a non-human VA gene segment. rearranged human linked operably to a constant region sequence of the human or mouse or rat light chain. In one embodiment, the mouse comprises a plurality of non-rearranged human VK gene segments (eg, two or more human VK segments and one or more human JK segments) or a plurality of non-rearranged human VA gene segments. (for example, two or more VA segments of human and one or more human JA segments). In one embodiment, non-rearranged human VK or non-rearranged human VK gene segments are operably linked to a constant region sequence at a locus of the endogenous immunoglobulin light chain.

In one embodiment, the mouse further comprises a modification that causes a locus of the endogenous light chain k and / or a locus of the endogenous light chain A to be non-functional (s). In one embodiment, the mouse comprises a blocked expression or a deletion of a locus of the endogenous mouse light chain k and / or a locus of the mouse endogenous light chain A.

In one aspect, there is provided a method for maintaining a mouse strain, in which the mouse strain comprises a replacement of a mouse immunoglobulin heavy chain sequence with one or more human immunoglobulin light chain sequences. In one embodiment, said one or more sequences of the human immunoglobulin light chain are human immunoglobulin VK and / or JK gene segments.

In one embodiment, the mouse strain comprises a deletion of one or more segments of the VH gene, DH, and / or mouse JH. In one embodiment, the mouse further comprises one or more human VL gene segments and one or more human JL gene segments. In one embodiment, the mouse comprises at least 6, at least 16, at least 30, or at least 40 segments of the human VK gene and at least five segments of the JK gene. In a specific embodiment, the gene segments of the human light chain are operably linked to a gene of the constant region. In one embodiment, the gene of the constant region is a gene of the mouse constant region. In one embodiment, the constant region gene comprises a mouse constant region gene sequence that is selected from a CH1, a hinge, a CH2, a CH3, and / or a CH4 or a combination thereof .

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

In one embodiment, the method comprises obtaining cells at from male or female homozygous or female mice heterozygotes for the sequence of the human heavy chain, and use said cells as donor cells or the nuclei coming from them as donor nuclei, and use the cells or nuclei to elaborate genetically modified animals using the host cells and / or carrying out the gestation of cells and / or nuclei in surrogate mothers.

In one embodiment, only male mice that are heterozygous for replacement at the heavy chain locus are crossed with female mice. In a specific embodiment, female mice are homozygous, heterozygous, or wild type with respect to a locus of the replaced heavy chain.

In one embodiment, the mouse further comprises a replacement of the variable sequences of the light chain A and / or k at a locus of the endogenous immunoglobulin light chain with heterologous immunoglobulin light chain sequences. In one embodiment, the heterologous immunoglobulin light chain sequences are variable sequences of the human immunoglobulin light chain A and / or k.

In one embodiment, the mouse further comprises a transgene at a different locus of an endogenous immunoglobulin locus, in which the transgene comprises a sequence encoding a rearranged or non-rearranged heterologous light chain A ok sequence (eg, VL not rearranged and JL not rearranged, or VJ rearranged) linked in operable form (for those not rearranged) or merged (for the rearranged) to a sequence of the region constant of the immunoglobulin light chain. In one embodiment, the sequence of the heterologous light chain h or k is human. In one embodiment, the sequence of the constant region is selected from rodent, human, and non-human primate. In one embodiment, the sequence of the constant region is selected from mouse, rat, and hamster. In one embodiment, the transgene comprises a non-immunoglobulin promoter that controls the expression of the light chain sequences. In a specific embodiment, the promoter is an active promoter from the transcription point of view. In a specific embodiment, the promoter is a ROSA26 promoter.

In one aspect, a fertile mouse comprising a modification of an endogenous ADAM6 gene is provided, in which the mouse comprises an ectopic sequence that confers ADAM6 function to the mouse, and in which the mouse comprises a segment in its germline. of the non-rearranged immunoglobulin light chain gene operably linked to a nucleic acid sequence encoding an immunoglobulin heavy chain sequence.

In one aspect, a fertile mouse comprising a modification of a locus of endogenous ADAM6 is provided, in which the modification makes the locus of ADAM6 non-functional, and in which the mouse expresses an immunoglobulin-binding protein comprising a domain variable of the human immunoglobulin light chain contiguous with a constant sequence of the heavy chain.

In one embodiment, the immunoglobulin binding protein further comprises a variable domain of the light chain of human cognate immunoglobulin fused to a constant sequence of the light chain.

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

In one aspect, a mouse is provided, comprising a locus of the immunoglobulin heavy chain comprising a replacement of one or more variable region gene segments of the immunoglobulin heavy chain (VH), gene diversity segments of heavy chain (DH), and heavy chain binding gene (JH) segments at a locus of the endogenous immunoglobulin heavy chain with one or more light chain variable region (VL) gene segments and one or more gene segments of the light chain binding region (JL), in which the mouse is capable of expressing an ADAM6 protein.

In one aspect, a mouse is provided, comprising a locus of the immunoglobulin heavy chain comprising a replacement of all or substantially all segments of the VH, DH, and JH gene with one or more segments of the VL gene and one or more segments JL gene to form a sequence of segment VL gene into a locus of the endogenous heavy chain (locus VLH), in which the locus VLH is capable of recombining with CH gene endogenous to form a rearranged gene is derived from a VL gene segment, a JL gene segment, and an endogenous CH gene.

In one embodiment, the VL segments are human VL. In one embodiment, the JL segments are human JL segments. In a specific mode, segments VL and JL are segments VL of human and JL of human.

In one embodiment, all or substantially all of the VH, DH, and JH gene segments are replaced with at least six segments of the human VK gene and at least one segment of the JK gene. In one embodiment, all or substantially all of the VH, DH, and JH gene segments are replaced with at least 16 segments of the human VK gene (human VK) and at least one segment of the JK gene. In one embodiment, all or substantially all of the VH, DH, and H gene segments are replaced with at least 30 segments of the human VK gene and at least one segment of the JK gene. In one embodiment, all or substantially all of the VH, DH, and JH gene segments are replaced with at least 40 segments of the human VK gene and at least one segment of the JK gene. In one embodiment, said at least one segment of the JK gene comprises two, three, four, or five segments of the human JK gene.

In one embodiment, the VL segments are human VK segments. In one embodiment, the human VK segments comprise 4-1, 5-2, 7-3, 2-4, 1 -5, and 1-6. In one embodiment, the human VK segments comprise 3-7, 1 -8, 1 -9, 2-10, 3-11, 1 -12, 1 -13, 2-14, 3-15, and 1 - . 1-16 In one embodiment, the human VK segments comprise 1-17, 2-18, 2-19, 3-20, 6-21, 1 -22, 1-23, 2-24, 3-25, 2-26, 1 -27, 2-28, 2-29, and 2 -30. In one embodiment, the human VK segments comprise 3-31, 1-32, 1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and 2-40. .

In one embodiment, the VL segments are VK segments of human and comprise 4-1, 5-2, 7-3, 2-4, 1-5, 1-6, 3-7, 1-8, 1-9, 2-10, 3- 11, 1-12, 1-13, 2-14, 3-15, and 1-16. In one embodiment, the VK segments further comprise 1 -17, 2-18, 2-19, 3-20, 6-21, 1 -22, 1-23, 2-24, 3-25, 2-26, 1 -27, 2-28, 2-29, and 2-30. In one embodiment, the VK segments further comprise 3-31, 1 -32, 1 -33, 3-34, 1 -35, 2-36, 1-37, 2-38, 1-39, and 2-40.

In one embodiment, the VL segments sori human VA segments and comprise a cluster fragment (cluster) A of the human light chain A locus. In a specific embodiment, the fragment of cluster A of the human light chain A locus extends from hVA3-27 to hVA3-1.

In one embodiment, the VL segments comprise a fragment of cluster B of the locus of human light chain A. In a specific embodiment, the fragment of cluster B of the human light chain A locus extends from hVA5-52 to hVA1-40.

In one embodiment, the VL segments comprise a sequence of the variable region of the light chain A human genomic fragment comprising a cluster A and a genomic fragment cluster B. In one embodiment, the sequence of the variable region chain light A of human comprises at least one segment of cluster A gene and at least one segment of cluster B gene.

In one embodiment, the VL segments comprise at least one gene segment of cluster B and at least one segment of cluster C gene.

In one embodiment, the VL segments comprise hVA3-1, 4- 3, 2-8, 3-9, 3-10, 2-11, and 3-12. In a specific embodiment, the VL segments comprise a contiguous sequence of the human light chain A locus ranging from VA3-12 to VA3-1. In one embodiment, the contiguous sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hVAs. In a specific embodiment, the hVAs include 3-1, 4-3, 2-8, 3-9, 3-10, 2-1 1, and 3-12. In a specific embodiment, the hVAs comprise a contiguous sequence of human A locus ranging from VA3-12 to VA3-1.

In one embodiment, the hVAs comprise 13 to 28 or more hVAs. In a specific modality, the hVAs 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 hVAs comprise a contiguous sequence of human A locus ranging from VA3-27 to VA3-1.

In one embodiment, the VL segments comprise 29 to 40 hVAs. In a specific embodiment, the VL segments comprise a contiguous sequence of human A locus ranging from VA3-29 to VA3-1, and a contiguous sequence of human A locus ranging from VA5-52 to VA1-40. In a specific embodiment, all or substantially all sequences between hVA1-40 and hVA3-29 in the genetically modified mouse consists essentially of a human A sequence of approximately 959 bp found in Nature (eg, in the human population) towards the 3 'end of the hVA1 -40 gene segment (downstream of the 3' untranslated portion), a restriction enzyme site (e.g., Pl-Scel), followed by a human A sequence of approximately 3.431 bp towards the 5 'end of the hVA3-29 gene segment found in Nature.

In one embodiment, the JK is human and is selected from the group consisting of J k 1, JK2, JK3, JK4, JK5, and a combination thereof. In a specific embodiment, the JK comprises JK1 through JK5.

In one embodiment, the VL segments are human VA segments, and the JK gene segment comprises a recombination signal sequence (RSS) having a 12-mer spacer, in which the RSS is juxtaposed at the upstream end of the JK gene segment. In one embodiment, the VL gene segments are human VA and the VI_H locus comprises two or more JK gene segments, each comprising an RSS having a 12-mer spacer in which the RSS is juxtaposed at the current end above each JK gene segment.

In a specific embodiment, the VL segments comprise contiguous human k-gene segments spanning the human k-locus from VK4-1 to VK2-40, and the JL segments comprise contiguous gene segments spanning the human k-locus from JK1 up to JK5.

In one embodiment, in which the segments VL are segments VA and no segment DH is present between the segments VL and the segments J, the segments VL are flanked towards the 3 'end. { that is, juxtaposed on the downstream side) with RSS of 23-mers, and the JK S¡ segments were present or the JA segments if present are flanked to the 5 'end (i.e., juxtaposed on the upstream side) with 12-mer RSS.

In one embodiment, in which the V gene segments are VK gene segments and no DH gene segment is present between the V gene segments and the J gene segments, the VK gene segments are each juxtaposed on the side downstream with a 12-mer RSS, and the JK segments if present or the JA segments if present are each juxtaposed on the upstream side with a 23-mer RSS.

In one embodiment, the mouse comprises a rearranged gene that is derived from a VL gene segment, a JL gene segment, and an endogenous CH gene. In one embodiment, the rearranged gene is somatically used. In one embodiment, the rearranged gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N additions. In one embodiment, the N additions and / or somatic mutations observed in the rearranged gene derived from the VL segment and the JL segment are 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or at least 5 times more than the number of N additions and / or somatic mutations observed in a variable domain of the rearranged light chain (derived from the same VL gene segment and the same JL gene segment) that is rearranged at a locus of the endogenous light chain. In one embodiment, the rearranged gene is in a B cell that specifically binds an antigen of interest, in which the B cell binds the antigen of interest with a KD in the range lower or lower nanomolar (for example, a KD of 10 nanomolar or lower). In a specific embodiment, the VL segment, the JL segment, or both, are segments of the human gene. In a specific embodiment, segments VL and JL are human k-gene segments. In one embodiment, the mouse CH gene is selected from IgM, IgD, IgG, IgA and IgE. In a specific embodiment, mouse IgG is selected from I g G1, IgG2A, IgG2B, IgG2C and IgG3. In another specific embodiment, the mouse IgG is IgG 1.

In one embodiment, the mouse comprises a cell B, in which the B cell expresses from a locus on a chromosome of the B cell a binding protein consisting essentially of four polypeptide chains, in which the four chains of polypeptides consist essentially of (a) two identical polypeptides comprising an endogenous CH region fused to a VL; and, (b) two identical polypeptides comprising an endogenous CL region fused to a VL region that is cognate with respect to the VL region that is fused to the mouse CH region, and, in one embodiment, is a VL region (eg example, a human k) of human. In one embodiment, the VL region fused to the endogenous CH region is a human VL region. In a specific embodiment, the human VL region fused to the mouse CH region is a VK region. In a specific embodiment, the human VL region fused to the mouse CH region is identical to a V region encoded by a nucleotide sequence of the rearranged human germline light chain. In a specific modality, the human VL region fused to the CH region of mouse comprises two, three, four, five, six, or more somatic hypermutations. In one embodiment, the human VL region fused to the mouse CH region is encoded by a rearranged gene comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

In one embodiment, at least 50% of all IgG molecules expressed by the mouse comprise a polypeptide comprising a CH region of IgG isotype and a VL region, in which the length of said polypeptide is not longer than 535 , 530, 525, 520, or 515 amino acids. In one embodiment, at least 75% of all IgG molecules comprise the polypeptide recited in this paragraph. In one embodiment, at least 80%, 85%, 90%, or 95% of all IgG molecules comprise the polypeptide recited in this paragraph. In a specific embodiment, all IgG molecules expressed by the mouse comprise a polypeptide that is not longer than the length of the polypeptide recited in this paragraph.

In one embodiment, the mouse expresses a binding protein comprising a first polypeptide comprising an endogenous CH region fused to a variable domain encoded by a rearranged V gene segment of human and a J gene segment but not a DH gene segment. , and a second polypeptide comprising an endogenous CL region fused to a V domain encoded by a human V rearranged gene segment and a J gene segment but not a DH gene segment, and the binding protein specifically binds an antigen with an affinity in the micromolar, nanomolar, or picomolar range. In one modality, segment J is a J segment of human (for example, a segment of human k gene). In one embodiment, the human V segment is a human VK segment. In one embodiment, the variable domain that is fused with the endogenous CH region comprises a greater number of somatic hypermutations than the variable region that is fused with the endogenous CL region; in a specific embodiment, the variable region fused to the endogenous CH region comprises approximately 1.5, 2, 3, 4, or 5 times or more somatic hypermutations than the V region fused to the endogenous CL region; in a specific embodiment, the V region fused to the mouse CH region comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more somatic hypermutations than the V region fused with the CL region of mouse. In one embodiment, the V region fused to the mouse CH region is encoded by a rearranged gene comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

In one embodiment, the mouse expresses a binding protein comprising a first variable domain of the light chain (VL1) fused to a constant region sequence of the immunoglobulin heavy chain and a second variable domain of the light chain (VL2) fused to a constant region of the immunoglobulin light chain, in which VL1 comprises a number of somatic hypermutations that is approximately 1.5 to about 5 times higher or higher than the number of somatic hypermutations present in VL2. In one embodiment, the number of somatic hypermutations in VL1 is approximately 2 up to about 4 times higher than in VL2. In a modality the number of somatic hypermutations in VL1 is approximately 2 to approximately 3 times higher than in VL2. In one embodiment, VL1 is encoded by a sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

In one aspect, there is provided a genetically modified mouse that expresses an immunoglobulin consisting essentially of the following polypeptides: first two identical polypeptides each consisting essentially of a CH region fused to a variable domain that is derived from gene segments which consist essentially of a VL gene segment and a JL gene segment, and a few seconds two identical polypeptides each consisting essentially of a CL region fused to a variable domain that is derived from gene segments consisting essentially of a segment VL and a segment Ju.

In a specific embodiment, the two identical polypeptides having the CH region have a CH CH region.

In a specific embodiment, the two identical polypeptides having the CL region have a mouse CL region.

In one embodiment, the variable domain fused to the CL region is a variable domain that is cognate with the variable domain fused to the CH region.

In one embodiment, the variable domain that is fused with the endogenous CH region comprises a larger number of somatic hypermutations than the variable domain that is fused with the Cj region. endogenous in a specific modality, the domain variable fused with the endogenous CH region comprises approximately 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times or more somatic hypermutations than the variable domain fused to the endogenous CL region. In one embodiment, the variable domain fused to the endogenous CL region is encoded by a gene comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N additions.

In one embodiment, one or more of the segments V and the segments J are human gene segments. In a specific embodiment, both the V segments and the J segments are human k-gene segments. In another specific embodiment, both the V segments and the J segments are human A gene segments. In one embodiment, segments V and segments J are independently selected from segments of human A and human k gene A. In a specific embodiment, the segments V are segments VK and the segments J are segments JA. In another specific embodiment, the segments V are segments VA and the segments J are segments JK.

In one embodiment, one or more of the variable domains fused to the CL region and the variable domains fused to the CH region are human variable domains. In a specific embodiment, the human variable domains are human VK domains. In another specific embodiment, the human variable domains are VA domains. In one embodiment, human domains are independently selected from human VK domains and human VA domains. In a specific embodiment, the human variable domain fused to the CL region is a VA domain of human and the human variable domain fused with the CH region is a human VK domain. In another embodiment, the human variable domain fused to the CL region is a human VK domain and the human variable domain fused to the CH is a human VA domain.

In one embodiment, the VL gene segment of the first two identical polypeptides is selected from a human VA segment and a human VK segment. In one embodiment, the VL segment of the second two identical polypeptides is selected from a human VA segment and a human VK segment. In a specific embodiment, the VL segment of the first two identical polypeptides is a human VK segment and the VL segment of the second two identical polypeptides is selected from a human VK segment and a human VA segment. In a specific embodiment, the VL segment of the first two identical polypeptides is a human VA segment and the VL segment of the second two identical polypeptides is selected from a human VA segment and a human VK segment. In a specific embodiment, the human VL segment of the first two identical polypeptides is a human VK segment, and the human VL segment of the second two identical polypeptides is a human VK segment.

In one embodiment, the mouse IgG comprises a binding protein made in response to an antigen, in which the protein of The binding comprises a polypeptide consisting essentially of a variable domain and a CH region, in which the variable domain is encoded by a nucleotide sequence consisting essentially of a rearranged VL segment and a rearranged J segment, and in which the binding protein specifically binds an epitope of the antigen with a KD in the micromolar, nanomolar, or picomolar range.

In one aspect, a mouse is provided, in which all or substantially all of the IgGs made by the mouse in response to an antigen comprise a heavy chain comprising a variable domain, in which the variable domain is encoded by a rearranged gene derived from gene segments consisting essentially of a segment of gene V and a segment of gene J. In one embodiment, the rearranged gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

In one embodiment, segment V is a segment V of a light chain. In one embodiment, the light chain is selected from a light chain k and a light chain l. In a specific embodiment, the light chain is a light chain K. In a specific embodiment, segment V is a human V segment. In a specific embodiment, segment V is a human VK segment and segment J is a human JK segment.

In one embodiment, segment J is a segment J of a light chain. In one embodiment, the light chain is selected from a light chain k and a light chain A. In a specific embodiment, the light chain is a light chain K. In one embodiment specific, segment J is a segment J of human. In other mode, segment J is a segment J of a heavy chain (ie, a JH). In a specific embodiment, the heavy chain has its origin in mouse. In another specific embodiment, the heavy chain is of human origin.

In one embodiment, the variable domain of the heavy chain that is made from no more than one segment V and one segment J is a somatically mutated variable domain.

In one embodiment, the variable domain of the heavy chain that is made from no more than one segment V and one segment J is fused to a CH region of the mouse.

In a specific embodiment, all or substantially all of the IgGs made by the mouse in response to an antigen comprise a variable domain that is derived from no more than one human V segment and no more than one human J segment, and the The variable domain is fused to a constant region of mouse IgG, and the IgG further comprises a light chain comprising a human Vt domain fused to a mouse CL region. In a specific embodiment, the VL domain fused to the mouse CL region is derived from a human VK segment and a human JK segment. In a specific embodiment, the VL domain fused to the mouse CL region is derived from a human VA segment and a human JA segment.

In one aspect, there is provided a mouse that makes an IgG comprising a first CDR3 in a polypeptide comprising a CH region and a second CDR3 in a polypeptide comprising a CL region, in which both the first CDR3 and the second CDR3 are each derived independently from no more than two gene segments, in which the two gene segments essentially consist of a VL gene segment and a JL gene segment. In one embodiment, the CDR3 in the polypeptide comprising the CH region comprises a sequence that is derived from a nucleotide sequence of CDR3 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

In one embodiment, the VL segment and the JL segment are human gene segments. In one embodiment, segment VL and segment JL are segments of gene K. In one embodiment, segment VL and segment JL are segments of gene l.

In one aspect, there is provided a mouse that makes an IgG comprising a first CDR3 in a first polypeptide comprising a CH region and a second CDR3 in a second polypeptide comprising a CL region, in which both the first CDR3 and the second CDR3 each comprise an amino acid sequence in which more than 75% of the amino acids are derived from a segment of gene V. In one embodiment, the CDR3 in the polypeptide comprising the CH region comprises a sequence that is derived from a nucleotide sequence of CDR3 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

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

In one embodiment, no more than two amino acids of the first CDR3 are derived from a different gene segment to the V-gene segment of the light chain. In one embodiment, no more than two amino acids of the second CDR3 are derived from a different gene segment to the V segment of the light chain. In a specific embodiment, no more than two amino acids of the first CDR3 and no more than two amino acids of the second CDR3 are derived from a gene segment other than the V segment of the light chain. In one embodiment, no CDR3 of the IgG comprises an amino acid sequence derived from a segment of gene D. In one embodiment, the CDR3 of the first polypeptide does not comprise a sequence derived from a segment D.

In one embodiment, segment V is a segment of human V gene. In a specific embodiment, segment V is a human VK gene segment.

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

In one embodiment, the first CDR3 consists essentially of amino acids derived from a V-gene segment of the human light chain and a J-gene segment from the light chain of human, and the second CDR3 consists essentially of amino acids derivatives? from a segment of gene V of the light chain of human and a segment of gene J of the light chain of human. In one embodiment, the first CDR3 is derived from a nucleic acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions. In one embodiment, the first CDR3 is derived from no more than two gene segments, wherein said no more than two gene segments are a human VK gene segment and a human JK gene segment; and the second CDR3 is derived from no more than two gene segments, in which said no more than two gene segments are a human VK gene segment and a J gene segment that are selected from a segment Human JK, a human JA segment, and a human JH segment. In one embodiment, the first CDR3 is derived from no more than two gene segments, in which said no more than two gene segments are a human VA segment and a J segment that are selected from a JK segment of human, a segment JA of human, and a segment JH of human.

In one aspect, there is provided a mouse that makes an IgG that does not contain an amino acid sequence derived from a DH gene segment, in which the IgG comprises a first polypeptide having a first VL domain fused to a CL region of mouse and a second polypeptide having a second VL domain fused to a mouse CH region, in which the first VL domain and the second VL domain are not identical. In one embodiment, the first and second VL domains are derived from different V segments. In another embodiment, the first and second VL domains are derived from different J segments. In one embodiment, the first and second VL domains are derived from identical V and J segments, in which the second VL domain comprises a higher number of somatic hypermutations compared to the first VL domain.

In one embodiment, the first and second VL domains are independently selected from human and mouse VL domains. In one embodiment, the first and second VL domains are independently selected from VK and VA domains. In a specific embodiment, the first VL domain is selected from one VK domain and one VA domain, and the second VL domain is a VK domain. In another specific embodiment, the VK domain is a human VK domain.

In one aspect, a mouse is provided, in which all or substantially all of the IgGs made by the mouse consist essentially of a light chain having a first human VL domain fused to a mouse CL domain, and a heavy chain having a second human VL domain fused to a mouse CH domain.

In one embodiment, the human VL domain fused to the mouse CH domain is a human VK domain.

In one embodiment, the first and second human VL domains are not identical.

In one aspect, a mouse is provided, in which at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or approximately 100% the immunoglobulin G made by the mouse consists essentially of a dimer of (a) a first polypeptide consisting essentially of an immunoglobulin VL domain and an immunoglobulin CL region; and, (b) a second polypeptide of no more than 535 amino acids in length, wherein the second polypeptide consists essentially of a CH region and a V domain lacking a sequence derived from a DH- En gene segment. embodiment, the second polypeptide is approximately 435-535 amino acids in length. In a specific embodiment, the second polypeptide is approximately 435-530 amino acids in length. In a specific embodiment, the second polypeptide is approximately 435-525 amino acids in length. In a specific embodiment, the second polypeptide is about 435-520 amino acids in length. In a specific embodiment, the second polypeptide is about 435-515 amino acids in length.

In one embodiment, in approximately 90% or more of the IgG made by the mouse the second polypeptide is no more than about 535 amino acids in length.

In one embodiment, in about 50% or more of the IgG made by the mouse the second polypeptide is no more than about 535 amino acids in length. In one embodiment, in about 50% or more of the immunoglobulin G made by the mouse the second polypeptide is no more than about 530, 525, 520, 515, 510, 505, 500, 495, 490, 485, 480, 475 , 470, 465, 460, 455, or 450 amino acids in length. In one modality, approximately 60%, 70%, 80%, 90%, or 95% or more of the IgG made by the mouse is of the recited length. In a specific embodiment, all or substantially all of the IgGs made by the mouse are of the recited length.

In one embodiment, the V domain of the second polypeptide is a VL domain. In a specific embodiment, the V domain of the second polypeptide is selected from a VK domain and a VA domain. In a specific embodiment, the V domain of the second polypeptide is a human VK or VA domain.

In one aspect, a mouse is provided which expresses from a nucleotide sequence in its germline a polypeptide comprising a variable sequence of the light chain (eg, a sequence of V and / or J), a DH sequence, and a constant region of the heavy chain.

In one embodiment, the mouse expresses the polypeptide from an endogenous heavy chain locus comprising a replacement of all or substantially all of the functional endogenous heavy chain variable locus gene segments with a plurality of human gene segments in the locus of the endogenous heavy chain.

In one embodiment, the polypeptide comprises a Vu sequence derived from a segment of the VA gene or a segment of the VK gene, the polypeptide comprises a CDR3 derived from a segment of the DH gene, and the polypeptide comprises a sequence derived from of a JH or JA or JK gene segment.

In one embodiment, the mouse comprises an immunoglobulin locus of the endogenous heavy chain comprising a replacement of all functional VH gene segments with one or more segments of the VA gene of the human light chain in which said one or more segments V of human are each juxtaposed on the downstream side a spaced 23-mer recombination signal (RSS) sequence, in which the VA segments are operably linked to a human or mouse DH segment having a juxtaposed upstream and downstream an RSS spaced 12-mer; the DH gene segment is operably linked with a J-segment juxtaposed upstream with a 23-mer spaced RSS that is suitable for recombination with the spaced 12-mer RSS, which is juxtaposed to the DH gene segment; wherein the segments V, DH, and J are operably linked to a nucleic acid sequence encoding a constant region of the heavy chain.

In one embodiment, the mouse comprises an endogenous heavy chain immunoglobulin locus comprising a replacement of all functional VH gene segments with one or more human VK gene segments each juxtaposed on the downstream side with a sequence of 12-mer spaced recombination signal (RSS), in which the V segments are operably linked to a DH human or mouse DH segment which is juxtaposed both upstream and downstream with a 23-mer spaced RSS; the DH segment is operably linked with a J segment juxtaposed on the upstream side with an RSS 12-mer spaced that is appropriate to combine with the spaced 23-mer RSS that is juxtaposed to the DH segment; wherein the V, DH, and gene segments are operably linked to a nucleic acid sequence encoding a constant region of the heavy chain.

In one embodiment, the constant region of the heavy chain is a constant region of the endogenous heavy chain. In one embodiment, the nucleic acid sequence encodes a sequence that is selected from a CH1, a hinge, a CH2, a CH3, and a combination thereof. In one embodiment, one or more of the CH1, hinge, CH2, and CH3 are human.

In one embodiment, the mouse comprises an immunoglobulin locus of the endogenous heavy chain comprising a replacement of all functional VH gene segments with a plurality of segments of the human VA or VK gene each juxtaposed downstream with spaced-apart RSS. -numbers, a plurality of human DH segments juxtaposed both upstream and downstream by a spaced 12-mer RSS, a plurality of human J segments (JH or JA or JK) juxtaposed both upstream and downstream with an RSS 23-mer spaced, in which the locus comprises a sequence of the endogenous constant region that is selected from CH1, hinge, CH2, CH3, and a combination thereof. In a specific embodiment, the mouse comprises all or substantially all of the VA or VK segments functional, all or substantially all DH segments functional human, and all or substantially all segments JH or JA or JK.

In one embodiment, the mouse expresses an antigen binding protein comprising (a) a polypeptide comprising a human light chain sequence linked to a constant sequence of the heavy chain comprising a mouse sequence; and (b) a polypeptide comprising a variable region of the human light chain linked to a constant sequence of the human or mouse light chain. In a specific embodiment, the sequence of the light chain is a sequence of the human light chain, and after exposure to a protease that is capable of cutting an antibody in a Fe and a Fab, a fully human Fab comprising at least four CDRs of the light chain, wherein said at least four CDRs of the light chain are selected from A sequences, K sequences, and a combination thereof. In one embodiment, the Fab comprises at least five CDRs of the light chain. In one embodiment, the Fab comprises six CDRs of the light chain. In one embodiment, at least one CDR of the Fab comprises a sequence derived from a segment VA or a segment VK, and said at least one CDR further comprises a sequence derived from a segment D. In one embodiment, said at least one CDR is a CDR3 and the CDR is derived from a human VK segment, a human D segment, and a human JK segment.

In one embodiment, the polypeptide comprises a region variable derived from a human VA or VK gene segment, a human DH gene segment, and a human JH or JA or JK gene segment. In a specific embodiment, the constant sequence of the heavy chain is derived from a human CH1 sequence and a mouse CH2 sequence and a mouse CH3 sequence.

In one aspect, a mouse is provided comprising in its germ line a segment of the VK or human reactivated VA gene operably linked to a segment of human J gene and a sequence of the constant region of the heavy chain, in the wherein the mouse expresses a VL binding protein comprising a human VK domain fused to a constant region of the heavy chain and a human VL domain fused to a constant domain of the light chain. In one embodiment, the human VL domain comprises a human VL rearranged gene segment that is selected from a human VK gene segment and a human VA gene segment. In a specific embodiment, the human VL domain further comprises a human-reassembled JL gene segment that is selected from a human JK gene segment and a human JA gene segment.

In one aspect, a mouse is provided that expresses an immunoglobulin protein from a locus of the modified endogenous heavy chain in its germline, in which the locus of the modified endogenous heavy chain lacks a V-gene segment of the functional mouse heavy chain and the locus comprises non-rearranged light chain V gene segments and J gene segments not rearranged, in which the non-rearranged light chain V gene segments and non-rearranged J gene segments are operably linked to a sequence of the heavy chain constant region; wherein the immunoglobulin protein consists essentially of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an immunoglobulin light chain sequence and a constant sequence of the immunoglobulin heavy chain, and the second polypeptide comprises a variable domain of the immunoglobulin light chain and a constant region of the light chain.

In one aspect, a mouse expressing an immunoglobulin protein, in which the immunoglobulin protein lacks a variable immunoglobulin heavy chain domain, is provided., and the immunoglobulin protein comprises a first variable domain derived from a light chain gene, and a second variable domain derived from a light chain gene, in which the first variable domain and the second variable domain they are cognate with respect to each other, in which the first and second variable domains of the light chain are not identical, and in which the first and second variable domains of the light chain are associated and when they are associated, they specifically link a antigen of interest.

In one aspect, a mouse is provided which expresses from immunoglobulin protein segments comprising non-rearranged gene segments in its germline comprising variable regions that are completely derived from gene segments consisting of essentially of non-rearranged human gene segments, in which the immunoglobulin protein comprises a constant sequence of the immunoglobulin light chain and a constant sequence of the immunoglobulin heavy chain which are selected from the group consisting of a CH1, a hinge, a CH2, a CH3, and a combination thereof.

In one aspect, a mouse is provided that expresses from immunoglobulin protein comprising variable regions, in which all CDR3s of all variable regions are generated in their entirety from non-rearranged gene segments in their germline. of V and J gene segments of the light chain, and optionally one or more somatic hypermutations, for example, one or more N additions.

In one aspect, a mouse is provided that expresses a somatically mutated immunoglobulin protein derived from nonrecognized human immunoglobulin light chain variable region segments in the germline of the mouse, in which the The immunoglobulin protein lacks a CDR comprising a sequence derived from a segment of the D gene, in which the immunoglobulin protein comprises a first CDR3 in a variable domain of the light chain fused to a constant region of the chain light, comprises a second CDR3 in a variable domain of the light chain fused to a constant region of the heavy chain, and in which the second CDR3 is derived from a variable region sequence of the rearranged light chain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

In one aspect, there is provided a mouse as described in the present application, wherein the mouse comprises a locus of the functionally silenced light chain that is selected from a locus A, a K locus, and a combination thereof . In one embodiment, the mouse comprises a deletion of a locus A and / or a locus K, in whole or in part, such that locus A and / or locus k is non-functional.

In one aspect, a mouse embryo is provided, comprising a cell comprising a modified immunoglobulin locus as described in the present application. In one embodiment, the mouse is a chimera and at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the cells of the embryo comprise a modified immunoglobulin locus as described in the present application. In one embodiment, at least 96%, 97%, 98%, 99%, or 99.8% of the cells of the embryo comprise a modified immunoglobulin locus as described in the present application. In one embodiment, the embryo comprises a host cell and a cell derived from a donor ES cell, in which the cell derived from the donor ES cell comprises a modified immunoglobulin locus as described in the present application. In one embodiment, the embryo is a host embryo in a stage of 2, 4, 8, 16, 32, or 64 cells, or a blastocyst, and further comprises a donor cell comprising a modified immunoglobulin locus as described in the present invention. request.

In one aspect, a mouse or an elaborate cell is provided using a nucleic acid construct as described in the present application.

In one aspect, an elaborated mouse is provided using a cell as described in the present application. In one embodiment, the cell is a mouse ES cell.

In one aspect, mouse cells and mouse embryos are provided, including but not limited to ES cells, pluripotent cells, and induced pluripotent cells, which comprise genetic modifications as described in the present application. Cells that are XX and cells that are XY are provided. Also provided are cells comprising a core containing a modification as described in the present application, for example, a modification introduced into a cell by pronuclear injection. Also provided are cells, embryos, and mice comprising a virally introduced ADAM6 gene, e.g., cells, embryos, and mice comprising a transduction construct comprising an ADAM6 gene that is functional in the mouse.

In one aspect, a cell or tissue derived from a mouse is provided as described in the present application. In one embodiment, the cell or tissue is derived from the spleen, lymph node, or bone marrow of a mouse as described in the present application. 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.

In one embodiment, the tissue is selected from tissue connective, muscle tissue, nervous tissue and epithelial tissue. In a specific modality, the tissue is reproductive tissue.

In one embodiment, the cell and / or tissue derived from a mouse as described in the present application is isolated for use in one or more ex vivo tests. In various embodiments, said one or more ex vivo tests include measurements of physical, thermal, electrical, mechanical or optical properties, a surgical procedure, measurements of interactions of different tissue types, the development of techniques for image formation, or a combination of the same.

In aspect, the use of a cell or tissue derived from a mouse as described in the present application is provided to make an antibody. In one aspect, the use of a cell or tissue derived from a mouse as described in the present application is provided to make a hybridoma or quadroma.

In one aspect, a non-human cell comprising a chromosome or fragment thereof of a non-human animal as described in the present application. In one embodiment, the non-human cell comprises a nucleus of a non-human animal as described in the present application. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as a result of a nuclear transfer.

In one aspect, a core derived from a mouse is provided as described in the present application. In one embodiment, the nucleus is a diploid cell that is not a cell 6.

In one aspect, a mouse cell is genetically provided modified, in which the cell is unable to express a heavy chain comprising endogenous rearranged immunoglobulin heavy chain gene segments, and the cell comprises a functional ADAM6 gene encoding a mouse ADAM6 protein or functional fragment thereof . In one embodiment, the cell further comprises an insertion of gene segments of the human immunoglobulin light chain. In a specific embodiment, the human immunoglobulin light chain gene segments are VK and / or JK gene segments that are operably linked to constant regions of the mouse heavy chain such that after rearrangement they encode for a variable domain of the functional light chain fused to a constant domain of the mouse heavy chain.

In one aspect, a genetically modified mouse cell is provided; in which the cell lacks a functional endogenous ADAM6 locus, the cell comprises an ectopic nucleotide sequence that codes for a mouse ADAM6. In one embodiment, the cell further comprises a modification of a variable region sequence of the endogenous immunoglobulin heavy chain. In a specific embodiment, the modification of the variable region sequence of the endogenous immunoglobulin heavy chain comprises a deletion that is selected from a deletion of a mouse VH gene segment, a deletion of a DH gene segment of mouse, a deletion of a mouse JH gene segment, and a combination of the same. In a specific embodiment, the mouse comprises a replacement of one or more VH, DH, and / or mouse immunoglobulin JH sequences with a human immunoglobulin sequence. In a specific embodiment, the human immunoglobulin sequence is selected from a human VH, a human VL, a human DH, a human JH, a human JL, and a combination thereof.

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

In one aspect, a mouse B cell is provided, in which the mouse B cell comprises a rearranged immunoglobulin gene, in which the B cell comprises on a B cell chromosome a nucleic acid sequence encoding a ADAM6 protein or ortholog or homologue or fragment thereof that is functional in a male mouse. In one embodiment, the mouse B cell comprises two alleles of the nucleic acid sequence. In one embodiment, the rearranged immunoglobulin gene comprises a sequence of the rearranged immunoglobulin light chain contiguous with a constant sequence of the heavy chain. In one embodiment, the sequence of the light chain is a sequence K; in one embodiment, the sequence of the light chain is a sequence A.

In one embodiment, the nucleic acid sequence is in a nucleic acid molecule (e.g., a B cell chromosome) that is contiguous with the immunoglobulin locus of the rearranged heavy chain of the mouse.

In one embodiment, the nucleic acid sequence is in a nucleic acid molecule (e.g., a cell 6 chromosome) that is distinct from the nucleic acid molecule comprising the immunoglobulin locus of the mouse rearranged heavy chain.

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

In one aspect, a somatic mouse cell is provided, comprising a chromosome comprising a locus of the modified immunoglobulin heavy chain, and a nucleic acid sequence encoding a mouse ADAM6 or ortholog or homologue or fragment thereof. which is functional in a male mouse. In one embodiment, the nucleic acid sequence is on the same chromosome as the locus of the modified immunoglobulin heavy chain. 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 an individual 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 embodiment, the modified immunoglobulin heavy chain locus comprises a gene segment of the rearranged immunoglobulin light chain operably linked to a constant region sequence of the heavy chain.

In one aspect, a mouse germ cell is provided, comprising a nucleic acid sequence encoding a mouse ADAM6 (or homologous or orthologous or functional fragment thereof) on a germ cell chromosome, wherein the nucleic acid sequence encoding the mouse ADAM6 (or homologous or orthologous or functional fragment thereof) is at a position on the chromosome that is different from a position on a chromosome of a wild-type mouse germ cell, in wherein the mouse further comprises a modification comprising an immunoglobulin gene fragment of the non-rearranged light chain (a VK and / or VA and / or VK and JK and / or VA and JA) operably linked to a sequence of the constant region of the heavy chain. In one embodiment, the nucleic acid sequence is in a mouse immunoglobulin locus. In one embodiment, the nucleic acid sequence is on the same germ cell chromosome as that of a mouse immunoglobulin locus. In one embodiment, the nucleic acid sequence is on a chromosome different from the germ cell than that of the mouse immunoglobulin locus. In one embodiment, the mouse immunoglobulin locus comprises a replacement of at least one mouse immunoglobulin sequence with at least one non-mouse immunoglobulin sequence. In a specific modality, said at least one non-mouse immunoglobulin sequence is a human immunoglobulin sequence.

In one aspect, a pluripotent, induced pluripotent, or totipotential cell derived from a mouse is provided as described in the present application. In a specific embodiment, the cell is a mouse embryonic stem cell (ES).

In one aspect, a cell is provided, comprising a modified immunoglobulin locus as described in the present application. In one embodiment, the cell is selected from a totipotential cell, a pluripotential cell, an induced pluripotent stem cell (iPS), and an ES cell. In a specific embodiment, the cell is a mouse cell, for example, a mouse ES cell. In one embodiment, the cell is homozygous for the modified immunoglobulin locus.

In one aspect, a cell is provided, comprising a nucleic acid sequence encoding a first polypeptide comprising a first somatically mutated human VK or VA sequence fused to a constant region sequence of the human heavy chain.

In one embodiment, the cell further comprises a second polypeptide chain comprising a second somatically mutated VK or VA sequence of human fused to a constant region sequence of the human light chain.

In one embodiment, the human VK OR VA sequence of the first polypeptide is cognate with the human VK or VA sequence of the second pol i peptide.

In one embodiment, the VK O VA of the first polypeptide and the human VK or VA of the second polypeptide when associated specifically bind an antigen of interest. In a specific embodiment, the first polypeptide comprises a variable domain consisting essentially of a human VK, and the second polypeptide comprises a variable domain consisting of a human VK that is cognate with the human VK of the first polypeptide, and the The sequence of the human constant region is an IgG sequence.

In one embodiment, the cell is selected from a CHO cell, a COS cell, a 293 cell, a HeLa cell, and a human retinal cell that expresses a viral nucleic acid sequence (e.g., a PERC.6 cell). ™.

In one aspect, a rodent somatic cell (eg, mouse) is provided, comprising a chromosome comprising a genetic modification as described in the present application.

In one aspect, a rodent germ cell (e.g., mouse) is provided, comprising a nucleic acid sequence comprising a genetic modification as described in the present application.

In one aspect, a pluripotent, induced pluripotent, or totipotential cell knocked out from a rodent (e.g., mouse) is provided as described in the present application. In a specific embodiment, the cell is a mouse embryonic stem cell (ES).

In one aspect, the use of a cell as described in the present application is provided for the manufacture of a rodent (e.g., a mouse), a cell, or a therapeutic protein (e.g., an antibody or other binding protein). antigen). In one embodiment, the use of a cell as described in the present application for the manufacture of a therapeutic protein is provided, wherein the therapeutic protein comprises a human variable domain. In a specific embodiment, the human variable domain comprises rearranged VK and JK gene segments.

In one aspect, a rodent (e.g., a mouse) that is made using a target vector, nucleotide construct, or cell is provided as described in the present application.

In one aspect, the use of a target vector as described in the present application is provided for the manufacture of a rodent (e.g., a mouse) or a cell (e.g., a mouse ES cell, a fibroblast of mouse, etc.). In one embodiment, the vector for choice of target comprises a human genomic fragment containing non-rearranged immunoglobulin gene segments. In a specific embodiment, the non-rearranged immunoglobulin gene segments include segments of the V and J gene. In a specific embodiment, the non-rearranged immunoglobulin gene segments include V, D, and J gene segments.

In one aspect, a nucleic acid construct is provided, which comprises a homology arm towards the 5 'end and a homology arm towards the 3 'end, in which the homology arm towards the 5' end comprises a sequence that is identical or substantially identical to a variable region sequence of the human immunoglobulin heavy chain, the homology arm towards the 3 'end it comprises a sequence that is identical or substantially identical to a sequence of the human or mouse immunoglobulin variable region, and is arranged between the arms of homology towards the 5' end and towards the 3 'end is a sequence comprising a nucleotide sequence encoding a mouse ADAM6 protein. In a specific embodiment, the sequence encoding the mouse ADAM6 gene is operably linked to a mouse promoter with which mouse ADAM6 is ligated in a wild-type mouse.

In one aspect, a vector for targeting is provided, comprising (a) a nucleotide sequence that is identical or substantially identical to a nucleotide sequence of the human variable region gene segment; and, (b) a nucleotide sequence encoding a mouse ADAM6 or ortholog or homologue or fragment thereof that is functional in a mouse.

In one embodiment, the vector for targeting also comprises a promoter operably linked to the sequence encoding the mouse ADAM6. In a specific embodiment, the promoter is a mouse ADAM6 promoter.

In one aspect, a nucleotide construct is provided to modify a variable locus of the immunoglobulin heavy chain of mouse, wherein the construct comprises at least one site-specific recombinase recognition site and a sequence encoding an ADAM6 protein or ortholog or homologue or fragment thereof that is functional in a mouse.

In one aspect, a nucleic acid construct comprising a human DH gene segment juxtaposed upstream and downstream with a 23-mer spaced RSS is provided. In a specific embodiment, the nucleic acid construct comprises a homology arm that is homologous to a human genomic sequence comprising human VK gene segments. In one embodiment, the construction for target selection comprises all or substantially all of the human DH gene segments each juxtaposed upstream and downstream with a 23-mer spaced RSS.

In one aspect, a nucleic acid construct comprising a human JK gene segment juxtaposed upstream with a spaced 12-mer RSS is provided. In a specific embodiment, the nucleic acid construct comprises a first homology arm containing homology to a human genomic DH gene sequence that is juxtaposed upstream and downstream with a 23-mer spaced RSS. In one embodiment, the nucleic acid construct comprises a second homology arm that contains homology to a human genomic J sequence of human or that contains homology to a constant region sequence of the mouse heavy chain or that contains homology to a J-C intergenic sequence towards the 5 'end of a heavy chain sequence of the mouse constant region.

In one aspect, a nucleic acid construct is provided comprising a VA segment of human juxtaposed downstream with a 23-mer spaced RSS, a human DH segment juxtaposed upstream and downstream with a spaced RSS of 12-mers, and a human J segment that is selected from a JK segment juxtaposed upstream with a 23-mer spaced RSS, a human JA segment juxtaposed upstream with a spaced 23-mer RSS, and a human JH segment juxtaposed upstream with an RSS spaced 23-mer. In one embodiment, the construct comprises a homology arm that contains homology to a mouse constant region sequence, an intergenic J-C mouse sequence, and / or a human VA sequence.

In one embodiment, the nucleic acid construct comprises a variable region sequence of the human light chain A comprising a fragment of cluster A of the human light chain A locus. In a specific embodiment, the fragment of cluster A of the human light chain A locus extends from hVA3-27 to hVA3-1.

In one embodiment, the nucleic acid construct comprises a variable region sequence of the human light chain A comprising a fragment of cluster B of the human light chain A locus. In a specific embodiment, the fragment of cluster B of the human light chain A locus extends from hVA5-52 to I A1-40.

In one embodiment, nucleic acid construct comprises a variable region sequence of the human light chain 1 comprising a genomic fragment of cluster A and a genomic fragment of cluster B. In one embodiment, the sequence of the variable region of the light chain of human comprises at least one segment of cluster A gene and at least one segment of cluster B gene.

In one embodiment, the sequence of the variable region of the human light chain l comprises at least one gene segment of cluster B and at least one segment of cluster C gene.

In one aspect, a nucleic acid construct is provided, comprising a DH segment of human juxtaposed upstream and downstream with a 23-mer spaced RSS normally found in Nature flanking any of a JK segment, a JH segment, a segment VA, or a segment VH. In one embodiment, the nucleic acid construct comprises a first arm of homology homologous to an intergenic region V-J of human or homologous to a human genomic sequence comprising a segment of human V gene. In one embodiment, the nucleic acid construct comprises a second arm of homology homologous to a constant region sequence of the human or mouse heavy chain. In a specific embodiment, the sequence of the human or mouse heavy chain constant region is selected from a CH1, hinge, CH2, CH3, and a combination thereof.

In one embodiment, the nucleic acid construct comprises a human J gene segment flanked upstream with a 12-mer RSS. In one embodiment, the nucleic acid construct comprises a second homology arm that contains homology to a J gene segment flanked upstream with a 12-mer RSS. In one embodiment, the J gene segment is selected from a human JK, a human JA, and a human JH.

In one aspect, a nucleic acid construct is provided comprising a DH segment of human juxtaposed upstream and downstream with a 23-mer spaced RSS, and a site-specific recombinase recognition sequence, eg, a recognized sequence. by a site-specific recombinase such as a Cre protein, a Flp protein, or a Dre protein.

In one aspect, a nucleic acid construct is provided comprising a human VA segment or a human VK segment, a DH segment juxtaposed upstream and downstream with a spaced 12-mer RSS or a spaced 23-mer RSS , and a human J segment with a spaced RSS of 12-mers or an RSS spaced of 23-mers, in which the spaced RSS of 12-mers or the RSS spaced of 23-mers is positioned immediately 5 'to segment J of human (ie, with respect to the direction of transcription). In one embodiment, the construct comprises a juxtaposed 3 'human VA with a 23-mer spaced RSS, a human DH segment juxtaposed upstream and downstream with a spaced 12-mer RSS, and a human JK segment. juxtaposed 5 'with an RSS spaced 23-mer. In one embodiment, the construct comprises a juxtaposed 3 'human VK with a spaced 12-mer RSS, a human DH segment juxtaposed upstream and downstream with a 23-mer spaced RSS, and a juxtaposed human J segment. 5 'with an RSS spaced 12-mer.

In one aspect, a vector for target selection is provided, comprising (a) a first arm for choice of target and a second arm for target selection, in which the first and second arms for target selection are selected from independently from arms for choice of human target and mouse, in which the arms for choice of target direct the vector to a gene locus of the V region of endogenous or modified immunoglobulin; and, (b) a contiguous sequence of human VL gene segments or a contiguous sequence of human VL gene segments and at least one human JK gene segment, in which the contiguous sequence is selected from the group consisting of (i) hVK4-1 to hVKl -6 and JK1, (ii) hVK4-1 to hVx1 -6 and JK1 to JK2, (iii) hVx4-1 to hVx1 -6 and JK1 to JK3, (iv) hVx4 -1 to hVx1 -6 and JK1 to JK4, (V) hVx4-1 to hVKl -6 and JK1 to JK5, (vi) hVK3-7 to hVKl-16, (vii) hVxl-17 to hVK2-30, (viii ) hVx3-31 to hVK2-40, and (ix) a combination thereof.

In one embodiment, the arms for targeting targeting the vector to an endogenous or modified immunoglobulin locus are identical or substantially identical to a sequence in the endogenous or modified immunoglobulin locus.

In one aspect, a method for making a genetically modified mouse is provided, which comprises replacing one or more immunoglobulin heavy chain gene segments towards the 5 'end (with respect to transcription of the heavy chain gene segments). immunoglobulin) from an endogenous mouse ADAM6 locus with one or more light chain gene segments and / or human immunoglobulin heavy chain, and replace one or more immunoglobulin gene segments towards the 3 'end (with respect to to the transcription of the immunoglobulin heavy chain gene segments) of the mouse ADAM6 locus with one or more heavy chain gene segments or the human immunoglobulin light chain. In one embodiment, said one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments towards the 5 'end of an endogenous mouse ADAM6 locus include V gene segments. Human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments towards the 5 'end of an endogenous mouse ADAM6 locus include V and D gene segments. In one embodiment, said one or more gene segments of human immunoglobulin replacing one or more endogenous immunoglobulin gene segments towards the 3 'end of an endogenous ADAM6 locus of the mouse include J gene segments. In one embodiment, said one or more human immunoglobulin gene segments that replace one or more segments of endogenous immunoglobulin gene towards the 3 'end of an endogenous mouse ADAM6 locus include D and J gene segments. In one embodiment, said one or more human immunoglobulin gene segments replacing one or more gene segments of endogenous immunoglobulin towards the 3 'end of an endogenous mouse ADAM6 locus include segments of V, D and J gene.

In one aspect, a method for making a genetically modified mouse is provided, which comprises replacing one or more immunoglobulin heavy chain gene segments towards the 5 'end (with respect to transcription of the heavy chain gene segments). immunoglobulin) from an endogenous ADAM6 locus of the mouse with one or more segments of the human immunoglobulin light chain gene, and replace one or more immunoglobulin gene segments towards the 3 'end (with respect to the transcription of the immunoglobulin heavy chain gene segments) of the mouse ADAM6 locus with one or more segments of the human immunoglobulin light chain. In one embodiment, said one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments towards the 5 'end of an endogenous mouse ADAM6 locus include V gene segments., human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments towards the 5 'end of an endogenous mouse ADAM6 locus include V and J gene segments. In one embodiment, said one or more segments of human immunoglobulin gene that replace one or more endogenous immunoglobulin gene segments toward the 3 'end of an endogenous mouse ADAM6 locus include J gene segments. In one embodiment, said one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments towards the 3 'end of an endogenous mouse ADAM6 locus include V and J gene segments. In one embodiment, said one or more human immunoglobulin gene segments replacing one or more segments of endogenous immunoglobulin gene towards the 3 'end of an endogenous mouse ADAM6 locus include V gene segments.

In a specific embodiment, the V gene segments are VL gene segments · In another specific embodiment, the J gene segments are JL gene segments.

In one embodiment, said one or more segments of the immunoglobulin heavy chain gene to the 5 'end and / or to the 3' end of the ADAM6 gene are replaced in a pluripotent, induced pluripotent, or totipotential cell to form a progenitor cell genetically modified; the genetically modified progenitor cell is introduced into a host; and, the host comprising the genetically modified progenitor cell is subjected to gestation to form a mouse comprising a genome derived from the genetically modified progenitor cell. In one modality, the host is an embryo. In a specific embodiment, the host is selected from a pre-morula (e.g., in the 8 or 4 cell stage), a tetraploid embryo, an aggregate of embryonic cells, or a blastocyst, of mouse.

In one aspect, there is provided a method for making a genetically modified mouse, which comprises replacing a mouse nucleotide sequence comprising a mouse immunoglobulin gene segment and a mouse ADAM6 nucleotide sequence (or ortholog or homologue or fragment). of the same functional in a male mouse) with a sequence comprising a human immunoglobulin gene segment to form a first chimeric locus, then inserting a sequence comprising a sequence encoding mouse ADAM6 (or a sequence that encodes for an ortholog or homologue or functional fragment thereof) in the sequence comprising the human immunoglobulin gene segment to form a second chimeric locus.

In one embodiment, the second chimeric locus comprises a variable gene segment of the human Immunoglobulin (VH) heavy chain. In one embodiment, the second chimeric locus comprises a segment of the variable gene of the human immunoglobulin light chain (VL). In a specific embodiment, the second chimeric locus comprises a human VH gene segment or a human VL gene segment operably linked to a human DH gene segment and a human JH gene segment. In a specific embodiment, the second chimeric locus comprises a human VL gene segment operably linked to a human JH gene segment or a human JL gene segment. In a further specific embodiment, the second chimeric locus is bound in a operable at a third chimeric locus comprising a human CH1 sequence, or a human CH1 and human hinge sequence, fused to a mouse CH2 + CH3 sequence.

In one aspect, there is provided a method for modifying an immunoglobulin locus of a mouse heavy chain, comprising: (a) making a first modification of an immunoglobulin locus of the mouse heavy chain that results in a reduction or deletion of endogenous mouse ADAM6 activity in a male mouse; and, (b) making a second modification to add a nucleic acid sequence that confers on the mouse ADAM6 activity that is functional in a male mouse.

In one embodiment, the first modification comprises the addition of a human immunoglobulin sequence or the replacement of a mouse immunoglobulin sequence with a human immunoglobulin sequence.

In one embodiment, the human immunoglobulin sequence is a heavy chain sequence. In one embodiment, the human immunoglobulin sequence is a sequence of the light chain.

In one embodiment, the first and second modifications are made in an individual ES cell, and the individual ES cell is introduced into a host embryo to make the mouse.

In one aspect, a progeny of a mouse pairing is provided as described in the present application with a second mouse that is a wild type or genetically modified mouse.

In one aspect, there is provided the use of a mouse comprising an ectopic nucleotide sequence comprising a locus or sequence of mouse ADAM6 for making a male-fertile mouse, wherein the use comprises matching the mouse comprising the sequence of ectopic nucleotide comprising the locus or sequence of ADAM6 of mouse with a mouse lacking a locus or functional endogenous ADAM6 sequence, and obtaining a progeny that is a female capable of producing progeny having the locus or sequence of ectopic ADAM6 or which is a male comprising the locus or sequence of ectopic ADAM6, and the male exhibits a fertility that is approximately the same as a fertility exhibited by a male wild type mouse.

In one aspect, the use of a mouse as described in the present application is provided to introduce an ectopic ADAM6 sequence into a mouse that lacks a functional endogenous ADAM6 sequence, in which the use comprises matching the mouse as shown in FIG. described in the present application with the mouse lacking the functional endogenous ADAM6 sequence.

In one aspect, the use of genetic material from a mouse as described in the present application is provided to make a mouse having an ectopic ADAM6 sequence. In one embodiment, the use comprises nuclear transfer using a nucleus of a cell of a mouse as described in the present application. In one embodiment, the use comprises cloning a cell of a mouse as it is described in the present application to produce a derived animal to from the cell. In one embodiment, the use comprises using a sperm or an ovum of a mouse as described in the present application in a method for making a mouse comprising the ectopic ADAM6 sequence.

In one aspect, there is provided a method for making a male-fertile mouse comprising a locus of the modified immunoglobulin heavy chain, comprising fertilizing a first mouse germ cell comprising a modification of an immunoglobulin locus of the heavy chain endogenous with a second mouse germ cell comprising an ADAM6 gene or ortholog or homolog or fragment thereof that is functional in a male mouse; form a fertilized cell; allow the fertilized cell to develop as an embryo; and, carry out the gestation of the embryo in a substitute to obtain a mouse.

In one modality, fertilization is achieved by matching a male mouse and a female mouse. In one embodiment, the female mouse comprises the ADAM6 gene or ortholog or homologue or fragment thereof. In one embodiment, the male mouse comprises the ADAM6 gene or ortholog or homologue or fragment thereof.

In one aspect, the use is provided of a nucleic acid sequence encoding a mouse ADAM6 protein or an ortholog or homologue thereof or a functional fragment of the corresponding ADAM6 protein to restore or increase the fertility of a mouse having a genome comprising a modification of a locus of the Immunoglobulin heavy chain, in which the modification reduces or eliminates the function of endogenous ADAM6.

In one embodiment, the nucleic acid sequence is integrated into the mouse genome in 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 locus of the heavy chain. In one embodiment, the nucleic acid sequence is integrated into the mouse genome at a position different from that of an endogenous immunoglobulin locus.

In one aspect, a method for making a genetically modified mouse is provided, which comprises replacing at one locus of the endogenous heavy chain one or more immunoglobulin heavy chain gene segments of a mouse with one or more gene segments of the mouse. human immunoglobulin light chain. In one embodiment, the replacement is all or substantially all segments of the functional mouse immunoglobulin heavy chain (i.e., the VH, DH, and JH segments) with one or more functional human light chain segments (ie. say, the segments VL and JL) · In one embodiment, the replacement is of all or substantially all segments VH, DH, and JH of the functional mouse heavy chain with all or substantially all of the VA or VK segments of human and at least one segment JA or JK. In a specific embodiment, the replacement includes all or substantially all of the functional JA or JK human segments.

In one aspect, a method is provided for making a mouse expressing a polypeptide comprising a sequence derived from a VA or VK segment and / or human immunoglobulin JA or JK fused to a constant region of the mouse heavy chain. , which comprises replacing the endogenous heavy chain immunoglobulin variable segments (VH, DH, and JH) with at least one segment VA or VK of human and at least one segment JA or JK of human, in which replacement is in a pluripotent, induced pluripotent, or totipotential mouse cell to form a genetically modified mouse progenitor cell; the genetically modified mouse progenitor cell is introduced into a mouse host; and, the mouse host comprising the genetically modified progenitor cell is subjected to gestation to form a mouse comprising a genome derived from the genetically modified mouse progenitor cell. In one modality, the host is an embryo. In a specific embodiment, the host is selected from a pre-mouse morula (e.g., 8 or 4 cell stage), a tetraploid embryo, an embryonic cell aggregate, or a blastocyst.

In one aspect, there is provided a method for making a genetically modified mouse as described in the present application, comprising introducing a nucleic acid containing a modification as described in the present application into a cell by nuclear transfer, and maintaining the cell under appropriate conditions (for example, including culturing the cell and carrying out the gestation of a embryo comprising the cell in a surrogate mother) to be developed as a mouse as described in the present application.

In one aspect, a method for making a modified mouse is provided, which comprises modifying as described in the present application a mouse ES cell or pluripotent or pluripotent or induced pluripotent mouse cell to include one or more variable gene segments of the non-rearranged immunoglobulin light chain operably linked to a constant sequence of the immunoglobulin heavy chain, culturing the ES cell, introducing the ES cell cultured into a host embryo to form a chimeric embryo, and introducing the chimeric embryo into a mouse appropriate host to develop as a modified mouse. In one embodiment, said one or more gene segments of the variable region of the non-rearranged immunoglobulin light chain are segments of human A or human k gene. In one embodiment, said one or more gene segments of the variable region of the non-rearranged immunoglobulin light chain comprise human VA or human VK segments and one or more segments JA, JK, O JH. In one embodiment, the constant gene sequence of the heavy chain is a human sequence that is selected from CH1, hinge, CH2, CH3, and a combination thereof. In one embodiment, said one or more variable gene segments of the non-rearranged immunoglobulin light chain replace all or substantially all of the functional endogenous heavy chain variable region segments at the locus of the endogenous heavy chain, and the sequence Heavy chain constant is a mouse sequence comprising a CH1, a hinge, a CH2, and a CH3.

In one aspect, constructs of nucleic acids, cells, embryos, mice, and methods for making proteins comprising one or more immunoglobulin sequences of the variable region of the light chain ky / ol and a sequence of the constant region of the chain are provided. immunoglobulin heavy, including proteins comprising a variable domain of the human light chain lok and a constant region sequence of the human or mouse heavy chain.

In one aspect, a nucleotide sequence encoding an immunoglobulin variable region made in a mouse is provided as described in the present application.

In one aspect, an amino acid sequence of the variable region of the heavy chain or light chain of an antibody made in a mouse is provided as described in the present application.

In one aspect, a nucleotide sequence of the variable region of the heavy chain or light chain encoding a variable region of an antibody made in a mouse is provided as described in the present application.

In one aspect, there is provided an antibody or antigen-binding fragment thereof (eg, Fab, F (ab) 2, scFv) made in a mouse as described in the present application.

In one aspect, binding proteins are disclosed which they comprise variable domains of immunoglobulin that are derived from immunoglobulin variable domains of the light chain (ie, kappa (K) and / or lambda (l)), but not from immunoglobulin variable domains of the heavy chain of full length Methods and compositions for making binding proteins, including genetically modified mice, are also provided.

In one aspect, there is provided a method for making an antigen-binding protein that does not comprise a variable domain of the immunoglobulin heavy chain, comprising a step of immunizing a mouse as described in the present application with an antigen of interest, and keeping the mouse under conditions that allow it to make an antigen-binding protein that specifically binds the antigen of interest.

In one aspect, there is provided a method for making an antigen-binding protein comprising a first variable domain of the immunoglobulin light chain contiguous with a sequence of the heavy chain constant regions, and a second variable domain of the light chain of immunoglobulin that is contiguous with a constant sequence of the light chain, comprising a step of immunizing a mouse as described in the present application with an antigen of interest, and keeping the mouse under conditions that allow it to make a binding protein an antigen that specifically binds the antigen of interest.

In one aspect, there is provided a method for making an antigen-binding protein comprising a variable domain of the light chain of immunoglobulin contiguous with a sequence of the constant regions of the heavy chain, in which the antigen-binding protein lacks an immunoglobulin light chain comprising a step of immunizing a mouse as described in the present application comprising blocking the expression of an endogenous kyl locus (or comprising a locus ky / o non-functional endogenous A locus) with an antigen of interest, and keeping the mouse under conditions that allow it to make an antigen-binding protein that binds specifically the antigen of interest.

In one aspect, the use of a mouse as described in the present application is provided to make a nucleotide sequence of the immunoglobulin variable region. In one embodiment, the nucleotide sequence of the variable region comprises segments of the rearranged VL, DH and JL gene. In one embodiment, the nucleotide sequence of the variable region comprises rearranged VL, DH and JH gene segments. In one embodiment, the nucleotide sequence of the variable region comprises segments of the rearranged VK and JK gene. In one embodiment, the nucleotide sequence of the variable region comprises segments of VA and JA rearranged gene.

In one aspect, the use of a mouse as described in the present application is provided to make a fully human Fab or a fully human F (ab) 2. In one embodiment, the fully human Fab or a fully human F (ab) 2 comprises rearranged VL, DH and JL gene segments. In one modality, the Fab completely human or a fully human F (ab) 2 comprises VL, DH and JH gene segments rearranged. In one embodiment, the fully human Fab or a fully human F (ab) 2 comprises rearranged VK and JK gene segments. In one embodiment, the fully human Fab or a fully human F (ab) 2 comprises rearranged VA and JA gene segments.

In one aspect, the use of a mouse as described in the present application is provided to make a fully human Fab (comprising a first VL of human fused with a constant region of the human light chain, and a second human VL). fused to a sequence of the constant region of the human heavy chain) or a fully human F (ab) 2.

In one aspect, the use of a mouse as described in the present application is provided to make an immortalized cell line. In one embodiment, the immortalized cell line comprises a nucleic acid sequence encoding a human VA or VK domain operably linked to a nucleic acid sequence comprising a mouse constant region nucleic acid sequence. In one embodiment, the immortalized cell line expresses an antibody comprising a human variable domain. In a specific embodiment, the human variable domain is a variable domain of the light chain.

In one aspect, the use of a mouse as described in the present application is provided to make a hybridoma or quadroma. In one embodiment, the hybridoma or quadroma expresses a polypeptide that comprises a variable human domain that binds an antigen from interest.

In one aspect, the use of a mouse as described in the present application is provided to make a phage library containing variable regions of the human light chain. In one embodiment, the variable regions of the light chain are human VK regions.

In one aspect, the use of a mouse as described in the present application is provided to generate a variable region sequence for making a human antigen-binding protein, comprising (a) immunizing a mouse as described herein request with an antigen of interest, (b) isolate a lymphocyte from the immunized mouse of (a), (c) expose the lymphocyte to one or more labeled antibodies, (d) identify a lymphocyte that is capable of binding to the antigen of interest, and (e) amplifying one or more nucleic acid sequences of the variable region of the human light chain from the lymphocyte thus generating a sequence of the variable region.

In one embodiment, the lymphocyte is derived from the spleen of the mouse. In one embodiment, the lymphocyte is derived from a mouse lymph node. In one embodiment, the lymphocyte is derived from the bone marrow of the mouse.

In one embodiment, the labeled antibody is an antibody conjugated with fluorophore. In one embodiment, said one or more antibodies conjugated with fluorophore are selected from an IgM, an IgG, and / or a combination thereof.

In one embodiment, the lymphocyte is a B cell.

In one embodiment, said one or more nucleic acid sequences of the variable region comprise a sequence of the variable region of the light chain. In a specific embodiment, the sequence of the variable region of the light chain is a sequence of the variable region of the immunoglobulin light chain k. In one embodiment, said one or more nucleic acid sequences of the variable region is a sequence of the variable region of the light chain A.

In one embodiment, the use of a mouse as described in the present application is provided to generate one or more sequences of the variable region of the light chain k to make a human antigen binding protein., which comprises (a) immunizing a mouse as described in the present application with an antigen of interest, (b) isolating the spleen from the immunized mouse of (a), (c) exposing the B lymphocytes from the spleen to one or more labeled antibodies, (d) identifying a B lymphocyte of (c) that is capable of binding to the antigen of interest, and (e) amplifying a nucleic acid sequence of the variable region of the light chain k from the B lymphocyte generating in this way the sequence of the variable region of the light chain K.

In one embodiment, the use of a mouse as described in the present application is provided to generate a sequence of the variable region of the light chain k to make a human antigen-binding protein, comprising (a) immunizing a mouse as described in the present application with an antigen of interest, (b) isolating one or more lymph nodes from the immunized mouse of (a), (c) exposing the B lymphocytes from said one or more ganglia lymphatics to one or more labeled antibodies, (d) identifying a B lymphocyte of (c) that is capable of binding to the antigen of interest, and (e) amplifying a nucleic acid sequence of the variable region of the light chain k from the B lymphocyte thus generating the sequence of the variable region of the light chain K.

In one embodiment, the use of a mouse as described in the present application is provided to generate a sequence of the variable region of the light chain k to make a human antigen-binding protein, comprising (a) immunizing a mouse as described in the present application with an antigen of interest, (b) isolating bone marrow from the immunized mouse from (a), (c) exposing the B lymphocytes from the bone marrow to one or more labeled antibodies, (d) identifying a B lymphocyte of (c) that is capable of binding to the antigen of interest; and (e) amplifying a nucleic acid sequence of the variable region of the light chain k from the B lymphocyte, thereby generating the sequence of the region variable of the light chain K. In various embodiments, said one or more labeled antibodies are selected from an IgM, an IgG, and / or a combination thereof.

In various embodiments, the use of a mouse as described in the present application is provided to generate a sequence of the variable region of the light chain k to make a human antigen-binding protein, which further comprises fusing the sequence of the region variable of the light chain k amplified to sequences of the constant region of the heavy chain or light chain of human u optionally a sequence of the variable region of the human heavy chain, expressing the fused sequences in a cell, and recovering the expressed sequences thereby generating a human antigen-binding protein.

In various embodiments, the constant regions of the human heavy chain are selected from IgM, I g D, IgA, I g E and IgG. In various specific embodiments, IgG is selected from IgG 1, IgG 2, IgG 3 and IgG 4. In various embodiments, the constant region of the human heavy chain comprises a CH1, a hinge, a CH2, a CH3, a CH4, or a combination thereof. In various embodiments, the constant region of the light chain is a constant region k of immunoglobulin. 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 cell, a SHSY5Y cell (human neuroblastoma), a Saos-2 cell, a Vero cell, a CHO cell, a GH3 cell, a PC12 cell, a human retinal cell (e.g., a PER.C6 cell) ™), and an MC3T3 cell. In a specific embodiment, the cell is a CHO cell.

In one aspect, there is provided a method for generating a variable region of the human light chain specific against an antigen of interest, comprising the steps of immunizing a mouse as described in the present application with the antigen, isolating at least one cell from the mouse that produces a variable region of the chain light of human specific against the antigen, generate at least a cell that produces a human antigen-binding protein comprising the variable region of the light chain specific against the antigen, and culturing at least one cell that produces the human antigen-binding protein, and obtaining said antigen-binding protein human In one embodiment, the variable region of the human light chain is a human VK region.

In various embodiments, said at least one cell isolated from the mouse that produces a variable region of the specific light chain against the antigen is a splenocyte or a B cell.

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

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

In one aspect, the use of a mouse as described in the present application is provided to make a nucleic acid sequence encoding an immunoglobulin variable region or fragment thereof. In one embodiment, the nucleic acid sequence is used to make a human antibody or antigen-binding fragment thereof. In one embodiment, the mouse is used to make an antigen-binding protein that is selected from an antibody, a multispecific antibody (e.g., a bispecific antibody), a scFv, a bis-scFv, a diabody, a triabody , a tetrabody, a V-NAR, a VHH, a VL, an F (ab), an F (ab) 2, a DVD (ie, variable domain antigen binding protein) dual), an SVD (ie, individual variable domain antigen binding protein), or a bispecific T cell coupler (BiTE).

In one aspect, the use of the mouse is provided as described in the present application for the manufacture of a medicament (eg, 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.

In one aspect, the use of a mouse as described in the present application is provided to make a nucleic acid sequence encoding a first variable sequence of human light chain immunoglobulin (VL1) that is cognate with a second sequence. Immunoglobulin variable of human light chain (VL2), in which VL1 fused to a human immunoglobulin light chain constant region (polypeptide 1) is expressed with Vu2 fused to a constant region of the immunoglobulin heavy chain (polypeptide 2), as a dimer of polypeptide 1 / polypeptide 2, to form an antibody VL1 -VL2.

In one aspect, the use of a mouse as described in the present application for making a nucleic acid sequence encoding a variable sequence of the human immunoglobulin light chain that is fused to an immunoglobulin heavy chain sequence of human, in which the nucleic acid sequence encodes a human V-CH polypeptide, in the which the human VL-CH polypeptide is expressed as a dimer, and in the which dimer are expressed in the absence of an immunoglobulin light chain (e.g., in the absence of a human A or human k light chain). In one embodiment, the VL-CH dimer specifically binds an antigen of interest in the absence of a l light chain and in the absence of a K. light chain.

In one aspect, the use of a mouse as described in the present application to make a nucleic acid sequence encoding all or a portion of an immunoglobulin variable domain. In one embodiment, the immunoglobulin variable domain is a human VA or human VK domain.

In one aspect, the use of a nucleic acid construct as described in the present application is provided for the manufacture of a mouse, a cell, or a therapeutic protein (e.g., an antibody or other antigen-binding protein).

In one aspect, the use of a nucleic acid sequence from a mouse as described in the present application is provided to make a cell line for the manufacture of a human therapeutic agent. In one embodiment, the human therapeutic agent is a binding protein comprising a variable sequence of the human light chain (e.g., derived from a VA segment of human or human VK) fused to a constant sequence of the chain heavy of human. In one embodiment, the human therapeutic agent comprises a first polypeptide that is a human immunoglobulin A or k light chain, and a second polypeptide which comprises a variable VA sequence of human or VK of human fused with a constant sequence of the human heavy chain.

In one aspect, an expression system is provided, comprising a mammalian cell transfected with a DNA construct encoding a polypeptide comprising a somatically mutated human VL domain fused to a human CH domain.

In one embodiment, the expression system further comprises a nucleotide sequence that encodes an immunoglobulin VL domain fused to a human CL domain, in which the VL domain fused to the human CL domain is a light chain cognate with the VL domain fused with the CH domain of human.

In one embodiment, the mammalian cell is selected from a CHO cell, a COS cell, a Vero cell, a 293 cell, and a retinal cell that expresses a viral gene (e.g., a PER.C6 ™ cell).

In one aspect, there is provided a method for making a binding protein, comprising obtaining a nucleotide sequence coding for a VL domain from a gene encoding a VL region fused to a CH region from a cell of a mouse as described in the present application, and cloning the nucleotide sequence encoding the frame VL region sequence with a gene encoding a human CH region to form a human binding protein sequence, expressing the sequence of human binding protein in an appropriate cell.

In one embodiment, the mouse has been immunized with a antigen of interest, and the VL region fused to the CH region specifically binds (e.g., with a KD in the micromolar, nanomolar, or picomolar range) an epitope of the antigen of interest. In one embodiment, the nucleotide sequence encoding the VL region fused to the CH region is somatically mutated in the mouse.

In one embodiment, the appropriate cell is selected from a B cell, a hybridoma, a quadroma, a CMO cell, a COS cell, a 293 cell, a HeLa cell, and a human retinal cell that expresses an acid sequence viral nucleic acid (for example, a PERC.6 ™ cell).

In one embodiment, the CH region comprises an isotype of human IgG. In a specific embodiment, human IgG is selected from an I gG 1, IgG2, and IgG4. In another specific embodiment, human IgG is IgG1. In another specific embodiment, human IgG is IgG4. In another specific embodiment, human IgG4 is a modified IgG4. In one embodiment, the modified IgG4 comprises a substitution in the hinge region. In a specific embodiment, the modified IgG4 comprises a substitution at the amino acid residue 228 relative to a wild-type IgG4 from human, numbered according to the Kabat EU numbering index. In a specific embodiment, the substitution at amino acid residue 228 is a substitution S228P, numbered according to the Kabat EU numbering index.

In one embodiment, the cell further comprises a nucleotide sequence that codes for a VL domain of a chain light that is cognate with the VL domain fused to the CH region, and the method further comprises expressing the nucleotide sequence encoding the cognate VL domain fused to a human CK O CA domain.

In one aspect, there is provided a method for making a bispecific antigen-binding protein, comprising exposing a first mouse as described in the present application to a first antigen of interest and identifying a sequence of a first human VL domain that ligates specifically to the first antigen of interest; exposing a second mouse as described in the present application to a second antigen of interest and identifying a sequence of a second human VL domain that specifically binds the second antigen of interest; in which the first VL domain of human does not bind to the second antigen of interest, and the second VL domain of human does not bind to the first antigen of interest; and fusing the sequence of the first VL domain of human to a first constant sequence of the heavy chain to form a first antigen-binding polypeptide, and fusing the sequence of the second VL domain of human to a second constant sequence of the heavy chain to form a second antigen-binding polypeptide; and, employing the first and second antigen-binding polypeptides in a bispecific binding protein.

In one embodiment, the antigen-binding protein further comprises a first immunoglobulin light chain comprising a variable k or A domain of human that is cognate with the first VL domain of human, and a second light chain of human immunoglobulin. immunoglobulin comprising a variable domain k or l of human that is cognate with the second VL domain of human.

In one embodiment, the first constant sequence of the heavy chain is identical to the second constant sequence of the heavy chain. In one embodiment, the first constant sequence of the heavy chain comprises a modification that reduces or eliminates the binding of the first constant region of the heavy chain to protein A, and the second constant sequence of the heavy chain links protein A.

In one embodiment, the first and second human VL domains comprise a sequence derived from a human VK gene segment and a human JK gene segment. In one embodiment, the first and second human VL domains comprise a sequence derived from a human VA gene segment and a human JA gene segment. In one embodiment, the first and second human VL domains comprise a sequence derived from a human DH gene segment. In one embodiment, the first and second human VL domains comprise a sequence derived from a human JH gene segment. In a specific embodiment, the first and second human VL domains comprise a sequence derived from a human DH gene segment and a human JH gene segment. In a specific embodiment, the first and second human VL domains comprise a sequence derived from a human DH gene segment and a human JK gene segment.

In one embodiment, the first human VL domain comprises a sequence derived from a human VK gene segment and a human JK gene segment and the second human VL domain comprises a sequence derived from a gene segment. VA of human and a segment of human JA gene. In one embodiment, the first human VL domain comprises a sequence derived from a human VA gene segment and a human JA gene segment and the second human VL domain comprises a sequence derived from a gene segment. Human VK and a human JK gene segment.

In one aspect, an immunoglobulin variable region (VR) is provided (eg, comprising a VL sequence of human fused to a JL, or JH, or DH and JH, O DH and JL of human) made in a mouse as is described in the present application. In a specific embodiment, the immunoglobulin VR is derived from a germline human gene segment that is selected from a VK segment and a VA segment, in which the VR is encoded by a rearranged sequence from the mouse in which the rearranged sequence is somatically hypermutated. In one embodiment, the rearranged sequence comprises 1 to 5 somatic hypermutations. In one embodiment, the rearranged sequence comprises at least 6, 7, 8, 9, or 10 somatic hypermutations. In one embodiment, the rearranged sequence comprises more than 10 somatic hypermutations. In one embodiment, the rearranged sequence is fused to one or more sequences of the constant region of the heavy chain of human or mouse (for example, which are selected from a CH1, hinge, CH2, CH3 of human or mouse, and a combination thereof).

In one aspect, an amino acid sequence of the immunoglobulin variable domain of a binding protein made in a mouse is provided as described in the present application. In one embodiment, the VR is fused to one or more sequences of the human or mouse heavy chain constant region (eg, which are selected from a CH1, hinge, CH2, CH3, human or mouse and a combination thereof).

In one aspect, a variable domain of the light chain encoded by a nucleic acid sequence derived from a mouse is provided as described in the present application.

In one aspect, there is provided an antibody or antigen-binding fragment thereof (eg, Fab, F (ab) 2i scFv) made in a mouse as described in the present application, or derived from a sequence made in a mouse as described in the present application.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates schemes (not to scale) of the locus of the mouse heavy chain (upper part) and the locus of the human light chain k (lower part). The locus of the mouse heavy chain is approximately 3 Mb in length and contains approximately 200 segments of heavy chain variable gene (VH), 13 heavy chain diversity (DH) gene segments and 4 heavy chain binding gene segments (JH) as well as increments (Enh) and constant regions of the heavy chain (CH). The locus of the human light chain k is duplicated in the distal and proximal contiguous of opposite polarity that encompass approximately 440 kb and 600 kb, respectively. Between the two contiguous are approximately 800 kb of DNA that is believed to be free of segments of the VK gene. The human light chain k locus contains approximately 76 VK gene segments, 5 JK gene segments, an intronic increment (Enh) and an individual constant region (CK).

Figure 2 shows an example of target selection strategy for the progressive insertion of 40 human VK gene segments and five human JK gene segments at the locus of the mouse heavy chain which results in a modified locus of the mouse immunoglobulin heavy chain comprising human VK and JK gene segments operably linked to constant regions of the mouse immunoglobulin heavy chain. Cassettes for selection of hygromycin (hyg) and neomycin (neo) are shown with recombinase recognition sites (R1, R2, etc.).

Figure 3 shows an example strategy for target selection for the progressive insertion of a plurality of human VA gene segment and a single human JA gene segment at the locus of the mouse heavy chain. Cassettes for selection of hygromycin (hyg) and neomycin (neo) are shown with sites of Recombinase recognition (R1, R2, etc.).

Figure 4 shows an example strategy for target selection for the progressive insertion of a plurality of segments of the human VA gene and four segments of the human JA gene into the locus of the mouse heavy chain. Cassettes for selection of hygromycin (hyg) and neomycin (neo) are shown with recombinase recognition sites (R1, R2, etc.).

Figure 5 shows an example strategy for target selection for the progressive insertion of segments of human VA gene, human DH and human JH at the locus of the mouse heavy chain. Cassettes for selection of hygromycin (hyg) and neomycin (neo) are shown with recombinase recognition sites (R1, R2, etc.).

Figure 6 shows an example strategy for target selection for the progressive insertion of segments of human VA gene, human DH and human JK at the locus of the mouse heavy chain. Cassettes for selection of hygromycin (hyg) and neomycin (neo) are shown with recombinase recognition sites (R1, R2, etc.).

Figure 7 shows the steps to clone a genomic fragment encoding the mouse ADAM6 genes from a VD intergenic region of the mouse immunoglobulin heavy chain and the engineering steps to modify the genomic fragment for insertion into a locus of the modified immunoglobulin heavy chain.

Figure 8 shows a target selection strategy for the insertion of a genomic fragment encoding the mouse ADAM6 genes in the intergenic VK-JK region of a modified mouse immunoglobulin heavy chain locus containing gene segments. VK and JK of human bound operably to constant regions of the mouse immunoglobulin heavy chain.

Figure 9 shows a target selection strategy for the insertion of a genomic fragment encoding the mouse ADAM6 genes upstream (5 ') of human VK gene segments (ie, hVK2-40) from a locus Modified from the mouse immunoglobulin heavy chain containing human VK and JK gene segments operably linked to constant regions of the mouse immunoglobulin heavy chain.

DETAILED DESCRIPTION OF THE INVENTION This invention is not limited to particular methods, and the experimental conditions described, since many methods and conditions may vary. It should also be understood that the terminology used in the present application is for the purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is defined by the claims.

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

The phrase "substantial" or "substantially" when used to refer to a number of gene segments (eg, "substantially all" the V gene segments) includes both functional and non-functional gene segments and includes, in various embodiments , for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of all gene segments; in various embodiments, "substantially all" the gene segments include, for example, at least 95%, 96%, 97%, 98%, or 99% of the functional gene segments (ie, no pseudogenes).

The term "replacement" includes wherein a DNA sequence is placed in a genome of a cell in such a manner to replace a sequence within the genome with a heterologous sequence (eg, a human sequence in a mouse), at the locus of the genomic sequence. The DNA sequence placed in this way may include one or more regulatory sequences that are part of the source DNA used to obtain the sequence thus placed (for example, promoters, enhancers, 5 'or 3' untranslated regions, appropriate recombination signal sequences, etc.). For example, in various embodiments, replacement is a substitution of an endogenous sequence with a heterologous sequence that results in the production of a gene product from the DNA sequence placed in this manner (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 function similar to that of a protein encoded by the endogenous genomic sequence (e.g., the endogenous genomic sequence encodes an immunoglobulin gene or domain) , and the DNA fragment codes for one or more human immunoglobulin genes or domains). In various embodiments, an endogenous gene or fragment thereof is replaced with a corresponding human gene or fragment thereof. A corresponding human gene or fragment thereof is a human gene or fragment that is an ortholog, a homolog, or is substantially identical or the same in structure and / or function, as the endogenous gene or fragment thereof that was replaced.

The term "contiguous" includes reference to the appearance 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 interrupted by another nucleic acid sequence . For example, a rearranged V (D) J sequence is "contiguous" with a gene sequence of the constant region, although the final codon of the sequence V (D) J is not immediately followed by the first codon of the sequence of the constant region. In another example, two sequences of V gene segments are "contiguous" if they occur in the same genomic fragment, although these may be separated by sequence that does not code for a codon of the V region, for example, these may be separated by a regulatory sequence, for example, a promoter sequence or other non-coding sequence. In one embodiment, a contiguous sequence includes a genomic fragment containing accommodated genomic sequences as found in a wild-type genome.

The phrase "derived from" when used with respect to a variable region "derived from" a gene or gene segment referred to includes the ability to trace the sequence back to a segment or segments of the non-gene. Individual rearrangements that were rearranged to form a gene that expresses the variable domain (which explains, where applicable, splicing differences and somatic mutations).

The phrase "functional" when used with respect to a gene segment of the variable region or segment of the binding gene refers to use in a repertoire of expressed antibody; for example, in humans the gene segments VA 3-1, 4-3, 2-8, etc. They are functional, while the gene segments VA 3-2, 3-4, 2-5, etc. They are non-functional.

A "heavy chain locus" includes a location in a chromosome, for example, a mouse chromosome, in which the DNA sequences of the variable region of the heavy chain (VH), the heavy chain diversity (DH), the binding of heavy chain (JH), and heavy chain constant (CH) · The phrase "bispecific binding protein" includes a binding protein that is capable of selectively binding two or more epitopes. Bispecific binding proteins comprise two different polypeptides comprising a first variable domain of the light chain (VL1) fused to a first CH region and a second variable domain of the light chain (VL2) fused to a second CH region. In general, the first and second CH regions are identical, or these differ in one or more amino acid substitutions (e.g., as described in the present application). VL1 and VL2 specifically bind different epitopes - either in two different molecules (for example, antigens) or in the same molecule (for example, in the same antigen). If a bispecific binding protein selectively binds two different epitopes (a first epitope and a second epitope), the affinity of VL1 for the first epitope will generally be at least one to two or three or four orders of magnitude lower than the affinity of VL1 for the second epitope, and vice versa with respect to VL2. The epitopes recognized by the bispecific binding protein may be on the same or a different target (eg, on the same antigen or on a different antigen). Bispecific binding proteins can be made, for example, by combining a VL1 and a VL2 that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding VL sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences that encode different CH regions, and such sequences can be expressed in a cell that expresses a chain of immunoglobulin, or they can be expressed in a cell that does not express an immunoglobulin light chain. A typical bispecific binding protein has two heavy chains each having three CDRs of the light chain, followed by a CH1 domain (N-terminal to C-terminal), a hinge, a CH2 domain, and a CH3 domain, and a chain light immunoglobulin which may not confer antigen binding specificity but may be associated with each heavy chain, or which may be associated with each heavy chain and which may bind one or more of the epitopes linked by VL1 and / or VL2, or that can be associated with each heavy chain and allow binding or assist in the binding of one or both heavy chains to one or both epitopes.

Therefore, two general types of bispecific binding proteins are (1) VL1 -CH (dimer), and (2) VL1-CH: light chain + VL2-CH: light chain, in which the light chain is the same or it is different. In any case, the CH (that is, the constant region of the heavy chain) can be differentially modified (for example, to differentially bind protein A, to increase the serum half-life, etc. ) as described in the present application, or it may be the same.

The term "cell", when used in connection with expressing a sequence, includes any cell that is appropriate to express a recombinant nucleic acid sequence. The cells include those of prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., Etc.), mycobacterial cells, fungal cells, yeast cells (e.g. , S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (for example, SF-9, SF-21, insect cells infected with baculovirus, 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 (e.g., CHO K1, DXB-1 1 CHO, Veggie-CHO), COS (e.g., COS-7), cell from the retina, Vero, CV1, renal (eg, HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (eg, BHK21) , Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2 / 0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from 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 (e.g., a PER.C6 ™ cell).

The term "cognate", when used in the sense of "cognate with", for example, a first VL domain that is "cognate with" a second VL domain, is intended to include the reference to the relationship between two VL domains from the same binding protein made by a mouse in accordance with the invention. For example, a mouse that is genetically modified according to an embodiment of the invention, for example, a mouse having a locus of the heavy chain in which the VH, DH, and JH regions are replaced with Vi regions. and JL, make antibody-like binding proteins having two identical polypeptide chains made from the same mouse CH region (eg, an IgG isotype) fused to a first human VL domain, and two identical polypeptide chains made from the same mouse CL region fused to a second human VL domain. During clonal selection in the mouse, the first and second human VL domains are selected by the clonal selection procedure to appear together in the context of an individual antibody-like binding protein. Therefore, the first and second VL domains that appear together, as a result of the clonal selection procedure, in an individual antibody type molecule are referred to as "cognates". In contrast, a VL domain that appears in a first antibody-like molecule and a VL domain that appears in a second antibody-like molecule are not cognate, unless the first and second antibody-like molecules have identical heavy chains (ie, a less than the VL domain fused to the first region of the chain human constant and the VL domain fused to the second region of the heavy chain of humans are identical).

The phrase "determining region of complementarity", or the term "CDR," includes an amino acid sequence encoded by a nucleic acid sequence of the immunoglobulin genes of an organism that normally (i.e., in a wild type animal) appears between two regions of base structure in a variable region of a light chain or a heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). A CDR can be encoded by, for example, a germline sequence or a rearranged or non-rearranged sequence, and, for example, by a B cell or a T cell not affected by treatment or mature. In some circumstances (for example, for a CDR3), the CDRs can be encoded by two or more sequences (eg, germline sequences) that are not contiguous (eg, in a non-rearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, for example, as a result of splicing or connecting the sequences (e.g., recombination of VDJ to form a CDR3 of the heavy chain).

The phrase "gene segment", or "segment" includes reference to a segment of immunoglobulin V gene (light or heavy) or D or J (light or heavy), which includes sequences not rearranged at the immunoglobulin loci ( in for example, humans and mice) that can participate in a rearrangement (mediated by, for example, endogenous recombinases) to form a rearranged V / J or V / D / J sequence. Unless otherwise indicated, segments V, D, and J comprise recombination signal sequences (RSS) that allow V / J recombination or V / D / J recombination in accordance with regulation 12/23. Unless otherwise indicated, the segments also comprise sequences with which they are associated in Nature or functional equivalents thereof (eg, for promoter (s) and leader (s) of segments V).

The phrase "heavy chain" or "immunoglobulin heavy chain" includes a constant region sequence of the immunoglobulin heavy chain from any organism, and unless otherwise specified includes a variable domain of the heavy chain (VH) . The VH domains include three CDRs of the heavy chain and four regions of the base structure (FR) regions, unless otherwise specified. Fragments of the heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain consists essentially of, after the variable domain (from N-terminal to C-terminal), a CH1 domain, a hinge, a CH2 domain, a CH3 domain, and optionally a CR4 domain (e.g., in the case of IgM or IgE) and a transmembrane domain (M) (for example, in the case of membrane-bound immunoglobulin in lymphocytes). A constant region of the heavy chain is a region of a heavy chain that extends (from the N-terminal side to the C-terminal side) from outside the FR4 to the C-terminus of the heavy chain. The constant regions of the heavy chain with minor deviations, for example, truncations of one, two, three or several amino acids from the C-terminus, would be encompassed by the phrase "constant region of the heavy chain", as well as constant regions of the heavy chain with sequence modifications, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions . The amino acid substitutions can be made in one or more positions that are selected from, for example (with reference to the UE numbering of an immunoglobulin constant region, eg, a constant region of human IgG), 228, 233 , 234, 235, 236, 237, 238, 239, 241, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 31 1, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

For example, and not by way of limitation, a constant region of the heavy chain can be modified to exhibit increased serum half-life (as compared to the same constant region of the heavy chain without modification or recited modifications) and have a modification at position 250 (for example, E or Q); 250 and 428 (for example, L or F); 252 (for example, L / Y / F / W or T), 254 (for example, S or T), and 256 (for example, S / R / Q / E / D or T); or a modification at 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y); or a modification in 250 and / or 428; or a modification to 307 or 308 (for example, 308F, V308F), and 434. In another example, the modification may comprise a 428L modification (for example, example, M428L) and modification 434S (e.g., N434S); a modification 428L, 259I (e.g., V259I), and a modification 308F (e.g., V308F); a modification 433K (e.g., H433) and a modification 434 (e.g., 434Y); a modification 252, 254, and 256 (e.g., 252Y, 254T, and 256E); a modification 250Q and 428L (for example, T250Q and M428L); a modification 307 and / or 308 (for example, 308F or 308P).

The phrase "light chain" includes a sequence of the constant region of the immunoglobulin light chain (CL) from any organism, and unless otherwise specified includes the light chains k and l of human. The variable domains of the light chain (VL) typically include three CDRs of the light chain and four regions of the base structure (FR) regions, unless otherwise specified. In general terms, a full-length light chain (VL + CL) includes, from the amino terminus to the carboxyl terminus, a VL domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a region CL. The light chains (VL + CL) that can be used with this invention include those, for example, that do not selectively bind any one of a first or second (in the case of bispecific binding proteins) epitopes selectively linked by the binding protein (eg, the epitope or epitopes selectively linked by the VL domain fused to the CH domain) · The VL domains that do not selectively bind the epitope or epitopes bound by the VL that is fused to the CH domain include those that can be identified by screening regarding the chains light ones most commonly used in existing antibody libraries (wet or in silico libraries), in which the light chains do not substantially interfere with the affinity and / or selectivity of the epitope-binding domains of the binding proteins. Suitable light chains include those that can bind (alone or in combination with their cognate VL fused to the CH region) an epitope that is specifically bound by the VL fused to the CH region.

The phrase "micromolar range" is intended to mean 1 -999 micromolar; the phrase "nanomolar interval" is intended to mean 1-999 nanomolar; the phrase "picomolar interval" is intended to mean 1 -999 picomolar.

The term "non-human animals" is intended to include any non-human animals such as skulls, bone 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. Appropriate non-human animals are selected from the family of rodents including rat and mouse. In one embodiment, non-human animals are mice.

The mouse as a genetic model has been greatly improved through tansgenic and bloqe expression technologies, which have allowed the study of the effects of over-expression or targeted deletion of specific genes. Despite all its advantages, the mouse still has genetic obstacles that make it a model imperfect for human diseases and an imperfect platform to test human therapeutic agents or to elaborate them. First, although approximately 99% of human genes have a mouse homologue (Waterston et al. (2002), Initial sequencing and comparative analysis of the mouse genome, Nature 420, 520-562), potential therapeutic agents often fail to present cross reaction, or cross-react inappropriately, with mouse orthologs of the intended human targets. To eliminate this problem, the selected target genes can be "humanized", ie, the mouse gene can be deleted and replaced by the corresponding human orthologous gene sequence (e.g., US 6,586,251, US 6,596,541 and US 7, 105,348 , incorporated in the present application for reference). Initially, efforts to humanize mouse genes using a strategy of "expression blocking plus transgenic humanization" involves crossing a mouse carrying a deletion (i.e., blocking expression) of the endogenous gene with a mouse carrying an integrated human transgene. randomly (see, for example, Bril et al. (2006), Tolerance to factor VIII in a transgenic mouse expressing human factor VIII cDNA carrying an Arg (593) to Cys substitution, Thromb Haemost 95: 341-347, Homanics et al. (2006), Production and characterization of murine models of classic and intermediary maple syrup urine disease, BMC Med Genet 7:33, Jamsai et al. (2006), A humanized BAC transgenic / knockout mouse model for HbE / beta-thalassemia , Genomics 88 (3): 309-15; Pan et al. (2006), Different role for mouse and human CD3delta / epsilon heterodimer N preT cell receiver (preTCR) function: human CD3delta / epsilon heterodimer restores the detective preTCR function in CD3gamma- and CD3gammadelta-deficent mice, Mol Immunol 43: 1741-1750). But such efforts were hampered by size limitations; conventional expression blocking technologies were not sufficient to directly replace large mouse genes with their large human genomic counterparts. A simple direct homologous replacement strategy, in which an endogenous mouse gene is directly replaced by the human counterpart gene in the same precise genetic location of the mouse gene (i.e., in the endogenous mouse locus), rarely it is attempted due to technical difficulties. Until now, direct replacement efforts involve laborious and complicated procedures, thereby limiting the length of genetic material that could be handled and the precision with which it can be manipulated.

The human immunoglobulin transgenes introduced in an exogenous manner are rearranged in the B precursor cells in the mice (Alt et al. (1985), Immunoglobulin genes in transgenic mice, Trends Genet 1: 231-236). This discovery was exploited by designing mice using the expression-plus-transgenic blocking strategy to express human antibodies (Green et al. (1994), Antigen-specific human monoclonal antibodies from human engineered with human Ig heavy and light chain YACs, Nat Genet 7: 13-21; Lonberg, N. (2005), Human antibodies from transgenic animáis Nat Biotechnol 23: 1 1 17-1 125; Lonberg et al. (1994), Antigen-specific human antibodies from mice comprising four distinct genetic modifications, Nature 368: 856-859; Jakobovits et al. (2007), From XenoMouse technology to panitumumab, the first completely human antibody product from transgenic mice, Nat Biotechnol 25: 1 134-1143). Endogenous immunoglobulin heavy chain and light chain k mouse loci are inactivated in these mice by targeted deletion of small but critical portions of each endogenous locus, followed by introduction of human immunoglobulin gene loci as large integrated transgenes in the form random, as previously described, or minichromosomes (Tomizuka et al. (2000), Double trans-chromosomic mice: maintenance of two individual human chromosome fragment containing Ig heavy and kappa loci and expression of completely human antibodies, PNAS USA 97: 722- 727). These mice represent an important advance in genetic engineering; The fully human monoclonal antibodies isolated therefrom provided promising therapeutic potential to treat a variety of human diseases (Gibson et al. (2006), Randomized phase III trial results of panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody , in metastatic colorectal cancer, Clin Colorectal Cancer 6: 29-31; Jakobovits et al., 2007; Kim et al. (2007), Clinical efficacy of zanolimumab (HuMax-CD4): two Phase II studies in refractory cutaneous T-cell lymphoma, Blood 109 (11): 4655-62, Lonberg, 2005, Maker et al. (2005), Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2: a phase l / ll study , Ann Surg Oncol 12: 1005-1016; McClung et al. (2006), Denosumab in postmenopausal women with low bone mineral density, N Engl J Med 354: 821-831). But, as discussed above, these mice exhibit compromised B cell development and immune deficiencies when compared to wild-type mice. Such problems potentially limit the ability of mice to support a vigorous humoral response and, therefore, generate fully human antibodies against some antigens. The deficiencies may be due to: (1) inefficient functionality due to the random introduction of human immunoglobulin transgenes and resulting incorrect expression due to a lack of control elements towards the 5 'end and towards the 3' end (Garrett et al. al. (2005), Chromatin architecture near a potential 3 'end of the Igh locus involves modular regulation of histone modifications during B-cell development and in vivo occupancy at CTCF sites, Mol Cell Biol 25: 1511-1525; Manís et al. (2003), Elucidation of a downstream boundary of the 3 'IgH regulatory region, Mol Immunol 39: 753-760; Pawlitzky et al. (2006), Identification of a candidate regulatory element within the 5 'flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A, J Immunol 176: 6839-6851 ); (2) inefficient interactions between species between the constant human domains and the mouse components of the B cell receptor signaling complex on the cell surface, which may impair the signaling processes required for maturation, proliferation, and normal survival of B cells (Hombach et al. (1990), Molecular components of the B-cell antigen receptor complex of the IgM class, Nature 343: 760-762); and (3) inefficient interactions between species between soluble human immunoglobulins and mouse Fe receptors that could reduce affinity selection (Rao et al., 2002). Differential expression of the inhibitory IgG Fe receptor FcgammaRIIB on germinal center cells: implications for selection of high-affinity B cells, J Immunol 169: 1859-1868) and serum immunoglobulin concentrations (Brambell et al. (1964), A Theoretical Model of Gamma-Globulin Catabolism, Nature 203: 1352-1354; Junghans and Anderson, (1996), The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor, PNAS USA 93: 5512-5516, Rao et al., 2002, Hjelm et al. (2006), Antibody- mediated regulation of the immune response, Scand J Immunol 64: 177-184, Nimmerjahn and Ravetch, (2007), Fc-receptors as regulators of immunity, Adv Immunol 96: 179-204). These deficiencies can be corrected by in situ humanization of only the variable regions of the mouse immunoglobulin loci within their natural locations at the endogenous loci of the heavy and light chains. This would effectively result in mice making "inverted chimeric" antibodies (ie, human V: mouse C) that would be capable of normal interactions and selection with the mouse environment based on retaining the mouse constant regions. In addition, said inverted chimeric antibodies can be easily reformatted into fully human antibodies for therapeutic purposes.

Genetically modified animals that comprise an insertion or replacement at the locus of the heavy chain of Endogenous immunoglobulin with heterologous immunoglobulin sequences (eg, from another species) can be made in conjunction with insertions or replacements at endogenous immunoglobulin light chain loci or in conjunction with immunoglobulin light chain transgenes (e.g. , transgenes of the chimeric or completely completely human mouse chimeric immunoglobulin chain, etc.) · The species from which the heterologous immunoglobulin sequences are derived can vary widely. Examples of heterologous immunoglobulin sequences include human sequences.

Immunoglobulin variable region nucleic acid sequences, for example, segments V, D, and / or J, in various embodiments are obtained from a human animal or a non-human animal. Non-human animals suitable for providing segments V, D, and / or J include, for example, bone 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., cows, bulls, buffaloes), deer, camels, ferrets and rodents and non-human primates (e.g., chimpanzees, orangutans, gorillas, marmosets, rhesus babumos monkeys). Appropriate non-human animals are selected from the family of rodents including rats, mice, and hamsters. In one embodiment, non-human animals are mice.

As is evident from the context, various non-human animals as a source of variable domains or gene segments of the variable region (e.g., sharks, rays, mammals, e.g., camels, rodents such as mice and rats).

In accordance with the context, non-human animals are also used as sources of sequences of the constant region that will be used in connection with variable sequences or segments, for example, rodent constant sequences in transgenes linked in operable variable sequences can be used human or non-human (eg, variable sequences of human or non-human primate operably linked to, eg, constant rodent sequences), for example, mouse or rat or hamster). Therefore, in various embodiments, human V, D, and / or J segments are operably linked to gene sequences of the rodent constant region (e.g., mouse or rat or hamster). In some embodiments, the human V, D, and / or J segments (or one or more rearranged VDJ or VJ genes) are operably linked or fused to a mouse, rat, or hamster constant region gene sequence. in, for example, a transgene integrated in a locus that is not an endogenous immunoglobulin locus.

In a specific embodiment, a mouse is provided comprising a replacement of VH, DH, and JH gene segments at a locus of the endogenous immunoglobulin heavy chain with one or more human VL gene segments and one or more gene segments. JL of human, in which said one or more segments of VL gene of human and one or more JL gene segments are operably linked to a endogenous immunoglobulin heavy chain gene; wherein the mouse comprises a transgene at a different locus of an endogenous Immunoglobulin locus, in which the transgene comprises a segment of human VL gene and human JL not rearranged or rearranged operably linked to a mouse constant region or rat or human. In various embodiments, said one or more human VL gene segments include segments of human VK gene or human VA. In one embodiment, said one or more human JL gene segments include human JK gene segments or human JA segments.

We describe a method for a large in situ genetic replacement of the variable genes of the mouse germline immunoglobulin heavy chain with variable genes of the human germline immunoglobulin light chain while maintaining the capacity of the mice to generate offspring. Specifically, we describe the precise replacement of variable gene loci of the mouse heavy chain with those of the variable gene of the human light chain while leaving constant mouse regions intact. As a result, mice expressing immunoglobulin-like binding proteins have been created in the context of endogenous constant regions. The variable regions of the human light chain are ligated to the constant regions of the mouse heavy chain to form human-mouse chimeric immunoglobulin loci that rearrange and express unique immunoglobulin-like molecules. The immunoglobulin-like molecules expressed are "inverted chimeras", that is, they comprise variable region sequences of human and mouse constant region sequences.

The engineering introduction of human immunoglobulin sequences into the genome of a mouse, even at precise locations, for example, at endogenous mouse immunoglobulin loci, may present certain challenges due to the divergent evolution of the immunoglobulin loci between the mouse and the human. For example, intergenic sequences interspersed within immunoglobulin loci are not identical between mice and humans and, in some circumstances, may not be functionally equivalent. The differences between mice and humans at their immunoglobulin loci can even result in abnormalities in humanized mice, particularly when certain portions of the endogenous mouse immunoglobulin heavy chain loci are humanized or manipulated. Some modifications in the loci of the mouse immunoglobulin heavy chain are deleterious. Harmful modifications may include, for example, loss of the ability of the modified mice to mate and produce offspring. In various embodiments, the engineering introduction of human immunoglobulin sequences into the genome of a mouse includes methods that maintain the endogenous sequences that are deleterious when they are absent in the modified mouse strains. Exemplary deleterious effects may include inability to propagate the modified strains, loss of function of the essential genes, inability to express polypeptides, and so on. These harmful effects can be directly or indirectly related with the modification introduced by engineering in the mouse genome.

Despite the almost wild type humoral immune function observed in mice with immunoglobulin loci replaced, there are other challenges encountered when using a direct replacement of the immunoglobulin that are not found in some strategies that use transgenes integrated in a random manner. Differences in the genetic composition of the immunoglobulin loci between mice and humans have led to the discovery of sequences beneficial for the propagation of mice with replaced immunoglobulin gene segments. Specifically, mouse ADAM genes located within the locus of the endogenous immunoglobulin heavy chain are optimally present in mice with immunoglobulin loci replaced, due to their participation in fertility. [00395] [00395] A precise in situ replacement of six megabases of the variable regions of the immunoglobulin loci of the mouse heavy chain (VH-DH-JH) with variable gene loci of the light chain of the mouse is carried out. human immunoglobulin (VL-JL), while leaving the mouse flanking and functional sequences within the hybrid loci intact, including all the genes of the mouse constant chain and the transcriptional control regions of the locus (Figure 2 - Figure 6). Engineering steps are performed to maintain the mouse sequences that confer on the mouse the ability to mate and produce offspring in a manner comparable to that of a wild type mouse (Figure 7 - Figure 9). Specifically, approximately half of a megabase of the human immunoglobulin k light chain locus containing the proximal arm (ie, 40 functional human VK gene segments and 5 human JK gene segments) and the mouse ADAM6 genes they are introduced through vectors for choice of chimeric BAC targets in mouse ES cells using the genetic engineering technology VELOCIGENE® (see, for example, US Patent No. 6,586,251 and Valenzuela et al., 2003, High-throughput engineering of the mouse genome coupled with high-resolution expresslon analysis, Nat Biotechnol 21: 652-659) ..

Genomic location and function of mouse ADAM6 Male mice lacking the ability to express any functional ADAM6 protein exhibit a severe defect in the ability of mice to mate and generate offspring. Mice lack the ability to express a functional ADAM6 protein due to a replacement of all or substantially all of the variable gene segments of the mouse immunoglobulin heavy chain with variable gene segments of the human light chain. The loss of ADAM6 function results because the ADAM6 locus is located within a region of the variable gene locus of the endogenous immunoglobulin heavy chain, proximal to the 3 'end of the upstream VH gene segment of the locus of the segments of DH gene. In order to cross mice that are homozygous for a replacement of all or substantially all the variable gene segments of the endogenous heavy chain with variable gene segments of the human light chain, it is generally a problematic strategy to join females and males that are each homozygous for replacement and expect a productive mating . Successful litters are relatively rare and the average litter size is very low. In contrast, heterozygous males have been used for replacement to mate with homozygous females for replacement to generate progeny that are heterozygous for replacement, then a homozygous mouse is reproduced from them. The inventors have determined that the probable cause of the loss in fertility in male mice is the absence of a functional ADAM6 protein in male homozygous mice.

In various aspects, male mice comprising a damaged (ie, non-functional or marginally functional) ADAM6 gene exhibit a reduction or elimination of fertility. Because in mice (and other rodents) the ADAM6 gene is located at the locus of the immunoglobulin heavy chain, the inventors determined that in order to propagate the mice, or create and maintain a strain of mice, which comprises modifications to a locus of the endogenous immunoglobulin heavy chain, several modified cross or propagation schemes are employed. The low fertility, or infertility, of male homozygous mice for a replacement of the locus of the variable gene of the endogenous immunoglobulin heavy chain makes it difficult to maintain such modification in a mouse strain. In various embodiments, maintaining the strain comprises avoiding the infertility problems exhibited by homozygous male-gender mice for a replacement.

In one aspect, a method for maintaining a mouse strain is provided as described in the present application. The mouse strain need not comprise an ectopic ADAM6 sequence, and in various embodiments, the mouse strain is homozygous or heterozygous for a blockade in the expression (eg, a blockade in functional expression) of ADAM6.

The mouse strain comprises a modification of a locus of the endogenous immunoglobulin heavy chain 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 the fertility of a male mouse comprising the modification; in a specific embodiment, the modification reduces or eliminates the fertility of a male mouse that is homozygous for the modification.

In one embodiment, the mouse strain is homozygous or heterozygous for a blockage of expression (e.g., a functional expression block) or a deletion of an ADAM6 gene.

In one embodiment, the mouse strain is maintained by isolation of a cell from a mouse that is homozygous or heterozygous for the modification, and using the donor cell in the host embryo, and performing the gestation of the host embryo and the donor cell in a surrogate mother, and obtain from the surrogate mother a progeny that understands the genetic modification. In one embodiment, the donor cell is an ES cell. In one embodiment, the donor cell is a pluripotential cell, for example, an induced pluripotent cell.

In one embodiment, the mouse strain is kept isolating from a mouse that is homozygous or heterozygous for modification a nucleic acid sequence comprising the modification, and introducing the nucleic acid sequence into a host nucleus, and effecting the gestation of a a cell comprising the nucleic acid sequence and the host nucleus in a suitable animal. In one embodiment, the nucleic acid sequence is introduced into an embryo of the host oocyte.

In one embodiment, the mouse strain is maintained by isolating a nucleus from a mouse that is homozygous or heterozygous for modification, and introducing the nucleus into a host cell, and performing the gestation of the host cell and nucleus in an appropriate animal. to obtain a progeny that is homozygous or heterozygous for the modification.

In one embodiment, the mouse strain is maintained using in vitro fertilization (IVF) of a mouse of female gender (wild type, homozygous for modification, or heterozygous for modification) using a spermatozoon from a male mouse comprising the genetic modification. In one embodiment, the male mouse is heterozygous for genetic modification. In one embodiment, the male mouse is homozygous for genetic modification.

In one embodiment, the mouse strain is maintained by crossing a male mouse that is heterozygous for genetic modification with a female mouse to obtain progeny that comprises the genetic modification, to identify a male progeny and a female progeny which comprises the genetic modification, and employing a male that is heterozygous for genetic modification in a cross with a female that is wild type, homozygous, or heterozygous for genetic modification, to obtain progeny comprising the genetic modification. In one embodiment, the step of crossing a heterozygous male for genetic modification with a wild-type female, a heterozygous female for genetic modification, or a homozygous female for genetic modification is repeated in order to maintain the genetic modification in the strain of mouse.

In one aspect, there is provided a method for maintaining a mouse strain comprising replacing a variable gene locus of the endogenous immunoglobulin heavy chain with one or more human immunoglobulin light chain sequences, and optionally one or more human DH gene segments, comprising crossing the mouse strain to generate heterozygous male-gender mice, in which male heterozygous mice breed to maintain genetic modification in the strain. In a specific embodiment, the strain is not maintained by any crossing of a homozygous male with a wild-type female, or a homozygous or heterozygous female for genetic modification.

The ADAM6 protein is a member of the Desintegrin and Metalloproteinase (ADAM) family of proteins, which is a large family with diverse functions including cell adhesion. Some members of the ADAM family are involved in spermatogenesis and fertilization. For example, ADAM2 codes for a subunit of the fertilin protein, which is involved in sperm-egg interactions. ADAM3, or ciritestine, appears to be necessary for the sperm to bind to the zona pellucida. The absence of either ADAM2 or ADAM3 results in infertility. It has been postulated that ADAM2, ADAM3, and ADAM6 form a complex on the surface of mouse sperm.

In humans, an ADAM6 gene, as reported by a pseudogene, is located between the VH gene segments of human VH1 -2 and VH6-1. In mice, there are two ADAM6 genes -ADAM6a and ADAM6b- that are located in an intergenic region between the mouse VH and DH gene segments, and are oriented in transcription orientation opposite to that of the mouse segments. surrounding immunoglobulin gene. In mice, a functional ADAM6 locus is apparently required for normal fertilization. Then, a locus or functional ADAM6 sequence refers to a locus or sequence of ADAM6 that can complement, or rescue, the drastically reduced fertilization exhibited in male-gender mice with missing or damaged endogenous ADAM6 loci.

The position of the intergenic sequence in mice encoding ADAM6 and ADAM6b makes the intergenic sequence susceptible to modification when an endogenous heavy chain is modified. When the segments of the VH gene are deleted or replaced, or when the segments of the DH gene are deleted or replaced, there is a high probability that a resulting mouse exhibits a severe deficit in fertility. To compensate for the deficit, the mouse is modified to include a nucleotide sequence that codes for a protein that complements the loss of ADAM6 activity due to a modification of the endogenous ADAM6 locus. In various embodiments, the complementary nucleotide sequence is one that codes for a mouse ADAM6a, a mouse ADAM6b, or a homologue or ortholog or functional fragment thereof that rescues the fertility deficit. In various embodiments, the complementary nucleotide sequence codes for a mouse ADAM6a protein as indicated in SEQ ID NO: 1, and / or codes for a mouse ADAM6b protein as indicated in SEQ ID NO: 2. Alternatively, methods can be used suitable for preserving the endogenous ADAM6 locus, while making the endogenous immunoglobulin heavy chain sequences flanking the mouse ADAM6 locus incapable of rearrangement to code for a variable region of the functional endogenous heavy chain. Examples of alternative methods include manipulation of large portions of the mouse chromosomes that position the endogenous immunoglobulin heavy chain variable region loci such that they are capable of rearrangement to code for a variable region of the heavy chain that is operably linked to a constant gene of the endogenous heavy chain. In various embodiments, the methods include inversions and / or translocations of mouse chromosomal fragments containing endogenous immunoglobulin heavy chain gene segments.

The nucleotide sequence that rescues fertility can be placed in any appropriate position. This can be placed in an intergenic region (eg, between the V and J gene segments or upstream of the V gene segments), or at any appropriate position in the genome (ie, ectopically). In one embodiment, the nucleotide sequence can be introduced into a transgene that is randomly integrated into the mouse genome. In one embodiment, the sequence can be maintained in episomal form, ie, in a separate nucleic acid instead of in a mouse chromosome. The positions Suitable positions include positions that are permissive or active from the transcription point of view, 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). The nucleotide sequences for target selection for transcriptionally active loci are described, for example, in US 7,473,557, incorporated in the present application for reference.

Alternatively, the nucleotide sequence that rescues fertility may be coupled with an inducible promoter to facilitate optimal expression in appropriate cells and / or tissues, e.g., reproductive tissues. Examples of inducible promoters include promoters activated by physical means (e.g., heat shock promoter) and / or chemicals (e.g., IPTG or tetracycline).

In addition, nucleotide expression can be linked to other genes to achieve expression at specific stages of development or within specific tissues. Said expression can be achieved by placing the nucleotide sequence in operable linkage with the promoter of a gene expressed at a specific stage of development. For example, immunoglobulin sequences from a species that are engineered into the genome of a host species are placed in operable linkage with a promoter sequence of a CD19 gene (a specific B cell gene) from the Host species. In this way the specific expression of cell B is achieved in the precise stages of development when the immunoglobulins are expressed.

Even another method for achieving robust expression of an inserted nucleotide sequence is to use a constitutive promoter. Examples of constitutive promoters include SV40, CMV, UBC, EF1A, PGK and CAGG. In a similar way, the desired nucleotide sequence is placed in operable linkage with a selected constitutive promoter, which provides high level of expression of the protein or proteins encoded by the nucleotide sequence.

The term "ectopic (a)" is intended to include a displacement, or placement in a position that is not normally found in Nature (eg, placement of a nucleic acid sequence in a position that is not the same position in the that the nucleic acid sequence is found in a wild-type mouse). The term in various modalities is used in the sense that its goal is outside of its normal, or appropriate, position. For example, the phrase "an ectopic nucleotide sequence coding for ..." refers to a nucleotide sequence that appears in a position in which 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 homologue or fragment thereof that provides the same benefit or similar fertility benefit in male mice), the sequence can be placed in a different position in the mouse genome than that in which it is normally found in a wild-type mouse. In such cases, the novel sequence junctions of the mouse sequence will be created by placing the sequence in a different position in the mouse genome than in a wild-type mouse. A functional homologue or ortholog of mouse ADAM6 is a sequence that confers a rescue of the loss of fertility (e.g., loss of the ability of a male mouse to generate offspring by mating) observed in an ADAM6 mouse. Functional homologs or orthologs include proteins that have at least about 89% identity or more, eg, up to 99% identity, with the amino acid sequence of ADAM6a and / or with the amino acid sequence of ADAM6b, and that they can complement, or rescue the ability for successful mating of a mouse having a genotype that includes a deletion or blockade of expression of ADAM6a and / or ADAM6b.

The ectopic position can be anywhere (eg, such as with random insertion of a transgene containing a mouse ADAM6 sequence), or it can be, for example, in an approaching position (but not precisely the same as) to 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., within an immunoglobulin locus modified but between different gene segments, or in a different position in a mouse V-D intergenic sequence). An example of an ectopic placement is to maintain the position normally found in wild type mice within the locus of the endogenous immunoglobulin heavy chain while making surrounding endogenous heavy chain gene segments unable to rearrange to code for a functional heavy chain containing a constant region of the endogenous heavy chain. In this example, this can be achieved by inverting the chromosomal fragment containing the variable loci of the endogenous immunoglobulin heavy chain, for example, by using site-specific recombination sites designed, positioned at positions flanking the locus of the variable region. Therefore, after recombination the loci of the variable region of the endogenous heavy chain are placed at a great distance away from the genes of the constant region of the endogenous heavy chain, thereby preventing rearrangement so that they encode for a functional heavy chain containing a constant region of the endogenous heavy chain. Other exemplary methods for achieving functional silencing of endogenous immunoglobulin heavy chain variable gene loci while maintaining a functional ADAM6 locus will be apparent to those skilled in the art after reading this description and / or in combination with methods known in the art. With this placement of the locus of the heavy chain endogenously, the endogenous ADAM6 genes are maintained and the immunoglobulin heavy chain locus is functionally silenced.

Another example of an ectopic placement is placement within a locus of the modified immunoglobulin heavy chain. For example, a mouse comprising a replacement of one or more endogenous VH gene segments with human VL gene segments, in which the replacement removes an endogenous ADAM6 sequence, can be designed to have a mouse ADAM6 sequence. located within the sequence containing the human VL gene segments. The resulting modification will generate a mouse (ectopic) ADAM6 sequence within a human gene sequence, and the placement (ectopic) of the mouse ADAM6 sequence within the human gene sequence can be approximated to the position of the human ADAM6 pseudogene (i.e. between two V segments) or can be approximated to the position of the mouse ADAM6 sequence (i.e., within the VD intergenic region). The resulting sequence junctions created by the binding of a mouse (ectopic) ADA 6 sequence within or adjacent to a human gene sequence (e.g., an immunoglobulin light chain gene sequence) within the line The germline of the mouse will be novel compared to the same position or similar positions in the genome of a wild-type mouse.

In various embodiments, non-human animals lacking an ADAM6 or ortholog or homologous thereof are provided, in which the absence makes the animal non-human infertile, or substantially reduces the non-human animal's fertility. In various embodiments, the absence of ADAM6 or orthologous or homologous thereof is due to a modification of a locus of the endogenous immunoglobulin heavy chain. A substantial reduction in fertility is, for example, a reduction in fertility (for example, frequency of crosses, litters per litter, litters per year, etc.) of approximately 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 homologue or functional fragment thereof that is functional in a male individual of the non-human animal, in which the ADAM6 gene supplemented or ortholog Its homolog or functional fragment rescues the reduction in fertility in its entirety or in a substantial part. A substantial part 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 as compared to a locus of the heavy chain unmodified (ie, an animal without a modification to the ADAM6 gene or ortholog or homologous thereof).

The sequence that confers the genetically modified animal (ie, the animal lacking a functional ADAM6 or ortholog or homologue thereof, due, for example, to a modification of a locus of the immunoglobulin heavy chain), in various embodiments, is selected from an ADAM6 gene or ortholog or homologue or the same. For example, in a mouse, the loss of the ADAM6 function is rescued by adding, in one embodiment, a mouse ADAM6 gene. In one embodiment, the loss of ADAM6 function in the mouse is rescued by adding an ortholog or homolog of a closely related species to the mouse, eg, a rodent, eg, a mouse of a different strain or species, a rat of any species, a rodent; in which the addition of the ortholog or mouse homolog rescues the loss of fertility due to loss of ADAM6 function or loss of an ADAM6 gene. Orthologs and homologs from other species, in various modalities, are selected from a phylogenetically related species and, in various embodiments, exhibit a percent identity with endogenous ADAM6 (or ortholog) which is approximately 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, or 97% or more, and that rescues the loss of fertility related to ADAM6 or (in an animal that is not a mouse) with ADAM6 ortholog . For example, in a genetically modified male rat that lacks the ADAM6 function (eg, a rat with a variable region of the endogenous immunoglobulin heavy chain replaced with a variable region of the human immunoglobulin heavy chain, or a blockage of expression in the rat immunoglobulin heavy chain region), loss of fertility in the rat it is rescued by the addition of a rat ADAM6 or, in some embodiments, an ortholog of a rat ADAM6 (eg, an ADA 6 orthologue from another strain or rat species, or, in one embodiment, from a mouse).

Therefore, in various embodiments, genetically modified animals that do not exhibit fertility or that exhibit a reduction in fertility due to modification of a nucleic acid sequence encoding an ADAM6 protein (or ortholog or homologue thereof) or a regulatory region operably linked to the nucleic acid sequence comprises a nucleic acid sequence that complements, or restores, the loss in fertility wherein the nucleic acid sequence that complements or restores the loss in fertility comes from a different strain of the same species or of a phylogenetically related species. In various embodiments, the complementary nucleic acid sequence is an ortholog or homologue of ADAM6 or functional fragment thereof. In various modalities, the ortholog or homolog of ADAM6 or functional fragment of the same complementary one comes from a non-human animal that is closely related to the genetically modified animal that has the fertility defect. For example, in cases where the genetically modified animal is a mouse of a particular strain, an ortholog or homologue of ADAM6 or functional fragment thereof can be obtained from a mouse of another strain, or from a mouse from a related species. In one modality, in cases where the genetically modified animal includes the fertility defect of the order Rodentia, the orthologue or homologue of ADAM6 or functional fragment of it comes from another animal of the order Rodentia. In one embodiment, the genetically modified animal comprising the defect of a fertility is from a Myomoropha suborder (eg, gerbils, jumping mice, mouse-like hamsters, hamsters, rats and New World mice, voles, mice and true rats, gerbils) , spiny mouse, crested rats (Lophiomys imhausi), climbing mice (Dendromus insignis), bag mice (Chaetodipus), white-tailed rats, Malagasy rats and mice, spiny dormice, mole rats, bamboo rats (Rhizomys sinensis), zokor (Myospalax aspalax)), and the ortholog or homolog of ADAM6 or functional fragment thereof is selected from an animal of the order Rodentia, or from the suborder Myomorpha.

In one embodiment, the genetically modified animal comes from the Dipodoidea superfamily, and the orthologue or homolog from ADAM6 or functional fragment thercomes from the Muroidea superfamily. In one embodiment, the genetically modified animal comes from the Muroidea superfamily, and the ortholog or homologue from ADAM6 or functional fragment thercomes from the Dipodoidea superfamily.

In one embodiment, the genetically modified animal is a rodent. In one embodiment the rodent is selected from the Muroidea superfamily, and the orthologue or homologue from ADAM6 is derived of a different species within the Muroidea superfamily. In one embodiment, the genetically modified animal comes from a family that is selected from Calomyscidae (e.g., mouse-type hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true rats and mice, gerbils, spiny mice, crestados mice), nesomyidae (climbing mice, mice bags, rats white glue, rats and malgaches mice), platacanthomyidae (spiny lirones), and Spalacidae (eg mole rats, bamboo rats, and Myospalax aspalax) ·, and the orthologue or homologue of ADAM6 is selected from a different species of the same family. In a specific embodiment, the genetically modified rodent is selected from a true mouse or rat (family Muridae), and the orthologue or homologue from ADAM6 comes from a species that is selected from a gerbil, spiny mouse, or crested rat . In one embodiment, the genetically modified mouse comes from a member of the Muridae family, and the orthologue or homologue from ADAM6 comes from a different species of the Muridae family. In a specific embodiment, the genetically modified rodent is a mouse of the family Muridae, and the orthologue or homologue of ADAM6 comes from a rat, gerbil, spiny mouse, or crested rat of the family Muridae.

In various embodiments, one or more rodent ADAM6 orthologs or homologs or functional fragments of the same as a rodent in a family restores fertility to a rodent genetically modified in the same family that lacks an ortholog or homolog ADAM6 (eg Cricetidae (eg, hamsters, rats and mice New World voles); Muridae (for example, rats and true mice, gerbils, spiny ratonesw , crested rats)).

In various embodiments, orthologs, ADAM6 homologs, and fragments therare evaluated for functionality by establishing whether the ortholog, homolog, or fragment restores or does not restore fertility to a genetically modified non-human male animal that lacks the activity of ADAM6 (for example, a rodent, for example, a mouse or rat, comprising a blockade of the expression of ADAM6 or its ortholog). In various embodiments, the functionality is defined as the ability of a sperm from a genetically modified animal lacking an endogenous or ortholog or homolog therADAM6 to migrate into the oviduct and fertilize an egg from the same species of genetically modified animal.

In various aspects, mice comprising deletions or replacements of the variable region locus of the endogenous heavy chain or portions thercontaining an ectopic nucleotide sequence encoding a protein that confers fertility benefits similar to those of ADAM6 can be made. of mouse (for example, an ortholog or a homologue or a fragment therthat is functional in a male mouse). The ectopic nucleotide sequence may include a sequence of nucleotide encoding a protein that is a homologue or ortholog of ADAM6 (or fragment ther of a different mouse strain or a different species, eg, a different rodent species, and that confers a benefit on fertility, example, increased number of litters through a specified period of time and / or increased number of litters per litter, and / or the ability of a sperm of a male mouse to travel through the oviduct of a mouse to fertilize a mouse ovule.

In one embodiment, ADAM6 is a homologue or ortholog that is at least 89% to 99% identical to a mouse ADAM6 protein (eg, 89% to 99% identical to mouse ADAM6a or mouse ADAM6b). In one embodiment, the ectopic nucleotide sequence codes for one or more proteins that are independently selected from a protein at least 89% identical to mouse ADAM6a, a protein at least 89% identical to mouse ADAM6b, and a combination of them. In one embodiment, the homologue or ortholog is a rat, hamster, mouse, or guinea pig protein that is or is modified to be approximately 89% or more identical to a mouse ADAM6a and / or mouse ADAM6b. In one embodiment, the homologue or ortholog is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a mouse ADAM6a and / or a mouse ADAM6b. In a specific embodiment, mouse ADAM6 comprises SEQ ID NO: 1 or a functional fragment thereof, and mouse ADAM6b comprises SEQ ID NO: 2 or a functional fragment thereof.

In one aspect, non-human animals are provided, wherein the non-human animals comprise (a) an insertion of one or more segments of human VL and JL gene upstream of a constant region of the non-human immunoglobulin heavy chain, (b) an insertion of one or more human VL and JL gene segments upstream of a constant region of the non-human immunoglobulin light chain, and (c) a nucleotide sequence encoding an ADAM6 protein or a functional fragment Of the same. In one embodiment, the constant regions of the heavy chain and / or non-human light chain are rodent constant regions (eg, selected from constant regions of mouse, rat or hamster). In one embodiment, the constant region of the non-human light chain is a constant region of a rodent. In a specific embodiment, the constant region of the light chain is a mouse CK region or a rat CK region. In a specific embodiment, the constant region of the light chain is a mouse CA region or a rat CK region. In one embodiment, the human VL and JL gene segments are VK and JK gene segments. In one embodiment, the human VL and JL gene segments are VA and JA gene segments. In one embodiment, the non-human animal also comprises one or more human DH gene segments present between the human VL and JL gene segments. Suitable non-human animals include rodents, for example mice, rats and hamsters. In one embodiment, the rodent is a mouse or a rat.

In one embodiment, the non-human animal comprises at least six up to at least 40 segments of the human VK gene and at least one to at least five segments of the human J K gene. In a specific embodiment, the non-human animal comprises six human VK gene segments and five human JK gene segments. In a specific embodiment, the non-human animal comprises 16 human VK gene segments and five human JK gene segments. In a specific embodiment, the non-human animal comprises 30 human VK gene segments and five human J K gene segments. In a specific embodiment, the non-human animal comprises 40 segments of human VK gene and five segments of human JK gene. In various embodiments, human JK gene segments are selected from JK1, JK2, JK3, JK4, JK5, and a combination thereof.

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 in the same publication as compared to a non-human wild-type ADAM6 locus. In one embodiment, the non-human animal is a mouse and the nucleotide sequence codes for a mouse ADAM6 protein or functional fragment thereof and is present at an ectopic location in the genome of the non-human animal. In one embodiment the non-human animal is a mouse and the nucleotide sequence codes for a mouse ADAM6 protein or functional fragment thereof and is present within the segments of the immunoglobulin gene. In a specific embodiment, the immunoglobulin gene segments are gene segments of the heavy chain of the non-human animal. In a specific embodiment, the immunoglobulin gene segments are gene segments of the light chain of another species. In one embodiment, the light chain gene segments are gene segments of the human light chain k. In one embodiment, the mouse comprises an ectopic contiguous sequence comprising one or more non-rearranged endogenous heavy chain gene segments, and the ADAM6 sequence is within the contiguous ectopic sequence.

In one embodiment, the non-human animal lacks a VL gene segment and / or a JL gene segment of endogenous immunoglobulin at a locus of the endogenous immunoglobulin light chain. In one embodiment, the non-human animal comprises endogenous immunoglobulin Vu 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 endogenous immunoglobulin VK and JK gene segments are replaced with one or more human VK and JK gene segments. In one embodiment, all or substantially all of the endogenous immunoglobulin VA and JA gene segments are deleted in whole or in part. In one embodiment, all or substantially all of the endogenous immunoglobulin VL and JL gene segments are intact in the non-human animal and the non-human animal comprises one or more segments of VK gene of human and one or more human JK gene segments inserted between endogenous immunoglobulin VL and / or JL gene segments and a constant region of the endogenous immunoglobulin light chain. In a specific embodiment, intact endogenous immunoglobulin VL and JL gene segments become incapable of rearrangement to form a VL domain of an antibody in the non-human animal, In one embodiment, the locus of the endogenous immunoglobulin light chain of the animal non-human is a locus of the light chain of immunoglobulin. In one embodiment, the endogenous immunoglobulin VL and JL gene segments are the VK and JK gene segments.

In one aspect, derived cells and / or tissues are provided from non-human animals as described in the present application, wherein the cells and / or tissues comprise (a) an insertion of one or more segments of the VK gene and JK of human upstream of a constant region of the non-human immunoglobulin light chain, (b) an insertion of one or more human VK and JK gene segments upstream of a constant region of the non-human immunoglobulin heavy chain, and (c) a nucleotide sequence that encodes an ADAM6 protein or a functional fragment thereof. In one embodiment, the constant regions of the heavy and / or non-human light chain are constant mouse regions. In one embodiment, the constant regions of the heavy and / or non-human light chain are constant rat regions. In one embodiment, the constant regions of the heavy and / or non-human light chain are constant regions of hamster.

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 in the same location as compared to a non-human wild-type ADAM6 locus. In one embodiment the non-human cell and / or tissue is derived from a mouse and the nucleotide sequence codes for a mouse ADAM6 protein or functional fragment thereof and is present at an ectopic location. In one embodiment, the non-human cell and / or tissue is derived from a mouse and the nucleotide sequence codes for a mouse ADAM6 protein or functional fragment thereof and is present within the immunoglobulin gene segments. In a specific modality, the immunoglobulin gene segments are heavy chain gene segments. In a specific embodiment, the immunoglobulin gene segments are light chain gene segments. In one embodiment, a contiguous sequence of endogenous heavy chain gene segments are placed in ectopic form in the non-human animal, in which the contiguous sequence of endogenous heavy chain gene segments placed in ectopic form comprises a ADAM6 that is functional in the mouse (for example, in a male mouse).

In one aspect, the use of a non-human animal is provided as described in the present application to make a protein from antigen binding, in which the non-human animal expresses (a) an antibody comprising (i) an immunoglobulin light chain comprising a human VK domain and a non-human light chain constant region and (ii) a chain heavy immunoglobulin comprising a human VK domain and a non-human constant region; and (b) an ADAM6 protein or functional fragment thereof. In one embodiment, the antigen-binding protein is from human. In one embodiment, the non-human animal is a rodent and the non-human constant regions are constant rodent regions. In a specific modality, the rodent is a mouse.

In one aspect, a non-human cell or tissue derived from a non-human animal is provided as described in the present application. In one embodiment, the non-human cell or tissue comprises one or more segments of the human immunoglobulin VK gene and at least one human immunoglobulin JK gene segment contiguous with a constant region gene of the immunoglobulin light chain. human and one or more human VK gene segments and one or more human JK gene segments contiguous with an immunoglobulin heavy chain constant region gene, in which the cell or tissue expresses an ADAM6 protein or functional fragment Of the same. In one embodiment, the gene of the constant region of the non-human light chain is a mouse CK.

In one embodiment, the nucleotide sequence encoding the ADAM6 protein or functional fragment thereof is ectopic.

In one embodiment, the nucleotide sequence that codes for the ADAM6 protein or functional fragment thereof is located in a position that is the same as in a non-human wild-type 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.

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

In one aspect, the use of a cell or tissue derived from a non-human animal as described in the present application is provided to make a hybridoma or quadroma.

In one aspect, there is provided a non-human cell comprising a modified genome as described in the present application, in which the non-human cell is an oocyte, a host embryo, or a fusion of a non-human animal cell as described in the present application and a cell from a different non-human animal.

In one aspect, the use of a cell or tissue derived from a non-human animal as described in the present application is provided to make a human antigen-binding protein. In one embodiment, the human antigen-binding protein comprises a human VK domain isolated from a non-human animal as described in the present application.

In one aspect, there is provided a method for making an antigen-binding protein that binds to an antigen of interest, wherein the method comprises (a) exposing a non-human animal as describes in the present application an antigen of interest, (b) isolating one or more B lymphocytes from the non-human animal, wherein said one or more B lymphocytes express a VL binding protein that binds the antigen of interest, and ( c) identifying a nucleic acid sequence encoding a VL domain of the VL binding protein that binds the antigen of interest, in which the VL binding protein comprises a human VK domain and a constant domain of the chain non-human light and a human VK domain and a non-human heavy chain constant domain, and (d) use the nucleic acid sequence of (c) with a human immunoglobulin constant region nucleic acid sequence to make a human antigen binding protein that binds the antigen of interest.

In one embodiment, the constant domain of the non-human light chain of the VL binding protein is a mouse CK. In one embodiment, the constant domain of the non-human heavy chain of the VL binding protein is a mouse Cy. In one embodiment, the non-human animal is a mouse.

In one aspect, a fertile male mouse comprising a modification in a locus of the immunoglobulin heavy chain is provided, in which the male fertile mouse comprises an ectopic ADAM6 sequence that is functional in the male mouse .

Ectopic ADAM6 at modified immunoglobulin heavy chain loci Developments in gene targeting, for example, the development of bacterial artificial chromosomes (BACs), currently allow the recombination of relatively large genomic fragments. Engineering with BAC has provided the ability to make large deletions, and large insertions, in mouse ES cells.

Mice that make human antibodies (ie, human variable regions) have been available for some time now. Although these represent an important advance in the development of human therapeutic antibodies, these mice display a number of significant abnormalities that limit their usefulness. For example, these display committed B-cell development. The compromised development may be due to a variety of differences between the transgenic mice and the wild-type mice.

Human antibodies may not interact optimally with pre-cell B or mouse B cell receptors on the surface of mouse cells that signal for maturation, proliferation, or survival during clonal selection. Fully human antibodies may not interact optimally with a mouse Fe receptor system; mice express Fe receptors that do not display a one-to-one correspondence with human Fe receptors. Finally, several mice that elaborate fully human antibodies do not include all genuine mouse sequences, for example, enhancer elements towards the 3 'end and other control elements of the locus, which might be necessary for the development of wild-type B cell.

Mice that make fully human antibodies usually comprise endogenous immunoglobulin loci that are somehow disabled, and human transgenes comprising variable gene segments and immunoglobulin constants are introduced at a random location in the mouse genome. As long as the endogenous locus is sufficiently disabled so as not to rearrange the gene segments to form a functional immunoglobulin gene, the goal of making fully human antibodies in said mouse can be achieved - although with committed development of B cell.

Although bound to make fully human antibodies from the locus of the human transgene, generating human antibodies in a mouse is apparently a disadvantaged process. In some mice, the process is so disadvantaged that it results in the formation of variable human heavy chains / chimeric mouse constant (but not light chains) through the trans switching mechanism. Through this mechanism, the transcripts that code for fully human antibodies undergo isotype switching in trans from the human isotype to a mouse isotype. The process is in trans, because the completely human transgene is located separately from the locus endogenous that retains an undamaged copy of a gene of the constant region of the mouse heavy chain. Although in these mice the commutation in trans is easily evident, the phenomenon remains insufficient to rescue the development of cell B, which remains frankly damaged. In any case, the trans-switched antibodies made in said mice retain completely human light chains, because the phenomenon of switching in trans apparently does not occur with respect to the light chains; trans switching presumably is based on switching the sequences in the endogenous loci used (albeit in different form) in the normal isotype switching in cis. Therefore, even when mice genetically engineered to make fully human antibodies select a trans switching mechanism to make antibodies with constant mouse regions, the strategy remains insufficient to rescue normal B cell development.

A primary concern in the development of antibody-based human therapeutic agents is to make a sufficiently large diversity of human immunoglobulin variable region sequences to identify useful variable domains that specifically recognize particular epitopes and link them with a desirable affinity, usually - but not always - with high affinity. Prior to the development of VELOCIMMUNE® mice (described in the present application), there was no indication that mice expressing variable regions of human with constant mouse regions could exhibit any significant differences of the mice that make human antibodies from a transgene. That assumption, however, was incorrect.

VELOCIMMUNE® mice, which contain a precise replacement of variable regions of mouse immunoglobulin with variable regions of human immunoglobulin at the endogenous loci, display a striking and remarkable similarity to wild-type mice with respect to the development of cell B. In a surprising and impressive development, the VELOCIMMUNE® mice showed a wild-type response, essentially normal towards immunization that differed only in a significant aspect of the wild-type mice - the variable regions generated in response to the immunization are completely human.

VELOCIMMUNE® mice contain a large-scale, precise replacement of germline variable regions of the mouse immunoglobulin heavy chain (IgH) and the immunoglobulin light chain (eg, light chain k, I g K) with regions corresponding human immunoglobulin variables, at the endogenous loci. In total, approximately six megabases of mouse loci are replaced with approximately 1.5 megabases of human genomic sequence. This precise replacement results in a mouse with hybrid immunoglobulin loci that elaborates heavy and light chains that have variable regions of human and a constant mouse region. The precise replacement of mouse segments VH-DH-JH and VK-JK leaves the flanking mouse sequences intact and functional in hybrid immunoglobulin loci. The mouse humoral immune system works like that of a wild-type mouse. The development of cell B is not impeded in any significant aspect and a rich diversity of variable human regions in the mouse is generated after challenge with antigen.

VELOCIMMUNE® mice are possible because the immunoglobulin gene segments for the heavy and light chains k are similarly rearranged in humans and mice, which does not mean that their loci are the same or even close-clearly these they are not. However, the loci are sufficiently similar that the humanization of the locus of the variable gene of the heavy chain can be achieved by replacing approximately three million base pairs of the contiguous mouse sequence containing all the segments of the VH gene, DH, and JH with approximately one million bases of the contiguous human genomic sequence that basically covers the equivalent sequence of a human immunoglobulin locus.

In some embodiments, the additional replacement of certain mouse constant region gene sequences with human gene sequences (eg, the replacement of the mouse CH1 sequence with the human CH1 sequence, and the replacement of the CL sequence of mouse with the human CL sequence) results in mice with hybrid immunoglobulin loci that make antibodies having variable regions of human and partially human constant regions, suitable for, eg, making fully human antibody fragments, for example, Fab's completely human. 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 is indistinguishable from that of wild type mice, and display normal cell populations at all stages of normal B cell development and lymphoid organ structures - even in cases where the mice lack a Complete repertoire of gene segments from the human variable region. Immunization of these mice results in robust humoral responses that display a wide diversity of use of variable gene segments.

The precise replacement of gene segments of the mouse germline variable region allows to make mice that have partially human immunoglobulin loci. Because partially human immunoglobulin loci rearrange, hypermute, and commute normally, the partially human immunoglobulin loci generate antibodies in a mouse that comprise variable regions of the human. The nucleotide sequences encoding the variable regions can be identified and cloned, then fused (e.g., in an in vitro system) with any sequences of choice, e.g., any isotype of immunoglobulin appropriate for a particular use, which results in an antibody or antigen-binding protein derived entirely from human sequences.

Humanization on a large scale using methods of Recombination engineering is used to modify mouse embryonic stem (ES) cells to create unique immunoglobulin heavy chain loci by accurately replacing up to three megabases of the immunoglobulin locus of the mouse heavy chain that includes essentially all gene segments VH, DH, and mouse JH with up to a half-megabase segment of the human genome comprising one of two repeats that encode essentially all segments of the human VK and JK gene. Additionally, a segment of up to half a megabase of the human genome is used which comprises one of two repeats that encode essentially all of the human VK and JK gene segments to replace a three megabase segment of the light chain locus k mouse immunoglobulin containing essentially all mouse VK and JK gene segments. Mice with said replaced immunoglobulin loci may comprise an alteration or deletion of the mouse ADAM6 locus, which is normally located between the VH gene segment more towards the 3 'end and the DH gene segment more towards the 5' end. in the locus of the mouse immunoglobulin heavy chain. Alteration in this region can lead to reduction or elimination of functionality of the mouse ADAM6 locus.

Described are mice comprising the loci replaced as described above, and also comprising an ectopic nucleic acid sequence encoding a mouse ADAM6, in which the mice exhibit essentially normal fertility.

In one embodiment, the ectopic nucleic acid sequence is placed between a human VL gene segment and a human JL gene segment or upstream of a human VL gene segment further towards the 5 'end at the locus of the modified endogenous heavy chain. The transcription direction of the ADAM6 genes may be opposite (Figure 7) or the same (Figure 8) with respect to the transcription direction of the surrounding human VL gene segments. Although the examples in the present application show the fertility rescue by placing the ectopic sequence between the indicated human VL and JL gene segments or upstream of a human VL gene segment further towards the 5 'end, the experts in the art they will recognize that the placement of the ectopic sequence at any permissive locus from the point of view of appropriate transcription in the mouse genome (or even extrachromosomally) could be expected to similarly restore fertility in a male mouse. In various embodiments, the ectopic nucleic acid sequence is selected from SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, in which the ectopic sequence codes for one or more ADAM6 proteins, in which said one or more ADAM6 proteins comprise SEQ ID NO: 1, SEQ ID NO: 2 or a combination thereof.

The phenomenon of supplementing a mouse lacking a functional ADAM6 locus with an ectopic sequence comprising a mouse ADAM6 gene or ortholog or homologue or functional fragment thereof is a general method that can be applied to rescue any mice with loci. of non-functional endogenous ADAM6 or minimally functional Therefore, a large number of mice comprising an ADAM6-altering modification of the immunoglobulin heavy chain locus can be rescued with the compositions and methods of the invention. Accordingly, the invention comprises mice with a wide variety of modifications of the immunoglobulin heavy chain loci that compromise the function of endogenous ADAM6. In this description some examples are provided (not limiting). In addition to the mice described, compositions and methods related to ADAM6 can be used in many applications, for example, when a locus of the heavy chain is modified in a wide variety of ways.

In one aspect, there is provided a mouse comprising an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or ortholog or homologue or functional fragment thereof), a replacement of all or substantially all segments of mouse VH gene with one or more human VL gene segments, a replacement of all or substantially all of the DH gene segments and mouse JH gene segments with human JL gene segments; in which the mouse lacks a CH 1 region and / or hinge region. In one embodiment, the mouse makes an individual variable domain binding protein that is a dimer of the immunoglobulin chains selected from: (a) human VL-mouse CH1-mouse CH2-mouse CH3; (b) Human VL - mouse hinge - mouse CH2 - mouse CH3; and, (c) human VL - mouse CH2 - mouse CH3.

In one aspect, the nucleotide sequence of rescuing the Fertility is placed within a variable region sequence of the human immunoglobulin light chain (e.g., between segments of human VK4-1 and JK1 gene) in a mouse that has a replacement of all or substantially all segments of variable gene of the mouse immunoglobulin heavy chain (mVH's, mDH's, and mJH's) with one or more with one or more variable gene segments of the human immunoglobulin light chain k (hVx's and hJK's), and the mouse comprises in addition a replacement of all or substantially all segments of the variable of the mouse immunoglobulin light chain k (mVK's, mJK's) with one or more variable gene segments of the human immunoglobulin light chain k (hVK's and hJK's).

In one aspect, a functional mouse ADAM6 (or orthologous or homologous or functional fragment thereof) locus can be placed in the middle of human VL gene segments or upstream of a human VL gene segment more towards the 5 'end, in which human VL gene segments replace the endogenous VH gene segments. In one embodiment, all or substantially all mouse VH gene segments are deleted and replaced with one or more human VL gene segments, and the mouse ADAM6 locus is placed immediately adjacent to the 5 'end of the gene segments. VL of human more towards the 5 'end, or between two segments of human VL gene. In a specific embodiment, the mouse ADAM6 locus is positioned between two VL gene segments near the 3 'terminal end of the inserted human VL gene segments. In a modality Specifically, the arrangement of the human VL gene segments is then the following (from the 5 'end to the 3' end with respect to the transcription direction of the human VL gene segments): Human VK5-2 locus of mouse ADAM6 - human VK4-1. In a specific embodiment, the arrangement of the human VL gene segments is then the following (from the 5 'end to the 3' end with respect to the transcription direction of the human VL gene segments): ADAM6 locus of mouse - VK2-40 of human, wherein human VK2-40 is the human VL gene segment further towards the 5 'end at the modified immunoglobulin heavy chain locus. In one embodiment, the orientation of one or more of mouse ADAM6b ADAM6b ADAM6b from the mouse ADAM6 locus is opposite with respect to the transcription direction compared to the orientation of the human VL gene segments. In one embodiment, the orientation of one or more of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is the same with respect to the transcription direction as compared to the orientation of human VL gene segments.

In one aspect, a functional mouse ADAM6 (or orthologous or homologous or functional fragment thereof) locus can be placed between a human VL gene segment and a human JL gene segment (i.e. ntergenics between the human VL gene segment towards the 3 rd end and the JL gene segment towards the 5 'end), in which the human VL and JL gene segments replace the endogenous V H gene segments. In one modality, all or substantially all mouse VH gene segments are deleted and replaced with one or more human VL gene segments and one or more segments of the human JL gene, and the mouse ADAM6 locus is placed immediately adjacent to the human end. 'of the human VL gene segment more towards a 3' end and immediately adjacent to the 5 'end of the human JL gene segment more towards the 5' end. In a specific embodiment, said one or more human VL gene segments and one or more human JL gene segments are the VK and JK gene segments. In a specific embodiment, the arrangement of the human VL gene segments is then the following (from the 5 'end to the 3' end with respect to the transcription direction of the human VL gene segments): VK4-1 of human-locus of mouse ADAM6-human JK1. In one embodiment, the orientation of one or more of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is opposite to the direction of transcription compared to the orientation of human VL gene segments. In one embodiment, the orientation of one or more of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is the same with respect to the transcription direction as compared to the orientation of human VL gene segments.

A modified mouse with one or more human VL gene segments (eg, VK or VA segments) that replace all or substantially all of the endogenous VH gene segments can be modified either to maintain the endogenous ADAM6 locus, as described earlier, for example, using a vector for choice of target having a homology arm towards the 3 'end that includes a locus of mouse ADAM6 or functional fragment thereof, or to replace a damaged ADA 6 locus of mouse with an ectopic sequence positioned between two VL gene segments of human or between the human VL gene segments and a DH gene segment (either human or mouse, eg, VA + m / hDH), or a segment of the J gene (either human or mouse) , for example, VK + JH). In one embodiment, the replacement includes two or more human VL gene segments, and the mouse ADAM6 locus or functional fragment thereof is positioned between the two VL gene segments most towards the 3 'end. In a specific embodiment, the arrangement of the human VL gene segments is then the following (from the 5 'end to the 3' end with respect to the transcription direction of the human gene segments); VL3'-1 from human - mouse ADAM6-VL3 'locus of human. In one embodiment, the orientation of one or more of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is opposite to the direction of transcription compared to the orientation of human VL gene segments. Alternatively, the mouse ADAM6 locus can be placed in the intergenic region between the human VL gene segment more towards the 3 'end and the JL gene segment more towards the 5' end.

In one aspect, a mouse is provided with a replacement of one or more endogenous VH gene segments, and comprising at least one endogenous DH gene segment. In said mouse, the modification of the endogenous VH gene segments may comprise a modification of one or more of the VH gene segments more towards the 3 'end, but not the DH gene segment further towards the 5' end, where care is taken so that the modification of said one or more VH gene segments more towards the 3 'end does not alter or make the locus of non-functional endogenous ADAM6. For example, in one embodiment the mouse comprises a replacement of all or substantially all of the endogenous VH gene segments with one or more human VL gene segments, and the mouse comprises one or more endogenous DH gene segments and an ADAM6 locus endogenous functional.

In another embodiment, the mouse comprises the modification of VH gene segments more towards the endogenous 3 'end, and a modification of one or more endogenous DH gene segments, and the modification is carried out in order to maintain the integrity of the locus of endogenous ADAM6 to the extent that the endogenous ADAM6 locus remains functional. In one example, such modification is effected in two steps: (1) replacing the VH gene segments more towards the endogenous 3 'end with one or more human VL gene segments using a target vector with a homology arm towards the 5 'end and a homology arm towards the 3' end in which the homology arm towards the 3 'end includes all or a portion of a functional locus of mouse ADAM6; (2) then replacing an endogenous DH gene segment with a target vector having a 5 'homology arm that includes all or a functional portion of a mouse ADM6 locus.

In various aspects, use mice that contain a Ectopic sequence encoding a mouse ADAM6 protein or an ortholog or homologue or functional homologue thereof are useful in cases where the modifications alter the function of mouse endogenous ADAM6. The likelihood of altering the function of mouse endogenous ADAM6 is high when modifications are made to the mouse immunoglobulin loci, in particular when the variable regions of the mouse immunoglobulin heavy chain and the surrounding sequences are modified. Therefore, said mice provide particular benefit when making mice with immunoglobulin heavy chain loci that are deleted in whole or in part, are fully or partially humanized, or are replaced (for example with VKO sequences). VA) in whole or in part. The methods for making the described genetic modifications for the mice described below are known to those skilled in the art.

Mice that contain an ectopic sequence encoding a mouse ADAM6 protein, or a substantially identical or similar protein that confers the fertility benefits of a mouse ADAM6 protein, are particularly useful in conjunction with modifications to a variable gene locus of the mouse immunoglobulin heavy chain that alter or eliminate the endogenous ADAM6 sequence. Although they are described primarily in connection with mice expressing antibodies with variable regions of human and mouse constant regions, said mice are useful in connection with any genetic modifications that alter the genes of Endogenous ADAM6. Those skilled in the art will recognize that this covers a wide variety of genetically modified mice that contain modifications of the variable gene loci of the mouse immunoglobulin heavy chain. These include, for example, mice with a deletion or replacement of all or a portion of the mouse immunoglobulin heavy chain gene segments, independently of other modifications. The non-limiting examples are described below.

In some aspects, genetically modified mice comprising a mouse ectopic gene, rodent, or another ADAM6 gene (or ortholog or homologue or fragment) functional in a mouse, and one or more gene segments of the variable region and / are provided. or human immunoglobulin constant. In various embodiments, other orthologs or homologs or fragments of functional ADAM6 gene in a mouse may include sequences from bovine, canine, primate, rabbit or other non-human sequences.

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 mouse VH gene segments with one or more segments of human VL gene; a replacement of all or substantially all segments of the mouse DH and JH gene with one or more human JL gene segments.

In one embodiment, the mouse further comprises a replacement of a mouse CH1 nucleotide sequence with a nucleotide sequence of human CR 1. 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 locus of the immunoglobulin light chain (VL and JL) with a variable locus of the human immunoglobulin light chain. In one embodiment, the mouse further comprises a replacement of a nucleotide sequence of the constant region of the mouse immunoglobulin light chain with a nucleotide sequence of the human immunoglobulin light chain constant region. In a specific embodiment, the V_, J L, and CL are sequences of the immunoglobulin light chain k. In a specific embodiment, the mouse comprises a sequence of the mouse CH2 immunoglobulin constant region and a mouse CH3 fused to a human hinge sequence and a human CH1 sequence, such that the immunoglobulin loci of human mouse are rearranged to form a gene encoding a binding protein comprising (a) a heavy chain having a variable region of human, a human CH1 region, a human hinge region, and a mouse CH2 region and a mouse CH3 region; and (b) a gene encoding an immunoglobulin light chain comprising a human variable domain and a human constant region.

In one aspect, a mouse comprising an ectopic ADAM6 sequence encoding an ADAM6 protein is provided. functional, a replacement of all or substantially all mouse VH gene segments with one or more human Vu gene segments, and optionally a replacement of all or substantially all of the DH gene segments and / or JH gene segments with one or more human DH gene segments and / or human JH gene segments, or optionally a replacement of all or substantially all of the DH gene segments and JH gene segments with one or more human JL gene segments.

In one embodiment, the mouse comprises a replacement of all or substantially all of the mouse VH, DH, and JH gene segments with one or more VL gene segments, one or more DH gene segments, and one or more gene segments J (e.g., JK OR JA), in which the gene segments are operably linked to a mouse hinge region, in which the mouse forms a 5'-containing, rearranged immunoglobulin chain gene. towards 3 'in the transcription direction, VL of human - DH of human or mouse - J of human or mouse - hinge of mouse - CH2 of mouse - CH3 of mouse. In one embodiment, region J is a human JK region. In one embodiment, the J region is a human JH region. In one embodiment, region J is a human JA region. In one embodiment, the human VL region is selected from a human VA region and a human VK region.

In specific embodiments, the mouse expresses an individual variable domain antibody having a mouse or human constant region and a variable region derived from starting from a VK of human, a DH of human and a J K of human; a human VK, a human DH, and a human JH; a VA of human, a DH of human, and a JA of human; a human VA, a human DH, and a human JH; a human VK, a human DH, and a human JA; a VA of human, a DH of human, and a JK of human. In specific mode, the recombination recognition sequences are modified to allow productive rearrangements to occur between the recited V, D, and J gene segments or between the recited V and J gene segments.

In one aspect, there is provided a mouse comprising an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or ortholog or homologue or functional fragment thereof), a replacement of all or substantially all segments of mouse VH gene with one or more human VL gene segments, a replacement of all or substantially all of the DH gene segments and the mouse JH gene segments with human JL gene segments; in which the mouse lacks a region of CH1 and / or hinge.

In one embodiment, the mouse lacks a sequence encoding a CH1 domain - In one embodiment, the mouse lacks a sequence encoding a hinge region. In one embodiment, the mouse lacks a sequence coding for a CH1 domain and a hinge region.

In a specific embodiment, the mouse expresses a binding protein comprising a variable domain of the chain of human immunoglobulin (I or K) fused to a mouse CH2 domain that is linked to a mouse CH3 domain.

In one aspect, there is provided a mouse comprising an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or ortholog or homologue or functional fragment thereof), a replacement of all or substantially all segments of mouse VH gene with one or more human VL gene segments, a replacement of all or substantially all segments of the mouse DH and JH gene with segments of the human JL gene.

In one embodiment, the mouse comprises a deletion of a gene sequence of the immunoglobulin heavy chain constant region encoding a CH1 region, a hinge region, a CH1 region and a hinge region, or a CH1 region and a hinge region and a CH2 region.

In one embodiment, the mouse makes an individual variable domain binding protein comprising a homodimer that is selected from the following: (a) human VL-mouse CH1-mouse CH2-mouse CH3; (b) Human VL - mouse hinge - mouse CH2 - mouse CH3; (c) Human VL - Mouse CH2 - Mouse CH3.

In one aspect, a non-human animal is provided, comprising a locus of the modified immunoglobulin heavy chain, in which the modified immunoglobulin heavy chain locus comprises a sequence of non-human ADAM6 or ortholog or homologue thereof.

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

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

In one embodiment, the non-human animal is selected from a mouse, a rat, and a hamster and the ortholog or homologue of ADAM6 is from a non-human animal that is selected from a mouse, a rat, and a hamster . In a specific embodiment, the non-human animal is a mouse and the ortholog or homologue of ADAM6 is from an animal that is selected from a different mouse species, a rat, and a hamster. In specific embodiment, the non-human animal is a rat, and the ortholog or homologue of ADAM6 is from a rodent that is selected from a different rat species, a mouse, and a hamster. In a specific embodiment, the non-human animal is a hamster, and the ortholog or homologue of ADAM6 is from a rodent that is selected from a different species of hamster, a mouse, and a rat.

In a specific embodiment, the non-human animal is from the Myomorpha suborder, and the ADAM6 sequence is from an animal that is selected from a rodent of the Dipodoidea superfamily and a rodent from the Muroidea superfamily. In a specific embodiment, the rodent is a mouse of the Muroidea superfamily, and the orthologue or homologue of ADAM6 is of a mouse or a rat or a hamster of the Muroidea superfamily.

In one embodiment, the modified immunoglobulin heavy chain locus comprises one or more human VL gene segments and one or more human JL gene segments. In a specific embodiment, said one or more human VL gene segments and one or more human J gene segments are operably linked to one or more genes of the human, chimeric and / or rodent constant region (eg. example, mouse or rat). In one embodiment, the genes of the constant region are mouse. In one embodiment, the genes of the constant region are rat. In one embodiment, the genes of the constant region are hamster. In one embodiment, the genes of the constant region comprise a sequence that is selected from a hinge, a CH2, a CH3, and a combination thereof. In specific mode, the genes of the constant region comprise a hinge sequence, one of CH2, and one of CH3. In one embodiment, the human VL and JL gene segments are human VK and J K gene segments.

In one embodiment, the non-human ADAM6 sequence is contiguous with a sequence of the human immunoglobulin light chain. In one embodiment, the non-human ADAM6 sequence is positioned within a sequence of the human immunoglobulin light chain. In a specific embodiment, the human immunoglobulin light chain sequence comprises a segment of V and / or J gene.

In one modality, the non-human ADAM6 sequence is juxtaposed with a segment of gene V. In one embodiment, the sequence of non-human ADAM6 is positioned between two segments of gene V. In one embodiment, the sequence of non-human ADAM6 is juxtaposed between a segment of V gene and a segment of J. In one embodiment, the mouse ADAM6 sequence is juxtaposed between two segments of gene J.

In one aspect, a genetically modified non-human animal is provided, comprising a B cell expressing a VL domain of human cognate with a human VL domain of an immunoglobulin locus, in which the non-human animal expresses a non-human protein non-immunoglobulin type from the immunoglobulin locus. In one embodiment, the non-immunoglobulin type non-human protein is an ADAM protein. In a specific embodiment, the ADAM protein is an ADAM6 protein or homologous or ortholog or functional fragment thereof.

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

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

In one embodiment, human VL domains are linked directly or through a linker to an immunoglobulin constant domain sequence. In a specific embodiment, the sequence of the constant domain comprises a sequence that is selected from a hinge, a CH2, a CH3, and a combination thereof. In a specific embodiment, the human VL domain is selected from a VK domain OR a VA domain.

In various embodiments, human VL domains are human VK domains.

In one aspect, a genetically modified non-human animal is provided, which comprises in its germ line a human immunoglobulin sequence, in which the sperm of a male non-human animal is characterized by an in vivo migration defect. In one embodiment, the in vivo migration defect comprises a disability of the sperm of the male non-human animal to migrate from a uterus through an oviduct of a female non-human animal of the same species. In one embodiment, the non-human animal lacks a nucleotide sequence encoding an ADAM6 protein or functional fragment thereof. In a specific embodiment, the ADAM6 protein or functional fragment thereof includes an ADAM6a protein and / or an ADAM6b protein or functional fragments thereof. In one embodiment, the non-human animal is a rodent. In a specific modality, the rodent is selected to from a mouse, a rat, and a hamster.

In one aspect, a non-human animal is provided, comprising a human immunoglobulin sequence contiguous with a non-human sequence encoding an ADAM6 protein or ortholog or homologue 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.

In one embodiment, the human immunoglobulin sequence is a sequence of the immunoglobulin light chain. In one embodiment, the immunoglobulin sequence comprises one or more VL gene segments. In one embodiment, the human immunoglobulin sequence comprises one or more segments of the Ju gene. In one embodiment, the human immunoglobulin sequence comprises one or more segments of the VL gene and one or more segments of the JL gene. In various embodiments, the human Vu and Ju segments are the VK and JK gene segments.

In one aspect, a mouse is provided with a locus of the disabled endogenous immunoglobulin heavy chain, comprising a locus of endogenous ADAM6 disabled or deleted, in which the mouse comprises a nucleic acid sequence expressing an antibody of human or mouse or human / mouse or other chimeric antibody. In one embodiment, the nucleic acid sequence is present in an integrated transgene that is randomly integrated into the mouse genome. In one modality, the The nucleic acid sequence is in an episome (eg, a chromosome) not found in a wild-type mouse.

In one aspect, a mouse is provided with a locus of the disabled endogenous immunoglobulin heavy chain, comprising a functional endogenous ADA 6 locus, in which the mouse comprises a nucleic acid sequence expressing a human or mouse antibody. or from human / mouse or other chimeric antibody. In one embodiment, the nucleic acid sequence is present at the locus of the endogenous immunoglobulin heavy chain in an upstream position of one or more endogenous heavy chain constant region genes. In one embodiment, the nucleic acid sequence is present in an integrated transgene that 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.

Bispecific binding proteins The binding proteins described in the present application, and the nucleotide sequences that encode them, can be used to make multispecific binding proteins, for example, bispecific binding proteins. In this regard, a first polypeptide consisting essentially of a first VL domain fused to a CH region can be associated with a second polypeptide consisting essentially of a second VL domain. merged with a CH region. In cases where the first VL domain and the second VL domain specifically bind a different epitope, a bispecific binding molecule can be made using the two VL domains. The CH region may be the same or it may be different. In one embodiment, for example, one of the CH regions can be modified to eliminate a protein A binding determinant, while the other constant region of the heavy chain is not modified. This particular arrangement simplifies the isolation of the bispecific binding protein from, for example, a mixture of homodimers (for example, homodimers of the first or second polypeptides).

In one aspect, the methods and compositions described in the present application are used to make bispecific binding proteins. In this regard, a first VL that is fused to a CH region and a second VL that is fused to a CH region are each cloned independently in frame with a human IgG sequence of the same isotype (e.g. gG 1, IgG2, IgG3, or IgG4 from human). The first VL specifically binds a first epitope, and the second VL specifically binds a second epitope. The first and second epitopes can be on different antigens, or on the same antigen.

In one embodiment, the IgG isotype of the CH region fused to the first VL and the IgG isotype of the CH region fused to the second VL are the same isotype, but differ in that an isotype of IgG comprises at least one amino acid substitution. In a embodiment, said at least one amino acid substitution makes the heavy chain bearing the substitution incapable or substantially incapable of binding protein A compared to the heavy chain lacking the substitution.

In one embodiment, the first CH region comprises a first CH3 domain of a human IgG that is selected from IgG 1, IgG2, and IgG4; and the second CH region comprises a second CH3 domain of a human IgG which is selected from I g G1, IgG2, and IgG4, wherein the second CH3 domain comprises a modification that reduces or eliminates the binding of the second CH3 domain to protein A.

In one embodiment, the second domain CH3 comprises a modification 435R, numbered according to the Kabat UE index. In another embodiment, the second domain CH3 further comprises a modification 436F, numbered in accordance with the Kabat UE index.

In one embodiment, the second CH3 domain is that of a human IgG1 comprising a modification that is selected from the group consisting of D356E, L358M, N384S, K392N, V397M, and V4221, numbered according to the EU index of Kabat.

In one embodiment, the second CH3 domain is that of a human IgG2 comprising a modification that is selected from the group consisting of N384S, K392N, and V4221, numbered according to the Kabat EU index.

In one embodiment, the second CH3 domain is that of a human IgG4 comprising a modification that is selected from the group consisting of Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221, numbered according to the index Kabat EU.

In one embodiment, the binding protein comprises CH regions having one or more modifications as recited in the present application, in which the constant region of the binding protein is non-immunogenic or substantially non-immunogenic in a human. In a specific embodiment, the CH regions comprise amino acid sequences that do not have an immunogenic epitope in a human. In another specific embodiment, the binding protein comprises a CH region that is not found in a wild type human heavy chain, and the CH region does not comprise a sequence that generates a T cell epitope.

EXAMPLES The following examples are provided to describe how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors consider to be their invention. Unless stated otherwise, the temperature is indicated in degrees Celsius, and the pressure is or is close to atmospheric pressure.

EXAMPLE 1 Introduction of gene segments of the human light chain at a locus of the heavy chain Several constructions are made for choice of target using genetic engineering technology VELOCIGENE® (see, for example, US Patent No. 6,586,251 and Valenzuela et al. (2003), High-throughput engineering of the mouse, genome coupled with high-resolution expression analysis , Nat Biotechnol 21: 652-659) to modify the genomic libraries of the Bacterial Artificial Chromosome (BAC) of mouse genomes. Mouse BAC DNA is modified by homologous recombination to inactivate the endogenous heavy chain locus through targeted deletion of VH, DH and JH gene segments for subsequent insertion of light chain k-line gene sequences Human germline not rearranged (for example, see upper part of Figure 2).

Briefly, the locus of the mouse heavy chain is removed in two successive target selection events using recombinase-mediated recombination. The first target selection event includes an address at the 5 'end of the mouse heavy chain locus using a target vector comprising from 5' to 3 'a mouse 5' homology arm, a site of recombinase recognition, a neomycin cassette and a 3 'homology arm. The arms of 5 'homology and 3 'contain the 5' sequence of the mouse heavy chain locus. The second event for target selection includes an address at the 3 'end of the mouse heavy chain locus in the region of the JH gene segments using a second target vector containing from 5' to 3 'an arm of 5 'mouse homology, a 5' recombinase recognition site, a second recombinase recognition site, a hygromycin cassette, a third recombinase recognition site, and a 3 'homology arm of mouse. The arms of 5 'and 3' homology contain the sequence flanking the mouse JH gene and 5 'segments of the intronic enhancer and constant regions. ES positive cells containing a locus of the modified heavy chain targeted with both vectors for target selection (as described above) are confirmed by karyotype determination. The DNA is then isolated from the doubly chosen ES cells as target and subjected to treatment with a recombinase thereby mediating the deletion of the genomic DNA from the mouse heavy chain locus between the 5 'recombinase recognition site in the the first vector for choice of target and the 5 'recombinase recognition site in the second vector for choice of target, leaving a single recombinase recognition site and the hygromycin cassette flanked by two recombinase recognition sites (upper part of Figure 2). Therefore a locus of the modified mouse heavy chain containing intact CH genes is created for Progressively insert human germline k gene segments in a precise manner using target vectors described below.

Four separate target vectors are designed to progressively insert 40 human VK gene segments and five human JK gene segments at the locus of the mouse inactivated heavy chain (described above) using standard molecular techniques recognized in the field (Figure 2). The human k-gene segments used to design the four constructs for target selection are found naturally in the proximal proximal locus of the human germline light chain k (bottom of Figure 1 and Table 1).

A human genomic fragment of ~ 10,499 bp containing the first six segments of human VK gene and five segments of human JK gene is designed to contain a Pl-Scel 431 bp site towards the 3 'end (3') of the human JK5 gene segment. Another Pl-Scel site is designed at the 5 'end of a ~7,852 bp genomic fragment containing the mouse heavy chain intron enhancer, the IgM switching region (Sp) and the IgM gene of the locus of the heavy mouse chain. This mouse fragment is used as a 3 'homology arm by ligation to the human fragment of ~ 10.5 kb, which creates a 3' splice containing, from 5 'to 3', ~ 110.5 kb of locus genomic sequence of the light chain k of human that contains the first six consecutive VK gene segments and five JK gene segments, one Pl-Scel site, -7.852 bp of the mouse heavy chain sequence containing the mouse intronic enhancer, Sp and the mouse IgM constant gene . Towards the 5 'end (5') from the human VK1 -6 gene segment there is an additional 3,710 pb of human k-sequence before the start of the mouse 5 'homology arm, which contains 19,752 bp of mouse genomic DNA corresponding to the 5 'sequence of the locus of the mouse heavy chain. Between the 5 'homology arm and the start of the human k-sequence there is a neomycin cassette flanked by three recombinase recognition sites (see Vector for Choice of White 1, Figure 2). The vector for final target selection for the first insertion of human sequence k from 5 'to 3' includes a 5 'homology arm containing -20 kb of 5' mouse genomic sequence of the heavy chain locus, a first recombinase recognition site (R1), a neomycin cassette, a second recombinase recognition site (R2), a third recombinase recognition site (R3), -110.5 kb human genomic sequence k containing the first six consecutive human VK gene segments and five human JK gene segments, a Pl-Scel site, and a 3 'homology arm containing -8 kb of mouse genomic sequence including the intronic enhancer, Sp and the constant gene of Mouse IgM (Figure 2, Vector for Choice of White 1). Homologous recombination with this vector for choice of target creates a locus of the heavy chain of modified mouse containing six human VK gene segments and five human JK gene segments operably linked to the endogenous heavy chain constant genes which, after recombination, leads to the formation of a hybrid heavy chain ( that is, a human VK domain and a mouse CH region).

TABLE 1 Introduction of ten additional human VK gene segments at a locus of the chain to hybrid A second vector for target selection is designed for the introduction of 10 additional human VK gene segments at the locus of the modified mouse heavy chain described above (see Figure 2, Vector for Choice of White 2). A 140,058 bp human genomic fragment containing 12 consecutive human VK gene segments from the locus of the human light chain k is designed with a 5 'homology arm containing the 5' mouse genomic sequence of the human locus. mouse heavy chain and a 3 'homology arm containing human genomic k sequence. Towards the 5 'end (5') from the human VK1 -16 gene segment there are 10, 170 additional pb of human sequence k before the start of the mouse 5 'homology arm, which is the same arm of 5' homology used for the construction of the Vector for Choice of Target 1 (Figure 2). Between the 5 'homology arm and the start of the human k-sequence there is a hygromycin cassette flanked by recombinase recognition sites. The 3 'homology arm includes an overlap of 31, 165 bp of human genomic VK sequence corresponding to the 5' equivalent end of the ~ 10.5 kb fragment of the human genomic k sequence of the Vector for Choice of White 1 (Figure 2 ). The vector for final target selection for the insertion of 10 additional human VK gene segments, from 5 'to 3', includes a 5 'homology arm containing ~ 20 kb of mouse 5' genomic sequence from the locus of the heavy chain, a first recombinase recognition site (R1), a hygromycin cassette, a second recombinase recognition site (R2) and ~ 140 kb human genomic sequence containing 12 consecutive human VA gene segments, of which ~ 31 kb they overlap with the 5 'end of the human sequence k of the Vector for Choice of White 1 and serve as the 3' homology arm for this construction for target selection. Homologous recombination with this vector for choice of target creates a locus of the modified mouse heavy chain containing 16 human VK gene segments and five human JK gene segments operably linked to the constant genes of the heavy chain of mouse which, after recombination, leads to the formation of a hybrid heavy chain.

Introduction of fourteen additional human VK gene segments at a locus of the hybrid heavy chain A third vector for target selection is designed for the introduction of 14 additional human VK gene segments to the modified mouse heavy chain locus described above (Figure 2, Vector for Choice of Target 3). A human 160,579 bp genomic fragment containing 15 consecutive human VK gene segments with a 5 'homology arm containing the 5' mouse genomic sequence of the mouse heavy chain locus and a homology arm is designed. 'which contains human genomic k sequence. Towards the 5 'end (5') from the human VK2-30 gene segment there are 14,687 bp of additional human k sequence before the start of the mouse 5 'homology arm, which is the same 5' homology arm used for the two vectors for previous white choice (described above, see also Figure 2). Between the 5 'homology arm and the start of the human k-sequence there is a neomycin cassette flanked by recombinase recognition sites. The 3 'homology arm includes an overlap of 21,275 bp of human genomic sequence k corresponding to the 5' end of the ~ 140 kb fragment of the human genomic k sequence of the Vector for Choice of White 2 (Figure 20) . The vector for final target selection for insertion of 14 additional human VK gene segments, from 5 'to 3' includes a 5 'homology arm containing ~ 20 kb of 5' mouse genomic sequence of the chain locus mouse heavy, a first recombinase recognition site (R1), a neomycin cassette, a second recombinase recognition site (R2) and ~ 161 kb human genomic k sequence containing 15 human VK gene segments, of which ~ 21 kb overlap with the 5 'end of the human sequence k of the Vector for Choice of White 2 and serve as the homology arm 3' for this construction for choice of target. Homologous recombination with this vector for choice of target creates a locus of the modified mouse heavy chain containing 30 human VK gene segments and five human JK gene segments operably linked to the constant genes of the human heavy chain. mouse which, after recombination, leads to the formation of a chimeric heavy chain k.

Introduction of ten additional human VK gene segments at a locus of the hybrid heavy chain A fourth vector for target selection is designed for the introduction of 10 additional human VK gene segments to the modified mouse heavy chain locus described above (Figure 2, Vector for Choice of Target 4). A 90,398 bp human genomic fragment containing 16 consecutive human VK gene segments with a 5 'homology arm containing the mouse 5' genomic sequence of the mouse heavy chain locus and a homology arm is designed. 'containing human genomic VK sequence. Towards the 5 'end (5') from the human VK2-40 gene segment there are an additional 8.484 pb of human k sequence before the start of the mouse 5 'homology arm, which is the same arm of 5' homology than the previous target selection vectors (described above, Figure 2). Between the 5 'homology arm and the start of the human k-sequence there is a hygromycin cassette flanked by recombinase recognition sites. The 3 'homology arm includes an overlap of 61, 615 bp of human genomic VK sequence corresponding to the 5 'end of the ~ 160 kb fragment of the human genomic k sequence of the Vector for White 3 Choice (Figure 2). The vector for final target selection for the insertion of 10 additional human VK gene segments, from 5 'to 3', includes a 5 'homology arm containing ~ 20 kb of mouse 5' genomic sequence from the locus of the mouse heavy chain, a first recombinase recognition site (R1), a hygromycin cassette, a second recombinase recognition site (R2) and ~ 90 kb human genomic k sequence containing 16 human VK gene segments , of which ~ 62 kb overlap with the 5 'end of the human sequence k of the Vector for Choice of White 3 and serve as the homology arm 3' for this construction for choice of target. Homologous recombination with this vector for choice of target creates a locus of the modified mouse heavy chain containing 40 human VK gene segments and five human JK gene segments operably linked to the mouse heavy chain genes which, after recombination, leads to the formation of a chimeric heavy chain k (bottom of Figure 2).

Using a similar strategy as described above, other combinations of variable domains of the human light chain are constructed in the context of constant regions of the mouse heavy chain. Additional light chain variable domains can be obtained from VA and JA gene segments (Figure 3 and Figure 4).

The human A light chain locus extends over 1,000 kb and contains about 80 genes that code for variable (V) or junction (J) segments. Among the 70 segments of the VA gene of the locus of the human light chain A, anywhere from 30-38 there appear to be functional segments of the compliance with published reports. The 70 VA sequences are arranged in three clusters, which all contain different members of different V gene family groups (clusters A, B and C). Within the locus of human light chain A, more than half of all VA domains are encoded by the gene segments 1 -40, 1 -44, 2-8, 2-14, and 3-21. There are seven JA gene segments, of which only four are considered as generally functional JA gene segments - JA1, JA2, JA3, and JA7. In some alleles, a qumto pair of JA-CA gene segments reported to be a pseudogene (CA6). The incorporation of multiple human JA gene segments in a locus of the hybrid heavy chain, as described in the present application, is constructed by de novo synthesis. In this way, a genomic fragment containing multiple human JA gene segments in germline configuration with multiple human VA gene segments is designed and allows normal V-J recombination in the context of a constant region of the heavy chain.

The coupling of variable domains of the light chain with constant regions of the heavy chain represents a potentially rich source of diversity to generate unique VL binding proteins with VL regions of human in non-human animals. Exploitation of this diversity of the locus of human light chain A (or human locus k as described above) in mice results in the design of unique hybrid heavy chains and gives rise to another dimension of binding proteins for the immune repertoire of genetically modified animals and their subsequent use as a next generation platform for the generation of therapeutics.

Additionally, segments of the human DH and JH (O JK) gene can be incorporated with either human VK or VA gene segments to construct novel hybrid loci that will give origin, after recombination, to novel designed variable domains (Figure 5). and 6). In the latter case, designing combinations of gene segments that are not normally contained in an individual locus will require specific attention to the recombination signal (RSS) sequences that are associated with respective gene segments so that the normal recombination when these are combined at an individual locus. For example, it is known that recombination V (D) J is guided by conserved non-coding DNA sequences., known as heptamer and nonamer sequences that are adjacent to each gene segment at the precise location where recombination takes place. Among said non-coding DNA sequences are non-conserved spacer regions that are 12 or 23 base pairs (bp) in length. In general terms, recombination occurs only in gene segments located on the same chromosome and said gene segments flanked by a spacer of 12 bp can be linked to a gene segment flanked by a 23 bp spacer, ie. Rule 12/23, although the union of two of the DH gene segments (each flanked by 12 bp spacers) in a small proportion of antibodies. To allow recombination between gene segments that do not normally have compatible spacers (for example, VK and a DH or DH and JA), compatible, unique spacers are synthesized at adjacent locations with the desired gene segments for the construction of hybrid heavy chains unique ones that allow successful recombination to form unique heavy chains containing variable regions of the light chain.

Therefore, using the strategy outlined above for the incorporation of human k light chain gene segments at a locus of the endogenous heavy chain allows the use of other combinations of human light chain A gene segments as well as also specific human heavy chain gene segments (e.g., DH and JH) and combinations thereof.

EXAMPLE 2 Identification of targeted ES cells and generation of genetically modified mice that carry segments of the human light chain gene at a locus of the endogenous heavy chain The engineered BAC DNA elaborated in the previous Examples is used to electroporate mouse ES cells into cells ES modified to generate chimeric mice expressing VL binding proteins (ie, human k light chain gene segments operably linked to constant regions of the mouse heavy chain). Directed ES cells containing an insertion of non-rearranged light chain gene segments from human are identified by a quantitative PCR test, TAQMAN® (Lie, YS, and Petropoulos, CJ (1998) Advances quantitative PCR technology : 5 'nuclease assays, Curr Opin Biotechnol 9 (1): 43-48). Specific primer sets and probes are designed to detect the insertion of human k sequences and associated selection cassettes, the loss of mouse heavy chain sequences and the retention of mouse sequences flanking the locus of the endogenous heavy chain.

ES cells carrying the human k light chain gene segments can be transfected with a construct that expresses a recombinase in order to eliminate any unwanted selection cassette introduced by the insertion of the target-containing construction containing segments of human k gene. Optionally, mice carrying a locus of the designed heavy chain containing the human k light chain gene segments can be crossed with a mouse strain that removes FLPe (see, for example, Rodriguez, Cl et al. 2000) High-efficiency deletor mice show that FLPe is an alternative to Cre-loxP. Nature Genetics 25: 139-140; US 6,774,279) in order to eliminate any cassette flanked with Frt introduced by the vector for choice of target that is not eliminated, for example, in the cell stage ES or in the embryo. Optionally, the selection cassette is retained in the mice.

The ES-directed cells described above are used as ES donor cells and introduced into an 8-cell mouse embryo using the VELOCIMOUSE® method (supra). Mice carrying a locus of the modified heavy chain carrying human VK and JK gene segments operably linked to the mouse immunoglobulin heavy chain constant region genes are identified by genotyping using a modification of the allele test (Valenzuela et al, supra) that detects the presence and / or absence of the cassette sequences, the human VK and JK gene segments and the endogenous heavy chain sequences.

The offspring are subjected to genotype determination and a heterozygous offspring for a locus of the modified heavy chain containing human k light chain gene segments operably linked to the endogenous immunoglobulin heavy chain constant genes of mouse select to characterize the repertoire of the immunoglobulin heavy chain.

EXAMPLE 3 Propagation of mice expressing V binding proteins To create a new generation of VL-binding proteins, mice carrying the non-rearranged human gene segments of k can be crossed with another mouse containing a deletion of the opposite or non-directed endogenous heavy chain allele (i.e. heterozygous mouse for modification). In this way, the obtained progeny will express only hybrid heavy chains as described in Example 1. The crossing is carried out using standard techniques recognized in the field and, alternatively, by commercial companies, for example, The Jackson Laboratory. Mouse strains carrying a locus of the modified heavy chain are screened for the presence of single heavy chains containing variable domains of the human light chain.

Alternatively, mice carrying the non-rearranged human k-gene segments can be optimized at the locus of the mouse heavy chain by crossing with other mice containing one or more deletions at the loci of the mouse light chain (K and l). In this way, the obtained progeny will only express antibodies only with single human heavy chain k as described in Example 1. Crossing is carried out in a similar manner using standard techniques recognized in the field and, alternatively, by commercial companies, for example, The Jackson Laboratory. Mouse strains carrying a locus of the modified heavy chain and one or more deletions of the loci of the mouse light chain are screened for the presence of single heavy chains containing human VK domains and constant domains of the mouse heavy chain and absence of endogenous light chains.

Mice carrying a locus of the modified heavy chain (described above) are also crossed with mice that contain a locus replacement of the variable gene of the endogenous heavy chain k with the locus of the variable gene of the human heavy chain k (see US 6,596,541, Regeneron Pharmaceuticals, The VELOCIMMUNE® Humanized Mouse Technology). The VELOCIMMUNE® Humanized Mouse includes, in part, a genome comprising variable regions of the human light chain k operably linked to loci of the variable constant region of the endogenous light chain k such that the mouse produces antibodies comprising a variable domain of the human light chain k and a constant domain of the mouse heavy chain in response to antigenic stimulation. The DNA encoding the variable regions of the light chains of the antibodies can be isolated and operably linked to the DNA encoding the constant regions of the human light chain. The DNA can then be expressed in a cell that can express the fully human light chain of the antibody. After an appropriate breeding program, mice are obtained that carry a replacement of the endogenous light chain with the locus of the human light chain k and a locus of the modified heavy chain according to Example 1. Unique VL binding proteins containing somatically mutated VK domains can be isolated after immunization with an antigen of interest.

EXAMPLE 4 Re-engineering of ADAM genes within a locus of the modified heavy chain Mice with modified immunoglobulin heavy chain loci in which the endogenous variable region gene segments (i.e., VDJ) have been replaced and / or deleted lack expression of endogenous ADAM6 genes. In particular, male mice comprising said modifications of the immunoglobulin heavy chain loci demonstrate a reduction in fertility. This Example demonstrates two methods for re-designing the ability to express ADAM6 in mice with the heavy chain loci modified according to Example 1, to thereby enable the maintenance of the modified mouse strains using normal breeding methods .

Re-engineering of the ADAM6 genes within the A locus of the heavy chain of modified immunoglobulin containing human VK and JK gene segments to contain a genomic fragment encoding mouse ADAM6a and ADAM6b by homologous recombination using BAC DNA. This is achieved using the genetic engineering technology VELOCIGENE® (supra) in a series of six steps including the modification of BAC DNA containing mouse and human sequences that produce a vector for final target selection containing gene segments. VK and JK of human contiguous with mouse ADAM6 genes and constant regions of the mouse heavy chain.

A mouse BAC clone (VI 149) containing, from 5 'to 3', a unique restriction site (l-Ceul), mouse Adam6a and Adam6b genes, a regulatory element of IGCR1 (Guo et al. , 201 1), immunoglobulin DH and H gene segments, an Em enhancer, and an IgM constant region gene as a starting material for re-designing ADAM6 genes within a locus of the modified heavy chain containing segments of VL and JL gene (Figure 7). VI149 is modified by bacterial homologous recombination (BHR) to remove all segments of the DH and JH gene and the IgM gene from approximately 53 bp 5 'of the most distal segment D (DFLI6.1) to the 3' end of the BAC. This region is replaced with a spectomycin resistance cassette (pSV0000) containing a unique Ascl site at its 5 'end to produce the BAC VI413 clone.

Additional BHR modifications are made to create BAC clones containing the mouse Adam6a and Adam6b genes, as well as the IGCR1 element. The first BAC clone is created by replacing a 47199 bp region between Adam6a and Adam6b with a neomycin resistance cassette flanked with Frt with unique l-Ceul (5 ') and Ascl (3') restriction sites (pLMa0294). This deletion extends the region from 4779 bp 3 'from the CDS of Adam6b to 290 bp 5' of the CDS of Adam6b. The resulting BAC clone is named VI421. The second BAC clone is created by inserting the same cassette flanked with Frt between Adam6a and Adam6b at position 4782 bp 3 'of the CDS of Adam6a CDS in VI413 to produce VI422.

The clone of BAC V1421 contains, from 5 'to 3', a single l-Ceul site, Adam6a including 751 bp 5 'and 4779 bp 3' of the CDS, the neomycin resistance cassette flanked with Frt, Adam6b including 290 bp 5 'and 7320 bp 3' of the CDS, IGCR1, and a unique Ascl site (SEQ ID NO: 3).

The clone of BAC VI422 contains, from 5 'to 3', a single l-Ceul site, Adam6a which includes 751 bp 5 'and 4779 bp 3' of the CDS, the hygromycin resistance cassette flanked with Frt, Adam6b which includes 47490 bp 5 'and 7320 bp 3' of the CDS, IGCR1, and a unique Ascl site (SEQ ID NO: 4).

The re-engineering of the ADAM6 genes is achieved by inserting VI421 and VI422 into the intergenic region of the modified version of the Target Vector for Choice 1 (Figure 2) as described in Example 1. The Vector for Choice of White 1 is modified by two steps of BHR to insert the fragments of mouse ADAM6 from VI421 and VI422. The first step of BHR, the neomycin cassette from the Vector for Choice of White 1 is deleted with a hygromycin cassette (pLMa0100). The resulting BAC clone is named VI425, which contains, from 5 'to 3', a hygromycin resistance cassette, the four most proximal human VK segments, an intergenic VK-JK region of 23.552 bp, and the five segments JK of human, which are functionally linked to an arm of 3 'mouse homology of 8 kb containing the Em enhancer and the mouse IgM constant region gene. For the second BHR, VI425 is modified to replace 740 bp within the intergenic VK-JK region with a chloramphenicol resistance cassette flanked by unique l-Ceul and Ascl restriction sites (pDBa0049, Figure 8). The location of the 740 bp deletion is from 16,858 to 17,597 bp 3 'of the most proximal human VK gene segment (VK4-1). The resulting BAC clone from both BHRs is named VI426 (Figure 8).

The DNA fragment containing the mouse ADAM6 genes from VI421 and VI422 is used independently to replace the chloramphenicol cassette of VI426 by digestion with l-Ceul / Ascl and relegation of compatible ends. Figure 8 shows the vectors for final target selection, named VI429 and VI428, respectively. Each is used to electroporate into ES cells previously modified with the Vector for Choice of White 4 (as described in Example 1, see Figure 2) to allow recombination with the modified single heavy chain locus according to Example 1 and the insertion of the DNA fragment encoding mouse ADAM6 genes. Positive colonies are selected with neomycin.

Re-engineering of ADAM6 genes flanking gene segments of the human light chain A locus of the modified immunoglobulin heavy chain containing human VK and JK gene segments located towards the 5 'end of all endogenous heavy chain constant regions is re-designed to contain a genomic fragment encoding ADAM6a and Mouse ADAM6b by homologous recombination using BAC DNA. This is achieved using the genetic engineering technology VELOCIGENE © (supra) in a series of steps that includes the modification of BAC DNA containing mouse and human sequences that produce a vector for final target selection containing VK gene segments. and JK of human contiguous with mouse ADAM6 genes and constant regions of the mouse heavy chain.

The Vector for Choice of White 4 made in accordance with Example 1 (see Figure 2 and the upper part of Figure 9) is modified by BHR to replace the hygromycin cassette flanked with Frt with a chloramphenicol cassette containing restriction sites Ascl (5 ') and l-Ceul (3') unique (pLMa0231; Figure 9). The Vector for Choice of Target 4 contains, from 5 'to 3', a distal mouse IgH homology arm of ~ 20 kb, a hygromycin resistance cassette flanked with Frt, and human VK2-40 gene segments. up to human VK3-25.

Next, a BAC clone named VI444 is used to insert a DNA fragment coding for mouse ADAM6 genes at the 5 'position of the human VK gene segments of the BAC clone VI477 by digestion with Ascl / I- Ceul and relegation of compatible ends. Clone VI444 contains, from 5 'to 3', a single l-Ceul site, the Adam6a gene including 751 bp 5 'and 4779 bp 3' of the CDS, a neomycin resistance cassette flanked with Frt, the Adam6b gene which includes 290 bp 5 'and 1633 bp 3' of the CDS, and a single Ascl site (SEQ ID NO: 5). The resulting BAC clone used as the target vector for the insertion of mouse ADAM6 genes towards the 5 'end of the human VK gene segments is named VI478, which, in contrast to VI421 and VI422, positions the mouse ADAM6 genes in V1478 in reverse orientation (i.e., the same transcription direction relative to human VK gene segments; Figure 9). The vector for final target selection for the insertion of mouse ADAM6 genes at the distal end of the human JK gene segments contains, from 5 'to 3', the distal mouse IgH homology arm of -20 kb , a unique Ascl site, mouse Adam6b, a neomycin resistance cassette flanked with Frt, mouse Adam6a, a unique l-Ceul site, and the VK2- gene segments 40 from human to VK3-25 from human. This vector for choice of target is used to electroporate in ES cells previously modified with the Vector for Choice of Target 4 (Figure 2) to allow recombination with the locus of the modified single heavy chain according to Example 1 and the insertion of the DNA fragment that codes for mouse ADAM6 genes. Positive colonies are selected with neomycin.

Selection and confirmation of ES cells chosen as target Each of the final target vectors (described above) is used to electroporate mouse ES cells to create modified ES cells comprising an ectopically placed mouse genomic sequence comprising mouse ADAM6a and ADAM6b sequences within the locus of the modified heavy chain containing mouse VK and JK gene segments. ES positive cells containing the ectopic mouse genomic fragment within the locus of the modified heavy chain are identified by a quantitative PCR test using TAQMAN ™ probes (Líe and Petropoulos (1998), supra).

The ES cells chosen as target described above are used as donor ES cells and are introduced into a mouse embryo in an 8-cell stage using the VELOCIMOUSE® mouse engineering method (see, eg, patent E.U.A. Nos. 7,6598,442; 7,576,259; and 7,294,754). Mice carrying a locus of the modified heavy chain containing human k light chain gene segments and an ectopic mouse genomic sequence comprising mouse ADAM6a and ADA 6b sequences are identified by genotyping using a modification of the allele test (Valenzuela et al., 2003) that detects the presence of mouse ADAM6 and ADAM6b genes within the locus of the modified heavy chain as well as sequences of the human light chain k.

The offspring are subjected to genotype determination and a heterozygous offspring for a modified heavy chain locus containing an ectopic mouse genomic fragment comprising mouse ADAM6a and ADAM6b sequences is selected to characterize the expression and fertility of the mouse ADAM6 gene and Fertility

Claims (32)

1. CLAIMS 1. - A non-human animal comprising (a) an insertion of one or more human VL gene segments and one or more human JL gene segments towards the 5 'end of a constant region of the non-human immunoglobulin light chain, (b) an insertion of one or more human Vu gene segments and one or more human JL gene segments towards the 5 'end of a constant region of the non-human immunoglobulin heavy chain, and (c) a nucleotide sequence encoding an ADAM6 protein or a functional fragment thereof, wherein the ADAM6 protein is expressed from an ectopic ADAM6 nucleic acid sequence. 2. - The non-human animal according to claim 1, wherein the constant regions of the heavy and / or non-human light chain are rodent constant regions. 3. - The non-human animal according to claim 1 or 2, wherein the constant region of the light chain is a mouse CK. 4. - The non-human animal according to any of the preceding claims, wherein the human VL and JL gene segments towards the 5 'end of the constant region of the Non-human immunoglobulin light chain are human VK and JK gene segments. 5. - The non-human animal according to any of the preceding claims, wherein the human VL and JL gene segments towards the 5 'end of the constant region of the non-human immunoglobulin heavy chain are VK and JK gene segments of human. 6. The non-human animal according to any one of the preceding claims, wherein the nucleotide sequence encoding an ADAM6 protein or functional fragment thereof is present in the same location as compared to a non-human wild-type ADAM6 locus. 7. - The non-human animal according to any of the preceding claims, wherein the nucleotide sequence encoding an ADAM6 protein or functional fragment thereof is present within immunoglobulin gene segments. 8. - The non-human animal according to claim 7, wherein the immunoglobulin gene segments are gene segments of the human light chain k. 9. - The non-human animal according to claim 7 or 8, wherein the immunoglobulin gene segments are endogenous heavy chain gene segments of the non-human animal. 10. - The non-human animal in accordance with any of the preceding claims, wherein 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. 1 1.- a genetically modified non-human animal comprising (a) one or more non-rearranged human VK gene segments and one or more non-rearranged human JK gene segments at a locus of the endogenous immunoglobulin heavy chain of the non-human animal, (b) one or more non-rearranged human VK gene segments and one or more non-rearranged human JK gene segments at a locus of the endogenous immunoglobulin light chain of the non-human animal, wherein the non-human animal is capable of expressing an ADAM6 protein or functional fragment thereof. 12. - A cell obtained from the non-human animal according to claim 1 or 1. 13. - The cell according to claim 12, wherein the cell is a B cell. 14. - A hybridoma made from the B cell according to claim 13. 15. - The cell according to claim 12, wherein the non-human animal is a rodent. 16. - The cell according to claim 15, in where the rodent is selected from a mouse and a rat. 17. - A method is provided for making an antigen-binding protein that binds to an antigen of interest, wherein the method comprises (a) exposing a non-human animal according to claim 1 or 11 to an antigen of interest, (b) isolating one or more B lymphocytes from the non-human animal, wherein said one or more B lymphocytes express a VL binding protein that binds to the antigen of interest, and (c) identifying a nucleic acid sequence encoding a VL domain of the VL binding protein that binds to said antigen of interest, wherein the VL binding protein comprises a human VK domain and a constant domain of the non-human light chain and a human VK domain and a constant domain of the non-human heavy chain, and (d) employing the nucleic acid sequence of (c) with a nucleic acid sequence of the human immunoglobulin constant region to make an antigen-binding protein of human that binds to the antigen of interest. 18. - The method according to claim 17, wherein the constant region of the non-human light chain of the VL binding protein is a mouse CK and the constant region of the non-human heavy chain is a constant region of the chain heavy mouse 19. - The method according to claim 17 or 18, where the non-human animal is a mouse. 20. A rodent comprising one or more human immunoglobulin light chain gene segments operably linked to a non-human immunoglobulin heavy chain constant region gene, wherein the rodent expresses one or more ADAM6 proteins. 21. - The rodent according to claim 20, wherein said one or more human immunoglobulin light chain gene segments are human VK and JK gene segments. 22. - The rodent according to claim 20 or 21, wherein the gene of the constant region of the non-human immunoglobulin heavy chain is a gene of the constant region of the mouse or rat heavy chain. 23. - The rodent according to any of claims 20-22, wherein the gene of the constant region of the immunoglobulin heavy chain comprises a CH1 and / or a hinge region. 24. - The rodent according to any of claims 20-23, wherein the rodent comprises deleting, or replacing, one or more endogenous immunoglobulin heavy chain gene sequences. 25. - The rodent according to any of claims 20-24, wherein the endogenous VH, DH, and JH gene segments are unable to rearrange to form a V / D / J sequence rearranged. 26. - The rodent according to any of claims 20-25, wherein the rodent comprises a deletion of an endogenous ADAM6 gene and further comprises an ectopic mouse ADAM6 gene. 27. - The rodent according to any of claims 20-26, wherein the rodent is selected from a mouse or a rat. 28. - A method for modifying an immunoglobulin locus of the heavy chain of a rodent, comprising: (a) making a first modification of an immunoglobulin locus of the rodent heavy chain that results in a reduction or elimination of endogenous ADAM6 activity in a male rodent, where the first modification includes the insertion of a segment of gene that is selected from one or more human VL gene segments, one or more human JL gene segments, and a combination thereof; Y, (b) making a second modification to add a nucleic acid sequence to the rodent that confers on the rodent ADAM6 activity that is functional in a male rodent. 29. - The method according to claim 28, wherein the first modification further comprises the insertion of one or more human DH gene segments that are capable of rearrange with one or more VL gene segments and one or more JL gene segments. 30. The method according to claim 28, wherein said one or more VL gene segments and one or more human Ju gene segments are the VK and JK gene segments or the VA and JA gene segments. 31. - The method according to any of claims 28-30, wherein the nucleic acid sequence that confers on the rodent ADAM6 activity that is functional in a male rodent is contiguous with said one or more human VL gene segments. and / or said one or more human JL gene segments. 32. - The method according to any of claims 28-31, wherein the first and second modifications are made in an individual ES cell, and the individual ES cell is introduced into a host embryo to manufacture the rodent.