Non-human animals having limited lambda light chain repertoire expressed from kappa locus and uses thereof

Abstract

To provide non-human animals having a limited lambda light chain repertoire expressed from the kappa locus and uses thereof.SOLUTION: The present disclosure provides, among other things, genetically modified non-human animals whose germline genome comprises a modified endogenous immunoglobulin κ light chain locus into which a single rearranged human immunoglobulin λ light chain variable region is inserted and operably linked to a non-human Cλ gene segment, where the single rearranged human immunoglobulin λ light chain variable region comprises 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 include human immunoglobulin λ light chain variable domains expressed from the single rearranged human immunoglobulin λ light chain variable region or a somatically hypermutated version thereof.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 857,712, filed June 5, 2019, which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy created on May 20, 2020 is named 2010794-2050_SL.txt and is 47,576 bytes in size. [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 encoding, in whole or in part, human antibodies has revolutionized the field of human therapeutic monoclonal antibodies for the treatment of various diseases. There remains a need for the development of improved in vivo systems for generating human monoclonal antibodies that maximize the human antibody repertoire in host genetically modified animals. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides genetically modified rodents. In some embodiments, the provided genetically modified rodents 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 present 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 a genetically modified rodent described herein. In some embodiments, the third genetically modified rodent is a progeny of the first genetically modified rodent and the second genetically modified rodent.

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

[0007] In some embodiments, all immunoglobulin λ light chains expressed by provided genetically modified rodent B cells comprise human immunoglobulin λ light chain variable domains expressed from a limited human λ light chain variable region repertoire. In some embodiments, all immunoglobulin λ light chains expressed by provided genetically modified rodent B cells comprise human immunoglobulin λ light chain variable domains expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatically hypermutated version thereof. In some embodiments, all immunoglobulin light chains expressed by provided genetically modified rodent B cells comprise human immunoglobulin λ light chain variable domains expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatically hypermutated version thereof. In some embodiments, all heavy chains expressed by genetically modified rodent B cells comprise a human immunoglobulin heavy chain variable domain and a rodent immunoglobulin heavy chain constant domain.

[0008] In some embodiments, a genetically modified rodent is provided that comprises a modified endogenous rodent immunoglobulin light chain locus that comprises a limited human λ light chain variable region repertoire. In some embodiments, a genetically modified rodent is provided that comprises a modified endogenous rodent immunoglobulin λ light chain locus that comprises a limited human λ light chain variable region repertoire. In some embodiments, a genetically modified rodent is provided that comprises a modified endogenous rodent immunoglobulin κ light chain locus that comprises a limited human λ light chain variable region repertoire. In some embodiments, the germline genome of the genetically modified rodent 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 rodent 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 the genetically modified rodent comprises a modified endogenous immunoglobulin κ locus comprising two alleles. In some embodiments, a first allele comprises a limited human λ light chain variable region repertoire, and a second allele comprises a limited human κ light chain variable region repertoire. In some embodiments, the germline genome of the genetically modified rodent, and rodent cells and tissues therefrom, described herein comprises a first modified endogenous immunoglobulin κ light chain locus allele comprising a single rearranged human immunoglobulin λ light chain variable region operably linked to a rodent Cλ gene segment, 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 genetically modified rodents described herein, and rodent cells or tissues therefrom, comprise a second modified endogenous immunoglobulin κ light chain locus allele comprising a single rearranged human immunoglobulin κ light chain variable region operably linked to a rodent Cκ gene segment, wherein the single rearranged human immunoglobulin κ light chain variable region comprises a human Vκ gene segment and a human Jκ gene segment. In some embodiments, such non-human animals or 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, 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, the single rearranged human immunoglobulin κ light chain variable region is Vκ3-20 / Jκ1 and the single rearranged human immunoglobulin λ light chain variable region is Vλ1-51 / Jλ2 or Vλ2-14 / Jλ2.

[0010] In some embodiments, genetically modified rodents are provided that comprise a limited repertoire of human λ light chain variable regions operably linked to light chain constant region gene segments. In some embodiments, genetically modified rodents are provided that comprise a limited repertoire of human λ light chain variable regions operably linked to Cκ gene segments. In some embodiments, genetically modified rodents are provided that comprise a limited repertoire of human λ light chain variable regions operably linked to Cλ gene segments.

[0011] In some embodiments, a genetically modified rodent is provided that comprises a modified endogenous immunoglobulin κ light chain locus that comprises a single rearranged human immunoglobulin λ light chain variable region operably linked to a rodent Cλ gene segment, wherein the single rearranged human immunoglobulin λ light chain variable region comprises a human Vλ gene segment and a human Jλ gene segment.

[0012] In some embodiments, the 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. In some embodiments, the 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 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 genetically modified rodent provided lacks a rodent Cκ gene at the modified endogenous immunoglobulin κ light chain locus.

[0014] In some embodiments, provided genetically modified rodents have 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 segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J H In some embodiments, one or more unrearranged human V gene segments are included. H gene segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J H The gene segments are operably linked to one or more rodent immunoglobulin heavy chain constant region genes.

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

[0016] In some embodiments, the genetically modified rodent provided has one or more endogenous V H gene segment, one or more endogenous D H Gene segment, one or more endogenous J H one or more unrearranged human V gene segments in place of the V gene segments, or a combination thereof H gene segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J HIn some embodiments, the genetically modified rodent has a germline genome comprising one or more endogenous V gene segments. H gene segment, one or more endogenous D H gene segment and one or more endogenous J H one or more unrearranged human Vs each replacing a gene segment H gene segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J H They have a germline genome that includes gene segments.

[0017] In some embodiments, one or more unrearranged 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 unrearranged 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 7-27, or any combination thereof. In some embodiments, one or more unrearranged 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.

[0018] In some embodiments, (i) one or more unrearranged 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 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; and (ii) one or more unrearranged 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 7-27, or any combination thereof; and (iii) one or more unrearranged 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, provided genetically modified rodents have a germline genome that includes 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, genetically modified rodents are provided that have a germline genome comprising a modified endogenous immunoglobulin heavy chain locus that lacks a functional endogenous rodent Adam6 gene. In some embodiments, genetically modified rodents are provided that have a germline genome comprising one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, genetically modified rodents are provided that express one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, genetically modified rodents are provided that have a germline genome comprising one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof contained on the same chromosome as the modified endogenous immunoglobulin heavy chain locus. In some embodiments, genetically modified rodents are provided that have a germline genome comprising a modified endogenous immunoglobulin heavy chain locus comprising one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, genetically modified rodents are provided that have a germline genome comprising one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof in place of a human Adam6 pseudogene. In some embodiments, genetically modified rodents are provided that have a germline genome comprising one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof that replace a human Adam6 pseudogene.

[0021] In some embodiments, the genetically modified rodent provided comprises a first and a second human V H One or more human V gene segments H gene segments, as well as the first human V HGene segments and the second human V H In some embodiments, the first human V has a germline genome comprising one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof, between the first and second gene segments. 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, the one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are selected from the group consisting of human V H Gene segments and human D H Between gene segments.

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

[0024] In some embodiments, the rodent Cλ gene is or comprises a mouse Cλ gene, hi some embodiments, the rodent Cλ gene is or comprises a mouse Cλ1 gene.

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

[0026] In some embodiments, the rodent Cλ gene is or comprises a rat Cλ gene.

[0027] In some embodiments, 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. In some embodiments, 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.

[0028] In some embodiments, the genetically modified mice provided herein comprise a functional endogenous immunoglobulin λ light chain locus. In some embodiments, the genetically modified mice provided herein comprise 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 a portion of the endogenous immunoglobulin λ light chain locus. In some embodiments, the endogenous Vλ gene segments, endogenous Jλ gene segments, and endogenous Cλ genes are deleted in whole or in part.

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

[0030] The present disclosure provides a rodent embryo comprising a genetic modification 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 operably linked to a rodent Cλ gene segment, 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 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 genome of a rodent embryo comprises one or more unrearranged human V operably linked to one or more endogenous immunoglobulin heavy chain constant region genes. H gene segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J H A rodent embryo is provided, comprising a modified endogenous immunoglobulin heavy chain locus comprising a gene segment, hi some embodiments, the genome of the rodent embryo is homozygous for the modified endogenous immunoglobulin heavy chain locus.

[0032] The present disclosure provides genetically modified rodent B cells described herein. In some embodiments, the genetically modified rodent B cells described herein comprise a single rearranged human immunoglobulin λ light chain variable region or a somatically hypermutated version thereof at a modified endogenous κ light chain locus.

[0033] In some embodiments, the present disclosure provides a genetically modified rodent B cell described herein that comprises a single rearranged human immunoglobulin λ light chain variable region or a somatically hypermutated version thereof at a modified endogenous κ light chain locus. In some embodiments, the genetically modified rodent B cell described herein comprises one or more unrearranged human VHVs at a modified endogenous heavy chain locus. H Human V gene segment H gene segment, one or more unrearranged human D H Human D gene segment H gene segment, and one or more unrearranged human J H Human J gene segment H It comprises rearranged human immunoglobulin heavy chain variable regions derived from gene segments.

[0034] The present disclosure provides hybridomas generated from the genetically modified rodent B cells described herein.

[0035] The present disclosure provides a population of B cells from a single genetically modified rodent as described herein. In some embodiments, all of the antibodies expressed by the population of 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 somatically hypermutated version thereof. In some embodiments, the antibodies expressed by the population of B cells comprise multiple human immunoglobulin heavy chain variable domains expressed from at least two different rearranged human immunoglobulin heavy chain variable regions or somatically hypermutated versions thereof.

[0036] The present disclosure provides stem cells, such as embryonic stem (ES) cells, comprising the genetic modifications described herein. In some embodiments, the stem cells (e.g., ES cells) comprise a modified endogenous immunoglobulin κ light chain locus comprising a single rearranged human immunoglobulin λ light chain variable region operably 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. In some embodiments, the 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. In some embodiments, the 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 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) contain one or more unrearranged human V operably linked to one or more endogenous immunoglobulin heavy chain constant region genes. H gene segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J H It includes a modified endogenous immunoglobulin heavy chain locus comprising a gene segment.

[0038] Mammalian cells expressing antibodies are provided by the present disclosure. In some embodiments, the antibody expressed by the mammalian cells comprises a heavy chain comprising a human immunoglobulin heavy chain variable domain and a light chain comprising 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 a genetically modified rodent described herein.

[0039] In some embodiments, the antibody is prepared by a method comprising: (a) exposing a genetically modified rodent described herein to an antigen of interest; (b) maintaining the genetically modified rodent under conditions sufficient for the genetically modified rodent to generate an immune response to the antigen of interest; and (c) recovering from the genetically modified rodent: (i) an antibody that binds to the antigen of interest, (ii) nucleotides encoding a human light chain variable domain, a human heavy chain variable domain, a light chain, or a heavy chain of the antibody that binds to the antigen of interest, or (iii) a cell that expresses the antibody that binds to the antigen of interest, wherein the antibody of (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, a method of producing an antibody comprises (a) exposing a genetically modified rodent described herein to an antigen; (b) allowing the genetically modified rodent to mount an immune response to the antigen; and (c) isolating from the genetically modified rodent an antibody specific for the antigen, a B cell expressing the antibody specific for the antigen, or one or more nucleotide sequences encoding the antibody specific for the antigen. In some embodiments, the antibody is a bispecific antibody.

[0041] In some embodiments, a method of making an antibody comprises: (a) expressing in a mammalian cell said antibody comprising two human immunoglobulin λ light chains and two human immunoglobulin heavy chains, wherein each human immunoglobulin λ light chain comprises a human immunoglobulin λ light chain variable domain, and each human immunoglobulin heavy chain comprises a human immunoglobulin heavy chain variable domain, and 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 a genetically modified rodent described herein; and (b) obtaining the antibody. In some embodiments, the antibody is a bispecific antibody.

[0042] In some embodiments, a method of making 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 from the first genetically modified rodent that express a first antibody specific for the first epitope of the first antigen and determining a first human immunoglobulin heavy chain variable domain of the first antibody; (d) isolating B cells from the second genetically modified rodent that express a second antibody specific for the second epitope of the second antigen and determining a second human immunoglobulin heavy chain variable domain of the second antibody; and (e) isolating a nucleic acid encoding the first human immunoglobulin heavy chain variable domain. (f) operably linking 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 the second human heavy chain; and (g) expressing in a mammalian cell (i) the first nucleotide sequence; (ii) the second nucleotide sequence; and (iii) a third nucleotide sequence comprising a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutated version thereof operably linked to a human immunoglobulin λ light chain constant region.

[0043] In some embodiments, provided herein are methods of making a bispecific antibody, the method comprising: (a) expressing in a mammalian cell: (i) a first nucleotide sequence comprising a first human immunoglobulin heavy chain variable region operably linked to a first human immunoglobulin constant region; (ii) a second nucleotide sequence comprising a second human immunoglobulin heavy chain variable region operably linked to a second human immunoglobulin constant region; and (iii) a third nucleotide sequence comprising a human immunoglobulin λ light chain variable region operably 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 a first antibody in a first genetically modified rodent described herein that has been immunized with a first epitope of a first antigen, and 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 from, isolated from, or identical to a second antibody in a second genetically modified rodent described herein that has been immunized with a second epitope of a second antigen, wherein the second antibody specifically binds to the second epitope of the second antigen. In some embodiments, the human immunoglobulin λ light chain variable region of the third nucleotide is a single rearranged human immunoglobulin λ light chain variable region or a somatically hypermutated 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 comprises the steps of: (a) exposing a genetically modified rodent described herein to an antigen of interest; (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) operably linking 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 an antigen of interest; 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, a human immunoglobulin heavy chain variable domain produced by the method of this paragraph is provided.

[0048] In some embodiments, a method for producing a collection of human immunoglobulin heavy chain variable domains comprises (a) exposing a genetically modified rodent described herein to an antigen of interest, and (b) isolating a collection of human immunoglobulin heavy chain variable domains from the genetically modified rodent. In some embodiments, the collection of human immunoglobulin heavy chain variable domains each binds to a human immunoglobulin λ light chain variable domain expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutated 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 comprises: (a) exposing a genetically modified rodent described herein to an antigen of interest; (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) operably linking 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 producing a human immunoglobulin λ light chain variable domain comprises (a) exposing a genetically modified rodent described herein to an antigen of interest; 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, a human immunoglobulin λ light chain variable domain produced by the method of this paragraph is provided.

[0051] In some embodiments, a method for generating a nucleotide sequence encoding a human immunoglobulin heavy chain comprises the steps of: (a) exposing a genetically modified rodent described herein to an antigen of interest; (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) operably linking 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 generating a nucleotide sequence comprising a human immunoglobulin heavy chain variable region comprises (a) exposing a genetically modified rodent described herein to an antigen of interest; 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 comprising a human immunoglobulin heavy chain variable region produced by the method of this paragraph is provided.

[0053] In some embodiments, a method for generating a nucleotide sequence encoding a human immunoglobulin λ light chain comprises: (a) exposing a genetically modified rodent described herein to an antigen of interest; (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) operably linking 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 generating a nucleotide sequence comprising a human immunoglobulin λ light chain variable region comprises: (a) exposing a genetically modified rodent described herein to an antigen of interest; and (b) obtaining a human immunoglobulin λ light chain variable region that specifically binds to the antigen and encodes the human immunoglobulin λ light chain variable domain sequence of an antibody produced by the genetically modified rodent. In some embodiments, a nucleotide sequence comprising 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, which comprises: (i) a 5′ homology arm comprising a nucleotide sequence corresponding to a 5′ target sequence in an endogenous rodent κ light chain locus; (ii) a single rearranged human immunoglobulin λ light chain variable region comprising a Vλ gene segment and a Jλ gene segment; (iii) a rodent Cλ gene segment; and (iv) a 3′ homology arm comprising a nucleotide sequence corresponding to a 3′ target sequence in an endogenous rodent κ light chain locus. In some embodiments, the 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. In some embodiments, the 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 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 methods of generating a genetically modified rodent described herein include: (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 rodent ES cell; and (b) generating a rodent using the rodent ES cell generated in step (a).

[0057] In some embodiments, the method of generating a genetically modified rodent described herein comprises: (a) introducing a single rearranged human immunoglobulin λ light chain variable region operably 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 a rodent using the rodent ES cell generated in step (a). In some embodiments, the genome of the rodent ES cell contains one or more unrearranged human Vλ light chain variable regions operably linked to one or more endogenous immunoglobulin heavy chain constant region genes at the modified endogenous immunoglobulin heavy chain locus. H gene segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J H In some embodiments, the genome of the rodent ES cell further comprises one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof.

[0058] In some embodiments, a method of generating a genetically modified rodent ES cell comprises 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 of generating a genetically modified rodent ES cell comprises introducing a single rearranged human immunoglobulin λ light chain variable region operably 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 the rodent ES cell comprises one or more unrearranged human Vλ light chain variable regions operably linked to one or more endogenous immunoglobulin heavy chain constant region genes at the modified endogenous immunoglobulin heavy chain locus. H gene segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J H In some embodiments, the genome of the rodent ES cell further comprises one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof.

[0059] In some embodiments, a method of making a genetically modified rodent described herein comprises: (a) modifying an endogenous immunoglobulin κ light chain locus in the germline genome of the rodent to comprise a single rearranged human immunoglobulin λ light chain variable region operably linked to a rodent Cλ gene segment, such that all immunoglobulin λ light chains expressed by B cells of the genetically modified rodent comprise human immunoglobulin λ light chain variable domains expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatic hypermutated version thereof, 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 method further comprises (b) modifying an endogenous immunoglobulin heavy chain locus in the germline genome of the rodent to one or more unrearranged human V operably linked to one or more rodent immunoglobulin heavy chain constant region genes such that all heavy chains expressed by B cells of the genetically modified rodent comprise a human immunoglobulin heavy chain variable domain and a rodent immunoglobulin heavy chain constant domain. H gene segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J H In some embodiments, step (b) further comprises modifying the endogenous immunoglobulin heavy chain locus to further comprise one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, step (a) and / or step (b) are performed in rodent ES cells.

[0060] In some embodiments, the 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. In some embodiments, the 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 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 a rodent Cκ gene.

[0062] In some embodiments, one or more unrearranged human V H gene segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J HA gene segment can be derived from one or more endogenous V H gene segment, one or more endogenous D H Gene segment, one or more endogenous J H In some embodiments, the unrearranged human V gene segments are substituted for one or more unrearranged human V gene segments, or combinations thereof. H gene segment, one or more unrearranged human D H gene segment, and one or more unrearranged human J H A gene segment can be derived from 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.

[0063] In some embodiments, one or more unrearranged 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 unrearranged 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 7-27, or any combination thereof. In some embodiments, one or more unrearranged 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, the 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 a functional endogenous rodent Adam6 gene. In some embodiments, the germline genome of the genetically modified rodent comprises one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are expressed by the genetically modified rodent. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are contained on the same chromosome as the modified endogenous immunoglobulin heavy chain locus. In some embodiments, the modified endogenous immunoglobulin heavy chain locus comprises one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are in place of the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof replace 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 The one or more nucleotide sequences comprising gene segments and encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are selected from the group consisting of a first human V H Gene segments and the second human V H 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, the one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are selected from the group consisting of human V H Gene segments and human D H Between gene segments.

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

[0069] In some embodiments, the rodent Cλ gene is or comprises a mouse Cλ gene, hi some embodiments, the rodent Cλ gene is or comprises a mouse Cλ1 gene.

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

[0071] In some embodiments, the rodent Cλ gene is or comprises a rat Cλ gene.

[0072] In some embodiments, 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. In some embodiments, 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. In some embodiments, the germline genome of the genetically modified rodent described herein further comprises 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 a portion of the endogenous immunoglobulin λ light chain locus. In some embodiments, the endogenous Vλ gene segments, endogenous Jλ gene segments, and endogenous Cλ genes are deleted in whole or in part.

[0073] The drawings contained herein, consisting of the following figures, are for illustration purposes only and not for limitation: [Brief explanation of the drawings]

[0074] [Figure 1A] Figure 1 shows a diagram, not to scale, of an exemplary embodiment of a strategy for constructing a targeting vector (described in Examples 1 and 4) used in generating non-human animal embodiments according to the present disclosure. Unless a label in the figure indicates otherwise (e.g., for a selection cassette, loxP site, etc.), solid shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates an Frt recombinase system site; "UB-NEO" indicates a neomycin resistance gene with a UB promoter; "enh" indicates an enhancer; "Ei" indicates an intronic enhancer; "3'E" indicates a 3' enhancer; "SPEC" indicates a spectinomycin resistance gene; "SD" indicates a splice donor site; "CDS" indicates a coding sequence; "lox" indicates a lox2372 site; "loxP" indicates a lox P site; and "UB-HYG" indicates a hygromycin resistance gene with a UB promoter. [Figure 1B]Figure 1 shows a diagram, not to scale, of an exemplary embodiment of a strategy for constructing a targeting vector (described in Examples 1 and 4) used in generating non-human animal embodiments according to the present disclosure. Unless a label in the figure indicates otherwise (e.g., for a selection cassette, loxP site, etc.), solid shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates an Frt recombinase system site; "UB-NEO" indicates a neomycin resistance gene with a UB promoter; "enh" indicates an enhancer; "Ei" indicates an intronic enhancer; "3'E" indicates a 3' enhancer; "SPEC" indicates a spectinomycin resistance gene; "SD" indicates a splice donor site; "CDS" indicates a coding sequence; "lox" indicates a lox2372 site; "loxP" indicates a lox P site; and "UB-HYG" indicates a hygromycin resistance gene with a UB promoter. [Figure 2A] Figure 1 shows a diagram, not to scale, of an exemplary embodiment of a strategy for constructing a targeting vector (described in Examples 7 and 10) used in generating non-human animal embodiments according to the present disclosure. Unless a label in the figure indicates otherwise (e.g., for a selection cassette, loxP site, etc.), solid shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates an Frt recombinase system site; "UB-NEO" indicates a neomycin resistance gene with a UB promoter; "enh" indicates an enhancer; "Ei" indicates an intronic enhancer; "3'E" indicates a 3' enhancer; "SPEC" indicates a spectinomycin resistance gene; "SD" indicates a splice donor site; "CDS" indicates a coding sequence; "lox" indicates a lox2372 site; "loxP" indicates a lox P site; and "UB-HYG" indicates a hygromycin resistance gene with a UB promoter. [Figure 2B]Figure 1 shows a diagram, not to scale, of an exemplary embodiment of a strategy for constructing a targeting vector (described in Examples 7 and 10) used in generating non-human animal embodiments according to the present disclosure. Unless a label in the figure indicates otherwise (e.g., for a selection cassette, loxP site, etc.), solid shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates an Frt recombinase system site; "UB-NEO" indicates a neomycin resistance gene with a UB promoter; "enh" indicates an enhancer; "Ei" indicates an intronic enhancer; "3'E" indicates a 3' enhancer; "SPEC" indicates a spectinomycin resistance gene; "SD" indicates a splice donor site; "CDS" indicates a coding sequence; "lox" indicates a lox2372 site; "loxP" indicates a lox P site; and "UB-HYG" indicates a hygromycin resistance gene with a UB promoter. [Figure 3] FIG. 1 shows a diagram, not to scale, of the insertion of targeting vector A (described in Example 1) into the modified Igκ light chain locus of a rodent embryonic stem (ES) cell clone (described in Example 2), which was used in the generation of rodent embodiments according to the present disclosure. The diagram includes the approximate locations (indicated by dashes in circles) of various probes used to confirm that the embryonic stem (ES) cell clone was positive for a given exemplary sequence. Unless labels in the diagram indicate otherwise (e.g., for selection cassettes, loxP sites, etc.), solid shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates an Frt recombinase system site; "UB-NEO" indicates a neomycin resistance gene with a UB promoter; "Ei" indicates an intronic enhancer; "3'E" indicates a 3' enhancer; and "SD" indicates a splice donor site. "Het" indicates heterozygous mice and "ho" indicates homozygous mice. [Figure 4]FIG. 1 shows a diagram, not to scale, of the insertion of targeting vector B (described in Example 4) into the modified Igκ light chain locus of a rodent embryonic stem (ES) cell clone (described in Example 5), which ES cell clone was used in the generation of rodent embodiments according to the present disclosure. The diagram includes the approximate locations (indicated by dashes in circles) of various probes used to confirm that the embryonic stem (ES) cell clone was positive for a given exemplary sequence. Unless labels in the diagram indicate otherwise (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 an 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; "SD" indicates a splice donor site; "loxP" indicates a lox P site; and "UB-HYG" indicates a hygromycin resistance gene with a UB promoter. "Het" indicates a heterozygous mouse, and "ho" indicates a homozygous mouse. [Figure 5]FIG. 1 shows a diagram, not to scale, of the insertion of targeting vector C (described in Example 7) into the modified Igκ light chain locus of a rodent embryonic stem (ES) cell clone (described in Example 8), which was used in the generation of rodent embodiments according to the present disclosure. The diagram includes the approximate locations (indicated by dashes in circles) of various probes used to confirm that the embryonic stem (ES) cell clone was positive for a given exemplary sequence. Unless labels in the diagram indicate otherwise (e.g., for selection cassettes, loxP sites, etc.), solid shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "FRT" indicates an Frt recombinase system site; "UB-NEO" indicates a neomycin resistance gene with a UB promoter; "Ei" indicates an intronic enhancer; "3'E" indicates a 3' enhancer; and "SD" indicates a splice donor site. "Het" indicates heterozygous mice and "ho" indicates homozygous mice. [Figure 6]FIG. 1 shows a diagram, not to scale, of the insertion of targeting vector D (described in Example 10) into the modified Igκ light chain locus of a rodent embryonic stem (ES) cell clone (described in Example 11), which ES cell clone was used in the generation of rodent embodiments according to the present disclosure. The diagram includes the approximate locations (indicated by dashes in circles) of various probes used to confirm that the embryonic stem (ES) cell clone was positive for a given exemplary sequence. Unless labels in the diagram indicate otherwise (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 an 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; "SD" indicates a splice donor site; "loxP" indicates a lox P site; and "UB-HYG" indicates a hygromycin resistance gene with a UB promoter. "Het" indicates a heterozygous mouse, and "ho" indicates a homozygous mouse. [Figure 7]

[0023] Figure 2 shows the nucleotide sequence of mouse Cκ (SEQ ID NO: 25). The coding sequence is shown in bold, the 3' untranslated region is non-bold. [Figure 8] The nucleotide sequence of rat Cκ (SEQ ID NO: 27) is shown. The coding sequence is shown in bold, the 3′ untranslated region is non-bold. [Figure 9A]The nucleotide sequence of the modified Vλ1-51 / Jλ2 vector (SEQ ID NO: 31) is shown. Restriction enzyme site sequences—AscI (at the 5' end of the sequence) and PI-SceI (at the 3' end of the sequence)—are shown in uppercase italic letters; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); The 5'UTR sequence of Vλ1-51 is shown in uppercase, italicized, bold letters; the rearranged Vλ1-51 / Jλ2 sequence comprises: Vλ1-51 exon 1 sequence is shown in uppercase (non-bold) letters, within which the Vλ1-51 exon 1 start codon is also italicized and underlined; Vλ1-51 intron 1 sequence is shown in lowercase, bold letters; Vλ1-51 exon 2 sequence is shown in uppercase, bold, and underlined (solid underline) letters, and Jλ2 sequence is shown in uppercase, bold, and underlined (dashed underline) letters; and the human Jκ5-Cκ intron sequence is shown in lowercase, italicized letters. [Figure 9B] The nucleotide sequence of the modified Vλ1-51 / Jλ2 vector (SEQ ID NO: 31) is shown. Restriction enzyme site sequences—AscI (at the 5' end of the sequence) and PI-SceI (at the 3' end of the sequence)—are shown in uppercase italic letters; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); The 5'UTR sequence of Vλ1-51 is shown in uppercase, italicized, bold letters; the rearranged Vλ1-51 / Jλ2 sequence comprises: Vλ1-51 exon 1 sequence is shown in uppercase (non-bold) letters, within which the Vλ1-51 exon 1 start codon is also italicized and underlined; Vλ1-51 intron 1 sequence is shown in lowercase, bold letters; Vλ1-51 exon 2 sequence is shown in uppercase, bold, and underlined (solid underline) letters, and Jλ2 sequence is shown in uppercase, bold, and underlined (dashed underline) letters; and the human Jκ5-Cκ intron sequence is shown in lowercase, italicized letters. [Figure 9C]The nucleotide sequence of the modified Vλ1-51 / Jλ2 vector (SEQ ID NO: 31) is shown. Restriction enzyme site sequences—AscI (at the 5' end of the sequence) and PI-SceI (at the 3' end of the sequence)—are shown in uppercase italic letters; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); The 5'UTR sequence of Vλ1-51 is shown in uppercase, italicized, bold letters; the rearranged Vλ1-51 / Jλ2 sequence comprises: Vλ1-51 exon 1 sequence is shown in uppercase (non-bold) letters, within which the Vλ1-51 exon 1 start codon is also italicized and underlined; Vλ1-51 intron 1 sequence is shown in lowercase, bold letters; Vλ1-51 exon 2 sequence is shown in uppercase, bold, and underlined (solid underline) letters, and Jλ2 sequence is shown in uppercase, bold, and underlined (dashed underline) letters; and the human Jκ5-Cκ intron sequence is shown in lowercase, italicized letters. [Figure 10]

[0033] Figure 3 shows the nucleotide sequence of the rearranged Vλ1-51 / Jλ2 variable region including the Vλ1-51 intron (SEQ ID NO:32). Vλ1-51 exon 1 sequence is indicated by uppercase letters, with the Vλ1-51 exon 1 start codon also italicized and underlined; Vλ1-51 intron 1 sequence is indicated by lowercase bold letters; Vλ1-51 exon 2 sequence is indicated by uppercase bold and underlined (solid underline) letters; and Jλ2 sequence is indicated by uppercase bold and underlined (dashed underline) letters. [Figure 11] Figure 3 shows the nucleotide sequence of the rearranged Vλ1-51 / Jλ2 variable region without the Vλ1-51 intron (SEQ ID NO:33). The Vλ1-51 coding sequence is shown in uppercase letters, the Vλ1-51 start codon is also italicized and underlined; the Jλ2 sequence is shown in uppercase letters with a dashed underline. [Figure 12]The amino acid sequence of the rearranged Vλ1-51 / Jλ2 variable domain including the signal peptide (SEQ ID NO: 34) is shown. The bold italicized text indicates 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 site sequences—AscI (at the 5' end of the sequence) and PI-SceI (at the 3' end of the sequence)—are shown in uppercase italic letters; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); The 51 5'UTR sequence is indicated by capital italicized bold letters; the rearranged Vλ2-14 / Jλ2 sequence includes: Vλ2-14 exon 1 sequence is indicated by capital (non-bold) letters, within which the Vλ2-14 exon 1 start codon is also italicized and underlined; Vλ2-14 intron 1 sequence is indicated by lowercase bold letters; Vλ2-14 exon 2 sequence is indicated by capital, bold, and underlined (solid underline) letters, and Jλ2 sequence is indicated by capital, bold, and underlined (dashed underline) letters; and the human Jκ5-Cκ intron sequence is indicated by lowercase italic letters. [Figure 13B]The nucleotide sequence of the modified Vλ2-14 / Jλ2 vector (SEQ ID NO: 36) is shown. Restriction enzyme site sequences—AscI (at the 5' end of the sequence) and PI-SceI (at the 3' end of the sequence)—are shown in uppercase italic letters; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); The 51 5'UTR sequence is indicated by capital italicized bold letters; the rearranged Vλ2-14 / Jλ2 sequence includes: Vλ2-14 exon 1 sequence is indicated by capital (non-bold) letters, within which the Vλ2-14 exon 1 start codon is also italicized and underlined; Vλ2-14 intron 1 sequence is indicated by lowercase bold letters; Vλ2-14 exon 2 sequence is indicated by capital, bold, and underlined (solid underline) letters, and Jλ2 sequence is indicated by capital, bold, and underlined (dashed underline) letters; and the human Jκ5-Cκ intron sequence is indicated by lowercase italic letters. [Figure 13C] The nucleotide sequence of the modified Vλ2-14 / Jλ2 vector (SEQ ID NO: 36) is shown. Restriction enzyme site sequences—AscI (at the 5' end of the sequence) and PI-SceI (at the 3' end of the sequence)—are shown in uppercase italic letters; the Vλ1-51 promoter sequence is shown in lowercase (non-bold); The 51 5'UTR sequence is indicated by capital italicized bold letters; the rearranged Vλ2-14 / Jλ2 sequence includes: Vλ2-14 exon 1 sequence is indicated by capital (non-bold) letters, within which the Vλ2-14 exon 1 start codon is also italicized and underlined; Vλ2-14 intron 1 sequence is indicated by lowercase bold letters; Vλ2-14 exon 2 sequence is indicated by capital, bold, and underlined (solid underline) letters, and Jλ2 sequence is indicated by capital, bold, and underlined (dashed underline) letters; and the human Jκ5-Cκ intron sequence is indicated by lowercase italic letters. [Figure 14]

[0033] Figure 3 shows the nucleotide sequence of the rearranged Vλ2-14 / Jλ2 variable region including the Vλ2-14 intron (SEQ ID NO:37). Vλ2-14 exon 1 sequence is indicated by uppercase letters, with the Vλ2-14 exon 1 start codon also italicized and underlined; Vλ2-14 intron 1 sequence is indicated by lowercase bold letters; Vλ1-51 exon 2 sequence is indicated by uppercase bold and underlined (solid underline) letters; and Jλ2 sequence is indicated by uppercase bold and underlined (dashed underline) letters. [Figure 15]

[0033] Figure 3 shows the nucleotide sequence of the rearranged Vλ2-14 / Jλ2 variable region without the Vλ2-14 intron (SEQ ID NO:38). The Vλ2-14 sequence is shown in uppercase letters, and the Vλ2-14 start codon is also italicized and underlined; the Jλ2 sequence is shown in uppercase letters with a dashed underline. [Figure 16] 1 shows the amino acid sequence of the rearranged Vλ2-14 / Jλ2 variable domain including the signal peptide (SEQ ID NO: 39). The bold italicized text indicates the sequence of the signal peptide. DETAILED DESCRIPTION OF THE INVENTION

[0075] A brief description of selected sequences 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 The immunoglobulin Facts Book is available from the Immunoglobulin System website at www.imgt.org, or from LeFranc, MP., The Immunoglobulin Facts Book, Academic Press, May 23, 2001 (referred to herein as "LeFranc 2001").

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

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

[0078] Nucleotide sequence of mouse Cκ (SEQ ID NO: 25) (reproduced in Figure 7):

[0079] GGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGG CAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCC ACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAGagacaaaggtcctgagacgccaccaccagctccccagctccatcctatcttcccttctaaggtcttggaggcttcc ccacaagcgacctaccactgttgcggtgctccaaacctcctccccacctccttctcctcctcctccctttccttggcttttatcatgctaatatttgcagaaaatattcaataaagtgagtctttgcacttga

[0080] With respect to SEQ ID NO: 25, the following applies: -coding sequences are indicated by bold letters; The -3' untranslated region is in non-bold.

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

[0082] ADAAPTVSIFPPSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0083] Nucleotide sequence of rat Cκ (SEQ ID NO: 27) (reproduced in Figure 8):

[0084] GGGCTGATGCTGCACCAACTGTATCTATCTTCCCACCATCCACGGAACAGTTAGCAACTGGAGGTGCCTCAGTCGTGTGCCTCATGAACAACTTCTATCCCAGAGACATCAGTGTCAAGTGGAAGATTGA TGGCACTGAACGACGAGATGGTGTCCTGGACAGTGTTACTGATCAGGACAGCAAAGACAGCACGTACAGCATGAGCAGCACCCTCTCGTTGACCAAGGCTGACTATGAAAGTCATAACCTCTATACCTGT GAGGTTGTTCATAAGACATCATCCTCACCCGTCGTCAAGAGCTTCAACAGGAATGAGTGTTAGACCCAAAGGTCCTGAGGTGCCACCTGCTCCCCAGCTCCTTCCAATCTTCCCTCCTAAGGTCTTGGAG ACTTCCCCACAAGCGACCTACCACTGTTGCGGTGCTCCAAACCTCCTCCCCACCTCATCCTCCTTCCTTTCCTTGGCTTTGATCATGCTAATATTTGGGGAATATTAAATAAAGTGAATCTTTGCACTTGA

[0085] With respect to SEQ ID NO: 27, the following applies: -coding sequences are indicated by bold letters; The -3' untranslated region is in non-bold.

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

[0087] ADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVKWKIDGTERRDGVLDSVTDQDSKDSTYSMSSTLSLTKADYESHNLYTCEVVHKTSSSPVVKSFNRNEC

[0088] Nucleotide sequence of human Cκ (SEQ ID NO:29):

[0089] GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGA GAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

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

[0091] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0092] Nucleotide sequence of the modified Vλ1-51 / Jλ2 vector (SEQ ID NO: 31) (shown schematically in FIG. 1A and reproduced in FIGS. 9A-9C):

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

[0094] With respect to SEQ ID NO: 31, the following applies: - restriction enzyme site sequences - AscI (5' end of the sequence) and PI-SceI (3' end of the sequence) - are shown in capital italic letters; -Vλ1-51 promoter sequence is indicated by lowercase (non-bold) letters; - The 5'UTR sequence of Vλ1-51 is indicated by capital italicized bold letters; - The rearranged Vλ1-51 / Jλ2 sequence contains: o The exon 1 sequence of Vλ1-51 is indicated by capital letters (non-bold), of which the exon 1 start codon of Vλ1-51 is additionally italicized and underlined; o Intron 1 sequence of Vλ1-51 is indicated by lowercase bold letters; o The exon 2 sequence of Vλ1-51 is indicated by bold, capitalized, and underlined (solid underline) letters; o Jλ2 sequence is indicated by capitalized, bold, and underlined (dashed underline) letters; - Human Jκ5-Cκ intron sequence is indicated by lowercase italic letters.

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

[0096] [ka]

[0097] With respect to SEQ ID NO: 32, the following applies: -Vλ1-51 exon 1 sequence is indicated by capital letters, and the Vλ1-51 exon 1 start codon is additionally italicized and underlined; -Intron 1 sequence of Vλ1-51 is indicated by lowercase bold letters; -Vλ1-51 exon 2 sequence is indicated by bold, capitalized, and underlined (solid underline) letters; The -Jλ2 sequence is indicated by capitalized, bold, and underlined (dashed underline) letters.

[0098] Nucleotide sequence of the Vλ1-51 intronless rearranged Vλ1-51 / Jλ2 variable region (SEQ ID NO:33) (reproduced in Figure 11):

[0099] [ka]

[0100] With respect to SEQ ID NO: 33, the following applies: -Vλ1-51 coding sequence is indicated by capital letters, and the Vλ1-51 start codon is additionally italicized and underlined; The -Jλ2 sequence is shown in uppercase with a dashed underline.

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

[0102] [ka]

[0103] With respect to SEQ ID NO: 34, the bold italic text indicates the sequence of the signal peptide.

[0104] Amino acid sequence of rearranged Vλ1-51 / Jλ2 variable domain without signal peptide (SEQ ID NO:35):

[0105] QSVLTQPPSVSAAPGQKVTISSCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKLTVL

[0106] Nucleotide sequence of the modified Vλ2-14 / Jλ2 vector (SEQ ID NO:36) (shown schematically in FIG. 2A and reproduced in FIGS. 13A-13C): [ka] [ka]

[0107] With respect to SEQ ID NO: 36, the following applies: - restriction enzyme site sequences - AscI (5' end of the sequence) and PI-SceI (3' end of the sequence) - are shown in capital italic letters; -Vλ1-51 promoter sequence is indicated by lowercase (non-bold) letters; - The 5'UTR sequence of Vλ1-51 is indicated by capital italicized bold letters; - The rearranged Vλ2-14 / Jλ2 sequence contains: o The exon 1 sequence of Vλ2-14 is indicated by capital letters (non-bold), of which the exon 1 start codon of Vλ2-14 is additionally italicized and underlined; o Intron 1 sequence of Vλ2-14 is indicated by lowercase bold letters; o The exon 2 sequence of Vλ2-14 is indicated by bold, capitalized, and underlined (solid underline) letters; o Jλ2 sequence is indicated by capitalized, bold, and underlined (dashed underline) letters; o Human Jκ5-Cκ intron sequences are indicated by lowercase italic letters.

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

[0109] With respect to SEQ ID NO: 37, the following applies: -Vλ2-14 exon 1 sequence is indicated by capital letters, and the Vλ2-14 exon 1 start codon is additionally italicized and underlined; -Vλ2-14 intron 1 sequence is indicated by lowercase bold letters; -The exon 2 sequence of Vλ1-51 is indicated by bold, capitalized, and underlined letters (solid underline); The -Jλ2 sequence is indicated by capitalized, bold, and underlined (dashed underline) letters.

[0110] Nucleotide sequence of the Vλ2-14 intronless rearranged Vλ2-14 / Jλ2 variable region (SEQ ID NO:38) (reproduced in Figure 15):

[0111] [ka]

[0112] With respect to SEQ ID NO: 38, the following applies: -Vλ2-14 sequence is indicated by capital letters, the Vλ2-14 start codon is additionally italicized and underlined; The -Jλ2 sequence is shown in uppercase with a dashed underline.

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

[0114] [ka]

[0115] For SEQ ID NO: 39, the bold italics indicate the sequence of the signal peptide.

[0116] Amino acid sequence of rearranged Vλ2-14 / Jλ2 variable domain without signal peptide (SEQ ID NO:40):

[0117] QSALTQPASVGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVVFGGGTKLTVL

[0118] definition The scope of the present invention is defined by the claims appended hereto and is not limited by the specific embodiments described herein. Those skilled in the art who read this specification will recognize various modifications that are equivalent to such described embodiments or that may otherwise be within the scope of the claims. Generally, terms used herein follow their understood meanings in the art unless expressly stated otherwise. Clear definitions of certain terms are provided below; the meaning of these and other terms in specific instances throughout this specification will be apparent to those skilled in the art from the context. Additional definitions for the following and other terms are provided throughout this specification. Patent and non-patent references cited within this specification, or relevant portions thereof, are incorporated herein by reference in their entirety.

[0119] The use of ordinal terms such as "first," "second," "third," etc. to modify claim elements in the claims does not, of itself, imply any priority, precedence, or order of one claim element over another, or the chronological order in which acts of a method are performed, but is merely used as a marker to distinguish one claim element having a given name from another element having the same name (but using ordinal terms).

[0120] The articles "a" and "an," as used herein, should be understood to include plural references unless clearly indicated to the contrary. A claim or description including "or" between one or more members of a group is considered to be satisfied when one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise apparent from the context. In some embodiments, exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. In some embodiments, more than one, or all group members are present in, employed in, or otherwise relevant to a given product or process. The invention should be understood to encompass all incarnations, combinations, and permutations of one or more limitations, elements, clauses, descriptive language, etc. from one or more of the enumerated claims that are introduced in any other claim dependent on the same base claim (or any other claim, if relevant), unless otherwise indicated or unless it is apparent to one of ordinary skill in the art that a contradiction or inconsistency would result. When elements are presented as a list (e.g., a Markush group or similar format), it is to be understood that each subgroup of the elements is also disclosed, and that any element(s) can be removed from the group. Typically, when an embodiment or aspect is referred to as "comprising" certain elements, features, etc., it should be understood that a given embodiment or aspect "consists of" or "consists essentially of" such elements, features, etc. For purposes of simplicity, those embodiments have not been specifically set forth in so many words in every instance herein. It should also be understood that any embodiment or aspect can be expressly excluded from the scope of the claims, regardless of whether a specific exclusion is described herein.

[0121] Administration: As used herein, includes administration of a composition (e.g., an antigen or antibody) to a subject or system (e.g., to a cell, organ, tissue, organism, or related component or series thereof). One of skill in the art will understand that the route of administration can vary depending, for example, on the subject or system to which the composition is administered, the nature of the composition, the purpose of the administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., to a human or rodent) can be bronchial (including by bronchial instillation), buccal, enteral, intracutaneous, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, and / or intravitreal. In some embodiments, administration can include intermittent dosing. In some embodiments, administration can include continuous dosing (e.g., perfusion) for at least a selected period of time.

[0122] Antigen-binding protein: As used herein, refers to any protein or polypeptide that specifically binds to at least one antigen of interest. Antigen-binding proteins include, but are not limited to, antibodies, heavy chains, light chains (e.g., lambda or kappa light chains), heavy chain variable domains, light chain variable domains (e.g., lambda or kappa light chain variable domains), and single-chain variable fragments (ScFvs). In some embodiments, antigen-binding proteins are multispecific and can specifically bind to more than one epitope or antigen.

[0123] Approximately: When applied to one or more values ​​of interest, includes values ​​similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within ±10% of (higher or lower than) the stated reference value, unless otherwise stated or apparent from the context (except where such number would exceed 100% of the possible values).

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

[0125] Equivalent: As used herein, refers to two or more substances, entities, states of affairs, sets of states, etc. that may not be identical to one another, but that are sufficiently similar to permit a comparison between them so that reasonable conclusions can be made based on observed differences or similarities. Those of skill in the art will understand, in context, what degree of identity is required for two or more such substances, entities, states of affairs, sets of states, etc. to be considered equivalent in any given situation.

[0126] Conservative: As used herein, the term "conservative amino acid substitution" refers to examples including the replacement of an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Typically, conservative amino acid substitutions do not substantially alter the functional property of interest of a protein, such as the ability of a receptor to bind to a ligand. Examples of groups of amino acids with 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); aromatic 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 substitutions 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, conservative amino acid substitutions can be the substitution of any native residue in a protein with alanine, for example, as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made that have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445 (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, refers to the art-recognized meaning of "control," which is a standard against which results are compared. Typically, controls are used to enhance the integrity of an experiment by isolating a variable in order to draw conclusions about such a variable. In some embodiments, a control is a reaction or assay performed simultaneously with a test reaction or assay to provide a basis for comparison. "Control" also includes "control animals." "Control animals" may have a modification described herein, a different modification than those described herein, or no modification (i.e., wild-type animals). In one experiment, the "test" parameter (e.g., the variable being tested) is applied. In another experiment, the "control," the variable being tested, is not applied. In some embodiments, a control is a historical control (i.e., of a test or assay already performed, or of a quantity or result already known). In some embodiments, a control is or includes a printed or otherwise kept record. A control may be a positive or negative control.

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

[0129] Determining, measuring, assessing, evaluating, assaying, and analyzing: are used interchangeably herein to refer to any form of measurement, including determining whether an element is present or absent. These terms include both quantitative and / or qualitative determinations. Assays can be relative or absolute. "Assaying for the presence of" can be determining the amount of something present and / or determining whether it is present or absent.

[0130] Endogenous promoter: As used herein refers to a promoter that is naturally associated with an endogenous gene, e.g., in the wild-type organism.

[0131] Modified: As used herein, generally refers to aspects that have been manipulated by the hand of man. For example, in some embodiments, a polynucleotide may be considered "modified" if it has been manipulated by the hand of man so that two or more sequences that are not naturally linked to each other in that order are directly linked to each other in the modified polynucleotide. In some embodiments, a modified polynucleotide may include a regulatory sequence that is found in nature in operable association with a first coding sequence but not in operable association with a second coding sequence, and that has been linked by the hand of man into operable association with the second coding sequence. Alternatively, or additionally, in some embodiments, first and second nucleic acid sequences, each encoding polypeptide elements or domains that are not naturally linked to each other, may be linked to each other in a single modified polynucleotide. Similarly, in some embodiments, a cell or organism may be considered "modified" if its genetic information has been altered (e.g., new genetic material not previously present has been introduced, or pre-existing genetic material has been altered or removed). As is common practice and understood by those of skill in the art, progeny of a modified polynucleotide or cell are typically still referred to as "modified," even if actual manipulation was performed on the predecessor entity. Furthermore, as will be understood by those of skill in the art, a variety of methodologies are available by which the "modifying" described herein can be achieved. For example, in some embodiments, "modifying" can include the selection or design (e.g., of nucleic acid sequences, polypeptide sequences, cells, tissues, and / or organisms) through the use of a computer system programmed to perform analyses or comparisons, or otherwise analyze, recommend, and / or select sequences, alterations, etc. Alternatively, or additionally, in some embodiments, "modifying" can include in vitro chemical synthesis methodologies and / or recombinant nucleic acid techniques, such as nucleic acid amplification (e.g., via polymerase chain reaction), hybridization, mutation, transformation, transfection, etc., and / or the use of any of a variety of control mating methodologies.As will be appreciated by those skilled in the art, a variety of established such techniques (e.g., for 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. 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, R.W. and S.B. Primrose, Blackwell Science, Inc., 1994 (incorporated herein by reference in their entireties).

[0132] Functional: As used herein, refers to a form or fragment of an entity (e.g., a gene or gene segment) that exhibits a particular property (e.g., forms part of a coding sequence) and / or exhibits an activity. For example, in the context of immunoglobulins, variable regions are encoded by unique gene segments (i.e., V, D, and / or J) that are assembled (or recombined) to form a functional coding sequence. When present in a genome, gene segments are organized in clusters, although variation exists. A "functional" gene segment is one that appears in an expressed sequence (i.e., a variable region) and for which the corresponding genomic DNA has been isolated (i.e., cloned) and identified by sequence. While some immunoglobulin gene segment sequences contain open reading frames and are considered functional, even though they do not appear in the expressed repertoire, other immunoglobulin gene segment sequences contain mutations (e.g., point mutations, insertions, deletions, etc.) that result in stop codons and / or truncated sequences that subsequently render such gene segment sequences unable to exhibit the property(ies) and / or activity(ies) associated with the non-mutated sequence(s), such sequences do not appear in the expressed sequence and are therefore classified as pseudogenes.

[0133] Gene: As used herein, refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene comprises coding sequence (i.e., a sequence that encodes a specific product). In some embodiments, a gene comprises non-coding sequence. In some specific embodiments, a gene can include both coding (e.g., exons) and non-coding (e.g., intron) sequences. In some embodiments, a gene can include, for example, 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 purposes of clarity, we recognize that the term "gene," as used in this disclosure, typically refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof; the term can optionally encompass regulatory sequences, as will be clear from the context to one of skill in the art. 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 encodes a polypeptide.

[0134] Genetically modified non-human animal or genetically modified non-human animal: Used interchangeably herein, refers to any non-naturally occurring non-human animal (e.g., a rodent, e.g., a rat or mouse) in which one or more of the non-human animal's cells contain, in whole or in part, a heterologous nucleic acid and / or gene encoding a polypeptide of interest. For example, in some embodiments, a "genetically modified non-human animal" or a "genetically modified non-human animal" refers to a non-human animal containing a transgene or transgene construct described herein. In some embodiments, the heterologous nucleic acid and / or gene is introduced into the cell directly or indirectly by introduction into a precursor cell using deliberate genetic engineering, e.g., by microinjection or infection with a recombinant virus. The term genetic engineering does not include classical breeding techniques, but rather covers the introduction of a recombinant DNA molecule(s). This molecule may be integrated into a chromosome. The phrases "genetically modified non-human animal" or "genetically modified non-human animal" refer to animals that are heterozygous or homozygous for heterologous nucleic acids and / or genes and / or that have single or multiple copies of heterologous nucleic acids and / or genes.

[0135] Germline configuration: As used herein, refers to the arrangement of sequences (e.g., gene segments) found in the endogenous germline genome of a wild-type animal (e.g., mouse, rat, or human). Examples of germline configurations of immunoglobulin gene segments can be found, for example, in LeFranc, MP., The Immunoglobulin Facts Book, Academic Press, May 23, 2001 (referred to herein as "LeFranc 2001"): Exemplary organizations of human heavy chain variable region gene segments and human heavy chain constant region genes can be found in LeFranc 2001, p. 47; Exemplary organizations of human lambda light chain variable region gene segments and human lambda light chain constant region genes can be found in LeFranc 2001, p. 61; Exemplary organizations of human kappa light chain variable region gene segments and human kappa light chain constant region genes can be found at p. 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, 2002. nd Edition, Academic Press, 2015 (Lucas); Exemplary configurations of mouse λ light chain variable region gene segments and mouse λ light chain constant region genes are described in LeFranc, MP et al., Chapter 4: Immunoglobulin Lambda (IGL) Genes of Human and Mouse, Molecular Biology of B Cells, 1 st Edition, Academic Press, 2004 (LeFranc 2004); Exemplary configurations of mouse kappa light chain variable region gene segments and mouse kappa light chain constant region genes are described 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 are incorporated herein by reference.

[0136] Germline genome: As used herein, refers to the genome found in embryonic cells (e.g., gametes, e.g., sperm or eggs) used in the formation of an animal. 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 a modification in its germline genome is considered to have the modification in all 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., a 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., a mouse, rat, or human). Representative germline sequences for immunoglobulin gene segments can be found, for example, in LeFranc 2001: Human V that may be utilized 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 of human D gene segments and representative germline amino acid sequences of human D gene segments that may be utilized in some embodiments described herein can be found on pages 98-100 of LeFranc 2001; Human J that may be utilized 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 of human Vλ gene segments and representative germline amino acid sequences of human Vλ gene segments that can be utilized in some embodiments of the 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 utilized in some of the non-human animal embodiments 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] Heterologous: As used herein, refers to a substance or entity from a different source. For example, when used with reference to a polypeptide, gene, or gene product present in a particular cell or organism, the term clarifies that the relevant polypeptide, gene, or gene product: 1) has been modified by the hand of man; 2) has been introduced into the cell or organism (or its precursor) via the hand of man (e.g., via genetic modification); and / or 3) is not naturally produced by or present in the relevant cell or organism (e.g., the relevant cell type or organism type). "Heterologous" also includes polypeptides, genes, or gene products that are normally present in a particular native cell or organism but that have been altered or modified, for example, by mutation or placement under the control of a non-endogenous regulatory element (e.g., a promoter) with which they are not naturally associated, in some embodiments.

[0139] Host cell: As used herein, refers to a cell into which a nucleic acid or protein has been introduced. Those of skill in the art reading this disclosure will understand that such terms are used to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the phrase "host cell." In some embodiments, a host cell is or includes a prokaryotic or eukaryotic cell. Generally, a host cell is any cell suitable for receiving and / or producing a heterologous nucleic acid or protein, regardless of the kingdom of life to which the cell is assigned. Exemplary cells include prokaryotic and eukaryotic cells (unicellular or multicellular), bacterial cells (e.g., strains of Escherichia coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, etc.), and the like. The cell may be a human cell, such as a Pichia pombe, Pichia pastoris, or Pichia methanolica cell, a plant cell, an insect cell (e.g., SF-9, SF-21, baculovirus-infected insect cell, Trichoplusia ni, etc.), a non-human animal cell, a human cell, or a cell fusion, such as, for example, a hybridoma or quadroma. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is 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 cell comprises one or more viral genes, e.g., a retinal cell expressing a viral gene (e.g., a PER.C6® cell). In some embodiments, the host cell is or comprises an isolated cell. In some embodiments, the host cell is part of a tissue. In some embodiments, the host cell is part of an organism.

[0140] Identity: When used herein in connection with sequence comparison, refers to identity as 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, identity as described herein is determined using ClustalW v.1.83 (slow) alignment with an open gap penalty of 10.0, an extend gap penalty of 0.1, and a Gonnet similarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008).

[0141] In place of: As used herein, refers to a positional replacement in which a first nucleic acid sequence is located at the position of a second nucleic acid sequence in a chromosome (e.g., where the second nucleic acid sequence was previously (e.g., originally) located in the chromosome, e.g., at the endogenous locus of the second nucleic acid sequence). The phrase "in place of" does not require that the second nucleic acid sequence be removed, e.g., from a locus or chromosome. In some embodiments, the second nucleic acid sequence and the first nucleic acid sequence are equivalent to each other, e.g., in that 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 sequence comprises one or more of a promoter, an enhancer, a splice donor site, a splice acceptor site, an intron, an exon, an untranslated region (UTR); in some embodiments, the first and / or second nucleic acid sequence comprises one or more coding sequences. In some embodiments, the first nucleic acid sequence is a homolog or variant (e.g., mutant) of the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence is an ortholog or homolog of the second sequence. In some embodiments, the first nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, including when the first nucleic acid sequence is or comprises a human nucleic acid sequence, the second nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse or rat sequence). In some embodiments, including when the first nucleic acid sequence is or comprises a human nucleic acid sequence, the second nucleic acid sequence is or comprises 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 sequences so positioned may include one or more regulatory sequences (e.g., promoters, enhancers, 5' or 3' untranslated regions, etc.) that were part of the source nucleic acid sequence used to obtain the so positioned sequence.For example, in various embodiments, the first nucleic acid sequence is a substitution of an endogenous sequence with a heterologous sequence that results in the production of a gene product from the nucleic acid sequence (including the heterologous sequence) so arranged, but does not result in expression of the endogenous sequence; the first nucleic acid sequence is of an endogenous genomic sequence having a nucleic acid sequence encoding a polypeptide having a similar function as the polypeptide encoded by the endogenous sequence (e.g., the endogenous genomic sequence encodes a non-human variable region polypeptide in whole or in part, and the DNA fragment encodes one or more human variable region polypeptides in whole or in part). In various embodiments, a human immunoglobulin gene segment or fragment thereof is in place of an endogenous non-human immunoglobulin gene segment or fragment.

[0142] In vitro: As used herein, refers to events that take place in an artificial environment, e.g., in a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

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

[0144] Isolated: As used herein, refers to a substance or entity that is (1) separated from at least some of the components with which it was associated when originally produced (either in nature and / or in an experimental setting), and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can 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 with which they were originally associated. In some embodiments, isolated substances are separated from 10% to 100%, 15% to 100%, 20% to 100%, 25% to 100%, 30% to 100%, 35% to 100%, 40% to 100%, 45% to 100%, 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, or 99% to 100% of other components with which they were originally associated. In some embodiments, isolated substances are separated from 10% to 100%, 10% to 99%, 10% to 98%, 10% to 97%, 10% to 96%, 10% to 95%, 10% to 90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 55%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15% of other components with which they were originally associated. In some embodiments, isolated substances are separated from 11% to 99%, 12% to 98%, 13% to 97%, 14% to 96%, 15% to 95%, 20% to 90%, 25% to 85%, 30% to 80%, 35% to 75%, 40% to 70%, 45% to 65%, 50% to 60%, or 55% to 60% of other components with which they were originally associated. In some embodiments, isolated substances are 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 greater than about 99% pure.In some embodiments, an isolated substance is 80%-99%, 85%-99%, 90%-99%, 95%-99%, 96%-99%, 97%-99%, or 98%-99% pure. In some embodiments, an isolated substance is 80%-99%, 80%-98%, 80%-97%, 80%-96%, 80%-95%, 80%-90%, or 80%-85% pure. In some embodiments, an isolated substance is 85%-98%, 90%-97%, or 95%-96% pure. In some embodiments, a substance is "pure" if it is substantially free of 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 certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the isolation or purity of the substance is calculated without including 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: a) it is not associated with some or all of the components that naturally accompany it in its native state due to factors of its origin or source; b) it is substantially free from other polypeptides or nucleic acids of the same species as the species that naturally produces it; or c) it is expressed by, or otherwise associated with, components from, a cell or other expression system that is not of the species that naturally produces it. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system other than that in which it naturally occurs is considered an "isolated" polypeptide. Alternatively, or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is naturally associated; and / or b) with which it was associated when originally produced.

[0145] Locus or loci: As used herein, refers to the location(s) of a gene, DNA sequence, polypeptide-encoding sequence, or location on a chromosome in an organism's genome. For example, an "immunoglobulin locus" can 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 an organism's genome identified with respect to where such a sequence is located. An "immunoglobulin locus" of an immunoglobulin gene segment can include regulatory elements, including, but not limited to, an enhancer, a promoter, 5' and / or 3' regulatory sequences or regions, or combinations thereof. An "immunoglobulin locus" can also include intergenic DNA, e.g., DNA normally present or occurring 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 can demonstrate physical co-localization of similar loci when compared across different species. Such loci can be described as having shared synteny.

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

[0147] Non-human animal: As used herein, refers to any vertebrate organism that is not a human. In some embodiments, the non-human animal is a cyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian, a reptile, a mammal, or a bird. In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, the non-human animal is a rodent, such as a rat or a mouse.

[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, a "nucleic acid" is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As is clear from the context, in some embodiments, a "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or a nucleoside); in some embodiments, a "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a "nucleic acid" is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a "nucleic acid" is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a "nucleic acid" in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a "nucleic acid" is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and contain peptide bonds instead of phosphodiester bonds in the backbone. Alternatively, or additionally, in some embodiments, a "nucleic acid" has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, a "nucleic acid" is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, a "nucleic acid" is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intervening bases, and combinations thereof). In some embodiments, a "nucleic acid" comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In some embodiments, a "nucleic acid" has a nucleotide sequence that encodes a functional gene product, e.g., an RNA or a polypeptide. In some embodiments, a "nucleic acid" comprises one or more introns. In some embodiments, a "nucleic acid" comprises one or more exons. In some embodiments, a "nucleic acid" is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, a "nucleic acid" is at least, for example, but not limited to, 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 residues in length. In some embodiments, the "nucleic acid" is single-stranded; in some embodiments, the "nucleic acid" is double-stranded.In some embodiments, a "nucleic acid" has a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. In some embodiments, a "nucleic acid" has enzymatic activity.

[0149] Operably linked: As used herein, refers to the juxtaposition of components in a relationship that permits the components described to function in their intended manner (e.g., when the components are in the correct tissue, cell type, cellular activity, etc.). For example, one or more V H a gene segment, one or more D gene segments, and one or more J gene segments H The gene segment is V H , D, and J HA gene segment is "operably linked" to a heavy chain constant region if it can be spliced ​​to the heavy chain constant region at the appropriate time during B cell development, regardless of whether splicing occurs in, for example, cells outside the immune system (e.g., embryonic cells). A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences that are contiguous with a gene of interest and expression control sequences that act in trans or at a distance to control a gene of interest (or sequence of interest). The term "expression control sequence" includes polynucleotide sequences necessary to affect the expression and processing of coding sequences to which it is ligated. "Expression control sequences" include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, e.g., splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance polypeptide stability; and, if desired, sequences that enhance polypeptide secretion. The nature of such control sequences will vary depending on the host organism. For example, in prokaryotes, such control sequences usually include a promoter, ribosomal binding site, and transcription termination sequence, whereas in eukaryotes, such control sequences typically include a promoter and a transcription termination sequence. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0150] Polypeptide: As used herein, refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that contains portions that occur naturally separately from each other (i.e., from two or more different organisms, e.g., human and non-human portions). In some embodiments, a polypeptide has an amino acid sequence that is modified in that it has been designed and / or generated through the work of the hand of man. In some embodiments, a polypeptide has an amino acid sequence that is encoded by a sequence that does not occur in nature (e.g., a sequence that is modified in that it has been designed and / or generated through the work of the hand of man to encode said polypeptide).

[0151] Rearranged: As used herein, the term refers to a DNA sequence comprising two or more immunoglobulin gene segments joined together (directly or indirectly), where the joined gene segments together comprise a DNA sequence encoding an immunoglobulin variable region. The two or more immunoglobulin gene segments of the rearranged DNA sequence are no longer functionally associated with a recombination signal sequence (RSS) and thus cannot undergo further rearrangement. Those skilled in the art will recognize that although two or more immunoglobulin gene segments of a rearranged DNA sequence may not be capable of further rearrangement, this does not mean that other immunoglobulin gene segments within the same locus cannot undergo, for example, secondary rearrangements. Those skilled in the art will understand that rearranged gene segments (e.g., those in a rearranged immunoglobulin variable region) can be joined together via the natural VDJ recombination process. Those skilled in the art will also understand that rearranged gene segments (e.g., those in a rearranged immunoglobulin variable region) can be modified to be joined together, for example, by joining the gene segments using standard recombinant techniques. A rearranged immunoglobulin variable region typically comprises two or more joined immunoglobulin gene segments. For example, a rearranged immunoglobulin λ light chain variable region can comprise a Vλ gene segment joined with a Jλ gene segment. A rearranged immunoglobulin heavy chain variable region can comprise a joined Vλ gene segment. H Gene segment, D gene segment, J H The rearranged immunoglobulin variable region may include gene segments. Those skilled in the art will also understand that all or substantially all intergenic sequences are typically removed between immunoglobulin gene segments in rearranged immunoglobulin variable regions. Those skilled in the art will further understand that the rearranged sequences may include, inter alia, introns in the gene segments.

[0152] Recombinant: As used herein, refers to a molecule (e.g., DNA, RNA, or polypeptide) formed by laboratory methods of genetic recombination (e.g., cloning) to combine genetic material from multiple sources (e.g., organisms, tissues, cells, genomes, or portions of genomes). In some embodiments, a recombinant polypeptide designed, modified, prepared, expressed, produced, or isolated by recombinant means includes, for example, a polypeptide expressed using a recombinant expression vector transfected into a host cell, a polypeptide isolated from a recombinant combinatorial human polypeptide library (Hoogenboom, H.R., 1997, TIB Tech. 15:62-70; Azzazy, H. and W.E. Highsmith, 2002, Clin. Biochem. 35:425-45; Gavilondo, J.V. and J.W. Larrick, 2002, BioTechniques 29:128-45; Hoogenboom H., and P. Chames, 2000, Immunol. Today 21:371-8, which are incorporated herein by reference in their entireties), an antibody isolated from an animal (e.g., a mouse) that has been genetically modified to contain human immunoglobulin genes (e.g., Taylor, L.D. 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 al., 2014, Proc. Natl. Acad. Sci. USA 111(14):5153-8 (each of which is incorporated herein by reference in its entirety)) or 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 such selected sequence elements result, for example, from mutagenesis (e.g., in vivo or in vitro) of known sequence elements from natural or synthetic (e.g., artificial) sources. For example, in some embodiments, the recombinant polypeptide comprises a sequence found in the genome of a target organism source (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide has an amino acid sequence that results from mutagenesis (e.g., in vitro or in vivo, e.g., in a non-human animal), such that the amino acid sequence of the recombinant polypeptide is a sequence that originates from and is related to the polypeptide sequence, but that may not naturally occur in the genome of the non-human animal in vivo.

[0153] Reference: As used herein, refers to a standard or control substance, animal, cohort, individual, population, sample, sequence, or value to which a substance, animal, cohort, individual, population, sample, sequence, or value of interest is compared. In some embodiments, the reference substance, animal, cohort, individual, population, sample, sequence, or value is tested and / or determined substantially simultaneously with the testing or determination of the substance, animal, cohort, individual, population, sample, sequence, or value of interest. In some embodiments, the reference substance, animal, cohort, individual, population, sample, sequence, or value is a prior reference, optionally embodied in a tangible medium. In some embodiments, a reference can refer to a control. "Reference" also includes "reference animal." A "reference animal" may have a modification described herein, a modification different from those described herein, or may have no modification (i.e., a wild-type animal). Typically, as will be understood by one of skill in the art, the reference material, animal, cohort, individual, population, sample, sequence or value is determined or characterized under conditions equivalent to those used to determine or characterize the material, animal (e.g., mammal), cohort, individual, population, sample, sequence or value of interest.

[0154] Replacement: As used herein, refers to a process in which a "replacement" nucleic acid sequence (e.g., a gene) found in a host locus (e.g., in a genome) is removed from that locus and a different "replacement" nucleic acid is placed in its place. In some embodiments, the replaced 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 replaced nucleic acid sequence comprises one or more of a promoter, enhancer, splice donor site, splice acceptor site, intron, exon, untranslated region (UTR); in some embodiments, the replacement nucleic acid sequence comprises one or more coding sequences. In some embodiments, the replacement nucleic acid sequence is a homolog or variant (e.g., mutant) of the nucleic acid sequence it replaces. In some embodiments, the replacement nucleic acid sequence is an ortholog or homolog of the sequence it replaces. In some embodiments, the replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments in which the replacement nucleic acid sequence is or comprises a human nucleic acid sequence, the replaced nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse or rat sequence). In some embodiments in which the replacement nucleic acid sequence is or comprises a human nucleic acid sequence, the replaced nucleic acid sequence is or comprises a human sequence. In some embodiments, the replacement nucleic acid sequence is a variant or mutant of the sequence it replaces (i.e., a sequence that contains one or more sequence differences, e.g., substitutions, compared to the sequence it replaces). The nucleic acid sequence so positioned may include one or more regulatory sequences (e.g., promoters, enhancers, 5' or 3' untranslated regions, etc.) that were part of the source nucleic acid sequence used to obtain the sequence so positioned.For example, in various embodiments, the replacement is of an endogenous sequence with a heterologous sequence that results in the production of a gene product from the nucleic acid sequence (including the heterologous sequence) so arranged, but does not result in expression of the endogenous sequence; or the replacement is of an endogenous genomic sequence with a nucleic acid sequence that encodes a polypeptide having a similar function as the polypeptide encoded by the endogenous sequence (e.g., the endogenous genomic sequence encodes a non-human variable region polypeptide in whole or in part, and the DNA fragment encodes one or more human variable region polypeptides in whole or in part). In various embodiments, an endogenous non-human immunoglobulin gene segment, or fragment thereof, is replaced with a human immunoglobulin gene segment, or fragment thereof.

[0155] Substantially: As used herein, refers to a quantitative condition indicating the whole or nearly whole extent or degree of a feature or characteristic of interest. Those skilled in the biological arts understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent 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 "substantially similar" if they contain similar residues (e.g., amino acids or nucleotides) at corresponding positions. As understood in the art, similar residues can be identical residues (see substantial identity below); similar residues can also be non-identical residues with roughly equivalent structural and / or functional characteristics. For example, as is well known by those skilled in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "non-polar" side chains. Substituting one amino acid for another of the same type can often be considered a "conservative" substitution. Exemplary 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 any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, S. F. et al., 1990, J. Mol. Biol., 215(3):403-10; Altschul, S. F. et al., 1996, Meth. Enzymol. 266:460-80; Altschul, S. F. et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, A. D. and B. F. F. Feuillette (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 (incorporated herein by reference in their entireties). In addition to identifying similar sequences, the above programs typically provide an indication of the degree of similarity. In some embodiments, two sequences are considered to be substantially similar if at least, for example, but not limited to, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are similar (e.g., identical or include conservative substitutions) over the relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence (e.g., the sequence of a gene, gene segment, sequence encoding a domain, polypeptide, or domain). In some embodiments, the relevant stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues.In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues. In some embodiments, the relevant stretch comprises contiguous residues along the complete sequence. In some embodiments, the relevant stretch comprises non-contiguous residues along the complete sequence, e.g., non-contiguous residues held together by a folded conformation of the polypeptide or portion thereof.

[0158] Substantial identity: 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 to be "substantially identical" if they contain identical residues (e.g., amino acids or nucleotides) at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms available in commercially available computer programs, including, for example, BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, S. F. et al., 1990, J. Mol. Biol., 215(3):403-10; Altschul, S. F. et al., 1996, Meth. Enzymol. 266:460-80; Altschul, S. F. et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, A. D. and B. F. F. Huellette (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 which is incorporated herein by reference in its entirety). In addition to identifying identical sequences, the above programs typically provide an indication 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 over the relevant stretch of residues, which in some embodiments is the complete sequence.In some embodiments, the relevant stretch of residues is, for example and without limitation, 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 comprises a sequence identical or substantially identical to a sequence in a target cell, tissue, or animal, providing for integration of the targeting construct into a location within the genome of the cell, tissue, or animal via homologous recombination. Targeting regions that use site-specific recombinase recognition sites (e.g., loxP or Frt sites) are also included and described herein. In some embodiments, the targeting constructs described herein further comprise a specific nucleic acid sequence or gene of interest, a selectable marker, control and / or regulatory sequences, and other nucleic acid sequences that enable recombination mediated through the exogenous addition of proteins that assist or facilitate recombination, including such sequences. In some embodiments, the targeting constructs described herein further comprise a gene of interest, in whole or in part, which is a heterologous gene that in whole or in part encodes a polypeptide having a similar function 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 nucleic acid sequence engineered by the hand of man. For example, in some embodiments, the targeting construct (or targeting vector) may be constructed to contain a modified or recombinant polynucleotide that contains two or more sequences that are not linked together in their natural order but have been engineered by the hand of man so that they are directly linked to each other 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, in whole or in part) that has been introduced into a cell by the hand of man, for example, by the methods described herein. A transgene can be partially or entirely heterologous, i.e., foreign, to the genetically modified animal or cell into which it is introduced. A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, e.g., introns or promoters, that may be necessary for expression of a selected nucleic acid sequence.

[0161] Unrearranged: As used herein, refers to a DNA sequence comprising two or more immunoglobulin gene segments that have not undergone a recombination event or are otherwise not joined, and therefore comprise an intergenic sequence(s) between them. Those skilled in the art will understand that unrearranged V and J gene segments may be associated with intact recombination signal sequences (RSSs). An unrearranged D gene segment may be flanked by two intact recombination signal sequences (RSSs). Those skilled in the art will further understand that unrearranged gene segments may comprise, inter alia, introns.

[0162] Vector: As used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, vectors are capable of extrachromosomal replication and / or expression of nucleic acids to which they are linked in a host cell, such as a eukaryotic and / or prokaryotic cell. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors."

[0163] Wild-type: As used herein, refers to an entity having a structure and / or activity found in nature in a "normal" (as opposed to mutant, diseased, altered, modified, etc.) state or situation. Those skilled in the art will appreciate that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles). (Mode for Carrying Out the Invention)

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

[0165] The present disclosure recognizes that generating antibodies comprising human λ light chain variable domains in non-human animals has previously presented challenges when expression of λ light chains in a given non-human animal is low. For example, mice utilize significantly more κ light chains than λ light chains (i.e., at a κ:λ ratio of approximately 95:5). The present disclosure further recognizes that generating antibodies comprising universal light chains (e.g., light chains capable of binding to multiple heavy chains) by limiting the light chain variable region repertoire of a non-human animal can be challenging because it eliminates some of the most powerful diversity-generating mechanisms for generating high-affinity antibodies, e.g., combinatorial diversity, junctional diversity, and secondary rearrangement. It also recognizes that "mix and match" antibodies can be used to generate antibodies that are "mix and match" (e.g., "mix and match"). In light of these realizations, there remains a need in the art for platforms and methods for generating human λ light chain variable regions in non-human animals from limited human λ light chain variable regions.

[0166] The present disclosure provides the insight that non-human animals (e.g., rodents, e.g., rats or mice) containing a limited human λ light chain variable region repertoire at the κ light chain locus can effectively generate high-affinity, antigen-specific antibodies. This result is unexpected because the limited human λ light chain variable region repertoire in non-human animals forces the non-human animals to utilize λ light chain variable region sequences. The use of λ light chain variable region sequences goes against the natural preferences of a given non-human animal. As noted above, the use of a limited human λ light chain variable region repertoire in non-human animals also eliminates many of the natural mechanisms used to generate high-affinity, antigen-specific antibodies.

[0167] The present disclosure provides genetically modified non-human animals (e.g., rodents, e.g., rats or mice) that express human immunoglobulin λ light chain variable domains, wherein the non-human animals have a limited human λ light chain variable region repertoire (comprising one or two human Vλ gene segments). In some embodiments, the present disclosure provides genetically modified non-human animals that express human immunoglobulin λ light chain variable domains, wherein the non-human animals have a limited human λ light chain variable repertoire and a human immunoglobulin heavy chain variable domain. Biological systems for generating human λ light chain variable domains expressed from a limited human λ light chain variable region repertoire in association with a diverse repertoire of affinity-matured human heavy chain variable domains are also provided. Methods for making antigen-binding proteins comprising 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-chain variable fragments (ScFvs)) are provided. In some embodiments, the methods involve immunizing a non-human animal described herein with an antigen of interest. In some embodiments, the methods involve using immunoglobulin variable region gene sequences of a non-human animal (e.g., a rodent, e.g., a rat or mouse) described herein in an antigen binding protein (e.g., a binding protein that specifically binds to an antigen of interest). Methods include methods for producing human immunoglobulin heavy chain and / or lambda light chain variable domains suitable for use in making 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-matured human heavy chains expressed by such non-human animals, and the multiple heavy chain variable regions are capable of specifically binding different epitopes. In some embodiments, the non-human animals described herein express and select suitable affinity-matured human immunoglobulin heavy chain variable domains derived from a repertoire of unrearranged human heavy chain variable region gene segments, and the affinity-matured human heavy chain variable domains associate with and express human λ light chain variable domains derived from the non-human animal's limited human immunoglobulin λ light chain variable region gene repertoire. The present disclosure provides insight that human λ light chain variable domains and human heavy chain variable domains (together with the encoding human λ light chain variable regions and human heavy chain variable regions) can be utilized in the generation of multispecific antibodies, particularly bispecific antibodies.

[0169] Antibody repertoires 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) is composed of two identical heavy chains and two identical light chains, each of which has two structural components: a variable domain and a constant domain. The heavy and light chain variable regions vary 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 comprise the 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 different in antibodies of different isotypes. The following table summarizes the nine antibody isotypes in mice and humans. [Table 3]

[0170] Additional isotypes have been identified in other species. Isotypes confer distinctive biological properties to antibodies due to different structural features among different isotypes and are found in different locations (cells, tissues, etc.) within an animal's body. Initially, B cells produce IgM and IgD, which have identical antigen-binding regions. Upon activation, B cells switch to a different isotype through a process called class switching, which involves changing the constant region of the antibody produced by the B cell while retaining the antigen specificity of the original antibody (B cell) by keeping the variable region the same.

[0171] Two separate loci (Igκ and Igλ) contain gene segments that, when rearranged, encode the light chains of antibodies and exhibit both allelic and isotypic exclusion. + vs. λ +B cell expression ratios vary between 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 opposite of that observed in mice and rats. Several studies have been conducted to elucidate the possible reasons behind these observed ratios, and both the complexity of the locus (i.e., the number of gene segments, particularly V gene segments) and the efficiency of gene segment rearrangement have been proposed as reasons. The human immunoglobulin λ light chain locus stretches over 1,000 kb and contains approximately 70 Vλ gene segments (29-33 functional) and seven Jλ-Cλ gene segment pairs (4-5 functional) organized in three clusters (see, e.g., Figure 1 of U.S. Pat. No. 9,006,511, incorporated herein by reference in its entirety). The majority of Vλ regions observed in expressed antibody repertoires are encoded by gene segments contained within the closest cluster, designated Cluster A. The mouse immunoglobulin λ light chain locus, depending on the lineage, is notably different from the human locus, containing several Vλ and Jλ gene segments organized in two distinct gene clusters (see, e.g., Figure 2 of U.S. Pat. No. 9,006,511, incorporated herein by reference in its entirety).

[0172] The development of therapeutic antibodies for the treatment of various human diseases has largely focused on the creation of modified non-human animal strains, particularly modified rodent strains, that carry varying amounts of genetic material in their genomes that correspond to human immunoglobulin genes (reviewed, e.g., in Bruggemann, M. et al., 2015, Arch. Immunol. Ther. Exp. 63:101-8, which is incorporated herein by reference in its entirety). Early efforts in generating such genetically modified rodent strains focused on the incorporation of portions of human immunoglobulin loci, which, as such, could support gene segment recombination and the production of fully human heavy and / or light chains while having 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. D. et al., 1992, Nucl. Acids Res. 20:6287-6295; Davies, N. P. et al., 1993, Biotechnol. 11:911-4; Green, L. 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 which is incorporated by reference in its entirety). In particular, several efforts have involved the incorporation of human immunoglobulin λ light chain sequences (see, e.g., U.S. Patent Application Publication Nos. 2002 / 0088016A1, 2003 / 0217373A1, and 2011 / 0236378A1; U.S. Patent Nos. 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 which is incorporated herein by reference in its entirety). Such efforts have focused on randomly integrating yeast artificial chromosomes containing human Vλ, Jλ, and Cλ sequences, thereby generating mouse strains that express fully human immunoglobulin λ light chains (i.e., human Vλ and Cλ domains).More recent efforts have employed similar strategies using constructs that also contain human Vλ, Jλ, and Cλ sequences (Osborn, MJ et al., 2013, J. Immunol. 190:1481-90; Lee, EC. et al., 2014, Nat. Biotech. 32(4):356-63, each of which is incorporated herein by reference in its entirety).

[0173] Yet other approaches have involved the specific insertion of human Vλ and Jλ gene segments into endogenous rodent immunoglobulin light chain loci (κ and λ) such that the human Vλ and Jλ gene segments are operably linked to endogenous immunoglobulin light chain constant region genes (see, e.g., U.S. Pat. Nos. 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 by reference in their entireties). In some embodiments of such animals, all of the human Vλ gene segments from clusters A and B and either one or four human Jλ gene segments were inserted into 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, with the light chains containing human Vλ domains for either endogenous Cκ and Cλ regions (see, e.g., Table 7 and Figures 11-13 of U.S. Pat. No. 9,006,511, incorporated herein by reference in its entirety). In particular, mice with modified immunoglobulin κ light chain loci bearing human Vλ and Jλ gene segments demonstrated a human lambda to endogenous lambda ratio of approximately 1:1 in the splenic compartment (as measured by the IgCκ to IgCλ ratio) (see, e.g., Table 4 of U.S. Pat. No. 9,006,511, incorporated herein by reference in its entirety). Indeed, both modified mouse strains (i.e., modified immunoglobulin κ or modified immunoglobulin λ light chain loci) demonstrated that human Vλ domains can be expressed from endogenous immunoglobulin light chain loci in rodents that normally exhibit a large bias in light chain expression (see above). The present disclosure provides the recognition that alternative modified immunoglobulin light chain locus structures can be generated to maximize expression of human λ light chain variable domains from the limited human λ light chain variable region repertoire.Such alternative, modified immunoglobulin light chain locus structures offer the potential for unique antibody repertoires due to their design.

[0174] The present 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 operably linked to a rodent Cλ gene segment. 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, all immunoglobulin λ light chains expressed by B cells of the genetically modified rodent comprise human immunoglobulin λ light chain variable domains expressed from a single rearranged human immunoglobulin λ light chain variable region or a somatically hypermutated version thereof. In some embodiments, the modified endogenous immunoglobulin κ light chain locus comprises a single rearranged human immunoglobulin λ light chain variable region operably linked to a non-human or human immunoglobulin λ or immunoglobulin κ light chain constant region gene. In some embodiments, expression of such a light chain may be achieved by insertion of the single rearranged human immunoglobulin λ light chain variable region into an endogenous immunoglobulin κ light chain locus (or allele). In some embodiments, the provided non-human animals are modified such that expression of the endogenous immunoglobulin λ light chain variable region is inactivated (e.g., by gene deletion). In some embodiments, the provided non-human animals are modified such that expression of the endogenous immunoglobulin κ light chain variable region is inactivated (e.g., by insertion, replacement, or substitution).

[0175] Universal light chain Previous efforts to generate useful multispecific antigen-binding proteins, e.g., bispecific antibodies, have been hampered by a variety of problems that often share a common paradigm: in vitro selection or engineering of sequences to rationally modify, or modify through trial and error, suitable formats for pairing heterodimeric bispecific human immunoglobulins. Unfortunately, most, if not all, in vitro modification approaches generally provide suitable ad hoc solutions for individual molecules, if at all.

[0176] In vivo methods have been developed to use complex organisms to select appropriate pairings capable of resulting in human therapeutics (see, e.g., U.S. Pat. No. 10,143,186, incorporated by reference in its entirety). However, no nonhuman animal has previously been generated capable of generating a robust immune response, including the generation of a high-affinity universal λ light chain with a human immunoglobulin λ light chain variable domain at sufficient titer levels. Typically, native nonhuman sequences are often not a good source for human therapeutic sequences. For at least that reason, generating nonhuman heavy chain immunoglobulin variable domains paired with universal human light chains has limited practical utility. More in vitro modification efforts will likely be undertaken in a trial-and-error process to humanize nonhuman heavy chain variable sequences in the hope of retaining epitope specificity and affinity and the ability to bind common human light chains, although the outcome is uncertain. At the end of such a process, the final product may retain some of the specificity and affinity and associate with the universal light chain, but ultimately, immunogenicity in humans may pose a significant risk.

[0177] Therefore, suitable non-human animals for producing human therapeutics will contain an appropriately broad repertoire of human heavy chain variable region gene segments in place 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 comprising human variable domains and non-human constant domains). The heavy chain locus should be capable of undergoing class switching and somatic hypermutation, so that an appropriately broad repertoire of heavy chain variable domains is available for the non-human animal to select from 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 a universal light chain for multiple heavy chains have practical utility. 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 identical light chains. This is particularly useful in generating bispecific antibodies. For example, such non-human animals can be immunized with a first immunogen to generate B cells that express an antibody that specifically binds to a first epitope. The non-human animal (or a non-human animal containing the same genetic modifications to the heavy chain and lambda light chain loci) can be immunized with a second immunogen to generate B cells that express an antibody that specifically binds to a second epitope. The variable heavy chain region can be cloned from the B cell and expressed with the same heavy chain constant region and the same light chain in a cell to generate a bispecific antibody, where the light chain component of the bispecific antibody has been selected by the non-human animal to associate with and express the light chain component. Non-human animals that express universal κ light chains have been developed (see, e.g., U.S. Pat. No. 10,143,186, incorporated by reference in its entirety). However, there remains a need for the development of non-human animals that can express universal λ light chains, including human λ light chain variable domains.

[0179] The present disclosure provides modified non-human animals (e.g., rodents, e.g., rats or mice) for producing immunoglobulin λ light chains that pair favorably with a fairly diverse family of heavy chains, including heavy chains whose variable regions deviate from germline sequences, e.g., heavy chains that have been affinity matured or somatically hypermutated. In various embodiments, the non-human animals described herein are modified to express and pair a human λ light chain variable domain with a human heavy chain variable domain that contains somatic mutations, thereby enabling a route to high-affinity binding proteins (e.g., antibodies) suitable for use as human therapeutics.

[0180] The genetically modified non-human animals (e.g., rodents, e.g., rats or mice) described herein, through the long and complex process of antibody selection within an organism, make biologically relevant choices in pairing 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 wide diversity of human heavy chain variable domain options. When administered an immunogen, the non-human animals described herein can maximize the number of solutions in their repertoire to generate antibodies against the immunogen that are largely or exclusively limited by the number or light chain options in their repertoire. In various embodiments, this includes enabling the non-human animals to achieve suitable and compatible somatic mutation of light chain variable domains that are nevertheless compatible with a relatively diverse range of human heavy chain variable domains, including particularly 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 can be modified to render their ability to generate or rearrange native non-human λ and / or κ light chain variable domains non-functional or substantially non-functional. In some embodiments, this can be achieved by deleting the λ and / or κ light chain variable region gene segments of the non-human animal. In some embodiments, the endogenous non-human locus can then be engineered with suitable exogenous human λ light chain variable region sequence(s) operably linked to the endogenous non-human light chain constant domain(s). In some embodiments, the exogenous human variable region gene segments are unrearranged (e.g., two Vλ gene segments and one or more Jλ gene segments), and can rearrange and splice into the endogenous non-human light chain constant region gene to form a rearranged reverse chimeric light chain gene (human variable, non-human constant). In some embodiments, the exogenous human variable gene segments are rearranged (e.g., one Vλ gene segment and one Jλ gene segment) and can be spliced ​​into an endogenous non-human light chain constant region gene to form a reverse chimeric light chain gene comprising a human variable region and a non-human constant region. In some embodiments, the exogenous human variable gene segments are rearranged (e.g., one Vλ gene segment and one Jλ gene segment) and can be spliced ​​into an exogenous human light chain constant region gene to form a humanized light chain gene comprising a human variable region and a human constant region. In various embodiments, the light chain variable region is capable of somatic hypermutation. In various embodiments, an appropriate enhancer(s) is retained in the non-human animal. Enhancers have been reported to maximize the ability of light chain variable regions to acquire somatic mutations. For example, in modifying the κ locus of a non-human animal by replacing endogenous non-human animal κ variable region gene segments with human λ variable region gene segments, the non-human κ intronic enhancer and the non-human κ 3' enhancer are functionally maintained or not disrupted. Although embodiments in which enhancers are removed or disrupted are contemplated by the present disclosure, such embodiments would be expected to reduce or eliminate somatic hypermutation.In such embodiments, somatic hypermutation of the light chain variable region is reduced, for example, compared to a light chain variable region comprising 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 constant) light chains associated with diverse reverse-chimeric (human variable, non-human constant) 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 operably linked to the endogenous non-human κ constant region gene. In some embodiments, the non-human κ intronic enhancer and the non-human κ 3' enhancer are maintained. Without being bound by any one theory, the enhancer may, among other things, maximize somatic hypermutation of the human λ light chain variable region gene segment. In various embodiments, the non-human animal also comprises a non-functional λ light chain locus, or a deletion thereof, or a deletion that renders the locus incapable of making λ light chains.

[0183] In various embodiments, genetically modified non-human animals are provided that lack endogenous non-human light chain variable gene segments and comprise a light chain variable region locus that comprises human variable gene segments operably linked to non-human Cλ gene segments, in some embodiments a rearranged human immunoglobulin λ light chain variable region, wherein the locus is capable of undergoing somatic hypermutation, and the locus expresses a light chain comprising a human immunoglobulin λ light chain variable region linked to a non-human Cλ gene segment. Thus, in various embodiments, the locus comprises a non-human κ 3' enhancer that correlates with normal, or wild-type, levels of somatic hypermutation.

[0184] In various embodiments, the genetically modified non-human animals, when immunized with an antigen of interest, generate B cells that exhibit diverse rearrangements of human immunoglobulin heavy chain variable regions that express and function with one or two rearranged light chains. In some embodiments, the human λ light chain variable regions comprise somatic hypermutations. In some embodiments, each human λ light chain variable region comprises one to five somatic hypermutations. In various embodiments, the light chains expressed by the non-human animals described herein are capable of associating with and expressing any heavy chain comprising a human immunoglobulin heavy chain variable region expressed in the non-human animal.

[0185] Provided non-human animals, cells and tissues Provided are non-human animals that express (e.g., whose B cells express) antibodies containing light chains comprising a human λ light chain variable domain derived from (i) one or two unrearranged Vλ gene segments and one or more unrearranged Jλ gene segments, or (ii) a single rearranged 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. It will be understood that in various embodiments described herein, the genetically modified non-human animal is a rodent, e.g., a rat or a mouse, and the non-human elements (enhancers, constant regions, etc.) described herein are rodent, e.g., rat or mouse elements. Suitable examples of non-human animals described herein include, but are not limited to, rodents, e.g., rats or mice, particularly mice.

[0186] The present disclosure provides improved in vivo systems for identifying and developing new antigen-binding proteins, antibodies, antibody components (e.g., antigen-binding portions and / or compositions or formats comprising same), and / or antibody-based therapeutics that can be used, for example, in the treatment of a variety of diseases affecting humans. Furthermore, the present disclosure encompasses the recognition that non-human animals (e.g., rodents, e.g., rats or mice) with modified immunoglobulin loci, e.g., modified immunoglobulin κ light chain loci comprising a limited λ light chain variable region repertoire, are useful. In some embodiments, the non-human animals described herein provide improved in vivo systems for the development of antibodies and / or antibody-based therapeutics for administration to humans. In some embodiments, the non-human animals described herein provide improved in vivo systems for the development of antibodies and / or antibody-based therapeutics containing human λ light chain variable domains characterized by improved and / or different performance (e.g., expression and / or occurrence in antigen-specific antibody repertoires) compared to antibodies and / or antibody-based therapeutics obtained from existing in vivo systems containing human Vλ region sequences.

[0187] The present disclosure provides, inter alia, a non-human animal (e.g., a rodent, e.g., a rat or mouse), non-human (e.g., a rodent, e.g., a rat or mouse) cell, or non-human (e.g., a rodent, e.g., a rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include a limited human λ light chain variable region repertoire. In some embodiments, the sequences of the limited human λ light chain variable region repertoire are operably linked to a non-human light chain constant region. In some embodiments, the non-human light chain constant region is a rodent (e.g., a 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 sequences of the limited human λ light chain variable region repertoire are operably linked to a non-human (e.g., a rodent, e.g., a rat or mouse) Cκ. In some embodiments, sequences of a limited human λ light chain variable region repertoire are operably 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) is in place of an endogenous non-human Cκ.

[0188] The present disclosure provides, inter alia, a non-human animal (e.g., a rodent, e.g., a rat or a mouse), a non-human (e.g., a rodent, e.g., a rat or a mouse) cell, or a non-human (e.g., a rodent, e.g., a rat or a mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include two unrearranged human Vλ gene segments and one or more unrearranged human Jλ gene segments. In some embodiments, the two unrearranged 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. In some embodiments, the two unrearranged 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 two unrearranged 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 one or more unrearranged human Jλ gene segments are selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, the one or more unrearranged human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, the one or more unrearranged human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.In some embodiments, the two unrearranged human Vλ gene segments and one or more unrearranged human Jλ gene segments are operably 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 two unrearranged human Vλ gene segments and one or more unrearranged human Jλ gene segments are operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cκ. In some embodiments, the two unrearranged human Vλ gene segments and one or more unrearranged human Jλ gene segments are operably 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., rodent, e.g., rat or mouse, e.g., mouse Cλ, e.g., mouse Cλ1) is in place of the endogenous non-human (e.g., rodent, e.g., rat or mouse) Cκ.

[0189] The present disclosure provides, inter alia, a non-human animal (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cell, or non-human (e.g., rodent, e.g., rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include two unrearranged human Vλ gene segments and four unrearranged human Jλ gene segments operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cκ. In some embodiments, the two unrearranged 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 unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7.

[0190] The present disclosure provides, inter alia, a non-human animal (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cell, or non-human (e.g., rodent, e.g., rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include two unrearranged human Vλ gene segments and five unrearranged human Jλ gene segments operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cκ. In some embodiments, the two unrearranged 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 unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0191] The present disclosure provides, inter alia, a non-human animal (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cell, or non-human (e.g., rodent, e.g., rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include two unrearranged human Vλ gene segments and four unrearranged human Jλ gene segments operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cλ (e.g., Cλ1). In some embodiments, the two unrearranged 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 unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7.

[0192] The present disclosure provides, inter alia, a non-human animal (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cell, or non-human (e.g., rodent, e.g., rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include two unrearranged human Vλ gene segments and five unrearranged human Jλ gene segments operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cλ (e.g., Cλ1). In some embodiments, the two unrearranged 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 unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0193] The present disclosure provides, inter alia, a non-human animal (e.g., a rodent, e.g., a rat or a mouse), a non-human (e.g., a rodent, e.g., a rat or a mouse) cell, or a non-human (e.g., a rodent, e.g., a rat or a mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include one unrearranged human Vλ gene segment and one or more unrearranged human Jλ gene segments. In some embodiments, one unrearranged 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. In some embodiments, one unrearranged 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 unrearranged 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 one or more unrearranged human Jλ gene segments are selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, the one or more unrearranged human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, the one or more unrearranged human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.In some embodiments, one unrearranged human Vλ gene segment and one or more unrearranged human Jλ gene segments are operably 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 unrearranged human Vλ gene segment and one or more unrearranged human Jλ gene segments are operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cκ. In some embodiments, one unrearranged human Vλ gene segment and one or more unrearranged human Jλ gene segments are operably 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., rodent, e.g., rat or mouse, e.g., mouse Cλ, e.g., mouse Cλ1) is in place of the endogenous non-human (e.g., rodent, e.g., rat or mouse) Cκ.

[0194] The present disclosure provides, inter alia, a non-human animal (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cell, or non-human (e.g., rodent, e.g., rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include one unrearranged human Vλ gene segment and four unrearranged human Jλ gene segments operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cκ. In some embodiments, the one unrearranged 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 unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, one unrearranged human Vλ gene segment is Vλ1-51 and the four unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, one unrearranged human Vλ gene segment is Vλ2-14 and the four unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7.

[0195] The present disclosure provides, inter alia, a non-human animal (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cell, or non-human (e.g., rodent, e.g., rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include one unrearranged human Vλ gene segment and five unrearranged human Jλ gene segments operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cκ. In some embodiments, the one unrearranged 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 unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, one unrearranged human Vλ gene segment is Vλ1-51 and the five unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, one unrearranged human Vλ gene segment is Vλ2-14 and the five unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0196] The present disclosure provides, inter alia, a non-human animal (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cell, or non-human (e.g., rodent, e.g., rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include one unrearranged human Vλ gene segment and four unrearranged human Jλ gene segments operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cλ. In some embodiments, the one unrearranged 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 unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, one unrearranged human Vλ gene segment is Vλ1-51 and the four unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7. In some embodiments, one unrearranged human Vλ gene segment is Vλ2-14 and the four unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, and Jλ7.

[0197] The present disclosure provides, inter alia, a non-human animal (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cell, or non-human (e.g., rodent, e.g., rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include one unrearranged human Vλ gene segment and five unrearranged human Jλ gene segments operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cλ. In some embodiments, the one unrearranged 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 unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, one unrearranged human Vλ gene segment is Vλ1-51 and the five unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7. In some embodiments, one unrearranged human Vλ gene segment is Vλ2-14 and the five unrearranged human Jλ gene segments are Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0198] The present disclosure provides, inter alia, a non-human animal (e.g., a rodent, e.g., a rat or a mouse), non-human (e.g., a rodent, e.g., a rat or a mouse) cell, or non-human (e.g., a rodent, e.g., a rat or a mouse) tissue 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. 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 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. 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 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 the 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 the single rearranged human λ light chain variable region is Vλ1-51. In some embodiments, the human Vλ gene segment of the single rearranged human λ light chain variable region is Vλ2-14.In some embodiments, the human Jλ gene segment of the 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 the single rearranged human λ light chain variable region is Jλ1. In some embodiments, the human Jλ gene segment of the single rearranged human λ light chain variable region is Jλ2. In some embodiments, the human Jλ gene segment of the single rearranged human λ light chain variable region is Jλ3. In some embodiments, the single rearranged human λ light chain variable region is operably 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 operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cκ. In some embodiments, a single rearranged human λ light chain variable region is operably 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) is in place of an endogenous non-human (e.g., rodent, e.g., rat or mouse) Cκ.

[0199] The present disclosure provides, inter alia, a non-human animal (e.g., a rodent, e.g., a rat or mouse), non-human (e.g., a rodent, e.g., a rat or mouse) cell, or non-human (e.g., a rodent, e.g., a rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include a single rearranged human λ light chain variable region operably linked to a mouse Cκ, wherein the single rearranged human λ light chain variable region comprises 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] The present disclosure provides, inter alia, a non-human animal (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cell, or non-human (e.g., rodent, e.g., rat or mouse) tissue having an endogenous immunoglobulin κ light chain locus modified to include a single rearranged human λ light chain variable region operably linked to a non-human (e.g., rodent, e.g., rat or mouse) Cλ, wherein the single rearranged human λ light chain variable region comprises 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] The present disclosure provides, inter alia, a mouse, mouse cell, or mouse tissue having an endogenous immunoglobulin κ light chain locus modified to comprise a single rearranged human λ light chain variable region operably linked to a mouse Cλ, wherein the single rearranged human λ light chain variable region comprises a human Vλ gene segment and a human Jλ gene segment. In some embodiments, the human Vλ gene segment comprises Vλ1-51. In some embodiments, the human Vλ gene segment comprises Vλ2-14. In some embodiments, the human Jλ gene segment comprises 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] The present disclosure provides, inter alia, a genetically modified mouse, mouse cell, or mouse tissue, 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 mouse Cλ1 gene segment, the single rearranged human immunoglobulin λ light chain variable region comprising a human Vλ1-51 gene segment and a human Jλ2 gene segment, and all immunoglobulin λ light chains expressed by 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 somatically hypermutated version thereof.

[0203] The present disclosure provides, inter alia, a genetically modified mouse, mouse cell, or mouse tissue, 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 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 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 somatically hypermutated version thereof.

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

[0205] In some embodiments, provided is a non-human animal (e.g., a rodent, e.g., a rat or a mouse), non-human (e.g., a rodent, e.g., a rat or a mouse) cell or non-human (e.g., a rodent, e.g., a rat or a mouse) tissue, whose germline genome comprises (a) a single rearranged human immunoglobulin λ light chain variable region, and (b) an endogenous immunoglobulin κ light chain locus comprising a Cλ gene, wherein (a) is operably linked to (b), and wherein the non-human animal lacks a non-human animal Cκ gene at the endogenous immunoglobulin κ light chain locus.

[0206] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse), non-human (e.g., a rodent, e.g., a rat or mouse) cell, or non-human (e.g., a rodent, e.g., a rat or mouse) tissue, whose genome comprises an endogenous immunoglobulin κ light chain locus comprising a single rearranged human immunoglobulin λ light chain variable region and an insertion of a Cλ gene, wherein the single rearranged human immunoglobulin λ light chain variable region is operably linked to the Cλ gene, and the Cλ gene is inserted in place of the non-human Cκ gene at the endogenous immunoglobulin κ light chain locus, is provided. In many embodiments of the non-human animal, non-human cell, or non-human tissue, the Cλ gene inserted in place of the non-human Cκ gene at the endogenous immunoglobulin κ light chain locus is a non-human or human Cλ gene. In some embodiments, the non-human Cλ gene is or comprises a mammalian Cλ gene selected from the group consisting of primate, caprine, ovine, porcine, canine, bovine, or rodent (e.g., rat or mouse) Cλ genes.

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

[0208] In some embodiments, the rodent Cλ gene is or comprises a mouse Cλ gene. In some embodiments, the mouse Cλ gene comprises a sequence 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 comprises a sequence substantially identical to 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 comprises SEQ ID NO: 1. In some certain embodiments, the mouse Cλ2 gene is or comprises SEQ ID NO: 2. In some certain embodiments, the mouse Cλ3 gene is or comprises SEQ ID NO: 3. In some certain embodiments, the mouse Cλ gene comprises a sequence identical to a mouse Cλ1 gene.

[0209] In some embodiments, the mouse Cλ gene comprises a sequence that is 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% 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 comprises a sequence that is 80% to 98%, 80% to 95%, 80% to 90%, or 80% to 85% 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 comprises a sequence that is 85% to 98%, 90% to 95%, or 88% to 93% identical to a 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 comprises a rat Cλ gene. In some embodiments, the rat Cλ gene comprises a sequence 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 comprises a sequence 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 certain embodiments, the rat Cλ1 gene is or comprises SEQ ID NO:7. In some certain embodiments, the rat Cλ2 gene is or comprises SEQ ID NO:8. In some certain embodiments, the rat Cλ3 gene is or comprises SEQ ID NO:9. In some certain embodiments, the rat Cλ4 gene is or comprises SEQ ID NO:10.

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

[0212] In some embodiments, the human Cλ gene comprises a sequence 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 comprises a sequence 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 comprises a sequence 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 certain embodiments, the human Cλ1 gene is or comprises SEQ ID NO: 15. In some certain embodiments, the human Cλ2 gene is or comprises SEQ ID NO: 16. In some certain embodiments, the human Cλ3 gene is or comprises SEQ ID NO: 17. In some certain embodiments, the human Cλ6 gene is or comprises SEQ ID NO: 18. In some certain embodiments, the human Cλ7 gene is or comprises SEQ ID NO: 18. In some certain embodiments, the human Cλ gene is or comprises a human Cλ2 gene.

[0213] In some embodiments, the human Cλ gene comprises a sequence that is 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 comprises a sequence that is 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 comprises a sequence that is 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 provided 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, the germline genome of said non-human animals, non-human cells, or non-human tissues contains one or more human V H gene segment, one or more human D H gene segment, and one or more human J H and further comprising an endogenous immunoglobulin heavy chain locus modified to include a human V gene segment. H , D H and J H The gene segment is operably linked to a non-human immunoglobulin heavy chain constant region at an endogenous immunoglobulin heavy chain locus (see, e.g., 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. Patent Nos. 6,596,541, 8,642,835, 8,697,940, and 8,791,323 (each of which is incorporated by reference herein in its entirety)).

[0215] In some embodiments, one or more human V H gene segment, one or more human D H A gene segment and one or more human J H The insertion of the gene segment may, in whole or in part, be H , D H and J H In place of or replacing a gene segment (e.g., a non-human V H , D H and J H The coding sequence of the gene segment was determined to be human V H , D H and J HIn 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 certain embodiments, the insertion is in a positional replacement or substitution with the coding sequence of a human V H , D H and J H In some embodiments, the immunoglobulin heavy chain loci described herein comprise human V gene segments, and the human non-coding DNA that naturally occurs between the gene segments and combinations thereof. 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 In some embodiments, the insertion comprises an insertion of a human V at the endogenous heavy chain locus. 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 Naturally occurring human non-coding DNA adjacent to 6-1, 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 naturally occurring human non-coding DNA adjacent to 7-27 and human J at the endogenous heavy chain locus H 1. J H 2. J H 3. J H 4. J H 5, or J H 6 and contains naturally occurring human non-coding DNA adjacent to it.

[0216] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse), non-human (e.g., a rodent, e.g., a rat or mouse) cell, or non-human (e.g., a rodent, e.g., a rat or mouse) tissue described herein comprises an Adam6 gene in its genome (e.g., its germline genome), which encodes an ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated by reference in its entirety). In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse), non-human (e.g., a rodent, e.g., a rat or mouse) cell, or non-human (e.g., a rodent, e.g., a rat or mouse) tissue described herein comprises a rodent (e.g., a mouse or rat) Adam6 gene in its genome (e.g., its germline genome), which encodes a rodent (e.g., a mouse or rat) ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety). In some embodiments, the ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof is expressed from the Adam6 gene. In some embodiments, the Adam6 gene in a genetically modified non-human animal described herein does not originate from that particular non-human animal (e.g., a mouse comprising a rat Adam6 gene or a mouse Adam6 gene obtained from another mouse strain). In some embodiments, the non-human animal described herein comprises an ectopic Adam6 gene. As used herein, "ectopic" Adam6 gene refers to an Adam6 gene that is in a different situation than the Adam6 gene appears in wild-type non-human animals. For example, the Adam6 gene can be located on a different chromosome, can be located at a different locus, or can be 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 comprise an inserted or integrated Adam6 gene.

[0217] In some embodiments, a non-human animal, non-human cell, or non-human tissue described herein comprises in its genome (e.g., in its germline genome) an insertion of one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof.

[0218] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse), non-human (e.g., a rodent, e.g., a rat or mouse) cell, or non-human (e.g., a rodent, e.g., a rat or mouse) tissue described herein comprises in its genome (e.g., in its germline genome) one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse), non-human (e.g., a rodent, e.g., a rat or mouse) cell, or non-human (e.g., a rodent, e.g., a rat or mouse) tissue described herein comprises in its genome (e.g., in its germline genome) a mouse Adam6a gene and / or a mouse Adam6b gene. In some embodiments, the non-human animals, non-human cells or non-human tissues described herein comprise one or more nucleotide sequences encoding mouse ADAM6a, its functional ortholog, functional homolog, or functional fragment, and / or mouse ADAM6b, its functional ortholog, functional homolog, or functional fragment.

[0219] In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted and / or located on the same chromosome as an endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted and / or located at a position such that the one or more nucleotide sequences encoding the one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are contiguous with a human immunoglobulin heavy chain variable region gene segment. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted and / or located at a position such that the one or more nucleotide sequences encoding the one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are adjacent to a human immunoglobulin heavy chain variable region gene segment. In some embodiments, the one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted and / or positioned at a location such that the one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are located between human immunoglobulin heavy chain variable region gene segments. In some embodiments, the one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are located between first and second human V H 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 HIn some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted and / or positioned in place of a human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted and / or positioned in place of a human Adam6 pseudogene. H Gene 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., rodent, e.g., rats or mice) cells, or non-human (e.g., rodent, e.g., rats or mice) tissues described herein contain an Adam6 gene that restores or improves ADAM6 activity. In some embodiments, the Adam6 gene restores ADAM6 activity to the level of a comparable non-human animal that contains a functional endogenous Adam6 gene. In some embodiments, the Adam6 gene improves ADAM6 activity to a level that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than the ADAM6 activity of a comparable non-human animal that does not contain a functional Adam6 gene.

[0221] In some embodiments, the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodent, e.g., rats or mice) cells, or non-human (e.g., rodent, e.g., rats or mice) tissues described herein comprise an Adam6 gene that, when expressed in a male non-human animal, restores or improves fertility. In some embodiments, the Adam6 gene restores fertility in the male non-human animal to the level of a comparable non-human animal that contains a functional endogenous Adam6 gene. In some embodiments, the Adam6 gene restores fertility in the male non-human animal such that the number of pups produced by mating with the male non-human animal is at least 70%, at least 80%, at least 90%, or at least 95% of the number of pups produced by a comparable mating with a comparable male non-human animal that does not contain a functional Adam6 gene. In some embodiments, the Adam6 gene enhances the reproductive performance of the male non-human animal such that the number of pups produced by mating with the male non-human animal is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than the number of pups produced by a similar mating with a comparable male non-human animal that does not contain a 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 lack of at least one endogenous non-human Adam6 gene reduces ADAM6 activity and / or fertility 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 comprise a 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 fertility in male rodents (e.g., mice or rats) lacking the endogenous non-human Adam6 gene.

[0223] In some embodiments of the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodent, e.g., rats or mice) cells, or non-human (e.g., rodent, 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 comprising a human heavy chain variable region gene segment described herein.

[0224] In some embodiments of the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodent, e.g., rats or mice) cells, or non-human (e.g., rodent, 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 comprising a human light chain variable gene segment (human variable λ light chain gene segment) described herein.

[0225] In some embodiments, the non-human animal (e.g., a rodent, e.g., a rat or mouse), non-human (e.g., a rodent, e.g., a rat or mouse) cell, or non-human (e.g., a rodent, e.g., a rat or mouse) tissue described herein comprises a first modified endogenous immunoglobulin κ light chain locus allele comprising a single rearranged human immunoglobulin λ light chain variable region operably linked to a rodent Cλ gene segment, 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 non-human animal, non-human cell, or non-human tissue comprises a second modified endogenous immunoglobulin κ light chain locus allele comprising a single rearranged human immunoglobulin κ light chain variable region operably linked to a rodent Cκ gene segment, wherein the single rearranged human immunoglobulin κ light chain variable region comprises a human Vκ gene segment and a human Jκ gene segment. In some embodiments, such a non-human animal or tissue 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, 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, the single rearranged human immunoglobulin κ light chain variable region is Vκ3-20 / Jκ1 or Vκ1-39 / Jκ5, and the single rearranged human immunoglobulin λ light chain variable region is Vλ1-51 / Jλ2 or Vλ2-14 / Jλ2.

[0226] In some embodiments of the provided non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodent, e.g., rats or mice) cells, or non-human (e.g., rodent, e.g., rats or mice) tissues, the endogenous immunoglobulin λ light chain locus is deleted in whole or in part. In some embodiments of the provided non-human animals, non-human cells, or non-human tissues, the endogenous immunoglobulin λ light chain locus is functionally silenced or otherwise non-functional (e.g., by gene targeting). In certain embodiments of the provided non-human animals, non-human cells, or non-human tissues, the non-human animals, non-human cells, or non-human tissues are homozygous for a 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., rodent, e.g., rats or mice) cells, or non-human (e.g., rodent, e.g., rats or mice) tissues described herein do not detectably express endogenous λ immunoglobulin light chains. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein do not detectably express endogenous κ immunoglobulin light chains. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein do not detectably express endogenous λ immunoglobulin light chains or endogenous κ immunoglobulin light chains.

[0228] In some embodiments, the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodent, e.g., rats or mice) cells, or non-human (e.g., rodent, 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 λ immunoglobulin light chains, endogenous κ immunoglobulin light chains, and endogenous immunoglobulin heavy chains.

[0229] In some embodiments, the non-human animals, cells, or tissues described herein have a genome that further comprises a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operably linked to a transcriptional control element (see, e.g., WO 2017 / 210586 and U.S. Publication No. 2017 / 0347633, each of which is incorporated by reference in its entirety).

[0230] In some embodiments, the transcriptional control element comprises a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin kappa light chain transcriptional control element, an immunoglobulin lambda 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 kappa light chain locus, the immunoglobulin lambda light chain locus, the immunoglobulin heavy chain locus, the RAG1 locus, or the RAG2 locus.

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

[0233] In some embodiments, a single rearranged human immunoglobulin κ light chain variable region is introduced into the endogenous immunoglobulin κ light chain locus in a manner that maintains the integrity of the non-human immunoglobulin κ light chain enhancer region (or enhancer sequence) (e.g., the non-human immunoglobulin κ intronic enhancer and / or the non-human immunoglobulin κ 3' enhancer) near the point of insertion. Thus, such non-human animals have a wild-type immunoglobulin κ light chain enhancer region (or enhancer sequence) operably linked to human and non-human immunoglobulin λ light chain sequences (e.g., human Vλ and Jλ gene segments and a non-human Cλ or CK) or operably linked to a human immunoglobulin λ light chain sequence (e.g., human Vλ and Jλ gene segments and a human Cλ or CK).

[0234] In some embodiments, the non-human immunoglobulin κ light chain locus that has been changed, 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 of two copies of said non-human immunoglobulin κ light chain locus, resulting in a non-human animal that is heterozygous for the human immunoglobulin κ light chain sequence. In some embodiments, a non-human animal is provided that is homozygous for an immunoglobulin κ light chain locus comprising 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 portions thereof) at 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 portions thereof) at 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) at the endogenous non-human immunoglobulin λ light chain locus are altered, moved, disrupted, deleted, or replaced such 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 with a targeting vector such that the non-human immunoglobulin λ light chain locus is functionally inactivated (i.e., incapable of producing a functional light chain of an antibody that is expressed and / or detectable in the antibody repertoire of a non-human animal described herein). Guidance for the inactivation of endogenous non-human immunoglobulin λ light chain loci is provided, for example, in U.S. Patent No. 9,006,511 (see, e.g., Figure 2), which is incorporated herein by reference in its entirety.

[0236] A modified immunoglobulin κ light chain locus or transgene (e.g., comprising a limited human λ light chain variable region repertoire described herein) or its expression product may be detected using a variety of methods, including, for example, PCR, Southern blot, restriction fragment length polymorphism (RFLP), allelic gain or loss assay, Western blot, FACS analysis, etc. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein are heterozygous for a modified immunoglobulin κ light chain locus described herein. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein are hemizygous for a modified immunoglobulin κ light chain locus described herein. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein contain one or more copies of a modified immunoglobulin κ light chain locus or transgene described herein. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein contain an endogenous immunoglobulin κ light chain locus modified as shown in the figures.

[0237] The present disclosure recognizes that the non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodent, e.g., rats or mice) cells, or non-human (e.g., rodent, 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 region gene segments, or somatic hypermutated variants thereof, produced by the non-human animals, cells, or tissues described herein are encoded by the human heavy chain and λ light chain variable region gene segments, respectively, contained in their genomes.

[0238] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse) is provided, the genome of which comprises a modified immunoglobulin κ light chain locus, and the non-human animal comprises B cells comprising 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 B cells of the non-human animal (e.g., a rodent, e.g., a rat or mouse) of the present disclosure have one, two, three, four, five, or more somatic hypermutations. Those skilled in the art are aware of methods for identifying the source of gene segments in mature antibody sequences. For example, various tools are available to assist in this analysis, including, by way of example, DNAPLOT, IMGT / V-QUEST, JOINSOLVER, SoDA, and Ab-origin.

[0239] The present disclosure provides, among other things, cells and tissues from the non-human animals described herein (e.g., rodents, e.g., rats, mice). In some embodiments, splenocytes (and / or other lymphoid tissues) from the non-human animals described herein are provided. In some embodiments, B cells from the non-human animals described herein are provided. In some embodiments, pro-B cells from the non-human animals described herein are provided. In some embodiments, pre-B cells from the non-human animals described herein are provided. In some embodiments, immature B cells from the non-human animals described herein are provided. In some embodiments, mature naive B cells from the non-human animals described herein are provided. In some embodiments, activated B cells from the non-human animals described herein are provided. In some embodiments, memory B cells from the non-human animals described herein are provided. In some embodiments, B-lineage lymphocytes from the non-human animals described herein are provided. In some embodiments, plasma or plasma cells from the non-human animals described herein are provided. In some embodiments, stem cells from the non-human animals described herein are provided. In some embodiments, the stem cells are embryonic stem cells. In some embodiments, embryonic cells from the non-human animals described herein are provided. In some embodiments, the germ cell is an oocyte. In some embodiments, the germ cell is a sperm cell. In some embodiments, sperm cells from a non-human animal described herein express one or more ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, any cell or tissue from a non-human animal described herein can be isolated. In some embodiments, isolated cells and / or isolated tissues from a non-human animal described herein are provided. In some embodiments, hybridomas are provided, wherein the hybridomas are produced using B cells from a non-human animal described herein. In some embodiments, the hybridomas are produced using B cells from a non-human animal immunized with an antigen of interest.In some embodiments, hybridomas are generated using B cells of the non-human animal that express antibodies that bind (eg, specifically bind) to an epitope on an antigen of interest.

[0240] Any non-human animal (e.g., a rodent, e.g., a rat or mouse) described herein can be immunized with one or more antigens of interest under conditions and for a time sufficient for the non-human animal to mount an immune response to the one or more antigens of interest. Those skilled in the art will know methods for immunizing non-human animals. Exemplary, 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] The present disclosure provides, inter alia, 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 comprising 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 comprise 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 comprise (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 an antigen of interest, and the antibodies expressed by the population of B cells generated in response to the antigen comprise (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 the human λ 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%, 90% of the B cells in a population of B cells generated in response to an antigen comprise somatically hypermutated human heavy chain variable region sequences and / or human λ light chain variable region sequences.

[0243] Specific Exemplary Embodiments—Immunoglobulin Kappa Light Chain Locus In some embodiments, a provided non-human animal (e.g., a rodent, e.g., a rat or mouse) comprises a modified endogenous immunoglobulin κ light chain locus comprising a single rearranged human immunoglobulin λ light chain variable region inserted upstream of and operably linked to a non-human or human Cλ gene segment, where the non-human or human Cλ gene segment has been 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 that appears in cluster A of the human immunoglobulin λ light chain locus. 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 human Vλ gene segments that appear in cluster B of the human immunoglobulin λ light chain locus. 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 human Vλ gene segments that appear 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- and 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 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) comprises 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 comprising 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 comprising 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 comprising a human Vλ gene segment selected from the group consisting of Vλ1-51 or Vλ2-14.In some embodiments, the modified immunoglobulin κ light chain locus (or allele) comprises a single rearranged human immunoglobulin λ light chain variable region comprising 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) comprises a single rearranged human immunoglobulin λ light chain variable region comprising 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) comprises a single rearranged human immunoglobulin λ light chain variable region comprising a human Jλ gene segment selected from the group consisting of Jλ1, Jλ2, Jλ3, and Jλ7.

[0244] The present disclosure recognizes that the non-human animals (e.g., rodents, e.g., rats or mice) described herein utilize human 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 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 somatic hypermutated variants thereof.

[0245] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse) is provided, the genome of which comprises a modified endogenous immunoglobulin κ light chain locus, and the non-human animal comprises B cells comprising 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 B cells of the non-human animal (e.g., a rodent, e.g., a rat or mouse) of the present disclosure have one, two, three, four, five, or more somatic hypermutations. Those skilled in the art are aware of methods for identifying the source of gene segments in mature antibody sequences. For example, various tools are available to assist in this analysis, including, by way of example, DNAPLOT, IMGT / V-QUEST, JOINSOLVER, SoDA, and Ab-origin.

[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 is present 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 is present 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, a non-human animal (e.g., a rodent, e.g., a rat or mouse) described herein comprises in its germline genome a limited human λ light chain variable region repertoire (e.g., a single rearranged human immunoglobulin λ light chain variable region) operably linked to one or more non-human immunoglobulin κ light chain enhancers (i.e., enhancer sequences or regions). In some certain embodiments, the limited human λ light chain variable region repertoire (e.g., a single rearranged human immunoglobulin λ light chain variable region) is operably linked to a murine immunoglobulin κ light chain intronic enhancer region (Igκ Ei or Eiκ). In some certain embodiments, the limited human λ light chain variable region repertoire (e.g., a single rearranged human immunoglobulin λ light chain variable region) is operably linked to a murine immunoglobulin κ light chain 3' enhancer region (Igκ 3'E or 3'Eκ). In certain embodiments, the limited human λ light chain variable region repertoire (e.g., a single rearranged human immunoglobulin λ light chain variable region) is operably linked to a murine Eiκ and operably linked 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, for example, a mouse Cλ gene or a rat Cλ gene. In some certain embodiments, the non-human Cλ gene of the modified immunoglobulin κ light chain locus (or allele) is or comprises a mouse Cλ gene from a genetic background including a 129 strain, a BALB / c strain, a C57BL / 6 strain, a crossed 129×C57BL / 6 strain, or a combination thereof.

[0249] In some embodiments, a non-human Cλ gene of a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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, a non-human Cλ gene of a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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, a non-human Cλ gene of a modified immunoglobulin κ light chain locus (or allele) described herein is or comprises the sequence of a mouse Cλ1 gene.

[0250] In some embodiments, a non-human Cλ domain encoded by a sequence located at a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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, a non-human Cλ domain encoded by a sequence located at a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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, a non-human Cλ gene encoded by a sequence located at a modified immunoglobulin κ light chain locus (or allele) described herein is or comprises a mouse Cλ1 domain polypeptide.

[0251] In some embodiments, a non-human Cλ gene of a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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 certain embodiments, a non-human Cλ gene of a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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 certain embodiments, a non-human Cλ gene of a modified immunoglobulin κ light chain locus (or allele) described herein is or comprises the sequence of a rat Cλ1 gene.

[0252] In some embodiments, a non-human Cλ domain encoded by a sequence located at a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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, a non-human Cλ domain encoded by a sequence located at a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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, a non-human Cλ domain encoded by a sequence located at a modified immunoglobulin κ light chain locus (or allele) described herein is or comprises a rat Cλ1 domain polypeptide.

[0253] In some embodiments, the human Cλ gene of the modified immunoglobulin κ light chain locus (or allele) comprises a human Cλ gene, such as, for example, a human Cλ1 gene, a human Cλ2 gene, a human Cλ3 gene, a human Cλ6 gene, or a human Cλ7 gene. In some certain embodiments, the human Cλ gene of the modified immunoglobulin κ light chain locus (or allele) is or comprises a human Cλ2 gene.

[0254] In some embodiments, a human Cλ gene of a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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, a human Cλ gene of a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence substantially identical 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, a human Cλ gene of a modified immunoglobulin κ light chain locus (or allele) described herein is or comprises the sequence of a human Cλ2 gene.

[0255] In some embodiments, a human Cλ domain encoded by a sequence located at a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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, a human Cλ domain encoded by a sequence located at a modified immunoglobulin κ light chain locus (or allele) described herein comprises a sequence 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, a human Cλ domain encoded by a sequence located at a modified immunoglobulin κ light chain locus (or allele) described herein is or comprises a human Cλ2 domain polypeptide.

[0256] Specific Exemplary Embodiments—Immunoglobulin Heavy Chain Locus In some embodiments, the provided non-human animal (e.g., rodent, e.g., rat or mouse) comprises a modified endogenous immunoglobulin κ light chain locus that includes a limited human immunoglobulin λ light chain variable region repertoire (e.g., a single rearranged human immunoglobulin λ light chain variable region) as described herein, arranged in germline configuration and operably linked to non-human (e.g., rodent, e.g., rat or mouse) immunoglobulin heavy chain constant region genes. H , D H and J H Further included are modified immunoglobulin heavy chain loci (or alleles) characterized by the presence of gene segments, enhancers, and regulatory regions. In some embodiments, the modified immunoglobulin heavy chain loci (or alleles) described herein comprise one or more human V operably linked to a non-human immunoglobulin heavy chain constant region. H gene segment, one or more human DH A gene segment and one or more human J H In certain embodiments, the modified immunoglobulin heavy chain locus (or allele) comprises at least one human V gene segment. 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 certain embodiments, the modified immunoglobulin heavy chain locus (or allele) comprises at least one 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 7-27, or any combination thereof. In some certain embodiments, the modified immunoglobulin heavy chain locus (or allele) comprises at least one 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] The present disclosure recognizes that the non-human animals (e.g., rodents, e.g., rats or mice) described herein utilize human heavy 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 heavy chain variable domains produced by the non-human animals described herein are encoded by human heavy chain variable region gene segments or somatic hypermutated variants thereof contained in their genomes.

[0258] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse) comprises B cells comprising human heavy chain variable region sequences and / or human λ light chain variable region sequences that have been somatically hypermutated. In some embodiments, the human heavy chain variable region sequences and / or human λ light chain variable region sequences present in B cells of a non-human animal (e.g., a rodent, e.g., a rat or mouse) of the present disclosure have one, two, three, four, five, or more somatic hypermutations. Those skilled in the art are aware of methods for identifying the source of gene segments in mature antibody sequences. For example, various tools are available to assist in this analysis, including, by way of example, DNAPLOT, IMGT / V-QUEST, JOINSOLVER, SoDA, and Ab-origin.

[0259] In some embodiments, the non-human immunoglobulin heavy chain constant region comprises one or more non-human immunoglobulin heavy chain constant region genes, such as, for example, immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin E (IgE), and immunoglobulin A (IgA). In certain embodiments, the non-human immunoglobulin heavy chain constant region comprises 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 operably 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 operably 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 operably linked to one or more non-human immunoglobulin heavy chain enhancers (or enhancer sequences) and one or more non-human immunoglobulin heavy chain control regions (or control sequences).

[0260] In some embodiments, a modified immunoglobulin heavy chain locus described herein does not contain an endogenous Adam6 gene. In some embodiments, a modified immunoglobulin heavy chain locus described herein does not contain an endogenous Adam6 gene (or sequence encoding Adam6) at the same germline genomic location as found in the germline genome of a wild-type non-human animal of the same species. In some embodiments, a modified immunoglobulin heavy chain locus described herein does not contain a human Adam6 pseudogene. In some embodiments, a modified immunoglobulin heavy chain locus described herein comprises an insertion of at least one nucleotide sequence encoding one or more non-human (e.g., rodent) Adam6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, the insertion is located outside of a modified immunoglobulin heavy chain locus described herein (for example, but not limited to, the 5'-most V H The gene segment), within a modified immunoglobulin heavy chain locus, or elsewhere in the germline genome of the non-human animal (for example, but not limited to, a randomly introduced non-human Adam6 coding sequence), cell, or tissue.

[0261] In various embodiments, the non-human animals (e.g., rodents, e.g., rats or mice) provided herein contain endogenous non-human V in antibody molecules. H In various embodiments, the non-human animals provided herein do not detectably express the endogenous non-human V region in antibody molecules. H Region (e.g., V H , D H and / or J HIn various embodiments, the non-human animals provided herein do not contain (or lack or contain deletions of) one or more nucleotide sequences encoding, in whole or in part, an endogenous non-human V. H , D H and J H and having a germline genome that comprises, in whole or in part, a deletion of a gene segment. In various embodiments, the non-human animals provided are capable of reproduction.

[0262] Guidance for the generation of targeting vectors, non-human (e.g., rodent, e.g., rat or mouse) cells and animals carrying such modified immunoglobulin heavy chain loci (or alleles) can be found in Macdonald (2014), U.S. Patent Nos. 6,596,541, 8,642,835, 8,697,940, and 8,791,323, each of which is incorporated by reference herein in its entirety. Those of skill in the art will be aware of a variety of techniques known in the art for achieving such genetic modifications and / or manipulations of non-human (e.g., mammalian) genomes or for preparing, providing, or manufacturing such sequences for introduction into the germline genome of a non-human animal.

[0263] Specific Exemplary Embodiments—Immunoglobulin Loci Combinations In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or mouse) provided herein comprises, in its germline genome, a modified endogenous immunoglobulin κ light chain locus comprising a limited human immunoglobulin λ light chain variable region repertoire (e.g., a single rearranged human immunoglobulin λ light chain variable region) described herein, and further comprises one or more additional immunoglobulin loci comprising human immunoglobulin gene segments or other human or humanized genes (e.g., a human gene encoding TdT) (e.g., via hybridization or multiple gene targeting strategies). Such non-human animals can be prepared as described above or using methods known in the art to achieve a 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., a human gene encoding TdT) at other immunoglobulin loci can be introduced via further alterations in the genome of cells (e.g., embryonic stem cells) harboring the above-described genetic modifications or, if desired, via breeding techniques known in the art with other genetically modified strains.

[0264] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or a mouse) provided herein comprises, in its germline genome, a modified endogenous immunoglobulin κ light chain locus that includes a limited human immunoglobulin λ light chain variable region repertoire (e.g., a single rearranged human immunoglobulin λ light chain variable region) described herein, and comprises, in its germline genome, one or more human V operably linked to one or more non-human animal immunoglobulin heavy chain constant region genes. H a human D gene segment, one or more human D gene segments, and one or more human J gene segments HThe non-human animal further comprises a modified endogenous immunoglobulin heavy chain locus comprising a gene segment. In some embodiments, the non-human animal is heterozygous or homozygous for a modified endogenous immunoglobulin κ light chain locus comprising a 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 homozygous for a modified endogenous immunoglobulin κ light chain locus comprising a limited human immunoglobulin λ light chain variable region repertoire described herein (e.g., a single rearranged human immunoglobulin λ light chain variable region) and homozygous for a modified endogenous immunoglobulin heavy chain locus described herein.

[0265] In some embodiments, a non-human animal (e.g., a rodent, e.g., a rat or a mouse) provided herein comprises, in its germline genome, a modified endogenous immunoglobulin κ light chain locus that comprises a limited human immunoglobulin λ light chain variable region repertoire (e.g., a single rearranged human immunoglobulin λ light chain variable region) as described herein, one or more human V operably linked to one or more non-human animal immunoglobulin heavy chain constant region genes. H a human D gene segment, one or more human D gene segments, and one or more human J gene segments HThe non-human animal comprises a modified endogenous immunoglobulin heavy chain locus comprising a gene segment, and further comprises a functionally inactivated (e.g., deleted in whole or in part, or otherwise rendered non-functional) 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 a 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 in whole or in part, or otherwise rendered non-functional) endogenous immunoglobulin λ light chain locus. In some embodiments, the non-human animal is homozygous for a modified endogenous immunoglobulin κ light chain locus that comprises a limited human immunoglobulin λ light chain variable region repertoire described herein (e.g., a single rearranged human immunoglobulin λ light chain variable region), is homozygous for a modified endogenous immunoglobulin heavy chain locus described herein, and is homozygous for a functionally inactivated (e.g., deleted in whole or in part or otherwise rendered non-functional) endogenous immunoglobulin λ light chain locus.

[0266] In some embodiments, the germline genome of a genetically modified non-human animal that is a rodent (e.g., a mouse or rat) comprises 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 genome of a genetically modified rodent (e.g., a 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 comprising a single rearranged human immunoglobulin λ light chain variable region operably linked to a rodent (e.g., mouse or rat) Cλ gene segment, 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 genetically modified rodents (e.g., mice or rats) described herein, and rodent (e.g., mouse or rat) cells or rodent (e.g., mouse or rat) tissues therefrom, comprise a second modified endogenous immunoglobulin κ light chain locus allele comprising a single rearranged human immunoglobulin κ light chain variable region operably 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 can 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, 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, the single rearranged human immunoglobulin κ light chain variable region is Vκ3-20 / Jκ1 and the 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 (e.g., a rodent, e.g., a rat or a mouse) provided is selected from the group consisting of: (a) a non-human animal and a non-human C. H Human V fused to domain sequence H a human V operably linked to one or more endogenous non-human immunoglobulin heavy chain constant regions to express an immunoglobulin heavy chain comprising a V domain sequence; H , D H and J H (b) a homozygous or heterozygous immunoglobulin heavy chain locus comprising a human Vλ gene segment; and (b) an immunoglobulin κ light chain locus comprising a single rearranged human immunoglobulin λ light chain operably linked to a non-human (e.g., rodent) immunoglobulin Cλ gene segment such that the non-human animal expresses an immunoglobulin light chain comprising a human Vλ domain sequence fused to the non-human Cλ domain sequence.

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

[0269] For example, as described herein, a non-human animal comprising a modified endogenous immunoglobulin κ light chain locus as described herein may further comprise (e.g., via crossbreeding or multiple gene targeting strategies) one or more of the modifications described in U.S. Pat. Nos. 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 by reference in its entirety).

[0270] Nucleic Acid Constructs 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 portion(s) 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), synthesized from germline sequences designed in silico based on published sequences available from GenBank or other publicly available databases (e.g., IMGT). Alternatively, bacterial artificial chromosome (BAC) libraries can provide 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 carry 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, which are incorporated herein by reference in their entireties). For example, human BAC libraries with an average insert size of 164 to 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 which 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, incorporated herein by reference in its entirety). Furthermore, YACs have previously been used to construct human immunoglobulin λ light chain locus transgenes (see, e.g., Popov, A. V. et al., 1996, Gene 177:195-201; Popov, A. V. et al., 1999, J. Exp. Med. 189(10):1611-19, each of which is incorporated herein by reference in its entirety). The entire immunoglobulin λ light chain locus (human or non-human) can be cloned and contained within several YACs. Regardless of the sequences contained, if multiple YACs are used and contain overlapping regions of similarity, they can be recombined in a yeast host strain to generate a single construct representing the entire locus or a desired portion of the locus (e.g., the region targeted by a targeting vector). The YAC arms can be additionally modified by refinement with a mammalian selection cassette to aid 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 constructing the modified immunoglobulin κ light chain loci described herein can be obtained from public databases (e.g., GenBank, IMGT, etc.) and / or published antibody sequences.

[0274] In certain 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 operably linked 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 CK, respectively) gene. In certain 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 operably linked 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 operably linked 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 unrearranged human Vλ and Jλ sequences further comprises intergenic DNA of human and / or murine origin, hi some embodiments, the intergenic 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, nucleic acid constructs can be prepared as part of a larger plasmid. Such preparation allows for cloning and selection of the correct construct in an efficient manner, as known in the art. Nucleic acid constructs containing the human immunoglobulin λ light chain sequences described herein, in whole or in part, can be placed between restriction sites on a plasmid so that they can be isolated from remaining plasmid sequences for integration into a desired non-human animal (e.g., a rodent, e.g., a rat or mouse).

[0277] Various methods used in preparing nucleic acid constructs (e.g., plasmids) and transforming host organisms are known in the art. For other suitable expression systems for both prokaryotes and eukaryotes, as well as general recombinant procedures, see "Principles of Gene Manipulation: An Introduction to Genetic Manipulation," 5th Ed., ed. By Old, R.W. and S.B. Primrose, 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 which is incorporated herein by reference in its entirety).

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

[0279] In some embodiments, the methods described herein use two, three, or more targeting vectors that are capable of recombining with each other. 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 comprise 5' and 3' homology arms. The 3' homology arm of the first targeting vector comprises a sequence that overlaps with the 5' homology arm of the second targeting vector (i.e., overlapping sequence), which allows for 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 corresponding segments (i.e., target sequences) within the target genome locus, which may promote homologous recombination of the first and second targeting vectors with the corresponding genome segments to modify the target genome 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., overlapping sequence), which may enable homologous recombination between the second and third LTVEC. The 5' homology arm of the first targeting vector and the 3' homology arm of the third targeting vector resemble corresponding segments (i.e., target sequences) within the target genome locus, which may promote homologous recombination of the first and third targeting vectors with the corresponding genome segments to modify the target genome locus.

[0282] A homology arm and a target sequence, or two homology arms, "correspond" or "correspond" to one another if the two regions share a sufficient level of sequence identity with each other so that they can act as substrates for a homologous recombination reaction. The sequence identity between a given target sequence and the corresponding homology arm (i.e., overlapping sequence) found in a targeting vector, or between two homology arms, can be any degree of sequence identity that allows homologous recombination to occur. By way of example only, the amount of sequence identity shared by the homology arms of a targeting vector (or a fragment thereof) and the target sequence of another targeting vector or the target sequence of a target genomic locus (or a fragment thereof) can be, for example, but 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] Moreover, the corresponding region of similarity (e.g., identity) between the homology arms 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 comprise a corresponding region of similarity that is, for example, but not limited to, about 5-10 kb, 5-15 kb, 5-20 kb, 5-25 kb, 5-30 kb, 5-35 kb, 5-40 kb, 5-45 kb, 5-50 kb, 5-55 kb, 5-60 kb, 5-65 kb, 5-70 kb, 5-75 kb, 5-80 kb, 5-85 kb, 5-90 kb, 5-95 kb, 5-100 kb, 100-200 kb, or 200-300 kb in length (e.g., as described elsewhere herein), such that the homology arm has sufficient similarity to undergo homologous recombination with corresponding target sequence(s) within a target genomic locus of a cell or within another targeting vector. In some embodiments, a given homology arm and / or corresponding target sequence comprises a corresponding region of similarity that is, for example, but not limited to, about 10-100 kb, 15-100 kb, 20-100 kb, 25-100 kb, 30-100 kb, 35-100 kb, 40-100 kb, 45-100 kb, 50-100 kb, 55-100 kb, 60-100 kb, 65-100 kb, 70-100 kb, 75-100 kb, 80-100 kb, 85-100 kb, 90-100 kb, or 95-100 kb in length (e.g., as described elsewhere herein), such that the homology arm has sufficient similarity to undergo homologous recombination with corresponding target sequence(s) within a target genomic locus of a cell or within another targeting vector.

[0284] The overlapping sequence between the 3' homology arm of a first targeting vector and the 5' homology arm of a second targeting vector, or between the 3' homology arm of a second targeting vector and the 5' homology arm of a third targeting vector, can be of any length sufficient to promote homologous recombination between the targeting vectors. For example, a given overlapping sequence of a homology arm may include a corresponding overlapping region that is about 1 to 5 kb, 5 to 10 kb, 5 to 15 kb, 5 to 20 kb, 5 to 25 kb, 5 to 30 kb, 5 to 35 kb, 5 to 40 kb, 5 to 45 kb, 5 to 50 kb, 5 to 55 kb, 5 to 60 kb, 5 to 65 kb, 5 to 70 kb, 5 to 75 kb, 5 to 80 kb, 5 to 85 kb, 5 to 90 kb, 5 to 95 kb, 5 to 100 kb, 100 to 200 kb, or 200 to 300 kb in length, such that the overlapping sequence of the homology arm has sufficient similarity to undergo homologous recombination with a corresponding overlapping sequence in another targeting vector. In some embodiments, a given overlapping sequence of a homology arm comprises an overlapping region that is about 1 to 100 kb, 5 to 100 kb, 10 to 100 kb, 15 to 100 kb, 20 to 100 kb, 25 to 100 kb, 30 to 100 kb, 35 to 100 kb, 40 to 100 kb, 45 to 100 kb, 50 to 100 kb, 55 to 100 kb, 60 to 100 kb, 65 to 100 kb, 70 to 100 kb, 75 to 100 kb, 80 to 100 kb, 85 to 100 kb, 90 to 100 kb, or 95 to 100 kb in length, such that the overlapping sequence of the homology arm has sufficient similarity to undergo homologous recombination with a corresponding overlapping sequence in another targeting vector. In some embodiments, the overlapping sequence is 1 to 5 kb (inclusive). In some embodiments, the overlapping sequence is about 1 kb to about 70 kb (inclusive). In some embodiments, the overlapping sequence is about 10 kb to about 70 kb (inclusive). In some embodiments, the overlapping sequence is about 10 kb to about 50 kb (inclusive). In some embodiments, the overlapping sequence is at least 10 kb. In some embodiments, the overlapping sequence is at least 20 kb.For example, the overlapping sequences may be about 1 kb to about 5 kb (inclusive), about 5 kb to about 10 kb (inclusive), about 10 kb to about 15 kb (inclusive), about 15 kb to about 20 kb (inclusive), about 20 kb to about 25 kb (inclusive), about 25 kb to about 30 kb (inclusive), about 30 kb to about 35 kb (inclusive), about 35 kb to about 40 kb (inclusive), about 40 kb to about 45 kb (inclusive), about 45 kb to about 50 kb (inclusive), about 50 kb to about 60 kb (inclusive), about 60 kb to about 70 kb (inclusive), about 70 kb to about 80 kb (inclusive), about The length can be 80 kb to about 90 kb (inclusive), about 90 kb to about 100 kb (inclusive), about 100 kb to about 120 kb (inclusive), about 120 kb to about 140 kb (inclusive), about 140 kb to about 160 kb (inclusive), about 160 kb to about 180 kb (inclusive), about 180 kb to about 200 kb (inclusive), about 200 kb to about 220 kb (inclusive), about 220 kb to about 240 kb (inclusive), about 240 kb to about 260 kb (inclusive), about 260 kb to about 280 kb (inclusive), or about 280 kb to about 300 kb (inclusive). To give just one example, the overlapping sequence can be about 20 kb to about 60 kb (inclusive). Alternatively, the overlapping sequence can be at least 1 kb, at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 120 kb, at least 140 kb, at least 160 kb, at least 180 kb, at least 200 kb, at least 220 kb, at least 240 kb, at least 260 kb, at least 280 kb, or at least 300 kb.In some embodiments, the overlapping sequence 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, the homology arms correspond to a locus native to the cell (e.g., a targeted locus), or alternatively, they may correspond to a region of a heterologous or exogenous segment of DNA integrated into the cell's genome (e.g., including a transgene, expression cassette, or heterologous or exogenous region of DNA). In some embodiments, the homology arms may correspond to a region on a targeting vector in the cell. In some embodiments, the homology arms of the targeting vector may correspond to a region of a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a human artificial chromosome, or any other modified region contained in a suitable host cell. Furthermore, the homology arms of the targeting vector may correspond to or be derived from a region of a BAC library, a cosmid library, or a P1 phage library. In certain embodiments, the homology arms of the targeting vector correspond to a locus that is native, heterologous, or exogenous to a prokaryote, yeast, bird (e.g., chicken), non-human mammal, rodent, human, rat, mouse, hamster, rabbit, pig, cow, deer, sheep, goat, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey), domesticated mammal, agricultural mammal, or any other organism of interest. In some embodiments, the homology arms correspond to a locus in a cell that exhibits limited susceptibility to targeting using conventional methods in the absence of nicks or double-strand breaks induced by nuclease agents (e.g., Cas proteins), or that has exhibited 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 a mouse genome of a first strain and the targeting arms correspond to a mouse genome of a second strain, where the first strain and the second strain are different. In certain embodiments, the homology arms correspond to genomes of the same animal or are from genomes of the same strain, e.g., the targeted genome is a mouse genome of a first strain and the targeting arms correspond to mouse genomes from the same mouse or from the same strain.

[0287] The homology arms of the targeting vector may have a length of, for example, 1 to 5 kb (inclusive), 5 to 10 kb (inclusive), 5 to 15 kb (inclusive), 5 to 20 kb (inclusive), 5 to 25 kb (inclusive), 5 to 30 kb (inclusive), 5 to 35 kb (inclusive), 5 to 40 kb (inclusive), 5 to 45 kb (inclusive), 5 to 50 kb (inclusive), 5 to 55 kb (inclusive), 5 to 60 kb (inclusive), It may be of any length sufficient to promote a homologous recombination event with the corresponding target sequence, including 5 to 65 kb (inclusive), 5 to 70 kb (inclusive), 5 to 75 kb (inclusive), 5 to 80 kb (inclusive), 5 to 85 kb (inclusive), 5 to 90 kb (inclusive), 5 to 95 kb (inclusive), 5 to 100 kb (inclusive), 100 to 200 kb (inclusive), or 200 to 300 kb (inclusive). In some embodiments, the homology arms of the targeting vector have a length of 1 to 100 kb (inclusive), 5 to 100 kb (inclusive), 10 to 100 kb (inclusive), 15 to 100 kb (inclusive), 20 to 100 kb (inclusive), 25 to 100 kb (inclusive), 30 to 100 kb (inclusive), 35 to 100 kb (inclusive), 40 to 100 kb (inclusive), 45 to 100 kb (inclusive), 50 to 100 kb (inclusive), 60 to 100 kb (inclusive), 70 to 100 kb (inclusive), 80 to 100 kb (inclusive), 90 to 100 kb (inclusive), 100 to 100 kb (inclusive), 110 to 100 kb (inclusive), 120 to 100 kb (inclusive), 130 to 100 kb (inclusive), 140 to 100 kb (inclusive), 150 to 100 kb (inclusive), 160 to 100 kb (inclusive), 170 to 100 kb (inclusive), 180 to 100 kb (inclusive), 190 to 100 kb (inclusive), 210 to 200 kb (inclusive), 220 to 200 kb (inclusive), 230 to 200 kb (inclusive), 240 to 200 kb (inclusive), 250 to 200 kb (inclusive), 260 to 200 kb (inclusive), 270 to 200 kb (inclusive), 280 to 280 kb (inclusive), 290 to 290 kb (inclusive), The targeting vectors may be up to 100 kb (inclusive), 55 to 100 kb (inclusive), 60 to 100 kb (inclusive), 65 to 100 kb (inclusive), 70 to 100 kb (inclusive), 75 to 100 kb (inclusive), 80 to 100 kb (inclusive), 85 to 100 kb (inclusive), 90 to 100 kb (inclusive), or 95 to 100 kb (inclusive), and have a length sufficient to promote a homologous recombination event with the corresponding target sequence. As described herein, larger targeting vectors can use longer targeting arms.

[0288] To facilitate modification of a target locus (e.g., modification of an immunoglobulin κ light chain locus, or modification of an already modified or altered immunoglobulin κ light chain locus), a nuclease agent (e.g., a CRISPR / Cas system) can be used in combination with a targeting vector. Such a nuclease agent can promote homologous recombination between the targeting vector and the target locus. When a nuclease agent is used in combination with a targeting vector, the targeting vector can include 5' and 3' homology arms corresponding to the 5' and 3' target sequence located sufficiently close to the nuclease cleavage site to promote the occurrence of a homologous recombination event between the target sequence and the homology arms upon a nick or double-strand break at the nuclease cleavage site. The term "nuclease cleavage site" includes a DNA sequence (e.g., a Cas9 cleavage site) at which a nick or double-strand break is created by a nuclease agent. A target sequence within a targeted locus corresponding to the 5' and 3' homology arms of a targeting vector is "located sufficiently close" to a nuclease cleavage site if it is at a distance such that, upon a nick or double-strand break at the recognition site, a homologous recombination event between the 5' and 3' target sequences and the homology arms is promoted. Thus, in certain embodiments, the target sequence corresponding to the 5' and / or 3' homology arms of a targeting vector is 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 immediately 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 vary, for example, the target sequence can be located 5' to the nuclease cleavage site, the target sequence can be located 3' to the recognition site, or the target sequence can flank the nuclease cleavage site.

[0290] The combined use of a targeting vector (including, for example, a large targeting vector) and a nuclease substance can 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 can be improved by 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, at least 10 times, or within a range formed by 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 contain homology arms corresponding to and derived from larger nucleic acid sequences than those typically used by other approaches intended to perform homologous recombination in cells. LTVECs can be, for example, at least 10 kb in length, or the sum of the 5' and 3' homology arms can be, for example, at least 10 kb. LTVECs also include targeting vectors that contain larger nucleic acid constructs than those typically used by other approaches intended to perform homologous recombination in cells. For example, LTVECs allow for the modification of large loci that cannot be accommodated by traditional plasmid-based targeting vectors due to their size limitations. For example, the targeted locus may be a cellular locus (i.e., the 5' homology arm and the 3' homology arm may correspond to the locus) that cannot be targeted using conventional methods, or that can be targeted only imprecisely or with significantly less efficiency, in the absence of a nick or double-strand break induced by a nuclease agent (e.g., a Cas protein).

[0292] In some embodiments, the methods described herein may use two or three LTVECs that can recombine with each other and with the target genomic locus in a three-way or four-way recombination event, allowing for the modification of large loci that cannot be achieved using a single LTVEC.

[0293] Examples of LTVEC include vectors derived from bacterial artificial chromosome (BAC), human artificial chromosome, or yeast artificial chromosome (YAC).LTVEC can be linear or circular.Examples of LTVEC and methods for producing it are described in, for example, Macdonald (2014), U.S. Patent Nos. 6,586,251, 6,596,541 and 7,105,348; and International Patent Application Publication No. WO2002 / 036789 (each of which is incorporated herein by reference in its entirety).

[0294] Methods for producing non-human animals are provided. Compositions and methods are provided for generating a non-human animal (e.g., a rodent, e.g., a rat or a mouse) whose germline genome comprises a modified immunoglobulin κ light chain locus that comprises one or more human immunoglobulin λ light chain sequences (e.g., human Vλ and Jλ gene segments) that comprise a human immunoglobulin λ light chain encoding sequence comprising particular polymorphic forms of human Vλ and Jλ segments (e.g., particular V and / or J alleles or variants), in place of a non-human immunoglobulin κ light chain sequence, including non-human or human immunoglobulin κ light chain sequences. The present invention also includes compositions and methods for generating non-human animals that express antibodies comprising immunoglobulin λ light chains containing human variable regions and non-human or human constant regions, constructed from an immunoglobulin κ light chain locus containing a single rearranged human immunoglobulin λ light chain variable region operably linked to a heterologous immunoglobulin λ light chain constant region gene, where the non-human or human immunoglobulin λ light chain constant region gene is placed in place of the non-human immunoglobulin κ light chain constant region gene normally present in the wild-type non-human immunoglobulin κ light chain locus. In some embodiments, compositions and methods for generating non-human animals that express such antibodies under the control of endogenous immunoglobulin κ enhancer(s) and / or endogenous immunoglobulin κ regulatory sequence(s) are also provided. In some embodiments, compositions and methods for generating non-human animals that express such antibodies under the control of heterologous immunoglobulin κ enhancer(s) and / or heterologous immunoglobulin κ regulatory sequence(s) are also provided.

[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., a rodent, e.g., a murine, e.g., a rat or mouse) or a human Cλ region gene), where the non-human or human immunoglobulin λ light chain constant region gene is placed in place of the non-human immunoglobulin κ light chain constant region gene normally occurring 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., a rodent (e.g., a murine, e.g., a rat or mouse) Cλ domain) or the presence of a light chain containing a human λ light chain variable domain and a human Cλ domain, and which is expressed both on the surface of B cells and in the serum of the non-human animal.

[0296] In some embodiments, the methods involve inserting genetic material containing a single rearranged human immunoglobulin λ light chain variable region into an immunoglobulin κ light chain locus (e.g., a wild-type, engineered, or modified immunoglobulin κ light chain locus of a non-human animal (e.g., a rodent, e.g., a rat or mouse)). In some embodiments, the methods involve inserting genetic material containing a single rearranged human immunoglobulin λ light chain variable region into an immunoglobulin κ light chain locus of an engineered or modified strain of a non-human animal (e.g., a rodent, e.g., a rat or mouse).

[0297] In some embodiments, the method comprises multiple insertions in a single ES cell clone, in some embodiments, the method comprises sequential insertions made in successive ES cell clones, in some embodiments, the method comprises a single insertion made in a modified ES cell clone.

[0298] In some embodiments, the method comprises inserting DNA(s) upstream of a non-human (e.g., rodent, e.g., rat or mouse) Cλ1 gene (or human Cλ2 gene), whereby said DNA insert(s) are operably linked to said non-human (e.g., rodent, e.g., rat or mouse) Cλ1 gene (or human Cλ2 gene), and the DNA insert(s) 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, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ and one or two human Vλ gene segments selected from the group consisting of Vλ3-3, Vλ4-3, Vλ5-3, Vλ6-3, Vλ7-3, Vλ8-3, Vλ9-3, Vλ10-3, Vλ11-3, Vλ12-3, Vλ13-3, Vλ14-3, Vλ15-3, Vλ16-3, Vλ17-3, Vλ18-3, Vλ19 ...In some embodiments, the method comprises inserting DNA(s) upstream of a non-human (e.g., rodent, e.g., rat or mouse) Cλ1 gene (or human Cλ2 gene), whereby said DNA insert(s) are operably linked to said non-human (e.g., rodent, e.g., rat or mouse) Cλ1 gene (or human Cλ2 gene), and the DNA insert(s) 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, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3- and a single rearranged human λ light chain variable region comprising a human Vλ gene segment selected from the group consisting of Vλ2-25, 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, 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, human immunoglobulin λ light chain sequences encoding human immunoglobulin λ light chain variable domains (i.e., sequences containing human Vλ and Jλ gene segments) can be separately modified to include codons optimized for expression in non-human animals (see, e.g., U.S. Patent Nos. 5,670,356 and 5,874,304, each of which is incorporated herein by reference). Codon-optimized sequences are modified sequences that preferably encode the same polypeptide (or a biologically active fragment of the full-length polypeptide having substantially the same activity as the full-length polypeptide) encoded by a non-codon-optimized parent polynucleotide. In some embodiments, human immunoglobulin λ light chain sequences encoding human immunoglobulin λ light chain variable domains can separately include sequences that have been altered to optimize codon usage for a particular cell type (e.g., rodent cells, e.g., rat or mouse cells). For example, the codons of each nucleotide sequence to be inserted into the genome of a non-human animal (e.g., a rodent, e.g., a rat or mouse) described herein can be optimized for expression in cells of the non-human animal. Such sequences may be described as codon-optimized sequences.

[0300] Insertion of a nucleotide sequence encoding a human immunoglobulin λ light chain variable domain results in expression of an antibody comprising a light chain having a human Vλ domain, with minimal modification of the germline genome of the non-human animal described herein, wherein the human immunoglobulin λ light chain variable domain is expressed from an endogenous modified immunoglobulin κ light chain locus. Methods for generating modified non-human animals (e.g., rodents, e.g., rats or mice), including knockouts and knockins, are known in the art (see, e.g., Gene Targeting: A Practical Approach, Joyner, ed., Oxford University Press, Inc., 2000, incorporated herein by reference in its entirety). For example, generation of genetically modified rodents can 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 a 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 of a randomly inserted modified light chain transgene in the germline genome of a rodent. In some embodiments, a nucleotide sequence encoding a 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 of an endogenous immunoglobulin κ light chain locus in the germline genome of a rodent; in some certain embodiments, the endogenous immunoglobulin κ light chain locus is altered, modified, or altered to contain human immunoglobulin λ gene segments (e.g., human V and J) operably linked to a mouse Cλ1 gene or operably linked to a 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 a 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 can be linearized and electroporated into rodent embryonic stem (ES) cells to generate rodents whose germline genome contains a modified immunoglobulin κ light chain locus. As described in the Examples section below, the rodent ES cells used in the targeting vector electroporation contained the modified immunoglobulin κ light chain locus previously described in U.S. Pat. No. 10,143,186 (incorporated herein by reference in its entirety). Positive rodent ES cell clones are confirmed using screening methods known in the art. Any remaining selection cassettes can be deleted via recombinase-mediated deletion if desired.

[0302] Alternatively, a human Cλ gene can be used in place of the mouse Cλ gene in the targeting vector. The targeting vector can be constructed in a similar manner to that described above (or in the examples below), except that a sequence encoding a human Cλ gene (e.g., Cλ2) is modified and introduced into the targeting vector. Such an approach enables the development of human antibody therapeutics, because the DNA encoding the light chain variable and constant regions is isolated together, thereby eliminating any subsequent cloning step of linking the human light chain constant region to prepare a fully human antibody.

[0303] The targeting vectors for constructing the modified immunoglobulin κ light chain loci described herein can be integrated into the germline genome of a non-human cell (e.g., a rodent (e.g., rat or mouse) embryonic stem cell). In some embodiments, the targeting vectors described herein contain a human V operably linked to one or more immunoglobulin heavy chain constant region genes. H , D H and J H Genomic DNA (e.g., multiple human V H , D H and J H In some embodiments, the targeting vectors described herein are integrated into a wild-type immunoglobulin κ light chain locus in the germline genome of a non-human (e.g., rodent, e.g., rat or mouse) cell that further contains a human V gene segment operably linked to one or more immunoglobulin heavy chain constant region genes (see, e.g., Macdonald (2014); U.S. Patent Nos. 6,596,541; 8,642,835; 8,697,940; and 8,791,323, each of which is incorporated herein by reference in its entirety). In some embodiments, the targeting vectors described herein contain a human V gene segment operably linked to one or more immunoglobulin heavy chain constant region genes. H , D H and J H Genomic DNA (e.g., multiple human V H , D H and J H and integrated into an altered or modified immunoglobulin κ light chain locus in the germline genome of a non-human cell further comprising a target gene segment (see, e.g., Macdonald (2014), U.S. Patent Nos. 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 by reference in its entirety).

[0304] The targeting vector is introduced into non-human (e.g., rodent, e.g., mouse or rat) embryonic stem cells by electroporation, whereby the sequences contained in the targeting vector confer the ability of the non-human (e.g., rodent, e.g., rat or mouse) cell or non-human animal (e.g., rodent, e.g., rat or mouse) to express an antibody having a light chain comprising 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, genetically modified non-human animals are generated in which a modified immunoglobulin κ light chain locus (e.g., an endogenous immunoglobulin κ light chain locus containing a human immunoglobulin λ light chain sequence (i.e., a rearranged human λ light chain variable region) operably linked to a rodent or human Cλ gene in place of the endogenous rodent Cκ gene) has been generated in the germline genome of the non-human animal. Antibodies featuring a light chain 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 said non-human animal. Where the endogenous immunoglobulin κ light chain locus in the germline genome of a non-human animal described herein is not targeted by a 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 the non-human animals described above provides modified non-human animal strains that produce antibodies comprising immunoglobulin λ light chains expressed from such modified immunoglobulin κ light chain loci having human Vλ domains and non-human (e.g., rodent, e.g., rat or mouse) or human Cλ domains. H , D H and J HWhen utilized in conjunction with the presence of a modified immunoglobulin heavy chain locus containing gene segments, modified non-human animal strains are generated that produce antibodies and antibody components for the development of human antibody-based therapeutics. Thus, a single modified non-human animal strain has been shown to have the potential to provide an alternative in vivo system for utilizing human Vλ domains for the development of new antibody-based drugs to treat human diseases.

[0306] In some embodiments, a method of making a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) comprises 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 (e.g., rodent, e.g., rat or mouse) embryonic stem (ES) cell. In some embodiments, a method of making a genetically modified non-human animal comprises introducing a non-human (e.g., rodent, e.g., 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 of making a genetically modified non-human animal (e.g., a rodent, e.g., rat or mouse) comprises generating the non-human animal using the non-human (e.g., rodent, e.g., rat or mouse) ES cell described above.

[0307] In some embodiments, a method of making a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) comprises 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. In some embodiments, a method of making a genetically modified non-human animal comprises 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 of making a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) comprises: (a) introduc...

Claims

[Claim 1] The invention described in the specification.

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