Non-human animals having a limited lambda light chain repertoire expressed from the kappa locus and their use

Abstract

The present disclosure provides, inter alia, a genetically modified non-human animal whose germline genome comprises a modified endogenous immunoglobulin κ light chain locus comprising a single rearranged human immunoglobulin λ light chain variable region operably linked to a non-human Cλ gene segment, the single rearranged human immunoglobulin λ light chain variable region comprising a human Vλ gene segment and a human Jλ gene segment. All immunoglobulin λ light chains expressed by B cells of the genetically modified non-human animal comprise a human immunoglobulin λ light chain variable domain expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatically hypermutated version thereof. Such animals, tissues from such animals, and cells from such animals represent effective platforms for generating antibodies, e.g., bispecific antibodies.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 62 / 857,712, filed on 5 June 2019, which is incorporated herein by reference.

[0002] Sequence List This application includes an array listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on May 20, 2020, is named 2010794-2050_SL.txt and has a size of 47,576 bytes. [Background technology]

[0003] Human antibodies are the fastest-growing class of therapeutic agents. Among the technologies currently used for their production, the development of genetically modified animals (e.g., rodents) modified with genetic material that codes for human antibodies, either whole or partially, has revolutionized the field of human therapeutic monoclonal antibodies for the treatment of various diseases. However, there is still a need for improved in vivo systems to produce human monoclonal antibodies that maximize the human antibody repertoire in genetically modified host animals. [Overview of the project] [Means for solving the problem]

[0004] This disclosure provides genetically modified rodents. In some embodiments, the genetically modified rodents provided are rats or mice. In some embodiments, all endogenous sequences are rat or mouse sequences. For example, in some embodiments, the genetically modified rodent is a rat, and all endogenous sequences are rat sequences. In some embodiments, the genetically modified rodent is a mouse, and all endogenous sequences are mouse sequences.

[0005] In some embodiments, the Disclosure provides a breeding colony of genetically modified rodents provided herein, comprising a first genetically modified rodent, a second genetically modified rodent, and a third genetically modified rodent, wherein the first, second, and third genetically modified rodents are each of the genetically modified rodents described herein. In some embodiments, the third genetically modified rodent is an offspring of the first genetically modified rodent and the second genetically modified rodent.

[0006] The provided genetically modified rodents have a germline genome containing a limited human λ light chain variable region repertoire. In some embodiments, the limited human λ light chain variable region repertoire may include one or two unreorganized human Vλ gene segments and one or more unreorganized human Jλ gene segments. In some embodiments, the limited human λ light chain variable region repertoire may include two unreorganized human Vλ gene segments and four unreorganized human Jλ gene segments. In some embodiments, the limited human λ light chain variable region repertoire may include two unreorganized human Vλ gene segments and five unreorganized human Jλ gene segments. In some embodiments, the limited human λ light chain variable region repertoire may include a single reorganized human immunoglobulin λ light chain variable region containing a human Vλ gene segment and a human Jλ gene segment.

[0007] In some embodiments, all immunoglobulin λ light chains expressed by the provided genetically modified rodent B cells include human immunoglobulin λ light chain variable domains expressed from a limited repertoire of human λ light chain variable regions. In some embodiments, all immunoglobulin λ light chains expressed by the provided genetically modified rodent B cells include human immunoglobulin λ light chain variable domains expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof. In some embodiments, all immunoglobulin light chains expressed by the genetically modified rodent B cells provided herein include human immunoglobulin λ light chain variable domains expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof. In some embodiments, all heavy chains expressed by the genetically modified rodent B cells include human immunoglobulin heavy chain variable domains and rodent immunoglobulin heavy chain constant domains.

[0008] In some embodiments, the provided genetically modified rodents include a modified endogenous rodent immunoglobulin light chain locus containing a limited human λ light chain variable region repertoire. In some embodiments, the provided genetically modified rodents include a modified endogenous rodent immunoglobulin λ light chain locus containing a limited human λ light chain variable region repertoire. In some embodiments, the provided genetically modified rodents include a modified endogenous rodent immunoglobulin κ light chain locus containing a limited human λ light chain variable region repertoire. In some embodiments, the germline genome of the genetically modified rodents is homozygous for the modified endogenous immunoglobulin light chain locus (e.g., a modified endogenous immunoglobulin λ or κ light chain locus). In some embodiments, the germline genome of the genetically modified rodents is heterozygous for the modified endogenous immunoglobulin light chain locus (e.g., a modified endogenous immunoglobulin λ or κ light chain locus).

[0009] In some embodiments, the germline genome of a genetically modified rodent includes a modified endogenous immunoglobulin κ locus comprising two alleles. In some embodiments, the first allele comprises a limited human λ light chain variable region repertoire, and the second allele comprises a limited human κ light chain variable region repertoire. In some embodiments, the germline genomes of the genetically modified rodents described herein, and the resulting rodent cells and rodent tissues, comprise a first modified endogenous immunoglobulin κ light chain locus allele comprising a single rearranged human immunoglobulin λ light chain variable region functionally linked to the rodent Cλ gene segment, the single rearranged human immunoglobulin λ light chain variable region comprising the human Vλ gene segment and the human Jλ gene segment. In some embodiments, the genetically modified rodents described herein, and the resulting rodent cells or rodent tissues, comprise a second modified endogenous immunoglobulin κ light chain locus allele containing a single rearranged human immunoglobulin κ light chain variable region functionally linked to a rodent Cκ gene segment, the single rearranged human immunoglobulin κ light chain variable region comprising a human Vκ gene segment and a human Jκ gene segment. In some embodiments, such non-human animals or non-human tissues may express a λ light chain from the first modified endogenous immunoglobulin κ light chain locus allele and a κ light chain from the second modified endogenous immunoglobulin κ light chain locus allele. In some embodiments, the single rearranged human immunoglobulin κ light chain variable region comprises Vκ3-20 or Vκ1-39, and the single rearranged human immunoglobulin λ light chain variable region comprises Vλ1-51 or Vλ2-14. In one embodiment, a single rearranged human immunoglobulin κ light chain variable region is Vκ3-20 / Jκ1, and a single rearranged human immunoglobulin λ light chain variable region is Vλ1-51 / Jλ2 or Vλ2-14 / Jλ2.

[0010] In some embodiments, the provided genetically modified rodents include a limited repertoire of human λ light chain variable regions functionally linked to a light chain constant region gene segment. In some embodiments, the provided genetically modified rodents include a limited repertoire of human λ light chain variable regions functionally linked to a Cκ gene segment. In some embodiments, the provided genetically modified rodents include a limited repertoire of human λ light chain variable regions functionally linked to a Cλ gene segment.

[0011] In some embodiments, the provided genetically modified rodents include a modified endogenous immunoglobulin κ light chain locus comprising a single rearranged human immunoglobulin λ light chain variable region functionally linked to the rodent Cλ gene segment, the single rearranged human immunoglobulin λ light chain variable region comprising the human Vλ gene segment and the human Jλ gene segment.

[0012] For several purposes, human Vλ gene segments are selected from the group consisting of Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. For several purposes, human Vλ gene segments are selected from the group consisting of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some embodiments, the human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the human Vλ gene segment is Vλ1-51 or Vλ2-14. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, the human Jλ gene segment is Jλ2.

[0013] In some embodiments, the provided genetically modified rodents lack the rodent Cκ gene at a modified endogenous immunoglobulin κ light chain locus.

[0014] In some embodiments, the provided genetically modified rodent has a germline genome comprising a modified endogenous immunoglobulin heavy chain locus. In some embodiments, the modified endogenous immunoglobulin heavy chain locus comprises one or more unrearranged human V H gene segments, one or more unrearranged human D H gene segments, and one or more unrearranged human J H gene segments. In some embodiments, the one or more unrearranged human V H gene segments, one or more unrearranged human D H gene segments, and one or more unrearranged human J H gene segments are operably linked to one or more rodent immunoglobulin heavy chain constant region genes.

[0015] In some embodiments, the provided genetically modified rodent has a germline genome comprising a modified endogenous immunoglobulin heavy chain locus comprising one or more unrearranged human V H gene segments, one or more unrearranged human D H gene segments, and one or more unrearranged human J H gene segments operably linked to one or more rodent immunoglobulin heavy chain constant region genes. In some embodiments, the provided genetically modified rodent has a germline genome that is homozygous for the modified endogenous immunoglobulin heavy chain locus.

[0016] In some embodiments, the provided genetically modified rodent has one or more non-rearranged human V H gene segments, one or more non-rearranged human D H gene segments, one or more non-rearranged human J H gene segments, or combinations thereof, instead of one or more endogenous V H gene segments, one or more endogenous D H gene segments, one or more endogenous J HIt has a germline genome that includes a gene segment. In some embodiments, the provided genetically modified rodents have one or more endogenous V H Gene segment, one or more endogenous D H Gene segment, and one or more endogenous J H One or more unreorganized human V genes that replace each gene segment H Gene segment, one or more unreorganized human D H Gene segment, and one or more unreorganized human J H It has a germline genome that includes gene segments.

[0017] In some embodiments, one or more unreorganized human V H The gene segment is V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, VH 1 - 8, V H 3 - 7, V H 2 - 5, V H 7 - 4 - 1, V H 4 - 4, V H 1 - 3, V H 1 - 2, V H 6 - 1, or any combination thereof. In some embodiments, one or more non - rearranged human D H gene segments are D H 1 - 1, D H 2 - 2, D H 3 - 3, D H 4 - 4, D H 5 - 5, D H 6 - 6, D H 1 - 7, D H 2 - 8, D H 3 - 9, D H 3 - 10, D H 5 - 12, D H 6 - 13, D H 2 - 15, D H 3 - 16, D H 4 - 17, D H 6 - 19, D H 1 - 20, D H 2 - 21, D H 3 - 22, D H 6 - 25, D H 1 - 26, D H 7 - 27, or any combination thereof. In some embodiments, one or more non - rearranged human J H gene segments are J H 1, J H 2, J H 3, J H 4, J H 5, J H 6, or any combination thereof.

[0018] In some embodiments, (i) one or more non - rearranged human V H gene segments are V H 3 - 74, V H 3 - 73, V H 3 - 72, V H 2 - 70, V H 1 - 69, V H3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1, or any combination thereof, including (ii) one or more unreorganized human D H The gene segment is D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, DH 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H (iii) one or more unreorganized human J H The gene segment is J H 1, J H 2, J H 3, J H 4, J H 5, J H 6, or any combination thereof.

[0019] In some embodiments, the provided genetically modified rodents have a germline genome containing one or more rodent immunoglobulin heavy chain constant region genes. In some embodiments, the one or more rodent immunoglobulin heavy chain constant region genes are one or more endogenous rodent immunoglobulin heavy chain constant region genes.

[0020] In some embodiments, the provided genetically modified rodents have a germline genome containing a modified endogenous immunoglobulin heavy chain locus lacking the functional endogenous rodent Adam6 gene. In some embodiments, the provided genetically modified rodents have a germline genome containing one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments. In some embodiments, the provided genetically modified rodents express one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments. In some embodiments, the provided genetically modified rodents have a germline genome containing one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments located on the same chromosome as the modified endogenous immunoglobulin heavy chain locus. In some embodiments, the provided genetically modified rodents have a germline genome comprising a modified endogenous immunoglobulin heavy chain locus containing one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments. In some embodiments, the provided genetically modified rodents have a germline genome comprising one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments in place of the human Adam6 pseudogene. In some embodiments, the provided genetically modified rodents have a germline genome comprising one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments replacing the human Adam6 pseudogene.

[0021] In some embodiments, the provided genetically modified rodents are first and second human V H One or more human V genes containing a gene segment H Genetic segment, and the first human V HGenetic segments and the second human V H The germline genome has one or more nucleotide sequences between gene segments that encode one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments. In some embodiments, the first human V H The gene segment is V H 1-2, and the second human V H The gene segment is V H The score is 6-1.

[0022] In some embodiments, one or more rodent ADAM6 polypeptides, one or more nucleotide sequences encoding a functional ortholog, functional homolog, or functional fragment thereof, are human V H Genetic segments and human D H It is located between gene segments.

[0023] In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of mouse Cλ1. In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of mouse Cλ2. In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of mouse Cλ3.

[0024] In some embodiments, the rodent Cλ gene is the mouse Cλ gene, or includes it. In some embodiments, the rodent Cλ gene is the mouse Cλ1 gene, or includes it.

[0025] In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of rat Cλ1. In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of rat Cλ2. In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of rat Cλ3. In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of rat Cλ4.

[0026] In some embodiments, the rodent Cλ gene is the rat Cλ gene, or includes it.

[0027] In some embodiments, a single rearranged human immunoglobulin λ light chain variable region replaces one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof.

[0028] In some embodiments, the genetically modified mice provided herein include a functional endogenous immunoglobulin λ light chain locus. In some embodiments, the genetically modified mice provided herein include an inactivated endogenous immunoglobulin λ light chain locus. In some embodiments, the endogenous immunoglobulin λ light chain locus is inactivated by deleting or inverting all or part of the endogenous immunoglobulin λ light chain locus. In some embodiments, the endogenous Vλ gene segment, the endogenous Jλ gene segment, and the endogenous Cλ gene are deleted as a whole or in part.

[0029] In some embodiments, the genetically modified mice provided herein do not detectably express the endogenous immunoglobulin κ light chain variable domain.

[0030] This disclosure provides rodent embryos comprising the genetic modifications described herein. In some embodiments, the rodent embryo has a genome comprising a modified endogenous immunoglobulin κ light chain locus comprising a single rearranged human immunoglobulin λ light chain variable region functionally linked to a rodent Cλ gene segment, the single rearranged human immunoglobulin λ light chain variable region comprising a human Vλ gene segment and a human Jλ gene segment. In some embodiments, the genome of the rodent embryo is homozygous for the modified endogenous immunoglobulin κ light chain locus. In some embodiments, the human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, the human Vλ gene segment is Vλ1-51 or Vλ2-14. In some embodiments, the human Jλ gene segment is Jλ2.

[0031] In some embodiments, the rodent embryo is one or more unreorganized human V genomes functionally linked to one or more endogenous immunoglobulin heavy chain constant region genes. H Gene segment, one or more unreorganized human D H Gene segment, and one or more unreorganized human J H A rodent embryo is provided that contains a modified endogenous immunoglobulin heavy chain locus including a gene segment. In some embodiments, the genome of the rodent embryo is homozygous for the modified endogenous immunoglobulin heavy chain locus.

[0032] This disclosure provides genetically modified rodent B cells as described herein. In some embodiments, the genetically modified rodent B cells described herein include a single rearranged human immunoglobulin λ light chain variable region of a modified endogenous κ light chain locus or a somatic hypermutant version thereof.

[0033] In some embodiments, the disclosure provides genetically modified rodent B cells as described herein, comprising a single rearranged human immunoglobulin λ light chain variable region of a modified endogenous κ light chain locus or a somatic hypermutant version thereof. In some embodiments, the genetically modified rodent B cells as described herein comprise one or more unrearranged human V cells at a modified endogenous heavy chain locus. H Human V gene segment H Gene segment, one or more unreorganized human D H Human D gene segment H Gene segment, and one or more unreorganized human J H Human J gene segment H It contains a rearranged human immunoglobulin heavy chain variable region derived from a gene segment.

[0034] This disclosure provides hybridomas generated from genetically modified rodent B cells as described herein.

[0035] This disclosure provides a population of B cells from a single genetically modified rodent as described herein. In some embodiments, all antibodies expressed by the population of B cells include a human immunoglobulin λ light chain variable domain expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof. In some embodiments, the antibodies expressed by the population of B cells include multiple human immunoglobulin heavy chain variable domains expressed from at least two different rearranged human immunoglobulin heavy chain variable regions or somatic hypermutant versions thereof.

[0036] This disclosure provides stem cells, such as embryonic stem (ES) cells, including the genetic modifications described herein. In some embodiments, the stem cells (e.g., ES cells) include a modified endogenous immunoglobulin κ light chain locus containing a single rearranged human immunoglobulin λ light chain variable region functionally linked to a rodent Cλ gene segment. In some embodiments, the genome of the stem cells (e.g., ES cells) is homozygous for the modified endogenous immunoglobulin κ light chain locus. For several purposes, human Vλ gene segments are selected from the group consisting of Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. For several purposes, human Vλ gene segments are selected from the group consisting of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some embodiments, the human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, the human Vλ gene segment is Vλ1-51 or Vλ2-14. In some embodiments, the human Jλ gene segment is Jλ2.

[0037] In some embodiments, stem cells (e.g., ES cells) are functionally linked to one or more endogenous immunoglobulin heavy chain constant region genes, and one or more unreorganized human V cells are connected to one or more endogenous immunoglobulin heavy chain constant region genes. H Gene segment, one or more unreorganized human D H Gene segment, and one or more unreorganized human J H It contains a modified endogenous immunoglobulin heavy chain locus that includes a gene segment.

[0038] Mammalian cells expressing antibodies are provided by this disclosure. In some embodiments, the antibodies expressed by the mammalian cells comprise a heavy chain containing a human immunoglobulin heavy chain variable domain and a light chain containing a human immunoglobulin λ light chain variable domain, wherein the human immunoglobulin heavy chain variable domain, the human immunoglobulin λ light chain variable domain, or both are identified from, isolated from, or identical to, the genetically modified rodents described herein.

[0039] In some embodiments, the antibody is prepared by a method comprising the steps of (a) exposing genetically modified rodents described herein to a target antigen; (b) maintaining the genetically modified rodents under conditions sufficient to produce an immune response to the target antigen; and (c) recovering from the genetically modified rodents (i) an antibody that binds to the target antigen, (ii) a human light chain variable domain, a human heavy chain variable domain, a light chain, or a nucleotide encoding a heavy chain of an antibody that binds to the target antigen, or (iii) cells expressing an antibody that binds to the target antigen, wherein the antibody in (c) comprises a human heavy chain variable and a human λ light chain variable domain. In some embodiments, the antibody is a bispecific antibody.

[0040] In some embodiments, the method for producing antibodies includes (a) exposing genetically modified rodents described herein to an antigen; (b) enabling the genetically modified rodents to elicit an immune response to the antigen; and (c) isolating from the genetically modified rodents an antibody specific to the antigen, B cells expressing the antibody specific to the antigen, or one or more nucleotide sequences encoding the antibody specific to the antigen. In some embodiments, the antibody is a bispecific antibody.

[0041] In some embodiments, a method for producing an antibody includes (a) expressing an antibody comprising two human immunoglobulin λ light chains and two human immunoglobulin heavy chains in mammalian cells, each human immunoglobulin λ light chain comprising a human immunoglobulin λ light chain variable domain, and each human immunoglobulin heavy chain comprising a human immunoglobulin heavy chain variable domain, wherein the amino acid sequence of at least one of the human immunoglobulin heavy chain variable domains, at least one of the λ light chain variable domains, or a combination thereof has been identified or isolated from genetically modified rodents as described herein; and (b) obtaining an antibody. In some embodiments, the antibody is a bispecific antibody.

[0042] In some embodiments, a method for producing a bispecific antibody comprises: (a) contacting a first genetically modified rodent described herein with a first epitope of a first antigen; (b) contacting a second genetically modified rodent described herein with a second epitope of a second antigen; (c) isolating B cells expressing a first antibody specific to the first epitope of the first antigen from the first genetically modified rodent and determining the first human immunoglobulin heavy chain variable domain of the first antibody; (d) isolating B cells expressing a second antibody specific to the second epitope of the second antigen from the second genetically modified rodent and determining the second human immunoglobulin heavy chain variable domain of the second antibody; and (e) determining the nucleus encoding the first human immunoglobulin heavy chain variable domain. A method is provided comprising: (f) functionally ligating an otidol sequence to a nucleotide sequence encoding a first human immunoglobulin constant domain to generate a first nucleotide sequence encoding a first human heavy chain; (g) functionally ligating a nucleotide sequence encoding a second human immunoglobulin heavy chain variable domain to a nucleotide sequence encoding a second human immunoglobulin constant domain to generate a second nucleotide sequence encoding a second human heavy chain; and (iii) expressing in mammalian cells a third nucleotide sequence comprising (i) the first nucleotide sequence; (ii) the second nucleotide sequence; and (iii) a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof functionally ligated to the human immunoglobulin λ light chain constant region.

[0043] In some embodiments, a method for producing a bispecific antibody is provided herein, the method comprising expressing in mammalian cells (a) a first nucleotide sequence comprising (i) a first human immunoglobulin heavy chain variable region functionally linked to a first human immunoglobulin constant region; (ii) a second nucleotide sequence comprising a second human immunoglobulin heavy chain variable region functionally linked to a second human immunoglobulin constant region; and (iii) a third nucleotide sequence comprising a human immunoglobulin λ light chain variable region functionally linked to a human immunoglobulin λ light chain constant region. In some embodiments, the first human immunoglobulin heavy chain variable region encodes a first human heavy chain variable domain identified, isolated, or identical to that first human heavy chain variable domain identified from a first antibody in a first genetically modified rodent described herein immunized with a first epitope of a first antigen, wherein the first antibody specifically binds to the first epitope of the first antigen. In some embodiments, the second human immunoglobulin heavy chain variable region encodes a second human heavy chain variable domain identified, isolated, or identical to that of a second antibody in a second genetically modified rodent described herein immunized with a second epitope of a second antigen, the second antibody specifically binding to the second epitope of the second antigen. In some embodiments, the third nucleotide human immunoglobulin λ light chain variable region is a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof.

[0044] In some embodiments, the first genetically modified rodent and the second genetically modified rodent are the same genetically modified rodent. In some embodiments, the first genetically modified rodent and the second genetically modified rodent are different genetically modified rodents.

[0045] In some embodiments, the first antigen and the second antigen are the same antigen, and the first epitope and the second epitope are different epitopes. In some embodiments, the first antigen and the second antigen are different antigens.

[0046] In some embodiments, a method for producing a human immunoglobulin heavy chain includes (a) exposing a genetically modified rodent described herein to a target antigen; (b) obtaining a human immunoglobulin heavy chain variable domain sequence of an antibody that specifically binds to the antigen and is produced by the genetically modified rodent; and (c) functionally ligating the human immunoglobulin heavy chain variable domain sequence to a human immunoglobulin heavy chain constant domain sequence to form a human immunoglobulin heavy chain. In some embodiments, a human immunoglobulin heavy chain produced by the method of this paragraph is provided.

[0047] In some embodiments, a method for producing a human immunoglobulin heavy chain variable domain comprises (a) exposing a genetically modified rodent described herein to a target antigen; and (b) obtaining a human immunoglobulin heavy chain variable domain sequence of an antibody that specifically binds to the antigen and is produced by the genetically modified rodent. In some embodiments, the human immunoglobulin heavy chain variable domain produced by the method of this paragraph is provided.

[0048] In some embodiments, a method for preparing a collection of human immunoglobulin heavy chain variable domains includes (a) exposing genetically modified rodents described herein to a target antigen, and (b) isolating a collection of human immunoglobulin heavy chain variable domains from the genetically modified rodents. In some embodiments, each collection of human immunoglobulin heavy chain variable domains is bound to a human immunoglobulin λ light chain variable domain expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof, and the human λ light chain variable domain paired with any one of the human immunoglobulin heavy chain variable domains in the collection binds to the antigen.

[0049] In some embodiments, a method for producing a human immunoglobulin λ light chain includes (a) exposing a genetically modified rodent described herein to a target antigen; (b) obtaining a human immunoglobulin λ light chain variable domain sequence of an antibody that specifically binds to the antigen and is produced by the genetically modified rodent; and (c) functionally ligating the human immunoglobulin λ light chain variable domain sequence to a human immunoglobulin λ light chain constant domain sequence to form a human immunoglobulin λ light chain. In some embodiments, a human immunoglobulin λ light chain produced by the method of this paragraph is provided.

[0050] In some embodiments, a method for generating human immunoglobulin λ light chain variable domains comprises (a) exposing a genetically modified rodent described herein to a target antigen; and (b) obtaining a human immunoglobulin λ light chain variable domain sequence of an antibody that specifically binds to the antigen and is produced by the genetically modified rodent. In some embodiments, human immunoglobulin λ light chain variable domains generated by the method of this paragraph are provided.

[0051] In some embodiments, a method for producing a nucleotide sequence encoding a human immunoglobulin heavy chain includes (a) exposing a genetically modified rodent described herein to a target antigen; (b) obtaining a human immunoglobulin heavy chain variable region that specifically binds to the antigen and encodes a human immunoglobulin heavy chain variable domain sequence of an antibody produced by the genetically modified rodent; and (c) functionally ligating the human immunoglobulin heavy chain variable region to a human immunoglobulin heavy chain constant region sequence to form a nucleotide sequence encoding a human immunoglobulin heavy chain. In some embodiments, a nucleotide sequence encoding a human immunoglobulin heavy chain produced by the method of this paragraph is provided.

[0052] In some embodiments, a method for producing a nucleotide sequence containing a human immunoglobulin heavy chain variable region comprises (a) exposing a genetically modified rodent described herein to a target antigen; and (b) obtaining a human immunoglobulin heavy chain variable region that specifically binds to the antigen and encodes a human immunoglobulin heavy chain variable domain sequence of an antibody produced by the genetically modified rodent. In some embodiments, a nucleotide sequence containing a human immunoglobulin heavy chain variable region produced by the method of this paragraph is provided.

[0053] In some embodiments, a method for producing a nucleotide sequence encoding a human immunoglobulin λ light chain includes (a) exposing a genetically modified rodent described herein to a target antigen; (b) obtaining a human immunoglobulin λ light chain variable region that specifically binds to the antigen and encodes a human immunoglobulin λ light chain variable domain sequence of an antibody produced by the genetically modified rodent; and (c) functionally ligating the human immunoglobulin λ light chain variable region to a human immunoglobulin λ light chain constant region sequence to form a nucleotide sequence encoding a human immunoglobulin λ light chain. In some embodiments, a nucleotide sequence encoding a human immunoglobulin λ light chain produced by the method of this paragraph is provided.

[0054] In some embodiments, a method for producing a nucleotide sequence containing a human immunoglobulin λ light chain variable region comprises (a) exposing a genetically modified rodent described herein to a target antigen; and (b) obtaining a human immunoglobulin λ light chain variable region that specifically binds to the antigen and encodes a human immunoglobulin λ light chain variable domain sequence of an antibody produced by the genetically modified rodent. In some embodiments, a nucleotide sequence containing a human immunoglobulin λ light chain variable region produced by the method of this paragraph is provided.

[0055] In some embodiments, a targeting vector is provided. In some embodiments, the targeting vector comprises (i) a 5' homology arm containing a nucleotide sequence corresponding to a 5' target sequence at the endogenous rodent κ light chain locus; (ii) a single rearranged human immunoglobulin λ light chain variable region containing a Vλ gene segment and a Jλ gene segment; (iii) a rodent Cλ gene segment; and (iv) a 3' homology arm containing a nucleotide sequence corresponding to a 3' target sequence at the endogenous rodent κ light chain locus. For several purposes, human Vλ gene segments are selected from the group consisting of Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. For several purposes, human Vλ gene segments are selected from the group consisting of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some embodiments, the human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the human Vλ gene segment is Vλ1-51 or Vλ2-14. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, and Jλ7.In some embodiments, the human Jλ gene segment is Jλ2.

[0056] In some embodiments, the method for producing genetically modified rodents described herein includes (a) introducing a single rearranged human immunoglobulin λ light chain variable region, comprising a human Vλ gene segment and a human Jλ gene segment, into a modified endogenous immunoglobulin κ light chain locus in the genome of rodent ES cells; and (b) generating rodents using the rodent ES cells produced in step (a).

[0057] In some embodiments, the method for producing genetically modified rodents described herein includes (a) introducing a single rearranged human immunoglobulin λ light chain variable region functionally linked to a rodent Cλ gene segment into a modified endogenous immunoglobulin κ light chain locus in the genome of a rodent ES cell, wherein the single rearranged human immunoglobulin λ light chain variable region comprises a human Vλ gene segment and a human Jλ gene segment; and (b) generating rodents using the rodent ES cells produced in step (a). In some embodiments, the genome of the rodent ES cell comprises one or more unrearranged human Vλ light chain variable regions functionally linked to one or more endogenous immunoglobulin heavy chain constant region genes at a modified endogenous immunoglobulin heavy chain locus. H Gene segment, one or more unreorganized human D H Gene segment, and one or more unreorganized human J H It includes a gene segment. In some embodiments, the genome of a rodent ES cell further includes one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments.

[0058] In some embodiments, a method for producing genetically modified rodent ES cells includes the step of introducing a single rearranged human immunoglobulin λ light chain variable region, comprising a human Vλ gene segment and a human Jλ gene segment, into a modified endogenous immunoglobulin κ light chain locus in the genome of a rodent ES cell. In some embodiments, a method for producing genetically modified rodent ES cells includes the step of introducing a single rearranged human immunoglobulin λ light chain variable region functionally linked to a rodent Cλ gene segment into a modified endogenous immunoglobulin κ light chain locus in the genome of a rodent ES cell, wherein the single rearranged human immunoglobulin λ light chain variable region comprises a human Vλ gene segment and a human Jλ gene segment. In some embodiments, the genome of a rodent ES cell includes one or more unrearranged human Vλ light chain variable regions functionally linked to one or more endogenous immunoglobulin heavy chain constant region genes at a modified endogenous immunoglobulin heavy chain locus. H Gene segment, one or more unreorganized human D H Gene segment, and one or more unreorganized human J H It includes a gene segment. In some embodiments, the genome of a rodent ES cell further includes one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments.

[0059] In some embodiments, the method for producing the genetically modified rodents described herein includes (a) modifying the endogenous immunoglobulin κ light chain locus in the germline genome of a rodent to include a single rearranged human immunoglobulin λ light chain variable region functionally linked to the rodent Cλ gene segment, such that all immunoglobulin λ light chains expressed by the B cells of the genetically modified rodent include a human immunoglobulin λ light chain variable domain expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof, wherein the single rearranged human immunoglobulin λ light chain variable region includes the human Vλ gene segment and the human Jλ gene segment. In some embodiments, the method involves (b) linking one or more unreorganized human B cells to one or more rodent immunoglobulin heavy chain loci in the rodent germline genome, so that all heavy chains expressed by genetically modified rodent B cells include a human immunoglobulin heavy chain variable domain and a rodent immunoglobulin heavy chain constant domain. H Gene segment, one or more unreorganized human D H Gene segment, and one or more unreorganized human J H The process further includes modifying the gene segment to include a gene segment. In some embodiments, step (b) includes modifying the endogenous immunoglobulin heavy chain locus to further include one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments of one or more nucleotide sequences. In some embodiments, steps (a) and / or (b) are carried out in rodent ES cells.

[0060] For several purposes, human Vλ gene segments are selected from the group consisting of Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. For several purposes, human Vλ gene segments are selected from the group consisting of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some embodiments, the human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the human Vλ gene segment is Vλ1-51 or Vλ2-14. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, the human Jλ gene segment is Jλ2.

[0061] In some embodiments, the endogenous immunoglobulin κ light chain locus lacks the rodent Cκ gene.

[0062] In some embodiments, one or more unreorganized human V H Gene segment, one or more unreorganized human D H Gene segment, and one or more unreorganized human J HThe gene segment contains one or more endogenous V H Gene segment, one or more endogenous D H Gene segment, one or more endogenous J H A substitute for a gene segment, or a combination thereof. In some embodiments, one or more unreorganized human V H Gene segment, one or more unreorganized human D H Gene segment, and one or more unreorganized human J H The gene segment contains one or more endogenous V H Gene segment, one or more endogenous D H Gene segment, and one or more endogenous J H Each gene segment is replaced accordingly.

[0063] In some embodiments, one or more unreorganized human V H The gene segment is V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1, or any combination thereof. In some embodiments, one or more unreorganized human D H The gene segment is D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H Includes 7-27, or any combination thereof. In some embodiments, one or more unreorganized human J H The gene segment is J H 1, J H 2, J H 3, J H 4, J H 5, J H 6, or any combination thereof.

[0064] In some embodiments, one or more rodent immunoglobulin heavy chain constant region genes are one or more endogenous rodent immunoglobulin heavy chain constant region genes.

[0065] In some embodiments, the endogenous immunoglobulin heavy chain locus lacks the functional endogenous rodent Adam6 gene. In some embodiments, the germline genome of the genetically modified rodent contains one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments. In some embodiments, one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are expressed by the genetically modified rodent. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are located on the same chromosome as the modified endogenous immunoglobulin heavy chain locus. In some embodiments, the modified endogenous immunoglobulin heavy chain locus contains one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments. In some embodiments, one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments, are replaced by one or more nucleotide sequences that encode the human Adam6 pseudogene.

[0066] In some embodiments, one or more human V H The gene segments are the first and second human V H Including a gene segment, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are first human V H Genetic segments and the second human V H It lies between the gene segments. In some embodiments, the first human V H The gene segment is V H 1-2, and the second human V HThe gene segment is V H The score is 6-1.

[0067] In some embodiments, one or more rodent ADAM6 polypeptides, one or more nucleotide sequences encoding a functional ortholog, functional homolog, or functional fragment thereof, are human V H Genetic segments and human D H It is located between gene segments.

[0068] In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of mouse Cλ1. In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of mouse Cλ2. In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of mouse Cλ3.

[0069] In some embodiments, the rodent Cλ gene is the mouse Cλ gene, or includes it. In some embodiments, the rodent Cλ gene is the mouse Cλ1 gene, or includes it.

[0070] In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of rat Cλ1. In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of rat Cλ2. In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of rat Cλ3. In some embodiments, the rodent Cλ gene has sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to those of rat Cλ4.

[0071] In some embodiments, the rodent Cλ gene is the rat Cλ gene, or includes it.

[0072] In some embodiments, a single rearranged human immunoglobulin λ light chain variable region replaces one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof. In some embodiments, a single rearranged human immunoglobulin λ light chain variable region replaces one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof. In some embodiments, the genetically modified rodent germline genome described herein further includes an inactivated endogenous immunoglobulin λ light chain locus. In some embodiments, the endogenous immunoglobulin λ light chain locus is inactivated by deleting or inverting all or part of the endogenous immunoglobulin λ light chain locus. In some embodiments, the endogenous Vλ gene segment, the endogenous Jλ gene segment, and the endogenous Cλ gene are deleted as a whole or in part.

[0073] The drawings included in this specification, consisting of the following figures, are for illustrative purposes only and not for limitation. [Brief explanation of the drawing]

[0074] [Figure 1A] The figures show exemplary embodiments, not to scale, of the strategies for constructing the targeting vectors (described in Examples 1 and 4) used in generating the non-human animal embodiments of this disclosure. Unless otherwise indicated by the labels in the figures (e.g., for selection cassettes, loxP sites, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates the Frt recombinase system site; "UB-NEO" indicates the neomycin resistance gene with the UB promoter; "enh" indicates the enhancer; "Ei" indicates the intron enhancer; "3'E" indicates the 3' enhancer; "SPEC" indicates the spectinomycin resistance gene; "SD" indicates the splice donor site; "CDS" indicates the coding sequence; "lox" indicates the lox2372 site; "loxP" indicates the lox P site; and "UB-HYG" indicates the hygromycin resistance gene with the UB promoter. [Figure 1B]The figures show exemplary embodiments, not to scale, of the strategies for constructing the targeting vectors (described in Examples 1 and 4) used in generating the non-human animal embodiments of this disclosure. Unless otherwise indicated by the labels in the figures (e.g., for selection cassettes, loxP sites, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates the Frt recombinase system site; "UB-NEO" indicates the neomycin resistance gene with the UB promoter; "enh" indicates the enhancer; "Ei" indicates the intron enhancer; "3'E" indicates the 3' enhancer; "SPEC" indicates the spectinomycin resistance gene; "SD" indicates the splice donor site; "CDS" indicates the coding sequence; "lox" indicates the lox2372 site; "loxP" indicates the lox P site; and "UB-HYG" indicates the hygromycin resistance gene with the UB promoter. [Figure 2A] The figures show exemplary embodiments, not to scale, of the strategies for constructing the targeting vectors (described in Examples 7 and 10) used in generating the non-human animal embodiments of this disclosure. Unless otherwise indicated by the labels in the figures (e.g., for selection cassettes, loxP sites, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates the Frt recombinase system site; "UB-NEO" indicates the neomycin resistance gene with the UB promoter; "enh" indicates the enhancer; "Ei" indicates the intron enhancer; "3'E" indicates the 3' enhancer; "SPEC" indicates the spectinomycin resistance gene; "SD" indicates the splice donor site; "CDS" indicates the coding sequence; "lox" indicates the lox2372 site; "loxP" indicates the lox P site; and "UB-HYG" indicates the hygromycin resistance gene with the UB promoter. [Figure 2B]The figures show exemplary embodiments, not to scale, of the strategies for constructing the targeting vectors (described in Examples 7 and 10) used in generating the non-human animal embodiments of this disclosure. Unless otherwise indicated by the labels in the figures (e.g., for selection cassettes, loxP sites, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates the Frt recombinase system site; "UB-NEO" indicates the neomycin resistance gene with the UB promoter; "enh" indicates the enhancer; "Ei" indicates the intron enhancer; "3'E" indicates the 3' enhancer; "SPEC" indicates the spectinomycin resistance gene; "SD" indicates the splice donor site; "CDS" indicates the coding sequence; "lox" indicates the lox2372 site; "loxP" indicates the lox P site; and "UB-HYG" indicates the hygromycin resistance gene with the UB promoter. [Figure 3] The figure shows a non-scaled diagram of the insertion of targeting vector A (described in Example 1) into a modified Igκ light chain locus of a rodent embryonic stem (ES) cell clone (described in Example 2), the ES cell clone used in the generation of the rodent embodiments according to this disclosure. The figure includes the approximate locations (indicated by circled dashes) of various probes used to confirm that the embryonic stem (ES) cell clone is positive for a given exemplary sequence. Unless otherwise indicated by the labels in the figure (e.g., for selection cassette, loxP site, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates a Frt recombinase system site; "UB-NEO" indicates a neomycin resistance gene with a UB promoter; "Ei" indicates an intron enhancer; "3'E" indicates a 3' enhancer; and "SD" indicates a splice donor site. "Het" indicates a heterozygous mouse, and "ho" indicates a homozygous mouse. [Figure 4]The figure shows a non-scaled diagram of the insertion of targeting vector B (described in Example 4) into a modified Igκ light chain locus of a rodent embryonic stem (ES) cell clone (described in Example 5), the ES cell clone used in the generation of a rodent embodiment according to this disclosure. The figure includes the approximate locations (indicated by circled dashes) of various probes used to confirm that the embryonic stem (ES) cell clone is positive for a given exemplary sequence. Unless otherwise indicated by the labels in the figure (e.g., for selection cassette, loxP site, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates the Frt recombinase system site; "UB-NEO" indicates the neomycin resistance gene with the UB promoter; "Ei" indicates the intron enhancer; "3'E" indicates the 3' enhancer; "SD" indicates the splice donor site; "loxP" indicates the lox P site; and "UB-HYG" indicates the hygromycin resistance gene with the UB promoter. "Het" indicates a heterozygous mouse, and "ho" indicates a homozygous mouse. [Figure 5]The figure shows a non-scaled diagram of the insertion of targeting vector C (described in Example 7) into a modified Igκ light chain locus of a rodent embryonic stem (ES) cell clone (described in Example 8), the ES cell clone used in the generation of the rodent embodiments according to this disclosure. The figure includes the approximate locations (indicated by circled dashes) of various probes used to confirm that the embryonic stem (ES) cell clone is positive for a given exemplary sequence. Unless otherwise indicated by the labels in the figure (e.g., for selection cassette, loxP site, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates a Frt recombinase system site; "UB-NEO" indicates a neomycin resistance gene with a UB promoter; "Ei" indicates an intron enhancer; "3'E" indicates a 3' enhancer; and "SD" indicates a splice donor site. "Het" indicates a heterozygous mouse, and "ho" indicates a homozygous mouse. [Figure 6]The figure shows a non-scaled diagram of the insertion of targeting vector D (described in Example 10) into a modified Igκ light chain locus of a rodent embryonic stem (ES) cell clone (described in Example 11), the ES cell clone used in generating the rodent embodiments according to this disclosure. The figure includes the approximate locations (indicated by circled dashes) of various probes used to confirm that the embryonic stem (ES) cell clone is positive for a given exemplary sequence. Unless otherwise indicated by the labels in the figure (e.g., for selection cassette, loxP site, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates the Frt recombinase system site; "UB-NEO" indicates the neomycin resistance gene with the UB promoter; "Ei" indicates the intron enhancer; "3'E" indicates the 3' enhancer; "SD" indicates the splice donor site; "loxP" indicates the lox P site; and "UB-HYG" indicates the hygromycin resistance gene with the UB promoter. "Het" indicates a heterozygous mouse, and "ho" indicates a homozygous mouse. [Figure 7] The nucleotide sequence of mouse Cκ (SEQ ID NO: 25) is shown. The coding sequence is shown in bold, and the 3' untranslated region is not in bold. [Figure 8] The nucleotide sequence of rat Cκ (SEQ ID NO: 27) is shown. The coding sequence is shown in bold, and the 3' untranslated region is not in bold. [Figure 9A]The nucleotide sequence (SEQ ID NO: 31) of the modified Vλ1-51 / Jλ2 vector is shown. Restriction enzyme sites—AscI (5' end of the sequence) and PI-SceI (3' end of the sequence)—are shown in uppercase italics; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); Vλ1 The 5'UTR sequence of 51 is shown in uppercase italic bold; the rearranged Vλ1-51 / Jλ2 sequence includes: the exon 1 sequence of Vλ1-51 is shown in uppercase (non-bold), with the exon 1 start codon of Vλ1-51 further italicized and underlined; the intron 1 sequence of Vλ1-51 is shown in lowercase bold; the exon 2 sequence of Vλ1-51 is shown in uppercase bold and underlined (solid underline), and the Jλ2 sequence is shown in uppercase bold and underlined (dashed underline); the human Jκ5-Cκ intron sequence is shown in lowercase italic. [Figure 9B] The nucleotide sequence (SEQ ID NO: 31) of the modified Vλ1-51 / Jλ2 vector is shown. Restriction enzyme sites—AscI (5' end of the sequence) and PI-SceI (3' end of the sequence)—are shown in uppercase italics; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); Vλ1 The 5'UTR sequence of 51 is shown in uppercase italic bold; the rearranged Vλ1-51 / Jλ2 sequence includes: the exon 1 sequence of Vλ1-51 is shown in uppercase (non-bold), with the exon 1 start codon of Vλ1-51 further italicized and underlined; the intron 1 sequence of Vλ1-51 is shown in lowercase bold; the exon 2 sequence of Vλ1-51 is shown in uppercase bold and underlined (solid underline), and the Jλ2 sequence is shown in uppercase bold and underlined (dashed underline); the human Jκ5-Cκ intron sequence is shown in lowercase italic. [Figure 9C]The nucleotide sequence (SEQ ID NO: 31) of the modified Vλ1-51 / Jλ2 vector is shown. Restriction enzyme sites—AscI (5' end of the sequence) and PI-SceI (3' end of the sequence)—are shown in uppercase italics; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); Vλ1 The 5'UTR sequence of 51 is shown in uppercase italic bold; the rearranged Vλ1-51 / Jλ2 sequence includes: the exon 1 sequence of Vλ1-51 is shown in uppercase (non-bold), with the exon 1 start codon of Vλ1-51 further italicized and underlined; the intron 1 sequence of Vλ1-51 is shown in lowercase bold; the exon 2 sequence of Vλ1-51 is shown in uppercase bold and underlined (solid underline), and the Jλ2 sequence is shown in uppercase bold and underlined (dashed underline); the human Jκ5-Cκ intron sequence is shown in lowercase italic. [Figure 10] This shows the nucleotide sequence (SEQ ID NO: 32) of the rearranged Vλ1-51 / Jλ2 variable region, including the Vλ1-51 intron. The Vλ1-51 exon 1 sequence is shown in uppercase letters, with the Vλ1-51 exon 1 start codon further italicized and underlined; the Vλ1-51 intron 1 sequence is shown in lowercase and bold letters; the Vλ1-51 exon 2 sequence is shown in uppercase, bold, and underlined (solid underline); and the Jλ2 sequence is shown in uppercase, bold, and underlined (dashed underline). [Figure 11] The nucleotide sequence (SEQ ID NO: 33) of the rearranged Vλ1-51 / Jλ2 variable region without the Vλ1-51 intron is shown. The Vλ1-51 coding sequence is shown in uppercase, the Vλ1-51 start codon is further italicized and underlined; the Jλ2 sequence is shown in uppercase with a dashed underline. [Figure 12]The amino acid sequence (SEQ ID NO: 34) of the rearranged Vλ1-51 / Jλ2 variable domain containing the signal peptide is shown. The bolded italics indicate the sequence of the signal peptide. [Figure 13A] The nucleotide sequence of the modified Vλ2-14 / Jλ2 vector (SEQ ID NO: 36) is shown. Restriction enzyme sites—AscI (5' end of the sequence) and PI-SceI (3' end of the sequence)—are shown in uppercase italics; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); Vλ1 The 5'UTR sequence of 51 is shown in uppercase italic bold; the rearranged Vλ2-14 / Jλ2 sequence includes: the exon 1 sequence of Vλ2-14 is shown in uppercase (non-bold), in which the exon 1 start codon of Vλ2-14 is further italicized and underlined; the intron 1 sequence of Vλ2-14 is shown in lowercase bold; the exon 2 sequence of Vλ2-14 is shown in uppercase bold and underlined (solid underline), and the Jλ2 sequence is shown in uppercase bold and underlined (dashed underline); the human Jκ5-Cκ intron sequence is shown in lowercase italic. [Figure 13B]The nucleotide sequence of the modified Vλ2-14 / Jλ2 vector (SEQ ID NO: 36) is shown. Restriction enzyme sites—AscI (5' end of the sequence) and PI-SceI (3' end of the sequence)—are shown in uppercase italics; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); Vλ1 The 5'UTR sequence of 51 is shown in uppercase italic bold; the rearranged Vλ2-14 / Jλ2 sequence includes: the exon 1 sequence of Vλ2-14 is shown in uppercase (non-bold), in which the exon 1 start codon of Vλ2-14 is further italicized and underlined; the intron 1 sequence of Vλ2-14 is shown in lowercase bold; the exon 2 sequence of Vλ2-14 is shown in uppercase bold and underlined (solid underline), and the Jλ2 sequence is shown in uppercase bold and underlined (dashed underline); the human Jκ5-Cκ intron sequence is shown in lowercase italic. [Figure 13C] The nucleotide sequence of the modified Vλ2-14 / Jλ2 vector (SEQ ID NO: 36) is shown. Restriction enzyme sites—AscI (5' end of the sequence) and PI-SceI (3' end of the sequence)—are shown in uppercase italics; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); Vλ1 The 5'UTR sequence of 51 is shown in uppercase italic bold; the rearranged Vλ2-14 / Jλ2 sequence includes: the exon 1 sequence of Vλ2-14 is shown in uppercase (non-bold), in which the exon 1 start codon of Vλ2-14 is further italicized and underlined; the intron 1 sequence of Vλ2-14 is shown in lowercase bold; the exon 2 sequence of Vλ2-14 is shown in uppercase bold and underlined (solid underline), and the Jλ2 sequence is shown in uppercase bold and underlined (dashed underline); the human Jκ5-Cκ intron sequence is shown in lowercase italic. [Figure 14]This shows the nucleotide sequence (SEQ ID NO: 37) of the rearranged Vλ2-14 / Jλ2 variable region, including the Vλ2-14 intron. The Vλ2-14 exon 1 sequence is shown in uppercase letters, with the Vλ2-14 exon 1 start codon further italicized and underlined; the Vλ2-14 intron 1 sequence is shown in lowercase and bold letters; the Vλ1-51 exon 2 sequence is shown in uppercase, bold, and underlined (solid underline); and the Jλ2 sequence is shown in uppercase, bold, and underlined (dashed underline). [Figure 15] The nucleotide sequence (SEQ ID NO: 38) of the rearranged Vλ2-14 / Jλ2 variable region without the Vλ2-14 intron is shown. The Vλ2-14 sequence is shown in uppercase, with the Vλ2-14 start codon further underlined in italics; the Jλ2 sequence is shown in uppercase with a dashed underline. [Figure 16] The amino acid sequence (SEQ ID NO: 39) of the rearranged Vλ2-14 / Jλ2 variable domain containing the signal peptide is shown. The bolded italics indicate the sequence of the signal peptide. [Modes for carrying out the invention]

[0075] A brief description of the selected sequence in the sequence listing. Representative nucleotide and amino acid sequences of various human Vλ and Jλ gene segments that may be used in some embodiments of the non-human animals described herein are available from the International Immunogenetics Information System website, www.imgt.org, or from LeFranc, MP., The Immunoglobulin FactsBook, Academic Press, May 23, 2001 (hereinafter referred to as "LeFranc 2001").

[0076] The following are representative nucleotide and amino acid sequences of various mouse, rat, or human lambda constant regions or domains that may be used in some embodiments of non-human animals described herein. [Table 5] [Table 6] [Table 7] [Table 8] [Table 9-1] [Table 9-2] [Table 10]

[0077] The following are representative nucleotide and amino acid sequences of mouse, rat, or human kappa constant regions or domains that may be used in some embodiments of non-human animals described herein.

[0078] Mouse Cκ nucleotide sequence (SEQ ID NO: 25) (reproduced in Figure 7):

[0079] GGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGG CAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCC ACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAGagacaaaggtcctgagacgccaccaccagctccccagctccatcctatcttcccttctaaggtcttggaggcttcc ccacaagcgacctaccactgttgcggtgctccaaacctcctccccacctccttctcctcctcctccctttccttggcttttatcatgctaatatttgcagaaaatattcaataaagtgagtctttgcacttga

[0080] Regarding sequence number 25, the following applies: -The coding sequence is shown in bold; -3' Untranslated areas are not in bold.

[0081] Amino acid sequence of mouse Cκ (SEQ ID NO: 26):

[0082] ADAAPTVSIFPPSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0083] Rat Cκ nucleotide sequence (SEQ ID NO: 27) (reproduced in Figure 8):

[0084] GGGCTGATGCTGCACCAACTGTATCTATCTTCCCACCATCCACGGAACAGTTAGCAACTGGAGGTGCCTCAGTCGTGTGCCTCATGAACAACTTCTATCCCAGAGACATCAGTGTCAAGTGGAAGATTGA TGGCACTGAACGACGAGATGGTGTCCTGGACAGTGTTACTGATCAGGACAGCAAAGACAGCACGTACAGCATGAGCAGCACCCTCTCGTTGACCAAGGCTGACTATGAAAGTCATAACCTCTATACCTGT GAGGTTGTTCATAAGACATCATCCTCACCCGTCGTCAAGAGCTTCAACAGGAATGAGTGTTAGACCCAAAGGTCCTGAGGTGCCACCTGCTCCCCAGCTCCTTCCAATCTTCCCTCCTAAGGTCTTGGAG ACTTCCCCACAAGCGACCTACCACTGTTGCGGTGCTCCAAACCTCCTCCCCACCTCATCCTCCTTCCTTTCCTTGGCTTTGATCATGCTAATATTTGGGGAATATTAAATAAAGTGAATCTTTGCACTTGA

[0085] Regarding sequence number 27, the following applies: -The coding sequence is shown in bold; -3' Untranslated areas are not in bold.

[0086] Amino acid sequence of rat Cκ (SEQ ID NO: 28):

[0087] ADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVKWKIDGTERRDGVLDSVTDQDSKDSTYSMSSTLSLTKADYESHNLYTCEVVHKTSSSPVVKSFNRNEC

[0088] Human Cκ nucleotide sequence (SEQ ID NO: 29):

[0089] GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGA GAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0090] Amino acid sequence of human Cκ (SEQ ID NO: 30):

[0091] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0092] The nucleotide sequence of the modified Vλ1-51 / Jλ2 vector (SEQ ID NO: 31) (schematically shown in Figure 1A and reproduced in Figures 9A-9C):

[0093] [ka] [ka] [ka]

[0094] Regarding sequence number 31, the following applies: The restriction enzyme sites—AscI (5' end of the sequence) and PI-SceI (3' end of the sequence)—are indicated in uppercase italics; The -Vλ1-51 promoter sequence is indicated by lowercase (non-bold) letters; -Vλ1-51' 5'UTR sequence is shown in uppercase italics and bold; -The rearranged Vλ1-51 / Jλ2 sequence includes: The exon 1 sequence of Vλ1-51 is shown in uppercase (non-bold) letters, and the exon 1 start codon of Vλ1-51 is further underlined in italics; The intron 1 sequence of Vλ1-51 is shown in lowercase bold; The exon 2 sequence of Vλ1-51 is indicated by uppercase, bold, and underlined (solid underlined) letters. The Jλ2 sequence is indicated by uppercase, bold, and underlined (or dashed underlined) letters; -The human Jκ5-Cκ intron sequence is shown in lowercase italics.

[0095] The nucleotide sequence of the rearranged Vλ1-51 / Jλ2 variable region including the Vλ1-51 intron (SEQ ID NO: 32) (reproduced in Figure 10):

[0096] [ka]

[0097] Regarding sequence number 32, the following applies: The exon 1 sequence of -Vλ1-51 is shown in uppercase, and the exon 1 start codon of Vλ1-51 is further underlined in italics; The intron 1 sequence of -Vλ1-51 is indicated by lowercase bold letters; The exon 2 sequence of -Vλ1-51 is indicated by uppercase, bold, and underlined (solid underlined) letters. -Jλ2 sequences are indicated by uppercase, bold, and underlined (or dashed underlined) letters.

[0098] The nucleotide sequence of the rearranged Vλ1-51 / Jλ2 variable region without the Vλ1-51 intron (SEQ ID NO: 33) (reproduced in Figure 11):

[0099] [ka]

[0100] Regarding sequence number 33, the following applies: -The Vλ1-51 coding sequence is shown in uppercase, and the Vλ1-51 start codon is further underlined in italics; The -Jλ2 sequence is indicated by uppercase letters with a dashed underline.

[0101] The amino acid sequence of the rearranged Vλ1-51 / Jλ2 variable domain containing the signal peptide (SEQ ID NO: 34) (reproduced in Figure 12):

[0102] [ka]

[0103] Regarding Sequence ID No. 34, the italicized bold text indicates the sequence of the signal peptide.

[0104] Amino acid sequence of the rearranged Vλ1-51 / Jλ2 variable domain lacking the signal peptide (SEQ ID NO: 35):

[0105] QSVLTQPPSVSAAPGQKVTISSCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKLTVL

[0106] The nucleotide sequence of the modified Vλ2-14 / Jλ2 vector (SEQ ID NO: 36) (schematically shown in Figure 2A and reproduced in Figures 13A-13C): [ka] [ka]

[0107] Regarding sequence number 36, the following applies: The restriction enzyme sites—AscI (5' end of the sequence) and PI-SceI (3' end of the sequence)—are indicated in uppercase italics; The -Vλ1-51 promoter sequence is indicated by lowercase (non-bold) letters; -Vλ1-51' 5'UTR sequence is shown in uppercase italics and bold; -The rearranged Vλ2-14 / Jλ2 sequence includes: The exon 1 sequence of Vλ2-14 is shown in uppercase (non-bold) letters, and the exon 1 start codon of Vλ2-14 is further underlined in italics; The intron 1 sequence of Vλ2-14 is shown in lowercase bold; The exon 2 sequence of Vλ2-14 is indicated by uppercase, bold, and underlined (solid underlined) letters. The Jλ2 sequence is indicated by uppercase, bold, and underlined (or dashed underlined) letters; The human Jκ5-Cκ intron sequence is shown in lowercase italics.

[0108] The nucleotide sequence of the rearranged Vλ2-14 / Jλ2 variable region containing the Vλ2-14 intron (SEQ ID NO: 37) (reproduced in Figure 14): [ka] [ka]

[0109] Regarding sequence number 37, the following applies: The exon 1 sequence of -Vλ2-14 is shown in uppercase, and the exon 1 start codon of Vλ2-14 is further underlined in italics; The intron 1 sequence of -Vλ2-14 is shown in lowercase bold; The exon 2 sequence of -Vλ1-51 is indicated by uppercase, bold, and underlined (solid underlined) letters; -Jλ2 sequences are indicated by uppercase, bold, and underlined (or dashed underlined) letters.

[0110] The nucleotide sequence of the rearranged Vλ2-14 / Jλ2 variable region without the Vλ2-14 intron (SEQ ID NO: 38) (reproduced in Figure 15):

[0111] [ka]

[0112] Regarding sequence number 38, the following applies: The Vλ2-14 sequence is indicated by uppercase letters, and the Vλ2-14 start codon is further underlined in italics; The -Jλ2 sequence is indicated by uppercase letters with a dashed underline.

[0113] The amino acid sequence of the rearranged Vλ2-14 / Jλ2 variable domain containing the signal peptide (SEQ ID NO: 39) (reproduced in Figure 16):

[0114] [ka]

[0115] Regarding Sequence ID No. 39, the italicized bold text indicates the sequence of the signal peptide.

[0116] Amino acid sequence of the rearranged Vλ2-14 / Jλ2 variable domain lacking the signal peptide (SEQ ID NO: 40):

[0117] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVVFGGGTKLTVL

[0118] definition The scope of the present invention is defined by the claims appended herein and is not limited by the embodiments described herein. A person skilled in the art reading this specification will recognize a variety of modifications that are equivalent to or otherwise within the scope of the claims of such embodiments. Terms used herein generally follow their understood meanings in the art unless otherwise explicitly indicated. Clear definitions of the terms given herein are provided below; the meanings of these and other terms in specific examples throughout this specification will be evident to a person skilled in the art from the context. Additional definitions for the following and other terms are given throughout this specification. References to patents and non-patent literature or their relevant portions cited herein are incorporated herein by reference in their entirety.

[0119] The use of ordinal terms such as “first,” “second,” and “third” to modify elements of a claim in the claims does not in itself imply any priority, priority, or order of one element of a claim relative to another, or a temporal order in which the actions of the method are performed, but is merely used as a marker to distinguish one element of a claim having a given name from another element having the same name (if ordinal terms are used).

[0120] The articles “a” and “an,” as used herein, should be understood to include multiple references unless otherwise clearly indicated. Claims or statements containing “or” between one or more members of a group are considered satisfied if, unless otherwise indicated or otherwise evident from the context, one, more, or all members of the group are present, used, or otherwise related in a given product or process. In some embodiments, exactly one member of the group is present, used, or otherwise related in a given product or process. In some embodiments, multiple or all members of the group are present, used, or otherwise related in a given product or process. It should be understood that, unless otherwise indicated or otherwise evident to those skilled in the art, the present invention encompasses all types, combinations, and reinterpretations introduced in one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims into another claim (or any other claim, where relevant) dependent on the same basic claim. Where elements are presented as a list (e.g., a Markush group or similar format), each subgroup of the element is also disclosed, and any element(s) may be removed from the group. Generally, where an embodiment or aspect is referred to as "containing" a particular element, feature, etc., it should be understood that a given embodiment or aspect "consists of" or "essentially consists of" such element, feature, etc. For the sake of simplification, these embodiments are not explicitly shown in every case in a great many words as described herein. It should also be understood that any embodiment or aspect may be expressly excluded from the claims, whether or not a particular exclusion is described herein.

[0121] Administration: As used herein, this includes the administration of a composition (e.g., an antigen or antibody) to a subject or system (e.g., to cells, organs, tissues, organisms, or related components or sets of components thereof). Those skilled in the art will understand that the route of administration may vary depending on, for example, the subject or system to which the composition is administered, the characteristics of the composition, the purpose of administration, etc. For example, in a given embodiment, administration to an animal subject (e.g., a human or a rodent) may be bronchial (including by bronchial infusion), oral cavity, intestinal, interdermal, intra-arterial, intradermal, gastric, intramedullary, intramuscular, intra-nasal, intraperitoneal, intramedullary, intravenous, intraventricular, mucous membrane, nose, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal infusion), percutaneous, vaginal and / or vitreous. In some embodiments, administration may include intermittent dosing. In some embodiments, administration may include continuous dosing (e.g., perfusion) for at least a selected period.

[0122] Antigen-binding protein: As used herein, refers to any protein or polypeptide that specifically binds to at least one target antigen. Antigen-binding proteins include, but are not limited to, antibodies, heavy chains, light chains (e.g., λ or κ light chains), heavy chain variable domains, light chain variable domains (e.g., λ or κ light chain variable domains), and single-stranded variable fragments (ScFv). In some embodiments, antigen-binding proteins are multispecific and can specifically bind to two or more epitopes or antigens.

[0123] Approximately: When applied to one or more values, this includes values ​​similar to the described reference value. In a given embodiment, the term "approximately" or "about" refers to a range of values ​​that fall within ±10% of (higher or lower than) the described reference value, unless otherwise stated or evident from the context (unless such a number would exceed 100% of the possible values).

[0124] Biologically active: As used herein, this refers to the characteristic of any substance that is active in a biological system, in vitro or in vivo (e.g., in an organism). For example, a substance that has a biological effect within an organism when present in that organism is considered biologically active. In certain embodiments, if a protein or polypeptide is biologically active, the portion of the protein or polypeptide that shares at least one biological activity is typically referred to as the “biologically active” portion.

[0125] Equivalent: As used herein, equivalent means two or more substances, entities, situations, sets of states, etc., which may not be identical to one another but are similar enough to allow comparison between them so that conclusions can be reasonably drawn based on observed differences or similarities. A person skilled in the art will understand, in context, what degree of identity is required for two or more such substances, entities, situations, sets of states, etc., to be considered equivalent in any given situation.

[0126] Conservative: As used herein, when referring to a conservative amino acid substitution, it refers to examples of substitutions of an amino acid residue with another amino acid residue having a side-chain R group having similar chemical properties (e.g., charge or hydrophobicity). Typically, conservative amino acid substitutions do not substantially alter the functional properties of the protein in question, such as its ability to bind to a ligand or act as a receptor. Examples of amino acid groups having side chains with similar chemical properties include: aliphatic side chains, e.g., glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (Ile, I); aliphatic-hydroxyl side chains, e.g., serine (Ser, S) and threonine (Thr, T); amide-containing side chains, e.g., asparagine (Asn, N) and glutamine (Gln, Q); and aromatic side chains. These include aromatic side chains, such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains, such as lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains, such as aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfur-containing side chains, such as cysteine ​​(Cys, C) and methionine (Met, M). Conservative amino acid substituents include, for example, valine / leucine / isoleucine (Val / Leu / Ile, V / L / I), phenylalanine / tyrosine (Phe / Tyr, F / Y), lysine / arginine (Lys / Arg, K / R), alanine / valine (Ala / Val, A / V), glutamic acid / aspartic acid (Glu / Asp, E / D), and asparagine / glutamine (Asn / Gln, N / Q). In some embodiments, the conservative amino acid substitution may be a substitution of any native residue in a protein containing alanine, for example, as used in alanine scanning mutagenesis. In some embodiments, the conservative substitution has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445 (which is incorporated herein by reference in its entirety). In some embodiments, the substitution is a moderately conservative substitution, in which case the substitution has a non-negative value in the PAM250 log-likelihood matrix.

[0127] Control: As used herein, “control” refers to the meaning as understood in the art of “control,” which is a standard against which results are compared. Typically, controls are used to enhance the integrity of an experiment by isolating such variable in order to draw conclusions about that variable. In some embodiments, a control is a reaction or assay performed concurrently with the test reaction or assay to provide a comparison element. “Control” also includes “control animals.” “Control animals” may have modifications described herein, may have different modifications than those described herein, or may not have modifications (i.e., wild-type animals). In one experiment, a “test” parameter (e.g., the variable being tested) is applied. In another experiment, the “control,” which is the variable being tested, is not applied. In some embodiments, a control is an existing control (i.e., a test or assay already performed, or of a known quantity or result). In some embodiments, a control is or includes a printed or otherwise stored record. A control may be a positive control or a negative control.

[0128] Disruption, as used herein, refers to the result of a homologous recombination event in a DNA molecule (e.g., in an endogenous homologous sequence, e.g., a gene or gene locus). In some embodiments, disruption may achieve or represent an insertion, deletion, substitution, replacement, missense mutation, or frameshift of a DNA sequence(s), or any combination thereof. Insertions may include insertions of an entire gene or gene fragment, e.g., an exon, which may be of a non-endogenous origin (e.g., a heterologous sequence). In some embodiments, disruption may increase the expression and / or activity of a gene or gene product (e.g., a polypeptide encoded by the gene). In some embodiments, disruption may decrease the expression and / or activity of a gene or gene product. In some embodiments, disruption may alter the sequence of a gene or gene product (e.g., an encoded polypeptide). In some embodiments, disruption may cleave or fragment a gene or gene product (e.g., an encoded polypeptide). In some embodiments, disruption may elongate a gene or gene product. In some such embodiments, disruption may achieve the construction of a fusion polypeptide. In some embodiments, disruption may affect the level of a gene or gene product, but not its activity. In some embodiments, disruption may affect the activity of a gene or gene product, but not its level. In some embodiments, disruption may have no significant effect on the level of a gene or gene product. In some embodiments, disruption may have no significant effect on the activity of a gene or gene product. In some embodiments, disruption may have no significant effect on the level or activity of a gene or gene product.

[0129] Determining, measuring, assessing, evaluating, assaying, and analyzing: used interchangeably herein to refer to any form of measurement, including determining whether an element is present or not. These terms include both quantitative and / or qualitative determinations. Assays can be relative or absolute. "Assay on the presence" can mean determining the amount of something that is present and / or determining whether it is present or not.

[0130] Endogenous promoter: As used herein, this refers to a promoter that is naturally associated with an endogenous gene, for example, one found in wild-type organisms.

[0131] Modified: As used herein, this usually refers to an embodiment that has been manipulated by human hands. For example, in some embodiments, a polynucleotide may be considered “modified” if two or more sequences that are not linked to each other in their natural order are manipulated by human hands so that they are directly linked to each other in the modified polynucleotide. In some embodiments, a modified polynucleotide may include a regulatory sequence that is found functionally related to a first coding sequence in nature but not functionally related to a second coding sequence, and which has been manipulated by human hands to be functionally related to a second coding sequence. Alternatively or additionally, in some embodiments, first and second nucleic acid sequences encoding polypeptide elements or domains that are not linked to each other in nature, respectively, may be linked to each other in a single modified polynucleotide. Similarly, in some embodiments, a cell or organism may be considered “modified” if it has been manipulated so that its genetic information is altered (e.g., if new genetic material that was not previously present is introduced, or if previously present genetic material is altered or removed). As is common practice and as will be understood by those skilled in the art, modified polynucleotides or cellular offspring are typically referred to as “modified” even if the actual operation has been performed on a prior entity. Furthermore, as will be understood by those skilled in the art, a variety of methodologies are available through which the “modification” described herein can be achieved. For example, in some embodiments, “modification” may include the selection or design (e.g., nucleic acid sequences, polypeptide sequences, cells, tissues, and / or organisms) via the use of a computer system programmed to perform analysis or comparison, or otherwise to analyze, recommend, and / or select sequences, changes, etc. Alternatively or additionally, in some embodiments, “modification” may include the use of any of the following: in vitro chemical synthesis methodologies and / or recombinant nucleic acid technologies, e.g., nucleic acid amplification (e.g., via polymerase chain reaction), hybridization, mutation, transformation, transfection, etc., and / or a variety of control mating methodologies.As will be understood by those skilled in the art, a variety of established techniques for such purposes (e.g., recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection, etc.)) are well known in the art and are described in various general and more specific references cited and / or discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and Principles of Gene Manipulation: An Introduction to Genetic Manipulation, 5th Ed., ed. By Old, RW and SBPrimrose, Blackwell Science, Inc., 1994 (these in their entirety are incorporated herein by reference).

[0132] Functionality: As used herein, this refers to a form or fragment (e.g., a gene or gene segment) of an entity that exhibits specific properties (e.g., forms part of a coding sequence) and / or activity. For example, in the context of immunoglobulins, the variable region is encoded by its own gene segments (i.e., V, D, and / or J) constructed (or recombined) to form a functional coding sequence. Where present in the genome, gene segments are organized in clusters, but variations exist. A “functional” gene segment is a gene segment that appears in an expressed sequence (i.e., a variable region) and whose corresponding genomic DNA has been isolated (i.e., cloned) and identified by sequence. Some immunoglobulin gene segment sequences, while not appearing in the expressed repertoire, contain open reading frames and are considered functional, whereas other immunoglobulin gene segment sequences contain mutations (e.g., point mutations, insertions, deletions, etc.), resulting in stop codons and / or cleaved sequences, which subsequently prevent such gene segment sequences from exhibiting properties and / or activities associated with non-mutated sequences. Such sequences do not appear in the expressed sequences and are therefore classified as pseudogenes.

[0133] Gene: As used herein, a gene refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes a coding sequence (i.e., a sequence that codes for a particular product). In some embodiments, a gene includes a non-coding sequence. In some specific embodiments, a gene may include both coding (e.g., exons) and non-coding (e.g., introns) sequences. In some embodiments, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that can control or influence one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.). For the sake of clarity, we recognize that, as used in this disclosure, the term “gene” usually refers to a portion of a nucleic acid that codes for a polypeptide or a fragment thereof; the term may optionally include regulatory sequences, as will be apparent to those skilled in the art from the context. This definition is not intended to exclude the application of the term “gene” to non-protein-coding expression units, but rather to clarify that, in most cases, the term as used in this document refers to a nucleic acid that codes for a polypeptide.

[0134] Genetically modified non-human animals or genetically modified non-human animals: Used interchangeably herein, these terms refer to any non-human animal (e.g., a rodent, e.g., a rat or mouse) that does not exist in nature and in which one or more cells of the non-human animal contain, in whole or in part, a heterologous nucleic acid and / or gene encoding the polypeptide of interest. For example, in some embodiments, “genetically modified non-human animal” or “genetically modified non-human animal” refers to a non-human animal containing the transgene or transgene construct described herein. In some embodiments, the heterologous nucleic acid and / or gene is introduced into cells directly or indirectly by introduction into precursor cells using a well-thought-out genetic engineering technique, for example, by microinjection or infection with a recombinant virus. The term genetic engineering does not include classical mating techniques and rather refers to the introduction of recombinant DNA molecules(s). These molecules may be incorporated into chromosomes. The term “genetically modified non-human animal” or “genetically modified non-human animal” refers to an animal that is heterozygous or homozygous with respect to a different nucleic acid and / or gene, and / or an animal that has one or more copies of a different nucleic acid and / or gene.

[0135] Germline configuration: As used herein, this refers to the arrangement of sequences (e.g., gene segments) found in the endogenous germline genome of wild-type animals (e.g., mice, rats, or humans). An example of germline configuration of an immunoglobulin gene segment can be found, for example, in LeFranc, MP., The Immunoglobulin FactsBook, Academic Press, May 23, 2001 (referred to herein as "LeFranc 2001"): Exemplary configurations of human heavy chain variable region gene segments and human heavy chain constant region genes can be seen on page 47 of LeFranc 2001; Exemplary configurations of human λ light chain variable region gene segments and human λ light chain constant region genes can be seen on page 61 of LeFranc 2001; Exemplary configurations of human κ-light chain variable region gene segments and human κ-light chain constant region genes can be seen on page 53 of LeFranc 2001; Exemplary structures of mouse heavy chain variable region gene segments and mouse heavy chain constant region genes are described in Lucas, J. et al., Chapter 1: The Structure and Regulation of the Immunoglobulin Loci, Molecular Biology of B Cells, 2 nd It can be seen in Edition, Academic Press, 2015 (Lucas); Exemplary structures of mouse λ light chain variable region gene segments and mouse λ light chain constant region genes are shown in LeFranc, MP et al., Chapter 4: Immunoglobulin Lambda (IGL) Genes of Human and Mouse, Molecular Biology of B Cells, 1 st It can be seen in Edition, Academic Press, 2004 (LeFranc 2004); Exemplary structures of mouse κ light chain variable region gene segments and mouse κ light chain constant region genes can be found in Christele, MJ, et al., Nomenclature and Overview of the Mouse (Mus musculus and Mus sp.) Immunoglobulin Kappa (IGK) Genes, Exp Clin Immunogenet 2001, 18:255-279 (Christele); Each of the cited sections of LeFranc 2001, Lucas, LeFranc 2004, and Christele is incorporated herein by reference.

[0136] Germline genome: As used herein, this refers to the genome found in embryonic cells used in animal formation (e.g., gametes, e.g., sperm or eggs). The germline genome is the source of genomic DNA for cells in an animal. Thus, an animal (e.g., a mouse or rat) that has modifications in its germline genome is considered to have modifications in all of the genomic DNA of its cells.

[0137] Germline sequence: As used herein, refers to a DNA sequence found in the endogenous germline genome of a wild-type animal (e.g., mouse, rat, or human), or an RNA or amino acid sequence encoded by a DNA sequence found in the endogenous germline genome of an animal (e.g., mouse, rat, or human). Representative germline sequences of immunoglobulin gene segments can be found, for example, in LeFranc 2001: Human V may be used in some embodiments described herein. H Representative germline nucleotide sequences of gene segments and human V H Representative germline amino acid sequences of gene segments can be found on pages 107–234 of LeFranc 2001; Representative germline nucleotide sequences and representative germline amino acid sequences of human D gene segments that may be used in some embodiments described herein can be found on pages 98-100 of LeFranc 2001; Human J may be used in some embodiments described herein. H Representative germline nucleotide sequences of gene segments and human J H Representative germline amino acid sequences of gene segments can be found on page 104 of LeFranc 2001; Representative germline nucleotide sequences and representative germline amino acid sequences of human Vλ gene segments that may be used in some embodiments of non-human animals described herein can be found on pages 350-428 of LeFranc 2001; and Representative germline nucleotide sequences and representative germline amino acid sequences of human Jλ gene segments that may be used in some embodiments of non-human animals described herein can be found on page 346 of LeFranc 2001. Each of the cited sections of LeFranc 2001 is incorporated herein by reference.

[0138] Heterogeneous: As used herein, refers to a substance or entity from a different source. For example, when used in relation to a polypeptide, gene, or gene product present in a particular cell or organism, the term clarifies that the polypeptide, gene, or gene product in question is 1) modified by human hands; 2) introduced into a cell or organism (or its precursor) via human hands (e.g., via genetic modification); and / or 3) not produced in nature by, nor present in, the cell or organism in question (e.g., the cell type or organism type). Heterogeneous also includes polypeptides, genes, or gene products that are normally present in a particular native cell or organism but are not, for example, naturally occurring, and in some embodiments, altered or modified by mutation or arrangement under the control of non-endogenous regulatory elements (e.g., promoters).

[0139] Host cell: As used herein, refers to a cell into which a nucleic acid or protein has been introduced. A person skilled in the art who reads this disclosure will understand that such a term is used not only to refer to a specific cell of interest but also to the offspring of such a cell. Such offspring may not be identical to the parent cell in practice, because certain modifications may occur in subsequent generations due to either mutation or environmental influences, but they still fall within the scope of the phrase “host cell.” In some embodiments, the host cell is or includes a prokaryotic or eukaryotic cell. Typically, the host cell is any cell suitable for receiving and / or producing heterologous nucleic acids or proteins, regardless of the biological kingdom to which the cell is designated. Exemplary cells include those of prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., lineages such as Escherichia coli, Bacillus spp., Streptomyces spp.), Mycobacterium cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells.In some embodiments, the cells are eukaryotic and selected from the following cells: Chinese hamster ovary (CHO) (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cells contain one or more viral genes, for example, retinal cells expressing viral genes (e.g., PER.C6® cells). In some embodiments, the host cells are isolated cells or include them. In some embodiments, the host cells are part of a tissue. In some embodiments, the host cells are part of an organism.

[0140] Identity: When used herein in conjunction with sequence comparison, this refers to identity determined by a number of different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments, the identity described herein is determined using ClustalW v.1.83(slow) alignment with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a Gonnet similarity matrix (MACVECTOR® 10.0.2, MacVector Inc., 2008).

[0141] Instead of: As used herein, "instead of" refers to a positional substitution in which the first nucleic acid sequence is located at the position of the second nucleic acid sequence on the chromosome (e.g., the location where the second nucleic acid sequence was previously (e.g., originally) located on the chromosome, e.g., at the endogenous locus of the second nucleic acid sequence). The phrase "instead of" does not require that the second nucleic acid sequence be removed from, for example, a locus or chromosome. In some embodiments, the second nucleic acid sequence and the first nucleic acid sequence are equivalent in that, for example, the first and second sequences are homologous to each other, contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.), and / or have similar or identical sequences. In some embodiments, the first and / or second nucleic acid sequences include one or more of the following: promoter, enhancer, splice donor site, splice acceptor site, intron, exon, or untranslated region (UTR); in some embodiments, the first and / or second nucleic acid sequences include one or more coding sequences. In some embodiments, the first nucleic acid sequence is a homolog or variant (e.g., a mutant) of the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence is an orthologue or homolog of the second sequence. In some embodiments, the first nucleic acid sequence is or includes a human nucleic acid sequence. In some embodiments, including when the first nucleic acid sequence is or includes a human nucleic acid sequence, the second nucleic acid sequence is or includes a rodent sequence (e.g., a mouse or rat sequence). In some embodiments, including when the first nucleic acid sequence is or includes a human nucleic acid sequence, the second nucleic acid sequence is a human sequence. In some embodiments, the first nucleic acid sequence is a variant or mutant of the second sequence (i.e., a sequence containing one or more sequence differences, e.g., substitutions, compared to the second sequence). The nucleic acid sequence thus arranged may include one or more control sequences (e.g., promoters, enhancers, 5' or 3' untranslated regions, etc.) that are part of a nucleic acid sequence source used to obtain the sequence thus arranged.For example, in various embodiments, the first nucleic acid sequence is a substitution of the endogenous sequence with a heterologous sequence that results in the generation of a gene product from the nucleic acid sequence (including heterologous sequences) arranged in this manner, but does not result in the expression of the endogenous sequence; the first nucleic acid sequence is an endogenous genome sequence having a nucleic acid sequence that encodes a polypeptide having a similar function to the polypeptide encoded by the endogenous sequence (for example, the endogenous genome sequence encodes a non-human variable region polypeptide, either whole or partially, and the DNA fragment encodes one or more human variable region polypeptides, either whole or partially). In various embodiments, a human immunoglobulin gene segment or a fragment thereof is used instead of the endogenous non-human immunoglobulin gene segment or fragment.

[0142] In vitro: As used herein, this refers to events that occur in an artificial environment, such as a test tube or reaction vessel, or in a cell culture, rather than within a multicellular organism.

[0143] In vivo: As used herein, refers to events occurring within multicellular organisms such as humans and / or non-human animals. In the context of cell-based systems, the term may be used to refer to events occurring within living cells (as opposed to, for example, in vitro systems).

[0144] Isolated: As used herein, means (1) a substance or entity that has been separated (either naturally or in a laboratory setting) from at least a portion of the components to which it was originally formed (when it was first produced), and / or (2) a substance or entity that has been designed, produced, prepared, and / or manufactured by human hands. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components to which they were originally formed. In some embodiments, the isolated substances are separated from 10%-100%, 15%-100%, 20%-100%, 25%-100%, 30%-100%, 35%-100%, 40%-100%, 45%-100%, 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 96%-100%, 97%-100%, 98%-100%, or 99%-100% of the other components they were originally related to. In some embodiments, the isolated substances are separated from 10%-100%, 10%-99%, 10%-98%, 10%-97%, 10%-96%, 10%-95%, 10%-90%, 10%-85%, 10%-80%, 10%-75%, 10%-70%, 10%-65%, 10%-60%, 10%-55%, 10%-50%, 10%-45%, 10%-40%, 10%-35%, 10%-30%, 10%-25%, 10%-20%, or 10%-15% of the other components they were originally related to. In some embodiments, the isolated substances are separated from 11%-99%, 12%-98%, 13%-97%, 14%-96%, 15%-95%, 20%-90%, 25%-85%, 30%-80%, 35%-75%, 40%-70%, 45%-65%, 50%-60%, or 55%-60% of the other components they were originally related to. In some embodiments, the isolated substances are ultrapure of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99%.In some embodiments, the isolated substance is 80%–99%, 85%–99%, 90%–99%, 95%–99%, 96%–99%, 97%–99%, or 98%–99% pure. In some embodiments, the isolated substance is 80%–99%, 80%–98%, 80%–97%, 80%–96%, 80%–95%, 80%–90%, or 80%–85% pure. In some embodiments, the isolated substance is 85%–98%, 90%–97%, or 95%–96% pure. In some embodiments, the substance is “pure” if it substantially contains no other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered “isolated” or even “pure” after being combined with a predetermined other component, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the degree of isolation or purity of the substance is calculated without such carriers or excipients. To give just one example, in some embodiments, a biological polymer such as a naturally occurring polypeptide or polynucleotide is considered “isolated” if, by factors of its inducing origin or source, it is not associated with some or all of the components that accompany it in its natural state; b) it substantially does not contain other polypeptides or nucleic acids of the same species that produces it in nature; or c) it is expressed by a cell or other expression system that is not of the species that produces it in nature, or otherwise associated with components from that cell or other expression system. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from that which produces it in nature is considered an “isolated” polypeptide. Alternatively, or additionally, in some embodiments, polypeptides subjected to one or more purification techniques may be considered “isolated” polypeptides to the extent that they are a) naturally associated and / or b) separated from other components that were associated with them when they were first produced.

[0145] Locus or loci: As used herein, refers to the location(s) of a gene, DNA sequence, polypeptide-coding sequence, or location on a chromosome in the genome of an organism. For example, “immunoglobulin locus” may refer to an immunoglobulin gene segment (e.g., V, D, J, or C), an immunoglobulin gene segment DNA sequence, the location of a sequence encoding an immunoglobulin gene segment, or the location of an immunoglobulin gene segment on a chromosome in the genome of an organism identified in relation to the location of such sequence. An “immunoglobulin locus” of an immunoglobulin gene segment may include, but not limited to, enhancers, promoters, 5' and / or 3' regulatory sequences or regions, or combinations thereof. An “immunoglobulin locus” may include intergeneric DNA, e.g., DNA that is normally present or appears between gene segments in a wild-type locus. Those skilled in the art will understand that chromosomes, in some embodiments, contain hundreds or even thousands of genes, and when compared between different species, physical colocalization of similar loci can be demonstrated. Such loci may be described as having shared synteny.

[0146] Naturally occurring: When used herein in reference to a biological element (e.g., a nucleic acid sequence), this means that the biological element can be found in a cell or organism (e.g., an animal) in the absence of modification (e.g., gene modification). That is, a sequence that naturally occurs in a particular situation and / or location is not present in that situation and / or location as a result of modification (e.g., gene modification). For example, a sequence that naturally occurs adjacent to the human Jκ1 gene segment at the endogenous human immunoglobulin kappa light chain locus is a sequence that can be found in humans, in the absence of gene modification, adjacent to the human Jκ1 gene segment at the endogenous human immunoglobulin kappa light chain locus. In some embodiments, a sequence may be obtained, induced, and / or isolated from a site where it naturally occurs in a cell or organism. In some embodiments, a cell or organism is not the direct source of a sequence that naturally occurs in a cell or organism. For example, the corresponding sequence in a cell or organism may be identified, and then produced or replicated, by mechanisms known in the art.

[0147] Non-human animals: As used herein, this refers to any vertebrate organism that is not human. In some embodiments, non-human animals are cyclostomes, bony fish, cartilaginous fish (e.g., sharks or rays), amphibians, reptiles, mammals, and birds. In some embodiments, non-human animals are mammals. In some embodiments, non-human mammals are primates, goats, sheep, pigs, dogs, cattle, or rodents. In some embodiments, non-human animals are rodents, e.g., rats or mice.

[0148] Nucleic acid: As used herein, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, “nucleic acid” refers to a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As is evident from the context, in some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” is or comprises RNA; in some embodiments, “nucleic acid” is or comprises DNA. In some embodiments, “nucleic acid” is one or more native nucleic acid residues, comprising or consisting thereof. In some embodiments, “nucleic acid” is one or more nucleic acid analogs, comprising or consisting thereof. In some embodiments, a nucleic acid analog differs from “nucleic acid” in that it does not utilize a phosphodiester backbone. For example, in some embodiments, “nucleic acid” is one or more “peptide nucleic acids” known in the art and having peptide bonds instead of phosphodiester bonds in their backbone, comprising or consisting thereof. Alternatively, or additionally, in some embodiments, the “nucleic acid” has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds rather than phosphodiester bonds. In some embodiments, the “nucleic acid” is one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), and comprises or consists of them.In some embodiments, “nucleic acid” is one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof), and comprises or consists of such. In some embodiments, the "nucleic acid" comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in natural nucleic acids. In some embodiments, the "nucleic acid" has a nucleotide sequence encoding a functional gene product, e.g., RNA or polypeptide. In some embodiments, the "nucleic acid" comprises one or more introns. In some embodiments, the "nucleic acid" comprises one or more exons. In some embodiments, the "nucleic acid" is prepared by one or more of the following: isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in recombinant cells or systems, and chemical synthesis. In some embodiments, “nucleic acid” is at least, for example, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residue lengths. In some embodiments, the "nucleic acid" is single-stranded; in some embodiments, the "nucleic acid" is double-stranded.In some embodiments, the "nucleic acid" has a nucleotide sequence comprising at least one element that encodes a polypeptide or is a complement to a sequence that encodes a polypeptide. In some embodiments, the "nucleic acid" has enzymatic activity.

[0149] Functionally linked: As used herein, this refers to the juxtaposition of components that are in a relationship that allows the components described to function in their intended manner (e.g., when the components are in the appropriate tissue, cell type, cell activity, etc.). For example, one or more V H A gene segment, one or more D gene segments, and one or more J H The gene segment is V H , D, and J HA gene segment is "functionally ligated" to the heavy chain constant region if it can be spliced ​​into the heavy chain constant region at an appropriate time in B cell development, regardless of whether splicing occurs in extraimmune cells (e.g., embryonic cells). A control sequence "functionally ligated" to a coding sequence is ligated in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequence. "Functionally ligated" sequences include both expression control sequences that are continuous with the gene of interest and expression control sequences that act trans or asynchronously to control the gene (or sequence of interest) of interest. The term "expression control sequence" includes polynucleotide sequences necessary to influence the expression and processing of the coding sequence being ligated. "Expression control sequences" include appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals, e.g., splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that improve polypeptide stability; and, if desired, sequences that improve polypeptide secretion. The characteristics of such control sequences vary depending on the host organism. For example, in prokaryotes, such control sequences typically include a promoter, ribosome binding site, and transcription termination sequence, whereas in eukaryotes, such control sequences typically include a promoter and transcription termination sequence. The term “control sequence” is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as a leader sequence and a fusion partner sequence.

[0150] Polypeptide: As used herein, refers to any polymer chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has an amino acid sequence that does not exist naturally. In some embodiments, the polypeptide has an amino acid sequence containing parts that exist separately from each other in nature (i.e., from two or more different organisms, e.g., human and non-human parts). In some embodiments, the polypeptide has an amino acid sequence that is modified in that it is designed and / or produced through the action of the human hand. In some embodiments, the polypeptide has an amino acid sequence that is encoded by a sequence that does not exist naturally (e.g., a sequence that is modified in that it is designed and / or produced through the action of the human hand to encode the polypeptide).

[0151] Reorganized: As used herein, by including two or more immunoglobulin gene segments that are conjugated together (directly or indirectly), the conjugated gene segments represent a DNA sequence that together has a DNA sequence encoding the variable region of an immunoglobulin. Two or more immunoglobulin gene segments of a reorganized DNA sequence are no longer associated with the function of a recombinant signal sequence (RSS) and thus cannot undergo further reorganization. While two or more immunoglobulin gene segments of a reorganized DNA sequence may not be able to undergo further reorganization, those skilled in the art will recognize that this does not mean that other immunoglobulin gene segments within the same locus cannot undergo, for example, secondary reorganization. Those skilled in the art will understand that a reorganized gene segment (e.g., in a reorganized immunoglobulin variable region) can be conjugated together via a natural VDJ recombination process. Those skilled in the art will also understand that a reorganized gene segment (e.g., in a reorganized immunoglobulin variable region) can be modified to be conjugated together, for example, by conjugating gene segments using standard recombination techniques. A rearranged immunoglobulin variable region typically contains two or more conjugated immunoglobulin gene segments. For example, a rearranged immunoglobulin λ light chain variable region may contain a Jλ gene segment and a conjugated Vλ gene segment. A rearranged immunoglobulin heavy chain variable region may contain a conjugated V H Gene segment, D gene segment, J H This may include gene segments. Those skilled in the art will also understand that all or substantially all intergenetic sequences are typically removed between immunoglobulin gene segments in the rearranged immunoglobulin variable region. Those skilled in the art will further understand that the rearranged sequences in gene segments may, among other things, include introns.

[0152] Recombination: As used herein, recombination refers to a molecule (e.g., DNA, RNA, or polypeptide) formed by a laboratory method of genetic recombination (e.g., cloning) to combine genetic material from multiple sources (e.g., organisms, tissues, cells, genomes, or parts of genomes).In some embodiments, recombinant polypeptides designed, modified, prepared, expressed, produced, or isolated by recombinant means include, for example, polypeptides expressed using recombinant expression vectors transfected into host cells, polypeptides isolated from recombinant combinatorial human polypeptide libraries (Hoogenboom, HR, 1997, TIB Tech. 15:62-70; Azzazy, H. and WE Highsmith, 2002, Clin. Biochem. 35:425-45; Gavilondo, JV and JW Arrick, 2002, BioTechniques 29:128-45; Hoogenboom H., and P. Chames, 2000, Immunol. Today 21:371-8 (the whole of these is incorporated herein by reference)), antibodies isolated from animals (e.g., mice) genetically modified to contain human immunoglobulin genes (e.g., Taylor, L et al., 1992, Nucl. Acids Res.20:6287-95;Kellermann,SA.and LLGreen,2002,Curr.Opin.Biotechnol.13:593-7;Little,M.et al.,2000,Immunol.Today 21:364-70;Osborn,MJet al.,2013,J.Immunol.190:1481-90;Lee,EC.et al.,2014,Nat.Biotech.32(4):356-63;Macdonald,LEet al.,2014,Proc.Natl.Acad.Sci.USA111(14):5147-52;Murphy,AJet Polypeptides prepared, expressed, constructed or isolated by al., 2014, Proc. Natl. Acad. Sci. USA 111(14):5153-8 (each of which is incorporated herein by reference in whole) or by any other means including splicing selected sequence elements together. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico.In some embodiments, one or more of these selected sequence elements are the result of mutagenesis (e.g., in vivo or in vitro) of known sequence elements from, for example, natural or synthetic (e.g., artificial) sources. For example, in some embodiments, the recombinant polypeptide contains sequences found in the genome of the biological source of interest (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide has amino acid sequences resulting from mutagenesis (e.g., in vitro or in vivo, e.g., in non-human animals), so that the amino acid sequence of the recombinant polypeptide is of origin and related to the polypeptide sequence, but may not be naturally present in the genome of non-human animals in vivo.

[0153] Reference: As used herein, a reference substance, animal, cohort, individual, population, sample, sequence, or value refers to a standard or control substance, animal, cohort, individual, population, sample, sequence, or value on which the subject substance, animal, cohort, individual, population, sample, sequence, or value is compared. In some embodiments, the reference substance, animal, cohort, individual, population, sample, sequence, or value is tested and / or determined substantially concurrently with the test or determination of the subject substance, animal, cohort, individual, population, sample, sequence, or value. In some embodiments, the reference substance, animal, cohort, individual, population, sample, sequence, or value is a past reference optionally embodied in a tangible medium. In some embodiments, a reference may refer to a control. "Reference" also includes "reference animal." The "reference animal" may have modifications described herein, modifications different from those described herein, or may not have modifications (i.e., a wild-type animal). Typically, as would be understood by those skilled in the art, the reference substance, animal, cohort, individual, population, sample, sequence, or value is determined or characterized under conditions equivalent to those used to determine or characterize the substance, animal (e.g., mammal), cohort, individual, population, sample, sequence, or value in question.

[0154] Substitution, as used herein, refers to the process by which a “replacement” nucleic acid sequence (e.g., a gene) found at a host locus (e.g., in the genome) is removed from that locus and a different “replacement” nucleic acid is placed in its place. In some embodiments, the replacement nucleic acid sequence and the replacement nucleic acid sequence are equivalent to each other in that they are homologous to each other, contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.), and / or have similar or identical sequences. In some embodiments, the replacement nucleic acid sequence includes one or more of the following: promoter, enhancer, splice donor site, splice acceptor site, intron, exon, or untranslated region (UTR); in some embodiments, the replacement nucleic acid sequence includes one or more coding sequences. In some embodiments, the replacement nucleic acid sequence is a homolog or variant (e.g., a mutant) of the replacement nucleic acid sequence. In some embodiments, the replacement nucleic acid sequence is an orthologue or homolog of the replacement sequence. In some embodiments, the replacement nucleic acid sequence is or includes a human nucleic acid sequence. In some embodiments where the replacement nucleic acid sequence is a human nucleic acid sequence, or includes one, the replacement nucleic acid sequence is a rodent sequence (e.g., a mouse or rat sequence). In some embodiments where the replacement nucleic acid sequence is a human nucleic acid sequence, or includes one, the replacement nucleic acid sequence is a human sequence. In some embodiments, the replacement nucleic acid sequence is a variant or variant of the replacement sequence (i.e., a sequence containing one or more sequence differences, e.g., substitutions, compared to the replacement sequence). The thus-configured nucleic acid sequence may include one or more control sequences (e.g., promoters, enhancers, 5' or 3' untranslated regions, etc.) that are part of a nucleic acid sequence source used to obtain the thus-configured sequence.For example, in various embodiments, the substitution is the replacement of an endogenous sequence with a heterologous sequence that results in the generation of a gene product from the nucleic acid sequence (including heterologous sequences) thus arranged, but does not result in the expression of the endogenous sequence; the substitution is an endogenous genomic sequence having a nucleic acid sequence that encodes a polypeptide having a similar function to the polypeptide encoded by the endogenous sequence (for example, the endogenous genomic sequence encodes a non-human variable region polypeptide, either whole or partially, and the DNA fragment encodes one or more human variable region polypeptides, either whole or partially). In various embodiments, an endogenous non-human immunoglobulin gene segment or a fragment thereof is replaced with a human immunoglobulin gene segment or a fragment thereof.

[0155] Substantially, as used herein, refers to a quantitative state indicating the whole or nearly whole range or degree of the feature or characteristic in question. Those skilled in the art in the biological field will understand that biological and chemical phenomena are, if not entirely, rarely completed and / or carried out completely or achieve or avoid absolute results. The term “substantially” is therefore used herein to capture the inherent potential lack of completeness in many biological and chemical phenomena.

[0156] Substantial similarity: As used herein, refers to a comparison between amino acid or nucleic acid sequences. As understood by those skilled in the art, two sequences are generally considered "substantial similar" if they contain similar residues (e.g., amino acids or nucleotides) at their corresponding positions. As understood in the art, similar residues may be identical residues (see substantial identity below), and similar residues may also be non-identical residues having roughly equivalent structural and / or functional characteristics. For example, as is well known by those skilled in the art, given amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids and / or having "polar" or "nonpolar" side chains. Substituting one amino acid for another of the same type can often be considered a "conservative" substitution. Typical amino acid classifications are summarized in the table below. [Table 1] [Table 2]

[0157] As is well known in the art, amino acid or nucleic acid sequences can be compared using a variety of algorithms available in commercially available computer programs, such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary programs of this nature are described in Altschul, SF et al., 1990, J.Mol.Biol., 215(3):403-10; Altschul, SF et al., 1996, Meth.Enzymol.266:460-80; Altschul, SF et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, AD and BFF Uellette (eds.) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.) Bioinformatics Methods and Protocols, Methods in Molecular Biology, Vol.132, Humana Press, 1998 (these in their entirety are incorporated herein by reference). In addition to identifying similar sequences, the above programs typically provide an indicator of the degree of similarity. In some embodiments, two sequences are considered substantially similar if at least, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of their corresponding residues are similar (e.g., identical or including conservative substitutions) across the associated extension of the residues. In some embodiments, the associated extension is a complete sequence (e.g., a gene sequence, gene segment, sequence encoding a domain, polypeptide, or domain). In some embodiments, the associated extension is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, or more residues.In some embodiments, the associated elongation consists of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues. In some embodiments, the associated elongation includes continuous residues along the complete sequence. In some embodiments, the associated elongation includes discontinuous residues along the complete sequence, such as discontinuous residues joined together by the folded conformation of a polypeptide or a portion thereof.

[0158] Substantially identical: As used herein, this refers to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered "substantially identical" if they contain identical residues (e.g., amino acids or nucleotides) at their corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of the various algorithms available in commercially available computer programs, such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary programs of this nature are described in Altschul, SF et al., 1990, J.Mol.Biol., 215(3):403-10; Altschul, SF et al., 1996, Meth.Enzymol.266:460-80; Altschul, SF et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, AD and BFFOuellette (eds.) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.) Bioinformatics Methods and Protocols, Methods in Molecular Biology, Vol.132, Humana Press, 1998 (each of these is incorporated herein by reference in its entirety). In addition to identifying identical sequences, the above programs typically provide an indicator of the degree of identity. In some embodiments, two sequences are considered substantially identical if at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical across the associated extensions of the residues. In some embodiments, the associated extensions of the residues are complete sequences.In some embodiments, the associated extension of the residue is, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.

[0159] Targeting construct or targeting vector: as used herein, refers to a polynucleotide molecule containing a targeting region. The targeting region contains a sequence identical or substantially identical to a sequence in a target cell, tissue, or animal, providing integration of the targeting construct into a location within the genome of the cell, tissue, or animal via homologous recombination. Targeting regions that are targeted using site-specific recombinase recognition sites (e.g., loxP or Frt sites) are also included and described herein. In some embodiments, the targeting construct described herein further includes a specific target nucleic acid sequence or gene, a selectable marker, a control and / or regulatory sequence, and other nucleic acid sequences that enable recombination mediated by the exogenous addition of proteins that assist or facilitate recombination containing such sequences. In some embodiments, the targeting construct described herein further includes a target gene, either whole or in part, which is a heterologous gene that whole or in part encodes a polypeptide having a function similar to the protein encoded by the endogenous sequence. In some embodiments, the targeting construct described herein further comprises, in whole or in part, a humanized gene of interest, which in whole or in part encodes a polypeptide having a function similar to that of the polypeptide encoded by the endogenous sequence. In some embodiments, the targeting construct (or targeting vector) may comprise a human-engineered nucleic acid sequence. For example, in some embodiments, the targeting construct (or targeting vector) may be constructed to comprise a modified or recombinant polynucleotide comprising two or more sequences that are not linked together in their natural order but have been human-engineered to be directly linked to one another in the modified or recombinant polynucleotide.

[0160] Transgene or transgene construct: As used herein, refers to a nucleic acid sequence (e.g., encoding a polypeptide of interest, either whole or in part) introduced into a cell by human means, for example, by the method described herein. A transgene may be partially or entirely heterologous, i.e., foreign, to the genetically modified animal or cell into which it is introduced. A transgene may include one or more transcriptional regulatory sequences and any other nucleic acids, such as introns or promoters, which may be necessary for the expression of the selected nucleic acid sequence.

[0161] Non-reorganized: As used herein, non-reorganized refers to a DNA sequence comprising two or more immunoglobulin gene segments that have not undergone a recombination event or are otherwise not conjugated, and therefore contain an intergenetic sequence(s) between them. Those skilled in the art will understand that non-reorganized V and J gene segments may be associated with intact recombination signal sequences (RSS). A non-reorganized D gene segment may be flanked by two intact recombination signal sequences (RSS). Those skilled in the art will further understand that non-reorganized gene segments may, among other things, contain introns.

[0162] Vector: As used herein, a vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. In some embodiments, vectors enable extrachromosomal replication and / or expression of the nucleic acids to which they are linked in a host cell, such as a eukaryotic and / or prokaryotic cell. A vector capable of inducing the expression of a functionally linked gene is referred to herein as an “expression vector.”

[0163] Wild-type: As used herein, "wild-type" refers to an entity having the structure and / or activity naturally occurring in a "normal" state or circumstances (as opposed to mutant, diseased, altered, modified, etc.). Those skilled in the art will understand that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles). (Modes for carrying out the invention)

[0164] This disclosure provides insight that endogenous antibody development mechanisms in non-human animals, including immunoglobulin chain pairing and affinity maturation, can be utilized to produce antigen-specific high-affinity antibodies containing exogenous human immunoglobulin sequences. Such animals can produce normal and robust immune responses, which can be utilized, for example, to produce human antibody therapeutics. This disclosure recognizes that genetically modified non-human animals provide an effective and efficient platform for producing antibodies containing human variable domains, including human heavy chain, κ light chain, and λ light chain domains. This disclosure further recognizes that genetically modified non-human animals can successfully utilize human heavy chain, κ light chain, and λ light chain variable region gene segments to produce affinity-matured human heavy chain, κ light chain, and λ light chain variable domains.

[0165] This disclosure recognizes that the generation of antibodies containing human λ light chain variable domains in non-human animals has previously presented challenges when λ light chain expression is low in a given non-human animal. For example, mice utilize significantly more κ light chains than λ light chains (i.e., with a κ:λ ratio of approximately 95:5). This disclosure further recognizes that the generation of antibodies containing universal light chains (e.g., light chains capable of binding to multiple heavy chains) by limiting the repertoire of light chain variable regions in non-human animals can be challenging because it excludes some of the most potent diversity generation mechanisms for high-affinity antibody generation, such as combinatorial diversity, conjugation diversity, and secondary rearrangement. It also minimizes diversity generation effects that would result from "mix and match" heavy and light chain pairing. From these perspectives, there is still a need in the art for platforms and methods for generating human λ light chain variable regions in non-human animals from a limited human λ light chain variable region.

[0166] This disclosure provides the insight that non-human animals (e.g., rodents, e.g., rats or mice) containing a limited repertoire of human λ light chain variable regions at the κ light chain locus can effectively produce high-affinity antigen-specific antibodies. This result is unexpected because the limited repertoire of human λ light chain variable regions in non-human animals would not allow them to utilize λ light chain variable region sequences. The use of λ light chain variable region sequences goes against the natural preference tendencies of a given non-human animal. As described above, the use of a limited repertoire of human λ light chain variable regions in non-human animals also ostracizes many natural mechanisms used to produce high-affinity antigen-specific antibodies.

[0167] This disclosure provides genetically modified non-human animals (e.g., rodents, e.g., rats or mice) expressing human immunoglobulin λ light chain variable domains, wherein the non-human animals have a limited human λ light chain variable region repertoire (including one or two human Vλ gene segments). In some embodiments, this disclosure provides genetically modified non-human animals expressing human immunoglobulin λ light chain variable domains, wherein the non-human animals have a limited human λ light chain variable repertoire and human immunoglobulin heavy chain variable domains. Also provided are biological systems for generating human λ light chain variable domains expressed from a limited human λ light chain variable region repertoire that associate with a diverse repertoire of affinity-matured human heavy chain variable domains. Methods are provided for producing antigen-binding proteins containing human immunoglobulin variable domains (e.g., antibodies, heavy chains, light chains (e.g., λ light chains), heavy chain variable domains, light chain variable domains (e.g., λ light chain variable domains), single-stranded variable fragments (ScFv)). In some embodiments, the method includes immunizing a non-human animal described herein with a target antigen. In some embodiments, the method includes using an immunoglobulin variable region gene sequence of a non-human animal described herein (e.g., a rodent, e.g., a rat or mouse) in an antigen-binding protein (e.g., a binding protein that specifically binds to a target antigen). The method includes a method for producing human immunoglobulin heavy chain and / or λ light chain variable domains suitable for use in the production of multispecific antigen-binding proteins.

[0168] Genetically modified non-human animals (e.g., rodents, e.g., rats or mice) are provided that express a limited repertoire of human λ light chain variable domains from a limited repertoire of human λ light chain variable region gene segments. In some embodiments, the non-human animals described herein are genetically modified to include a single rearranged human λ light chain variable region sequence (Vλ / Jλ sequence). In some embodiments, the non-human animals described herein are genetically modified to include only one or two human unrearranged λ light chain variable region gene segments. In some embodiments, the non-human animals described herein are genetically modified to include only one or two unrearranged human Vλ gene segments and one or more unrearranged human Jλ gene segments. In certain embodiments, the non-human animals described herein include four or five unrearranged human Jλ gene segments. The rearranged human λ light chain variable domains expressed by the non-human animals described herein are capable of pairing with multiple affinity-mature human heavy chains expressed by such non-human animals, and the multiple heavy chain variable regions are capable of specifically binding to different epitopes. In some embodiments, the non-human animals described herein express and select preferred affinity-matured human immunoglobulin heavy chain variable domains derived from a repertoire of unreorganized human heavy chain variable region gene segments, and these affinity-matured human heavy chain variable domains associate with and express human λ light chain variable domains derived from a limited repertoire of human immunoglobulin λ light chain variable region genes in the non-human animals. This disclosure provides insight that human λ light chain variable domains and human heavy chain variable domains (along with the encoding human λ light chain variable regions and human heavy chain variable regions) can be utilized in the production of multispecific antibodies, particularly bispecific antibodies.

[0169] Antibody repertoire in non-human animals Immunoglobulins (also called antibodies) are large (approximately 150 kD) Y-shaped glycoproteins produced by B cells of the host immune system to neutralize pathogens (e.g., viruses, bacteria, etc.). Each immunoglobulin (Ig) consists of two identical heavy chains and two identical light chains, each having two structural components: a variable domain and a constant domain. The heavy and light chain variable regions differ in antibodies produced by different B cells, but are the same for all antibodies produced by a single B cell or B cell clone. Together, the heavy and light chain variable regions of each antibody contain an antigen-binding region (or antigen-binding site). Immunoglobulins can exist in different types called isotypes or classes based on the heavy chain constant region (or domain) they contain. The heavy chain constant region is the same in all antibodies of the same isotype, but differs in antibodies of different isotypes. The table below summarizes the nine antibody isotypes in mouse and human. [Table 3]

[0170] Further isotypes have been identified in other species. Isotypes confer characteristic biological properties to antibodies due to different structural features between different isotypes and are found in different locations (cells, tissues, etc.) within the animal body. Initially, B cells produce IgM and IgD, which have identical antigen-binding domains. When activated, B cells switch to different isotypes through a process called class switching, which involves a change in the constant region of the antibody produced by the B cell, while preserving the antigen specificity of the original antibody (B cell) by keeping the variable region the same.

[0171] The two separate gene loci (Igκ and Igλ), when rearranged, contain gene segments encoding the antibody light chain and exhibit both allele and isotype exclusion. + Relative to λ +B cell expression ratios vary across species. For example, humans exhibit a ratio of approximately 60:40 (κ:λ). In mice and rats, a ratio of 95:5 (κ:λ) is observed. Interestingly, the κ:λ ratio observed in cats (5:95) is the reverse of that in mice and rats. Several studies have been conducted to elucidate the possible reasons behind these observed ratios, with both locus complexity (i.e., the number of gene segments, particularly V gene segments) and the efficiency of gene segment rearrangement being proposed as rationale. The human immunoglobulin λ light chain locus extends beyond 1,000 kb and contains approximately 70 Vλ gene segments (29–33 functionalities) and 7 Jλ-Cλ gene segment pairs (4–5 functionalities) organized into three clusters (see, e.g., Figure 1 of U.S. Patent No. 9,006,511, which is incorporated herein by reference in its entirety). The majority of Vλ regions observed in the expressed antibody repertoire are encoded by gene segments contained within the nearest cluster (referred to as cluster A). The mouse immunoglobulin λ light chain locus, depending on the strain, differs particularly from the human locus and contains several Vλ and Jλ gene segments organized into two distinct gene clusters (see, for example, Figure 2 of U.S. Patent No. 9,006,511, which is incorporated herein by reference in its entirety).

[0172] The development of therapeutic antibodies for the treatment of various human diseases has generally focused on the creation of modified non-human animal strains, particularly modified rodent strains, that possess varying amounts of genetic material in their genomes corresponding to human immunoglobulin genes (for example, reviewed in Bruggemann, M. et al., 2015, Arch.Immunol.Ther.Exp.63:101-8 (the whole of which is incorporated herein by reference)).Early efforts in creating such genetically modified rodent lines have focused on incorporating parts of the human immunoglobulin locus, which in itself can support recombination of gene segments, as well as the generation of fully human heavy and / or light chains while possessing inactivated endogenous immunoglobulin loci (e.g., Bruggemann, M. et al., 1989, Proc. Nat. Acad. Sci. USA 86: 67-09-13; Bruggemann, M. et al., 1991, Eur. J. Immunol. 21: 1323-6; Taylor, L. et al., 1992, Nucl. Acids Res. 20: 6287-6295; Davies, N. et al., 1993, Biotechnol. 11: 911-4; Green, L. et al., 1994, Nat. Genet. 7: 13-21; Lonberg, N. et al. al.,1994,Nature 368:856-9;Taylor,LDet al.,1994,Int.Immunol.6:579-91;Wagner,SDet al.,1994,Eur.J.Immunol.24:2672-81;Fishwild,DMet al.,1996,Nat.Biotechnol.14:845-51;Wagner,SDet al.,1996,Genomics 35:405-14;Mendez,MJet al.,1997,Nat.Genet.15:146-56;Green,LLet al.,1998,J.Exp.Med.188:483-95;Xian,J.et al.,1998,Transgenics 2:333-43;Little,M.et al., 2000, Immunol. Today 21:364-70; Kellermann, SA and LL Green, 2002, Cur. Opin. Biotechnol. 13:593-7 (each of these is incorporated by reference as a whole).In particular, several efforts have involved the incorporation of human immunoglobulin λ light chain sequences (see, for example, U.S. Patent Applications Publications 2002 / 0088016A1, 2003 / 0217373A1, and 2011 / 0236378A1; U.S. Patents 6,998,514 and 7,435,871; Nicholson, IC et al., 1999, J.Immunol. 163:6898-906; Popov, AV et al., 1999, J.Exp.Med. 189(10):1611-19 (each of these is incorporated herein by reference in whole)). Such efforts have focused on randomly incorporating yeast artificial chromosomes containing human Vλ, Jλ, and Cλ sequences to create mouse lines that express fully human immunoglobulin λ light chains (i.e., human Vλ and Cλ domains). More recent efforts employ similar strategies using constructs that also contain human Vλ, Jλ, and Cλ sequences (Osborn, MJet al., 2013, J.Immunol. 190:1481-90; Lee, EC. et al., 2014, Nat.Biotech. 32(4):356-63 (each of these is incorporated herein by reference in whole)).

[0173] Further efforts included specifically inserting the human Vλ and Jλ gene segments into the endogenous rodent immunoglobulin light chain loci (κ and λ) so that the human Vλ and Jλ gene segments could be functionally linked to the endogenous immunoglobulin light chain constant region genes (see, for example, U.S. Patents 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662, and 9,163,092 (all of which are incorporated herein by reference in their entirety)). In some embodiments of such animals, all of the human Vλ gene segments from clusters A and B and one or four of the human Jλ gene segments were inserted into the endogenous immunoglobulin κ and immunoglobulin λ light chain loci. Several different human Vλ and Jλ gene segments demonstrated proper rearrangement at both modified rodent immunoglobulin light chain loci to form functional light chains expressed in the rodent antibody repertoire, the light chains containing the human Vλ domain for either the endogenous Cκ or Cλ region (see, e.g., Table 7 and Figures 11-13 of U.S. Patent No. 9,006,511, which is incorporated herein by reference in its entirety). In particular, mice with modified immunoglobulin κ light chain loci possessing human Vλ and Jλ gene segments demonstrated a human lambda to endogenous lambda ratio of approximately 1:1 in the splenic compartment (measured by the IgCκ to IgCλ ratio) (see, e.g., Table 4 of U.S. Patent No. 9,006,511, which is incorporated herein by reference in its entirety). In fact, both modified mouse strains (i.e., modified immunoglobulin κ or modified immunoglobulin λ light chain locus) demonstrated that human Vλ domains can be expressed from endogenous immunoglobulin light chain loci in rodents, which typically exhibit a large bias in light chain expression (see above). This disclosure provides the recognition that alternative modified immunoglobulin light chain locus structures can be generated to maximize the expression of human λ light chain variable domains from a limited repertoire of human λ light chain variable regions.Such alternative, modified immunoglobulin light chain locus structures offer the capability for a unique antibody repertoire resulting from their design.

[0174] This disclosure provides a non-human animal whose germline genome contains a modified endogenous immunoglobulin κ light chain locus comprising a single rearranged human immunoglobulin λ light chain variable region functionally linked to a rodent Cλ gene segment. The single rearranged human immunoglobulin λ light chain variable region comprises the human Vλ gene segment and the human Jλ gene segment. In some embodiments, all immunoglobulin λ light chains expressed by genetically modified rodent B cells comprise a human immunoglobulin λ light chain variable domain expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof. In some embodiments, the modified endogenous immunoglobulin κ light chain locus comprises a single rearranged human immunoglobulin λ light chain variable region functionally linked to a non-human or human immunoglobulin λ or immunoglobulin κ light chain constant region gene. In some embodiments, such light chain expression can be achieved by insertion of the single rearranged human immunoglobulin λ light chain variable region into the endogenous immunoglobulin κ light chain locus (or allele). In some embodiments, the provided non-human animal is modified so that the expression of the endogenous immunoglobulin λ light chain variable region is inactivated (e.g., by gene deletion). In some embodiments, the provided non-human animal is modified so that the expression of the endogenous immunoglobulin κ light chain variable region is inactivated (e.g., by insertion, replacement, or substitution).

[0175] Universal Light Chain Past efforts to produce useful multispecific antigen-binding proteins, such as bispecific antibodies, have often been hampered by a common paradigm: the diverse problems of in vitro sequence selection or manipulation for rationally modifying, or modifying, heterodimer bispecific human immunoglobulins in a suitable form for pairing, or through trial and error. Unfortunately, most, if not all, in vitro modification approaches offer, generally, ad-hoc solutions for individual molecules, if any at all.

[0176] In vivo methods have been developed to use complex organisms to select appropriate pairings that can yield human therapeutic agents (see, e.g., U.S. Patent No. 10,143,186, which is incorporated in its entirety by reference). However, no non-human animal capable of generating a robust immune response, including the production of high-affinity universal λ light chains with human immunoglobulin λ light chain variable domains at sufficient titer levels, has been produced to date. Native non-human sequences are generally not good sources for human therapeutic sequences. For at least this reason, generating non-human heavy chain immunoglobulin variable domains that pair with universal human light chains has limited practical applicability. More in vitro modification efforts will be made in the trial-and-error process of attempting to humanize non-human heavy chain variable sequences, hoping to retain epitope specificity and affinity, as well as the ability to bind to common human light chains, although the outcomes are uncertain. At the end of such a process, the final product may retain some specificity and affinity and be able to associate with universal light chains, but ultimately, immunogenicity in humans may still pose a significant risk.

[0177] Therefore, a suitable non-human animal for producing human therapeutic agents would contain a sufficiently broad repertoire of human heavy chain variable region gene segments instead of endogenous non-human heavy chain variable region gene segments. The human heavy chain variable region gene segments should be capable of rearranging and splicing with endogenous non-human heavy chain constant regions to form reverse chimeric heavy chains (i.e., heavy chains containing human variable domains and non-human constant domains). Heavy chain loci should be capable of undergoing class switching and somatic hypermutation, thereby making a sufficiently broad repertoire of heavy chain variable domains available to the non-human animal for selecting those that can associate with human λ light chain variable domains encoded by a limited repertoire of human λ light chain variable regions.

[0178] Non-human animals that select universal light chains for multiple heavy chains have practical applications. In various embodiments, antibodies expressed in non-human animals that can express only universal light chains have heavy chains that can associate with and express the same or substantially the same light chain. This is particularly useful in the production of bispecific antibodies. For example, such a non-human animal can be immunized with a first immunosource to produce B cells that express antibodies that specifically bind to a first epitope. A non-human animal (or a non-human animal containing the same genetic modification to its heavy chain and λ light chain loci) can be immunized with a second immunosource to produce B cells that express antibodies that specifically bind to a second epitope. A variable heavy chain region can be cloned from B cells and expressed together with the same heavy chain constant region and the same light chain in cells for the production of bispecific antibodies, in which case the light chain component of the bispecific antibody is selected by the non-human animal to associate with and express the light chain component. Non-human animals expressing universal κ light chains have been developed (see, for example, U.S. Patent No. 10,143,186, which is incorporated in its entirety by reference). However, there is still a need to develop non-human animals capable of expressing universal λ light chains, including human λ light chain variable domains.

[0179] This disclosure provides modified non-human animals (e.g., rodents, e.g., rats or mice) for generating immunoglobulin λ light chains that suitably pair with a considerably diverse family of heavy chains, including heavy chains whose variable regions deviate from germline sequences, e.g., affinity-matured or somatically hypermutated heavy chains. In various embodiments, the non-human animals described herein are modified to express and pair human λ light chain variable domains together with human heavy chain variable domains, including somatic mutations, thereby enabling a pathway to high-affinity binding proteins (e.g., antibodies) suitable for use as human therapeutic agents.

[0180] The genetically modified non-human animals described herein (e.g., rodents, e.g., rats or mice) undergo a long and complex process of antibody selection within the organism to make a biologically appropriate selection in the pairing of a diverse collection of human heavy chain variable domains with a limited number of light chain options. To achieve this, the non-human animals are modified to present a limited number of human λ light chain variable domain options in conjunction with a broad diversity of human heavy chain variable domain options. When administered an immunogen, the non-human animals described herein can generate antibodies against the immunogen, largely or exclusively limited by the number or light chain options in their repertoire, to maximize the number of solutions in their repertoire. In various embodiments, this involves enabling the non-human animal to achieve suitable and compatible somatic mutations of light chain variable domains, which nevertheless conform to a relatively diverse range of human heavy chain variable domains (in particular, including somatically hypermutated human heavy chain variable domains).

[0181] To achieve a limited repertoire of human λ light chain options, the non-human animals described herein may be modified to be non-functional or substantially non-functional in their ability to produce or reconstruct native non-human λ and / or κ light chain variable domains. In some embodiments, this may be achieved by deleting the λ and / or κ light chain variable region gene segment of the non-human animal. In some embodiments, the endogenous non-human locus may then be modified with a preferred exogenous human λ light chain variable region sequence(s) functionally linked to the endogenous non-human light chain constant domain. In some embodiments, the exogenous human variable region gene segment may be unreorganized (e.g., two Vλ gene segments and one or more Jλ gene segments) and may be reorganized and spliced ​​into the endogenous non-human light chain constant region gene to form a reorganized reverse chimeric light chain gene (human variable, non-human constant). In some embodiments, the exogenous human variable gene segment may be rearranged (e.g., one Vλ gene segment and one Jλ gene segment) and spliced ​​into an endogenous non-human light chain constant region gene to form a reverse chimeric light chain gene containing both the human variable region and the non-human constant region. In some embodiments, the exogenous human variable gene segment may be rearranged (e.g., one Vλ gene segment and one Jλ gene segment) and spliced ​​into an exogenous human light chain constant region gene to form a humanized light chain gene containing both the human variable region and the human constant region. In various embodiments, the light chain variable region is capable of somatic hypermutation. In various embodiments, appropriate enhancers are retained in non-human animals. Enhancers have been reported to maximize the light chain variable region's ability to acquire somatic mutations. For example, in modifying the κ locus of a non-human animal by replacing the endogenous non-human animal κ variable region gene segment with a human λ variable region gene segment, the non-human κ intron enhancer and the non-human κ3' enhancer are functionally maintained or not disrupted. Embodiments in which enhancers are removed or disrupted are contemplated by this disclosure, but such embodiments would be expected to reduce or eliminate somatic hypermutation.In such embodiments, somatic hypermutation in the light chain variable region is reduced compared to, for example, a light chain variable region containing one or more endogenous non-human enhancers.

[0182] Genetically modified non-human animals are provided that express a limited repertoire of reverse chimeric (human variable, non-human stationary) light chains associated with diverse reverse chimeric (human variable, non-human stationary) heavy chains. In various embodiments, the endogenous non-human κ light chain variable region gene segment is deleted or replaced with a single (or two) human λ light chain variable region gene segment functionally linked to the endogenous non-human κ stationary region gene. In some embodiments, non-human κ intron enhancers and non-human κ3' enhancers are maintained. Without being bound to any one theory, the enhancers may, among other things, maximize somatic hypermutation of the human λ light chain variable region gene segment. In various embodiments, the non-human animals also include a non-functional λ light chain locus, or a deletion thereof, or a deletion that makes it impossible for that locus to produce a λ light chain.

[0183] In various embodiments, a genetically modified non-human animal is provided that lacks an endogenous non-human light chain variable gene segment and includes a human variable gene segment functionally linked to a non-human Cλ gene segment, and in some embodiments, includes a light chain variable region locus containing a rearranged human immunoglobulin λ light chain variable region, wherein the locus is capable of undergoing somatic hypermutation and expresses a light chain containing a human immunoglobulin λ light chain variable region linked to a non-human Cλ gene segment. Thus, in various embodiments, the locus includes a non-human κ3' enhancer that correlates with normal or wild-type levels of somatic hypermutation.

[0184] In various embodiments, genetically modified non-human animals, when immunized with the target antigen, produce B cells that express one or two rearranged light chains and exhibit diverse rearrangements of the functional human immunoglobulin heavy chain variable region. In some embodiments, the human λ light chain variable region contains somatic hypermutations. In some embodiments, each human λ light chain variable region contains one to five somatic hypermutations. In various embodiments, the light chains expressed by the non-human animals described herein can associate with and express any heavy chain containing the human immunoglobulin heavy chain variable region expressed in the non-human animal.

[0185] Non-human animals, cells, and tissues provided (i) one or two unreorganized Vλ gene segments and one or more unreorganized Jλ gene segments, or (ii) a non-human animal expressing an antibody (e.g., expressed by its B cells) containing a light chain comprising a human λ light chain variable domain derived from a single reorganized human λ light chain variable region in place of a non-human immunoglobulin κ light chain variable region sequence at an endogenous non-human λ light chain locus in the germline genome of the non-human animal. In the various embodiments described herein, the genetically modified non-human animal will be understood to be a rodent, e.g., a rat or mouse, and the non-human elements described herein (enhancers, constant regions, etc.) will be rodent, e.g., rat or mouse elements. Preferred examples of non-human animals described herein include, but are not limited to, rodents, e.g., rat or mouse, and especially mice.

[0186] This disclosure provides an improved in vivo system for identifying and developing novel antigen-binding proteins, antibodies, antibody components (e.g., antigen-binding moieties and / or compositions or forms comprising them), and / or antibody-based therapeutics that can be used, for example, in the treatment of a variety of diseases affecting humans. Furthermore, this disclosure encompasses the recognition that non-human animals (e.g., rodents, e.g., rats or mice) having modified immunoglobulin loci, e.g., modified immunoglobulin κ light chain loci including a limited λ light chain variable region repertoire, are useful. In some embodiments, the non-human animals described herein provide an improved in vivo system for developing antibodies and / or antibody-based therapeutics for administration to humans. In some embodiments, the non-human animals described herein provide an improved in vivo system for developing antibodies and / or antibody-based therapeutics containing human λ light chain variable domains that feature improved and / or different performance (e.g., expression and / or appearance in antigen-specific antibody repertoire) compared to antibodies and / or antibody-based therapeutics obtained from existing in vivo systems containing human Vλ region sequences.

[0187] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include a limited human λ light chain variable region repertoire. In some embodiments, sequences of the limited human λ light chain variable region repertoire are functionally linked to a non-human light chain constant region. In some embodiments, the non-human light chain constant region is a rodent (e.g., mouse or rat) light chain constant region. In some embodiments, the non-human light chain constant region is a κ or λ light chain constant region. In some embodiments, sequences of the limited human λ light chain variable region repertoire are functionally linked to a non-human (e.g., rodents, e.g., rats or mice) Cκ. In some embodiments, a limited repertoire of human λ light chain variable regions is functionally linked to a non-human (e.g., rodent, e.g., rat or mouse) Cλ (e.g., Cλ1). In some embodiments, the non-human λ light chain constant region (e.g., mouse Cλ, e.g., mouse Cλ1) replaces the endogenous non-human Cκ.

[0188] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include two unreorganized human Vλ gene segments and one or more unreorganized human Jλ gene segments. For several purposes, two unreorganized human Vλ gene segments are selected from the group consisting of Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. For several purposes, two unreorganized human Vλ gene segments are selected from the group consisting of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some embodiments, two unreorganized human Vλ gene segments are selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, one or more unreorganized human Jλ gene segments are selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, one or more unreorganized human Jλ gene segments include Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, one or more unreorganized human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.In some embodiments, two unreorganized human Vλ gene segments and one or more unreorganized human Jλ gene segments are functionally linked to a non-human light chain constant region. In some embodiments, the non-human light chain constant region is a rodent (e.g., mouse or rat) light chain constant region. In some embodiments, the non-human light chain constant region is a κ or λ light chain constant region. In some embodiments, two unreorganized human Vλ gene segments and one or more unreorganized human Jλ gene segments are functionally linked to a non-human (e.g., rodent, e.g., rat or mouse) Cκ. In some embodiments, two unreorganized human Vλ gene segments and one or more unreorganized human Jλ gene segments are functionally linked to a non-human (e.g., rodent, e.g., rat or mouse) Cλ (e.g., Cλ1). In some embodiments, the non-human λ light chain constant region (e.g., rodents, e.g., rats or mice, e.g., mouse Cλ, e.g., mouse Cλ1) is replaced by the endogenous non-human (e.g., rodents, e.g., rats or mice) Cκ.

[0189] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include two unreorganized human Vλ gene segments and four unreorganized human Jλ gene segments functionally linked to non-human (e.g., rodents, e.g., rats or mice) Cκ. In some embodiments, the two unreorganized human Vλ gene segments are selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the four unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7.

[0190] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include two unreorganized human Vλ gene segments and five unreorganized human Jλ gene segments functionally linked to non-human (e.g., rodents, e.g., rats or mice) Cκ. In some embodiments, the two unreorganized human Vλ gene segments are selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the five unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0191] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include two unreorganized human Vλ gene segments and four unreorganized human Jλ gene segments functionally linked to non-human (e.g., rodents, e.g., rats or mice) Cλ (e.g., Cλ1). In some embodiments, the two unreorganized human Vλ gene segments are selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the four unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7.

[0192] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include two unreorganized human Vλ gene segments and five unreorganized human Jλ gene segments functionally linked to non-human (e.g., rodents, e.g., rats or mice) Cλ (e.g., Cλ1). In some embodiments, the two unreorganized human Vλ gene segments are selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the five unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0193] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include one unreorganized human Vλ gene segment and one or more unreorganized human Jλ gene segments. For several purposes, a single unreorganized human Vλ gene segment is selected from the group consisting of Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. For several purposes, a single unreorganized human Vλ gene segment is selected from the group consisting of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some embodiments, one unreorganized human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, one or more unreorganized human Jλ gene segments are selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, one or more unreorganized human Jλ gene segments include Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, one or more unreorganized human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.In some embodiments, one unreorganized human Vλ gene segment and one or more unreorganized human Jλ gene segments are functionally linked to a non-human light chain constant region. In some embodiments, the non-human light chain constant region is a rodent (e.g., mouse or rat) light chain constant region. In some embodiments, the non-human light chain constant region is a κ or λ light chain constant region. In some embodiments, one unreorganized human Vλ gene segment and one or more unreorganized human Jλ gene segments are functionally linked to a non-human (e.g., rodent, e.g., rat or mouse) Cκ. In some embodiments, one unreorganized human Vλ gene segment and one or more unreorganized human Jλ gene segments are functionally linked to a non-human (e.g., rodent, e.g., rat or mouse) Cλ (e.g., Cλ1). In some embodiments, the non-human λ light chain constant region (e.g., rodents, e.g., rats or mice, e.g., mouse Cλ, e.g., mouse Cλ1) is replaced by the endogenous non-human (e.g., rodents, e.g., rats or mice) Cκ.

[0194] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include one unreorganized human Vλ gene segment and four unreorganized human Jλ gene segments functionally linked to non-human (e.g., rodents, e.g., rats or mice) Cκ. In some embodiments, the one unreorganized human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the four unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, one unreorganized human Vλ gene segment is Vλ1-51, and four unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, one unreorganized human Vλ gene segment is Vλ2-14, and four unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7.

[0195] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include one unreorganized human Vλ gene segment and five unreorganized human Jλ gene segments functionally linked to non-human (e.g., rodents, e.g., rats or mice) Cκ. In some embodiments, the one unreorganized human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the five unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, one unreorganized human Vλ gene segment is Vλ1-51, and five unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, one unreorganized human Vλ gene segment is Vλ2-14, and five unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0196] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include one unreorganized human Vλ gene segment and four unreorganized human Jλ gene segments functionally linked to a non-human (e.g., rodents, e.g., rats or mice) Cλ. In some embodiments, the one unreorganized human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the four unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, one unreorganized human Vλ gene segment is Vλ1-51, and four unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, one unreorganized human Vλ gene segment is Vλ2-14, and four unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7.

[0197] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include one unreorganized human Vλ gene segment and five unreorganized human Jλ gene segments functionally linked to a non-human (e.g., rodents, e.g., rats or mice) Cλ. In some embodiments, the one unreorganized human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the five unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, one unreorganized human Vλ gene segment is Vλ1-51, and five unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, one unreorganized human Vλ gene segment is Vλ2-14, and five unreorganized human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0198] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., mouse) tissues having an endogenous immunoglobulin κ light chain locus modified to include a single rearranged human λ light chain variable region (V / J) comprising a human Vλ gene segment and a human Jλ gene segment. For several purposes, a single rearranged human λ light chain variable region human Vλ gene segment is selected from the group consisting of Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. For several purposes, a single rearranged human λ light chain variable region human Vλ gene segment is selected from the group consisting of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some embodiments, the human Vλ gene segment of a single rearranged human λ light chain variable region is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the human Vλ gene segment of a single rearranged human λ light chain variable region is Vλ1-51. In some embodiments, the human Vλ gene segment of a single rearranged human λ light chain variable region is Vλ2-14.In some embodiments, the human Jλ gene segment of a single rearranged human λ light chain variable region is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, the human Jλ gene segment of a single rearranged human λ light chain variable region is Jλ1. In some embodiments, the human Jλ gene segment of a single rearranged human λ light chain variable region is Jλ2. In some embodiments, the human Jλ gene segment of a single rearranged human λ light chain variable region is Jλ3. In some embodiments, the single rearranged human λ light chain variable region is functionally linked to a non-human light chain constant region. In some embodiments, the non-human light chain constant region is a rodent (e.g., mouse or rat) light chain constant region. In some embodiments, the non-human light chain constant region is a κ or λ light chain constant region. In some embodiments, the single rearranged human λ light chain variable region is functionally linked to a non-human (e.g., rodent, e.g., rat or mouse) Cκ. In some embodiments, a single reorganized human λ light chain variable region is functionally linked to a non-human (e.g., rodent, e.g., rat or mouse) Cλ (e.g., Cλ1). In some embodiments, the non-human λ light chain constant region (e.g., mouse Cλ, e.g., mouse Cλ1) replaces the endogenous non-human (e.g., rodent, e.g., rat or mouse) Cκ.

[0199] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include a single rearranged human λ light chain variable region functionally linked to mouse Cκ, wherein the single rearranged human λ light chain variable region includes a human Vλ gene segment and a human Jλ gene segment. In some embodiments, the human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, the human Vλ gene segment is Vλ1-51, and the human Jλ gene segment is Jλ2. In some embodiments, the human Vλ gene segment is Vλ2-14, and the human Jλ gene segment is Jλ2.

[0200] This disclosure provides, in particular, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues having an endogenous immunoglobulin κ light chain locus modified to include a single rearranged human λ light chain variable region functionally linked to a non-human (e.g., rodents, e.g., rats or mice) Cλ, the single rearranged human λ light chain variable region comprising a human Vλ gene segment and a human Jλ gene segment. In some embodiments, the human Vλ gene segment is selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, the human Vλ gene segment is Vλ1-51, and the human Jλ gene segment is Jλ2. In some embodiments, the human Vλ gene segment is Vλ2-14, and the human Jλ gene segment is Jλ2.

[0201] This disclosure provides, in particular, mice, mouse cells, or mouse tissues having an endogenous immunoglobulin κ light chain locus modified to include a single rearranged human λ light chain variable region functionally linked to mouse Cλ, the single rearranged human λ light chain variable region including a human Vλ gene segment and a human Jλ gene segment. In some embodiments, the human Vλ gene segment includes Vλ1-51. In some embodiments, the human Vλ gene segment includes Vλ2-14. In some embodiments, the human Jλ gene segment includes Jλ2. In some embodiments, the human Vλ gene segment is Vλ1-51 and the human Jλ gene segment is Jλ2. In some embodiments, the human Vλ gene segment is Vλ2-14 and the human Jλ gene segment is Jλ2.

[0202] This disclosure provides, in particular, a genetically modified mouse, mouse cells, or mouse tissue, wherein its germline genome comprises a modified endogenous immunoglobulin κ light chain locus containing a single rearranged human immunoglobulin λ light chain variable region functionally linked to a mouse Cλ1 gene segment, the single rearranged human immunoglobulin λ light chain variable region comprising the human Vλ1-51 gene segment and the human Jλ2 gene segment, and all immunoglobulin λ light chains expressed by the B cells of the genetically modified mouse comprise human immunoglobulin λ light chain variable domains expressed from the single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof.

[0203] This disclosure provides, in particular, a genetically modified mouse, mouse cells, or mouse tissue, wherein its germline genome comprises a modified endogenous immunoglobulin κ light chain locus containing a single rearranged human immunoglobulin λ light chain variable region functionally linked to a mouse Cλ1 gene segment, the single rearranged human immunoglobulin λ light chain variable region comprising a human Vλ2-14 gene segment and a human Jλ2 gene segment, and all immunoglobulin λ light chains expressed by the B cells of the genetically modified mouse comprise human immunoglobulin λ light chain variable domains expressed from the single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof.

[0204] In some embodiments, the non-human animals provided (e.g., rodents, e.g., rats or mice) are characterized by the expression of antibodies from the endogenous immunoglobulin κ light chain locus in the germline genome of the non-human animals, the antibodies comprising (1) a human Vλ domain and (2) a non-human Cλ domain. In some embodiments, the non-human animals provided are characterized by improved use (e.g., about twice as much, but not limited to) of the human Vλ region from a modified immunoglobulin κ light chain locus containing a limited human λ light chain variable region repertoire compared to one or more reference modified non-human animals.

[0205] In some embodiments, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues are provided, the germline genome comprising (a) a single rearranged human immunoglobulin λ light chain variable region, and (b) an endogenous immunoglobulin κ light chain locus containing the Cλ gene, wherein (a) is functionally linked to (b), and the non-human animals lack the non-human Cκ gene at the endogenous immunoglobulin κ light chain locus.

[0206] In some embodiments, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues are provided, the genome of which comprises an endogenous immunoglobulin κ light chain locus including a single rearranged human immunoglobulin λ light chain variable region and an insertion of a Cλ gene, the single rearranged human immunoglobulin λ light chain variable region functionally ligated to the Cλ gene, and the Cλ gene being inserted in place of a non-human Cκ gene at the endogenous immunoglobulin κ light chain locus. In many embodiments of non-human animals, non-human cells or non-human tissues, the Cλ gene inserted in place of a non-human Cκ gene at the endogenous immunoglobulin κ light chain locus is either a non-human or human Cλ gene. In some embodiments, the non-human Cλ gene is a mammalian Cλ gene selected from the group consisting of primate, goat, sheep, pig, dog, cattle, or rodent (e.g., rat or mouse) Cλ genes, or includes such a gene.

[0207] In some embodiments, the non-human Cλ gene is a rodent Cλ gene, or includes one.

[0208] In some embodiments, the rodent Cλ gene is or includes the mouse Cλ gene. In some embodiments, the mouse Cλ gene contains a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to a mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3. In some embodiments, the mouse Cλ gene contains a sequence that is substantially identical or identical to a mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3. In some embodiments, the mouse Cλ1 gene is or includes SEQ ID NO: 1. In some predetermined embodiments, the mouse Cλ2 gene is or includes SEQ ID NO: 2. In some predetermined embodiments, the mouse Cλ3 gene is or includes SEQ ID NO: 3. In some predetermined embodiments, the mouse Cλ gene contains the same sequence as the mouse Cλ1 gene.

[0209] In some embodiments, the mouse Cλ gene contains sequences that are 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% identical to the mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3. In some embodiments, the mouse Cλ gene contains sequences that are 80% to 98%, 80% to 95%, 80% to 90%, or 80% to 85% identical to the mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3. In some embodiments, the mouse Cλ gene contains sequences that are 85% to 98%, 90% to 95%, or 88% to 93% identical to the mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3.

[0210] In some embodiments, the rodent Cλ gene is or includes the rat Cλ gene. In some embodiments, the rat Cλ gene contains sequences that are at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to a rat Cλ gene selected from the group consisting of rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes. In some embodiments, the rat Cλ gene contains sequences that are substantially identical or identical to a rat Cλ gene selected from the group consisting of rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes. In some predetermined embodiments, the rat Cλ1 gene is or includes sequence number 7. In some predetermined embodiments, the rat Cλ2 gene is or includes sequence number 8. In some predetermined embodiments, the rat Cλ3 gene is or includes sequence number 9. In some predetermined embodiments, the rat Cλ4 gene is or includes sequence number 10.

[0211] In some embodiments, the rat Cλ gene contains sequences that are 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% identical to rat Cλ genes selected from the group consisting of rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes. In some embodiments, the rat Cλ gene contains sequences that are 80% to 98%, 80% to 95%, 80% to 90%, or 80% to 85% identical to rat Cλ genes selected from the group consisting of rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes. In some embodiments, the rat Cλ gene contains sequences that are 85% to 98%, 90% to 95%, or 88% to 93% identical to rat Cλ genes selected from the group consisting of rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes.

[0212] In some embodiments, the human Cλ gene contains a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to a human Cλ gene selected from the group consisting of human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes. In some embodiments, the human Cλ gene contains a sequence that is substantially identical or identical to a human Cλ gene selected from the group consisting of human Cλ, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes. In some embodiments, the human Cλ gene contains a sequence that is identical to a human Cλ gene selected from the group consisting of human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes. In some predetermined embodiments, the human Cλ1 gene is or includes sequence number 15. In some predetermined embodiments, the human Cλ2 gene is or includes sequence number 16. In some predetermined embodiments, the human Cλ3 gene is or includes sequence number 17. In some predetermined embodiments, the human Cλ6 gene is or includes SEQ ID NO: 18. In some predetermined embodiments, the human Cλ7 gene is or includes SEQ ID NO: 18. In some predetermined embodiments, the human Cλ gene is or includes the human Cλ2 gene.

[0213] In some embodiments, the human Cλ gene contains sequences that are 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% identical to a human Cλ gene selected from the group consisting of human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes. In some embodiments, the human Cλ gene contains sequences that are 80% to 98%, 80% to 95%, 80% to 90%, or 80% to 85% identical to a human Cλ gene selected from the group consisting of human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes. In some embodiments, the human Cλ gene contains sequences that are 85% to 98%, 90% to 95%, or 88% to 93% identical to a human Cλ gene selected from the group consisting of human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes.

[0214] In some embodiments of the non-human animal (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cells or non-human (e.g., rodent, e.g., rat or mouse) tissue provided, the germline genome of the non-human animal, non-human cell or non-human tissue is one or more human V H Gene segment, one or more human D H Gene segment, and one or more human J H Further comprising an endogenous immunoglobulin heavy chain locus modified to include a gene segment, human V H , D H and J H The gene segment is functionally linked to the non-human immunoglobulin heavy chain constant region at the endogenous immunoglobulin heavy chain locus (see, for example, Macdonald, LE, et al., “Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes,” Proc. Natl. Acad. Sci. USA, 111(14):5147-5152 (April 8, 2014), U.S. Patents 6,596,541, 8,642,835, 8,697,940 and 8,791,323 (each of which is incorporated herein by reference in whole)).

[0215] In some embodiments, one or more human V H Gene segment, one or more human D H Genetic segment and one or more human J H The insertion of a gene segment may occur, either entirely or partially, in non-human V H , D H and J H A substitute for, or replaces, a gene segment (e.g., non-human V H , D H and J H The coding sequence of the gene segment is human V H , D H and J H(Positionally replace or substitute in the coding sequence of a gene segment). In some embodiments, the non-human immunoglobulin heavy chain constant region is or comprises an endogenous non-human immunoglobulin heavy chain constant region. In many embodiments, the non-human immunoglobulin heavy chain constant region (e.g., endogenous) comprises one or more non-human immunoglobulin heavy chain constant region genes or gene segments (e.g., IgM, IgD, IgG, IgE, IgA, etc.). In some predetermined embodiments, the insertion is human V H , D H and J H The gene segments and the combinations thereof include naturally occurring human non-coding DNA. In some predetermined embodiments, the immunoglobulin heavy chain locus described herein is human V H Gene segment V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1, or any combination thereof, Human D H Gene segment D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H 7-27, or any combination thereof, and human J H Gene segment J H 1, J H 2, J H 3, J H 4, J H 5, J H 6. The insertion includes insertion of any combination thereof. In some predetermined embodiments, the insertion is at the endogenous heavy chain locus of human V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, or V H Adjacent to 6-1 is naturally occurring human non-coding DNA, human D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, or DH Adjacent to 7-27 are naturally occurring human non-coding DNA, and at the endogenous heavy chain locus, human J H 1, J H 2, J H 3, J H 4, J H 5, or J H It contains naturally occurring human non-coding DNA adjacent to 6.

[0216] In some embodiments, the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein include the Adam6 gene in its genome (e.g., its germline genome), which encodes the ADAM6 polypeptide, its functional orthologue, functional homolog, or functional fragment (see, for example, U.S. Patents 8,642,835 and 8,697,940, each of which is incorporated herein by reference in whole). In some embodiments, the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein contain the rodent (e.g., mouse or rat) Adam6 gene in its genome (e.g., its germline genome), which encodes the rodent (e.g., mouse or rat) ADAM6 polypeptide, its functional orthologue, functional homolog, or functional fragment (see, for example, U.S. Patents 8,642,835 and 8,697,940, each of which is incorporated herein by reference in whole). In some embodiments, the ADAM6 polypeptide, its functional orthologue, functional homolog, or functional fragment is expressed from the Adam6 gene. In some embodiments, the Adam6 gene in the genetically modified non-human animals described herein does not originate from that particular non-human animal (e.g., a rat Adam6 gene or a mouse containing a mouse Adam6 gene obtained from another mouse strain). In some embodiments, the non-human animals described herein include an ectopic Adam6 gene. “Ectopic” Adam6 gene, as used herein, refers to an Adam6 gene in a different context than that which appears in wild-type non-human animals. For example, the Adam6 gene may be located on a different chromosome, at a different locus, or adjacent to a different sequence.An exemplary ectopic Adam6 gene is a mouse Adam6 gene located within a human immunoglobulin sequence (e.g., a human heavy chain variable region gene segment). In some embodiments, the non-human animals described herein include an inserted or incorporated Adam6 gene.

[0217] In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein include insertions of one or more nucleotide sequences in their genome (e.g., in their germline genome) that encode one or more non-human Adam6 polypeptides, functional orthologues, functional homologs, or functional fragments thereof.

[0218] In some embodiments, the non-human animal (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein include one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologues, functional homologs, or functional fragments thereof in their genome (e.g., in their germline genome). In some embodiments, the non-human animal (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein include the mouse Adam6a gene and / or the mouse Adam6b gene in their genome (e.g., in their germline genome). In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein include one or more nucleotide sequences encoding mouse ADAM6a, its functional orthologue, functional homolog, or functional fragment, and / or mouse ADAM6b, its functional orthologue, functional homolog, or functional fragment.

[0219] In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are inserted and / or located on the same chromosome as the endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are inserted and / or located in a position contiguous with the human immunoglobulin heavy chain variable region gene segment. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are inserted and / or located in a position adjacent to the human immunoglobulin heavy chain variable region gene segment. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are inserted and / or positioned between human immunoglobulin heavy chain variable region gene segments. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are inserted and / or positioned between first and second human V H It is inserted and / or positioned between gene segments. In some embodiments, the first human V H The gene segment is human V H 1-2, and the second human V H The gene segment is human V H6-1. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are inserted in place of and / or positioned in place of the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are human V H Genetic segments and human D H It is inserted between gene segments.

[0220] In some embodiments, the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein contain an Adam6 gene that restores or enhances ADAM6 activity. In some embodiments, the Adam6 gene restores ADAM6 activity to the level of an equivalent non-human animal containing a functional endogenous Adam6 gene. In some embodiments, the Adam6 gene enhances ADAM6 activity to at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times the level of an equivalent non-human animal not containing a functional Adam6 gene.

[0221] In some embodiments, the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein contain the Adam6 gene, which, when expressed in male non-human animals, restores or enhances fertility. In some embodiments, the Adam6 gene restores fertility in male non-human animals to the level of equivalent non-human animals containing the functional endogenous Adam6 gene. In some embodiments, the Adam6 gene restores fertility in male non-human animals such that the number of offspring produced by mating with a male non-human animal is at least 70%, at least 80%, at least 90%, or at least 95% of the number of offspring produced by equivalent mating with an equivalent male non-human animal that does not contain the functional Adam6 gene. In some embodiments, the Adam6 gene enhances the reproductive capacity of male non-human animals such that the number of offspring produced by mating with a male non-human animal is at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times the number of offspring produced by similar mating with an equivalent male non-human animal that does not contain the functional Adam6 gene.

[0222] In some embodiments, the non-human immunoglobulin heavy chain loci described herein lack at least one endogenous non-human Adam6 gene. In some embodiments, the absence of at least one endogenous non-human Adam6 gene reduces ADAM6 activity and / or reproductive capacity in male rodents (e.g., mice or rats) lacking the endogenous non-human Adam6 gene. In some embodiments, the non-human immunoglobulin heavy chain loci described herein include the disruption of at least one endogenous non-human Adam6 gene. In some embodiments, the disruption of at least one endogenous non-human Adam6 gene reduces ADAM6 activity and / or reproductive capacity in male rodents (e.g., mice or rats) lacking the endogenous non-human Adam6 gene.

[0223] In some embodiments of non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein, the non-human animals, non-human cells or non-human tissues are homozygous or heterozygous for a modified endogenous immunoglobulin heavy chain locus containing a human heavy chain variable region gene segment described herein.

[0224] In some embodiments of non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein, the non-human animals, non-human cells or non-human tissues are homozygous or heterozygous for a modified endogenous immunoglobulin κ light chain locus containing a human light chain variable gene segment (human variable λ light chain gene segment) as described herein.

[0225] In some embodiments, the non-human animal (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissue described herein comprises a first modified endogenous immunoglobulin κ light chain locus allele containing a single rearranged human immunoglobulin λ light chain variable region functionally linked to a rodent Cλ gene segment, the single rearranged human immunoglobulin λ light chain variable region containing a human Vλ gene segment and a human Jλ gene segment. In some embodiments, the non-human animal, non-human cell or non-human tissue comprises a second modified endogenous immunoglobulin κ light chain locus allele containing a single rearranged human immunoglobulin κ light chain variable region functionally linked to a rodent Cκ gene segment, the single rearranged human immunoglobulin κ light chain variable region containing a human Vκ gene segment and a human Jκ gene segment. In some embodiments, such non-human animals or non-human tissues may express a λ light chain from a first modified endogenous immunoglobulin κ light chain locus allele and a κ light chain from a second modified endogenous immunoglobulin κ light chain locus allele. In some embodiments, a single rearranged human immunoglobulin κ light chain variable region comprises Vκ3-20 or Vκ1-39, and a single rearranged human immunoglobulin λ light chain variable region comprises Vλ1-51 or Vλ2-14. In one embodiment, a single rearranged human immunoglobulin κ light chain variable region is Vκ3-20 / Jκ1 or Vκ1-39 / Jκ5, and a single rearranged human immunoglobulin λ light chain variable region is Vλ1-51 / Jλ2 or Vλ2-14 / Jλ2.

[0226] In some embodiments of the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues provided, the endogenous immunoglobulin λ light chain locus is deleted in whole or in part. In some embodiments of the non-human animals, non-human cells or non-human tissues provided, the endogenous immunoglobulin λ light chain locus is functionally silenced or otherwise non-functional (e.g., by gene targeting). In some predetermined embodiments of the non-human animals, non-human cells or non-human tissues provided, the non-human animals, non-human cells or non-human tissues are homozygous for the functionally silenced or otherwise non-functional endogenous immunoglobulin λ light chain locus described herein.

[0227] In some embodiments, the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein do not detectably express endogenous immunoglobulin λ light chains. In some embodiments, the non-human animals, non-human cells or non-human tissues described herein do not detectably express endogenous immunoglobulin κ light chains. In some embodiments, the non-human animals, non-human cells or non-human tissues described herein do not detectably express endogenous immunoglobulin λ light chains or endogenous immunoglobulin κ light chains.

[0228] In some embodiments, the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein do not detectably express endogenous immunoglobulin heavy chains. In some embodiments, the non-human animals, non-human cells or non-human tissues described herein do not detectably express endogenous immunoglobulin λ light chains, endogenous immunoglobulin κ light chains, and endogenous immunoglobulin heavy chains.

[0229] In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein have a genome further comprising nucleic acid sequences encoding exogenous terminal deoxynucleotidyltransferases (TdTs) functionally linked to transcriptional control elements. (See, for example, WO2017 / 210586 and U.S. Publication No. 2017 / 0347633, each of which is incorporated herein by reference in whole.)

[0230] In some embodiments, the transcriptional control element includes a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin κ light chain transcriptional control element, an immunoglobulin λ light chain transcriptional control element, or any combination thereof.

[0231] In some embodiments, the nucleic acid sequence encoding exogenous TdT is located at the immunoglobulin κ light chain locus, immunoglobulin λ light chain locus, immunoglobulin heavy chain locus, RAG1 locus, or RAG2 locus.

[0232] In some embodiments, TdT is human TdT. In some embodiments, TdT is a short isoform of TdT (TdTS).

[0233] In some embodiments, a single rearranged human immunoglobulin λ light chain variable region is introduced into the endogenous immunoglobulin κ light chain locus in a manner that preserves the integrity of a non-human immunoglobulin κ light chain enhancer region (or enhancer sequence) near the insertion site (e.g., a non-human immunoglobulin κ intron enhancer and / or a non-human immunoglobulin κ3' enhancer). Thus, such a non-human animal has a wild-type immunoglobulin κ light chain enhancer region (or enhancer sequence) functionally ligated to human and non-human immunoglobulin λ light chain sequences (e.g., human Vλ and Jλ gene segments, and non-human Cλ or Cκ) or to human immunoglobulin λ light chain sequences (e.g., human Vλ and Jλ gene segments, and human Cλ or Cκ).

[0234] In some embodiments, a non-human immunoglobulin κ light chain locus altered, moved, disrupted, deleted, substituted, or modified with one or more human immunoglobulin λ light chain sequences described herein is a murine immunoglobulin κ light chain locus. In some embodiments, one or more human immunoglobulin λ light chain sequences described herein are inserted into one copy (i.e., an allele) of the non-human immunoglobulin κ light chain locus out of two copies of the non-human immunoglobulin κ light chain locus, resulting in a non-human animal that is heterozygous with respect to the human immunoglobulin κ light chain sequences. In some embodiments, a non-human animal is provided that is homozygous with respect to an immunoglobulin κ light chain locus containing one or more human immunoglobulin λ light chain sequences described herein.

[0235] In some embodiments, one or more endogenous non-human immunoglobulin λ light chain sequences (or parts thereof) of the endogenous non-human immunoglobulin λ light chain locus are not deleted. In some embodiments, one or more endogenous non-human immunoglobulin λ light chain sequences (or parts thereof) of the endogenous non-human immunoglobulin λ light chain locus are deleted. In some embodiments, one or more endogenous non-human immunoglobulin λ light chain sequences (e.g., V, J, and / or C or any combination thereof) of the endogenous non-human immunoglobulin λ light chain locus are altered, moved, disrupted, deleted, or replaced so that the non-human immunoglobulin λ light chain locus is functionally silenced. In some embodiments, one or more endogenous non-human immunoglobulin λ light chain sequences (e.g., V, J, and / or C or any combination thereof) of an endogenous non-human immunoglobulin λ light chain locus are altered, moved, disrupted, deleted, or replaced by a targeting vector so that the non-human immunoglobulin λ light chain locus is functionally inactivated (i.e., it becomes impossible to generate functional light chains of antibodies expressed and / or detectable in the antibody repertoire of non-human animals described herein). Guidance for inactivating endogenous non-human immunoglobulin λ light chain loci is provided, for example, in U.S. Patent No. 9,006,511 (see, for example, Figure 2) (which is incorporated herein by reference in its entirety).

[0236] Modified immunoglobulin κ light chain loci or transgenes (e.g., including the limited human λ light chain variable region repertoire described herein) or their expression products can be detected using a variety of methods, including, for example, PCR, Southern blotting, restriction fragment length polymorphism (RFLP), allele acquisition or loss assays, Western blotting, and FACS analysis. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein are heterozygous with respect to the modified immunoglobulin κ light chain loci described herein. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein are semizygous with respect to the modified immunoglobulin κ light chain loci described herein. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein contain one or more copies of the modified immunoglobulin κ light chain loci or transgenes described herein. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein contain the modified endogenous immunoglobulin κ light chain loci as shown in the drawings.

[0237] This disclosure recognizes that non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells or non-human (e.g., rodents, e.g., rats or mice) tissues described herein utilize human heavy chain and λ light chain variable region gene segments contained in their genomes in their antibody selection and production mechanisms (e.g., recombination and somatic hypermutation). Thus, in various embodiments, the human immunoglobulin human heavy chain and λ light chain variable domains produced by the non-human animals, non-human cells or non-human tissues described herein are encoded by human heavy chain and λ light chain variable region gene segments contained in their genomes, respectively, or their somatic hypermutant variants.

[0238] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse) is provided, whose genome includes a modified immunoglobulin κ light chain locus, and the non-human animal includes B cells containing somatically hypermutated human heavy chain variable region sequences and / or human λ light chain variable region sequences. In some embodiments, the human heavy chain variable region sequences and / or human λ light chains present in the B cells of the non-human animal (e.g., a rodent, e.g., a rat or mouse) of the present disclosure have 1, 2, 3, 4, 5, or more somatic hypermutations. Those skilled in the art know methods for identifying gene segment sources in mature antibody sequences. For example, various tools such as DNAPLOT, IMGT / V-QUEST, JOINSOLVER, SoDA, and Ab-origin are available to assist in this analysis.

[0239] This disclosure provides, in particular, cells and tissues from non-human animals (e.g., rodents, e.g., rats, mice) as described herein. In some embodiments, splenocytes (and / or other lymphoid tissues) from non-human animals as described herein are provided. In some embodiments, B cells from non-human animals as described herein are provided. In some embodiments, pro-B cells from non-human animals as described herein are provided. In some embodiments, pre-B cells from non-human animals as described herein are provided. In some embodiments, immature B cells from non-human animals as described herein are provided. In some embodiments, mature naive B cells from non-human animals as described herein are provided. In some embodiments, activated B cells from non-human animals as described herein are provided. In some embodiments, memory B cells from non-human animals as described herein are provided. In some embodiments, B lineage lymphocytes from non-human animals as described herein are provided. In some embodiments, plasma or plasma cells from non-human animals as described herein are provided. In some embodiments, stem cells from non-human animals as described herein are provided. In some embodiments, the stem cells are embryonic stem cells. In some embodiments, embryonic cells from non-human animals as described herein are provided. In some embodiments, the embryonic cell is an oocyte. In some embodiments, the embryonic cell is a spermatid. In some embodiments, the spermatids from non-human animals described herein express one or more ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments. In some embodiments, any cells or tissues from non-human animals described herein can be isolated. In some embodiments, isolated cells and / or isolated tissues from non-human animals described herein are provided. In some embodiments, a hybridoma is provided, which is prepared using B cells from non-human animals described herein. In some embodiments, the hybridoma is prepared using B cells from non-human animals immunized with the antigen of interest.In some embodiments, hybridomas are prepared using B cells from non-human animals that express antibodies that bind (e.g., specifically bind) to an epitope on the target antigen.

[0240] Any non-human animal described herein (e.g., rodents, e.g., rats or mice) can be immunized with one or more target antigens under conditions and for a period of time sufficient to induce an immune response in the non-human animal to one or more target antigens. Those skilled in the art know methods for immunizing non-human animals. Exemplary and non-limiting methods for immunizing non-human animals can be found in U.S. Patent No. 7,582,298 (which is incorporated herein by reference in its entirety).

[0241] This disclosure provides, in particular, immunized non-human animals (e.g., rodents, e.g., rats or mice) as described herein, as well as cells and tissues isolated therefrom. In some embodiments, the non-human animals described herein generate a population of B cells in response to immunization with an antigen containing one or more epitopes. In some embodiments, the non-human animals generate a population of B cells that express antibodies that bind (e.g., specifically bind) to one or more epitopes of the antigen of interest. In some embodiments, the antibodies expressed by the population of B cells generated in response to the antigen include a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence and / or a lambda light chain having a human lambda light chain variable domain encoded by a human lambda light chain variable region sequence described herein. In some embodiments, the antibodies expressed by the population of B cells generated in response to the antigen include (i) a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence, and (ii) a lambda light chain having a human lambda light chain variable domain encoded by a human lambda light chain variable region sequence described herein.

[0242] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse) generates a population of B cells that express antibodies that bind to one or more epitopes of a target antigen, and the antibodies expressed by the population of B cells generated in response to the antigen include (i) a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence, and (ii) a lambda light chain having a human lambda light chain variable domain encoded by a human lambda light chain variable region sequence described herein. In some embodiments, the human heavy chain variable region sequence and / or human lambda light chain variable region sequence described herein are somatically hypermutated. In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90% of the B cells in a population of B cells generated in response to an antigen contain somatically hypermutated human heavy chain variable region sequences and / or human λ light chain variable region sequences.

[0243] Specific exemplary embodiment - immunoglobulin κ light chain locus In some embodiments, the provided non-human animal (e.g., a rodent, e.g., a rat or mouse) includes a modified endogenous immunoglobulin κ light chain locus comprising a single rearranged human immunoglobulin λ light chain variable region inserted upstream of a non-human or human Cλ gene segment and functionally linked thereto, wherein the non-human or human Cλ gene segment is inserted in place of the non-human Cκ gene. As described herein, such a modified endogenous immunoglobulin κ light chain locus further comprises a non-human immunoglobulin κ light chain enhancer region (or enhancer sequence). In some embodiments, the modified endogenous immunoglobulin κ light chain locus (or allele) comprises a single rearranged human immunoglobulin λ light chain variable region comprising a human Vλ gene segment appearing in cluster A of the human immunoglobulin λ light chain locus. In some embodiments, the modified endogenous immunoglobulin κ light chain locus (or allele) includes a single rearranged human immunoglobulin λ light chain variable region containing a human Vλ gene segment appearing in cluster B of the human immunoglobulin λ light chain locus. In some embodiments, the modified endogenous immunoglobulin κ light chain locus (or allele) includes a single rearranged human immunoglobulin λ light chain variable region containing a human Vλ gene segment appearing in cluster C of the human immunoglobulin λ light chain locus. In some embodiments, the modified endogenous immunoglobulin κ light chain locus (or allele) is Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-37, Vλ1-36, Vλ3- It contains a single rearranged human immunoglobulin λ light chain variable region, which includes a human Vλ gene segment selected from the group consisting of 27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1.In some embodiments, the modified κ light chain locus (or allele) contains a single rearranged human immunoglobulin λ light chain variable region comprising a human Vλ gene segment selected from the group consisting of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some embodiments, the modified κ light chain locus (or allele) contains a single rearranged human immunoglobulin λ light chain variable region comprising a human Vλ gene segment selected from the group consisting of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some embodiments, the modified κ light chain locus (or allele) comprises a single rearranged human immunoglobulin λ light chain variable region containing a human Vλ gene segment selected from the group consisting of Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, and Vλ2-14. In some embodiments, the modified κ light chain locus (or allele) comprises a single rearranged human immunoglobulin λ light chain variable region containing a human Vλ gene segment selected from the group consisting of Vλ1-51, Vλ1-40, and Vλ2-14. In some embodiments, the modified κ light chain locus (or allele) comprises a single rearranged human immunoglobulin λ light chain variable region containing a human Vλ gene segment selected from Vλ1-51 or Vλ2-14.In some embodiments, the modified immunoglobulin κ light chain locus (or allele) includes a single rearranged human immunoglobulin λ light chain variable region containing a human Jλ gene segment selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, the modified immunoglobulin κ light chain locus (or allele) includes a single rearranged human immunoglobulin λ light chain variable region containing a human Jλ gene segment selected from the group consisting of Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, the modified immunoglobulin κ light chain locus (or allele) includes a single rearranged human immunoglobulin λ light chain variable region containing a human Jλ2 gene segment.

[0244] This disclosure recognizes that non-human animals described herein (e.g., rodents, e.g., rats or mice) utilize human λ light chain variable region gene segments contained in their genomes in their antibody selection and production mechanisms (e.g., recombinant and somatic hypermutation). Thus, in various embodiments, the human immunoglobulin λ light chain variable domains produced by the non-human animals described herein are encoded by human λ light chain variable region gene segments contained in their genomes or by somatic hypermutant variants thereof.

[0245] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse) is provided, whose genome includes a modified endogenous immunoglobulin κ light chain locus, and the non-human animal includes B cells containing somatically hypermutated human heavy chain variable region sequences and / or human λ light chain variable region sequences. In some embodiments, the human heavy chain variable region sequences and / or human λ light chain variable region sequences present in the B cells of the non-human animal (e.g., a rodent, e.g., a rat or mouse) of the present disclosure have 1, 2, 3, 4, 5 or more somatic hypermutations. Those skilled in the art know methods for identifying gene segment sources in mature antibody sequences. For example, various tools such as DNAPLOT, IMGT / V-QUEST, JOINSOLVER, SoDA, and Ab-origin are available to assist in this analysis.

[0246] In many embodiments, the modified endogenous immunoglobulin κ light chain locus (or allele) contains a non-human immunoglobulin κ light chain enhancer region (or enhancer sequence) that appears in the wild-type immunoglobulin κ light chain locus (or allele). In some embodiments, the modified endogenous immunoglobulin κ light chain locus (or allele) contains a non-human immunoglobulin κ light chain enhancer region (or enhancer sequence) that appears in the wild-type immunoglobulin κ light chain locus (or allele) of a different species (e.g., a different rodent species).

[0247] In some embodiments, the non-human animals described herein (e.g., rodents, e.g., rats or mice) include in their germline genome a limited repertoire of human λ light chain variable regions (e.g., a single rearranged human immunoglobulin λ light chain variable region) functionally linked to one or more non-human immunoglobulin κ light chain enhancers (i.e., enhancer sequences or enhancer regions). In some predetermined embodiments, the limited repertoire of human λ light chain variable regions (e.g., a single rearranged human immunoglobulin λ light chain variable region) is functionally linked to a murine immunoglobulin κ light chain intron enhancer region (Igκ Ei or Eiκ). In some predetermined embodiments, the limited repertoire of human λ light chain variable regions (e.g., a single rearranged human immunoglobulin λ light chain variable region) is functionally linked to a murine immunoglobulin κ light chain 3' enhancer region (Igκ 3'E or 3'Eκ). In some predetermined embodiments, the limited human λ light chain variable region repertoire (e.g., a single rearranged human immunoglobulin λ light chain variable region) is functionally ligated to a murine Eiκ and functionally ligated to a murine 3'Eκ.

[0248] In some embodiments, the non-human Cλ gene of the modified endogenous immunoglobulin κ light chain locus (or allele) is a rodent Cλ gene, such as a mouse Cλ gene or a rat Cλ gene. In some predetermined embodiments, the non-human Cλ gene of the modified immunoglobulin κ light chain locus (or allele) is a mouse Cλ gene from a genetic background including the 129 line, BALB / c line, C57BL / 6 line, a crossed 129×C57BL / 6 line, or a combination thereof.

[0249] In some embodiments, the non-human Cλ gene of the modified immunoglobulin κ light chain locus (or allele) described herein contains a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 1 (mouse Cλ1), SEQ ID NO: 2 (mouse Cλ2), or SEQ ID NO: 3 (mouse Cλ3). In some embodiments, the non-human Cλ gene of the modified immunoglobulin κ light chain locus (or allele) described herein contains a sequence that is substantially identical or identical to SEQ ID NO: 1 (mouse Cλ1), SEQ ID NO: 2 (mouse Cλ2), or SEQ ID NO: 3 (mouse Cλ3). In some embodiments, the non-human Cλ gene of the modified immunoglobulin κ light chain locus (or allele) described herein is the sequence of the mouse Cλ1 gene, or contains it.

[0250] In some embodiments, the non-human Cλ domain encoded by a sequence located at the modified immunoglobulin κ light chain locus (or allele) described herein contains a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 4 (mouse Cλ1), SEQ ID NO: 5 (mouse Cλ2), or SEQ ID NO: 6 (mouse Cλ3). In some embodiments, the non-human Cλ domain encoded by a sequence located at the modified immunoglobulin κ light chain locus (or allele) described herein contains a sequence that is substantially identical or identical to SEQ ID NO: 4 (mouse Cλ1), SEQ ID NO: 5 (mouse Cλ2), or SEQ ID NO: 6 (mouse Cλ3). In some embodiments, the non-human Cλ gene encoded by a sequence located at the modified immunoglobulin κ light chain locus (or allele) described herein is or contains a mouse Cλ1 domain polypeptide.

[0251] In some embodiments, the non-human Cλ gene of the modified immunoglobulin κ light chain locus (or allele) described herein contains a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 7 (rat Cλ1), SEQ ID NO: 8 (rat Cλ2), SEQ ID NO: 9 (rat Cλ3), or SEQ ID NO: 10 (rat Cλ4). In some predetermined embodiments, the non-human Cλ gene of the modified immunoglobulin κ light chain locus (or allele) described herein contains a sequence that is substantially identical or identical to SEQ ID NO: 7 (rat Cλ1), SEQ ID NO: 8 (rat Cλ2), SEQ ID NO: 9 (rat Cλ3), or SEQ ID NO: 10 (rat Cλ4). In some predetermined embodiments, the non-human Cλ gene of the modified immunoglobulin κ light chain locus (or allele) described herein is the sequence of the rat Cλ1 gene, or contains it.

[0252] In some embodiments, the non-human Cλ domain encoded by a sequence located at the modified immunoglobulin κ light chain locus (or allele) described herein contains a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 11 (rat Cλ1), SEQ ID NO: 12 (rat Cλ2), SEQ ID NO: 13 (rat Cλ3), or SEQ ID NO: 14 (rat Cλ4). In some embodiments, the non-human Cλ domain encoded by a sequence located at the modified immunoglobulin κ light chain locus (or allele) described herein contains a sequence that is substantially identical or identical to SEQ ID NO: 11 (rat Cλ1), SEQ ID NO: 12 (rat Cλ2), SEQ ID NO: 13 (rat Cλ3), or SEQ ID NO: 14 (rat Cλ4). In some embodiments, the non-human Cλ domain encoded by a sequence located at the modified immunoglobulin κ light chain locus (or allele) described herein is or contains a rat Cλ1 domain polypeptide.

[0253] In some embodiments, the modified immunoglobulin κ light chain locus (or allele) human Cλ gene includes, for example, human Cλ1, human Cλ2, human Cλ3, human Cλ6, or human Cλ7 genes. In some predetermined embodiments, the modified immunoglobulin κ light chain locus (or allele) human Cλ gene is or includes the human Cλ2 gene.

[0254] In some embodiments, the modified immunoglobulin κ light chain locus (or allele) human Cλ gene described herein contains a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 15 (Human Cλ1), SEQ ID NO: 16 (Human Cλ2), SEQ ID NO: 17 (Human Cλ3), SEQ ID NO: 18 (Human Cλ6), or SEQ ID NO: 19 (Human Cλ7). In some embodiments, the modified immunoglobulin κ light chain locus (or allele) human Cλ gene described herein contains a sequence that is substantially identical to or identical to SEQ ID NO: 15 (Human Cλ1), SEQ ID NO: 16 (Human Cλ2), SEQ ID NO: 17 (Human Cλ3), SEQ ID NO: 18 (Human Cλ6), or SEQ ID NO: 19 (Human Cλ7). In some embodiments, the modified immunoglobulin κ light chain locus (or allele) human Cλ gene described herein is the sequence of the human Cλ2 gene, or contains it.

[0255] In some embodiments, the human Cλ domain encoded by a sequence located at the modified immunoglobulin κ light chain locus (or allele) described herein contains a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 20 (Human Cλ1), SEQ ID NO: 21 (Human Cλ2), SEQ ID NO: 22 (Human Cλ3), SEQ ID NO: 23 (Human Cλ6), or SEQ ID NO: 24 (Human Cλ7). In some embodiments, the human Cλ domain encoded by a sequence located at the modified immunoglobulin κ light chain locus (or allele) described herein contains a sequence that is substantially identical or identical to SEQ ID NO: 20 (Human Cλ1), SEQ ID NO: 21 (Human Cλ2), SEQ ID NO: 22 (Human Cλ3), SEQ ID NO: 23 (Human Cλ6), or SEQ ID NO: 24 (Human Cλ7). In some embodiments, the human Cλ domain encoded by a sequence located at the modified immunoglobulin κ light chain locus (or allele) described herein is a human Cλ2 domain polypeptide, or contains one.

[0256] Specific exemplary embodiments - immunoglobulin heavy chain locus In some embodiments, the non-human animal provided (e.g., rodents, e.g., rats or mice) includes a modified endogenous immunoglobulin κ light chain locus comprising the limited human immunoglobulin λ light chain variable region repertoire described herein (e.g., a single rearranged human immunoglobulin λ light chain variable region), arranged in a germline configuration, and functionally linked to multiple human V (e.g., rodents, e.g., rats or mice) immunoglobulin heavy chain constant region genes. H , D H and J H The modified immunoglobulin heavy chain locus (or allele) further comprises the presence of gene segments, enhancers, and regulatory regions. In some embodiments, the modified immunoglobulin heavy chain locus (or allele) described herein is one or more human V cells functionally linked to a non-human immunoglobulin heavy chain constant region. H Gene segment, one or more human DH Genetic segment and one or more human J H Includes a gene segment. In some predetermined embodiments, the modified immunoglobulin heavy chain locus (or allele) is at least human V H Gene segment V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1, or any combination thereof. In some predetermined embodiments, the modified immunoglobulin heavy chain locus (or allele) is at least human D H Gene segment D H1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H This includes 7-27, or any combination thereof. In some predetermined embodiments, the modified immunoglobulin heavy chain locus (or allele) is at least human J H Gene segment J H 1, J H 2, J H 3, J H 4, J H 5, J H 6, or any combination thereof.

[0257] This disclosure recognizes that non-human animals described herein (e.g., rodents, e.g., rats or mice) utilize human heavy chain variable region gene segments contained in their genomes in their antibody selection and production mechanisms (e.g., recombinant and somatic hypermutation). Thus, in various embodiments, the human immunoglobulin heavy chain variable domains produced by the non-human animals described herein are encoded by human heavy chain variable region gene segments contained in their genomes or by somatic hypermutant variants thereof.

[0258] In some embodiments, non-human animals (e.g., rodents, e.g., rats or mice) include B cells containing somatically hypermutated human heavy chain variable region sequences and / or human λ light chain variable region sequences. In some embodiments, the human heavy chain variable region sequences and / or human λ light chain variable region sequences present in the B cells of non-human animals (e.g., rodents, e.g., rats or mice) of the Disclosure have 1, 2, 3, 4, 5, or more somatic hypermutations. Those skilled in the art know methods for identifying gene segment sources in mature antibody sequences. For example, various tools such as DNAPLOT, IMGT / V-QUEST, JOINSOLVER, SoDA, and Ab-origin are available to assist in this analysis.

[0259] In some embodiments, the non-human immunoglobulin heavy chain constant region includes one or more non-human immunoglobulin heavy chain constant region genes, such as immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin E (IgE), and immunoglobulin A (IgA). In some predetermined embodiments, the non-human immunoglobulin heavy chain constant region includes rodent IgM, rodent IgD, rodent IgG3, rodent IgG1, rodent IgG2b, rodent IgG2a, rodent IgE, and rodent IgA constant region genes. In some embodiments, the human V H , D H and J H The gene segment is functionally linked to one or more non-human immunoglobulin heavy chain enhancers (i.e., enhancer sequences or enhancer regions). In some embodiments, the human V H , D H and J H The gene segment is functionally linked to one or more non-human immunoglobulin heavy chain regulatory regions (or regulatory sequences). In some embodiments, the human V H , D H and J HThe gene segment is functionally ligated to one or more non-human immunoglobulin heavy chain enhancers (or enhancer sequences) and one or more non-human immunoglobulin heavy chain regulatory regions (or regulatory sequences).

[0260] In some embodiments, the modified immunoglobulin heavy chain locus described herein does not contain the endogenous Adam6 gene. In some embodiments, the modified immunoglobulin heavy chain locus described herein does not contain the endogenous Adam6 gene (or a sequence encoding Adam6) at the same germline genomic location as found in the germline genomics of wild-type non-human animals of the same species. In some embodiments, the modified immunoglobulin heavy chain locus described herein does not contain the human Adam6 pseudogene. In some embodiments, the modified immunoglobulin heavy chain locus described herein includes the insertion of at least one nucleotide sequence encoding one or more non-human (e.g., rodent) Adam6 polypeptides, their functional orthologues, functional homologs, or functional fragments. In some embodiments, the insertion is outside the modified immunoglobulin heavy chain locus described herein (e.g., but not limited to the 5' end V H They may be located upstream of a gene segment, within modified immunoglobulin heavy chain loci, or in non-human animals (e.g., randomly introduced non-human Adam6 coding sequences), or at other locations in the germline genome of cells or tissues.

[0261] In various embodiments, the non-human animals provided herein (e.g., rodents, e.g., rats or mice) have endogenous non-human V in antibody molecules. H The region is not detected, either as a whole or partially. In various embodiments, the non-human animals provided herein have endogenous non-human V in the antibody molecule. H Domain (for example, V H , D H and / or J HIt does not contain (or lacks, or contains deletions thereof) one or more nucleotide sequences that encode ) in whole or in part. In various embodiments, the non-human animals provided herein are endogenous non-human V H , D H and J H The non-human animal has a germline genome that contains a deletion of a gene segment, either entirely or partially. In various embodiments, the non-human animal provided is fertile.

[0262] Guidance for the creation of targeting vectors, non-human (e.g., rodent, e.g., rat or mouse) cells and animals possessing such modified immunoglobulin heavy chain loci (or alleles) can be found in Macdonald (2014), U.S. Patents 6,596,541, 8,642,835, 8,697,940 and 8,791,323 (each of which is incorporated herein by reference in whole). Those skilled in the art are familiar with the diverse techniques known in the art for preparing, providing or manufacturing such sequences for achieving such gene modifications and / or manipulations of non-human (e.g., mammalian) genomes or for introduction into germline genomes of non-human animals.

[0263] Specific exemplary embodiments - combinations of immunoglobulin gene loci In some embodiments, non-human animals provided herein (e.g., rodents, e.g., rats or mice) include, in their germline genome, a modified endogenous immunoglobulin κ light chain locus containing the limited human immunoglobulin λ light chain variable region repertoire described herein (e.g., a single rearranged human immunoglobulin λ light chain variable region), and further include one or more additional immunoglobulin loci containing human immunoglobulin gene segments or other human or humanized genes (e.g., human genes encoding TdT) (e.g., via hybridization or multiple gene targeting strategies). Such non-human animals may be prepared as described above or by methods known in the art to achieve the desired modified genotype depending on the intended use of the non-human animal. Additional human immunoglobulin gene segments or other human or humanized genes (e.g., human genes encoding TdT) in other immunoglobulin loci may be introduced via further alteration of the genome of cells having the above-described genetic modification (e.g., embryonic stem cells) or, as desired, via hybridization techniques known in the art with other genetically modified lines.

[0264] In some embodiments, the non-human animals provided herein (e.g., rodents, e.g., rats or mice) have a modified endogenous immunoglobulin κ light chain locus in their germline genome that includes a limited repertoire of human immunoglobulin λ light chain variable regions described herein (e.g., a single rearranged human immunoglobulin λ light chain variable region), and one or more human V genes functionally linked to one or more non-human animal immunoglobulin heavy chain constant region genes in their germline genome. H Gene segment, one or more human D gene segments, and one or more human J HThe modified endogenous immunoglobulin heavy chain locus further includes a gene segment. In some embodiments, the non-human animal is heterozygous or homozygous for the modified endogenous immunoglobulin κ light chain locus, which includes the limited human immunoglobulin λ light chain variable region repertoire described herein (e.g., a single rearranged human immunoglobulin λ light chain variable region). In some embodiments, the non-human animal is heterozygous or homozygous for the modified endogenous immunoglobulin heavy chain locus, which includes the limited human immunoglobulin λ light chain variable region repertoire described herein (e.g., a single rearranged human immunoglobulin λ light chain variable region), and is homozygous for the modified endogenous immunoglobulin heavy chain locus, which is described herein.

[0265] In some embodiments, non-human animals provided herein (e.g., rodents, e.g., rats or mice) have modified endogenous immunoglobulin κ light chain loci in their germline genome that include a limited repertoire of human immunoglobulin λ light chain variable regions described herein (e.g., a single rearranged human immunoglobulin λ light chain variable region), and one or more human V κ light chain genes functionally linked to one or more non-human animal immunoglobulin heavy chain constant region genes. H Gene segment, one or more human D gene segments, and one or more human J HThe modified endogenous immunoglobulin heavy chain locus comprises a gene segment and further comprises a functionally inactivated (e.g., deleted as a whole or in part, or otherwise nonfunctional) endogenous immunoglobulin λ light chain locus. In some embodiments, the non-human animal is heterozygous or homozygous for a modified endogenous immunoglobulin κ light chain locus comprising the limited human immunoglobulin λ light chain variable region repertoire described herein (e.g., a single rearranged human immunoglobulin λ light chain variable region). In some embodiments, the non-human animal is heterozygous or homozygous for a modified endogenous immunoglobulin heavy chain locus described herein. In some embodiments, the non-human animal is heterozygous or homozygous for a functionally inactivated (e.g., deleted as a whole or in part, or otherwise nonfunctional) endogenous immunoglobulin λ light chain locus. In some embodiments, non-human animals are homozygous for modified endogenous immunoglobulin κ light chain loci, including a limited repertoire of human immunoglobulin λ light chain variable regions described herein (e.g., a single rearranged human immunoglobulin λ light chain variable region), homozygous for modified endogenous immunoglobulin heavy chain loci, and homozygous for functionally inactivated (e.g., deleted as a whole or in part, or otherwise nonfunctional) endogenous immunoglobulin λ light chain loci.

[0266] In some embodiments, the germline genome of a genetically modified non-human animal, such as a rodent (e.g., mouse or rat), comprises a modified endogenous immunoglobulin κ locus containing two alleles. In some embodiments, the first allele comprises a limited human λ light chain variable region repertoire, and the second allele comprises a limited human κ light chain variable region repertoire. In some embodiments, the germline genome of a genetically modified rodent (e.g., mouse or rat), rodent (e.g., mouse or rat) cell or rodent (e.g., mouse or rat) tissue described herein comprises a first modified endogenous immunoglobulin κ light chain locus allele containing a single rearranged human immunoglobulin λ light chain variable region functionally linked to a rodent (e.g., mouse or rat) Cλ gene segment, the single rearranged human immunoglobulin λ light chain variable region comprising a human Vλ gene segment and a human Jλ gene segment. In some embodiments, the genetically modified rodents (e.g., mice or rats) described herein, and the resulting rodent (e.g., mouse or rat) cells or rodent (e.g., mouse or rat) tissues, comprise a second modified endogenous immunoglobulin κ light chain locus allele containing a single rearranged human immunoglobulin κ light chain variable region functionally linked to a rodent (e.g., mouse or rat) Cκ gene segment, the single rearranged human immunoglobulin κ light chain variable region comprising a human Vκ gene segment and a human Jκ gene segment. In some embodiments, such rodent (e.g., mouse or rat) tissues may express a λ light chain from the first modified endogenous immunoglobulin κ light chain locus allele and a κ light chain from the second modified endogenous immunoglobulin κ light chain locus allele. In some embodiments, a single rearranged human immunoglobulin κ light chain variable region comprises Vκ3-20 or Vκ1-39, and a single rearranged human immunoglobulin λ light chain variable region comprises Vλ1-51 or Vλ2-14.In one embodiment, a single rearranged human immunoglobulin κ light chain variable region is Vκ3-20 / Jκ1, and a single rearranged human immunoglobulin λ light chain variable region is Vλ1-51 / Jλ2 or Vλ2-14 / Jλ2.

[0267] In some embodiments, the non-human animal provided (e.g., a rodent, e.g., a rat or mouse) is (a) a non-human animal that is a non-human C H Human V fused to the domain sequence H Human V globulins are functionally linked to one or more endogenous non-human immunoglobulin heavy chain constant regions to express an immunoglobulin heavy chain containing a domain sequence. H , D H and J H (b) A germline genome comprising a homozygous or heterozygous immunoglobulin heavy chain locus containing a gene segment; (b) an immunoglobulin κ light chain locus containing a single rearranged human immunoglobulin λ light chain functionally ligated to a non-human (e.g., rodent) immunoglobulin Cλ gene segment, such that the non-human animal expresses an immunoglobulin light chain containing a human Vλ domain sequence fused to a non-human Cλ domain sequence.

[0268] In some embodiments, the non-human animal provided (e.g., a rodent, e.g., a rat or mouse) is (a) a non-human animal that is a non-human C H Human V fused to the domain sequence H Human V globulins are functionally linked to one or more endogenous non-human immunoglobulin heavy chain constant regions to express an immunoglobulin heavy chain containing a domain sequence. H , D H and J H(b) an immunoglobulin κ light chain locus containing a homozygous or heterozygous immunoglobulin heavy chain locus containing a gene segment; (c) an immunoglobulin κ light chain locus containing a single rearranged human immunoglobulin λ light chain functionally linked to a non-human (e.g., rodent) immunoglobulin Cλ gene segment so that a non-human animal expresses an immunoglobulin light chain containing a human Vλ domain sequence fused to a non-human Cλ domain sequence; and (d) a germline genome containing a homozygous or heterozygous endogenous immunoglobulin λ light chain locus that is functionally inactivated or deleted as a whole or in part.

[0269] For example, a non-human animal containing a modified endogenous immunoglobulin κ light chain locus as described herein may further include one or more modifications described in U.S. Patents 8,642,835, 8,697,940, 9,006,511, 9,035,128, 9,066,502, 9,150,662 and 9,163,092 (each of which is incorporated in whole by reference) (e.g., via hybridization or multiple gene targeting strategies).

[0270] Nucleic acid construct Typically, a polynucleotide molecule containing a human immunoglobulin λ light chain sequence (e.g., a single rearranged human λ light chain variable region, or one or two unrearranged Vλ gene segments and at least one unrearranged Jλ gene segment), or a portion thereof, is ligated to (e.g., inserted into) a vector, preferably a DNA vector, for replication of the polynucleotide molecule in a host cell.

[0271] Human immunoglobulin λ light chain sequences can be cloned directly from known sequences or sources (e.g., libraries) or synthesized from germline sequences designed in silico based on publicly available sequences from GenBank or other publicly available databases (e.g., IMGT). Alternatively, bacterial artificial chromosome (BAC) libraries can provide the immunoglobulin DNA sequences of interest (e.g., human Vλ and Jλ sequences and combinations thereof). BAC libraries can contain insert sizes of 100–150 kb and can hold inserts as large as 300 kb (Shizuya, et al., 1992, Proc. Natl. Acad. Sci., USA 89:8794-8797; Swiatek, et al., 1993, Genes and Development 7:2071-2084; Kim, et al., 1996, Genomics 34 213-218 (all of which are incorporated herein by reference)). For example, human BAC libraries with an average insert size of 164–196 kb have been described (Osoegawa, K. et al., 2001, Genome Res. 11(3):483-96; Osoegawa, K. et al., 1998, Genomics 52:1-8, Article No. GE985423 (each of these is incorporated herein by reference in its entirety)). Human and non-human animal genomic BAC libraries have been constructed and are commercially available (e.g., ThermoFisher). Genomic BAC libraries can also serve as a source of immunoglobulin DNA sequences and transcriptional control regions.

[0272] Alternatively, immunoglobulin DNA sequences can be isolated, cloned, and / or transferred from yeast artificial chromosomes (YACs). For example, the nucleotide sequence of the human immunoglobulin λ light chain locus has been determined (see, e.g., Dunham, I. et al., 1999, Nature 402:489-95 (the whole is incorporated herein by reference)). Furthermore, YACs have long been used to construct human immunoglobulin λ light chain locus transgenes (see, e.g., Popov, A. et al., 1996, Gene 177:195-201; Popov, A. et al., 1999, J. Exp. Med. 189(10):1611-19 (each of these is incorporated herein by reference)). Entire immunoglobulin λ light chain loci (human or non-human) can be cloned and contained within some YACs. Regardless of the sequences they contain, if multiple YACs are used and contain overlapping similar regions, they can be recombined within the yeast host line to produce a single construct representing the entire locus or a desired portion of the locus (e.g., the region targeted by the targeting vector). The YAC arms may be further modified by improvements in mammalian selection cassettes to assist in introducing the construct into embryonic stem cells or embryos by methods known in the art and / or described herein.

[0273] The DNA and amino acid sequences of human immunoglobulin λ light chain gene segments for use in the construction of the modified immunoglobulin κ light chain locus described herein can be obtained from publicly available databases (e.g., GenBank, IMGT, etc.) and / or publicly available antibody sequences.

[0274] In some predetermined embodiments, a nucleic acid construct containing a human immunoglobulin λ light chain gene segment (e.g., a single rearranged human λ light chain variable region, or one or two unrearranged Vλ gene segments and at least one unrearranged Jλ gene segment) is functionally ligated to a human or non-human (e.g., rodent, e.g., rat or mouse) immunoglobulin λ or immunoglobulin κ light chain constant region (Cλ or Cκ, respectively) gene. In some predetermined embodiments, a nucleic acid construct containing a human immunoglobulin λ light chain gene segment (e.g., a single rearranged human λ light chain variable region, or one or two unrearranged Vλ gene segments and at least one unrearranged Jλ gene segment) is functionally ligated to one or more non-human (e.g., rodent, e.g., rat or mouse) immunoglobulin κ or immunoglobulin λ light chain enhancer regions (or enhancer sequences). In some embodiments, a nucleic acid construct containing a human immunoglobulin λ light chain gene segment (e.g., a single rearranged human λ light chain variable region, or one or two unrearranged Vλ gene segments and at least one unrearranged Jλ gene segment) is functionally ligated to a non-human (e.g., rodent, e.g., rat or mouse) or human Cλ region gene and a non-human immunoglobulin κ light chain enhancer region (or enhancer sequence).

[0275] In some embodiments, the nucleic acid construct containing non-reorganized human Vλ and Jλ sequences further comprises intergenetic DNA of human and / or murine origin. In some embodiments, the intergenetic DNA is, or comprises, a non-coding murine immunoglobulin κ light chain sequence, a non-coding human immunoglobulin κ light chain sequence, a non-coding murine immunoglobulin λ light chain sequence, a non-coding human immunoglobulin λ light chain sequence, or a combination thereof.

[0276] Nucleic acid constructs can be prepared using methods known in the art. For example, a nucleic acid construct can be prepared as part of a larger plasmid. Such preparations allow for the cloning and selection of precise constructs in an efficient manner as known in the art. Nucleic acid constructs containing, in whole or in part, human immunoglobulin λ light chain sequences described herein can be positioned between restriction sites on a plasmid so that they can be isolated from the residual plasmid sequence for incorporation into a desired non-human animal (e.g., a rodent, such as a rat or mouse).

[0277] Various methods are known in the art for the preparation of nucleic acid constructs (e.g., plasmids) and the transformation of host organisms. For other suitable expression systems for both prokaryotes and eukaryotic cells, as well as general recombination procedures, see Principles of Gene Manipulation: An Introduction to Genetic Manipulation, 5th Ed., ed. By Old, R.W. and S.P.B., Rimrose, Blackwell Science, Inc., 1994 and Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al., Cold Spring Harbor Laboratory Press: 1989 (each of these is incorporated herein by reference in its entirety).

[0278] Targeting vectors A targeting vector may be used to introduce a nucleic acid construct into a target genomic locus. A targeting vector may include a nucleic acid construct and homology arms flanking the nucleic acid construct; those skilled in the art are familiar with the diverse options and features generally applicable to the design, structure, and / or use of targeting vectors. For example, targeting vectors may be linear or circular in form, and they may be single-stranded or double-stranded. Targeting vectors may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). For ease of reference, homology arms are referred herein as 5' and 3' (i.e., upstream and downstream) homology arms. This terminology relates to the relative position of the homology arms to the nucleic acid construct within the targeting vector. The 5' and 3' homology arms correspond to regions within the targeted locus or regions within another targeting vector, which are referred herein as the “5' target sequence” and “3' target sequence,” respectively. In some embodiments, homology arms may also function as 5' or 3' target sequences.

[0279] In some embodiments, the methods described herein utilize two, three, or more targeting vectors that are recombinable with one another. In various embodiments, the targeting vectors are large targeting vectors (LTVECs), as described elsewhere herein. In some embodiments, the first, second, and third targeting vectors each include 5' and 3' homology arms. The 3' homology arm of the first targeting vector includes an overlapping sequence (i.e., overlapping sequence) with the 5' homology arm of the second targeting vector, which enables homologous recombination between the first and second LTVECs.

[0280] In the dual targeting method, the 5' homology arm of the first targeting vector and the 3' homology arm of the second targeting vector may resemble the corresponding segment (i.e., target sequence) within the target genomic locus, thereby promoting homologous recombination of the first and second targeting vectors with the corresponding genomic segment and potentially modifying the target genomic locus.

[0281] In the triple-targeting method, the 3' homology arm of the second targeting vector may contain a sequence that overlaps with the 5' homology arm of the third targeting vector (i.e., a duplicate sequence), which may enable homologous recombination between the second and third LTVECs. The 5' homology arm of the first targeting vector and the 3' homology arm of the third targeting vector are similar to the corresponding segment (i.e., target sequence) within the target genomic locus, which may facilitate homologous recombination of the first and third targeting vectors with the corresponding genomic segment and modify the target genomic locus.

[0282] A homology arm and a target sequence, or two homology arms, are considered "corresponding" or "corresponding" to each other if the two regions share a sufficient level of sequence identity with one another so that they can act as substrates for homologous recombination. The sequence identity between a given target sequence and the corresponding homology arm (i.e., duplicate sequence) found in the targeting vector, or between two homology arms, can be any degree of sequence identity that allows homologous recombination to occur. To give just one example, the amount of sequence identity shared by the homology arms of a targeting vector (or fragment thereof) and the target sequence (or fragment thereof) of another targeting vector or target sequence of a target genomic locus may be, for example, not limited to, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, such that the sequences undergo homologous recombination.

[0283] Furthermore, the homology arm and the corresponding region of similarity (e.g., identity) between it and the corresponding target sequence can be of any length sufficient to promote homologous recombination at the target genomic locus. For example, a given homology arm and / or corresponding target sequence may include a corresponding region of similarity such as, for example, but not limited to, approximately 5–10kb, 5–15kb, 5–20kb, 5–25kb, 5–30kb, 5–35kb, 5–40kb, 5–45kb, 5–50kb, 5–55kb, 5–60kb, 5–65kb, 5–70kb, 5–75kb, 5–80kb, 5–85kb, 5–90kb, 5–95kb, 5–100kb, 100–200kb, or 200–300kb (as described elsewhere in this specification) so that the homology arm has sufficient similarity to undergo homologous recombination with the corresponding target sequence(s) in the target genomic locus of the cell or in another targeting vector. In some embodiments, a given homology arm and / or corresponding target sequence includes a corresponding region of similarity such as, for example, but not limited to, having a length of about 10–100kb, 15–100kb, 20–100kb, 25–100kb, 30–100kb, 35–100kb, 40–100kb, 45–100kb, 50–100kb, 55–100kb, 60–100kb, 65–100kb, 70–100kb, 75–100kb, 80–100kb, 85–100kb, 90–100kb, or 95–100kb (as described elsewhere in this specification) so that the homology arm has sufficient similarity to undergo homologous recombination with the corresponding target sequence(s) in the target genomic locus of the cell or in another targeting vector.

[0284] The overlapping sequences of the 3' homology arm of the first targeting vector and the 5' homology arm of the second targeting vector, or the overlapping sequences of the 3' homology arm of the second targeting vector and the 5' homology arm of the third targeting vector, may be of any length sufficient to promote homologous recombination between the targeting vectors. For example, a given duplicate sequence of a homology arm may contain a corresponding duplicate region of approximately 1–5kb, 5–10kb, 5–15kb, 5–20kb, 5–25kb, 5–30kb, 5–35kb, 5–40kb, 5–45kb, 5–50kb, 5–55kb, 5–60kb, 5–65kb, 5–70kb, 5–75kb, 5–80kb, 5–85kb, 5–90kb, 5–95kb, 5–100kb, 100–200kb, or 200–300kb, such that the duplicate sequence of the homology arm has sufficient similarity to undergo homologous recombination with the corresponding duplicate sequence in another targeting vector. In some embodiments, a given duplicate sequence of a homology arm includes a duplicate region of approximately 1–100kb, 5–100kb, 10–100kb, 15–100kb, 20–100kb, 25–100kb, 30–100kb, 35–100kb, 40–100kb, 45–100kb, 50–100kb, 55–100kb, 60–100kb, 65–100kb, 70–100kb, 75–100kb, 80–100kb, 85–100kb, 90–100kb, or 95–100kb, such that the duplicate sequence of the homology arm has sufficient similarity to undergo homologous recombination with the corresponding duplicate sequence in another targeting vector. In some embodiments, the duplicate sequence is 1–5kb (including both ends). In some embodiments, the overlapping sequence is approximately 1kb to approximately 70kb (including both ends). In some embodiments, the overlapping sequence is approximately 10kb to approximately 70kb (including both ends). In some embodiments, the overlapping sequence is approximately 10kb to approximately 50kb (including both ends). In some embodiments, the overlapping sequence is at least 10kb. In some embodiments, the overlapping sequence is at least 20kb.For example, duplicate arrays are approximately 1kb to 5kb (including both ends), 5kb to 10kb (including both ends), 10kb to 15kb (including both ends), 15kb to 20kb (including both ends), 20kb to 25kb (including both ends), 25kb to 30kb (including both ends), 30kb to 35kb (including both ends), 35kb to 40kb (including both ends), 40kb to 45kb (including both ends), 45kb to 50kb (including both ends), 50kb to 60kb (including both ends), 60kb to 70kb (including both ends), 70kb to 80kb (including both ends), and so on. The size can be approximately 80kb to 90kb (including both ends), approximately 90kb to 100kb (including both ends), approximately 100kb to 120kb (including both ends), approximately 120kb to 140kb (including both ends), approximately 140kb to 160kb (including both ends), approximately 160kb to 180kb (including both ends), approximately 180kb to 200kb (including both ends), approximately 200kb to 220kb (including both ends), approximately 220kb to 240kb (including both ends), approximately 240kb to 260kb (including both ends), approximately 260kb to 280kb (including both ends), or approximately 280kb to 300kb (including both ends). To give just one example, a duplicate array can be approximately 20kb to 60kb (including both ends). Alternatively, the duplicated sequence may be at least 1kb, at least 5kb, at least 10kb, at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 35kb, at least 40kb, at least 45kb, at least 50kb, at least 60kb, at least 70kb, at least 80kb, at least 90kb, at least 100kb, at least 120kb, at least 140kb, at least 160kb, at least 180kb, at least 200kb, at least 220kb, at least 240kb, at least 260kb, at least 280kb, or at least 300kb.In some embodiments, the overlapping array may be at most 400kb, at most 350kb, at most 300kb, at most 280kb, at most 260kb, at most 240kb, at most 220kb, at most 200kb, at most 180kb, at most 160kb, at most 140kb, at most 120kb, at most 100kb, at most 90kb, at most 80kb, at most 70kb, at most 60kb, or at most 50kb.

[0285] In some embodiments, homology arms may correspond to loci native to the cell (e.g., the targeted locus), or alternatively, to regions of heterologous or exogenous segments of DNA integrated into the cell's genome (e.g., including transgenes, expression cassettes, or heterologous or exogenous regions of DNA). In some embodiments, homology arms may correspond to regions on the targeting vector in the cell. In some embodiments, homology arms of the targeting vector may correspond to regions of yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), human artificial chromosomes, or any other modified regions contained in a suitable host cell. Furthermore, homology arms of the targeting vector may correspond to or be derived from regions of a BAC library, cosmid library, or P1 phage library. In some predetermined embodiments, the homology arms of the targeting vector correspond to loci that are native, heterologous, or exogenous to prokaryotes, yeast, birds (e.g., chickens), non-human mammals, rodents, humans, rats, mice, hamsters, rabbits, pigs, cattle, deer, sheep, goats, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys), domesticated mammals, agricultural mammals, or any other organism of interest. In some embodiments, the homology arms correspond to loci in cells that exhibit limited sensitivity to targeting using conventional methods in the absence of nicks or double-strand breaks induced by a nuclease substance (e.g., Cas protein), or that exhibit relatively low levels of successful integration at the targeted site and / or significant levels of off-target integration. In some embodiments, the homology arms are designed to contain modified DNA.

[0286] In some embodiments, the 5' and 3' homology arms of the targeting vector(s) correspond to the targeted genome. Alternatively, the homology arms correspond to related genomes. For example, the targeted genome is the genome of a first strain of mouse, and the targeting arms correspond to the genome of a second strain of mouse, where the first and second strains are different. In certain embodiments, the homology arms correspond to the genome of the same animal or from the same strain; for example, the targeted genome is the genome of a first strain of mouse, and the targeting arms correspond to the genome of a mouse from the same mouse or from the same strain.

[0287] Homology arms of targeting vectors are, for example, 1-5kb (including both ends), 5-10kb (including both ends), 5-15kb (including both ends), 5-20kb (including both ends), 5-25kb (including both ends), 5-30kb (including both ends), 5-35kb (including both ends), 5-40kb (including both ends), 5-45kb (including both ends), 5-50kb (including both ends), 5-55kb (including both ends), 5-60kb (including both ends), The sequences may be of any length sufficient to facilitate homologous recombination events with the corresponding target sequence, including 5–65kb (inclusive), 5–70kb (inclusive), 5–75kb (inclusive), 5–80kb (inclusive), 5–85kb (inclusive), 5–90kb (inclusive), 5–95kb (inclusive), 5–100kb (inclusive), 100–200kb (inclusive), or 200–300kb (inclusive). In some embodiments, the homology arms of the targeting vector have lengths of 1-100kb (inclusive of both ends), 5-100kb (inclusive of both ends), 10-100kb (inclusive of both ends), 15-100kb (inclusive of both ends), 20-100kb (inclusive of both ends), 25-100kb (inclusive of both ends), 30-100kb (inclusive of both ends), 35-100kb (inclusive of both ends), 40-100kb (inclusive of both ends), 45-100kb (inclusive of both ends), and 50 The targeting arms are approximately 100kb (including both ends), 55-100kb (including both ends), 60-100kb (including both ends), 65-100kb (including both ends), 70-100kb (including both ends), 75-100kb (including both ends), 80-100kb (including both ends), 85-100kb (including both ends), 90-100kb (including both ends), or 95-100kb (including both ends), and are long enough to facilitate homologous recombination events with the corresponding target sequence. As described herein, larger targeting vectors may utilize longer targeting arms.

[0288] To facilitate modification of a target locus (e.g., modification of the immunoglobulin κ light chain locus, or modification of an already modified or altered immunoglobulin κ light chain locus), a nuclease substance (e.g., the CRISPR / Cas system) may be used in combination with a targeting vector. Such a nuclease substance may promote homologous recombination between the targeting vector and the target locus. When a nuclease substance is used in combination with a targeting vector, the targeting vector may include 5' and 3' homology arms corresponding to 5' and 3' target sequences located sufficiently close to the nuclease cleavage site to facilitate the occurrence of homologous recombination events between the target sequence and the homology arms upon nick or double-strand break at the nuclease cleavage site. The term “nuclease cleavage site” includes the DNA sequence (e.g., Cas9 cleavage site) on which nick or double-strand break is induced by the nuclease substance. The target sequences within the locus of the targeted gene corresponding to the 5' and 3' homology arms of the targeting vector are "sufficiently close" to the nuclease cleavage site if they are at a distance that facilitates the occurrence of homologous recombination events between the 5' and 3' target sequences and the homology arms upon nick or double-strand break at the recognition site. Thus, in a given embodiment, the target sequences corresponding to the 5' and / or 3' homology arms of the targeting vector are within at least one nucleotide of a given recognition site, or within at least 10 nucleotides to about 14 kb of a given recognition site. In some embodiments, the nuclease cleavage site is directly adjacent to at least one or both of the target sequences.

[0289] The spatial relationship between the target sequence corresponding to the homology arm of the targeting vector and the nuclease cleavage site can be diverse. For example, the target sequence may be located 5' relative to the nuclease cleavage site, the target sequence may be located 3' relative to the recognition site, or the target sequence may sandwich the nuclease cleavage site.

[0290] The combined use of a targeting vector (e.g., a large targeting vector) and a nuclease substance may result in improved targeting efficiency compared to the use of the targeting vector alone. For example, when a targeting vector is used in conjunction with a nuclease substance, the targeting efficiency of the targeting vector may be improved by at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or within a range formed from these integers, for example, 2 to 10 times, compared to the use of the targeting vector alone.

[0291] Some targeting vectors are “large targeting vectors” or “LTVECs,” which include targeting vectors that correspond to and contain homology arms derived from nucleic acid sequences larger than those typically used by other approaches intended to perform homologous recombination in cells. An LTVEC may, for example, be at least 10 kb in length, or the sum of the 5' and 3' homology arms may be at least 10 kb. LTVECs also include targeting vectors that contain nucleic acid constructs larger than those typically used by other approaches intended to perform homologous recombination in cells. For example, LTVECs enable modification of large loci that cannot be provided by traditional plasmid-based targeting vectors due to their size limitations. For example, a target locus may be a cellular locus that cannot be targeted using conventional methods, or can only be targeted inaccurately or with significantly lower efficiency, in the absence of nicks or double-strand breaks induced by a nuclease substance (e.g., Cas protein) (i.e., the 5' homology arm and 3' homology arm may correspond to that locus).

[0292] In some embodiments, the methods described herein may utilize two or three LTVECs capable of recombining with each other and with target genomic loci in a three-way or four-way recombination event. Such methods enable large locus modifications that cannot be achieved using a single LTVEC.

[0293] Examples of LTVECs include vectors derived from bacterial artificial chromosomes (BACs), human artificial chromosomes, or yeast artificial chromosomes (YACs). LTVECs may be linear or circular in form. Examples of LTVECs and methods for producing them are described, for example, in Macdonald (2014), U.S. Patents 6,586,251, 6,596,541, and 7,105,348; and International Patent Application Publication WO2002 / 036789 (each of which is incorporated herein by reference in whole).

[0294] Method for producing non-human animals A composition and method for producing a non-human animal (e.g., a rodent, e.g., a rat or mouse) is provided, wherein the germline genome comprises a modified immunoglobulin κ light chain locus that includes one or more human immunoglobulin λ light chain sequences (e.g., human Vλ and Jλ gene segments) encoding a sequence containing a specific polymorphic form of the human Vλ and Jλ segments (e.g., a specific V and / or J allele or variant) instead of a non-human immunoglobulin κ light chain sequence, and this includes a non-human or human Compositions and methods for producing non-human animals expressing antibodies comprising an immunoglobulin λ light chain containing a human variable region and a non-human or human constant region, constructed from an immunoglobulin κ light chain locus containing a single rearranged human immunoglobulin λ light chain variable region functionally linked to an immunoglobulin λ light chain constant region gene, wherein the non-human or human immunoglobulin λ light chain constant region gene is positioned in place of the non-human immunoglobulin κ light chain constant region gene that normally appears at the wild-type non-human immunoglobulin κ light chain locus. In some embodiments, compositions and methods are also provided for producing non-human animals expressing such antibodies under the control of endogenous immunoglobulin κ enhancers and / or endogenous immunoglobulin κ regulatory sequences. In some embodiments, compositions and methods are also provided for producing non-human animals expressing such antibodies under the control of heterologous immunoglobulin κ enhancers and / or heterologous immunoglobulin κ regulatory sequences.

[0295] The methods described herein involve inserting a single rearranged human immunoglobulin λ light chain variable region encoding a human immunoglobulin λ light chain variable domain upstream of a non-human or human immunoglobulin λ light chain constant region gene (e.g., rodents, e.g., murids, e.g., rats or mice) or a human Cλ region gene, wherein the non-human or human immunoglobulin λ light chain constant region gene is positioned in place of the non-human immunoglobulin κ light chain constant region gene that normally appears at the wild-type non-human immunoglobulin κ light chain locus, thereby expressing an antibody characterized by the presence of a light chain containing a human λ light chain variable domain and a non-human Cλ domain (e.g., rodents (e.g., murids, e.g., rats or mice) Cλ domain) or a light chain containing a human λ light chain variable and a human Cλ domain, and being expressed both on the surface of B cells and in the serum of non-human animals.

[0296] In some embodiments, the method involves inserting genetic material containing a single rearranged human immunoglobulin λ light chain variable region into the immunoglobulin κ light chain locus (e.g., wild-type, modified, or altered immunoglobulin κ light chain locus of a non-human animal (e.g., rodents, e.g., rats or mice)). In some embodiments, the method involves inserting genetic material containing a single rearranged human immunoglobulin λ light chain variable region into the immunoglobulin κ light chain locus of a modified or altered strain of a non-human animal (e.g., rodents, e.g., rats or mice).

[0297] In some embodiments, the method includes multiple insertions in a single ES cell clone. In some embodiments, the method includes sequential insertions performed in a successor ES cell clone. In some embodiments, the method includes a single insertion performed in a modified ES cell clone.

[0298] In some embodiments, the method includes DNA insertion(s) upstream of a non-human (e.g., rodent, e.g., rat or mouse) Cλ1 gene (or human Cλ2 gene), thereby functionally linking the DNA insertion(s) to the non-human (e.g., rodent, e.g., rat or mouse) Cλ1 gene (or human Cλ2 gene), where the DNA insertion(s) are Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ It comprises one or two human Vλ gene segments selected from the group consisting of 3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1, as well as one or more human Jλ gene segments selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7, wherein a non-human (e.g., rodent, e.g., rat or mouse) Cλ1 gene (or human Cλ2 gene) is positioned in place of the non-human (e.g., rodent, e.g., rat or mouse) Cκ gene at the endogenous immunoglobulin κ light chain locus.In some embodiments, the method includes DNA insertion(s) upstream of a non-human (e.g., rodent, e.g., rat or mouse) Cλ1 gene (or human Cλ2 gene), thereby functionally linking the DNA insertion(s) to the non-human (e.g., rodent, e.g., rat or mouse) Cλ1 gene (or human Cλ2 gene), where the DNA insertion(s) are Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3- It comprises a single rearranged human λ light chain variable region including a human Vλ gene segment selected from the group consisting of Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1, and a human Jλ gene segment selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7, wherein the non-human (e.g., rodent, e.g., rat or mouse) Cλ1 gene (or human Cλ2 gene) is located in place of the non-human (e.g., rodent, e.g., rat or mouse) Cκ gene at the endogenous immunoglobulin κ light chain locus.

[0299] Where appropriate, a human immunoglobulin λ light chain sequence encoding the human immunoglobulin λ light chain variable domain (i.e., a sequence containing the human Vλ and Jλ gene segments) may be separately modified to include codons optimized for expression in non-human animals (see, for example, U.S. Patents 5,670,356 and 5,874,304, each of which is incorporated herein by reference). The codon-optimized sequence is a modified sequence that preferably encodes the same polypeptide (or a biologically active fragment of a full-length polypeptide having substantially the same activity as the full-length polypeptide) encoded by the non-codon-optimized parent polynucleotide. In some embodiments, the human immunoglobulin λ light chain sequence encoding the human immunoglobulin λ light chain variable domain may separately include sequences modified to optimize codon usage frequency for a particular cell type (e.g., rodent cells, e.g., rat or mouse cells). For example, the codons of each nucleotide sequence inserted into the genome of a non-human animal (e.g., a rodent, e.g., a rat or mouse) described herein may be optimized for expression in cells of the non-human animal. Such sequences can be described as codon-optimized sequences.

[0300] Insertion of a nucleotide sequence encoding the human immunoglobulin λ light chain variable domain, using minimal modification of the germline genome of non-human animals as described herein, results in the expression of an antibody containing a light chain having the human Vλ domain, where the human immunoglobulin λ light chain variable domain is expressed from the endogenously modified immunoglobulin κ light chain locus. Methods for generating modified non-human animals (e.g., rodents, e.g., rats or mice), including knockout and knock-in, are known in the art (see, e.g., Gene Targeting: A Practical Approach, Joyner, ed., Oxford University Press, Inc., 2000 (which is incorporated herein by reference in its entirety)). For example, the generation of a genetically modified rodent may optionally include disruption of the locus of one or more endogenous rodent genes (or gene segments) and introduction of one or more heterologous genes (or gene segments or nucleotide sequences) into the rodent genome, in some embodiments, at the same location as the endogenous rodent genes (or gene segments). In some embodiments, a nucleotide sequence encoding the human Vλ domain is introduced upstream of a non-human (e.g., rodent, e.g., rat or mouse) or human immunoglobulin λ light chain constant region gene in a randomly inserted, modified light chain transgene in the germline genome of a rodent. In some embodiments, a nucleotide sequence encoding the human Vλ domain is introduced upstream of a non-human (e.g., rodent, e.g., rat or mouse) or human immunoglobulin λ light chain constant region gene in the endogenous immunoglobulin κ light chain locus in the germline genome of a rodent; in some predetermined embodiments, the endogenous immunoglobulin κ light chain locus is modified to contain human immunoglobulin λ gene segments (e.g., human V and J) functionally ligated to the mouse Cλ1 gene or the human Cλ2 gene.

[0301] Schematic diagrams (not to scale) of exemplary methods for constructing the modified immunoglobulin κ light chain locus described herein are provided in Figures 1–6. In particular, Figures 1–6 show exemplary strategies for constructing the modified immunoglobulin κ light chain locus characterized by the insertion of a nucleotide sequence containing a single rearranged human immunoglobulin λ light chain variable region, and the corresponding targeting vector. The targeting vector is linearized and electroporated into rodent embryonic stem (ES) cells so that a rodent with a germline genome containing the modified immunoglobulin κ light chain locus can be produced. As described in the Examples section below, the rodent ES cells used in the electroporation of the targeting vector contained the modified immunoglobulin κ light chain locus previously described in U.S. Patent No. 10,143,186 (which are incorporated herein by reference in their entirety). Positive rodent ES cell clones are confirmed using screening methods known in the art. Any remaining selective cassette may be deleted via recombinase-mediated deletion as desired.

[0302] Alternatively, a human Cλ gene may be used in the targeting vector instead of the mouse Cλ gene. The targeting vector can be constructed in the same manner as described above (or in the following examples), except that the sequence encoding the human Cλ gene (e.g., Cλ2) is modified and introduced into the targeting vector. Using such an approach makes it possible to develop human antibody therapeutics because the DNA encoding the variable and constant regions of the light chain is isolated together, thereby eliminating any subsequent cloning step of ligating the human light chain constant region for the preparation of a fully human antibody.

[0303] The targeting vectors for constructing modified immunoglobulin κ light chain loci described herein may be incorporated into the germline genome of non-human cells (e.g., rodent (e.g., rat or mouse) embryonic stem cells). In some embodiments, the targeting vectors described herein may be functionally linked to one or more immunoglobulin heavy chain constant region genes and human V H , D H and J H Genomic DNA (for example, multiple human V H , D H and J H The targeting vector is incorporated into the wild-type immunoglobulin κ light chain locus in the germline genome of non-human (e.g., rodents, e.g., rats or mice) cells further containing a gene segment (see, for example, Macdonald (2014), U.S. Patents 6,596,541, 8,642,835, 8,697,940 and 8,791,323 (each of which is incorporated herein by reference in whole)). In some embodiments, the targeting vector described herein is functionally linked to one or more immunoglobulin heavy chain constant region genes of human V H , D H and J H Genomic DNA (for example, multiple human V H , D H and J H It is incorporated into a modified or altered immunoglobulin κ light chain locus in the germline genome of a non-human cell that further contains a gene segment (see, for example, Macdonald (2014), U.S. Patents 6,596,541, 8,642,835, 8,697,940, 8,791,323, 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662 and 9,163,092 (each of which is incorporated herein by reference in whole)).

[0304] The targeting vector is introduced into non-human (e.g., rodent, e.g., mouse or rat) embryonic stem cells by electroporation, thereby resulting in the ability of non-human (e.g., rodent, e.g., rat or mouse) cells or non-human animals (e.g., rodents, e.g., rat or mouse) to express antibodies having a light chain containing a human immunoglobulin λ light chain variable domain and a non-human or human light chain constant (Cλ or Cκ) domain, the light chain being expressed from a modified endogenous immunoglobulin κ light chain locus. As described herein, a genetically modified non-human animal is produced in the germline genome of a non-human animal in which a modified immunoglobulin κ light chain locus is generated (e.g., an endogenous immunoglobulin κ light chain locus containing a human immunoglobulin λ light chain sequence functionally linked to a rodent or human Cλ gene instead of the endogenous rodent Cκ gene (i.e., a rearranged human λ light chain variable region)). Antibodies characterized by light chains having a human Vλ domain and a non-human or human Cλ domain are expressed on the surface of non-human animal B cells and in the serum of the said non-human animals. If the endogenous immunoglobulin κ light chain locus in the germline genome of the non-human animals described herein is not targeted by the targeting vector, the modified immunoglobulin κ light chain transgene is preferably inserted at a location other than the endogenous non-human animal immunoglobulin κ light chain locus (e.g., a randomly inserted transgene).

[0305] The generation of modified immunoglobulin κ light chain loci in non-human animals as described above provides a modified non-human animal line that produces antibodies containing immunoglobulin λ light chains expressed from such modified immunoglobulin κ light chain loci having a human Vλ domain and a non-human (e.g., rodent, e.g., rat or mouse) or human Cλ domain. Multiple human V H , D H and J HWhen utilized in conjunction with the presence of a modified immunoglobulin heavy chain locus containing a gene segment, modified non-human animal lines can be created to produce antibodies and antibody components for the development of human antibody-based therapeutics. Thus, a single modified non-human animal line has been found to have the capability to provide an alternative in vivo system for utilizing the human Vλ domain for the development of novel antibody-based drugs to treat human diseases.

[0306] In some embodiments, a method for producing a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) includes introducing a single rearranged human immunoglobulin λ light chain variable region, including human Vλ and human Jλ gene segments, into a modified endogenous immunoglobulin κ light chain locus in the genome of a non-human (e.g., a rodent, e.g., a rat or mouse) embryonic stem (ES) cell. In some embodiments, a method for producing a genetically modified non-human animal includes introducing a non-human (e.g., a rodent, e.g., a rat or mouse) Cλ gene segment into a modified endogenous immunoglobulin κ light chain locus in the genome of a non-human ES cell. In some embodiments, a method for producing a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) includes generating a non-human animal using the non-human (e.g., a rodent, e.g., a rat or mouse) ES cells described above.

[0307] In some embodiments, a method for producing a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) includes introducing a single rearranged human immunoglobulin λ light chain variable region, including human Vλ gene segments and human Jλ gene segments, into a modified endogenous immunoglobulin κ light chain locus in the genome of a non-human ES cell. In some embodiments, a method for producing a genetically modified non-human animal includes introducing a non-human Cλ gene segment into a modified endogenous immunoglobulin κ light chain locus in the genome of a non-human ES cell.

[0308] In some embodiments, a method for producing a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) includes the steps of (a) introducing a single rearranged human immunoglobulin λ light chain variable region, comprising a human Vλ gene segment and a human Jλ gene segment, into a modified endogenous immunoglobulin κ light chain locus in the genome of a non-human ES cell; and (b) introducing a non-human Cλ gene segment into a modified endogenous immunoglobulin κ light chain locus in the genome of a non-human ES cell.

[0309] In some embodiments, a method for producing a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) includes the steps of: (a) introducing a single rearranged human immunoglobulin λ light chain variable region, comprising a human Vλ gene segment and a human Jλ gene segment, into a modified endogenous immunoglobulin κ light chain locus in the genome of a non-human embryonic stem (ES) cell; (b) introducing a non-human Cλ gene segment into a modified endogenous immunoglobulin κ light chain locus in the genome of a non-human ES cell; and (c) generating a non-human animal using the non-human ES cells produced in steps (a) and (b).

[0310] In some embodiments, a method for creating a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) involves one or more unreorganized human V genes functionally linked to one or more endogenous immunoglobulin heavy chain constant region genes. H Gene segment, one or more unreorganized human D H Gene segment, and one or more unreorganized human J H This involves introducing a gene segment into a modified endogenous immunoglobulin heavy chain locus in the genome of non-human ES cells.

[0311] In some embodiments, a method for producing a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) is to (d) one or more unreorganized human V genes functionally linked to one or more endogenous immunoglobulin heavy chain constant region genes. HGene segment, one or more unreorganized human D H Gene segment, and one or more unreorganized human J H The process further includes introducing a gene segment into a modified endogenous immunoglobulin heavy chain locus in the genome of a non-hu...

Claims

1. A genetically modified rodent, whose germline genome is It comprises a modified endogenous immunoglobulin κ light chain locus containing a single rearranged human immunoglobulin λ light chain variable region functionally linked to the rodent Cλ gene segment, wherein the single rearranged human immunoglobulin λ light chain variable region contains the human Vλ gene segment and the human Jλ gene segment, and the rodent Cλ gene segment replaces the endogenous Cκ gene segment. The single rearranged human immunoglobulin λ light chain variable region replaces one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof. The modified endogenous immunoglobulin κ light chain locus contains one or more endogenous enhancers at their endogenous positions within the endogenous immunoglobulin κ light chain locus. The aforementioned gene locus is capable of undergoing somatic hypermutation, The genetically modified rodent wherein all immunoglobulin λ light chains expressed by the B cells of the genetically modified rodent include human immunoglobulin λ light chain variable domains expressed from the single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof.

2. The genetically modified rodent according to claim 1, wherein the germline genome of the genetically modified rodent is homozygous for the modified endogenous immunoglobulin κ light chain locus.

3. The genetically modified rodent according to claim 1, wherein the germline genome of the genetically modified rodent is heterozygous for the modified endogenous immunoglobulin κ light chain locus.

4. The aforementioned germline genome is One or more non-reorganized human V genes functionally linked to one or more rodent immunoglobulin heavy chain constant region genes. Ｈ Gene segment, one or more unreorganized human D Ｈ Gene segment, and one or more unreorganized human J Ｈ Further comprising a modified endogenous immunoglobulin heavy chain locus containing a gene segment, The genetically modified rodent according to any one of claims 1 to 3, wherein all heavy chains expressed by the B cells of the genetically modified rodent include a human immunoglobulin heavy chain variable domain and a rodent immunoglobulin heavy chain constant domain.

5. The genetically modified rodent according to claim 4, wherein the germline genome of the genetically modified rodent is homozygous for the modified endogenous immunoglobulin heavy chain locus.

6. The genetically modified rodent according to any one of claims 1 to 5, wherein the human Vλ gene segment comprises Vλ1-51, Vλ5-45, Vλ1-44, Vλ1-40, Vλ3-21, or Vλ2-14.

7. The genetically modified rodent according to any one of claims 1 to 6, wherein the human Vλ gene segment comprises Vλ1-51.

8. The genetically modified rodent according to any one of claims 1 to 6, wherein the human Vλ gene segment comprises Vλ2-14.

9. The genetically modified rodent according to any one of claims 1 to 8, wherein the human Jλ gene segment comprises Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7.

10. The genetically modified rodent according to any one of claims 1 to 9, wherein the human Jλ gene segment includes Jλ2.

11. the one or more non-rearranged human V Ｈ gene segments, one or more non-rearranged human D Ｈ gene segments, and one or more non-rearranged human J Ｈ gene segments are replaced by one or more endogenous V Ｈ gene segments, one or more endogenous D Ｈ gene segments, one or more endogenous J Ｈ The genetically modified rodent according to any one of claims 4 to 10, which is a substitute for the gene segments, or a combination thereof.

12. The one or more unreorganized human V Ｈ Gene segment, one or more unreorganized human D Ｈ Gene segment, and one or more unreorganized human J Ｈ The gene segment contains one or more endogenous V Ｈ Gene segment, one or more endogenous D Ｈ Gene segment, and one or more endogenous J Ｈ A genetically modified rodent according to any one of claims 4 to 11, wherein each gene segment is replaced by a different gene segment.

13. The genetically modified rodent according to claim 11 or 12, wherein the one or more rodent immunoglobulin heavy chain constant region genes are one or more endogenous rodent immunoglobulin heavy chain constant region genes.

14. (i) The one or more unreorganized human V Ｈ The gene segment is V Ｈ 3-74, V Ｈ 3-73, V Ｈ 3-72, V Ｈ 2-70, V Ｈ 1-69, V Ｈ 3-66, V Ｈ 3-64, V Ｈ 4-61, V Ｈ 4-59, V Ｈ 1-58, V Ｈ 3-53, V Ｈ 5-51, V Ｈ 3-49, V Ｈ 3-48, V Ｈ 1-46, V Ｈ 1-45, V Ｈ 3-43, V Ｈ 4-39, V Ｈ 4-34, V Ｈ 3-33, V Ｈ 4-31, V Ｈ 3-30, V Ｈ 4-28, V Ｈ 2-26, V Ｈ 1-24, V Ｈ 3-23, V Ｈ 3-21, V Ｈ 3-20, V Ｈ 1-18, V Ｈ 3-15, V Ｈ 3-13, V Ｈ 3-11, V Ｈ 3-9, V Ｈ 1-8, V Ｈ 3-7, V Ｈ 2-5, V Ｈ 7-4-1, V Ｈ 4-4, V Ｈ 1-3, V Ｈ 1-2, V Ｈ 6-1, or any combination thereof, (ii) The one or more unreorganized human D Ｈ The gene segment is D Ｈ 1-1, D Ｈ 2-2, D Ｈ 3-3, D Ｈ 4-4, D Ｈ 5-5, D Ｈ 6-6, D Ｈ 1-7, D Ｈ 2-8, D Ｈ 3-9, D Ｈ 3-10, D Ｈ 5-12, D Ｈ 6-13, D Ｈ 2-15, D Ｈ 3-16, D Ｈ 4-17, D Ｈ 6-19, D Ｈ 1-20, D Ｈ 2-21, D Ｈ 3-22, D Ｈ 6-25, D Ｈ 1-26, D Ｈ Including 7-27, or any combination thereof, (iii) The one or more unreorganized human J Ｈ The gene segment is J Ｈ 1. J Ｈ 2. J Ｈ 3. J Ｈ 4. J Ｈ 5. J Ｈ 6, including any combination thereof, A genetically modified rodent according to any one of claims 4 to 13.

15. The genetically modified rodent according to any one of claims 4 to 14, wherein the germline genome of the genetically modified rodent comprises one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologues, functional homologs, or functional fragments thereof.

16. The rodent Cλ gene has a sequence that is at least 90% identical to (i) the mouse Cλ1 gene having the sequence described in SEQ ID NO: 1, (ii) the mouse Cλ2 gene having the sequence described in SEQ ID NO: 2, or (iii) the mouse Cλ3 gene having the sequence described in SEQ ID NO: 3, according to any one of claims 1 to 15.

17. The genetically modified rodent according to any one of claims 1 to 16, wherein the rodent Cλ gene includes the mouse Cλ gene.

18. The genetically modified rodent according to any one of claims 1 to 16, wherein the rodent Cλ gene includes the mouse Cλ1 gene.

19. A genetically modified rodent according to any one of claims 1 to 18, further comprising an inactivated endogenous immunoglobulin λ light chain locus.

20. The genetically modified rodent according to any one of claims 1 to 19, wherein all immunoglobulin light chains expressed by the B cells of the genetically modified rodent comprise human immunoglobulin λ light chain variable domains expressed from the single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof.

21. The genetically modified rodent according to any one of claims 1 to 20, wherein the rodent is a rat or a mouse.

22. A genetically modified mouse, whose germline genome is The modified endogenous immunoglobulin κ light chain locus comprises a single rearranged human immunoglobulin λ light chain variable region functionally linked to the mouse Cλ1 gene segment, wherein the single rearranged human immunoglobulin λ light chain variable region comprises the human Vλ1-51 gene segment and the human Jλ2 gene segment, and the mouse Cλ1 gene segment replaces the endogenous Cκ gene segment. The single rearranged human immunoglobulin λ light chain variable region replaces one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof. The modified endogenous immunoglobulin κ light chain locus contains one or more endogenous enhancers at their endogenous positions within the endogenous immunoglobulin κ light chain locus. The aforementioned gene locus is capable of undergoing somatic hypermutation, The genetically modified mouse wherein all immunoglobulin λ light chains expressed by the B cells of the genetically modified mouse include a human immunoglobulin λ light chain variable domain expressed from the single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof.

23. A genetically modified mouse, whose germline genome is The modified endogenous immunoglobulin κ light chain locus comprises a single rearranged human immunoglobulin λ light chain variable region functionally linked to the mouse Cλ1 gene segment, wherein the single rearranged human immunoglobulin λ light chain variable region comprises the human Vλ2-14 gene segment and the human Jλ2 gene segment, and the mouse Cλ1 gene segment replaces the endogenous Cκ gene segment. The single rearranged human immunoglobulin λ light chain variable region replaces one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof. The modified endogenous immunoglobulin κ light chain locus contains one or more endogenous enhancers at their endogenous positions within the endogenous immunoglobulin κ light chain locus. The aforementioned gene locus is capable of undergoing somatic hypermutation, The genetically modified mouse wherein all immunoglobulin λ light chains expressed by the B cells of the genetically modified mouse include a human immunoglobulin λ light chain variable domain expressed from the single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof.

24. A method for producing antibodies, wherein the method is (a) Exposing a genetically modified rodent or mouse according to any one of claims 1 to 23 to an antigen; (b) enabling the genetically modified rodents to elicit an immune response to the antigen; and (c) Isolating an antibody specific to the antigen, a B cell expressing an antibody specific to the antigen, or one or more nucleotide sequences encoding an antibody specific to the antigen from the genetically modified rodent. The method, including the method described above.

25. A method for producing a bispecific antibody, wherein the method is (a) To produce a first genetically modified rodent or mouse having B cells that express a first antibody specific to the first epitope of the first antigen by contacting a genetically modified rodent or mouse according to any one of claims 4 to 23 with a first epitope of a first antigen, (b) To produce a second genetically modified rodent or mouse having B cells that express a second antibody specific to the second epitope of the second antigen, by contacting a genetically modified rodent or mouse according to any one of claims 4 to 23 with a second epitope of a second antigen. (c) Isolating B cells expressing a first antibody specific to the first epitope of the first antigen from the first genetically modified rodent or mouse, and determining the first human immunoglobulin heavy chain variable domain of the first antibody; (d) Isolating B cells expressing a second antibody specific to the second epitope of the second antigen from the second genetically modified rodent or mouse, and determining the second human immunoglobulin heavy chain variable domain of the second antibody; (e) Functionally linking the nucleotide sequence encoding the first human immunoglobulin heavy chain variable domain with the nucleotide sequence encoding the first human immunoglobulin constant domain to generate a first nucleotide sequence encoding the first human heavy chain; (f) Functionally linking the nucleotide sequence encoding the second human immunoglobulin heavy chain variable domain with the nucleotide sequence encoding the second human immunoglobulin constant domain to generate a second nucleotide sequence encoding the second human heavy chain; (g) In mammalian cells, (i) The first nucleotide sequence; (ii) the second nucleotide sequence described above; and (iii) A third nucleotide sequence comprising the single reorganized human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof, functionally linked to the constant region of the human immunoglobulin λ light chain. To express The method, including the method described above.

26. A method for creating genetically modified rodents, (a) The step of introducing a single rearranged human immunoglobulin λ light chain variable region comprising a human Vλ gene segment and a human Jλ gene segment into a modified endogenous immunoglobulin κ light chain locus in the genome of a rodent ES cell, wherein the single rearranged human immunoglobulin λ light chain variable region is functionally linked to a rodent Cλ gene segment, the rodent Cλ gene segment is a substitute for an endogenous Cκ gene segment, the single rearranged human immunoglobulin λ light chain variable region is a substitute for one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof, the modified endogenous immunoglobulin κ light chain locus comprises one or more endogenous enhancers at their endogenous positions in the endogenous immunoglobulin κ light chain locus, and the locus is capable of undergoing somatic hypermutation; and (b) A step of generating rodents using the rodent ES cells generated in step (a). The method, including the method described above.

27. A method for creating genetically modified rodents, (a) The step of introducing a single rearranged human immunoglobulin λ light chain variable region functionally linked to a rodent Cλ gene segment into a modified endogenous immunoglobulin κ light chain locus in the genome of a rodent ES cell, wherein the single rearranged human immunoglobulin λ light chain variable region comprises a human Vλ gene segment and a human Jλ gene segment, the rodent Cλ gene segment is a substitute for an endogenous Cκ gene segment, the single rearranged human immunoglobulin λ light chain variable region is a substitute for one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof, the modified endogenous immunoglobulin κ light chain locus comprises one or more endogenous enhancers at their endogenous positions in the endogenous immunoglobulin κ light chain locus, and the locus is capable of undergoing somatic hypermutation; and (b) A step of generating rodents using the rodent ES cells generated in step (a). The method, including the method described above.

28. A method for creating genetically modified rodents, (a) The modification step of modifying the endogenous immunoglobulin κ light chain locus in the germline genome of the rodent to include a single rearranged human immunoglobulin λ light chain variable region functionally linked to the rodent Cλ gene segment, wherein the single rearranged human immunoglobulin λ light chain variable region includes a human Vλ gene segment and a human Jλ gene segment, the rodent Cλ gene segment is a substitute for the endogenous Cκ gene segment, the single rearranged human immunoglobulin λ light chain variable region is a substitute for one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof, the modified endogenous immunoglobulin κ light chain locus includes one or more endogenous enhancers at their endogenous positions in the endogenous immunoglobulin κ light chain locus, and the locus is capable of undergoing somatic hypermutation. The method wherein all immunoglobulin λ light chains expressed by the genetically modified rodent B cells include human immunoglobulin λ light chain variable domains expressed from the single reorganized human immunoglobulin λ light chain variable region or a somatic hypermutant version thereof.

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