Non-human animals having engineered immunoglobulin lambda light chain locus

Abstract

To develop improved in vivo systems for generating human monoclonal antibodies that maximize human antibody repertoires in host transgenic animals.SOLUTION: In certain aspects, provided herein are improved in vivo systems for identifying and developing new antibody and antibody-based therapeutics that can be used for treatment of a variety of diseases that affect humans. In certain embodiments, non-human animals (e.g., rodents) provided herein, having engineered immunoglobulin loci, in particular, engineered immunoglobulin (Ig)λ light chain loci and / or otherwise expressing, producing or containing antibody repertoires characterized by light chains having human Vλ regions, are useful, for example, for exploiting the diversity of human λ sequences in the identification and development of new antibody-based therapeutics.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 417,845, filed November 4, 2016, and U.S. Provisional Patent Application No. 62 / 567,932, filed October 4, 2017, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Human antibodies are the fastest growing therapeutic class. Among the technologies currently used for their production, the development of transgenic animals (e.g., rodents) engineered with genetic material encoding all or part of a human antibody has revolutionized the field of human therapeutic monoclonal antibodies for the treatment of various diseases. Furthermore, there is a need for the development of improved in vivo systems for the production of human monoclonal antibodies that maximize the human antibody repertoire in host transgenic animals. Summary of the Invention [Means for solving the problem]

[0003] In certain aspects, provided herein are improved in vivo systems for the identification and development of new antibodies and antibody-based therapeutics that can be used to treat a variety of diseases affecting humans. As disclosed herein, in certain embodiments, non-human animals (e.g., rodents) provided herein that express, produce, or contain antibody repertoires with engineered immunoglobulin loci, particularly engineered immunoglobulin (Ig) λ light chain loci, and / or that otherwise feature light chains with human Vλ regions, are useful for exploiting the diversity of human λ sequences, e.g., in the identification and development of novel antibody-based therapeutics. In some embodiments, the non-human animals described herein provide improved in vivo systems for the development of antibodies and / or antibody-based therapeutics intended 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 Vλ domains, characterized by improved performance compared to antibodies and / or antibody-based therapeutics obtained from existing in vivo systems containing human Vλ region sequences.

[0004] In certain aspects, provided herein are non-human animals having an Igλ light chain locus containing engineered immunoglobulin variable and constant regions, and in some embodiments, further comprising an engineered regulatory region (or sequence). As described herein, in certain embodiments, the non-human animals provided contain in their germline genome an Igλ light chain locus comprising an engineered Igλ light chain variable region characterized by the presence of one or more human Vλ gene segments, one or more human Jλ gene segments, one or more human Cλ region genes, and a rodent Cλ region gene, wherein the human Vλ, Jλ, and Cλ gene segments are operably linked to each other and to the rodent Cλ region gene.

[0005] In some embodiments, the non-human animal provided comprises an Igλ light chain locus that comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 human Vλ gene segments.

[0006] In some embodiments, the provided non-human animal comprises an Igλ light chain locus that comprises between 5 and 25, between 5 and 24, between 5 and 23, between 5 and 22, between 5 and 21, between 5 and 20, between 5 and 19, between 5 and 18, between 5 and 17, between 5 and 16, between 5 and 15, between 5 and 14, between 5 and 13, between 5 and 12, between 5 and 11, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, or between 5 and 6 human Vλ gene segments. In some embodiments, the non-human animal provided is 10-70, 10-69, 10-68, 10-67, 10-66, 10-65, 10-64, 10-63, 10-62, 10-61, 10-60, 10-59, 10-58, 10-57, 10-56, 10-55, 10-54, 10-53, 10-52, 10-51, 10-50, 10-49, 10-48, 10-47, 10-46, 10-45, 10-44, 10-43, 10-42, 10-41, 10-40, 10- and an Igλ light chain locus comprising 39, 10-38, 10-37, 10-36, 10-35, 10-34, 10-33, 10-32, 10-31, 10-32, 10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, or 10-11 human Vλ gene segments.

[0007] In some embodiments, the non-human animals provided comprise an Igλ light chain locus that comprises between 6 and 25, between 7 and 25, between 8 and 25, between 9 and 25, between 10 and 25, between 11 and 25, between 12 and 25, between 13 and 25, between 14 and 25, between 15 and 25, between 16 and 25, between 17 and 25, between 18 and 25, between 19 and 25, between 20 and 25, between 21 and 25, between 22 and 25, between 23 and 25, or between 24 and 25 human Vλ gene segments. In some embodiments, the non-human animal provided is a mammalian animal selected from the group consisting of: 11-70, 12-70, 13-70, 14-70, 15-70, 16-70, 17-70, 18-70, 19-70, 20-70, 21-70, 22-70, 23-70, 24-70, 25-70, 26-70, 27-70, 28-70, 29-70, 30-70, 31-70, 32-70, 33-70, 34-70, 35-70, 36-70, 37-70, 38-70, 39-70, 40-70, and an Igλ light chain locus comprising 41-70, 42-70, 43-70, 44-70, 45-70, 46-70, 47-70, 48-70, 49-70, 50-70, 51-70, 52-70, 53-70, 54-70, 55-70, 56-70, 57-70, 58-70, 59-70, 60-70, 61-70, 62-70, 63-70, 64-70, 65-70, 66-70, 67-70, 68-70, or 69-70 human Vλ gene segments.

[0008] In some embodiments, the non-human animals provided comprise an Igλ light chain locus that comprises between 6 and 24, 7 and 23, 8 and 22, 9 and 21, 10 and 20, 11 and 19, 12 and 18, 13 and 17, 14 and 16, or 15 and 16 human Vλ gene segments. In some embodiments, the provided non-human animals comprise an Igλ light chain locus that comprises 11-69, 12-68, 13-67, 14-66, 15-65, 16-64, 17-63, 18-62, 19-61, 20-60, 21-59, 22-58, 23-57, 24-56, 25-55, 26-54, 27-53, 28-52, 29-51, 30-50, 31-49, 32-48, 33-47, 34-48, 35-47, 36-46, 37-45, 38-44, 39-43, 40-42, or 41-42 human Vλ gene segments.

[0009] In some embodiments, the non-human animals provided comprise an Igλ light chain locus comprising 5, 16, or 25 functional human Vλ gene segments. In some embodiments, the non-human animals provided comprise an Igλ light chain locus comprising 10, 27, or 40 human Vλ gene segments. In some embodiments, the human Vλ gene segments comprise contiguous human Vλ gene segments such that the human Vλ gene segments are present in a human Igλ light chain locus in a human cell.

[0010] In some embodiments, the non-human animals provided comprise an Igλ light chain locus, wherein the locus comprises at least five human Jλ gene segments (for example, but not limited to, five human Jλ gene segments, six human Jλ gene segments, seven human Jλ gene segments, eight human Jλ gene segments, etc.). In some embodiments, the non-human animals provided comprise an Igλ light chain locus, wherein the locus comprises at least four human Cλ region genes (for example, but not limited to, four human Cλ region genes, five human Cλ region genes, six human Cλ region genes, seven human Cλ region genes, eight human Cλ region genes, etc.). In certain embodiments, the non-human animals provided comprise an Igλ light chain locus, wherein the locus comprises at least 25 human Vλ gene segments, at least five Jλ gene segments, and at least four human Cλ region genes in endogenous Igλ light chain alleles. In some embodiments, the provided non-human animals comprise a single murine (e.g., mouse or rat) Cλ region gene (e.g., a mouse Cλ1 region gene or a mouse Cλ1 gene segment) at an endogenous non-human Igλ light chain locus, which in some embodiments further comprises a human Eλ region (or sequence) characterized by three sequence elements.

[0011] In some embodiments, the non-human animals provided contain human Vλ, Jλ, and Cλ gene segments at the endogenous non-human Igλ light chain locus in a natural, or germline, configuration. In some embodiments, the non-human animals provided contain human Vλ, Jλ, and Cλ gene segments at the endogenous non-human Igλ light chain locus in a configuration that does not naturally occur at the human immunoglobulin λ light chain locus in the germline genome of the human cell.

[0012] In some embodiments, the provided non-human animals contain a DNA sequence at an endogenous non-human Igλ light chain locus that includes a plurality of human Vλ, Jλ, and Cλ coding sequences interspersed (or juxtaposed, associated, etc.) with non-coding human immunoglobulin λ light chain sequences. In some embodiments, the provided non-human animals contain a DNA sequence at an endogenous non-human Igλ light chain locus that includes a plurality of human Vλ, Jλ, and Cλ coding sequences interspersed with non-coding non-human (e.g., murine) immunoglobulin λ light chain sequences.

[0013] In some embodiments, the provided non-human animals are characterized by antibody expression from an endogenous non-human Igλ light chain locus in the germline genome of the non-human animal, wherein the antibody contains a human Vλ domain and a human or non-human Cλ domain. In some embodiments, the provided non-human animals are characterized by increased usage of human Vλ regions from an engineered immunoglobulin λ light chain locus (e.g., a 60:40 κ:λ ratio) compared to one or more reference engineered or wild-type non-human animals (e.g., but not limited to, a 95:5 κ:λ ratio).

[0014] In some embodiments, a non-human animal, cell, or tissue is provided whose genome comprises an endogenous immunoglobulin λ light chain locus comprising an insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and one or more human Cλ gene segments, wherein the human Vλ, Jλ, and Cλ gene segments are operably linked to a non-human Cλ gene segment, and the endogenous immunoglobulin λ light chain locus further comprises one or more non-human immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ).

[0015] In some embodiments, a non-human animal, non-human cell, or non-human tissue is provided whose germline genome comprises an endogenous immunoglobulin λ light chain locus comprising: (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments, wherein (a) and (b) are operably linked to (c) a non-human Cλ gene segment, and the endogenous immunoglobulin λ light chain locus further comprises one or more non-human immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ).

[0016] In some embodiments, the endogenous immunoglobulin λ light chain locus provided herein further comprises three human Eλ. In some embodiments, the endogenous immunoglobulin λ light chain locus further comprises one human Eλ characterized by the presence of three sequence elements. In some embodiments, the endogenous immunoglobulin λ light chain locus further comprises one human Eλ characterized by the presence of three sequence elements that act (or function) in a modular manner.

[0017] In some embodiments, an endogenous immunoglobulin λ light chain locus provided herein comprises two non-human Eλ. In some embodiments, an endogenous immunoglobulin λ light chain locus comprises two rodent Eλ. In some embodiments, an endogenous immunoglobulin λ light chain locus provided herein comprises two mouse Eλ. In some embodiments, an endogenous immunoglobulin λ light chain locus provided herein comprises a mouse Eλ and a mouse Eλ3-1. In some embodiments, an endogenous immunoglobulin λ light chain locus provided herein does not contain (or lacks) mouse Eλ2-4. In some embodiments, an endogenous immunoglobulin λ light chain locus comprises two rat Eλ.

[0018] In some embodiments, the endogenous immunoglobulin λ light chain locus provided herein comprises a deletion of all or part of the endogenous Vλ and Jλ gene segments. In some embodiments, the endogenous immunoglobulin λ light chain locus provided herein comprises a deletion of the Vλ2-Vλ3-Jλ2-Cλ2 gene segments and the Vλ1-Jλ3-Cλ3-Jλ1 gene segments. In some embodiments, the endogenous immunoglobulin λ light chain locus comprises a deletion of the Vλ2-Vλ3-Jλ2-Cλ2-Jλ4P-Cλ4P gene segments and the Vλ1-Jλ3-Jλ3P-Cλ3-Jλ1 gene segments. In some embodiments, the endogenous immunoglobulin λ light chain locus provided herein comprises a deletion of non-human Eλ2-4. In some embodiments, an endogenous immunoglobulin λ light chain locus provided herein comprises a deletion of Vλ2, Vλ3, Jλ2, Cλ2, Jλ4P, Cλ4P, Eλ2-4, Vλ1, Jλ3, Jλ3P, Cλ3, and Jλ1. In some embodiments, an endogenous immunoglobulin λ light chain locus provided herein comprises Cλ1, Eλ, and Eλ3-1 as the only non-human gene segments or sequence elements present.

[0019] In some embodiments, an endogenous immunoglobulin λ light chain locus provided herein comprises human Vλ gene segments Vλ4-69 to Vλ3-1, at least human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, human Jλ gene segment Jλ7, and an insertion of a rodent Cλ1 gene segment. In some embodiments, an endogenous immunoglobulin λ light chain locus provided herein comprises human Vλ gene segments Vλ5-52 to Vλ3-1, at least human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, human Jλ gene segment Jλ7, and an insertion of a rodent Cλ1 gene segment. In some embodiments, an endogenous immunoglobulin λ light chain locus provided herein comprises an insertion of human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1, at least human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, human Jλ gene segment Jλ7, and a rodent Cλ1 gene segment. In some embodiments, the insertion includes naturally occurring human non-coding DNA between human Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1, naturally occurring human non-coding DNA between human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, and naturally occurring human non-coding DNA upstream (or 5') of human Jλ gene segment Jλ7.

[0020] In some embodiments, the non-human Cλ gene segment is or comprises a rodent Cλ gene segment. In some embodiments, the rodent Cλ gene segment is or comprises a murine (e.g., mouse or rat) Cλ gene segment. In some embodiments, the rodent Cλ gene segment is or comprises a rat Cλ gene segment. In some embodiments, the rodent Cλ gene segment is or comprises a mouse Cλ gene segment. In some embodiments, the rodent Cλ gene segment is a mouse Cλ1 gene segment.

[0021] In some embodiments, the mouse Cλ gene (or gene segment) comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical, or 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 Cλ1 gene is or comprises SEQ ID NO: 1. In some embodiments, the mouse Cλ2 gene is or comprises SEQ ID NO: 3. In some embodiments, the mouse Cλ3 gene is or comprises SEQ ID NO: 5. In some embodiments, the mouse Cλ gene comprises a sequence that is identical to a mouse Cλ1 gene.

[0022] In some embodiments, the mouse Cλ gene (or gene segment) comprises a sequence that is 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%, or 98% to 100% identical to a mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3.

[0023] In some embodiments, the mouse Cλ gene (or gene segment) comprises a sequence that is between 50% and 98%, 50% and 95%, 50% and 90%, 50% and 85%, 50% and 80%, 50% and 75%, 50% and 70%, 50% and 65%, 50% and 60%, or 50% and 55% identical to a mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3.

[0024] In some embodiments, the mouse Cλ gene (or gene segment) comprises a sequence that is 55% to 98%, 60% to 95%, 65% to 90%, 70% to 85%, or 75% to 80% identical to a mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3.

[0025] In some embodiments, the rat Cλ gene (or gene segment) comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical, or 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 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 embodiments, the rat Cλ1 gene is or comprises SEQ ID NO:7. In some embodiments, the rat Cλ2 gene is or comprises SEQ ID NO:9. In some embodiments, the rat Cλ3 gene is or comprises SEQ ID NO:11. In some embodiments, the rat Cλ4 gene is or comprises SEQ ID NO:13.

[0026] In some embodiments, the rat Cλ gene (or gene segment) comprises a sequence that is 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%, 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.

[0027] In some embodiments, the rat Cλ gene (or gene segment) comprises a sequence that is 50% to 98%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, or 50% to 55% identical to a rat Cλ gene selected from the group consisting of rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes.

[0028] In some embodiments, the rat Cλ gene (or gene segment) comprises a sequence that is 55% to 98%, 60% to 95%, 65% to 90%, 70% to 85%, or 75% to 80% identical to a rat Cλ gene selected from the group consisting of rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes.

[0029] In some embodiments of the non-human animal, non-human cell, or non-human tissue provided, the germline genome or genome of the non-human animal, non-human cell, or non-human tissue comprises (i) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H The gene segment is operably linked to a non-human immunoglobulin heavy chain constant region, or (ii) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H The gene segments further include a locus operably linked to a non-human immunoglobulin heavy chain constant region, and an endogenous immunoglobulin κ light chain locus comprising an insertion of one or more human Vκ gene segments and one or more human Jκ gene segments, wherein the human Vκ and Jκ gene segments are operably linked to a non-human immunoglobulin Cκ region.

[0030] 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 gene segment is a non-human V H , D HIn one embodiment, the insertion replaces a gene segment containing a human V H , D H , and J. H In some embodiments, the non-human immunoglobulin heavy chain constant region is an endogenous non-human immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain locus is a V H 3-74 to V H Human V of 6-1 H Gene segment, D H 1-1 to D H 7-27 Human D H gene segments, and human J H Gene segment J H 1-J H In one embodiment, the insertion comprises an insertion of human V H 3-74~V H Naturally occurring (occurring) human non-coding DNA between 6-1, human D H 1-1~D H Naturally occurring (occurring) human non-coding DNA between 7-27, and human J H 1-J H 6. Naturally occurring (occurring) human non-coding DNA is included between the V and V loci. In some embodiments, the immunoglobulin heavy chain locus is comprised of all functional human V loci. H Gene segments, all functional human D H gene segments, and fully functional human J H It involves the insertion of a gene segment.

[0031] In some embodiments, the immunoglobulin heavy chain locus lacks an endogenous non-human Adam6 gene. In some embodiments, the immunoglobulin heavy chain locus further comprises an insertion of one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides. In some embodiments, the one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides are selected from the group consisting of a first and a second human V HIn 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 H In some embodiments, the one or more nucleotide sequences encoding the one or more rodent Adam6 polypeptides are human V H Gene segments and human D H Inserted between gene segments In some embodiments, one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides are inserted in place of a human Adam6 pseudogene.

[0032] In some embodiments, the inserted one or more human Vκ gene segments and one or more human Jκ gene segments replace non-human Vκ and Jκ gene segments. In some embodiments, the insertion includes naturally occurring human non-coding DNA between human Vκ and Jκ gene segments and combinations thereof. In some embodiments, the non-human immunoglobulin Cκ region is an endogenous non-human Cκ region. In some embodiments, the immunoglobulin κ light chain locus includes an insertion of all or a portion of the proximal Vκ duplication of the human immunoglobulin κ light chain locus. In some embodiments, the immunoglobulin κ light chain locus includes an insertion of human Vκ gene segments Vκ2-40 to Vκ4-1 and human Jκ gene segments Jκ1-Jκ5. In some embodiments, the insertion includes naturally occurring human non-coding DNA between human Vκ2-40 to Vκ4-1 and naturally occurring human non-coding DNA between human Jκ1-Jκ5.

[0033] In some embodiments of the non-human animals, non-human cells, or non-human tissues provided herein, the non-human animals, non-human cells, or non-human tissues are heterozygous or homozygous for an immunoglobulin heavy chain locus described herein (e.g., an endogenous immunoglobulin heavy chain locus described herein).

[0034] In some embodiments of the non-human animals, non-human cells, or non-human tissues provided herein, the non-human animals, non-human cells, or non-human tissues are heterozygous or homozygous for an immunoglobulin κ light chain locus described herein (e.g., an endogenous immunoglobulin κ light chain locus described herein).

[0035] In some embodiments of the non-human animals, non-human cells, or non-human tissues provided herein, the non-human animals, non-human cells, or non-human tissues are heterozygous or homozygous for an immunoglobulin λ light chain locus described herein (e.g., an endogenous immunoglobulin λ light chain locus described herein).

[0036] In some embodiments of the non-human animals, non-human cells, or non-human tissues provided herein, the germline genome of the non-human animal, non-human cell, or non-human tissue further comprises an insertion of one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides, and the animal is heterozygous or homozygous for the insertion.

[0037] In some embodiments, the non-human cell is a non-human lymphocyte, hi some embodiments, the non-human cell is selected from a B cell, a dendritic cell, a macrophage, a monocyte, and a T cell.

[0038] In some embodiments, the non-human cells are non-human embryonic stem (ES) cells. In some embodiments, the non-human ES cells are rodent ES cells. In some embodiments, the rodent ES cells are mouse ES cells (e.g., from the 129 strain, C57BL strain, BALB / c strain, or a mixture thereof). In some embodiments, the rodent embryonic stem cells are mouse embryonic stem cells and are a mixture of the 129 strain and the C57BL strain. In some embodiments, the rodent embryonic stem cells are mouse embryonic stem cells and are a mixture of the 129 strain, the C57BL strain, and the BALB / c strain.

[0039] In some embodiments, the non-human ES cells described herein are used to generate non-human animals. In some embodiments, the non-human ES cells are mouse ES cells and are used to generate mice comprising the engineered immunoglobulin λ light chain locus described herein. In some embodiments, the non-human ES cells are rat ES cells and are used to generate rats comprising the engineered immunoglobulin λ light chain locus described herein.

[0040] In some embodiments, the non-human tissue is selected from fat, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, egg, and combinations thereof.

[0041] In some embodiments, immortalized cells are provided that are made, generated, produced, or obtained from the isolated non-human cells or tissues described herein.

[0042] In some embodiments, a non-human embryo is provided that is generated, produced, produced, or obtained from a non-human ES cell described herein. In some embodiments, the non-human embryo is a rodent embryo, in some embodiments, a mouse embryo, and in some embodiments, a rat embryo.

[0043] In some embodiments, a kit is provided that includes a non-human animal, non-human cell, non-human tissue, immortalized cell, non-human ES cell, or non-human embryo described herein.

[0044] In some embodiments, kits described herein are provided for use in the manufacture and / or development of therapeutic or diagnostic agents (eg, antibodies or fragments thereof).

[0045] In some embodiments, kits described herein are provided for use in the manufacture and / or development of agents (e.g., antibodies or fragments thereof) for the treatment, prevention, or amelioration of a disease, disorder, or condition.

[0046] In some embodiments, there is provided a method for producing a non-human animal whose germline genome comprises an engineered endogenous immunoglobulin λ light chain locus, the method comprising: (a) introducing a DNA fragment into a non-human embryonic stem cell, wherein the DNA fragment comprises a nucleotide sequence comprising: (i) one or more human Vλ gene segments, (ii) one or more human Jλ gene segments, and (iii) one or more human Cλ gene segments, wherein (i)-(iii) are operably linked to a non-human Cλ gene segment, and wherein the nucleotide sequence further comprises one or more human immunoglobulin λ light chain enhancers (Eλ); (b) obtaining the non-human embryonic stem cells produced in (a); and (c) generating a non-human animal using the non-human embryonic stem cells of (b).

[0047] In some embodiments, methods are provided for producing a non-human animal whose germline genome comprises an engineered endogenous immunoglobulin λ light chain locus, wherein the engineered endogenous immunoglobulin λ light chain locus comprises an insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and one or more human Cλ gene segments, wherein the human Vλ and Jλ gene segments are operably linked to non-human and / or human Cλ gene segments, and the endogenous immunoglobulin λ light chain locus further comprises one or more non-human immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ), the methods comprising: and modifying the germline genome of the non-human animal so that the genome contains an engineered immunoglobulin λ light chain locus, wherein the engineered locus comprises insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and one or more human Cλ gene segments, wherein the human Vλ and Jλ gene segments are operably linked to non-human and / or human Cλ gene segments, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ), thereby creating the non-human animal.

[0048] In some embodiments of the methods for producing non-human animals described herein, the one or more human Vλ gene segments comprise Vλ4-69 to Vλ3-1, Vλ5-52 to Vλ3-1, or Vλ3-27 to Vλ3-1. In some embodiments of the methods for producing non-human animals, the one or more human Vλ gene segments comprise Vλ5-52 to Vλ1-40 and / or Vλ3-27 to Vλ3-1. In some embodiments of the methods for producing non-human animals, the one or more human Vλ gene segments comprise naturally occurring human non-coding DNA between Vλ5-52 to Vλ1-40 and / or Vλ3-27 to Vλ3-1. In some embodiments of the methods for producing non-human animals, the one or more human Jλ gene segments and the one or more human Cλ gene segments comprise human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6, and a human Jλ7 gene segment. In some embodiments of the method for producing a non-human animal, the human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6 comprise naturally occurring human non-coding DNA between the human Jλ and Cλ gene segment pairs, and the human Jλ7 gene segment comprises naturally occurring human non-coding DNA upstream (or 5') of the human Jλ7.

[0049] In some embodiments of the methods for producing non-human animals provided herein, the insertion of human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1 comprises naturally occurring human non-coding DNA between the human Vλ gene segments, the insertion of human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6 comprises naturally occurring human non-coding DNA between the human Jλ-Cλ gene segment pairs, and the insertion of human Jλ7 gene segment comprises naturally occurring human non-coding DNA upstream (or 5') of human Jλ7.

[0050] In some embodiments of the methods of producing a non-human animal provided herein, the non-human Cλ gene segment is a rodent Cλ gene segment, and in some embodiments, a mouse Cλ1 gene segment.

[0051] In some embodiments of the methods for producing non-human animals provided herein, the DNA fragment further comprises one or more selectable markers. In some embodiments of the methods for producing non-human animals provided herein, the DNA fragment further comprises one or more site-specific recombination sites. In some embodiments of the methods for producing non-human animals provided herein, the DNA fragment further comprises one or more sets of site-specific recombination sites that recombine with the same recombinase. In some embodiments of the methods for producing non-human animals provided herein, the DNA fragment further comprises one or more sets of site-specific recombination sites that recombine with different recombinases.

[0052] In some embodiments of the methods of producing a non-human animal provided herein, the DNA fragments are selected from those whose germline genomes contain (i) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H The gene segment is operably linked to a non-human immunoglobulin heavy chain constant region, or (ii) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. HThe gene segments are introduced into a non-human embryonic stem cell comprising an endogenous immunoglobulin κ light chain locus operably linked to a non-human immunoglobulin heavy chain constant region, and an endogenous immunoglobulin κ light chain locus comprising an insertion of one or more human Vκ gene segments and one or more human Jκ gene segments, wherein the human Vκ and Jκ gene segments are operably linked to a non-human immunoglobulin Cκ region.

[0053] In some embodiments of the methods for producing a non-human animal provided herein, the DNA fragment is introduced into a non-human embryonic stem cell whose germline genome comprises (i) a wild-type endogenous immunoglobulin heavy chain locus, or (ii) a wild-type endogenous immunoglobulin heavy chain locus and a wild-type endogenous immunoglobulin κ light chain locus, and in this case the method further comprises mating the mouse produced, generated, produced, or obtained from the non-human embryonic stem cell with a second mouse.

[0054] In some embodiments of the methods of producing a non-human animal provided herein, modifying the germline genome of a non-human animal to include an engineered immunoglobulin λ light chain locus in the genome comprises determining whether the germline genome contains (i) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H The gene segment is operably linked to a non-human immunoglobulin heavy chain constant region, or (ii) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. HThe gene segments are carried in a non-human embryonic stem cell further comprising a locus operably linked to a non-human immunoglobulin heavy chain constant region, and an endogenous immunoglobulin κ light chain locus comprising an insertion of one or more human Vκ gene segments and one or more human Jκ gene segments, wherein the human Vκ and Jκ gene segments are operably linked to a non-human immunoglobulin Cκ region.

[0055] In some embodiments of the methods of producing a non-human animal provided herein, 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 gene segments results in one or more human V H Naturally occurring human non-coding DNA between gene segments, including one or more human D H naturally occurring human non-coding DNA between gene segments, and one or more human J H In some embodiments of the methods of producing a non-human animal provided herein, the insertion of one or more human Vκ gene segments and one or more human Jκ gene segments comprises naturally occurring human non-coding DNA between the one or more human Vκ gene segments and naturally occurring human non-coding DNA between the one or more human Jκ gene segments.

[0056] In some embodiments of the methods of producing a non-human animal provided herein, modifying the germline genome of the non-human animal to include an engineered immunoglobulin λ light chain locus in the germline genome is performed in a non-human embryonic stem cell whose germline genome includes (i) a wild-type endogenous immunoglobulin heavy chain locus, or (ii) a wild-type endogenous immunoglobulin heavy chain locus and a wild-type endogenous immunoglobulin κ light chain locus, and in which case the method further includes mating the mouse produced, generated, produced, or obtained from the non-human embryonic stem cell with a second mouse.

[0057] In some embodiments, the mice described herein have a germline genome that includes wild-type IgH and Igκ loci, homozygous or heterozygous humanized IgH and Igκ loci, where the homozygous or heterozygous humanized IgH locus contains an inserted rodent Adam6 coding sequence, or a homozygous or heterozygous humanized IgH locus (with or without an inserted Adam6 coding sequence) and a homozygous or heterozygous inactivated Igκ locus.

[0058] In some embodiments, a non-human animal is provided that is made, generated, produced, obtained, or obtainable from the methods described herein.

[0059] In some embodiments, methods are provided for producing antibodies in non-human animals, the methods comprising: (a) immunizing a non-human animal described herein with an antigen of interest, (b) maintaining the non-human animal under conditions sufficient for the rodent to mount an immune response to the antigen of interest, and (c) recovering from the non-human animal or non-human cell an antibody that binds to the antigen of interest. In some embodiments, the antibody comprises a human lambda light chain variable domain.

[0060] In some embodiments, methods are provided for producing nucleic acid encoding a human lambda light chain variable domain in a non-human animal, the methods comprising: (a) immunizing a non-human animal described herein with an antigen of interest, (b) maintaining the non-human animal under conditions sufficient for the rodent to mount an immune response to the antigen of interest, and (c) recovering nucleic acid encoding the human lambda light chain variable domain from the non-human animal or non-human cell. In some embodiments, the methods further comprise recovering nucleic acid encoding the human heavy chain variable domain from the non-human animal or non-human cell.

[0061] In some embodiments of the methods for producing antibodies or nucleic acids in non-human animals, the non-human cells are B cells. In some embodiments of the methods for producing antibodies or nucleic acids in non-human animals, the non-human cells are hybridomas.

[0062] In some embodiments of the methods for producing antibodies in non-human animals, the antibody that binds to the antigen of interest recovered from the rodent or rodent cell comprises a human heavy chain variable domain and a human lambda light chain variable domain.

[0063] In some embodiments of the method for producing antibodies or nucleic acids in a non-human animal, the human heavy chain variable domain is a rearranged human V H a gene segment, the segment being 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 and V H 6-1.

[0064] In some embodiments of the method of producing an antibody or nucleic acid in a non-human animal, the human lambda light chain variable domain comprises a rearranged human Vλ gene segment, the segment being 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, Vλ6-57, Vλ7-58, Vλ8-59, Vλ9-60, Vλ10-61, Vλ11-62, Vλ12-63, Vλ13-64, Vλ14-65, Vλ15-66, Vλ16-67, Vλ17-68, Vλ18-69, Vλ19-70, Vλ20-71, Vλ21-72, Vλ22-73, Vλ23-74, Vλ24-75, Vλ25-76, Vλ26-77, Vλ27-78, Vλ28-79, Vλ29-80, Vλ30-81, Vλ31-82, Vλ32-83, Vλ33-84, Vλ34-85, Vλ35-86, Vλ36-87, Vλ37-88, Vλ38-89, Vλ39-90, Vλ40-91, Vλ41-92, Vλ42-93, Vλ43-94, Vλ45-95, Vλ46-96, Vλ47-97, Vλ48-99, Vλ49-100, Vλ49-111, Vλ50-112, Vλ51-123, Vλ52-131, Vλ53-13 and Vλ4-3 and Vλ3-1.

[0065] In some embodiments, a method of inducing an antigen-specific immune response in a non-human animal is provided, the method comprising: (a) immunizing a non-human animal described herein with a target antigen; and (b) maintaining the non-human animal under conditions sufficient for the rodent to generate an immune response to the target antigen.

[0066] In some embodiments, a non-human animal is provided whose germline genome comprises a homozygous endogenous immunoglobulin λ light chain locus, the light chain locus being selected from the group consisting of: (i) human Vλ gene segments Vλ4-69 to Vλ3-1, Vλ5-52 to Vλ3-1, Vλ3-27 to Vλ3-1, or Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1; (ii) human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3 and (iii) a human Jλ gene segment, Jλ7, and (iv) an insertion of three human immunoglobulin λ light chain enhancers (or a human immunoglobulin λ light chain enhancer having three sequence elements), wherein (i) to (iv) are operably linked to each other, the insertion is upstream of the non-human Cλ gene segment, and the endogenous immunoglobulin λ light chain locus lacks endogenous non-human immunoglobulin Eλ2-4.

[0067] In some embodiments, a non-human animal is provided whose germline genome comprises a homozygous endogenous immunoglobulin λ light chain locus comprising: (i) human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1; (ii) human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6; (iii) human Jλ gene segment Jλ7; and (iv) three human immunoglobulin λ light chain enhancers (or a human immunoglobulin λ light chain enhancer having three sequence elements), wherein (i) to (iv) are operably linked to each other, and (i) to (iii) are non-human. The endogenous immunoglobulin λ light chain locus is upstream (or 5') of the Cλ gene segment, and the endogenous immunoglobulin λ light chain locus lacks endogenous non-human immunoglobulin Eλ2-4; the human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1 contain naturally occurring human non-coding DNA between the human Vλ gene segments; the human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6 contain naturally occurring human non-coding DNA between the human Jλ-Cλ gene segment pair; and the human Jλ gene segment Jλ7 contains naturally occurring human non-coding DNA upstream (or 5') of human Jλ7.

[0068] In some embodiments of the provided non-human animals, the non-human Cλ gene (or gene segment) is a mouse Cλ1 gene (or gene segment). In some embodiments of the provided non-human animals, the endogenous immunoglobulin λ light chain locus further comprises endogenous non-human immunoglobulin λ light chain enhancers Eλ and Eλ3-1. In some embodiments of the provided non-human animals, the endogenous immunoglobulin λ light chain locus comprises a deletion of the endogenous non-human Vλ2-Vλ3-Jλ2-Cλ2-Jλ4P-Cλ4P gene segment and the Vλ1-Jλ3-Jλ3P-Cλ3-Jλ1 gene segment.

[0069] In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein are provided for use in the manufacture and / or development of therapeutic or diagnostic agents (e.g., antibodies or fragments thereof).

[0070] In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein are provided for use in the manufacture of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition.

[0071] In some embodiments, there is provided the use of a non-human animal, non-human cell, or non-human tissue described herein in the manufacture and / or development of a drug or vaccine for use in medicine, e.g., for use as a pharmaceutical.

[0072] In some embodiments, there is provided the use of a non-human animal or a non-human cell described herein in the production and / or development of an antibody or fragment thereof.

[0073] In various embodiments, the non-human animals, cells, or tissues provided herein are rodents, rodent cells, or tissues, and in some embodiments, mice, mouse cells, or tissues, and in some embodiments, rats, rat cells, or tissues. In some embodiments, the mice, mouse cells, or tissues described herein comprise a genetic background comprising a 129 strain, a BALB / c strain, a C57BL / 6 strain, a mixed 129xC57BL / 6 strain, or a combination thereof.

[0074] As used herein, the terms "about" and "approximately" are used interchangeably. Any numbers used herein, whether about or approximately, are intended to cover any normal variations recognized by one of ordinary skill in the relevant art. The present invention provides, for example, the following items. (Item 1) The germline genome (a) one or more human Vλ gene segments; (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments; 1. A rodent comprising an endogenous immunoglobulin lambda light chain locus comprising: (a) and (b) are operably linked to (c) and a rodent Cλ gene segment; and A rodent, wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ). (Item 2) 2. The rodent of item 1, wherein the endogenous immunoglobulin λ light chain locus comprises two rodent Eλ. (Item 3) 3. The rodent of item 2, wherein the two rodent Eλ are mouse Eλ and mouse Eλ3-1. (Item 4) 4. The rodent of any one of items 1 to 3, wherein the endogenous immunoglobulin λ light chain locus comprises three human Eλ. (Item 5) the germline genome comprises: (i) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus comprising an insertion of a human V gene segment, H , D H , and J. H an endogenous immunoglobulin heavy chain locus in which the gene segment is operably linked to a rodent immunoglobulin heavy chain constant region; or (ii) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus comprising an insertion of a human V gene segment, H , D H , and J. H5. The rodent of any one of paragraphs 1 to 4, further comprising an endogenous immunoglobulin heavy chain locus wherein the gene segments are operably linked to a rodent immunoglobulin heavy chain constant region, and an endogenous immunoglobulin κ light chain locus comprising an insertion of one or more human Vκ gene segments and one or more human Jκ gene segments, wherein the human Vκ and Jκ gene segments are operably linked to a rodent immunoglobulin Cκ region. (Item 6) 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 is H , D H Item 6. The rodent of item 5, wherein the gene segment is replaced. (Item 7) The insertion is in the human V H , D H , and J. H 7. The rodent of item 6, comprising gene segments, as well as naturally occurring human non-coding DNA between the gene segments. (Item 8) 7. The rodent of item 5 or 6, wherein the insertion of one or more human Vκ gene segments and one or more human Jλ gene segments replaces rodent Vκ and Jκ gene segments. (Item 9) 9. The rodent of item 8, wherein the insertion comprises human Vκ and Jκ gene segments and naturally occurring human non-coding DNA between the combination. (Item 10) 9. The rodent according to any one of items 5 to 8, wherein the rodent immunoglobulin heavy chain constant region is an endogenous rodent immunoglobulin heavy chain constant region. (Item 11) 11. The rodent according to any one of items 5 to 10, wherein the rodent Cκ region is an endogenous rodent Cκ region. (Item 12) 10. The rodent of any one of paragraphs 1 to 9, wherein the endogenous immunoglobulin κ light chain locus comprises a deletion of all or part of an endogenous Vλ and Jλ gene segment. (Item 13) 13. The rodent of any one of items 1 to 12, wherein the rodent Cλ gene segment is a mouse Cλ□ gene segment. (Item 14) 14. The rodent of any one of items 5 to 13, wherein the immunoglobulin κ light chain locus comprises an insertion of all or part of the proximal Vκ duplication of the human immunoglobulin κ light chain locus. (Item 15) 15. The rodent of any one of paragraphs 5 to 14, wherein the immunoglobulin heavy chain locus lacks an endogenous rodent Adam6 gene. (Item 16) 16. The rodent of item 15, wherein the immunoglobulin heavy chain locus further comprises an insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides. (Item 17) 17. The rodent of any one of items 5 to 16, wherein the rodent is homozygous for the endogenous immunoglobulin heavy chain locus. (Item 18) 18. The rodent of items 5 to 17, wherein the rodent is homozygous for the endogenous immunoglobulin κ light chain locus. (Item 19) 19. The rodent of any one of paragraphs 1 to 18, wherein the rodent is homozygous for the endogenous immunoglobulin λ light chain locus. (Item 20) 20. The rodent of any one of items 1 to 19, wherein the rodent is a rat or a mouse. (Item 21) The germline genome (a) one or more human Vλ gene segments; (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments; an isolated rodent cell comprising an endogenous immunoglobulin lambda light chain locus comprising: (a) and (b) are operably linked to (c) and a rodent Cλ gene segment; and The isolated rodent cell, wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ). (Item 22) 22. The isolated rodent cell of claim 21, wherein the rodent cell is a rodent embryonic stem cell. (Item 23) 1. A method of producing a rodent whose germline genome comprises an engineered endogenous immunoglobulin lambda light chain locus, said method comprising: (a) introducing a DNA fragment into a rodent embryonic stem cell, said DNA fragment comprising: (i) one or more human Vλ gene segments; (ii) one or more human Jλ gene segments, and (iii) one or more human Cλ gene segments; a nucleotide sequence comprising wherein (i)-(iii) are operably linked to a rodent Cλ gene segment; and the nucleotide sequence further comprises one or more human immunoglobulin lambda light chain enhancers (Eλ); (b) obtaining the rodent embryonic stem cells produced in (a); and (c) generating a rodent using the rodent embryonic stem cells of (b). (Item 24) 1. A method of producing a rodent whose germline genome comprises an engineered endogenous immunoglobulin λ light chain locus, said engineered endogenous immunoglobulin λ light chain locus comprising an insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and one or more human Cλ gene segments, said human Vλ and Jλ gene segments operably linked to rodent or human Cλ gene segments, and said endogenous immunoglobulin λ light chain locus further comprising one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ), said method comprising: 1. A method comprising: modifying the germline genome of a rodent such that the germline genome of the rodent comprises an immunoglobulin λ light chain locus engineered to include an insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and one or more human Cλ gene segments, wherein the human Vλ and Jλ gene segments are operably linked to rodent or human Cλ gene segments, and the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ), thereby creating the rodent. (Item 25) 25. The method of item 23 or 24, wherein the one or more human Vλ gene segments comprise Vλ5-52 to Vλ1-40 and / or Vλ3-27 to Vλ3-1. (Item 26) 26. The method of item 25, wherein the one or more human Vλ gene segments comprise naturally occurring human non-coding DNA between human Vλ5-52 to Vλ1-40 and / or Vλ3-27 to Vλ3-1. (Item 27) 27. The method of any one of Items 26 to 26, wherein the one or more human Jλ gene segments and the one or more human Cλ gene segments comprise the human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, and the human Jλ gene segments. (Item 28) 28. The method of claim 27, wherein the human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6 comprise naturally occurring human non-coding DNA between the human Jλ and Cλ gene segment pairs, and the human Jλ gene segments comprise naturally occurring human non-coding DNA upstream (or 5') of the human Jλ. (Item 29) 29. The method of any one of items 23 to 28, wherein the rodent Cλ gene segment is a mouse Cλ gene segment. (Item 30) 30. The method of any one of items 23 to 29, wherein the endogenous immunoglobulin λ light chain locus comprises three human Eλ. (Item 31) 31. The method of any one of items 23 to 30, wherein the endogenous immunoglobulin λ light chain locus comprises two rodent Eλ. (Item 32) 32. The method of claim 31, wherein the two rodent Eλs are mouse Eλ and mouse Eλ3-1. (Item 33) 1. A method for producing antibodies in a rodent, comprising: (a) immunizing a rodent with an antigen of interest, said rodent comprising: (i) one or more human Vλ gene segments; (ii) one or more human Jλ gene segments, and (iii) one or more human Cλ gene segments; and having a germline genome that includes an endogenous immunoglobulin lambda light chain locus comprising wherein (i) and (ii) are operably linked to (iii) and a rodent Cλ gene segment; and the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ); (b) maintaining the rodent under conditions sufficient to cause the rodent to generate an immune response to the target antigen; and (c) recovering from said rodent or rodent cell an antibody that binds to said target antigen. (Item 34) 34. The method of any one of items 23 to 33, wherein the rodent is a rat or a mouse. (Item 35) The germline genome (i) human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1; (ii) human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6; (iii) Jλ7 of the human Jλ gene segment, and (iv) three human immunoglobulin lambda light chain enhancers; 1. A rodent comprising a homozygous endogenous immunoglobulin lambda light chain locus comprising: wherein (i) through (iv) are operably linked to each other, and (i) through (iii) are upstream of a rodent Cλ gene segment, wherein the endogenous immunoglobulin λ light chain locus lacks endogenous rodent immunoglobulin Eλ2-4; the human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1 comprise naturally occurring human non-coding DNA between the human Vλ gene segments; the human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6 comprise naturally occurring human non-coding DNA between the human Jλ-Cλ gene segment pairs; and A rodent, wherein the human Jλ gene segment Jλ7 comprises naturally occurring human non-coding DNA upstream (or 5′) of human Jλ7. (Item 36) 36. The rodent of item 35, wherein the rodent is a rat or a mouse. [Brief explanation of the drawings]

[0075] The drawings contained herein, consisting of the following figures, are for illustrative purposes only and not for limitation:

[0076] [Figure 1]Figure 1 shows a schematic, not to scale, illustration of an exemplary strategy for constructing an engineered endogenous Igλ light chain locus in a rodent, characterized by the presence of multiple human Vλ, Jλ, and Cλ coding sequences operably linked to each other and to a rodent Cλ region (or rodent Cλ gene). As shown, five separate targeting vectors (6286, 6571, 6596, 6597, and 6680) are shown, which carry various amounts of genetic material from the human Igλ light chain locus and are inserted sequentially into the endogenous rodent (e.g., mouse) Igλ light chain locus (shown above). The first targeting vector (6286) was constructed to contain a modular human Igλ enhancer (Eλ) region (or sequence) inserted downstream of the rodent Cλ1 region and characterized by three sequence elements. The second targeting vector (6571) was inserted upstream of the rodent Cλ1 region and engineered to contain five functional human Vλ gene segments, four functional human Jλ-Cλ gene segment pairs, and a human Jλ7 gene segment (human Jλ1-Cλ1-Jλ2-Cλ2-Jλ3-Cλ3-Jλ4-Cλ4-Jλ5-Cλ5-Jλ6-Cλ6-Jλ7). The third (6596) and fourth (6597) targeting vectors further contain a set of additional human Vλ gene segments (11 and 9, respectively), which are sequentially added to the total human Vλ gene segment content of the endogenous mouse Igλ light chain locus after successful targeting of the first targeting vector. Both targeting vectors contain an overlapping region (striped black box) at their 3' ends, which facilitates homologous recombination with the 5' end of the preceding targeting vector upon integration into the endogenous mouse Igλ light chain locus. An alternative fifth targeting vector (6680) is also shown, which has the same genetic material as the 6597 targeting vector, except that it contains a 5' homology arm with sequence identical to the 5' (or upstream) sequence of the rodent Vλ2 gene segment, thereby facilitating removal of the endogenous Vλ2-Vλ3-Jλ2-Cλ2-Jλ4P-Cλ4P-Eλ2-4-Vλ1-Jλ3-Jλ3P-Cλ3-Jλ1 gene segment upon homologous recombination with the targeting vector.Unless otherwise indicated, solid symbols indicate rodent gene segments and / or sequences, while open symbols indicate human gene segments and / or sequences. Site-specific recombination recognition sites (e.g., loxP, Frt) are also shown, which flank the selection cassettes (HYG: hygromycin resistance gene [HYGR] under the transcriptional control of the ubiquitin promoter; NEO: neomycin resistance gene [NEOR] under the transcriptional control of the ubiquitin promoter). The locations of selected nucleotide junctions are indicated by lines below each junction and are indicated by their respective SEQ ID NOs.

[0077] [Figure 2]Figure 2 shows a schematic, not to scale, representation of an example rodent Igλ light chain allele after sequential insertion of the targeting vector described in Example 1. 6597 allele: An Igλ light chain allele containing 25 functional human Vλ gene segments, four functional human Jλ-Cλ gene segment pairs, and a human Jλ7 gene segment, which are operably linked to a rodent Cλ region (e.g., a mouse Cλ1 region). The Igλ light chain locus further comprises endogenous Vλ-Jλ-Cλ gene segments, three endogenous Igλ enhancer regions (or sequences) (i.e., E2.4, E, and E3.1), and a modular human Igλ enhancer region (or sequence) characterized by three sequence elements. 6680 allele: An Igλ light chain allele after site-specific excision of the Vλ-Jλ-Cλ gene segments and Igλ enhancer Eλ2-4, which contains 25 functional human Vλ gene segments, four functional human Jλ-Cλ gene segment pairs, and a human Jλ7 gene segment, which are operably linked to a rodent Cλ region (e.g., a mouse Cλ1 region). The Igλ light chain locus further contains two (i.e., E and E3.1) endogenous Igλ enhancer regions (or sequences) and a modular human Igλ enhancer region (or sequence, see above). Unless otherwise indicated, solid symbols indicate rodent gene segments and / or sequences, while open symbols indicate human gene segments and / or sequences. Site-specific recombination recognition sites (e.g., Frt) are also shown, which flank the selection cassette (HYG: hygromycin resistance gene [HYGR] under the transcriptional control of the ubiquitin promoter). Dashed lines indicate the deleted region between the two depicted Igλ alleles. The locations of selected nucleotide junctions are indicated by lines below each junction and are each represented by a SEQ ID NO:

[0078] [Figure 3]Figure 3 shows a schematic, not to scale, diagram of another example of a strategy for constructing an engineered endogenous Igλ light chain locus in rodents, characterized by the presence of multiple human Vλ, Jλ, and Cλ coding sequences operably linked to each other and to a rodent Cλ region. As shown, two different targeting vectors carrying varying amounts of genetic material from the human Igλ light chain locus are shown, which are simultaneously inserted into an engineered rodent (e.g., mouse) Igλ light chain locus (shown above), which contains five human Vλ gene segments, a human Jλ-Cλ cluster, and a mouse Cλ1 gene. The 6596 targeting vector is modified to remove the neomycin selection cassette and incorporate duplicated sequences (striped black boxes) at the 5' and 3' ends to provide regions of homology and promote recombination with the corresponding human sequences. The second targeting vector was designed to contain an overlapping region (striped black box) at the 3' end of the construct, which shares sequence homology with the modified 6596 targeting vector (trimmed 6596 targeting vector), thereby promoting homologous recombination with the 5' end of the trimmed 6596 targeting vector. These two targeting vectors further contain a set of additional human Vλ gene segments (11 and 9, respectively), which are sequentially added to the total human Vλ gene segment content of the endogenous mouse Igλ light chain locus after successful targeting of the first targeting vector. The second targeting vector contains a 5' homology arm with sequence identical to the 5' (or upstream) sequence of the rodent Vλ2 gene segment, which upon homologous recombination with the targeting vector facilitates excision of the endogenous Vλ2-Vλ3-Jλ2-Cλ2-Jλ4P-Cλ4P-Eλ2-4-Vλ1-Jλ3-Jλ3P-Cλ3-Jλ1 gene segment. The two targeting vectors are co-electroporated with a guide RNA (gRNA), which facilitates integration of the engineered Igλ light chain locus, indicated by an arrow near the location of the targeting vector and each represented by a SEQ ID NO:Unless otherwise indicated, solid symbols indicate rodent gene segments and / or sequences, while open symbols indicate human gene segments and / or sequences. Site-specific recombination recognition sites (e.g., loxP, Frt) are also shown, which flank the selection cassettes (HYG: hygromycin resistance gene [HYGR] under the transcriptional control of the ubiquitin promoter; NEO: neomycin resistance gene [NEOR] under the transcriptional control of the ubiquitin promoter). The locations of selected nucleotide junctions are indicated by lines below each junction and are indicated by their respective SEQ ID NOs.

[0079] [Figure 4] Figure 4 shows schematic diagrams, not to scale, of examples of wild-type and engineered rodent Igλ light chain alleles employed in the experiments described in Example 3. Wild-type allele: A wild-type mouse Igλ light chain locus (see also, e.g., Figure 2 of U.S. Pat. No. 9,006,511). 6571 allele: An Igλ light chain allele containing five functional human Vλ gene segments, four functional human Jλ-Cλ gene segment pairs, and a human Jλ7 gene segment, which are operably linked to a rodent Cλ region (e.g., a mouse Cλ1 region). The Igλ light chain locus further comprises endogenous Vλ-Jλ-Cλ gene segments, three endogenous Igλ enhancer regions (or sequences), and a modular human Igλ enhancer region (or sequence, see above). 6597 allele: See above. 6680 allele: see above. The locations of selected nucleotide junctions are indicated by a line under each junction and are given as their respective SEQ ID NOs.

[0080] [Figure 5] Figures 5A and 5B show representative contour plots of single-cell gated splenocytes (A) showing CD19 (y-axis) and CD3 (x-axis) expression and absolute cell numbers per spleen (B) harvested from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT).

[0081] [Figure 6] 6A and 6B show representative contour plots showing mature and transitional B cells (A) among splenocytes gated on CD19+ showing expression of IgD (y-axis) and IgM (x-axis) and absolute cell numbers per spleen (B) harvested from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT). Splenic B cell subpopulations are indicated above each dot plot (e.g., mature, transitional, etc.).

[0082] [Figure 7] Figures 7A and 7B show representative contour plots showing the expression of mouse Igλ (mIgλ, y-axis), mouse Igκ (mIgκ, x-axis), or human Igλ (hIgλ, y-axis) in CD19+-gated splenocytes harvested from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT).

[0083] [Figure 8] Figures 8A and 8B show representative contour plots of bone marrow (A) gated on single cells showing expression of CD19 (y-axis) and CD3 (x-axis) and absolute cell numbers per femur (B) harvested from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT).

[0084] [Figure 9] 9A and 9B show representative contour plots of bone marrow (A) gated on CD19+IgMlowB220int showing expression of c-kit (y-axis) and CD43 (x-axis) and absolute cell numbers per femur (B) harvested from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT). Splenic B cell subpopulations are indicated on each dot plot (e.g., pro-B, pre-B, etc.).

[0085] [Figure 10]10A and 10B show representative contour plots of CD19+-gated bone marrow (A) showing IgM (y-axis) and B220 (x-axis) expression, and absolute cell numbers per femur (B) harvested from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT). Specific B cell subpopulations are indicated on each dot plot (e.g., immature, mature, pre-, and pro-B cells).

[0086] [Figure 11] Figures 11A and 11B show representative contour plots showing immature bone marrow (gated on CD19+IgM+B220int) showing expression of mouse Igλ (mIgλ, y-axis), mouse Igκ (mIgκ, x-axis), or human Igλ (hIgλ, y-axis) from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT).

[0087] [Figure 12] Figures 12A and 12B show representative contour plots showing mature bone marrow (gated on CD19+IgM+B220+) expression of mouse Igλ (mIgλ, y-axis), mouse Igκ (mIgκ, x-axis), or human Igλ (hIgλ, y-axis) from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT).

[0088] [Figure 13]Figure 13 shows representative mean percentages of Igκ-expressing (%κC) and human Igλ-expressing (%humanλC) B cells in spleen, immature bone marrow (immature BM), and mature bone marrow (mature BM) from selected engineered mouse strains described herein. Data are expressed as means, with standard deviations shown. 6680HO / VI HO / Adam6 HO: An engineered mouse strain containing a homozygous engineered Igλ light chain locus, designed to contain 25 functional human Vλ gene segments, four functional human Jλ-Cλ gene segment pairs, and a human Jλ7 gene segment, operably linked to a rodent Cλ region (e.g., a mouse Cλ1 region). The Igλ light chain locus further contains two endogenous Igλ enhancer regions (or sequences) and a modular human Igλ enhancer region (or sequence, see above). and the mouse strain comprises homozygous humanized IgH and Igκ loci, wherein the homozygous humanized IgH locus contained an inserted rodent Adam6 coding sequence (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940, the contents of which are incorporated by reference in their entireties). 6889HO / VI HO / Adam6 HO: An engineered mouse strain containing a homozygous engineered Igλ light chain locus, wherein the locus contains 25 functional human Vλ gene segments, four functional human Jλ-Cλ gene segment pairs, and a human Jλ7 gene segment, which are operably linked to a rodent Cλ region (e.g., a mouse Cλ1 region), and the Igλ light chain locus further comprises two endogenous Igλ enhancer regions (or sequences) and a modular human Igλ enhancer region (or sequence, see above). The mouse strains contained homozygous humanized IgH and Igκ loci, with the homozygous humanized IgH locus containing an inserted rodent Adam6 coding sequence (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940, the contents of which are incorporated by reference in their entireties). The number of mice in each genotype cohort included at least three and up to eight per group.

[0089] [Figure 14] Figures 14A and 14B show representative immunoblots (Western blots) of SDS-PAGE under non-reducing conditions using serum isolated from an engineered mouse homozygous for insertion of the 6680 targeting vector (6680HO) and a wild-type littermate (WT), demonstrating the expression of mouse (B, right image) or human (A, left image) lambda light chains. Each sample was loaded in a lane with 1.5 μl of serum. PHS: Pooled human serum (Labquip Ltd Cat. No. #9101A) with 0.25 μl. The molecular weight Kd is indicated to the right of each gel image.

[0090] [Figure 15A] Figure 15A shows the use of representative human Vλ (top) and human Jλ (bottom) gene segments in human Cλ priming sequences amplified from RNA isolated from splenocytes harvested from 6889HET mice (n=5).

[0091] [Figure 15B] FIG. 15B shows the use of representative human Vλ gene segments in mouse Cλ priming sequences amplified from RNA isolated from splenocytes harvested from 6889HET mice (n=5).

[0092] [Figure 15C] Figure 15C shows the use of representative human Vλ (top) and human Jλ (bottom) gene segments in human Cλ priming sequences amplified from RNA isolated from splenocytes harvested from 6889HO / VI HO / Adam6 HO mice (n=6).

[0093] [Figure 15D] FIG. 15D shows the use of representative human Vλ gene segments in mouse Cλ priming sequences amplified from RNA isolated from splenocytes harvested from 6889HO / VI HO / Adam6 HO mice (n=6).

[0094] [Figure 16] Figures 16A and 16B show representative total IgG titers (A) and antigen-specific IgG titers (B) in serum collected on days 0 and 22 from immunized mice heterozygous for insertion of the 6597 (6597HET, n=6) or 6680 (6680HET, n=6) targeting vectors, and from immunized wild-type controls (WT, n=6).

[0095] [Figure 17A] Figures 17A-C show representative human λ light chain (hIgλ, left), mouse λ light chain (mIgλ, middle), and mouse κ light chain (mIgκ, right) titers in antigen-specific IgG in serum on days 0 and 22 collected from immunized mice heterozygous for insertion of the 6597 (6597HET, n=6) or 6680 (6680HET, n=6) targeting vectors, and immunized wild-type controls (WT, n=6). [Figure 17B] Same as above. [Figure 17C] Same as above.

[0096] [Figure 18] Figures 18A and 18B show representative contour plots of single-cell gated splenocytes (left) showing CD19 (y-axis) and CD3 (x-axis) expression, and total B cells per spleen (right), harvested from mice homozygous for insertion of the 6889 targeting vector (6889HO VI HO Adam6 HO) and a reference engineered mouse (VI). 6889HO / VI HO / Adam6 HO: See above. VI: An engineered mouse strain containing homozygous humanized IgH and Igκ loci, where the homozygous humanized IgH locus contained an inserted rodent Adam6 coding sequence (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940, which are incorporated by reference in their entireties). Viable single-cell splenocytes were determined by activity staining (Thermo Fisher).

[0097] [Figure 19]Figure 19 shows representative contour plots showing expression of human Igλ (hIgλ, y-axis) and mouse Igκ (mIgκ, x-axis) in CD19+-gated splenocytes harvested from mice homozygous for insertion of the 6889 targeting vector (6889HO VI HO Adam6 HO) and a reference engineered mouse (VI). 6889HO / VI HO / Adam6 HO: see above. VI: see above.

[0098] [Figure 20] Figure 20 shows representative contour plots showing single-cell gated lymphocytes from bone marrow taken from the femur of a mouse homozygous for insertion of the 6889 targeting vector (6889HO VI HO Adam6 HO) and a reference engineered mouse (VI) showing expression of IgM (y-axis) and B220 (x-axis). 6889HO / VI HO / Adam6 HO: see above. VI: see above. Immature and mature B-cell subpopulations are indicated above each contour plot.

[0099] [Figure 21] Figure 21 shows representative contour plots showing immature bone marrow (gated on CD19+IgM+B220int, left column) and mature bone marrow (gated on CD19+IgM+B220+, right column) showing expression of human Igλ (hIgλ, y-axis) and mouse Igκ (mIgκ, x-axis) from mice homozygous for insertion of the 6889 targeting vector (6889HO VI HO Adam6 HO) and a reference engineered mouse (VI). 6889HO / VI HO / Adam6 HO: see above. VI: see above. DETAILED DESCRIPTION OF THE INVENTION

[0100] Brief description of selected sequences in the sequence listing Mouse Cλ1 DNA (SEQ ID NO: 1): GCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCTCTGAAGAGCTCGAGACTAAACAAGGCCACACTGGTGTGTACGATCACTGATTTCTACCCAGGTGTGGTGACAGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGTATG GAGACAACCCAGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGACCCTGACAGCAAGAGCATGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCATGAAGGTCACACTGTGGAGAAGAGTTTGTCCCGTGCTGACTGTTCC

[0101] Mouse Cλ1 amino acid (SEQ ID NO: 2): GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS

[0102] Mouse Cλ2 DNA (SEQ ID NO: 3): GTCAGCCCAAGTCCACTCCCACTCTCACCGTGTTTCCACCTTCCTCTGAGGAGCTCAAGGAAAACAAAGCCACACTGGTGTGTCTGATTTCCAACTTTTCCCGAGTGGTGTGACAGTGGCCTGGAAGGCAAATGGTACACCTATCACCCAGGGTGT GGACACTTCAAATCCCACCAAAGAGGGCAACAAGTTCATGGCCAGCAGCTTCCTACATTTGACATCGGACCAGTGGAGATCTCACAACAGTTTTACCTGTCAAGTTACACATGAAGGGACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTCTC

[0103] Mouse Cλ2 amino acid (SEQ ID NO: 4): GQPKSTPTLTVFPPSSEELKENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTKEGNKFMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL

[0104] Mouse Cλ3 DNA (SEQ ID NO: 5): GTCAGCCCAAGTCCACTCCCACACTCACCATGTTTCCACCTTCCCCTGAGGAGCTCCAGGAAAACAAAGCCACACTCGTGTGTCTGATTTCCAATTTTTCCCCAAGTGGTGTGACAGTGGCCTGGAAGGCAAATGGTACACCTATCACCCAGGGTGT GGACACTTCAAATCCCACCAAAGAGGACAACAAGTACATGGCCAGCAGCTTCTTACATTTGACATCGGACCAGTGGAGATCTCACAACAGTTTTACCTGCCAAGTTACACATGAAGGGACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTCTC

[0105] Mouse Cλ3 amino acid (SEQ ID NO: 6): GQPKSTPTLTMFPPSPEELQENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTKEDNKYMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL

[0106] Rat Cλ1 DNA (SEQ ID NO: 7): GTCAGCCCAAGTCCACTCCCACACTCACAGTATTTCCACCTTCAACTGAGGAGCTCCAGGGAAACAAAGCCACACTGGTGTGTCTGATTTCTGATTTCTACCCGAGTGATGTGGAAGTGGCCTGGAAGGCAAATGGTGCACCTATCTCCCAGGGTGT GGACACTGCAAATCCCACCAAACAGGGCAACAAATACATCGCCAGCAGCTTCTTACGTTTGACAGCAGAACAGTGGAGATCTCGCAACAGTTTTACCTGCCAAGTTACACATGAAGGGAACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTGTC

[0107] Rat Cλ1 amino acid (SEQ ID NO: 8): GQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKANGAPISQGVDTANPTKQGNKYIASSFLRLTAEQWRSRNSFTCQVTHEGNTVEKSLSPAECV

[0108] Rat Cλ2 DNA (SEQ ID NO: 9): ACCAACCCAAGGCTACGCCCTCAGTCACCCTGTTCCCACCTTCCTCTGAAGAGCTCAAGACTGACAAGGCTACACTGGTGTGTATGGTGACAGATTTCTACCCTGGTGTTATGACAGTGGTCTGGAAGGCAGATGGTACCCCTATCACTCAGGGTGT GGAGACTACCCAGCCTTTCAAACAGAACAACAAGTACATGGCTACCAGCTACCTGCTTTTGACAGCAAAAGCATGGGAGACTCATAGCAATTACAGCTGCCAGGTCACTCACGAAGAGAACACTGTGGAGAAGAGTTTGTCCCGTGCTGAGTGTTCC

[0109] Rat Cλ2 amino acid (SEQ ID NO: 10): DQPKATPSVTLFPPSSEELKTDKATLVCMVTDFYPGVMTVVWKADGTPITQGVETTQPFKQNNKYMATSYLLLTAKAWETHSNYSCQVTHEENTVEKSLSRAECS

[0110] Rat Cλ3 DNA (SEQ ID NO: 11): GTCAGCCCAAGTCCACTCCCACACTCACAGTATTTCCACCTTCAACTGAGGAGCTCCAGGGAAACAAAGCCACACTGGTGTGTCTGATTTCTGATTTCTACCCGAGTGATGTGGAAGTGGCCTGGAAGGCAAATGGTGCACCTATCTCCCAGGGTGT GGACACTGCAAATCCCACCAAACAGGGCAACAAATACATCGCCAGCAGCTTCTTACGTTTGACAGCAGAACAGTGGAGATCTCGCAACAGTTTTACCTGCCAAGTTACACATGAAGGGAACACTGTGGAAAAGAGTCTGTCTCCTGCAGAGTGTGTC

[0111] Rat Cλ3 amino acid (SEQ ID NO: 12): GQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKANGAPISQGVDTANPTKQGNKYIASSFLRLTAEQWRSRNSFTCQVTHEGNTVEKSLSPAECV

[0112] Rat Cλ4 DNA (SEQ ID NO: 13): ACCAACCCAAGGCTACGCCCTCAGTCACCCTGTTCCCACCTTCCTCTGAAGAGCTCAAGACTGACAAGGCTACACTGGTGTGTATGGTGACAGATTTCTACCCTGGTGTTATGACAGTGGTCTGGAAGGCAGATGGTACCCCTATCACTCAGGGTGT GGAGACTACCCAGCCTTTCAAACAGAACAACAAGTACATGGCTACCAGCTACCTGCTTTTGACAGCAAAAGCATGGGAGACTCATAGCAATTACAGCTGCCAGGTCACTCACGAAGAGAACACTGTGGAGAAGAGTTTGTCCCGTGCTGAGTGTTCC

[0113] Rat Cλ4 amino acid (SEQ ID NO: 14): DQPKATPSVTLFPPSSEELKTDKATLVCMVTDFYPGVMTVVWKADGTPITQGVETTQPFKQNNKYMATSYLLLTAKAWETHSNYSCQVTHEENTVEKSLSRAECS

[0114] definition The scope of the present invention is defined by the claims appended hereto, and is not limited by any embodiments described herein. Those skilled in the art will recognize, upon reading this specification, various modifications that may be equivalent to such described embodiments, or that may otherwise be within the scope of the claims.

[0115] Generally, terms used herein are conditioned on their art-understood meanings unless expressly indicated otherwise. Throughout this specification, explicit definitions of certain terms are provided below; the meaning of these and other terms in specific instances will be apparent to one of ordinary skill in the art from the context. Additional definitions for the following terms and other terms are set forth throughout this specification. All patent and non-patent references cited within this specification, or relevant portions thereof, are hereby incorporated by reference in their entirety.

[0116] Administration: As used herein, includes administration of a composition to a subject or system (e.g., a cell, organ, tissue, organism, or related component or set of components thereof). One of skill in the art will recognize 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 administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., a human or rodent) may be bronchial (including bronchial infusion), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including intratracheal infusion), transdermal, vaginal, and / or intravitreal administration. In some embodiments, administration may include intermittent dosing. In some embodiments, administration may include continuous dosing (e.g., perfusion) for at least a selected period of time.

[0117] Amelioration: As used herein, includes the prevention, reduction, or alleviation of a condition, or improvement of a subject's condition. Amelioration includes, but does not necessarily include, complete reversal or complete prevention of a disease, disorder, or symptom.

[0118] Approximately: When applied to one or more subject values, includes values ​​similar to a specified reference value. In certain embodiments, the term "approximately" or "about" refers to a value within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction of the specified reference value, unless otherwise specified or unless otherwise clear from the context (except where such number would exceed 100% of possible values).

[0119] Biologically active: As used herein, refers to any characteristic of an agent that has activity in a biological system, in vitro or in vivo (e.g., in an organism). For example, if an agent is present in an organism, it is considered to be biologically active if it has a biological effect in 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 generally referred to as a "biologically active" portion.

[0120] Equivalent: As used herein, refers to two or more agents, entities, circumstances, sets of conditions, etc. that may not be identical to one another, but that are sufficiently similar to permit a comparison between them, such that conclusions may reasonably be drawn based on the observed differences or similarities. A person of ordinary skill in the art will understand, within context, the degree of identity required for two or more such agents, entities, circumstances, sets of conditions, etc. to be considered equivalent under any given circumstances.

[0121] Conservative: As used herein, this refers to an example describing a conservative amino acid substitution, 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). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein of interest, such as the ability of a receptor to bind to a ligand. Examples of amino acid groups having side chains with similar chemical properties include: aliphatic side chains, such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (Ile, I); aliphatic-hydroxyl side chains, such as serine (Ser, S) and threonine (Thr, T); and amide-containing side chains, such as asparagine (Asn, N) and glutamine (Gln, Q). 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 for any native residue in a protein, including alanine, such as those 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. 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.

[0122] Control: As used herein, refers to the art meaning of "control," a standard against which results are compared. Generally, controls are used to increase 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 comparator. "Control" also includes "control animals." A "control animal" may have a modification described herein, a modification different from those described herein, or be unmodified (i.e., a wild-type animal). In one experiment, the "test" (i.e., the variable being tested) is administered. In a second experiment, which is the "control," the variable being tested is not administered. In some embodiments, a control is a historical control (i.e., of a previously performed test or assay, or a previously known amount or result). In some embodiments, a control is or includes a printed or otherwise kept record. A control may be a positive or negative control.

[0123]

[0124] Derived from: When used with reference to an unrearranged variable region and / or a rearranged variable region gene or variable domain "derived from" an unrearranged variable region gene segment, refers to the ability to trace the sequence of the rearranged variable region gene or variable domain back to the unrearranged variable region gene segments that rearranged to form the rearranged variable region gene that expresses the variable domain (accounting for splice differences, and somatic mutation, as appropriate). For example, a rearranged variable region gene that has undergone somatic mutation remains the fact that it was derived from an unrearranged variable region gene segment.

[0125] Disruption: As used herein, refers to the result of a homologous recombination event with a DNA molecule (e.g., with an endogenous homologous sequence, such as a gene or locus). In some embodiments, the disruption may result in or exhibit an insertion, deletion, substitution, exchange, missense mutation, or frameshift of a DNA sequence, or any combination thereof. The insertion may include the insertion of an entire gene or a fragment of a gene, such as 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 effect assembly of a fusion polypeptide. In some embodiments, the disruption may affect the level of the gene or gene product but not its activity. In some embodiments, the disruption may affect the activity of the gene or gene product but not its level. In some embodiments, the disruption may have no significant effect on the level of the gene or gene product. In some embodiments, the disruption may have no 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.

[0126] Determining, measuring, assessing, evaluating, testing, and analyzing: used interchangeably herein and refer to any form of measurement, including determining whether an element is present. 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.

[0127] Endogenous locus or endogenous gene: As used herein, refers to a genetic locus present in a parent or reference organism prior to the introduction of a disruption, deletion, substitution, change, or modification described herein. In some embodiments, the endogenous locus has a sequence that occurs in nature. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the endogenous locus is an engineered locus. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is a genetically engineered organism. In some embodiments, the reference organism is a laboratory-bred organism (wild-type or genetically engineered).

[0128] Endogenous promoter: As used herein refers to a promoter that is naturally associated with an endogenous gene, e.g., in a wild-type organism.

[0129] Engineered: As used herein, generally refers to something that has been manipulated by the hand of man. For example, in some embodiments, two or more sequences that are not linked together in natural order may be considered "engineered" when they have been manipulated by the hand of man to be directly linked to one another in an engineered polynucleotide. In certain such embodiments, an engineered polynucleotide may include regulatory sequences that are naturally operably associated with a first coding sequence but not with a second coding sequence, and that have been linked by the hand of man to be operably associated with the second coding sequence. Alternatively, or in addition, in some embodiments, first and second nucleic acid sequences, each encoding polypeptide elements or domains that are not linked to one another in nature, may be linked to one another in a single engineered polynucleotide. Similarly, in some embodiments, a cell or organism may be considered "engineered" when its genetic information has been altered (e.g., new genetic material not previously present has been introduced, or previously present genetic material has been altered or removed). As is common and understood by those of skill in the art, the progeny of an engineered polynucleotide or cell are typically considered to be "engineered," even if the actual manipulation was performed on a prior entity. Furthermore, as will be recognized by those of skill in the art, a variety of techniques are available through which the "manipulation" described herein can be performed. For example, in some embodiments, "manipulation" may include 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 analyze or compare, or otherwise analyze recommended and / or selected sequences.Alternatively, or in addition, in some embodiments, "manipulation" may involve the use of in vitro chemical synthesis techniques and / or recombinant nucleic acid techniques, such as nucleic acid amplification (e.g., via the polymerase chain reaction), hybridization, mutagenesis, transformation, transfection, and / or the use of any of a variety of controlled mating methods. As will be recognized by those skilled in the art, a variety of such established techniques (e.g., recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection, etc.)) are well known in the art and are described in various general and detailed references, which are cited and / or discussed throughout this specification. See, e.g., 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.

[0130] 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., forming part of a coding sequence) and / or activity. For example, in the context of immunoglobulins, variable domains are encoded by unique gene segments (i.e., V, D, and / or J) that are assembled (or recombined) to form functional coding sequences. When present in a genome, gene segments are organized into clusters, although diversity occurs. A "functional" gene segment is one that appears in an expressed sequence (i.e., a variable domain) and whose corresponding genomic DNA has been isolated (i.e., cloned) and identified by sequence. Some immunoglobulin gene segment sequences contain an open reading frame and are considered functional, but do not appear in the expression repertoire. Other immunoglobulin gene segment sequences, on the other hand, contain mutations (e.g., point mutations, insertions, deletions, etc.) that result in stop codons and / or truncated sequences, thereby rendering such gene segment sequences unable to subsequently perform the properties and / or activities associated with the non-mutated sequence. Such sequences do not appear in expressed sequences and are therefore categorized as pseudogenes.

[0131] 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 certain embodiments, a gene comprises both coding sequence (e.g., exon sequence) and non-coding sequence (e.g., intron sequence). In some embodiments, a gene may comprise one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that can control or influence, for example, one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.). For clarity, as used in this disclosure, the term "gene" generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof; the term may optionally encompass regulatory sequences, as would be clear from the context to one of ordinary skill in the art. This definition is not intended to exclude the application of the term "gene" to non-protein-coding expression units, but is intended to clarify that as used herein, the term often refers to a nucleic acid that encodes a polypeptide.

[0132] Heterologous: As used herein, refers to a subject or entity from a different origin. For example, when used in reference to a polypeptide, gene, or gene product present in a particular cell or organism, the term indicates that the related polypeptide, gene, or gene product: 1) has been manipulated by the hand of man; 2) has been introduced into the cell or organism (or a precursor thereof) via the hand of man; and / or 3) is not naturally produced or present in the related cell or organism (e.g., related 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 are altered or modified by mutation or substitution, e.g., not naturally associated with, and in some embodiments, under the control of non-endogenous regulatory elements (e.g., promoters).

[0133] Host cell: As used herein, refers to a cell into which a nucleic acid or protein has been introduced. Those skilled in the art will understand upon reading this disclosure 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 successive generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the term "host cell." In some embodiments, a host cell is or comprises 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 species to which the cell is designated. Examples of cells include prokaryotic and eukaryotic (unicellular or multicellular) cells, bacterial cells (e.g., strains of Escherichia coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusion products such as, for example, hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells.In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: 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 foregoing cells. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell (e.g., a PER.C6® cell) expressing a viral gene. 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.

[0134] Identity: As used herein in connection with sequence comparisons, refers to identity as determined by many 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 using a Gonnet similarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008), with a gap opening penalty of 10.0 and a gap extension penalty of 0.1.

[0135] In vitro: As used herein, refers to events that take place not within a multicellular organism, but in an artificial environment such as a test tube or reaction vessel, cell culture, etc.

[0136] In vivo: As used herein, refers to events that occur within a multicellular organism, such as, for example, a human and / or a non-human animal. In the context of cell-based systems, the term may also be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0137] Isolated: As used herein, refers to (1) a substance and / or entity that has been separated from at least some of the components with which it was associated when originally produced (either in natural and / or experimental environments) and / or (2) a substance and / or entity that has been designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components 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, the isolated material is 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, the isolated material is 80%-99%, 85%-99%, 90%-99%, 95%-99%, 96%-99%, 97%-99%, or 98%-99% pure. In some embodiments, the isolated material is 80%-99%, 80%-98%, 80%-97%, 80%-96%, 80%-95%, 80%-90%, or 80%-85% pure. In some embodiments, the isolated material is 85%-98%, 90%-97%, or 95%-96% pure. In some embodiments, a substance is "pure" when it is substantially free of other components. In some embodiments, as one of skill in the art will understand, a substance may still be considered "isolated" or even "pure" even 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 percent isolation or purity of the substance is calculated without including such carriers or excipients. By way of example, in some embodiments, a naturally occurring biopolymer, such as a polypeptide or polynucleotide, is considered "isolated" when: a) its origin or source is not associated with some or all of the components that naturally accompany it in its natural state in nature; b) it is substantially free from other polypeptides or nucleic acids of the same species that produces it in nature; or c) it is expressed by or otherwise associated with components from a cell or other expression system other than the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from that which produces it in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques can be considered an "isolated" polypeptide so long as it is separated from other components with which a) it is associated in nature and / or b) it was associated when originally produced.

[0138] Locus: As used herein, refers to a specific location of a gene (or key sequence), a DNA sequence, a sequence encoding a polypeptide, or a location on a chromosome in the genome of an organism. For example, an "immunoglobulin locus" can refer to a specific location of an immunoglobulin gene segment (e.g., V, D, J, or C), an immunoglobulin gene segment DNA sequence, a sequence encoding an immunoglobulin gene segment, or the location of an immunoglobulin gene segment on a chromosome in the genome of an organism identified as the location where such a sequence is located. An "immunoglobulin locus" can contain regulatory elements of the immunoglobulin gene segment, including, but not limited to, an enhancer, a promoter, 5' and / or 3' regulatory sequences or regions, or a combination thereof. An "immunoglobulin locus" may contain DNA that normally resides between gene segments of a wild-type locus, but the DNA itself lacks immunoglobulin gene segments (e.g., immunoglobulin DNA sequences naturally occurring between V and J gene segments, immunoglobulin DNA sequences naturally occurring between J gene segments and constant region genes, or immunoglobulin DNA sequences naturally occurring 3' to constant region genes, etc.). Those skilled in the art will understand that in some embodiments, chromosomes may contain hundreds or even thousands of genes and may show physical co-localization of similar loci when compared between different species. Such loci may be described as sharing synteny.

[0139] Non-human animal: As used herein, refers to any vertebrate 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.

[0140] 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 an oligonucleotide chain or a compound and / or substance that can be incorporated into an oligonucleotide chain via a phosphodiester bond. As is clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA, and in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a "nucleic acid" comprises or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a "nucleic acid" comprises or consists of one or more nucleic acid analogs. In some embodiments, nucleic acid analogs differ from "nucleic acids" in that they do 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 have 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 or is composed 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 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-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating 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, such as 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, 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, and in some embodiments, the "nucleic acid" is double-stranded.In some embodiments, a "nucleic acid" has a nucleotide sequence that encodes a polypeptide or that includes at least one element that is the complement of a sequence that encodes a polypeptide. In some embodiments, a "nucleic acid" has enzymatic activity.

[0141] Operably linked: As used herein, this refers to a juxtaposition in which the described components are in a relationship allowing them to function in their intended manner. For example, if an unrearranged variable region gene segment can be rearranged to form a rearranged variable region gene, and the rearranged variable region is linked to a constant region gene and expressed as a polypeptide chain of an antigen-binding protein, the unrearranged variable region gene segment is "operably linked" to a contiguous constant region gene. A control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" sequences include both expression control sequences contiguous with a gene of interest and expression control sequences that act in trans or remotely to regulate a gene (or sequence of interest) of interest. The term "expression control sequence" includes polynucleotide sequences necessary to affect the expression and processing of coding sequences to which they are linked. "Expression control sequences" include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak 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 generally include a promoter, ribosomal binding site, and transcription termination sequence, while in eukaryotes, such control sequences generally include a promoter and a transcription termination sequence. The term "control sequence" is intended to include elements whose presence is essential for expression and processing, and can also include additional elements whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0142] Physiological conditions: As used herein, the term refers to the art-understood meaning of the conditions under which cells and organisms live and / or reproduce. In some embodiments, the term includes external or internal environmental conditions that may occur in nature for an organism or cellular system. In some embodiments, physiological conditions are conditions found within the body of a human or non-human animal, particularly at and / or within a surgical site. Physiological conditions typically include, for example, temperatures ranging from 20-40°C, 1 atmosphere of pressure, pH 6-8, glucose concentrations of 1-20 mM, atmospheric oxygen concentrations, and gravity found on Earth. In some embodiments, laboratory conditions are manipulated and / or maintained to be physiological. In some embodiments, physiological conditions are conditions found within an organism.

[0143] 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 includes portions that occur in nature 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 engineered, in that it is designed and / or produced 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., has a sequence that is engineered, in that it is designed and / or produced through the work of the hand of man to encode said polypeptide).

[0144] Recombinant: As used herein, is intended to refer to a polypeptide that is designed, engineered, prepared, expressed, produced, or isolated by recombinant means, such as 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), an antibody isolated from an animal (e.g., a mouse) that is transgenic for 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), or any other means involving intersplicing of selected sequence elements. In some embodiments, one or more of such selected sequence elements are naturally occurring. In some embodiments, one or more of such selected sequence elements are designed in silico.In some embodiments, one or more such selected sequence elements result, for example, from mutagenesis (e.g., in vivo or in vitro) of known sequence elements of natural or synthetic origin. For example, in some embodiments, the recombinant polypeptide consists of a sequence found in the genome of a source organism of interest (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 in a non-human animal), and thus the amino acid sequence of the recombinant polypeptide is derived from a polypeptide sequence that may not naturally occur in the genome of the non-human animal in vivo.

[0145] Reference: As used herein, refers to a standard or control agent, animal, cohort, individual, population, sample, sequence, or value to which a subject agent, animal, cohort, individual, population, sample, sequence, or value is compared. In some embodiments, the reference agent, animal, cohort, individual, population, sample, sequence, or value is tested and / or determined substantially simultaneously with the testing or determination of the subject agent, animal, cohort, individual, population, sample, sequence, or value. In some embodiments, the reference agent, animal, cohort, individual, population, sample, sequence, or value is a known reference, optionally embodied in a tangible medium. In some embodiments, a reference may refer to a control. "Reference" also includes "reference animal." A "reference animal" may have a modification described herein, a different modification than those described herein, or may be unmodified (i.e., a wild-type animal). Typically, as will be understood by one of skill in the art, the reference agent, animal, cohort, individual, population, sample, sequence or value is determined or characterized under conditions equivalent to the conditions used to determine or characterize the subject agent, animal (e.g., mammal), cohort, individual, population, sample, sequence or value.

[0146] Replacement: As used herein, refers to a process through which a "replacement" nucleic acid sequence (e.g., a gene) found at 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, e.g., in that they are homologous to each other and / or contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.). 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), and 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 replaced nucleic acid sequence. In some embodiments, the replacement nucleic acid sequence is an ortholog or homolog of the replaced sequence. In some embodiments, the replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, the replaced nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse or rat sequence), including when the replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, the replaced nucleic acid sequence is or comprises a human sequence, including when the replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, the replacement nucleic acid sequence is a variant or mutant of the replaced sequence (i.e., a sequence that contains one or more sequence differences, e.g., a substitution, compared to the replaced sequence). The nucleic acid sequence so positioned may include one or more regulatory sequences (e.g., a promoter, an enhancer, a 5'- or 3'-untranslated region, etc.) that were part of the source nucleic acid sequence used to obtain the so positioned sequence. For example, in various embodiments, the replacement is the replacement of an endogenous sequence with a heterologous sequence resulting in the production of a gene product from the so positioned nucleic acid sequence (including the heterologous sequence), but not the replacement of expression of the endogenous sequence.Substitutions are of the endogenous genomic sequence with a nucleic acid sequence encoding a polypeptide having a similar function to the polypeptide encoded by the endogenous sequence (e.g., the endogenous genomic sequence encodes all or part of a non-human variable domain polypeptide and the DNA fragment encodes all or part of one or more human variable domain polypeptides). In various embodiments, an endogenous non-human immunoglobulin gene segment or fragment thereof is replaced with a human immunoglobulin gene segment or fragment thereof.

[0147] Substantially: As used herein, refers to a qualitative state of exhibiting all or nearly all extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the possible lack of completeness inherent in many biological and chemical phenomena.

[0148] Substantial homology: As used herein, refers to a comparison between amino acid sequences or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues with reasonably similar structural and / or functional characteristics. For example, as will be known to those skilled in the art, certain amino acids are commonly classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid with another amino acid of the same type is often considered a "homologous" substitution. Common amino acid classifications are summarized in the table below. [Table A] [Table B]

[0149] 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 and BLASTP for nucleotide sequences, gapped BLAST, and PSI-BLAST for amino acid sequences. Examples of such programs are 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. Feuilletette (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. In addition to identifying homologous sequences, the programs described above typically provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least, for example, but not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over the relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. 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 includes contiguous residues along the complete sequence. In some embodiments, the relevant stretch includes discontinuous residues along the complete sequence, e.g., non-adjacent residues brought together by the folded structure of a polypeptide or portion thereof. In some embodiments, the relevant section is at least, for example and without limitation, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[0150] Substantial identity: As used herein, refers to a comparison between amino acid sequences or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues 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, including those available in commercially available computer programs, such as BLASTN and BLASTP for nucleotide sequences, gapped BLAST, and PSI-BLAST for amino acid sequences. Examples of such programs are 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. al. (eds.) Bioinformatics Methods and Protocols, Methods in Molecular Biology, Vol. 132, Humana Press, 1998. In addition to identifying identical sequences, the programs described above often also provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 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. In some embodiments, the relevant stretch of residues is a complete sequence. In some embodiments, the relevant stretch of residues is, for example, but not limited to, at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[0151] Targeting construct or targeting vector: As used herein, refers to a polynucleotide molecule comprising a targeting region. The targeting region comprises a sequence identical or substantially identical to a sequence in a target cell, tissue, or animal, and integrates the targeting construct into a location within the genome of the cell, tissue, or animal by 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 nucleic acid sequence or gene of particular interest, a selectable marker, control and / or regulatory sequences, and other nucleic acid sequences that enable recombination mediated by the exogenous addition of proteins that assist or promote recombination involving such sequences. In some embodiments, the targeting constructs described herein further comprise all or a portion of a gene of interest, wherein the gene of interest is a heterologous gene encoding all or a portion of a polypeptide with a similar function to a protein encoded by the endogenous sequence. In some embodiments, the targeting constructs described herein further comprise all or a portion of a humanized gene of interest, wherein the humanized gene of interest encodes all or a portion of a polypeptide having a similar function 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 that has been engineered by human beings. For example, in some embodiments, the targeting construct (or targeting vector) may be constructed to comprise an engineered or recombinant polynucleotide that includes two or more sequences that are not linked to each other in nature but have been engineered by human beings to be directly linked to each other in the engineered or recombinant polynucleotide.

[0152] Transgene or transgene construct: As used herein, refers to a nucleic acid sequence (e.g., a nucleic acid sequence encoding all or part of a polypeptide of interest) that has been introduced into a cell by the hand of man, such as by a method described herein. A transgene may be partially or entirely heterologous, i.e., foreign to the transgenic animal or cell into which it is introduced. A transgene may include one or more transcriptional control sequences, e.g., introns or promoters, and any other nucleic acid that may be required for expression of a selected nucleic acid sequence.

[0153] Transgenic Animals, Transgenic Non-Human Animals, or Tg + : can be used interchangeably herein and refer to any non-naturally occurring non-human animal, in which one or more of the non-human animal's cells contain a heterologous nucleic acid and / or gene encoding all or part of a polypeptide of interest. For example, in some embodiments, "transgenic animal" or "transgenic non-human animal" refers to an animal or non-human animal that contains a transgene or transgene construct as described herein. In some embodiments, the heterologous nucleic acid and / or gene is introduced into the cell directly or indirectly by introduction into a progenitor cell via deliberate genetic manipulation, such as using microinjection or infection with a recombinant virus. The term genetic manipulation does not include traditional breeding techniques, but rather covers the introduction of a recombinant DNA molecule. This molecule may be integrated into a chromosome or may be extrachromosomally replicating DNA. The term "Tg + " includes animals that are heterozygous or homozygous for the heterologous nucleic acid and / or gene, and / or that have a single copy or multiple copies of the heterologous nucleic acid and / or gene.

[0154] Variant: As used herein, refers to an entity that exhibits substantial structural identity with a reference entity but that differs structurally from the reference entity in the presence or level of one or more chemical moieties relative to the reference entity. In many embodiments, a "variant" also differs functionally from the reference entity. Generally, whether a particular entity is properly considered a "variant" of a reference entity is based on its degree of structural identity with the reference entity. As will be understood by those skilled in the art, all biological or chemical reference entities possess certain characteristic structural elements. A "variant," by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. In a few examples, a polypeptide may have a characteristic sequence element, which is composed of multiple amino acids that have a specified position relative to one another in linear or three-dimensional space and / or that contributes to a specific biological function. Alternatively, a polypeptide may have a characteristic sequence element composed of multiple nucleotide residues that have a specified position relative to one another in linear or three-dimensional space. In another example, a "variant polypeptide" may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, a "variant polypeptide" exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with a reference polypeptide. Alternatively, or in addition, in some embodiments, a "variant polypeptide" does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, a reference polypeptide possesses one or more biological activities. In some embodiments, a "variant polypeptide" shares one or more biological activities of a reference polypeptide. In some embodiments, a "variant polypeptide" lacks one or more biological activities of a reference polypeptide. In some embodiments, a "variant polypeptide" exhibits a reduced level of one or more biological activities compared to a reference polypeptide.In many embodiments, a subject polypeptide is considered a "variant" of a parent or reference polypeptide if the subject polypeptide has the same amino acid sequence as the parent, except for minor sequence changes at specific positions. Typically, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the variant's residues are substituted compared to the parent. In some embodiments, a "variant" has, for example, but not limited to, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the parent. Often, a "variant" has very few (e.g., less than 5, 4, 3, 2, or 1) functional residues (i.e., residues participating in a particular biological activity) substituted. Furthermore, a "variant" typically has, for example, but not limited to, no more than 5, 4, 3, 2, or 1 additions or deletions, and often no additions or deletions compared to the parent. Furthermore, any additions or deletions will generally be less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and usually about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is one that occurs in nature. As one of skill in the art will appreciate, multiple variants of a particular polypeptide of interest may typically be found in nature, particularly when the polypeptide of interest is an infectious agent peptide.

[0155] Vector: As used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. In some embodiments, vectors are capable of extrachromosomal replication and / or expression of nucleic acids to which they are linked in host cells, such as eukaryotic and / or prokaryotic cells. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors."

[0156] Wild-type: As used herein, refers to an entity having a structure and / or activity that occurs in nature in a "normal" state or situation (as opposed to a mutant, pathological, altered, etc.). Those skilled in the art will appreciate that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0157] Detailed Description of Specific Embodiments In certain aspects, provided herein are engineered non-human animals having heterologous genetic material encoding, inter alia, human variable domains, and in some embodiments, human constant domains, the heterologous genetic material including human Vλ, Jλ, and Cλ gene sequences (i.e., gene segments) and other human sequences that result in the appropriate rearrangement and expression of antibodies having human and non-human portions, or antibodies having sequences that are substantially human or substantially mostly human. In various embodiments, the engineered non-human animals provided contain heterologous genetic material inserted in a manner such that antibodies containing human Vλ domains and light chains having human or non-human Cλ domains are expressed in the antibody repertoire of the non-human animal. Additionally, the engineered non-human animals provided contain heterologous genetic material inserted in a manner such that antibodies containing human Vλ domains and light chains having human or non-human Cλ domains are expressed from an engineered Igλ light chain locus, the light chain locus including human and non-human Igλ enhancer regions (or sequences) in the germline genome of the non-human animal.

[0158] Without wishing to be bound by any particular theory, it is expected that the non-human animal embodiments described herein will provide an improved in vivo system that utilizes the expression of antibodies containing human Vλ domains for the production of therapeutic antibodies. It is also expected that the non-human animal embodiments described herein, in some embodiments, will provide alternatively engineered Igλ light chain loci containing heterologous genetic material for the development of human antibody-based therapeutics (e.g., human monoclonal antibodies, multispecific binders, scFvs, fusion polypeptides, etc.) against disease targets associated with biased antibody responses (e.g., antibody responses characterized by a predominance of either κ or λ light chains). Thus, the non-human animal embodiments described herein are particularly useful for the development of human antibodies against targets (e.g., viruses) associated with poor immunogenicity, due in part to asymmetric antibody repertoires and / or antibody responses.

[0159] In particular, in certain aspects, the disclosure describes the production of non-human animals (e.g., rodents such as rats or mice) having a germline genome containing an engineered Igλ light chain locus, wherein in some embodiments, the light chain locus is characterized by the introduction of multiple human Vλ, Jλ, and Cλ gene sequences operably linked to a non-human Cλ region, thereby resulting in the expression of antibodies containing light chains comprising human Vλ domains and human or non-human Cλ domains. As described herein, such engineered Igλ light chain loci are generated, such that expression of antibodies containing light chains comprising human Vλ domains and human or non-human Cλ domains results from the engineered Igλ light chain locus in the germline genome of the non-human animal. In some embodiments, the germline genome of the provided non-human animals further comprises (1) a humanized IgH and Igκ locus or (2) a humanized IgH locus and a functionally silenced or otherwise non-functional Igκ light chain locus. As described herein, the non-human animals provided express an antibody repertoire that includes an Igλ light chain, which light chain comprises a human Vλ domain.

[0160] In some embodiments, the non-human animals described herein contain human and non-human Igλ light chain sequences within a single Igλ light chain locus. In some embodiments, the non-human animals described herein contain human and murine (e.g., mouse or rat) Igλ light chain sequences within the Igλ light chain locus. In many embodiments of the non-human animals described herein, the non-human Igλ light chain sequence is or contains a murine (e.g., mouse or rat) sequence.

[0161] In some embodiments, the Igλ light chain sequence comprises intergenic DNA that is of human and / or murine (e.g., mouse or rat) origin. In some embodiments, the Igλ light chain sequence is synthetic and comprises intergenic DNA based on a sequence of human or murine (e.g., mouse or rat) origin. In some embodiments, the intergenic DNA is of the same immunoglobulin locus within which the intergenic DNA is so positioned, inserted, positioned, or engineered (e.g., Igλ intergenic DNA within an Igλ light chain locus). In some embodiments, the non-human animals described herein contain an engineered Igλ light chain locus, wherein the light chain locus contains intergenic DNA that comprises an Igλ light chain sequence of non-human origin (e.g., a mouse or rat Igλ light chain sequence).

[0162] In various embodiments, the humanized IgH locus comprises multiple human V H , D H , and J. HThe humanized Igκ light chain locus contains multiple human Vκ and Jκ gene segments, which are operably linked to a non-human IgH constant region (e.g., an endogenous non-human IgH constant region, including one or more IgH constant region genes, such as IgM, IgG, etc.). In various embodiments, the humanized Igκ light chain locus contains multiple human Vκ and Jκ gene segments, which are operably linked to a non-human Igκ constant region. In some embodiments, the non-human animals provided have a germline genome that includes the immunoglobulin loci (or alleles) depicted in the figures provided herein (see, e.g., Figures 1, 2, 3, and / or 4). Such engineered non-human animals provide a source of human antibodies and human antibody fragments, and / or nucleic acids encoding such human antibodies and human antibody fragments, as well as an improved in vivo system suitable for utilizing human Vλ sequences for the production of human therapeutic antibodies.

[0163] As described herein, in certain embodiments, non-human animals are provided having genomes containing multiple human λ light chain gene segments (e.g., Vλ, Jλ, and Cλ) in place of non-human immunoglobulin λ light chain gene segments at endogenous immunoglobulin λ light chain loci, and the non-human animals contain human non-coding intergenic DNA between the human variable region gene segments. In some embodiments, the non-human animals provided herein contain multiple human heavy chain variable region gene segments (i.e., Vλ, Jλ, and Cλ) in place of non-human immunoglobulin λ light chain gene segments at endogenous immunoglobulin λ light chain loci. H , D H and J. H ) and kappa light chain variable region gene segments (e.g., Vκ and Jκ) to non-human heavy chain variable region gene segments (i.e., V H , D H and J. H) and κ light chain variable region gene segments (e.g., Vκ and Jκ) at the endogenous immunoglobulin heavy chain loci and κ light chain loci, respectively. In many embodiments, the human immunoglobulin gene segments (heavy and / or light chain) are engineered with human intergenic DNA (i.e., human non-coding immunoglobulin intergenic DNA) that is naturally associated with the gene segments (i.e., the non-coding genomic DNA is associated with the gene segments that naturally occur at the human immunoglobulin locus in a human cell). Such intergenic DNA can include, for example, promoters, leader sequences, and recombination signal sequences, which enable proper recombination and expression of the human gene segments in the context of the antibody variable domains. Those of skill in the art will understand, upon reading this disclosure, that non-human immunoglobulin loci also contain such non-coding intergenic DNA and that other human or non-human intergenic DNA can be utilized in the construction of such engineered immunoglobulin loci to similarly express human variable domains in the context of antibodies in non-human animals. Such analogously engineered immunoglobulin loci need only contain the human coding sequences (i.e., exons) of the desired gene segments, or combinations of human gene segments, to achieve expression of antibodies containing human variable domains.

[0164] Various aspects of certain embodiments are described in detail in the following sections, each of which may be applicable to any aspect or embodiment described herein. The use of sections is not intended to be limiting, and the use of "or" means "and / or" unless otherwise stated.

[0165] 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 foreign antigens (e.g., viruses, bacteria, etc.). Each immunoglobulin (Ig) consists of two identical heavy chains and two identical light chains, each with two structural components: a variable domain and a constant domain. While the heavy-chain variable domains differ in antibodies produced by different B cells, all antibodies produced by a single B cell or B-cell clone are identical. Both the heavy-chain variable domain and the light-chain variable domain of each antibody contain the antigen-binding region (or antigen-binding site). Immunoglobulins exist in various forms, designated isotypes or classes based on the heavy-chain constant region (or domain) they contain. The heavy-chain constant domain is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The following table summarizes the nine mouse and human antibody isotypes. [Table C]

[0166] Additional isotypes have been identified in other species. Different isotypes have different structural features that give antibodies specialized biological properties and are present in different locations (cells, tissues, etc.) within an animal's body. Initially, B cells produce IgM and IgD, which have identical antigen-binding domains. Upon activation, B cells switch to a different isotype through a process called class switching. Class switching involves changing the constant domain of the antibody produced by the B cell, while the variable domain remains unchanged, thereby preserving the antigen specificity of the original antibody (B cell).

[0167] Two distinct loci (Igκ and Igλ) contain gene segments encoding antibody light chains and exhibit both allelic and isotypic exclusion. + and λ +B cell expression ratios vary by species. For example, humans exhibit a ratio of approximately 60:40 (κ:λ). A ratio of 95:5 (κ:λ) has been observed in mice and rats. Interestingly, a κ:λ ratio of 5:95 has been observed in cats, which contrasts sharply with mice and rats. Several studies have explained the likely reasons behind these observed ratios, proposing both locus complexity (i.e., the number of gene segments, particularly V gene segments) and the efficiency of gene segment rearrangement as rationales. The human immunoglobulin λ light chain locus spans 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 into three clusters (see, for example, Figure 1 in U.S. Pat. No. 9,006,511). The majority of Vλ regions observed in the expressed antibody repertoire are encoded by gene segments contained within the most proximal cluster (i.e., cluster A). The mouse immunoglobulin λ light chain locus is significantly different from the human locus, containing only a few Vλ and Jλ gene segments, depending on the lineage, which are organized into two distinct gene clusters (see, e.g., Figure 2 of U.S. Pat. No. 9,006,511).

[0168] The development of therapeutic antibodies for the treatment of various human diseases has largely focused on the creation of genetically engineered nonhuman animal strains, particularly engineered rodent strains that carry varying amounts of genetic material corresponding to human immunoglobulin genes in their genomes (e.g., reviewed in Bruggemann, M. et al., 2015, Arch. Immunol. Ther. Exp. 63:101-8). Early attempts to create such transgenic rodent strains focused on the integration of portions of the human immunoglobulin locus.This results in the recombination of gene segments in themselves and the production of entirely human heavy and / or light chains, while the endogenous immunoglobulin loci are inactivated (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., 1994, Nature 368:856-9; Taylor, L. D. et al., 1994, Nature 368:856-9). 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; see Kellermann, SA and LL Green, 2002, Cur. Opin. Biotechnol. 13:593-7).In particular, several attempts have involved the integration of human Igλ light chain sequences (see, e.g., U.S. Patent Application Publications 2002 / 0088016 A1, 2003 / 0217373 A1, and 2011 / 0236378 A1; 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). Such attempts have focused on the random integration of yeast artificial chromosomes containing human Vλ, Jλ, and Cλ sequences, thereby generating mouse strains expressing fully human λ light chains (i.e., human variable and human constant). Similar attempts have been made recently 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).

[0169] Still other attempts have involved specifically inserting human Vλ and Jλ gene segments into endogenous rodent Ig light chain loci (κ and λ) and operably linking the human Vλ and Jλ gene segments to endogenous Ig light chain constant regions (see, e.g., U.S. Patent 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 such animals, all of the human Vλ gene segments of clusters A and B and any one to four of the human Jλ gene segments were inserted into the endogenous Igκ and Igλ light chain loci. As a result, we demonstrated that several different human Vλ and Jλ gene segments were properly rearranged at both engineered rodent Ig light chain loci to form functional human Vλ domains, which were expressed in the context of both Cκ and Cλ regions in the light chains of the rodent antibody repertoire (see, e.g., Table 7 and Figures 11-13 of U.S. Pat. No. 9,006,511). In particular, mice with an engineered Igκ light chain locus bearing human Vλ and Jλ gene segments exhibited an approximately 1:1 κ:λ ratio in the spleen (see, e.g., Table 4 of U.S. Pat. No. 9,006,511). Indeed, both engineered mouse strains (i.e., engineered Igκ or engineered Igλ light chain loci) demonstrated that human Vλ domains could be expressed from endogenous Ig light chain loci in rodents, which normally exhibit a large bias in light chain expression (see above). The present invention is based on the recognition that alternative engineered Ig light chain locus structures can be created to maximize the utilization of human Vλ and Jλ gene segments in antibody repertoires against therapeutic targets in non-human animals, particularly compared to non-human animals (e.g., mice and rats) that contain Igλ light chain loci that lack the complexity and stable quality normally associated with human Igλ light chain loci (i.e., present in human cells). Such selectively engineered Ig light chain locus structures provide unique antibody repertoire capabilities resulting from their design.

[0170] The present disclosure particularly describes the successful generation of non-human animals whose germline genomes contain an engineered endogenous Igλ light chain locus, where the light chain locus comprises a plurality of human Vλ, Jλ, and Cλ gene segments, which are operably linked to a non-human Igλ light chain constant region. In particular, the present disclosure specifically describes the successful generation of engineered non-human animals that express antibodies having human variable domains and non-human constant domains, where the antibodies comprise a light chain containing a human Vλ domain. As described herein, expression of such a light chain is achieved by inserting the plurality of human Vλ, Jλ, and Cλ gene segments into an endogenous Igλ light chain locus (or allele). Also as described herein, in some embodiments, the provided non-human animals are engineered such that expression of a light chain containing an endogenous Vλ domain is inactivated (e.g., by gene deletion). Thus, the present disclosure encompasses, in at least some embodiments, the development of improved in vivo systems for human antibody production by providing engineered non-human animals containing selectively engineered Igλ light chain loci that give rise to expressed antibody repertoires that contain human Vλ domains.

[0171] DNA insertion Typically, a polynucleotide molecule containing a human Igλ light chain sequence (e.g., Vλ, Jλ, Cλ and Igλ enhancer) or a portion thereof is inserted into a vector, preferably a DNA vector, for replicating the polynucleotide molecule in a host cell.

[0172] Human Igλ light chain sequences can be cloned directly from known sequences or sources (e.g., libraries), or can be synthesized from germline sequences designed in silico based on published sequences available from GeneBank or other publicly available databases (e.g., IMGT). Alternatively, bacterial artificial chromosome (BAC) libraries can also provide immunoglobulin DNA sequences of interest (e.g., human Vλ gene segments, human Jλ-Cλ gene segment pairs, human Eλ regions or sequences, and combinations thereof). BAC libraries can contain insert sizes of 100-150 kb, and can even contain 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 entirety). For example, human BAC libraries with an average insert size of 164-196 kb have been reported (Osoegawa, K. et al., 2001, Genome Res. 11(3):483-96; Osoegawa, K. et al., 1998, Genomics 52:1-8, Article No. GE985423). Human and mouse genomic BAC libraries have been constructed and are commercially available (for example, from Thermo Fisher Scientific). Genomic BAC libraries can also be used as a source of immunoglobulin DNA sequences and transcriptional regulatory regions.

[0173] Alternatively, immunoglobulin DNA sequences may be isolated, cloned, and / or transferred from yeast artificial chromosomes (YACs). For example, the nucleotide sequence of the human Ig λ light chain locus has been determined (see, e.g., Dunham, I. et al., 1999, Nature 402:489-95). Furthermore, YACs have been used in the past to assemble human Ig 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). The entire Ig λ light chain locus (human or rodent) can be cloned and contained within several YACs. If multiple YACs are used and contain regions of overlapping homology, 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 arms of the YAC may be further modified by refinement with a mammalian selection cassette to aid in the introduction of the construct into embryonic stem cells or embryos by methods known in the art and / or described herein.

[0174] The DNA and amino acid sequences of human Igλ light chain gene segments for use in constructing the engineered Igλ light chain loci described herein may be obtained from public databases (e.g., GeneBank, IMGT, etc.) and / or published antibody sequences. DNA inserts containing human Igλ light chain gene segments, in some embodiments, include one or more human Igλ light chain enhancer sequences (or regions). In some embodiments, the DNA inserts include a human Igλ light chain enhancer sequence (or region) that includes one or more sequence elements, e.g., one, two, three, etc. In some embodiments, the DNA inserts include a human Igλ light chain enhancer sequence (or region), which is also referred to as a human Eλ with three distinct sequence elements. Thus, in some embodiments, the human Eλ described herein are modular, with one or more sequence elements functioning together as an enhancer sequence (or region). In some embodiments, the DNA insert containing a human Igλ light chain enhancer sequence comprises a human Igλ light chain enhancer sequence, which is operably linked to a non-human Igλ light chain sequence (e.g., a non-human Igλ light chain constant region sequence). In some embodiments, the DNA insert containing a human Igλ light chain enhancer sequence comprises a human Igλ light chain enhancer sequence, which is operably linked to a non-human Igλ light chain sequence (e.g., a non-human Igλ light chain constant region sequence), and is operably linked to one or more human Vλ gene segments, one or more human Jλ-Cλ gene segment pairs, and / or one or more human Jλ gene segments. In some embodiments, the DNA insert containing a human Ig λ light chain enhancer sequence comprises a human Ig λ light chain enhancer sequence, which is operably linked to a non-human Ig λ light chain sequence (e.g., a non-human Ig λ light chain constant region sequence), one or more human Vλ gene segments, one or more human Jλ gene segments, and one or more human Cλ gene segments.

[0175] The DNA insert can be prepared using methods known in the art. For example, the DNA insert can be prepared as part of a larger plasmid. Such preparation allows for efficient cloning and selection of the correct construct, as is known in the art. The DNA insert containing all or part of the human Ig λ light chain sequence described herein can be positioned between convenient restriction enzyme sites on the plasmid, allowing it to be easily isolated from the remaining plasmid sequences for incorporation into the desired non-human animal.

[0176] The various methods used in the preparation of the plasmids and transformation of host organisms are known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombinant methods, 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 See Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al., Cold Spring Harbor Laboratory Press: 1989.

[0177] Targeting Vector A targeting vector can be used to introduce a DNA insert into a genomic target locus and can include a DNA insert and homologous arms flanking the DNA insert. A targeting vector can be linear or circular, and can be single-stranded or double-stranded. A targeting vector can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). For ease of reference, homologous arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homologous arms. This terminology refers to the relative position of the homologous arms to the DNA insert within the targeting vector. The 5' and 3' homologous arms correspond to regions within the targeting locus or within another targeting vector, and are referred to herein as the "5' target sequence" and "3' target sequence," respectively. In some embodiments, homologous arms can also function as 5' or 3' target sequences.

[0178] In some embodiments, the methods described herein utilize two, three, or more targeting vectors that can recombine with each other. In various embodiments, the targeting vectors are large targeting vectors (LTVECs) described elsewhere herein. In such embodiments, the first, second, and third targeting vectors each comprise a 5' and a 3' homologous arm. The 3' homologous arm of the first targeting vector comprises a sequence that overlaps with the 5' homologous arm of the second targeting vector (i.e., an overlapping sequence), allowing homologous recombination between the first and second LTVECs.

[0179] In the dual targeting method, the 5' homologous arm of the first targeting vector and the 3' homologous arm of the second targeting vector are homologous to corresponding segments (i.e., target sequences) within the target genome locus, which promotes homologous recombination between the first and second targeting vectors and the corresponding genome segments to modify the target genome locus.

[0180] In the triple targeting method, the 3' homologous arm of the second targeting vector contains a sequence that overlaps with the 5' homologous arm of the third targeting vector (i.e., overlapping sequence), which allows homologous recombination between the second and third LTVEC. The 5' homologous arm of the first targeting vector and the 3' homologous arm of the third targeting vector are homologous to corresponding segments (i.e., target sequences) within the target genome locus, which promotes homologous recombination between the first and third targeting vectors and the corresponding genome segments to modify the target genome locus.

[0181] A homologous arm and a target sequence, or two homologous arms, "correspond" or "are corresponding" to one another if the two regions share a sufficient level of sequence homology with each other to act as substrates for a homologous recombination reaction. The term "homologous" includes DNA sequences that are identical to or share sequence identity with the corresponding sequence. The sequence identity between a given target sequence and the corresponding homologous arm (i.e., overlapping sequence) or two homologous arms present in a targeting vector may be any degree of sequence identity that allows homologous recombination to occur. By way of example, the amount of sequence identity shared by a homologous arm (or fragment thereof) of a targeting vector and a target sequence of another targeting vector or a target sequence (or fragment thereof) of a target genomic locus can be, for example and without limitation, 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, thereby allowing the sequences to undergo homologous recombination.

[0182] Furthermore, the corresponding homologous region between the homologous arm and the corresponding target sequence may be a region of any length that is 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 homology region that is, for example but not limited to, at least 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 or longer (e.g., as described elsewhere herein), such that the homology arm has sufficient homology to undergo homologous recombination with a corresponding target sequence within a target genomic locus in a cell or in another targeting vector. In some embodiments, a given homology arm and / or corresponding target sequence comprises a corresponding region of homology that is, for example but not limited to, at least 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 or longer (e.g., as described elsewhere herein), such that the homology arm has sufficient homology to undergo homologous recombination with a corresponding target sequence within a target genomic locus in a cell or in another targeting vector.

[0183] The overlapping sequence of the 3' homologous arm of a first targeting vector and the 5' homologous arm of a second targeting vector, or the overlapping sequence of the 3' homologous arm of a second targeting vector and the 5' homologous arm of a third targeting vector, may be of any length sufficient to promote homologous recombination between the targeting vectors. For example, a given overlapping sequence of a homologous arm may comprise a corresponding overlapping region that is at least about 1-5 kb, 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 or longer, such that the overlapping region of the homologous arm has sufficient homology to undergo homologous recombination with a corresponding overlapping sequence in another targeting vector. In some embodiments, a given overlapping sequence of a homologous arm comprises an overlapping region that is at least about 1-100 kb, 5-100 kb, 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 or longer, such that the overlapping region of the homologous arm has sufficient homology to undergo homologous recombination with a corresponding overlapping sequence in another targeting vector. In some embodiments, the overlapping sequence is between 1 kb and 5 kb. In some embodiments, the overlapping sequence is between about 1 kb and about 70 kb. In some embodiments, the overlapping sequence is between about 10 kb and about 70 kb. In some embodiments, the overlapping sequence is between about 10 kb and about 50 kb. 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 or more to about 5 kb or less, about 5 kb or more to about 10 kb or less, about 10 kb or more to about 15 kb or less, about 15 kb or more to about 20 kb or less, about 20 kb or more to about 25 kb or less, about 25 kb or more to about 30 kb or less, about 30 kb or more to about 35 kb or less, about 35 kb or more to about 40 kb or less, about 40 kb or more to about 45 kb or less, about 45 kb or more to about 50 kb or less, about 50 kb or more to about 60 kb or less, about 60 kb or more to about 70 kb or less, about 70 kb or more to about 80 kb or less, about 80 kb or more The length may be from about 20 kb to about 90 kb, from about 90 kb to about 100 kb, from about 100 kb to about 120 kb, from about 120 kb to about 140 kb, from about 140 kb to about 160 kb, from about 160 kb to about 180 kb, from about 180 kb to about 200 kb, from about 200 kb to about 220 kb, from about 220 kb to about 240 kb, from about 240 kb to about 260 kb, from about 260 kb to about 280 kb, or from about 280 kb to about 300 kb. As an example, the overlapping sequence may be from about 20 kb to about 60 kb. Alternatively, the overlapping sequences may 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.

[0184] In some embodiments, the homologous arms may correspond to a locus native to the cell (e.g., a targeted locus), or they may correspond to a region of a heterologous or foreign DNA segment, such as a transgene, expression cassette, or heterologous or exogenous DNA region integrated into the cell genome. Alternatively, in some embodiments, the homologous arms may correspond to a region on a targeting vector in the cell. In some embodiments, the homologous 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 engineered region contained in a suitable host cell. Furthermore, the homologous 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 some embodiments, the homology arms of the targeting vector correspond to a locus derived from, heterologous to, or exogenous to a prokaryotic cell, 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), livestock, agricultural mammal, or any other organism of interest. In some embodiments, the homology arms correspond to a locus in a cell that cannot be targeted by conventional methods, or that can be targeted imprecisely or with significantly reduced efficiency in the absence of a nick or double-strand break induced by a nuclease agent (e.g., Cas protein). In some embodiments, the homology arms are derived from synthetic DNA.

[0185] In some embodiments, one of the 5' or 3' homology arms of a targeting vector corresponds to a targeted genomic locus, and the other of the 5' or 3' homology arm corresponds to a region on another targeting vector.

[0186] In some embodiments, the 5' and 3' homologous arms of the targeting vector correspond to the genome to be targeted. Alternatively, the homologous arms may be derived from related genomes. For example, the genome to be targeted is the genome of a first mouse strain, and the targeting arms are derived from the genome of a second mouse strain, in which case the first and second strains are different. In some embodiments, the homologous arms are derived from the genomes of the same animal or the genomes of the same strain. For example, the genome to be targeted is the genome of a first mouse strain, and the targeting arms are derived from the genome of the same mouse or mouse of the same mouse strain.

[0187] The homology arms of the targeting vector may be of any length sufficient to promote a homologous recombination event with the corresponding target sequence, for example, at least 1 kb to 5 kb, 5 kb to 10 kb, 5 kb to 15 kb, 5 kb to 20 kb, 5 kb to 25 kb, 5 kb to 30 kb, 5 kb to 35 kb, 5 kb to 40 kb, 5 kb to 45 kb, 5 kb to 50 kb, or longer. The length may be any length including, but not limited to, 5 kb to 55 kb, 5 kb to 60 kb, 5 kb to 65 kb, 5 kb to 70 kb, 5 kb to 75 kb, 5 kb to 80 kb, 5 kb to 85 kb, 5 kb to 90 kb, 5 kb to 95 kb, 5 kb to 100 kb, 100 kb to 200 kb, or 200 kb to 300 kb, or longer. In some embodiments, the homologous arms of the targeting vector are at least 1 kb to 100 kb, 5 kb to 100 kb, 10 kb to 100 kb, 15 kb to 100 kb, 20 kb to 100 kb, 25 kb to 100 kb, 30 kb to 100 kb, 35 kb to 100 kb, 40 kb to 100 kb, 45 kb to 100 kb, 50 kb to 100 kb The targeting vectors have a length sufficient to promote homologous recombination events with corresponding target sequences that are 55 kb to 100 kb, 60 kb to 100 kb, 65 kb to 100 kb, 70 kb to 100 kb, 75 kb to 100 kb, 80 kb to 100 kb, 85 kb to 100 kb, 90 kb to 100 kb, or 95 kb to 100 kb in length, or longer. As described herein, large targeting vectors can employ longer targeting arms.

[0188] Nuclease agents (e.g., CRISPR / Cas systems) can be employed in combination with targeting vectors to promote modification of target loci (e.g., Igλ light chain loci). Such nuclease agents can promote homologous recombination between the targeting vector and the target locus. When nuclease agents are employed in combination with targeting vectors, the targeting vector contains 5' and 3' homologous arms corresponding to the 5' and 3' target sequence positioned sufficiently close to the nuclease cleavage site to promote the occurrence of a homologous recombination event between the target sequence and the homologous arms upon a nick or double-strand break at the nuclease cleavage site. The term "nuclease cleavage site" includes DNA sequences where a nick or double-strand break is generated by a nuclease agent (e.g., a Cas9 cleavage site). Target sequences within a target locus corresponding to the 5' and 3' homology arms of a targeting vector are "located sufficiently close" to a nuclease cleavage site if the distance is such that, upon 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 facilitated. Thus, in some embodiments, the target sequences corresponding to the 5' and / or 3' homology arms of a targeting vector are within 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, a nuclease cleavage site is immediately adjacent to at least one or both of the target sequences.

[0189] The spatial relationship of the target sequence and the nuclease cleavage site corresponding to the homologous arms of the targeting vector 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 be adjacent to the nuclease cleavage site.

[0190] The use of a targeting vector (including, for example, a large targeting vector) in combination with a nuclease substance can increase targeting efficiency compared to the use of the targeting vector alone. For example, when a targeting vector is used in combination with a nuclease substance, the targeting efficiency of the targeting vector can be increased by 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, at least 10-fold, or within a range consisting of integers thereof, for example, 2-10-fold, compared to the use of the targeting vector alone.

[0191] Some targeting vectors are "large targeting vectors" or "LTVECs," which include targeting vectors containing homologous arms corresponding to or derived from nucleic acid sequences larger than those typically used in other methods aimed at achieving homologous recombination in cells. LTVECs may be, for example, at least 10 kb in length, or the combined total of the 5' and 3' homologous arms may be, for example, at least 10 kb. LTVECs also include targeting vectors containing DNA inserts larger than those typically used in other methods aimed at achieving homologous recombination in cells. For example, LTVECs enable the modification of large loci that cannot be accommodated in conventional plasmid-based targeting vectors due to size limitations. For example, the targeted locus may be a cellular locus that cannot be targeted by conventional methods, or a cellular locus that can be targeted imprecisely or with significantly lower efficiency in the absence of a nick or double-strand break induced by a nuclease agent (e.g., a Cas protein) (i.e., the 5' and 3' homologous arms can correspond to the locus).

[0192] In some embodiments, the methods described herein employ 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.

[0193] Examples of LTVEC include the vector derived from bacterial artificial chromosome (BAC), human artificial chromosome, or yeast artificial chromosome (YAC).Examples of LTVEC and its preparation method are described in, for example, United States Patent No. 6,586,251, No. 6,596,541 and No. 7,105,348, and International Patent Application Publication No. 2002 / 036789, each of which is incorporated herein by reference in its entirety.LTVEC can be linear or circular.

[0194] The LTVEC may be of any length, including, for example, about 20 kb to about 300 kb, about 20 kb to about 50 kb, about 50 kb to about 75 kb, about 75 kb to about 100 kb, about 100 kb to 125 kb, about 125 kb to about 150 kb, about 150 kb to about 175 kb, about 175 kb to about 200 kb, about 200 kb to about 225 kb, about 225 kb to about 250 kb, about 250 kb to about 275 kb, or about 275 kb to about 300 kb. Alternatively, the LTVEC may be at least 10 kb, at least 15 kb, at least 20 kb, at least 30 kb, at least 40 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 150 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb or more. The size of the LTVEC may, in some embodiments, be too large to screen for targeting events using conventional assays, such as Southern blotting or long-range (e.g., 1 kb to 5 kb) PCR.

[0195] In some embodiments, the LTVEC may be from about 5 kb to about 200 kb, from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 30 kb, from about 30 kb to about 40 kb, from about 40 kb to about 50 kb, from about 60 kb to about 70 kb, from about 80 kb to about 90 kb, from about 90 kb to about 100 kb, or from about 100 kb or less. and DNA insertions ranging from about 110 kb to about 110 kb, from about 120 kb to about 130 kb, from about 130 kb to about 140 kb, from about 140 kb to about 150 kb, from about 150 kb to about 160 kb, from about 160 kb to about 170 kb, from about 170 kb to about 180 kb, from about 180 kb to about 190 kb, or from about 190 kb to about 200 kb. In some embodiments, the DNA insertion may be in the range of, for example, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 250 kb, about 250 kb to about 300 kb, about 300 kb to about 350 kb, or about 350 kb to about 400 kb. In some embodiments, the LTVEC comprises a DNA insert in the range of, for example, about 400 kb to about 450 kb, about 450 kb to about 500 kb, about 500 kb to about 550 kb, about 550 kb to about 600 kb, about 600 kb to about 650 kb, about 650 kb to about 700 kb, about 700 kb to about 750 kb, or about 750 kb to about 800 kb.

[0196] In some embodiments, the combined length of the 5' homologous arm and the 3' homologous arm of the LTVEC is at least 10 kb, and in some embodiments, the 5' homologous arm of the LTVEC is in the range of about 1 kb to about 100 kb, and / or the 3' homologous arm of the LTVEC is in the range of about 1 kb to about 100 kb. The total length of the 5' and 3' homologous arms may be, for example, about 1 kb or more to about 5 kb or less, about 5 kb or more to about 10 kb or less, about 10 kb or more to about 20 kb or less, about 20 kb or more to about 30 kb or less, about 30 kb or more to about 40 kb or less, about 40 kb or more to about 50 kb or less, about 50 kb or more to about 60 kb or less, about 60 kb or more to about 70 kb or less, about 70 kb or more to about 80 kb or less, about 80 kb or more to about 90 kb or less, or about 90 kb or more to about 100 kb. It may be about 100 kb or more to about 110 kb or less, about 110 kb or more to about 120 kb or less, about 120 kb or more to about 130 kb or less, about 130 kb or more to about 140 kb or less, about 140 kb or more to about 150 kb or less, about 150 kb or more to about 160 kb or less, about 160 kb or more to about 170 kb or less, about 170 kb or more to about 180 kb or less, about 180 kb or more to about 190 kb or less, or about 190 kb or more to about 200 kb or less. Alternatively, each homology arm may in some embodiments be at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 30 kb, at least 40 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 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, or at least 200 kb.Similarly, the total length of the 5' and 3' homology arms may in some embodiments be at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 30 kb, at least 40 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 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, or at least 200 kb.

[0197] In some embodiments, the LTVEC and DNA insertion may extend from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, or from about 100 kb to about 150 kb, from about 150 kb to about 200 kb, from about 200 kb to about 300 kb, or from about 300 kb or more at the target locus. The vector is designed to enable deletion of up to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 600 kb, about 600 kb to about 700 kb, about 700 kb to about 800 kb, or about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, or about 2.5 Mb to about 3 Mb of endogenous sequence. Alternatively, the deletion may be from about 3Mb to about 4Mb, from about 4Mb to about 5Mb, from about 5Mb to about 10Mb, from about 10Mb to about 20Mb, from about 20Mb to about 30Mb, from about 30Mb to about 40Mb, from about 40Mb to about 50Mb, from about 50Mb to about 60Mb, from about 60Mb to about 70Mb, from about 70Mb to about 80Mb, from about 80Mb to about 90Mb, or from about 90Mb to about 100Mb. Alternatively, the deletion may be at least 10 kb, at least 20 kb, at least 30 kb, at least 40 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 150 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb or more.

[0198] In some embodiments, the LTVEC and DNA insert are designed to allow insertion of an exogenous nucleic acid sequence into a target locus in the range of, for example, from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, from about 150 kb to about 200 kb, from about 200 kb to about 250 kb, from about 250 kb to about 300 kb, from about 300 kb to about 350 kb, or from about 350 kb to about 400 kb. Alternatively, the insertion may in some embodiments be in the range of about 400 kb or more to about 450 kb or less, about 450 kb or more to about 500 kb or less, about 500 kb or more to about 550 kb or less, about 550 kb or more to about 600 kb or less, about 600 kb or more to about 650 kb or less, about 650 kb or more to about 700 kb or less, about 700 kb or more to about 750 kb or less, or about 750 kb or more to about 800 kb. Alternatively, the insertion may in some embodiments be at least 10 kb, at least 20 kb, at least 30 kb, at least 40 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 150 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb or more.

[0199] In yet another example, the DNA insertion and / or the region of the endogenous locus that is changed, deleted, targeted, modified, engineered, etc. is at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides, or at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb or more. In some embodiments, the DNA insertion and / or the region of the endogenous locus to be changed, deleted, targeted, modified, engineered, etc., is between nucleotides and 20 kb, between 200 nucleotides and 20 kb, between 300 nucleotides and 20 kb, between 400 nucleotides and 20 kb, between 500 nucleotides and 20 kb, between 600 nucleotides and 20 kb, between 700 nucleotides and 20 kb, between 800 nucleotides and 20 kb, between 900 nucleotides and 20 kb, between 1000 nucleotides and 20 kb, between 1200 nucleotides and 20 kb, between 1300 nucleotides and 20 kb, between 1400 nucleotides and 20 kb, between 1500 nucleotides and 20 kb, between 1600 nucleotides and 20 kb, between 1700 nucleotides and 20 kb, between 2100 nucleotides and 20 kb, between 2200 nucleotides and 20 kb, between 2300 nucleotides and 20 kb, between 2400 nucleotides and 20 kb, between 2500 nucleotides and 20 kb, between 2600 nucleotides and 20 kb, between 2700 nucleotides and 20 kb, between 2800 nucleotides and 20 kb, between 3000 nucleotides and 20 kb, between 32 ... ~20kb, 1kb~20kb, 2kb~20kb, 3kb~20kb, 4kb~20kb, 5kb~20kb, 6kb~20kb, 7kb~20kb, 8kb~20kb, 9kb~20kb, 10kb~20kb, 11kb~20kb, 12kb~20kb, 13kb~20kb, 14kb~20kb, 15kb~20kb, 16kb~20kb, 17kb~20kb, 18kb~20kb, or 19kb~20kb.In some embodiments, the DNA insertion and / or the region of the endogenous locus to be changed, deleted, targeted, modified, engineered, etc. may be between 100 nucleotides and 19 kb, between 100 nucleotides and 18 kb, between 100 nucleotides and 17 kb, between 100 nucleotides and 16 kb, between 100 nucleotides and 15 kb, between 100 nucleotides and 14 kb, between 100 nucleotides and 13 kb, between 100 nucleotides and 12 kb, between 100 nucleotides and 11 kb, between 100 nucleotides and 10 kb, between 100 nucleotides and 9 kb, between 100 nucleotides and 8 kb, between 100 nucleotides and 10 ... 100 nucleotides to 6 kb, 100 nucleotides to 5 kb, 100 nucleotides to 4 kb, 100 nucleotides to 3 kb, 100 nucleotides to 2 kb, 100 nucleotides to 1 kb, 100 nucleotides to 900 nucleotides, 100 nucleotides to 800 nucleotides, 100 nucleotides to 700 nucleotides, 100 nucleotides to 600 nucleotides, 100 nucleotides to 500 nucleotides, 100 nucleotides to 400 nucleotides, 100 nucleotides to 300 nucleotides, or 100 nucleotides to 200 nucleotides. In some embodiments, the DNA insertion and / or the region of the endogenous locus that is changed, deleted, targeted, modified, engineered, etc. is between 200 nucleotides and 19 kb, between 300 nucleotides and 18 kb, between 400 nucleotides and 17 kb, between 500 nucleotides and 16 kb, between 600 nucleotides and 15 kb, between 700 nucleotides and 14 kb, between 800 nucleotides and 13 kb, between 900 nucleotides and 12 kb, between 1 kb and 11 kb, between 2 kb and 10 kb, between 3 kb and 9 kb, between 4 kb and 8 kb, between 5 kb and 7 kb, or between 5 kb and 6 kb.

[0200] Non-human animals provided In one embodiment, a non-human animal is provided that expresses an antibody containing a light chain comprising all or a portion of a human Igλ light chain sequence, wherein the light chain results from the integration of genetic material corresponding to at least a portion of a human Igλ light chain locus, where the light chain locus encodes at least a human Vλ domain (i.e., a rearranged human Vλ-Jλ sequence) in place of the corresponding non-human Igλ light chain sequence in the germline genome of the non-human animal. Suitable examples described herein include, but are not limited to, rodents, particularly rats or mice.

[0201] In some embodiments, the human Igλ light chain sequence comprises genetic material from a human Igλ light chain locus, wherein the human Igλ light chain sequence encodes an immunoglobulin light chain comprising a coding portion of genetic material from a human Igλ light chain locus. In some embodiments, the human Igλ light chain sequences described herein comprise at least one human Vλ gene segment and at least one human Jλ gene segment, and one or more sequences (e.g., recombination signal sequences) necessary to promote rearrangement of the at least one human Vλ gene segment and the at least one human Jλ gene segment to form a functional rearranged human Vλ-Jλ sequence encoding a human Vλ domain. In many embodiments, the human Igλ light chain sequence comprises multiple human Vλ gene segments and one or more sequences necessary to promote rearrangement of the human Vλ gene segments and at least one human Jλ gene segment. In many embodiments, the human Igλ light chain sequences described herein are genomic sequences of a human Igλ light chain locus (e.g., isolated from a bacterial artificial chromosome and / or cloned) and contain multiple human Vλ gene segments in a germline configuration. In some embodiments, the human Igλ light chain sequence comprises human Vλ, Jλ, and Cλ sequences in a germline configuration (i.e., the human Vλ, Jλ, and Cλ sequences are present in an Igλ light chain locus in a human cell). In some embodiments, the human Igλ light chain sequence is or comprises a human sequence depicted in a figure (see, e.g., Figures 1-4). In some embodiments, the human Igλ light chain sequence encodes all or part of an Igλ light chain polypeptide, which is present in an immunoglobulin, particularly an immunoglobulin expressed by a human B cell. Also provided are non-human animals, embryos, cells, and targeting constructs for generating non-human animals, non-human embryos, and cells that contain the human Igλ light chain sequences in place of the corresponding non-human Igλ light chain sequences (e.g., endogenous rodent Igλ light chain loci).

[0202] In some embodiments, the human Igλ light chain sequence is inserted into the germline genome of the non-human animal in place of the corresponding non-human Igλ light chain sequence. In some embodiments, the human Igλ light chain sequence is inserted upstream of the non-human Igλ light chain sequence (e.g., the non-human Igλ light chain constant region sequence). In some embodiments, the human Igλ light chain sequence is inserted in the middle of one or more non-human Igλ light chain sequences, such that the human Igλ light chain sequence is juxtaposed with the non-human Igλ light chain sequence (see, e.g., Figures 1, 2, 3, and / or 4).

[0203] In some embodiments, one or more non-human Igλ light chain sequences (or portions thereof) at the non-human Igλ light chain locus are not deleted. In some embodiments, one or more non-human Igλ light chain sequences (e.g., Vλ, Jλ, and / or Cλ) at the non-human Igλ light chain locus are altered, moved, disrupted, deleted, or replaced with human Igλ light chain sequences (e.g., one or more human Vλ gene segments, one or more human Jλ gene segments, one or more human Cλ gene segments, or a combination thereof), particularly as described herein, and the human light chain sequences are operably linked to a non-human Igλ light chain constant region and one or more enhancers and / or regulators at the non-human Igλ light chain locus. In some embodiments, all or substantially all of the non-human Igλ light chain locus is replaced with one or more human Igλ light chain sequences (described herein), and the one or more human light chain sequences are operably linked to a non-human Igλ light chain constant region and one or more non-human Igλ light chain enhancers and / or regulatory elements of the non-human Igλ light chain locus. In some embodiments, one or more non-human Igλ light chain constant region genes are not deleted or replaced in the non-human animal comprising the human Igλ light chain sequences described herein. As a non-limiting example, in instances where a human Igλ light chain sequence is inserted into a non-human Igλ light chain locus, the insertion is made in such a way as to maintain the integrity of the non-human Igλ light chain sequence (e.g., the non-human Igλ light chain constant region and / or the non-human Igλ light chain enhancer region or sequence) near the insertion point. Thus, such a non-human animal has a wild-type Igλ light chain constant region. In some embodiments, the non-human Igλ light chain locus that is altered, moved, disrupted, deleted, replaced, or engineered with one or more human Igλ light chain sequences described herein is a murine (e.g., mouse or rat) Igλ light chain locus. In some embodiments, the human Igλ light chain sequence is inserted into one copy (i.e., allele) of the two copies of the non-human Igλ light chain locus, resulting in a non-human animal that is heterozygous for the human Igλ light chain sequence.In some embodiments, a non-human animal is provided that is homozygous for an Igλ light chain locus, wherein the light chain locus comprises a human Igλ light chain sequence described herein.

[0204] In some embodiments, the engineered non-human Igλ light chain loci described herein comprise human Vλ, Jλ, and Cλ gene segments, which are operably linked to a non-human Igλ light chain constant region and one or more non-human Igλ light chain enhancers and / or regulators. In some embodiments, the engineered non-human Igλ light chain loci described herein comprise human Vλ, Jλ, and Cλ gene segments, which are operably linked to a non-human Igλ light chain constant region, one or more non-human Igλ light chain enhancers and / or regulators, and one or more human Igλ light chain enhancers and / or regulators.

[0205] In some embodiments, a non-human animal contains an engineered Igλ light chain locus described herein, with the light chain locus randomly integrated into its genome (e.g., as part of a randomly integrated human Igλ light chain sequence). Such a non-human animal can thus be described as having a human Igλ light chain transgene containing multiple human Vλ, Jλ, and / or Cλ gene segments, configured such that the human Vλ, Jλ, and / or Cλ gene segments can rearrange and encode all or part of an Igλ light chain of an antibody in the expression repertoire of the non-human animal. The engineered Igλ light chain locus or transgene described herein can be detected using a variety of methods, including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or allelic gain or loss assays. In some embodiments, the non-human animal described herein is heterozygous for the engineered Igλ light chain locus described herein. In some embodiments, the non-human animals described herein are hemizygous for an engineered Igλ light chain locus described herein. In some embodiments, the non-human animals described herein comprise one or more copies of an engineered Igλ light chain locus or transgene described herein. In some embodiments, the non-human animals described herein comprise an Igλ light chain locus depicted in a figure (see, e.g., Figures 1, 2, 3, and / or 4).

[0206] In some embodiments, compositions and methods are provided for producing non-human animals whose germline genomes comprise an engineered Igλ light chain locus, the light chain locus comprising one or more human Igλ light chain sequences (e.g., human Vλ, Jλ, and / or Cλ gene segments) in place of non-human Igλ light chain sequences, the human light chain sequences comprising human Igλ light chain encoding sequences comprising particular polymorphic human Vλ, Jλ, and / or Cλ segments, including compositions and methods for producing non-human animals that express antibodies comprising Igλ light chains comprising human variable domains and human or non-human constant domains assembled from an Igλ light chain locus containing human Vλ, Jλ, and Cλ segments operably linked to a non-human Igλ light chain constant region. In some embodiments, compositions and methods are also provided for producing non-human animals that express such antibodies under the control of endogenous enhancers and / or endogenous regulatory sequences. In some embodiments, compositions and methods for producing non-human animals that express such antibodies under the control of heterologous enhancers and / or heterologous regulatory sequences are also provided.

[0207] In one embodiment, the methods described herein involve inserting a sequence encoding all or part of a human Igλ light chain upstream of a non-human Igλ light chain constant region (e.g., a murine Cλ region), thereby expressing an antibody characterized by the presence of a light chain containing at least a human Vλ domain, and in some embodiments, a light chain containing human Vλ and Cλ domains, and which is expressed both on the surface of B cells and in the serum of the non-human animal.

[0208] In some embodiments, the method involves sequential insertion of genetic material corresponding to a human Igλ light chain locus. In some embodiments, the genetic material corresponding to a human Igλ light chain locus can be synthetic or genomic (e.g., cloned from a bacterial artificial chromosome). In some embodiments, the genetic material corresponding to a human Igλ light chain locus can be designed from a published source and / or from a bacterial artificial chromosome, whereby the genetic material contains human Vλ, Jλ, and / or Cλ segments in an orientation different from that present in the human Igλ light chain locus, but where the material is still in an orientation such that the human Vλ, Jλ, and / or Cλ segments are rearranged to still encode a functional Igλ light chain. By way of example, genetic material corresponding to a human Igλ light chain locus can be designed using the guidance provided herein for constructing a human Igλ light chain sequence, where the human light chain sequence contains human Vλ, Jλ, and / or Cλ segments in an order and / or configuration different from that present in the human Igλ light chain locus in a human cell. In such instances, the content of human Vλ, Jλ, and / or Cλ segments will be identical to the corresponding segments in a human cell, but the order and organization will be different. When constructing a human Igλ light chain locus for the purposes of generating the non-human animals described herein, essential recombination signal sequences are provided so that the human sequences can properly rearrange to form a functional Igλ light chain. Guidance regarding the germline configuration of human Igλ light chain segments and sequences required for proper recombination can be found in Molecular Biology of B Cells, London: Elsevier Academic Press, 2004, Eds. Honjo, T., Alt, F.W., Neuberger, M. Chapters 4 (pp. 37-59) and 5 (pp. 61-82), which are incorporated herein by reference in their entirety.

[0209] In some embodiments, serial insertion comprises multiple insertions of portions of heterologous genetic material in a single ES cell clone, hi some embodiments, serial insertion comprises sequential insertions of portions of heterologous genetic material in successive ES cell clones.

[0210] In some embodiments, the method comprises inserting approximately 11,822 bp of DNA downstream of a murine (e.g., mouse or rat) Cλ1 region, whereby the DNA is operably linked to the murine (e.g., mouse or rat) Cλ1 region, and the DNA comprises one or more human Ig λ light chain enhancer regions (or sequences). In some embodiments, the method comprises inserting approximately 11,822 bp of DNA comprising three human Ig λ light chain enhancer regions (or sequences), whereby the three human Ig λ light chain enhancer regions (or sequences) are inserted downstream of the murine (e.g., mouse or rat) Cλ region.

[0211] In some embodiments, the method comprises inserting approximately 125,473 bp of DNA upstream of a murine (e.g., mouse or rat) Cλ1 region, whereby the DNA is operably linked to the murine (e.g., mouse or rat) Cλ1 region, the DNA comprising human Vλ gene segments Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, human Jλ-Cλ segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6, and human Jλ gene segment Jλ7. In some embodiments, the method comprises inserting approximately 11,822 bp of DNA comprising one or more human Ig λ light chain enhancer regions (or sequences), whereby the one or more human Ig λ light chain enhancer regions (or sequences) are inserted downstream of the murine (e.g., mouse or rat) Cλ1 region.

[0212] In some embodiments, the method includes inserting approximately 171,458 bp of DNA upstream of a murine (e.g., mouse or rat) Cλ1 region, whereby the DNA is operably linked to the murine (e.g., mouse or rat) Cλ1 region, and the DNA includes human Vλ gene segments Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, and Vλ3-27. In some embodiments, the method includes inserting approximately 171,458 bp of DNA upstream of a human Vλ3-10 gene segment operably linked to a murine (e.g., mouse or rat) Cλ1 region, the DNA including human Vλ gene segments Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, and Vλ3-27.

[0213] In some embodiments, the method includes inserting approximately 121,188 bp of DNA upstream of a murine (e.g., mouse or rat) Cλ1 region, whereby the DNA is operably linked to the murine (e.g., mouse or rat) Cλ1 region, and the DNA includes human Vλ gene segments Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, and Vλ5-52. In some embodiments, the methods include inserting approximately 121,188 bp of DNA upstream of a human Vλ3-27 gene segment operably linked to a murine (e.g., mouse or rat) Cλ1 region, the DNA including human Vλ gene segments Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, and Vλ5-52.

[0214] In some embodiments, the method includes inserting approximately 121,188 bp of DNA upstream of a murine (e.g., mouse or rat) Cλ1 region, whereby the DNA is operably linked to the murine (e.g., mouse or rat) Cλ1 region, the DNA comprising human Vλ gene segments Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, and Vλ5-52, and the DNA comprises a homology arm comprising sequences 5' of a mouse Vλ2 gene segment. In some embodiments, the methods include inserting approximately 121,188 bp of DNA upstream of a human Vλ3-27 gene segment operably linked to a murine (e.g., mouse or rat) Cλ1 region, the DNA comprising human Vλ gene segments Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, and Vλ5-52, and the DNA comprising homologous arms comprising sequences 5' of a mouse Vλ2 gene segment, thereby inducing a deletion of a mouse Igλ genomic sequence (e.g., an Igλ light chain locus) upon homologous recombination with the DNA fragment.

[0215] Insertion of additional human Vλ, Jλ, and / or Cλ gene segments can be performed using the methods described herein, thereby further supplementing the diversity of the engineered Igλ light chain locus. For example, in some embodiments, the method includes inserting approximately 300 kb of DNA upstream of a murine (e.g., mouse or rat) Cλ1 region, whereby the DNA is operably linked to the murine (e.g., mouse or rat) Cλ1 region, the DNA comprising the human Vλ gene segments Vλ10-54, Vλ6-57, Vλ4-60, Vλ8-61, and Vλ4-69. In such embodiments, the DNA is inserted upstream of a human Vλ5-52 gene segment operably linked to a murine (e.g., mouse or rat) Cλ1 region, the DNA comprising the human Vλ gene segments Vλ10-54, Vλ6-57, Vλ4-60, Vλ8-61, and Vλ4-69. In some embodiments, the DNA comprises a human VpreB gene. The additional human Vλ segments described above may be cloned directly from commercially available BAC clones and placed into smaller DNA fragments using recombinant techniques described herein or otherwise known in the art. Alternatively, the additional human Vλ gene segments described above may be synthesized into DNA fragments and added to the engineered Igλ light chain locus described above. Similarly, additional human Jλ and / or Cλ gene segments may be obtained from commercially available BAC clones or synthesized directly from published sequences. Furthermore, as described herein, an endogenous Igλ light chain enhancer region (or sequence) may be deleted from the engineered Igλ light chain locus. Diagrams illustrating the engineered Igλ light chain loci of non-human animals described herein are set forth in any one of Figures 1, 2, 3, and 4.

[0216] Where appropriate, a human Igλ light chain sequence encoding all or a portion of an Igλ light chain (i.e., a sequence comprising human Vλ, Jλ, and / or Cλ gene segments) may be separately modified to contain codons optimized for expression in a non-human animal (see, e.g., U.S. Patent Nos. 5,670,356 and 5,874,304). A codon-optimized sequence is preferably a synthetic sequence that encodes 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, a human Igλ light chain sequence encoding all or a portion of an Igλ light chain may separately contain a sequence altered to optimize codon usage for a particular cell type (e.g., rodent cell). For example, the codons of each nucleotide sequence inserted into the genome of a non-human animal (e.g., rodent) may be optimized for expression in the cells of that non-human animal. Such sequences are sometimes described as codon-optimized sequences.

[0217] In some embodiments, insertion of a nucleotide sequence encoding all or part of a human Igλ light chain utilizes minimal modification of the germline genome of the non-human animal described herein, resulting in expression of an antibody comprising a light chain that is all or partly human. Methods for generating genetically engineered non-human animals, including knockouts and knockins, are known in the art (see, e.g., Gene Targeting: A Practical Approach, Joyner, ed., Oxford University Press, Inc., 2000). For example, generation of a transgenic rodent may optionally include disrupting the locus of one or more endogenous rodent genes (or gene segments) and introducing 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 all or a portion of a human Igλ light chain is introduced upstream of a murine (e.g., mouse or rat) Igλ light chain constant region gene of a randomly inserted Igλ light chain transgene in the germline genome of a rodent. In some embodiments, a nucleotide sequence encoding all or a portion of a human Igλ light chain is introduced upstream of a murine (e.g., mouse or rat) Igλ light chain locus constant region gene of an endogenous Igλ light chain locus in the germline genome of a rodent. In some embodiments, the endogenous Igλ light chain locus is altered, modified, or engineered to contain human Igλ gene segments (e.g., Vλ, Jλ, and / or Cλ), which are operably linked to a rodent Cλ1 region.

[0218] Schematic diagrams (not to scale) of exemplary engineered Igλ light chain loci are provided in Figures 1-4. In particular, Figures 1 and 3 set forth exemplary strategies for constructing engineered Igλ light chain loci, which are characterized by the insertion of nucleotide sequences containing multiple human Vλ, Jλ, and / or Cλ segments. As illustrated in Figure 1, a DNA fragment containing a human Eλ sequence (or region) is inserted downstream of a rodent Cλ region via homologous recombination. This DNA fragment contains a neomycin selection cassette (e.g., a neomycin resistance gene [NEO] flanked by loxP recombination recognition sites) located 3' to the human Eλ sequence. R ]), which contains three human Eλ elements engineered downstream (or 3') of the rodent Cλ1 region. Also shown in Figure 1 is a DNA fragment containing a human Vλ segment, a set of human Jλ-Cλ segment pairs (e.g., human Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6), and the first portion of a human Jλ7 segment, inserted upstream of the rodent Cλ1 region via homologous recombination. As shown, a hygromycin selection cassette (e.g., a hygromycin resistance gene [HYG] flanked by Frt recombination recognition sites) is inserted upstream of the rodent Cλ1 region. R

[0014] is positioned on the 5' end of the targeting vector and upstream of the human Ig λ light chain sequence contained in the targeting vector. The hygromycin selection cassette is removed via homologous recombination using the next targeting vector, described in the Examples section below. The targeting vector is then electroporated into rodent embryonic stem (ES) cells to generate rodents whose germline genomes contain the engineered Ig λ light chain locus. Once positive rodent ES cell clones are identified, other illustrated targeting vectors are sequentially electroporated, confirming the completion of the construction of the engineered Ig λ light chain locus at each step (see Figure 2). The final targeting vector is designed with (6680 targeting vector) or without (6597 targeting vector) homology arms to direct the deletion of the endogenous Ig λ light chain segment via homologous recombination, potentially generating two engineered Ig λ light chain alleles (Figure 2). Any remaining selection cassettes may be further deleted via recombinase-mediated deletion if desired. An alternative strategy for inserting additional human Vλ gene segments into an engineered Igλ light chain locus using guide RNAs (gRNAs) is illustrated in Figure 3.

[0219] When a human Igλ light chain sequence is inserted upstream of a non-human Igλ light chain constant region of a BAC clone, a targeting vector for integration into the Igλ light chain locus is generated. The target BAC clone carrying the human Igλ light chain sequence for generating the targeting vector may contain 5' and / or 3' flanking genomic DNA of murine (e.g., mouse or rat) origin. Alternatively, or in addition, the target BAC clone carrying the human Igλ light chain sequence for generating the targeting vector may contain 5' and / or 3' flanking genomic DNA of human origin, thereby generating an overlapping region with the human Igλ light chain sequence. This method allows for sequential targeting of multiple engineered BAC clones (see, e.g., Figure 1). The final targeting vector is integrated into the Igλ light chain locus in the genome of a non-human cell (e.g., a rodent embryonic stem cell). In some embodiments, the targeting vectors described herein are integrated into the Igλ light chain locus of the germline genome of a non-human cell, and the cell contains a human V operably linked to one or more IgH constant region genes. H , D H , and J. H Genomic DNA (e.g., multiple V H , D H , and J. H and / or further comprising human Vκ and Jκ genomic DNA (e.g., containing multiple human Vκ and Jκ gene segments) operably linked to an Igκ constant region gene (see, e.g., U.S. Pat. Nos. 8,502,018, 8,642,835, 8,697,940, and 8,791,323, which are incorporated by reference in their entireties).

[0220] The targeting vector is introduced into rodent (e.g., mouse) embryonic stem cells by electroporation, thereby inserting the sequences contained in the targeting vector into the genome of the rodent embryonic stem cells, conferring the ability of the non-human cell or animal (e.g., mouse) to express antibodies having all or a portion of a human Igλ light chain. As described herein, transgenic rodents are generated in which an engineered Igλ light chain locus (e.g., an endogenous Igλ light chain locus containing a human Igλ light chain sequence operably linked to an endogenous rodent Cλ region as described herein) has been engineered into the germline of the rodent genome. Antibodies are expressed on the surface of rodent B cells and in the serum of the rodent, and the antibodies are characterized by a light chain having a human Vλ domain, and in some embodiments, human Vλ and Cλ domains. If the endogenous Igλ light chain locus in the germline of the rodent genome is not targeted by the targeting vector, the engineered Igλ light chain locus is preferably inserted at a location other than the endogenous rodent Igλ light chain locus (e.g., as a randomly inserted transgene).

[0221] By generating an engineered Igλ light chain locus in the non-human animal described above, an engineered rodent strain is provided that produces antibodies comprising an Igλ light chain expressed from the engineered Igλ light chain locus having human Vλ domains, and in some embodiments, human Vλ and Cλ domains. H , D H and J. H By taking advantage of the presence of an engineered IgH locus containing gene segments, engineered rodent strains can be generated that produce antibodies and antibody components for the development of human antibody-based therapeutics. It is therefore understood that a single engineered rodent strain has the potential to provide an alternative in vivo system that utilizes human Vλ domains for the development of new antibody-based pharmaceuticals to treat human diseases.

[0222] In some embodiments, the genome of a non-human animal described herein further contains one or more human immunoglobulin heavy and / or light chain variable regions (e.g., via breeding or multiple gene targeting strategies), such as those described in U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, and 8,791,323, all of which are incorporated by reference in their entireties. Alternatively, an engineered Igλ light chain locus described herein can be engineered into an embryonic stem cell that contains a humanized IgH and / or Igκ locus, or a non-human animal that contains an engineered Igλ light chain locus described herein can be bred with another non-human animal that contains a humanized IgH and / or Igκ locus. Various animals containing such humanized IgH and / or Igκ loci are known, such as the VELOCIMMUNE® line (see, e.g., U.S. Pat. Nos. 8,502,018 and / or 8,642,835, which are incorporated herein by reference in their entireties), XENOMOUSE TM phylogeny (e.g. Mendez, MJet al., 1997, Nat. Genetics 15(2):146-56 and Jakobovits, A. et al., 1995, Ann. NY Acad. Sci. 764:525-35). Homozygosity for the engineered Igλ light chain loci described herein can be achieved by subsequent breeding. Alternatively, in the case of randomly inserted engineered Igλ light chain transgenes (described above), rodent strains can be selected based specifically on expression of human Vλ domains from the transgene.

[0223] Alternatively, and / or in addition, in some embodiments, the germline genome of the non-human animal described herein further comprises a deleted, inactivated, functionally silenced, or otherwise non-functional endogenous Igκ light chain locus. Genetic modifications to delete or render non-functional the gene or gene locus may be performed using methods described herein and / or methods known in the art.

[0224] A transgenic founder non-human animal can be identified based on the presence of an engineered Igλ light chain locus in its germline genome and / or based on the expression of antibodies having all or part of a human Igλ light chain sequence in tissues or cells of the non-human animal. The transgenic founder non-human animal can then be used to breed with another non-human animal carrying an engineered Igλ light chain locus, thereby generating a population of non-human animals each carrying one or more copies of an engineered Igλ light chain locus. Transgenic non-human animals carrying the engineered Igλ light chain locus described herein can also be further bred to other transgenic non-human animals carrying other desired transgenes (e.g., human immunoglobulin genes).

[0225] In some embodiments, transgenic non-human animals can be generated containing a selection system that allows for controlled, directed, inducible, and / or cell-type-specific expression of a transgene or integrated sequence. For example, the non-human animals described herein can be engineered to contain a conditionally expressed sequence encoding all or part of a human Ig λ light chain of an antibody (reviewed, e.g., in Rajewski, K. et al., 1996, J. Clin. Invest. 98(3):600-3). Exemplary systems include the Cre / loxP recombinase system of bacteriophage P1 (see, e.g., Lakso, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6) and the FLP / Frt recombinase system of Saccharomyces cerevisiae (O'Gorman, S. et al., 1991, Science 251:1351-5). Such animals can be provided, for example, by constructing a "double" transgenic animal by mating two transgenic animals, one containing a transgene with a selected modification (e.g., an engineered Igλ light chain locus described herein) and the other containing a transgene encoding a recombinase (e.g., Cre recombinase).

[0226] The non-human animals described herein may often be prepared as described above or using methods known in the art to contain additional human, humanized, or otherwise engineered genes, depending on the intended use of the non-human animal. The genetic material of such human, humanized, or otherwise engineered genes may be introduced through further modification of the genome of cells (e.g., embryonic stem cells) carrying the above-described genetic modifications or alterations, or through breeding techniques known in the art with other genetically modified or engineered strains as desired. In some embodiments, the non-human animals described herein are prepared to further contain transgenic human IgH and / or Igκ light chain genes or gene segments (see, e.g., Murphy, AJ et al., (2014) Proc. Natl. Acad. Sci. USA 111(14):5153-5158; U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, 8,791,323, and U.S. Patent Application Publication No. 2013 / 0096287A1, which are incorporated by reference in their entireties).

[0227] In some embodiments, the non-human animals described herein may be generated by introducing a targeting vector described herein into cells from the modified strain. In one example, the targeting vector described above may be introduced into a VELOCIMMUNE® mouse. The VELOCIMMUNE® mouse expresses antibodies with fully human variable domains and mouse constant domains. In some embodiments, the non-human animals described herein are generated to further comprise human immunoglobulin genes (variable region genes and / or constant region genes). In some embodiments, the non-human animals described herein comprise an engineered Igλ light chain locus described herein and genetic material of heterologous (e.g., human) origin, wherein the genetic material encodes all or part of one or more human heavy chain and / or Igκ light chain variable domains.

[0228] For example, as described herein, a non-human animal comprising an engineered Igλ light chain locus described herein may further comprise (e.g., via breeding or multiple gene targeting strategies) one or more of the modifications described in the following documents: Murphy, AJ et al., (2014) Proc. Natl. Acad. Sci. USA 111(14):5153-8; Macdonald, LE et al., 2014, Proc. Natl. Acad. Sci. USA 111(14):5147-52, U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, and 8,791,323, all of which are incorporated by reference in their entirety. In some embodiments, rodents comprising an engineered Igλ light chain locus described herein are bred with rodents comprising humanized IgH and / or Igκ light chain variable region loci (see, e.g., U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, and / or 8,791,323, which are incorporated by reference in their entireties). In some embodiments, rodents comprising an engineered Igλ light chain locus described herein are bred with rodents comprising a humanized IgH variable region locus (see, e.g., U.S. Pat. Nos. 8,502,018, 8,642,835, 8,697,940, and / or 8,791,323, which are incorporated herein by reference in their entireties), and an inactivated endogenous Igκ light chain locus (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, which are incorporated herein by reference in their entireties).

[0229] Although embodiments describing the construction of an engineered Igλ light chain locus in a mouse (i.e., a mouse having an engineered Igλ light chain locus characterized by the presence of multiple human Vλ, Jλ, and Cλ gene segments operably linked to a mouse Cλ region, thereby expressing antibodies containing a human Igλ light chain), other non-human animals comprising an engineered Igλ light chain locus are also provided. Such non-human animals include any non-human animal that can be genetically modified to express the antibodies described herein, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For example, for non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are employed to generate non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of a non-ES cell (e.g., a fibroblast or an induced pluripotent cell) and using somatic cell nuclear transfer (SCNT) to introduce the genetically modified genome into a suitable cell, such as an enucleated oocyte, and gestation of the modified cell (e.g., a modified oocyte) into a non-human animal under conditions suitable for the formation of an embryo.

[0230] Methods for modifying the germline genome of non-human animals (e.g., the genomes of pigs, cows, rodents, chickens, etc.) include, for example, using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or Cas proteins (i.e., CRISPR / Cas systems) to contain the engineered Igλ light chain loci described herein. Guidance regarding methods for modifying the germline genome of non-human animals can be found, for example, in U.S. Patent Application Nos. 14 / 747,461 (filed June 23, 2015), 14 / 948,221 (filed November 20, 2015), and 14 / 974,623 (filed December 18, 2015), all three of which are incorporated herein by reference in their entireties.

[0231] In some embodiments, the non-human animals described herein are mammals. In some embodiments, the non-human animals described herein are small mammals, for example, of the Jerboidea or Murine superfamily. In some embodiments, the genetically modified animals described herein are rodents. In some embodiments, the rodents described herein are selected from mice, rats, and hamsters. In some embodiments, the rodents described herein are selected from the Murine superfamily. In some embodiments, the genetically modified animals described herein are from a family selected from the family Odontoidea (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, and voles), Muridae (pure-breed mice and rats, gerbils, spiny mice, and maned mice), Tetragnathidae (tree mice, rock mice, white-tailed rats, Madagascar rats and mice), Dormiceidae (e.g., spiny dormice), and Moleratidae (e.g., mole rats, bamboo rats, and plateau mole rats). In some embodiments, the genetically modified rodents described herein are selected from pure breeds of mice or rats (Muridae), gerbils, spiny mice, and maned mice. In some embodiments, the genetically modified mice described herein are from members of the Muridae family. In some embodiments, the non-human animals described herein are rodents. In some embodiments, the rodents described herein are selected from mice and rats. In some embodiments, the non-human animals described herein are mice.

[0232] In some embodiments, the non-human animal described herein is a rodent that is a mouse of a C57BL strain selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mice described herein are 129 strains selected from the group consisting of strains that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach, W. et al., 2000, Biotechniques 29(5):1024-1028, 1030, 1032). In some embodiments, the genetically modified mice described herein are a mix of the 129 strain and the C57BL / 6 strain. In some embodiments, the mice described herein are a mix of the 129 strains described herein, or a mix of the BL / 6 strains described herein. In some embodiments, the mixed 129 strain described herein is a 129S6 (129 / SvEvTac) strain. In some embodiments, the mice described herein are a BALB strain, e.g., a BALB / c strain. In some embodiments, the mice described herein are a mix of a BALB strain and another strain described herein.

[0233] In some embodiments, the non-human animal described herein is a rat. In some embodiments, the rat described herein is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strain described herein is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0234] Rat pluripotent and / or totipotent cells may be derived from any rat strain, including, for example, the ACI rat strain, the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or a Fischer rat strain, such as Fisher F344 or Fisher F6. Rat pluripotent and / or totipotent cells may also be derived from a strain derived from a mixture of two or more of the above strains. For example, rat pluripotent and / or totipotent cells may be derived from the DA strain or the ACI strain. The ACI rat strain has a black agouti coat, a white abdomen and paws, and is characterized by the RT1av1 haplotype. This strain is available from various sources, including Harlan Laboratories. An example of a rat ES cell line derived from an ACI rat is the ACI.G1 rat ES cell. The Dark Agouti (DA) rat strain has an agouti coat color and is characterized by the RT1av1 haplotype. Such rats are available from a variety of sources, including Charles River and Harlan Laboratories. Examples of rat ES cell lines derived from DA rats are the DA.2B rat ES cell line and the DA.2C rat ES cell line. In some embodiments, the rat pluripotent and / or totipotent cells are derived from an inbred rat strain (see, e.g., U.S. Patent Application Publication No. 2014-0235933A1, published August 21, 2014, which is incorporated herein by reference in its entirety).

[0235] Specific Exemplary Embodiments - Engineered IgH Locus In some embodiments, the non-human animals provided comprise an engineered Igλ light chain locus described herein and a plurality of human V H , D H , and J. H Further included are engineered IgH loci (or alleles) characterized by the presence of gene segments arranged in a germline configuration and operably linked to non-human IgH constant regions, enhancers, and regulatory regions. In some embodiments, the engineered IgH loci (or alleles) described herein comprise one or more human VH gene segments. H gene segment, one or more human D H a gene segment, and one or more human J H It comprises gene segments, which are operably linked to a non-human IgH constant region.

[0236] In some embodiments, the engineered IgH locus (or allele) comprises 5, 10, 15, 20, 25, 30, 35, 40 or more (e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, etc.) human V H In some embodiments, the engineered IgH locus (or allele) comprises a human V gene segment encompassing both ends of a naturally occurring human IgH locus. H 3-74 and Human V H Functional human V located between the 6-1 gene segments H In some embodiments, the engineered IgH 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, VH 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, and V H Including 6-1.

[0237] In some embodiments, the engineered IgH locus (or allele) comprises 5, 10, 15, 20, 25 or more (e.g., 26, 27, etc.) human D H In some embodiments, the engineered IgH locus (or allele) comprises a human D gene segment encompassing both ends of a naturally occurring human IgH locus. H 1-1 and D H Functional human D located between the 7-27 gene segment H In some embodiments, the engineered IgH locus (or allele) comprises at least one human D gene segment. H Gene segment D H 1-1, DH 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, and D H Including 7-27.

[0238] In some embodiments, the engineered IgH locus (or allele) contains 1, 2, 3, 4, 5, 6 or more functional human J H In some embodiments, the engineered IgH locus (or allele) comprises a human J gene segment encompassing both ends of a naturally occurring human IgH locus. H 1 and Human J H Functional human J sequences present among six gene segments H In some embodiments, the engineered IgH locus (or allele) comprises at least one human J gene segment. H Gene segment J H 1. J H 2. J H 3. J H 4. J H 5, and J H Includes 6.

[0239] In some embodiments, the non-human IgH constant region comprises one or more non-human IgH constant region genes, such as immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin E (IgE), and immunoglobulin A (IgA). In some embodiments, the non-human IgH 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 IgH enhancers (i.e., enhancer sequences or enhancer regions). H , D H , and J. H The gene segment is operably linked to one or more non-human IgH regulatory regions (or regulatory sequences). H , D H , and J. H The gene segment is operably linked to one or more non-human IgH enhancers (or enhancer sequences) and one or more non-human IgH regulatory regions (or regulatory sequences).

[0240] In some embodiments, the engineered IgH loci described herein do not contain an endogenous Adam6 gene. In some embodiments, the engineered IgH loci described herein do not contain an endogenous Adam6 gene (or Adam6 coding sequence) at the same germline genomic location as is present in the germline genome of a wild-type non-human animal of the same species. In some embodiments, the engineered IgH loci described herein do not contain a human Adam6 pseudogene. In some embodiments, the engineered IgH loci described herein comprise an insertion of at least one nucleotide sequence encoding one or more non-human (e.g., rodent) Adam6 polypeptides. The insertion is located outside (e.g., near the 5'-most V) of the engineered immunoglobulin heavy chain loci described herein.H The IgH locus may be located upstream of the gene segment, within the engineered IgH locus, or elsewhere in the germline genome of the non-human animal, cell, or tissue (e.g., a randomly introduced non-human Adam6-encoding sequence).

[0241] In various embodiments, the non-human animals, non-human cells, or non-human tissues provided herein contain endogenous non-human V in antibody molecules. H In various embodiments, the non-human animals, cells, or tissues provided herein do not detectably express all or part of the endogenous non-human V region in the antibody molecule. H Region (e.g., V H , D H and / or J H In various embodiments, the non-human animals, cells, or tissues provided herein do not contain (or lack, or contain a deletion of) one or more nucleotide sequences encoding all or part of an endogenous non-human V. H , D H and J. H and having a germline genome that includes a deletion of all or part of a gene segment. In various embodiments, the non-human animals provided are capable of reproduction.

[0242] Guidance regarding the generation of targeting vectors, non-human cells, and animals carrying such engineered IgH loci (or alleles) can be found, for example, in U.S. Patent Nos. 8,642,835 and 8,697,940, which are incorporated herein by reference in their entireties. Those skilled in the art will recognize a variety of techniques known in the art for achieving such genetic manipulation and / or treatment of non-humans (e.g., mammals) or for otherwise preparing, providing, or manufacturing such sequences for introduction into the germline genome of non-human animals.

[0243] Specific Exemplary Embodiments—Engineered Igκ Light Chain Locus In some embodiments, the provided non-human animals comprise an engineered Igλ light chain locus described herein and further comprise an engineered Igκ locus (or allele) characterized by the presence of multiple human Vκ and Jκ gene segments, which are arranged in a germline configuration and operably linked to a non-human Igκ light chain constant region, an Igκ enhancer and regulatory region. In some embodiments, the engineered Igκ light chain locus (or allele) comprises one or more human Vκ gene segments and one or more human Jκ gene segments, which are operably linked to a non-human Igκ constant region (CK).

[0244] In some embodiments, the engineered Igκ light chain locus (or allele) comprises at least the human Vκ gene segments present in the distal variable cluster (or distal arm, or distal overlap) of a naturally occurring human Igκ light chain locus. In some embodiments, the engineered Igκ light chain locus (or allele) comprises at least the human Vκ gene segments present in the proximal variable cluster (or proximal arm, or proximal overlap) of a naturally occurring human Igκ light chain locus. In some embodiments, the engineered Igκ light chain locus (or allele) comprises the human Vκ gene segments present in the distal and proximal variable clusters of a naturally occurring human Igκ light chain locus. In some embodiments, the engineered Igκ light chain locus (or allele) comprises all or substantially all of the functional human Vκ gene segments present between the human Vκ2-40 (or Vκ3D-7) and human Vκ4-1 gene segments, inclusive, of a naturally occurring human Igκ locus.

[0245] In some embodiments, the engineered Igκ light chain locus (or allele) comprises 5, 10, 15, 20, 25, 30, 35 or more (e.g., 36, 37, 38, 39, 40, etc.) human κ gene segments. ... Vκ3D-7, Vκ1D-8, Vκ1D-43, Vκ3D-11, Vκ1D-12, Vκ1D-13, Vκ3D-15, Vκ1D-16, Vκ1D-17, Vκ3D-20, Vκ6D-21, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-3 Vκ1-0, Vκ1D-33, Vκ1D-39, Vκ2D-40, Vκ2-40, Vκ1-39, Vκ1-33, Vκ2-30, Vκ2-28, Vκ1-27, Vκ2-24, Vκ6-21, Vκ3-20, Vκ1-17, Vκ1-16, Vκ3-15, Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8, Vκ1-6, Vκ1-5, Vκ5-2, and Vκ4-1. In some embodiments, the engineered Igκ light chain loci (or alleles) include at least human Vκ gene segments Vκ3D-7, Vκ1D-8, Vκ1D-43, Vκ3D-11, Vκ1D-12, Vκ1D-13, Vκ3D-15, Vκ1D-16, Vκ1D-17, Vκ3D-20, Vκ6D-21, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ1D-33, Vκ1D-39, and Vκ2D-40. In some embodiments, the engineered Igκ light chain loci (or alleles) include at least the following human Vκ gene segments: Vκ2-40, Vκ1-39, Vκ1-33, Vκ2-30, Vκ2-28, Vκ1-27, Vκ2-24, Vκ6-21, Vκ3-20, Vκ1-17, Vκ1-16, Vκ3-15, Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8, Vκ1-6, Vκ1-5, Vκ5-2, and Vκ4-1.

[0246] In some embodiments, the engineered Igκ light chain locus (or allele) comprises one, two, three, four, five, or more functional human Jκ gene segments. In some embodiments, the engineered Igκ light chain locus (or allele) comprises all, or substantially all, of the functional human Jκ gene segments present between, and including, the human Jκ1 and human Jκ5 gene segments of a naturally occurring human Igκ light chain locus. In some embodiments, the engineered Igκ light chain locus (or allele) comprises at least the human Jκ gene segments Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5.

[0247] In some embodiments, the human Vκ and Jκ gene segments are operably linked to one or more non-human Igκ enhancers (i.e., enhancer sequences or regions). In some embodiments, the human Vκ and Jκ gene segments are operably linked to one or more non-human Igκ light chain regulatory regions (or regulatory sequences). In some embodiments, the human Vκ and Jκ gene segments are operably linked to one or more non-human Igκ light chain enhancers (or enhancer sequences or regions) and one or more non-human Igκ light chain regulatory regions (or regulatory sequences).

[0248] In some embodiments, the non-human Cκ region of the engineered Igκ light chain locus (or allele) comprises a rodent Cκ region, e.g., a mouse Cκ region or a rat Cκ region. In some embodiments, the non-human Cκ region of the engineered Igκ light chain locus (or allele) is or comprises a mouse Cκ region derived from a genetic background comprising 129, BALB / c, C57BL / 6, a mixed 129xC57BL / 6 strain, or a combination thereof.

[0249] In some embodiments, the non-human animals provided comprise an engineered Igλ light chain locus described herein and further comprise an inactivated Igκ light chain locus (or allele).

[0250] In various embodiments, the provided non-human animals, non-human cells, or non-human tissues described herein do not detectably express all or a portion of an endogenous non-human Vκ region in antibody molecules. In various embodiments, the provided non-human animals, non-human cells, or non-human tissues described herein do not contain (or lack, or contain a deletion of) one or more nucleotide sequences encoding all or a portion of an endogenous non-human Vκ region in antibody molecules. In various embodiments, the provided non-human animals, non-human cells, or non-human tissues described herein have a germline genome that includes a deletion of all or a portion of endogenous non-human Vκ and Jκ gene segments.

[0251] Guidance regarding the generation of targeting vectors, non-human cells, and animals carrying such engineered Igκ light chain loci (or alleles) can be found, for example, in U.S. Patent Nos. 8,642,835 and 8,697,940, which are incorporated herein by reference in their entireties. Those skilled in the art will recognize a variety of techniques known in the art for achieving such genetic engineering and / or treatment of non-humans (e.g., mammals) or for otherwise preparing, providing, or manufacturing such sequences for introduction into the germline genome of non-human animals.

[0252] Specific Exemplary Embodiments—Engineered Ig λ Light Chain Locus In some embodiments, the provided non-human animals comprise an engineered Igλ light chain locus characterized by the presence of multiple human Vλ, Jλ, and Cλ gene segments, arranged in a germline configuration and inserted upstream of and operably linked to a non-human Cλ gene segment (or Cλ region gene). As described herein, such an engineered Igλ light chain locus further comprises one or more human Igλ light chain enhancer regions (or enhancer sequences). In some embodiments, the engineered Igλ light chain locus comprises one or more human Vλ gene segments and one or more human Jλ gene segments, which are operably linked to a non-human Igλ light chain constant (Cλ) region. In some embodiments, the engineered Igλ light chain locus (or allele) comprises human Vλ gene segments present in at least cluster A of the human Igλ light chain locus, in some embodiments present in cluster A and cluster B of the human Igλ light chain locus, and in some embodiments present in cluster A, cluster B, and cluster C of the human Igλ light chain locus.

[0253] In some embodiments, an engineered Igλ light chain locus (or allele) comprises 5, 10, 15, 20, 25, 30, or more (e.g., 31, 32, 33, 34, 35, etc.) human Vλ gene segments. In some embodiments, an engineered Igλ light chain locus (or allele) comprises all, or substantially all, of the functional human Vλ gene segments present between, inclusive, the human Vλ4-69 and human Vλ3-1 gene segments of a naturally occurring human Igλ locus. In some embodiments, an engineered Igλ light chain locus (or allele) comprises all, or substantially all, of the functional human Vλ gene segments present between, inclusive, the human Vλ5-52 and human Vλ3-1 gene segments of a naturally occurring human Igλ locus. In some embodiments, the engineered Igλ light chain locus (or allele) comprises all or substantially all of the functional human Vλ gene segments present between the human Vλ3-27 and human Vλ3-1 gene segments, inclusive, of a naturally occurring human Igλ locus. In some embodiments, the engineered Igλ light chain locus (or allele) comprises the human Vλ gene segments 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, In some embodiments, the engineered Ig λ light chain loci (or alleles) include at least functional human Vλ gene segments Vλ5-52 through Vλ1-40 and Vλ3-27 through Vλ3-1.

[0254] In some embodiments, an engineered Igλ light chain locus (or allele) comprises one, two, three, four, five, six, seven, or more functional human Jλ gene segments. In some embodiments, an engineered Igλ light chain locus (or allele) comprises all, or substantially all, of the functional human Jλ gene segments present between, and including, the human Jλ1 and human Jλ7 gene segments of a naturally occurring human Igλ light chain locus. In some embodiments, an engineered Igλ light chain locus (or allele) comprises at least the human Jλ gene segments Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0255] In some embodiments, an engineered Igλ light chain locus (or allele) comprises one, two, three, four, five, six, seven, or more functional human Cλ gene segments. In some embodiments, an engineered Igλ light chain locus (or allele) comprises all, or substantially all, of the functional human Cλ gene segments present between, and including, the human Cλ1 and human Cλ7 gene segments of a naturally occurring human Igλ light chain locus. In some embodiments, an engineered Igλ light chain locus (or allele) comprises at least the human Cλ gene segments Cλ1, Cλ2, Cλ3, and Cλ6.

[0256] In some embodiments, the engineered Igλ light chain locus (or allele) does not contain the same non-human Igλ light chain enhancer region (or enhancer sequence) as is present in the wild-type Igλ light chain locus (or allele). In some embodiments, the engineered Igλ light chain locus (or allele) lacks all or a portion of at least one non-human Igλ light chain enhancer region (or enhancer sequence) (e.g., Igλ enhancers 2-4 or Eλ2-4).

[0257] In some embodiments, the human Vλ and Jλ gene segments are operably linked to one or more non-human Igλ light chain enhancers (i.e., enhancer sequences or regions) and one or more human Igλ light chain regulatory regions (i.e., enhancer sequences or regions). In some embodiments, the human Vλ and Jλ gene segments are operably linked to one or more non-human Igλ light chain regulatory regions (or regulatory sequences). In some embodiments, the human Vλ and Jλ gene segments are operably linked to one or more non-human Igλ light chain enhancers (or enhancer sequences or regions), one or more human Igλ light chain enhancers (i.e., enhancer sequences or regions), and one or more non-human Igλ light chain regulatory regions (or regulatory sequences).

[0258] In some embodiments, the engineered Ig λ light chain loci (or alleles) described herein do not contain the human VpreB gene (or human VpreB gene coding sequence).

[0259] In some embodiments, the non-human Cλ region of an engineered Igλ light chain locus (or allele) comprises a rodent Cλ region, e.g., a mouse Cλ region or a rat Cλ region. In some embodiments, the non-human Cλ region of an engineered Igλ light chain locus (or allele) is or comprises a mouse Cλ region derived from a genetic background comprising 129, BALB / c, C57BL / 6, a mixed 129xC57BL / 6 strain, or a combination thereof.

[0260] In some embodiments, the non-human Cλ region of an engineered Igλ light chain locus (or allele) described herein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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:3 (mouse Cλ2), or SEQ ID NO:5 (mouse Cλ3). In some embodiments, the non-human Cλ region of an engineered Igλ light chain locus (or allele) described herein is or comprises the sequence of a mouse Cλ1 region.

[0261] In some embodiments, a non-human Cλ region encoded by a sequence located at an engineered Igλ light chain locus (or allele) described herein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:2 (mouse Cλ1), SEQ ID NO:4 (mouse Cλ2), or SEQ ID NO:6 (mouse Cλ3). In some embodiments, a non-human Cλ region encoded by a sequence located at an engineered Igλ light chain locus (or allele) described herein comprises a sequence that is substantially identical to, or identical to, SEQ ID NO:2 (mouse Cλ1), SEQ ID NO:4 (mouse Cλ2), or SEQ ID NO:6 (mouse Cλ3). In some embodiments, a non-human Cλ region encoded by a sequence located at an engineered Igλ light chain locus (or allele) described herein is or comprises a mouse Cλ1 region polypeptide.

[0262] In some embodiments, the non-human Cλ regions of the engineered Igλ light chain loci (or alleles) described herein comprise a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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:9 (rat Cλ2), SEQ ID NO:11 (rat Cλ3), or SEQ ID NO:13 (rat Cλ4). In some embodiments, the non-human Cλ regions of the engineered Igλ light chain loci (or alleles) described herein comprise a sequence that is substantially identical or identical to SEQ ID NO:7 (rat Cλ1), SEQ ID NO:9 (rat Cλ2), SEQ ID NO:11 (rat Cλ3), or SEQ ID NO:13 (rat Cλ4). In some embodiments, the non-human Cλ region of an engineered Igλ light chain locus (or allele) described herein is or comprises the sequence of a rat Cλ1 region.

[0263] In some embodiments, a non-human Cλ region encoded by a sequence located at an engineered Igλ light chain locus (or allele) described herein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:8 (rat Cλ1), SEQ ID NO:10 (rat Cλ2), SEQ ID NO:12 (rat Cλ3), or SEQ ID NO:14 (rat Cλ4). In some embodiments, a non-human Cλ region encoded by a sequence located at an engineered Igλ light chain locus (or allele) described herein comprises a sequence that is substantially identical or identical to SEQ ID NO:8 (rat Cλ1), SEQ ID NO:10 (rat Cλ2), SEQ ID NO:12 (rat Cλ3), or SEQ ID NO:14 (rat Cλ4). In some embodiments, the non-human Cλ region encoded by a sequence located in an engineered Igλ light chain locus (or allele) described herein is or comprises a rat Cλ1 region polypeptide.

[0264] In some embodiments, the engineered Ig λ light chain loci (or alleles) described herein are characterized by the presence of one or more unique nucleotide sequence junctions that result from the insertion of human genetic material corresponding to a human Ig λ light chain sequence (genomic or synthetic) at the endogenous locus in place of a non-human Ig λ light chain sequence. Exemplary nucleotide sequence junctions are set forth in SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129.

[0265] In some embodiments, an engineered Ig λ light chain locus (or allele) described herein comprises one or more of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129.

[0266] In some embodiments, the engineered Ig λ light chain loci (or alleles) described herein comprise SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, and SEQ ID NO:123.

[0267] In some embodiments, the engineered Ig λ light chain loci (or alleles) described herein comprise SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, and SEQ ID NO:123.

[0268] In some embodiments, the engineered Ig λ light chain loci (or alleles) described herein comprise SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:128, and SEQ ID NO:129.

[0269] In some embodiments, the engineered Ig λ light chain loci (or alleles) described herein comprise SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, and SEQ ID NO:127.

[0270] In some embodiments, the engineered Ig λ light chain loci (or alleles) described herein comprise SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, and SEQ ID NO:125.

[0271] Guidance regarding human Vλ, Jλ, and Cλ gene segments can be found, for example, in Lefranc, MP, 2000, Nomenclature of the human immunoglobulin lambda (IGL) genes, Current Protocols in Immunology, No. Supplement, 40:A.1 p.1-A.1 p.37. In particular, the present disclosure demonstrates that the presence of human Vλ and Jλ gene segments at Igλ light chain loci (or alleles) provides an increased diversity in the light chain repertoire of a non-human animal compared to the light chain diversity in the expressed antibody repertoire of a non-human animal that does not contain such engineered Igλ light chain alleles.

[0272] method In certain aspects, the non-human animals described herein can be used to generate human antibodies and / or nucleic acid sequences encoding human antibodies, wherein the human antibodies comprise variable domains derived from nucleic acid sequences encoded by the genetic material of cells of the non-human animals described herein. For example, the non-human animals described herein are immunized with an antigen of interest under conditions and for a time sufficient to cause the non-human animal to generate an immune response against the antigen. Antibodies are isolated from the non-human animals (or one or more cells, e.g., one or more B cells) so immunized and characterized using various assays that measure, for example, affinity, specificity, epitope mapping, ability to inhibit ligand-receptor interactions, ability to inhibit receptor activity, etc. In various embodiments, the antibodies produced by the non-human animals described herein comprise one or more human variable domains derived from one or more human variable region nucleotide sequences isolated from the non-human animals. In some embodiments, anti-drug antibodies (e.g., anti-idiotypic antibodies) can be generated in the non-human animals described herein.

[0273] In some embodiments, the non-human animals described herein provide improved in vivo systems and sources of biological material (e.g., cells) for producing human antibodies useful in various assays. In various embodiments, the non-human animals described herein are used to develop therapeutic agents that target a polypeptide of interest (e.g., a transmembrane polypeptide or a secreted polypeptide) and / or that modulate one or more activities associated with the polypeptide of interest and / or that modulate the interaction of the polypeptide of interest with other binding partners (e.g., ligands or receptor polypeptides). For example, in various embodiments, the non-human animals described herein are used to develop therapeutic agents that target one or more receptor polypeptides, modulate receptor polypeptides, and / or modulate the interaction of receptor polypeptides with other binding partners. In various embodiments, the non-human animals described herein are used to identify, screen, and / or develop candidate therapeutic agents (e.g., antibodies, siRNAs, etc.) that bind to one or more polypeptides of interest. In various embodiments, the non-human animals described herein are used to screen and develop candidate therapeutic agents (e.g., antibodies, siRNAs, etc.) that inhibit one or more polypeptide activities of interest or that inhibit the activity of one or more receptor polypeptides of interest. In various embodiments, the non-human animals described herein are used to determine the binding profile of antagonists and / or agonists of one or more subject polypeptides. In some embodiments, the non-human animals described herein are used to determine the epitopes of one or more candidate therapeutic antibodies that bind to one or more subject polypeptides.

[0274] In various embodiments, the non-human animals described herein are used to determine the pharmacokinetic profile of one or more human antibody candidates. In various embodiments, one or more non-human animals described herein and one or more control or reference non-human animals are each exposed to one or more human antibody candidates at various dosages (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg or more). Candidate therapeutic antibodies may be administered by any desired route of administration, including oral and parenteral routes of administration. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intrathecal, intrathecal, intraventricular, intracranial, intrapleural, or other routes of infusion. Non-injection routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, and ocular. Administration may also be by continuous infusion, topical administration, sustained release from an implant (gel, membrane, etc.), and / or intravenous injection. Blood is isolated from non-human animals (humanized and control) at various time points (e.g., 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or more). Various assays may be performed to determine the pharmacokinetic profile of the administered candidate therapeutic antibody using samples obtained from the non-human animals described herein, including, but not limited to, total IgG, anti-therapeutic antibody response, agglutination, etc.

[0275] In various embodiments, the non-human animals described herein are used to measure the therapeutic effect of inhibiting or modulating the activity of a subject polypeptide and the resulting cellular changes on gene expression or, in the context of a receptor polypeptide, on the density of receptor polypeptide on the cell surface of the non-human animal. In various embodiments, the non-human animals described herein or cells isolated therefrom are exposed to a candidate therapeutic agent, which binds to the subject polypeptide, and after a period of time, the effect on a particular cellular process associated with the subject polypeptide, such as ligand-receptor interaction or signal transduction, is analyzed.

[0276] In some embodiments, the non-human animals described herein express human antibody variable domains. Thus, cells, cell lines, and cell cultures can be generated to serve as a source of human antibody variable domains for use in binding assays and functional assays, such as assaying for antagonist or agonist binding or function, where the antagonist or agonist is specific for a human antigen of interest or for an epitope that functions in ligand-receptor interaction (binding). In various embodiments, cells isolated from the non-human animals described herein can be used to determine the epitope bound by a therapeutic candidate antibody or siRNA.

[0277] The cells from the provided non-human animals can be isolated and used ad hoc or maintained in culture for many generations. In various embodiments, the cells from the provided non-human animals are immortalized (e.g., by the use of a virus) and maintained in culture (e.g., in continuous culture) indefinitely.

[0278] In some embodiments, the non-human animals described herein provide an in vivo system for generating variants of human antibody variable domains (e.g., human Vλ domain variants) that bind to a polypeptide of interest. Such variants include human antibody variable domains that have desirable functionality, particularly functionalities such as reduced cross-reactivity to common epitopes shared by two or more variants of a polypeptide of interest. In some embodiments, the non-human animals described herein are used to generate human antibody variable domain panels containing a range of variant variable domains that are screened for desirable or improved functionality.

[0279] In certain embodiments, the non-human animals described herein provide an in vivo system for generating human antibody variable region libraries (e.g., human Vλ domain libraries). Such libraries provide a source of heavy and / or light chain variable region sequences that can be grafted onto different Fc regions based on desired effector functions, used as a source for affinity maturation of variable region sequences using techniques known in the art (e.g., site-directed mutagenesis, error-prone PCR, etc.), and / or used as a source of antibody components for the generation of antibody-based therapeutic molecules such as chimeric antigen receptors (i.e., molecules engineered using antibody components, e.g., scFvs), multispecific binders (e.g., bispecific binders), and fusion proteins (e.g., single-domain antibodies, scFvs, etc.).

[0280] In some aspects, the non-human animals described herein provide an in vivo system for the analysis and testing of drugs or vaccines. In various embodiments, a candidate drug or vaccine is delivered to one or more non-human animals described herein, and the non-human animals may then be monitored to calculate one or more of the immune response to the drug or vaccine, the safety profile of the drug or vaccine, or its effect on a disease or condition and / or on one or more symptoms of a disease or condition. Exemplary methods used to calculate the safety profile include measuring toxicity, optimal dose concentrations, antibody (i.e., anti-drug) responses, drug or vaccine efficacy, and potential risk factors. Such drugs or vaccines may be improved and / or developed in such non-human animals.

[0281] The efficacy of a vaccine can be calculated in a number of ways. Briefly, a non-human animal as described herein is immunized using methods known in the art and then exposed to the vaccine, or the vaccine is administered to an already infected non-human animal. The response of the non-human animal to the vaccine can be measured by monitoring the non-human animal (or cells isolated therefrom) and / or by performing one or more assays thereon to determine the efficacy of the vaccine. The response of the non-human animal to the vaccine is then compared to a control animal using one or more means known in the art and / or described herein.

[0282] The efficacy of the vaccine can also be calculated by virus neutralization assay. Briefly, non-human animals described herein are immunized, and serum is collected at various days after immunization. Serial dilutions of serum are pre-incubated with the virus, during which the antibodies in the serum specific for the virus bind to the virus. The virus / serum mixture is then added to permissive cells, and infectivity is calculated by plaque assay or microneutralization assay. If the antibodies in the serum neutralize the virus, the number of plaques or relative luciferase units will be lower than those in the control group.

[0283] In some embodiments, the non-human animals described herein produce human antibody variable domains, thus providing an in vivo system for the production of human antibodies for use in diagnostic applications (e.g., immunological, serological, microbiological, cytopathological applications, etc.). In various embodiments, the non-human animals described herein may be used to produce human antibody variable domains that bind to relevant antigenic sites for identifying cellular changes, such as the expression of specific cell surface markers indicative of pathological changes. Such antibodies can be conjugated to various chemical entities (e.g., radiotracers) and utilized in various in vivo and / or in vitro systems, if desired.

[0284] In some embodiments, the non-human animals described herein provide an improved in vivo system for the development and selection of human antibodies for use in tumors and / or infectious diseases. In various embodiments, the non-human animals described herein and control non-human animals (e.g., those with genetic modifications different from those described herein or those without the genetic modifications (i.e., wild-type)) can be implanted with tumors (or tumor cells) or infected with viruses (e.g., influenza, HIV, HCV, HPV, etc.). After implantation or infection, the non-human animals can be administered a candidate therapeutic agent. The tumor or virus can be allowed sufficient time to establish itself in one or more locations within the non-human animal before being administered a candidate therapeutic agent. Alternatively and / or additionally, immune responses can be monitored in such non-human animals, allowing characterization and selection of human antibodies that could potentially be developed as therapeutic agents.

[0285] kit In some embodiments, the present invention further provides packs or kits comprising one or more containers filled with at least one non-human animal, non-human cell, DNA fragment, targeting vector, or any combination thereof, as described herein. The kits may be used in any applicable method (e.g., research methods). Optionally, associated with such containers may be a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical and biological products, reflecting (a) regulatory approval for manufacture, use, or sale for human administration, (b) instructions for use, and / or (c) a contract governing the transfer of materials and / or biological products (e.g., non-human animals or non-human cells described herein) between two or more entities and combinations thereof.

[0286] Other features of certain embodiments will become apparent in the course of the following description of exemplary embodiments, which are given by way of illustration and are not intended to be limiting thereof.

[0287] Additional Exemplary Embodiments In exemplary embodiment 1, provided herein is a rodent whose germline genome comprises an endogenous immunoglobulin λ light chain locus, the light chain locus comprising (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments, wherein (a) and (b) are operably linked to (c) a rodent Cλ gene segment, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ).

[0288] In exemplary embodiment 2, provided herein is a rodent according to embodiment 1, wherein the endogenous immunoglobulin λ light chain locus comprises two rodent Eλ.

[0289] In exemplary embodiment 3, provided herein is a rodent according to embodiment 2, wherein the two rodent Eλ are mouse Eλ and mouse Eλ3-1.

[0290] In exemplary embodiment 4, provided herein is a rodent according to any one of embodiments 1-3, wherein the endogenous immunoglobulin λ light chain locus comprises three human Eλ.

[0291] In exemplary embodiment 5, the germline genome comprises: (i) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H The gene segment is operably linked to a rodent immunoglobulin heavy chain constant region, or (ii) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H Provided herein is a rodent according to any one of embodiments 1-4, wherein the gene segment further comprises an endogenous immunoglobulin κ light chain locus comprising an insertion of one or more human Vκ gene segments and one or more human Jκ gene segments at a locus operably linked to a rodent immunoglobulin Cκ region.

[0292] In exemplary embodiment 6, one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H Insertion of gene segments into rodent VH , D H Provided herein is a rodent according to embodiment 5, in which the gene segment is replaced.

[0293] In exemplary embodiment 7, the insertion is in a human V H , D H , and J. H Provided herein is a rodent according to embodiment 6, comprising gene segments, as well as naturally occurring human non-coding DNA between the gene segments.

[0294] In exemplary embodiment 8, provided herein is a rodent according to embodiment 5 or 6, wherein the insertion of one or more human Vκ gene segments and one or more human Jκ gene segments replaces the rodent's Vκ and Jκ gene segments.

[0295] In exemplary embodiment 9, provided herein is a rodent according to embodiment 8, wherein the insertion comprises human Vκ, and Jκ gene segments, and naturally occurring human non-coding DNA between the combination.

[0296] In exemplary embodiment 10, provided herein is a rodent according to any one of embodiments 5-8, wherein the rodent immunoglobulin heavy chain constant region is an endogenous rodent immunoglobulin heavy chain constant region.

[0297] In exemplary embodiment 11, provided herein is a rodent according to any one of embodiments 5-10, wherein the rodent Cκ region is an endogenous rodent Cκ region.

[0298] In exemplary embodiment 12, provided herein is a rodent of any one of embodiments 1-9, wherein the endogenous immunoglobulin λ light chain locus comprises a deletion of all or part of an endogenous Vλ and Jλ gene segment.

[0299] In exemplary embodiment 13, provided herein is a rodent as described in embodiment 12, wherein the endogenous immunoglobulin λ light chain locus comprises a deletion of the Vλ2-Vλ3-Jλ2-Cλ2 gene segments and the Vλ1-Jλ3-Cλ3-Jλ1 gene segments.

[0300] In exemplary embodiment 14, provided herein is a rodent as described in embodiment 12, wherein the endogenous immunoglobulin λ light chain locus comprises a deletion of the Vλ2-Vλ3-Jλ2-Cλ2-Jλ4P-Cλ4P gene segments and the Vλ1-Jλ3-Jλ3P-Cλ3-Jλ1 gene segments.

[0301] In exemplary embodiment 15, provided herein is a rodent according to any one of embodiments 1-14, wherein the rodent Cλ gene segment is a mouse Cλ1 gene segment.

[0302] In exemplary embodiment 16, provided herein is a rodent according to any one of embodiments 1-13, wherein the endogenous immunoglobulin λ light chain locus comprises a rodent Eλ2-4 deletion.

[0303] In exemplary embodiment 17, provided herein is a rodent according to any one of embodiments 1-16, wherein the rodent does not detectably express endogenous immunoglobulin lambda light chain.

[0304] In exemplary embodiment 18, the immunoglobulin heavy chain locus is a human V H Gene segment V H 3-74~V H 6-1, Human D H Gene segment D H 1-1~D H 7-27, and Human J H Gene segment J H 1~J H Provided herein is a rodent according to any one of embodiments 5 to 17, comprising an insertion of 6.

[0305] In exemplary embodiment 19, the insertion is in a human V H 3-74~V H Human non-coding DNA naturally occurring between 6-1, human D H 1-1~D H Naturally occurring human non-coding DNA between 7-27 and human J H 1~J H

[0036] Provided herein is a rodent according to embodiment 18, comprising naturally occurring human non-coding DNA between 6.

[0306] In exemplary embodiment 20, provided herein is a rodent according to any one of embodiments 5 to 19, wherein the immunoglobulin κ light chain locus comprises an insertion of all or part of the proximal Vκ duplication of the human immunoglobulin κ light chain locus.

[0307] In exemplary embodiment 21, provided herein is a rodent as described in embodiment 20, wherein the immunoglobulin κ light chain locus comprises an insertion of human Vκ gene segments Vκ2-40 to Vκ4-1 and human Jκ gene segments Jκ1-Jκ5.

[0308] In exemplary embodiment 22, provided herein is a rodent according to embodiment 21, wherein the insertion comprises naturally occurring human non-coding DNA between human Vκ2-40 and Vκ4-1, and naturally occurring human non-coding DNA between human Jκ1 and Jκ5.

[0309] In exemplary embodiment 23, provided herein is a rodent according to any one of embodiments 1 to 22, wherein the endogenous immunoglobulin λ light chain locus comprises an insertion of human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1, at least human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6, human Jλ gene segment Jλ7, and a rodent Cλ1 gene segment.

[0310] In exemplary embodiment 24, provided herein is a rodent as described in embodiment 23, wherein the insertion comprises naturally occurring human non-coding DNA between human Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1, naturally occurring human non-coding DNA between human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, and naturally occurring human non-coding DNA upstream (or 5') of human Jλ gene segment Jλ7.

[0311] In exemplary embodiment 25, provided herein is a rodent according to any one of embodiments 5-24, wherein the immunoglobulin heavy chain locus lacks an endogenous rodent Adam6 gene.

[0312] In exemplary embodiment 26, provided herein is a rodent described in any one of embodiments 5 to 25, wherein the immunoglobulin heavy chain locus further comprises an insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides.

[0313] In exemplary embodiment 27, the one or more nucleotide sequences are selected from a first and a second human V H Provided herein is a rodent according to embodiment 26, wherein the insertion occurs between gene segments.

[0314] In exemplary embodiment 28, provided herein is the rodent of embodiment 26, wherein the one or more nucleotide sequences are inserted in place of a human Adam6 pseudogene.

[0315] In exemplary embodiment 29, 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 H Provided herein is a rodent according to embodiment 27, wherein the rodent is 6-1.

[0316] In exemplary embodiment 30, the one or more nucleotide sequences are selected from the group consisting of human V H Gene segments and human D H Provided herein is a rodent according to embodiment 26, wherein the insertion occurs between gene segments.

[0317] In exemplary embodiment 31, provided herein is a rodent according to any one of embodiments 5-30, wherein the rodent is heterozygous or homozygous for an endogenous immunoglobulin heavy chain locus.

[0318] In exemplary embodiment 32, provided herein is a rodent according to any one of embodiments 5-31, wherein the rodent is heterozygous or homozygous for an endogenous immunoglobulin κ light chain locus.

[0319] In exemplary embodiment 33, provided herein is a rodent according to any one of embodiments 1-32, wherein the rodent is heterozygous or homozygous for an endogenous immunoglobulin lambda light chain locus.

[0320] In exemplary embodiment 34, provided herein is a rodent according to any one of embodiments 1-33, wherein the rodent is a rat or a mouse.

[0321] In exemplary embodiment 35, provided herein is an isolated rodent cell whose germline genome comprises an endogenous immunoglobulin λ light chain locus comprising: (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments, (i) wherein (a) and (b) are operably linked to (c) and a rodent Cλ gene segment, and (ii) the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ).

[0322] In exemplary embodiment 36, provided herein is an immortalized cell made from the rodent cell described in embodiment 35.

[0323] In exemplary embodiment 37, provided herein is an isolated rodent cell according to embodiment 35, wherein the rodent cell is a rodent embryonic stem cell.

[0324] In exemplary embodiment 38, provided herein is a rodent embryo produced from the rodent embryonic stem cell of embodiment 35.

[0325] In exemplary embodiment 39, provided herein is a method for producing a rodent whose germline genome comprises an engineered endogenous immunoglobulin λ light chain locus, the method comprising: (a) introducing a DNA fragment into a rodent embryonic stem cell, wherein the DNA fragment comprises a nucleotide sequence comprising: (i) one or more human Vλ gene segments, (ii) one or more human Jλ gene segments, and (iii) one or more human Cλ gene segments, wherein (i)-(iii) are operably linked to a rodent Cλ gene segment, and wherein the nucleotide sequence further comprises one or more human immunoglobulin λ light chain enhancers (Eλ); (b) obtaining the rodent embryonic stem cell produced in (a); and (c) producing a rodent using the rodent embryonic stem cell of (b).

[0326] In exemplary embodiment 40, provided herein is the method of embodiment 39, wherein the nucleotide sequence further comprises one or more human immunoglobulin lambda light chain enhancers (Eλ).

[0327] In exemplary embodiment 41, provided herein is a method of producing a rodent whose germline genome comprises an engineered endogenous immunoglobulin λ light chain locus, wherein the engineered endogenous immunoglobulin λ light chain locus comprises an insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and one or more human Cλ gene segments, wherein the human Vλ and Jλ gene segments are operably linked to rodent or human Cλ gene segments, and the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ). The method includes modifying the germline genome of a rodent so that the genome contains an engineered immunoglobulin λ light chain locus, wherein the engineered locus comprises insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and one or more human Cλ gene segments, wherein the human Vλ and Jλ gene segments are operably linked to rodent or human Cλ gene segments, and the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ), thereby generating the rodent.

[0328] In exemplary embodiment 42, provided herein is a method of embodiment 39 or 41, wherein the one or more human Vλ gene segments comprise Vλ5-52 to Vλ1-40 and / or Vλ3-27 to Vλ3-1.

[0329] In exemplary embodiment 43, provided herein is a method as described in embodiment 42, wherein the one or more human Vλ gene segments comprise naturally occurring human non-coding DNA between Vλ5-52 to Vλ1-40 and / or Vλ3-27 to Vλ3-1.

[0330] In exemplary embodiment 44, the method described in any one of embodiments 39 to 43 is provided herein, wherein the one or more human Jλ gene segments and the one or more human Cλ gene segments include human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6, and human Jλ7 gene segments.

[0331] In exemplary embodiment 45, the method described in embodiment 44 is provided herein, wherein the human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6 comprise naturally occurring human non-coding DNA between the human Jλ and Cλ gene segment pairs, and the human Jλ7 gene segment comprises naturally occurring human non-coding DNA upstream (or 5') of the human Jλ7.

[0332] In exemplary embodiment 46, provided herein is a method according to any one of embodiments 39-45, wherein the rodent Cλ gene segment is a mouse Cλ1 gene segment.

[0333] In exemplary embodiment 47, provided herein is a method of any one of embodiments 39-46, wherein the endogenous immunoglobulin λ light chain locus comprises three human Eλs.

[0334] In exemplary embodiment 48, provided herein is a method of any one of embodiments 39-46, wherein the endogenous immunoglobulin λ light chain locus comprises two rodent Eλ.

[0335] In exemplary embodiment 49, provided herein is the method of embodiment 48, wherein the two rodent Eλ are mouse Eλ and mouse Eλ3-1.

[0336] In exemplary embodiment 50, provided herein is a method according to any one of embodiments 38 and 42-49, wherein the DNA fragment further comprises one or more selectable markers.

[0337] In exemplary embodiment 51, provided herein is a method according to any one of embodiments 39 and 42-50, wherein the DNA fragment further comprises one or more site-specific recombination sites.

[0338] In exemplary embodiment 52, the DNA fragment of (a) is a fragment whose germline genome is identical to one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H The gene segment is a locus operably linked to a rodent immunoglobulin heavy chain constant region or one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H

[0033] Provided herein are methods according to any one of embodiments 39 and 42-51, wherein the gene segment is introduced into a rodent embryonic stem cell comprising an endogenous immunoglobulin κ light chain locus, the endogenous immunoglobulin κ light chain locus comprising an insertion of one or more human Vκ gene segments and one or more human Jκ gene segments, the human Vκ and Jκ gene segments being operably linked to a rodent immunoglobulin Cκ region.

[0339] In exemplary embodiment 53, the method of any one of embodiments 39 and 42-51 is provided herein, wherein the DNA fragment of (a) is introduced into a rodent embryonic stem cell whose germline genome comprises a wild-type endogenous immunoglobulin heavy chain locus, or a wild-type endogenous immunoglobulin heavy chain locus and a wild-type endogenous immunoglobulin κ light chain locus, and wherein the method further comprises mating the mouse produced from the non-human embryonic stem cell with a second mouse.

[0340] In exemplary embodiment 54, modifying the germline genome of a rodent to include an engineered endogenous immunoglobulin lambda light chain locus in the germline genome comprises modifying the germline genome to include one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H The gene segment is a locus operably linked to a rodent immunoglobulin heavy chain constant region or one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H

[0023] Provided herein is a method according to any one of embodiments 47-49, wherein the gene segment is performed in a rodent embryonic stem cell further comprising a locus operably linked to a rodent immunoglobulin heavy chain constant region, and an endogenous immunoglobulin κ light chain locus comprising an insertion of one or more human Vκ gene segments and one or more human Jκ gene segments, wherein the human Vκ and Jκ gene segments are operably linked to a rodent immunoglobulin Cκ region.

[0341] In exemplary embodiment 55, 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 result in one or more human V H Naturally occurring human non-coding DNA between gene segments, including one or more human D H naturally occurring human non-coding DNA between gene segments, and one or more human J H 55. The method of embodiment 52 or 54 is provided herein, comprising naturally occurring human non-coding DNA between the gene segments.

[0342] In exemplary embodiment 56, provided herein is a method according to embodiment 52 or 54, wherein the insertion of one or more human Vκ gene segments and one or more human Jκ gene segments comprises naturally occurring human non-coding DNA between the one or more human Vκ gene segments, and naturally occurring human non-coding DNA between the one or more human Jκ gene segments.

[0343] In exemplary embodiment 57, provided herein is a method according to any one of embodiments 41-49, wherein modifying the germline genome of the non-human animal to include an engineered immunoglobulin λ light chain locus in the germline genome is performed in a non-human embryonic stem cell whose germline genome includes a wild-type endogenous immunoglobulin heavy chain locus, or a wild-type endogenous immunoglobulin heavy chain locus and a wild-type endogenous immunoglobulin κ light chain locus, and wherein the method further includes mating the mouse produced from the non-human embryonic stem cell with a second mouse.

[0344] In exemplary embodiment 58, provided herein is a method of any one of embodiments 53 or 57, wherein the second mouse has a germline genome comprising wild-type IgH and Igκ loci.

[0345] In exemplary embodiment 59, provided herein is a method according to any one of embodiments 53 or 57, wherein the second mouse has a germline genome comprising homozygous or heterozygous humanized IgH and Igκ loci, and the homozygous or heterozygous humanized IgH loci contain an inserted rodent Adam6 coding sequence.

[0346] In exemplary embodiment 60, provided herein is a method according to any one of embodiments 53 or 57, wherein the second mouse has a germline genome comprising a homozygous or heterozygous humanized IgH locus and a homozygous or heterozygous inactivated Igκ locus.

[0347] In exemplary embodiment 61, provided herein is a method of raising antibodies in a rodent, the method comprising: (1) immunizing the rodent with an antigen of interest, wherein the rodent has a germline genome comprising an endogenous immunoglobulin λ light chain locus comprising (ai) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments, wherein (a) and (b) are linked to (c) and rodent Cλ genes. segment, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ); (2) maintaining the rodent under conditions sufficient for the rodent to mount an immune response to the target antigen; and (3) recovering from the rodent or rodent cells an antibody that binds to the target antigen.

[0348] In exemplary embodiment 62, the rodent is a mammalian animal, comprising one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H, and J. H The gene segment is a locus operably linked to a rodent immunoglobulin heavy chain constant region or one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H

[0019] Provided herein is the method of embodiment 61, wherein the gene segment has a germline genome further comprising a locus operably linked to a rodent immunoglobulin heavy chain constant region, and an endogenous immunoglobulin κ light chain locus comprising an insertion of one or more human Vκ gene segments and one or more human Jκ gene segments, wherein the human Vκ and Jκ gene segments are operably linked to a rodent immunoglobulin Cκ region.

[0349] In exemplary embodiment 63, provided herein is a method according to any one of embodiments 61 or 62, wherein the rodent cell is a B cell.

[0350] In exemplary embodiment 64, provided herein is a method according to any one of embodiments 61 or 62, wherein the rodent cell is a hybridoma.

[0351] In exemplary embodiment 65, the endogenous immunoglobulin λ light chain locus comprises an insertion of human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1, human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6, and human Jλ gene segment Jλ7, as described in any one of embodiments 61 to 64.

[0352] In exemplary embodiment 66, the method of embodiment 65 is provided herein, wherein the insertion comprises naturally occurring human non-coding DNA between human Vλs Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1, naturally occurring human non-coding DNA between human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, and naturally occurring human non-coding DNA upstream (or 5') of human Jλ gene segment Jλ7.

[0353] In exemplary embodiment 67, provided herein is a method according to any one of embodiments 61-66, wherein the rodent Cλ gene segment is a mouse Cλ1 gene segment.

[0354] In exemplary embodiment 68, the immunoglobulin heavy chain locus is a human V H Gene segment V H 3-74~V H 6-1, Human D H Gene segment D H 1-1~D H 7-27, and Human J H Gene segment J H 1~J H 6 insertion, and the human V H , D H , and J. H

[0023] 68. The method of any one of embodiments 62-67, wherein the gene segment is operably linked to an endogenous rodent immunoglobulin heavy chain constant region.

[0355] In exemplary embodiment 69, the insertion is in a human V H 3-74~V H Human non-coding DNA naturally occurring between 6-1, human D H 1-1~D H Naturally occurring human non-coding DNA between 7-27 and human J H 1~J H 69. The method of embodiment 68 is provided herein, comprising naturally occurring human non-coding DNA between 6.

[0356] In exemplary embodiment 70, the human V H , D H , and J. H The gene segment is derived from rodent V H , D H , and J. H

[00133] Provided herein is a method according to embodiment 68, in which a gene segment is replaced.

[0357] In exemplary embodiment 71, provided herein is a method according to any one of embodiments 62 to 70, wherein the immunoglobulin κ light chain locus comprises an insertion of human Vκ gene segments Vκ2-40 to Vκ4-1 and human Jκ gene segments Jκ1-Jκ5, and the human Vκ and Jκ gene segments are operably linked to an endogenous rodent immunoglobulin Cκ region.

[0358] In exemplary embodiment 72, the method of embodiment 71 is provided herein, wherein the insertion comprises a human non-coding DNA naturally occurring between human Vκ2-40 and Vκ4-1, and a human non-coding DNA naturally occurring between human Jκ1 and Jκ5.

[0359] In exemplary embodiment 73, provided herein is the method of embodiment 71, wherein the human Vκ and Jκ gene segments replace rodent Vκ and Jκ gene segments.

[0360] In exemplary embodiment 74, the method described in any one of embodiments 61 to 73 is provided herein, wherein the rodent germline genome further comprises an insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides.

[0361] In exemplary embodiment 75, provided herein is a method of any one of embodiments 62-74, wherein the immunoglobulin heavy chain locus lacks an endogenous rodent Adam6 gene.

[0362] In exemplary embodiment 76, the immunoglobulin heavy chain locus further comprises an insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides, as described in embodiment 75.

[0363] In exemplary embodiment 77, the one or more nucleotide sequences encoding the one or more rodent Adam6 polypeptides are selected from the group consisting of a first and a second human V H 77. The method of embodiment 76, wherein the nucleic acid sequence is inserted between gene segments.

[0364] In exemplary embodiment 78, 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 H 6-1.

[0365] In exemplary embodiment 79, provided herein is a method according to embodiment 76, wherein one or more nucleotide sequences encoding the one or more rodent Adam6 polypeptides are inserted in place of a human Adam6 pseudogene.

[0366] In exemplary embodiment 80, the one or more nucleotide sequences encoding the one or more rodent Adam6 polypeptides are selected from the group consisting of human V H Gene segments and human D H 77. The method of embodiment 76, wherein the nucleic acid sequence is inserted between gene segments.

[0367] In exemplary embodiment 81, provided herein is a method according to any one of embodiments 61 to 80, wherein the antibody that binds to the antigen of interest recovered from the rodent or rodent cell comprises a human heavy chain variable domain and a human lambda light chain variable domain.

[0368] In exemplary embodiment 82, the human heavy chain variable domain is a rearranged human V H a gene segment, the segment being 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 and V H 82. The method of embodiment 81, wherein the method is selected from the group consisting of: 6-1.

[0369] In exemplary embodiment 83, the human lambda light chain variable domain comprises a rearranged human Vλ gene segment, the segment being 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,

[00130] Provided herein is the method of embodiment 81 or 82, wherein the λ5-37, λ1-36, λ3-27, λ3-25, λ2-23, λ3-22, λ3-21, λ3-19, λ2-18, λ3-16, λ2-14, λ3-12, λ2-11, λ3-10, λ3-9, λ2-8, λ4-3, and λ3-1.

[0370] In exemplary embodiment 84, provided herein is a method according to any one of embodiments 39-83, wherein the rodent is a rat or a mouse.

[0371] In exemplary embodiment 85, provided herein is a rodent whose germline genome comprises a homozygous endogenous immunoglobulin λ light chain locus comprising: (i) human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1, (ii) human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, (iii) human Jλ gene segment Jλ7, and (iv) three human immunoglobulin λ light chain enhancers, wherein (i) through (iv) are operably linked to each other, and (i) through (iii) are rodent Cλ gene segments. and the endogenous immunoglobulin λ light chain locus lacks endogenous rodent immunoglobulin Eλ2-4, human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1 comprise naturally occurring human non-coding DNA between the human Vλ gene segments, human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6 comprise naturally occurring human non-coding DNA between the human Jλ-Cλ gene segment pair, and human Jλ gene segment Jλ7 comprises naturally occurring human non-coding DNA upstream (or 5') of human Jλ7.

[0372] In exemplary embodiment 86, provided herein is a rodent according to embodiment 85, wherein the rodent Cλ gene segment is a mouse Cλ1 gene segment.

[0373] In exemplary embodiment 87, provided herein is a rodent of embodiment 85 or 86, wherein the endogenous immunoglobulin λ light chain locus further comprises endogenous rodent immunoglobulin λ light chain enhancers Eλ and Eλ3-1.

[0374] In exemplary embodiment 88, provided herein is a rodent described in any one of embodiments 85 to 87, wherein the endogenous immunoglobulin λ light chain locus comprises a deletion of the endogenous rodent Vλ2-Vλ3-Jλ2-Cλ2-Jλ4P-Cλ4P gene segments and the Vλ1-Jλ3-Jλ3P-Cλ3-Jλ1 gene segments.

[0375] In exemplary embodiment 89, provided herein is a rodent according to any one of embodiments 85-88, wherein the rodent is a rat or a mouse.

[0376] In some embodiments, provided herein is a rodent whose germline genome comprises an endogenous immunoglobulin λ light chain locus, wherein the light chain locus comprises (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments, wherein (a) and (b) are operably linked to (c) a rodent Cλ gene segment, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ).

[0377] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises two rodent Eλ.

[0378] In some embodiments, the two rodent Eλ are a mouse Eλ and a mouse Eλ3-1.

[0379] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises three human Eλs.

[0380] In some embodiments, the germline genome comprises (i) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J Han endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H The gene segment is operably linked to a rodent immunoglobulin heavy chain constant region, or (ii) one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a gene segment, H , D H , and J. H The gene segments further include a locus operably linked to a rodent immunoglobulin heavy chain constant region, and an endogenous immunoglobulin κ light chain locus comprising an insertion of one or more human Vκ gene segments and one or more human Jκ gene segments, wherein the human Vκ and Jκ gene segments are operably linked to a rodent immunoglobulin Cκ region.

[0381] 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 Insertion of gene segments into rodent V H , D H Replace gene segments.

[0382] In some embodiments, the insertion includes a human V H , D H , and J. H It includes gene segments as well as naturally occurring human non-coding DNA between combinations thereof.

[0383] In some embodiments, the insertion of one or more human Vκ gene segments and one or more human Jκ gene segments replaces rodent Vκ and Jκ gene segments.

[0384] In some embodiments, the insertion includes human Vκ and Jκ gene segments and naturally occurring human non-coding DNA between the combinations.

[0385] In some embodiments, the rodent immunoglobulin heavy chain constant region is an endogenous rodent immunoglobulin heavy chain constant region.

[0386] In some embodiments, the rodent Cκ region is an endogenous rodent Cκ region.

[0387] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises a deletion of all or part of an endogenous Vλ and Jλ gene segment.

[0388] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises a deletion of the Vλ2-Vλ3-Jλ2-Cλ2 gene segments and the Vλ1-Jλ3-Cλ3-Jλ1 gene segments.

[0389] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises a deletion of the Vλ2-Vλ3-Jλ2-Cλ2-Jλ4P-Cλ4P gene segments and the Vλ1-Jλ3-Jλ3P-Cλ3-Jλ1 gene segments.

[0390] In some embodiments, the rodent Cλ gene segment is a mouse Cλ1 gene segment.

[0391] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises a rodent Eλ2-4 deletion.

[0392] In some embodiments, the rodent does not detectably express endogenous immunoglobulin λ light chains.

[0393] In some embodiments, the immunoglobulin heavy chain locus is a human V H Gene segment V H 3-74 to V H6-1, Human D H Gene segment D H 1-1 to D H 7-27, and Human J H Gene segment J H 1-J H Includes 6 insertions.

[0394] In some embodiments, the insertion is a human V H 3-74~V H Human non-coding DNA naturally occurring between 6-1, human D H 1-1~D H Naturally occurring human non-coding DNA between 7-27 and human J H 1~J H 6, including naturally occurring human non-coding DNA.

[0395] In some embodiments, the immunoglobulin κ light chain locus comprises an insertion of all or part of the proximal Vκ duplication of the human immunoglobulin light chain κ locus.

[0396] In some embodiments, the immunoglobulin κ light chain locus comprises an insertion of human Vκ gene segments Vκ2-40 through Vκ4-1 and human Jκ gene segments Jκ1-Jκ5.

[0397] In some embodiments, the insertion includes a human non-coding DNA that naturally occurs between human Vκ2-40 and Vκ4-1, and a human non-coding DNA that naturally occurs between human Jκ1 and Jκ5.

[0398] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises an insertion of human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1, at least human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6, human Jλ gene segment Jλ7, and a rodent Cλ1 gene segment.

[0399] In some embodiments, the insertions include naturally occurring human non-coding DNA between human Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1, naturally occurring human non-coding DNA between human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, and naturally occurring human non-coding DNA upstream (or 5') of human Jλ gene segment Jλ7.

[0400] In some embodiments, the immunoglobulin heavy chain locus lacks an endogenous rodent Adam6 gene.

[0401] In some embodiments, the immunoglobulin heavy chain locus further comprises an insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides.

[0402] In some embodiments, the one or more nucleotide sequences comprise a first and a second human V H It is inserted between gene segments.

[0403] In some embodiments, the one or more nucleotide sequences are inserted in place of the human Adam6 pseudogene.

[0404] 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 H The score is 6-1.

[0405] In some embodiments, the one or more nucleotide sequences are selected from the group consisting of human V H Gene segments and human D H It is inserted between gene segments.

[0406] In some embodiments, the rodent is heterozygous or homozygous for an endogenous immunoglobulin heavy chain locus.

[0407] In some embodiments, the rodent is heterozygous or homozygous for an endogenous immunoglobulin κ light chain locus.

[0408] In some embodiments, the rodent is heterozygous or homozygous for an endogenous immunoglobulin λ light chain locus.

[0409] In some embodiments, the rodent is a rat or a mouse.

[0410] In some embodiments, provided herein is an isolated rodent cell whose germline genome comprises an endogenous immunoglobulin λ light chain locus comprising: (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments, (i) wherein (a) and (b) are operably linked to (c) a rodent Cλ gene segment, and (ii) the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ).

[0411] In some embodiments, provided herein are immortalized cells made from the rodent cells provided herein.

[0412] In some embodiments, the rodent cells are rodent embryonic stem cells.

[0413] In some embodiments, provided herein are rodent embryos generated from the rodent embryonic stem cells provided herein.

[0414] In some embodiments, provided herein are methods for producing a rodent whose germline genome comprises an engineered endogenous immunoglobulin λ light chain locus, the method comprising: (a) introducing a DNA fragment into a rodent embryonic stem cell, wherein the DNA fragment comprises a nucleotide sequence comprising: (i) one or more human Vλ gene segments, (ii) one or more human Jλ gene segments, and (iii) one or more human Cλ gene segments, wherein (i)-(iii) are operably linked to a rodent Cλ gene segment, and wherein the nucleotide sequence further comprises one or more human immunoglobulin λ light chain enhancers (Eλ); (b) obtaining the rodent embryonic stem cells produced in (a); and (c) producing a rodent using the rodent embryonic stem cells of (b).

[0415] In some embodiments, the nucleotide sequence further comprises one or more human immunoglobulin lambda light chain enhancers (Eλ).

[0416] In some embodiments, provided herein are methods of producing a rodent whose germline genome comprises an engineered endogenous immunoglobulin λ light chain locus, wherein the engineered endogenous immunoglobulin λ light chain locus comprises an insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and one or more human Cλ gene segments, wherein the human Vλ and Jλ gene segments are operably linked to rodent or human Cλ gene segments, and the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ); The method includes modifying the germline genome of a rodent so that the genome contains an engineered immunoglobulin λ light chain locus, wherein the engineered locus comprises insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and one or more human Cλ gene segments, wherein the human Vλ and Jλ gene segments are operably linked to rodent or human Cλ gene segments, and the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ) and one or more human immunoglobulin λ light chain enhancers (Eλ), thereby generating the rodent.

[0417] In some embodiments, the one or more human Vλ gene segments include Vλ5-52 through Vλ1-40 and / or Vλ3-27 through Vλ3-1.

[0418] In some embodiments, the one or more human Vλ gene segments comprise naturally occurring human non-coding DNA between Vλ5-52 to Vλ1-40 and / or Vλ3-27 to Vλ3-1.

[0419] In some embodiments, the one or more human Jλ gene segments and the one or more human Cλ gene segments include human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6, and a human Jλ7 gene segment.

[0420] In some embodiments, the human Jλ-Cλ gene segment pairs Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6 comprise naturally occurring human non-coding DNA between the human Jλ and Cλ gene segment pairs, and the human Jλ7 gene segment comprises naturally occurring human non-coding DNA upstream (or 5') of the human Jλ7.

[0421] In some embodiments, the rodent Cλ gene segment is a mouse Cλ1 gene segment.

[0422] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises three human Eλs.

[0423] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises two rodent Eλ.

[0424] In some embodiments, the two rodent Eλ are a mouse Eλ and a mouse Eλ3-1.

[0425] In some embodiments, the DNA fragment further comprises one or more selectable markers.

[0426] In some embodiments, the DNA fragment further comprises one or more site-specific recombination sites.

[0427] In some embodiments, the DNA fragment of (a) is a fragment whose germline genome contains one or more human V H gene segment, one or more human D H a gene segment, and one or more human J H an endogenous immunoglobulin heavy chain locus containing an insertion of a ...

Claims

[Claim 1] An object, method, or system as described in the specification.

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