Non-human animals with engineered lambda light chain locus

Abstract

FIELD: biotechnology.SUBSTANCE: method comprising immunizing a genetically modified mouse with an antigen of interest, wherein the genetically modified mouse is characterized by a germline genome containing an engineered endogenous light λ -immunoglobulin chain locus that contains (i) one or more segments of the human V geneλ , (ii) one or more segments of the human J gene λ and (iii) one or more segments of the human C geneλ . In this case, the constructed endogenous light λ -immunoglobulin chain locus additionally contains one or more enhancers of mice light λ -immunoglobulin chain (Eλ) and one or more enhancers of human light λ -immunoglobulin chain (Eλ).EFFECT: effective for producing antibodies.21 cl, 21 dwg, 4 tbl, 6 ex

Description

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 417,845, filed November 4, 2016, and U.S. Provisional Patent Application Serial No. 62 / 567,932, filed October 10, 2017, each of which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0002] Human antibodies represent the fastest-growing class of therapeutic agents. Among the technologies currently used for their production, the development of transgenic animals (e.g., rodents) modified with genetic material encoding complete or partial human antibodies has revolutionized the field of therapeutic monoclonal human antibodies for the treatment of various diseases. There remains a need to develop improved in vivo systems for the production of monoclonal human antibodies that maximize the human antibody repertoires in transgenic host animals. SUMMARY OF THE INVENTION

[0003] In certain aspects, improved in vivo systems are provided herein for identifying and developing new antibody therapeutics 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 have engineered immunoglobulin loci, particularly engineered immunoglobulin (Ig) λ light chain loci, and / or that otherwise express, produce, or contain antibody repertoires characterized by light chains with human Vλ regions are useful, for example, in engineering the diversity of human Vλ sequences for the purposes of identifying and developing new antibody-based therapeutics.In some embodiments, the non-human animals described herein provide improved in vivo systems for developing antibodies and / or antibody-based therapeutics for administration to humans. In some embodiments, the non-human animals described herein provide improved in vivo systems for developing antibodies and / or antibody-based therapeutics that contain human Vλ domains, which have improved performance compared to antibodies and / or antibody-based therapeutics derived from existing in vivo systems that contain human Vλ region sequences.

[0004] In certain aspects, provided herein is a non-human animal with an Ig light chain λ locus that comprises engineered immunoglobulin variable and constant regions; in some certain embodiments, further comprises an engineered regulatory region (or sequence). As described herein, in certain embodiments, the provided non-human animals comprise in their germline genome an Ig light chain λ locus that comprises an engineered Ig 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 operably linked to said rodent Cλ region gene.

[0005] In some embodiments, the provided non-human animals comprise an Ig light λ 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 animals comprise an Ig light λ chain locus that comprises 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, or 5-6 segments of a human Vλ gene. In some embodiments, the provided non-human animals comprise an Ig λ light chain locus that comprises 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, 10-39, 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 segments of the human Vλ gene.

[0007] In some embodiments, the provided non-human animals comprise an Ig light λ chain locus that comprises 6-25, 7-25, 8-25, 9-25, 10-25, 11-25, 12-25, 13-25, 14-25, 15-25, 16-25, 17-25, 18-25, 19-25, 20-25, 21-25, 22-25, 23-25, or 24-25 segments of a human Vλ gene. In some embodiments, the provided non-human animals comprise an Ig λ light chain locus that comprises 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, 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 segments of the human Vλ gene.

[0008] In some embodiments, the provided non-human animals comprise an Ig light λ chain locus that comprises 6-24, 7-23, 8-22, 9-21, 10-20, 11-19, 12-18, 13-17, 14-16, or 15-16 segments of a human Vλ gene. In some embodiments, the provided non-human animals comprise an Ig λ light chain locus that comprises the 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 segments human Vλ gene.

[0009] In certain embodiments, the provided non-human animals comprise an Ig light λ chain locus that comprises 5, 16, or 25 functional segments of a human Vλ gene. In certain embodiments, the provided non-human animals comprise an Ig light λ chain locus that comprises 10, 27, or 40 segments of a human Vλ gene. In certain embodiments, the human Vλ gene segments comprise consecutive segments of a human Vλ gene such that said segments of a human Vλ gene are located in a human Ig light λ chain locus in a human cell.

[0010] In some embodiments, the provided non-human animals comprise an Ig light λ chain locus that comprises at least 5 human Jλ gene segments (e.g., without limitation, 5 human Jλ gene segments, 6 human Jλ gene segments, 7 human Jλ gene segments, 8 human Jλ gene segments, etc.). In some embodiments, the provided non-human animals comprise an Ig light λ chain locus that comprises at least 4 human Cλ region genes (e.g., without limitation, 4 human Cλ region genes, 5 human Cλ region genes, 6 human Cλ region genes, 7 human Cλ region genes, 8 human CX region genes, etc.).In certain embodiments, the provided non-human animals comprise an Ig λ light chain locus that comprises at least 25 human Vλ gene segments, at least 5 human Jλ gene segments, and at least 4 human Cλ region genes in an endogenous Ig λ light chain allele. In some embodiments, the provided non-human animals comprise only one murine (e.g., mouse or rat) Cλ region gene (e.g., mouse Cλ1 gene or mouse Cλ1 gene segment) in an endogenous non-human Ig λ light chain locus. In some embodiments, the said Ig λ light chain locus further comprises a human Eλ region (or sequence) that is characterized by three sequence elements.

[0011] In some embodiments, the provided non-human animals comprise segments of human Vλ, Jλ, and Cλ genes at an endogenous non-human Ig light λ chain locus in a natural or germline configuration. In some embodiments, the provided non-human animals comprise segments of human Vλ, Jλ, and Cλ genes at an endogenous non-human Ig light λ chain locus in a configuration that is not naturally present at the human immunoglobulin light λ chain locus of the germline genome of a human cell.

[0012] In some embodiments, the provided non-human animals comprise a DNA sequence at an endogenous non-human Ig light chain locus that includes a plurality of human Vλ, Jλ, and Cλ coding sequences interspersed (or located adjacent, linked, etc.) with a non-coding sequence of a human immunoglobulin light chain. In some embodiments, the provided non-human animals comprise a DNA sequence at an endogenous non-human Ig light chain locus that includes a plurality of human Vλ, Jλ, and Cλ coding sequences interspersed with a non-coding sequence of a non-human (e.g., mouse) immunoglobulin light chain λ.

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

[0014] In some embodiments, a non-human animal, a non-human cell, or a non-human 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 (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers.

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

[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 specific 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, the endogenous immunoglobulin λ light chain locus provided herein comprises two non-human Eλ. In some specific embodiments, the endogenous immunoglobulin λ light chain locus provides two rodent Eλ. In some specific embodiments, the endogenous immunoglobulin λ light chain locus provides herein comprises two mouse Eλ. In some specific embodiments, the endogenous immunoglobulin λ light chain locus provides herein comprises mouse Eλ and mouse Eλ3-1. In some specific embodiments, the endogenous immunoglobulin λ light chain locus provides herein does not comprise (or is missing) mouse Eλ2-4. In some specific embodiments, the 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 the endogenous Vλ and Jλ gene segments, in whole or in part. In some specific 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 specific 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 certain specific embodiments, the 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 certain specific embodiments, the endogenous immunoglobulin λ light chain locus provided herein comprises Cλ1, Eλ, and Eλ3-1, if only non-human gene segments or sequence elements are present.

[0019] In some embodiments, the endogenous immunoglobulin λ light chain locus provided herein comprises an insertion of human Vλ gene segments from Vλ4-69 to Vλ3-1, at least pairs of human Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6 gene segments, a human Jλ gene segment, Jλ7, and a rodent Cλ1 gene segment. In some embodiments, the endogenous immunoglobulin λ light chain locus provided herein comprises an insertion of human Vλ gene segments from Vλ5-52 to Vλ3-1, at least pairs of human Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6 gene segments, a human Jλ gene segment, Jλ7, and a rodent Cλ1 gene segment.In some embodiments, the endogenous immunoglobulin λ light chain locus provided herein comprises an insertion of human Vλ gene segments from Vλ5-52 to Vλ1-40 and from Vλ3-27 to Vλ3-1, at least pairs of human Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6 gene segments, a human Jλ gene segment, Jλ7, and a rodent Cλ1 gene segment. In some specific embodiments, the insert comprises human non-coding DNA that occurs naturally between human Vλ5-52 and Vλ1-40 and Vλ3-27 and Vλ3-1, human non-coding DNA that occurs naturally between pairs of human gene segments Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3 and Jλ6-Cλ6, and human non-coding DNA that occurs naturally upstream (or 5') of a human gene segment Jλ, 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 specific 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 Cλ gene comprises a sequence that is substantially identical or identical to a mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3. In some specific embodiments, the mouse Cλ1 gene is or comprises SEQ ID NO: 1. In some specific embodiments, the mouse CX2 gene is or comprises SEQ ID NO: 3. In some specific embodiments, the mouse Cλ3 gene is or comprises SEQ ID NO: 5.In some specific embodiments, the mouse Cλ gene comprises a sequence that is identical to the mouse Cλ1 gene.

[0022] In some embodiments, the mouse Cλ gene (or gene segment) comprises a sequence that is 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, or 98%-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 50%-98%, 50%-95%, 50%-90%, 50%-85%, 50%-80%, 50%-75%, 50%-70%, 50%-65%, 50%-60%, or 50%-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%-98%, 60%-95%, 65%-90%, 70%-85%, or 75%-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 a rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 gene. In some embodiments, the rat Cλ gene comprises a sequence that is substantially identical to or identical to a rat Cλ gene selected from the group consisting of rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4. In some specific embodiments, the rat Cλ4 gene is or comprises SEQ ID NO:7. In some specific embodiments, the rat Cλ2 gene is or comprises SEQ ID NO:9.In certain specific embodiments, the rat Cλ3 gene is or comprises SEQ ID NO: 11. In certain specific 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%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, or 98%-100% identical to a rat Cλ gene selected from the group consisting of a rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 gene.

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

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

[0029] In some embodiments of the provided non-human animal, non-human cell, or non-human tissue, the germline genome or genome of said non-human animal, non-human cell, or non-human tissue further comprises (i) an endogenous immunoglobulin heavy chain locus comprising an insertion of one or more segments of a human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J Hfunctionally linked to a non-human immunoglobulin heavy chain constant region; or (ii) an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J H functionally linked to a non-human constant region of an immunoglobulin heavy chain, and an endogenous immunoglobulin κ light chain locus containing 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 functionally linked to a non-human Cκ region of an immunoglobulin.

[0030] In some embodiments, the inserted one or more segments of the human V gene H, one or more segments of the human D gene H and one or more segments of the human J gene H replace segments of non-human V genes H , D H In certain embodiments, the insert comprises non-coding human DNA that occurs naturally between segments of human V H , D H and J H and combinations thereof. 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 comprises an insertion of human V gene segments. H from V H 3-74 to V H 6-1, segments of the human D gene H from D H 1-1 to D H 7-27 and segments of the human J gene H , J H 1-J H6. In certain embodiments, the insert comprises non-coding human DNA that occurs naturally between human V H 3-74 and V H 6-1, human non-coding DNA that occurs (is found) in nature between human D H 1-1 and D H 7-27, and human noncoding DNA, which occurs (is) naturally between human J H 1 and J H 6. In some embodiments, the immunoglobulin heavy chain locus comprises an insertion of all functional segments of the human V gene H , all functional segments of the human D gene H and all functional segments of the human J gene H .

[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, one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides are inserted between the first and second segments of the human V gene. H In some embodiments, the first segment of the human V gene H represents the human V H 1-2, and the second segment of the human V gene H represents the human V H6-1. In some embodiments, one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides are inserted between a segment of the human V gene H and a segment of the human D gene H 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 segments of a human Vκ gene and one or more segments of a human Jκ gene replace segments of the non-human Vκ and Jκ genes. In some specific embodiments, the insertion comprises non-coding human DNA that occurs naturally between segments of the human Vκ and Jκ genes and combinations thereof. In some embodiments, the non-human Cκ region of the immunoglobulin is an endogenous non-human Cκ region. In some embodiments, the immunoglobulin κ light chain locus comprises an insertion of a proximal duplication of Vκ, in whole or in part, of a human immunoglobulin κ light chain locus. In some embodiments, 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 to Jκ5.In some specific embodiments, the insert comprises human non-coding DNA that occurs naturally between human Vκ2-40 and Vκ4-1, and human non-coding DNA that occurs naturally between human JκI and Jκ5.

[0033] In some embodiments of the non-human animal, non-human cell, or non-human tissue provided herein, the non-human animal, non-human cell, or non-human tissue is 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 animal, non-human cell, or non-human tissue provided herein, the non-human animal, non-human cell, or non-human tissue is 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 animal, non-human cell, or non-human tissue provided herein, the non-human animal, non-human cell, or non-human tissue is 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 animal, non-human cell, or non-human tissue provided herein, the germline genome of said 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 wherein the animal is heterozygous or homozygous for said insertion.

[0037] In some embodiments, the non-human cell is a non-human lymphocyte. In 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 cell is a non-human embryonic stem (ES) cell. In some embodiments, the non-human ES cell is a rodent ES cell. In certain embodiments, the rodent ES cell is a mouse ES cell (e.g., from the 129 line, the C57BL line, the BALB / c line, or a cross thereof). In some specific embodiments, the rodent embryonic stem cell is a mouse embryonic stem cell and is a cross between the 129 and C57BL lines. In some specific embodiments, the rodent embryonic stem cell is a mouse embryonic stem cell and is a cross between the 129, C57BL, and BALB / c lines.

[0039] In some embodiments, the non-human ES cell described herein is used to produce a non-human animal. In certain embodiments, the non-human ES cell is a mouse ES cell and is used to produce a mouse comprising the engineered immunoglobulin λ light chain locus described herein. In certain embodiments, the non-human ES cell is a rat ES cell and is used to produce a rat comprising the engineered immunoglobulin λ light chain locus described herein.

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

[0041] In some embodiments, an immortalized cell is provided that is created, formed, produced, or obtained from an isolated non-human cell or tissue described herein.

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

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

[0044] In some embodiments, a kit described herein is provided for use in the manufacture and / or development of a medicament (e.g., an antibody or fragment thereof) for therapy or diagnosis.

[0045] In some embodiments, a kit described herein is provided for use in the manufacture and / or development of a medicament (e.g., an antibody or fragment thereof) for treating, preventing, or ameliorating a disease, disorder, or condition.

[0046] In some embodiments, a method is provided 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 that includes (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 (Eλ) enhancers; (b) obtaining a non-human embryonic stem cell created from (a);and (c) producing a non-human animal using a non-human embryonic stem cell from (b).;

[0047] In some embodiments, a method is 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 a non-human and / or human Cλ gene segment, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more non-human immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers;wherein the method comprises modifying the germline genome of a non-human animal such that it comprises an engineered immunoglobulin λ light chain locus that comprises an insertion of one or more segments of a human Vλ gene, one or more segments of a human Jλ gene, and one or more segments of a human Cλ gene, wherein the segments of the human Vλ and Jλ genes are operably linked to a segment of a non-human and / or human Cλ gene, and wherein the endogenous immunoglobulin X light chain locus further comprises one or more rodent immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers, thereby producing said non-human animal.

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

[0049] In some specific embodiments of the method for producing a non-human animal provided herein, the insert of the human Vλ gene segments from Vλ5-52 to Vλ1-40 and from Vλ3-27 to Vλ3-1 comprises human non-coding DNA that occurs naturally between the human Vλ gene segments, the insert of the pairs of human Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3 and Jλ6-Cλ6 gene segments comprises human non-coding DNA that occurs naturally between the pairs of human Jλ-Cλ gene segments, and the insert of the human Jλ7 gene segment comprises human non-coding DNA that occurs naturally upstream (or in the 5' direction) of human Jλ7.

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

[0051] In some embodiments of the method for producing a non-human animal provided herein, the DNA fragment further comprises one or more selection markers. In some embodiments of the method for producing a non-human animal, the DNA fragment further comprises one or more site-specific recombination sites. In some specific embodiments of the method for producing a non-human animal provided herein, the DNA fragment further comprises one or more sets of site-specific recombination sites that recombine with the same recombinase. In some specific embodiments of the method for producing a non-human animal, 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 method 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) an endogenous immunoglobulin heavy chain locus comprising an insertion of one or more segments of a human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J H functionally linked to a non-human immunoglobulin heavy chain constant region; or (ii) an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H, D H and J H functionally linked to a non-human constant region of an immunoglobulin heavy chain, and an endogenous immunoglobulin κ light chain locus containing 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 functionally linked to a non-human Cκ region of an immunoglobulin.

[0053] In some embodiments of the method 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) an endogenous wild-type immunoglobulin heavy chain locus or (ii) an endogenous wild-type immunoglobulin heavy chain locus and an endogenous wild-type immunoglobulin κ light chain locus; and wherein the method further comprises the step of crossing a mouse created, formed, produced, or obtained from said non-human embryonic stem cell with a second mouse.

[0054] In some embodiments of the method for producing a non-human animal provided herein, the modification of the germline genome of the non-human animal so that it comprises an engineered immunoglobulin λ light chain locus is performed in a non-human embryonic stem cell whose germline genome comprises (i) an endogenous immunoglobulin heavy chain locus comprising an insertion of one or more segments of a human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J H functionally linked to a non-human immunoglobulin heavy chain constant region; or (ii) an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H, one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J H functionally linked to a non-human constant region of an immunoglobulin heavy chain, and an endogenous immunoglobulin κ light chain locus containing 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 functionally linked to a non-human Cκ region of an immunoglobulin.

[0055] In some specific embodiments of the method for producing a non-human animal provided herein, inserting one or more segments of a human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene Hrefers to non-coding human DNA that occurs naturally between one or more segments of the human V gene H , human noncoding DNA that occurs naturally between one or more segments of the human D gene H , and human noncoding DNA that occurs naturally between one or more segments of the human J gene H In some specific embodiments of the method for producing a non-human animal provided herein, the insertion of one or more segments of a human Vκ gene and one or more segments of a human Jκ gene provides for non-coding human DNA that occurs naturally between one or more segments of a human Vκ gene and non-coding human DNA that occurs naturally between one or more segments of a human Jκ gene.

[0056] In some embodiments of the method for producing a non-human animal provided herein, the modification of the germline genome of the non-human animal so that it comprises an engineered immunoglobulin λ light chain locus is performed in a non-human embryonic stem cell, the germline genome of which comprises (i) an endogenous wild-type immunoglobulin heavy chain locus or (ii) an endogenous wild-type immunoglobulin heavy chain locus and an endogenous wild-type immunoglobulin κ light chain locus; and wherein the method further comprises the step of crossing a mouse created, formed, produced, or derived from said non-human embryonic stem cell with a second mouse.

[0057] In some embodiments, a mouse described herein has a germline genome comprising wild-type Ig H chain and Ig κ chain loci, homozygous or heterozygous humanized Ig H chain and Ig κ chain loci, wherein the homozygous or heterozygous humanized Ig H chain locus comprises an inserted rodent Adam6 coding sequence, or a homozygous or heterozygous humanized Ig H chain locus (with or without an inserted Adam6 coding sequence) and a homozygous or heterozygous inactivated Ig κ chain locus.

[0058] In some embodiments, a non-human animal is provided that is created, formed, produced, obtained, or obtainable by the method described herein.

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

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

[0061] In some embodiments of the method of producing an antibody or nucleic acid in a non-human animal, the non-human cell is a B cell. In some embodiments of the method of producing an antibody or nucleic acid in a non-human animal, the non-human cell is a hybridoma.

[0062] In some embodiments of the method for producing an antibody in a non-human animal, an antibody isolated from a rodent or rodent cell that binds an antigen of interest comprises a human heavy chain variable domain and a human lambda light chain variable domain.

[0063] In some embodiments of the method of producing an antibody or nucleic acid in a non-human animal, the human heavy chain variable domain comprises a rearranged segment of the human V gene H , selected from the group consisting of V H 3-74, VH 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 6-1.

[0064] In some embodiments of the method of producing an antibody or nucleic acid in a non-human animal, the human variable domain of a lambda light chain comprises a rearranged segment of a human Vλ gene selected from the group consisting of Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 and Vλ3-1.

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

[0066] In some embodiments, a non-human animal is provided whose germline genome comprises a homozygous endogenous immunoglobulin λ light chain locus comprising an insertion of (i) the 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) the human Jλ-Cλ gene segment pairs, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, (iii) the human Jλ gene segment, Jλ7, and (iv) three human immunoglobulin λ light chain enhancers (or human immunoglobulin λ light chain enhancer with three sequence elements); where (i)-(iv) are operably linked to each other and the insertion is upstream of a segment of the non-human Cλ gene, and where the endogenous immunoglobulin λ light chain locus lacks endogenous non-human Eλ2-4 immunoglobulin.

[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) pairs of human Jλ-Cλ gene segments, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, (iii) a human Jλ gene segment, Jλ7, and (iv) three human immunoglobulin light chain enhancers (or a human immunoglobulin light chain enhancer with three sequence elements);wherein (i)-(iv) are operably linked to each other, and (i)-(iii) are upstream (or 5') of the non-human Cλ gene segment, and wherein the endogenous immunoglobulin λ light chain locus lacks endogenous non-human Eλ2-4 immunoglobulin, the human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1 comprise human non-coding DNA that occurs naturally between the human Vλ gene segments, the pairs of human Jλ-Cλ gene segments, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, comprise human non-coding DNA that occurs naturally between the pairs of human Jλ-Cλ gene segments, and the human Jλ gene segment, Jλ7 contains human noncoding DNA that occurs naturally upstream (or 5') of human Jλ7.

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

[0069] In some embodiments, the non-human animal, non-human cell, or non-human tissue described herein is provided for use in the manufacture and / or development of a medicament (e.g., an antibody or fragment thereof) for therapy or diagnosis.

[0070] In some embodiments, the non-human animal, non-human cell, or non-human tissue described herein is provided for use in the manufacture of a medicament for treating, preventing, or ameliorating a disease, disorder, or condition.

[0071] Some embodiments provide for the use of a non-human animal, a non-human cell, or a non-human tissue described herein in the manufacture and / or development of a medicament or vaccine for use in medicine, such as for use as a drug.

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

[0073] In various embodiments, the non-human animal, non-human cell, or non-human tissue described herein is a rodent, a rodent cell, or rodent tissue; in some embodiments, a mouse, a mouse cell, or mouse tissue; in some embodiments, a rat, a rat cell, or rat tissue. In some embodiments, the mouse, mouse cell, or mouse tissue described herein comprises a genetic background that includes the 129 line, the BALB / c line, the C57BL / 6 line, the mixed 129xC57BL / 6 line, or combinations thereof.

[0074] As used in this application, the terms "approximately" and "approximately" are used interchangeably. All numbers used in this application, with or without the terms "approximately" or "approximately," are intended to encompass any normal variations understood by one skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The graphics included in this document, which consist of the following figures, are for illustrative purposes only and not for limitation.

[0076] Figure 1 shows a schematic, not to scale, of an illustrative strategy for constructing an engineered endogenous Ig λ light chain locus in a rodent characterized by the presence of multiple human Vλ, Jλ, and Cλ coding sequences that are operably linked to each other and operably linked to the rodent Cλ region (or rodent Cλ gene). As depicted, five separate targeting vectors (6286, 6571, 6596, 6597, and 6680) with varying amounts of genetic material from the human Ig λ light chain locus are sequentially inserted into the endogenous Ig λ light chain locus of a rodent (e.g., mouse) (shown at the top). The first targeting vector (6286) is inserted downstream of the rodent Cλ1 region and is designed to contain the modular enhancer region (or sequence) of the human Ig λ chain (Eλ), characterized by three sequence elements.The second targeting vector (6571) was inserted upstream of the rodent Cλ1 region and was designed to contain five functional human Vλ gene segments, four pairs of functional human Jλ-Cλ gene segments, 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 contained additional sets of additional human Vλ gene segments (eleven and nine, respectively), which are sequentially added to the total human Vλ gene segment content of the endogenous mouse Ig λ light chain locus following successful targeting of the first targeting vector. Both targeting vectors contained overlapping regions (shaded rectangles) at their 3' ends to facilitate homologous recombination with the 5' end of the previous targeting vector already integrated into the endogenous mouse Ig λ light chain locus.Also shown is an alternative fifth targeting vector (6680), which contains the same genetic material as targeting vector 6597, except that this alternative targeting vector contained a 5' homology arm with a sequence identical to the 5' (or upstream) sequence of the rodent Vλ2 gene segment, thereby facilitating the deletion of the endogenous Vλ2-Vλ3-Jλ2-Cλ2-Jλ4P-Cλ4P-Eλ2-4-Vλ1-Jλ3-Jλ3P-Cλ3-Jλ1 gene segments upon homologous recombination with the targeting vector. Unless otherwise noted, filled symbols represent rodent gene segments and / or sequences, while open symbols represent human gene segments and / or sequences. Also shown are site-specific recombination recognition regions (e.g., loxP, Frt) flanking selection cassettes (HYG: hygromycin resistance gene [HYG. R] under the transcriptional control of the ubiquitin promoter; NEO: neomycin resistance gene [NEO R ] under the transcriptional control of the ubiquitin promoter). Selected locations of nucleotide junction sites are marked with a line under each junction site, and each is designated by SEQ ID NO.

[0077] Figure 2 shows a schematic, not to scale, of exemplary rodent Ig λ light chain alleles after sequential insertion of the targeting vectors described in Example 1. Allele 6597: an Ig λ light chain allele that contains 25 functional human Vλ gene segments, four pairs of functional human Jλ-Cλ gene segments, and a human Jλ7 gene segment operably linked to a rodent Cλ region (e.g., a mouse Cλ1 region), and wherein the Ig λ light chain locus further contains endogenous Vλ-Jλ-Cλ gene segments, three (i.e., E2.4, E, and E3.1) endogenous Ig λ chain enhancer regions (or sequences), and a modular human enhancer region (or sequence) Ig λ chain, characterized by three sequence elements.Allele 6680: an Ig λ light chain allele following a site-specific deletion of the endogenous Vλ-Jλ-Cλ gene segments and the Ig λ chain enhancer, Eλ2-4, wherein the Ig λ light chain allele comprises 25 functional human Vλ gene segments, four pairs of functional human Jλ-Cλ gene segments and a human Jλ7 gene segment operably linked to a rodent Cλ region (e.g., a mouse Cλ1 region), wherein the Ig λ light chain locus additionally comprises two (i.e., E and E3.1) endogenous Ig λ chain enhancer regions (or sequences) and a modular human Ig λ chain enhancer region (or sequence, see above). Unless otherwise noted, filled symbols represent rodent gene segments and / or sequences, while open symbols represent human gene segments and / or sequences. Site-specific recombination recognition regions (e.g., Frt) flanking the selection cassettes (HYG: hygromycin resistance gene [HYG.R ] under the transcriptional control of the ubiquitin promoter). The dotted lines indicate the deleted region between the two illustrated Ig λ chain alleles. Selected nucleotide junction locations are indicated by a line below each junction, and each is designated by SEQ ID NO.

[0078] Figure 3 shows a schematic, not to scale, of an alternative illustrative strategy for constructing an engineered endogenous Ig light chain locus in a rodent characterized by the presence of multiple human Vλ, Jλ, and Cλ coding sequences that are operably linked to each other and operably linked to the rodent Cλ region. As depicted, two different targeting vectors with different amounts of genetic material from the human Ig light chain locus are simultaneously inserted into an engineered rodent (e.g., mouse) Ig light chain locus (shown at top), which contains five human Vλ gene segments, a human Jλ-Cλ cluster, and a mouse Cλ1 gene.Targeting vector 6596 is modified to remove the neomycin resistance selection cassette and introduce overlapping sequences (shaded rectangles) at the 5' and 3' ends, creating regions of homology to facilitate recombination with the corresponding human sequence. A second targeting vector is designed to contain an overlapping region at the 3' end of the construct (shaded rectangles) that shares sequence homology with the modified targeting vector 6596 (truncated targeting vector 6596), facilitating homologous recombination with the 5' end of the truncated targeting vector 6596.These two targeting vectors contain additional sets of additional human Vλ gene segments (eleven and nine, respectively) that are sequentially added to the total human Vλ gene segment content of the endogenous mouse Ig λ light chain locus following successful targeting of the first targeting vector. The second targeting vector contained a 5' homology arm with a sequence identical to the 5' sequence (or upstream) of the rodent Vλ2 gene segment, thereby facilitating the deletion of the endogenous Vλ2-Vλ3-Jλ2-Cλ2-Jλ4P-Cλ4P-Eλ2-4-Vλ1-Jλ3-Jλ3P-Cλ3-Jλ1 gene segments upon homologous recombination with the targeting vector. The two targeting vectors were co-electroporated using guide RNAs (gRNAs) to facilitate integration into the engineered Ig λ light chain locus, which are indicated by arrows near the location of each sequence and each is designated by SEQ ID NO.Unless otherwise noted, filled symbols represent rodent gene segments and / or sequences, while open symbols represent human gene segments and / or sequences. Also shown are site-specific recombination recognition regions (e.g., loxP, Frt) flanking the selection cassettes (HYG: hygromycin resistance gene [HYG. R ] under the transcriptional control of the ubiquitin promoter; NEO: neomycin resistance gene [NEO R ] under the transcriptional control of the ubiquitin promoter). Selected locations of nucleotide junction sites are marked with a line under each junction site, and each is designated by SEQ ID NO.

[0079] Figure 4 shows a schematic, not to scale, of a wild-type allele and an illustrative engineered allele of a rodent Ig λ light chain in rodents used in the experiments described in Example 3. Wild-type allele: a wild-type locus of a mouse Ig λ light chain (see also, for example, Figure 2 of U.S. Patent No. 9,006,511); Allele 6571: an Ig λ light chain allele that contains five functional human Vλ gene segments, four pairs of functional human Jλ-Cλ gene segments, and a human Jλ7 gene segment operably linked to a rodent Cλ region (e.g., a mouse Cλ1 region), and wherein the Ig λ light chain locus additionally contains endogenous Vλ-Jλ-Cλ gene segments, three endogenous Ig λ chain enhancer regions (or sequences), and a modular human Ig λ chain enhancer region (or sequence, see above). Allele 6597: see above; allele 6680: see above.Selected nucleotide junction locations are marked with a line below each junction, and each is designated by SEQ ID NO.

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

[0081] Figures 6A and 6B show representative contour plots delineating mature and transitional B cells in splenocytes gated for CD19 +(A) showing IgD (y-axis) and IgM (x-axis) expression and absolute cell numbers per spleen (B) collected from mice homozygous for the 6680 targeting vector insert (6680HO) and wild-type (WT) mice from the same litter. Specific B cell subsets are indicated in each dot plot (e.g., mature, transitional).

[0082] Figures 7A and 7B show representative contour plots indicating the expression of mouse Ig λ chain (mIgλ, y-axis), mouse Ig κ chain (mIgκ, x-axis), or human Ig λ chain (hIgλ, y-axis) in CD19-gated + splenocytes collected from mice homozygous for the 6680 targeting vector insert (6680HO) and wild-type (WT) mice from the same litter.

[0083] Figures 8A and 8B show representative contour plots depicting single-cell gated bone marrow (A) showing CD19 (y-axis) and CD3 (x-axis) expression and absolute cell number per femur (B) collected from mice homozygous for the 6680 targeting vector insert (6680HO) and wild-type (WT) mice from the same litter.

[0084] Figures 9A and 9B show representative contour plots indicating the gated CD19 + IgM low B220 int Bone marrow (A) showing c-kit (y-axis) and CD43 (x-axis) expression and absolute cell counts per femur (B) collected from mice homozygous for the 6680 targeting vector insert (6680HO) and wild-type (WT) mice from the same litter. Specific B cell subsets are indicated in each dot plot (e.g., pro-B cells, pre-B cells).

[0085] Figures 10A and 10B show representative contour plots indicating the gated CD19 + Bone marrow (A) showing IgM (y-axis) and B220 (x-axis) expression and absolute cell counts per femur (B) collected from mice homozygous for the 6680 targeting vector insert (6680HO) and wild-type (WT) mice from the same litter. Specific B cell subsets are indicated in each dot plot (e.g., immature, mature, pre-, and pro-B cells).

[0086] Figures 11A and 11B show representative contour plots depicting immature bone marrow (gated for CD19 + IgM + B220 int ), showing the expression of mouse Ig λ chain (mIgλ, y-axis), mouse Ig κ chain (mIgκ, x-axis), or human Ig λ chain (hIgλ, y-axis) in mice homozygous for the 6680 targeting vector insert (6680HO) and wild-type (WT) mice from the same litter.

[0087] Figures 12A and 12B show representative contour plots depicting mature bone marrow (gated for CD19 + IgM + B220 + ), showing the expression of mouse Ig λ chain (mIgλ, y-axis), mouse Ig κ chain (mIgκ, x-axis), or human Ig λ chain (hIgλ, y-axis) in mice homozygous for the 6680 targeting vector insert (6680HO) and wild-type (WT) mice from the same litter.

[0088] Figure 13 shows the representative mean percentage of Ig κ chain-expressing (% κ C) and human Ig λ chain-expressing (% hum λ C) B cells in the spleen, immature bone marrow (immature BM), and mature bone marrow (mature BM) of selected engineered mouse strains described herein. Data are presented as mean values ​​with standard deviations also indicated. 6680HO / VI HO / Adam6 HO: An engineered mouse strain containing a homozygous engineered Ig λ light chain locus engineered to contain 25 functional human Vλ gene segments, four pairs of functional human Jλ-Cλ gene segments, and a human Jλ7 gene segment operably linked to a rodent Cλ region (e.g., a mouse Cλ1 region), wherein the Ig λ light chain locus further comprises two endogenous Ig λ chain enhancer regions (or sequences) and a modular human enhancer region (or sequence, seeabove) Ig λ chain; and homozygous humanized Ig H chain and Ig κ chain loci, wherein the homozygous humanized Ig H chain locus contains an inserted rodent Adam6 coding sequence (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940; hereby incorporated by reference in their entireties); 6889HO / VI HO / Adam6 HO: An engineered mouse strain containing a homozygous engineered Ig λ light chain locus comprising 25 functional human Vλ gene segments, four pairs of functional human Jλ-Cλ gene segments, and a human Jλ7 gene segment operably linked to a rodent Cλ region (e.g., a mouse Cλ1 region), wherein the Ig λ light chain locus further comprises two endogenous Ig λ chain enhancer regions (or sequences) and a modular human enhancer region (or sequence, seeabove) Ig λ chain; and homozygous humanized Ig H chain and Ig κ chain loci, wherein the homozygous humanized Ig H chain locus contains an inserted rodent Adam6 coding sequence (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940; hereby incorporated by reference in their entireties). The number of mice for each genotype cohort shown was at least three and no more than eight animals per group.

[0089] Figures 14A and 14B show representative SDS-PAGE Western blots under non-reducing conditions using serum isolated from engineered mice homozygous for the 6680 targeting vector insert (6680HO) and wild-type (WT) mice from the same litter, expressing mouse (B, right panel) or human (A, left panel) λ light chains; each sample was loaded into the lanes at a volume of 1.5 μl serum. PHS: pooled human serum at a volume of 0.25 μl (Labquip Ltd, cat. no. 9101A). Molecular weight values ​​in kDa are indicated on the right side of each gel panel.

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

[0091] Figure 15B shows a representative usage of human Vλ gene segments in mouse Cλ-primed sequences amplified from RNA isolated from splenocytes collected from 6889HET mice (n=5).

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

[0093] Figure 15D shows a representative usage of human Vλ gene segments in mouse Cλ-primed sequences amplified from RNA isolated from splenocytes collected from 6889HO / VI HO / Adam6 HO mice (n=6).

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

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

[0096] Figures 18A and 18B show representative contour plots of single-cell gated splenocytes (left) demonstrating CD19 (y-axis) and CD3 (x-axis) expression and the total number of B cells per spleen (right) collected from mice homozygous for the 6889 targeting vector insert (6889HO VI HO Adam6 HO) and reference engineered mice (VI). 6889HO / VI HO / Adam6 HO: see above; VI: An engineered mouse line containing homozygous humanized Ig H chain and Ig κ chain loci, wherein the homozygous humanized Ig H chain locus contains an inserted rodent Adam6 coding sequence (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940; hereby incorporated by reference in their entireties). Viable single-cell splenocytes were determined by viability staining (Thermo Fisher).

[0097] Figure 19 shows representative contour plots indicating the expression of human Ig λ chain (hIgλ, y-axis) and mouse Ig κ chain (mIgκ, x-axis) in CD19-gated + splenocytes collected from mice homozygous for the 6889 targeting vector insert (6889HO VI HO Adam6 HO) and from reference engineered mice (VI). 6889HO / VI HO / Adam6 HO: see above; VI: see above.

[0098] Figure 20 shows representative contour plots of single-cell gated bone marrow lymphocytes showing IgM (y-axis) and B220 (x-axis) expression collected from femurs of mice homozygous for the 6889 targeting vector insert (6889HO VI HO Adam6 HO) and reference engineered mice (VI). 6889HO / VI HO / Adam6 HO: see above; VI: see above. Immature and mature B cell subsets are indicated in each contour plot.

[0099] Figure 21 shows representative contour plots indicating immature (gated by CD19 + IgM + B220 int , left column) and mature (gated for CD19 + IgM + B220 + , right column) bone marrow showing expression of human Ig λ chain (hIgλ, y-axis) and mouse Ig κ chain (mIgκ, x-axis) from mice homozygous for the 6889 targeting vector insert (6889HO VI HO Adam6 HO) and reference engineered mice (VI). 6889HO / VI HO / Adam6 HO: see above; VI: see above. BRIEF DESCRIPTION OF SELECTED SEQUENCES IN SEQUENCE LISTING

[00100] mouse Cλ1 DNA (SEQ ID NO:1):

[00101] Amino acid sequence of mouse Cλ1 (SEQ ID NO:2):

[00102] Mouse Cλ2 DNA (SEQ ID NO:3):

[00103] Amino acid sequence of mouse Cλ2 (SEQ ID NO:4):

[00104] Mouse Cλ3 DNA (SEQ ID NO:5):

[00105] Amino acid sequence of mouse Cλ3 (SEQ ID NO:6):

[00106] Rat Cλ1 DNA (SEQ ID NO: 7):

[00107] Rat Cλ1 amino acid sequence (SEQ ID NO:8):

[00108] Rat Cλ2 DNA (SEQ ID NO:9):

[00109] Rat Cλ2 amino acid sequence (SEQ ID NO:10):

[00110] Rat Cλ3 DNA (SEQ ID NO:11):

[00111] Amino acid sequence of rat Cλ3 (SEQ ID NO:12):

[00112] Rat Cλ4 DNA (SEQ ID NO:13):

[00113] Rat Cλ4 amino acid sequence (SEQ ID NO:14): embodiments described herein; those skilled in the art, upon review of this description, will become aware of various modifications that may be equivalent to such described embodiments or otherwise within the scope of the claims.

[00115] In general, the terms used herein have the meanings understood in the art unless otherwise clearly defined. Precise definitions of certain terms are provided below; the meanings of these and other terms in particular instances within this description will be clear to those skilled in the art from the context. Additional definitions for the following and other terms are set forth throughout the description. Patents and non-patent literature cited in references cited in this description or relevant portions thereof are herein incorporated by reference in their entirety.

[00116] Administration as used herein includes the administration of a composition to a subject or system (e.g., a cell, organ, tissue, organism, or their respective component or set of components). One of skill in the art will understand that the route of administration may vary, for example, depending on the subject or system to which the composition is to be 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 a rodent) can be bronchial (including via bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, transmucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including via intratracheal instillation), transdermal, vaginal, and / or vitreous. In some embodiments, administration can involve intermittent dosing. In some embodiments, administration can involve continuous dosing (e.g., perfusion) over at least a selected period of time.

[00117] Amelioration, as used herein, includes preventing, alleviating, or temporarily alleviating a condition or improving a condition in a subject. Amelioration includes, but is not necessarily, a complete cure or complete prevention of a disease, disorder, or condition.

[00118] "Approximately," when applied to one or more values ​​of interest, includes a value similar to the stated reference value. In some embodiments, the term "about" or "approximately" refers to a range of values ​​that are within 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 (greater or lesser) of the stated reference value, unless otherwise stated or otherwise obvious from the context (except in cases where such number would exceed 100% of the possible value).

[00119] Biologically active, as used herein, refers to the characteristic of any agent having activity in a biological system in vitro or in vivo (e.g., in an organism). For example, an agent that, when present in an organism, exerts a biological effect within that organism is considered biologically active. In particular embodiments in which a protein or polypeptide is biologically active, the portion of the protein or polypeptide that has at least one biological activity in common with the protein or polypeptide is generally referred to as the "biologically active" portion.

[00120] Comparable, as used herein, refers to two or more agents, entities, concomitant conditions, sets of conditions, etc., which may not be identical to each other, but which are sufficiently similar to allow comparisons between them such that reasonable inferences can be drawn from the observed differences or similarities. It will be clear to those skilled in the art from the context what degree of identity is required in any given circumstance for two or more such agents, entities, co-conditions, sets of conditions, etc., to be considered comparable.

[00121] Conservative, as used herein, refers to instances describing a conservative amino acid substitution, including the substitution of an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Typically, a conservative amino acid substitution will not substantially alter the functional properties of the protein of interest, such as the ability of a receptor to bind a ligand.Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains, such as those of glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (I1e, I); aliphatic side chains with hydroxyl groups, such as those of serine (Ser, S) and threonine (Thr, T); amide-containing side chains, such as those of asparagine (Asn, N) and glutamine (Gln, Q); aromatic side chains, such as those of phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains, such as those of lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains such as those of aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfur-containing side chains such as those of cysteine ​​(Cys, C) and methionine (Met, M).Conservative amino acid substitution groups 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), glutamate / aspartate (Glu / Asp, E / D), and asparagine / glutamine (Asn / Gln, N / Q). In some embodiments, a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, used, for example, in alanine-scanning mutagenesis. In some embodiments, a conservative substitution is made that has a positive value in the log-likelihood matrix PAM250 disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445. In some embodiments, the substitution is a moderately conservative substitution, wherein the substitution has a non-negative value in the log-likelihood matrix PAM250.

[00122] As used herein, a control refers to the art-understood meaning of "control," which is a standard against which results are compared. Controls are typically used to enhance the integrity of experiments by separating variables in order to draw conclusions about those variables. In some embodiments, a control is a reaction or assay conducted concurrently with a test reaction or assay to provide a comparison. A "control" also includes a "control animal." A "control animal" may have a modification described herein, a modification different from that described herein, or no modification (i.e., be a wild-type animal). In one experiment, a "test" (i.e., the variable being tested) is used. In a second experiment, the "control," the variable being tested is not used. In some embodiments, the control is a historical control (i.e.,a test or analysis previously performed, or a quantity or result known in advance). In some embodiments, the control is or includes a printed or otherwise stored record. The control may be a positive control or a negative control.

[00123]

[00124] Derived from, when used in relation to a rearranged variable region gene or variable domain, "derived from" a non-rearranged variable region and / or non-rearranged variable region gene segments, refers to the ability to trace the sequence of the rearranged variable region gene or variable domain to a group of non-rearranged variable region gene segments that have been rearranged to form a rearranged variable region gene that expresses a variable domain (taking into account, where applicable, splicing differences and somatic mutations).For example, the fact that a rearranged variable region gene has undergone a somatic mutation does not change the fact that it is derived from segments of a non-rearranged variable region gene.

[00125] Disruption, as used herein, refers to the result of a homologous recombination event involving a DNA molecule (e.g., involving an endogenous homologous sequence, such as a gene or gene locus). In some embodiments, the disruption may result in or be an insertion, deletion, substitution, replacement, missense mutation, or frameshift in a DNA sequence(s), or any combination thereof. Insertions may involve the insertion of entire genes or gene fragments, such as exons, which may have an origin different from that of the endogenous sequence (e.g., a heterologous sequence).In some embodiments, the disruption may result in an increase in the expression level and / or activity of a gene or gene product (e.g., a polypeptide encoded by the gene). In some embodiments, the disruption may result in a decrease in the expression level and / or activity of a gene or gene product. In some embodiments, the disruption may result in a change in the sequence of a gene or an encoded gene product (e.g., an encoded polypeptide). In some embodiments, the disruption may result in truncation or fragmentation of a gene or an encoded gene product (e.g., an encoded polypeptide). In some embodiments, the disruption may result in elongation of a gene or an encoded gene product. In some such embodiments, the disruption may result in assembly of a fusion polypeptide. In some embodiments, the disruption may affect the expression level, but not the activity, of a gene or gene product.In some embodiments, the disruption may affect the activity, but not the expression level, of a gene or gene product. In some embodiments, the disruption may not have a significant effect on the expression level of a gene or gene product. In some embodiments, the disruption may not have a significant effect on the activity of a gene or gene product. In some embodiments, the disruption may not have a significant effect on either the expression level or the activity of a gene or gene product.

[00126] Determining, measuring, establishing, assessing, analyzing, and assaying are used interchangeably herein to refer to any form of measurement and involve determining the presence or absence of an element. These terms include quantitative and / or also qualitative determinations. The assay may be relative or absolute.An "assay for the presence" may be a determination of the amount of something present and / or a determination of whether it is present or absent.

[00127] An endogenous locus or endogenous gene, as used herein, refers to a genetic locus found in a source or reference organism prior to the introduction of the disruption, deletion, substitution, alteration, or modification described herein. In some embodiments, the endogenous locus has a sequence found 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 an engineered organism.In some embodiments, the reference organism is a laboratory-bred organism (either wild-type or engineered).

[00128] Endogenous promoter, as used herein, refers to a promoter that is naturally associated, such as in a wild-type organism, with an endogenous gene.

[00129] Engineered, as used herein, refers generally to the aspect of human manipulation. For example, in some embodiments, a polynucleotide may be considered "engineered" if two or more sequences that are not linked together in this order in nature are manipulated by humans to be directly linked together in the engineered polynucleotide.In some such specific embodiments, the engineered polynucleotide may comprise a regulatory sequence that is found in nature in operative linkage with the first coding sequence, but not in operative linkage with the second coding sequence, linked by man to be operably linked to the second coding sequence. Alternatively or additionally, in some embodiments, the first and second nucleic acid sequences, each encoding polypeptide elements or domains that are not naturally linked to one another, may be linked to one another into a single engineered polynucleotide.Similarly, in some embodiments, a cell or organism may be considered "engineered" if it has been manipulated such that its genetic information is altered (e.g., new genetic material that was previously absent has been introduced, or previously present genetic material has been altered or deleted). As is common practice and understood by those skilled in the art, the progeny of an engineered polynucleotide or cell are generally still referred to as "engineered," even if the actual manipulation was performed on the precursor. Furthermore, as those skilled in the art will understand, a variety of techniques are available by which the "engineering" described herein can be accomplished.For example, in some embodiments, "designing" may involve selecting or designing (e.g., nucleic acid sequences, polypeptide sequences, cells, tissues, and / or organisms) by using computer systems programmed to analyze or compare, or otherwise provide for analysis, recommendation regarding, and / or selection, sequences, changes, etc.). Alternatively, or additionally, in some embodiments, "designing" may involve the use of in vitro chemical synthesis techniques and / or recombinant nucleic acid technologies such as, for example, amplification (e.g., by polymerase chain reaction), hybridization, mutation, transformation, transfection of nucleic acids, etc., and / or any of a variety of controlled mating techniques.As will be appreciated by those skilled in the art, many such conventional techniques (e.g., for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection, etc.)) are well known in the art and are described in various general and more specific literature references that are cited and / or discussed throughout this disclosure. 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, RW and SB Primrose, Blackwell Science, Inc., 1994.

[00130] Functional, as used herein, refers to a form or fragment of an entity (e.g., a gene or gene segment) that exhibits a particular property (e.g., forms part of a coding sequence) and / or activity.For example, in the case of immunoglobulins, variable domains are encoded by unique gene segments (i.e., V, D, and / or J) that are organized (or recombined) to form functional coding sequences. When present in the genome, gene segments are organized into clusters, although in reality, variation occurs. A "functional" gene segment is a gene segment that is present in the expressed sequence (i.e., the variable domain) for which the corresponding genomic DNA has been isolated (i.e., cloned) and sequence-identified. Some immunoglobulin gene segment sequences contain open reading frames and are considered functional even though they are not present in the expressed repertoire, while other immunoglobulin gene segment sequences contain mutations (e.g., point mutations, insertions, deletions, etc.).), resulting in the formation of a stop codon and / or a truncated sequence, which subsequently renders such gene segment sequences incapable of providing the property / properties and / or activity / activities associated with the unmutated sequence(s). Such sequences are not represented among the expressed sequences and are therefore classified as pseudogenes.

[00131] A gene, as used herein, refers to a DNA sequence on a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene comprises a coding sequence (i.e., a sequence that encodes a particular product). In some embodiments, a gene comprises a non-coding sequence.In some specific embodiments, a gene may comprise both a coding (e.g., exonic) and non-coding (e.g., intronic) sequence. In some embodiments, a gene may comprise one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intronic sequences, which, for example, may control or influence one or more aspects of gene expression (e.g., cell type-specific expression, inducible expression, etc.). For the purpose of clarity, the term "gene," as used herein, generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof; however, the term may optionally encompass regulatory sequences, as will be understood from the context by those skilled in the art.This definition is not intended to exclude the use of the term "gene" to non-protein-coding expression units, but rather to clarify that, in most cases, the term as used herein refers to a nucleic acid encoding a polypeptide.

[00132] Heterologous, as used herein, refers to an agent or entity from another source. For example, when used in relation to a polypeptide, gene, or gene product present in a particular cell or organism, the term clarifies that the corresponding polypeptide, gene, or gene product 1) was engineered by man; 2) was introduced into the cell or organism (or a precursor thereof) by man (e.g., by genetic engineering); and / or 3) is not naturally produced by or is not present in the corresponding cell or organism (e.g., the corresponding cell type or organism type)."Heterologous" also includes a polypeptide, gene, or gene product that is normally present in a particular native cell or organism but has been altered or modified, such as by mutation or by placement under the control of non-naturally occurring associated and, in some embodiments, non-endogenous regulatory elements (e.g., a promoter).

[00133] A host cell, as used herein, refers to a cell into which a nucleic acid or protein has been introduced. Those skilled in the art, upon reading this disclosure, will appreciate that such terms refer not only to the particular cell in question, but are also used to refer to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to the influence of either mutations or the environment, such progeny may not actually be identical to the original cell, but are still included within the scope of the phrase "host cell."In some embodiments, the 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 animal kingdom to which the cell belongs. Illustrative cells include prokaryotic and eukaryotic cells (unicellular or multicellular), bacterial cells (e.g., strains of Escherichia coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces 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 quadroms.In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (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, a tumor cell and a cell line derived from said cell. In some embodiments, the cell comprises one or more viral genes, such as a retinal cell that expresses a viral gene (e.g., a PER.C6® cell). In some embodiments, the host cell is or comprises an isolated cell.In some embodiments, the host cell is part of a tissue. In some embodiments, the host cell is part of an organism.

[00134] Identity, as used herein in connection with sequence comparison, refers to identity determined using a number of different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments, the identity values ​​described herein are determined using the ClustalW version 1.83 (slow) alignment, which uses a gap opening penalty of 10.0, a gap extension penalty of 0.1, and using the Gonnet similarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008).

[00135] In vitro, as used herein, refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., and not in a multicellular organism.

[00136] In vivo, as used herein, refers to events that occur in a multicellular organism, such as a human and / or a non-human animal. In the case of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[00137] Isolated, as used herein, refers to a substance and / or object that has been (1) separated from at least some of the components with which it was associated when originally produced (whether in nature and / or under experimental conditions), and / or (2) designed, produced, obtained, and / or manufactured by a human.The isolated substances and / or objects 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, the isolated agents are separated from 10%-100%), 15%-100%, 20%-100%, 25%-100%, 30%-100%, 35%-100%, 40%-100%, 45%-100%, 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 96%-100%, 97%-100%, 98%-100%, or 99%-100% of the other components with which they were originally associated.In some embodiments, the isolated agents are separated from 10%-100%, 10%-99%, 10%-98%, 10%-97%, 10%-96%, 10%-95%, 10%-90%, 10%-85%, 10%-80%, 10%-75%, 10%-70%, 10%-65%, 10%-60%, 10%-55%, 10%-50%, 10%-45%, 10%-40%, 10%-35%, 10%-30%, 10%-25%, 10%-20%, or 10%-15% of the other components with which they were originally associated. In some embodiments, the isolated means are separated from 11%-99%, 12%-98%, 13%-97%, 14%-96%, 15%-95%, 20%-90%, 25%-85%, 30%-80%, 35%-75%, 40%-70%, 45%-65%, 50%-60%, or 55%-60% of the other components with which they were originally associated. In some embodiments, the isolated means are about 80% pure, about 85% pure, about 90% pure, about 91% pure, about 92% pure, about 93% pure, about 94% pure, about 95% pure, about 96% pure, about 97% pure, about 98% pure, about 99% pure, or greater than about 99%.In some embodiments, the isolated agents are 80%-99%, 85%-99%, 90%-99%, 95%-99%, 96%-99%, 97%-99%, or 98%-99% pure. In some embodiments, the isolated agents are 80%-99%, 80%-98%, 80%-97%, 80%-96%, 80%-95%, 80%-90%, or 80%-85% pure. In some embodiments, the isolated agents are 85%-98%, 90%-97%, or 95%-96% pure. In some embodiments, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" after combination with certain other components, such as, for example, one or more carriers or excipients (e.g., a buffer, solvent, water, etc.); in such embodiments, the percent isolation or purity of the substance is calculated without taking into account such carriers or excipients.As just one example, in some embodiments, a biological polymer, such as a polypeptide or polynucleotide, occurring in nature is considered "isolated" if: a) it is, by reason of its origin or source of production, not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces them in nature; or c) it is expressed or otherwise associated with components in 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 other than the one that 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 may be considered an "isolated" polypeptide to the extent that it has been separated from other components: a) with which it is associated in nature and / or b) with which it was associated during its original production.

[00138] Locus or loci, as used herein, refer to a particular location(s) of a gene (or significant sequence), a DNA sequence encoding a polypeptide sequence, or a position on a chromosome of the genome of an organism.For example, an "immunoglobulin locus" may refer to a specific location of an immunoglobulin gene segment (e.g., V, D, J, or C), a DNA sequence of an immunoglobulin gene segment, a sequence encoding the immunoglobulin gene segment, or a position of an immunoglobulin gene segment on a chromosome of an organism's genome that has been identified as containing such a sequence. An "immunoglobulin locus" may comprise a regulatory element of an immunoglobulin gene segment, including, but not limited to, an enhancer, a promoter, a 5' and / or 3' regulatory sequence or region, or a combination thereof.An "immunoglobulin locus" may comprise DNA that is normally found between gene segments at a wild-type locus, but the DNA itself lacks an immunoglobulin gene segment (e.g., an immunoglobulin DNA sequence that is naturally found between a group of V gene segments and a group of J gene segments, an immunoglobulin DNA sequence that is naturally found between a group of J gene segments and a constant region gene, or an immunoglobulin DNA sequence that is naturally found at the 3' end of a constant region gene). Those skilled in the art will appreciate that, in some embodiments, chromosomes may contain several hundred or even several thousand genes and exhibit physical co-localization of similar genetic loci when compared across species. Such genetic loci may be described as having common synteny.

[00139] A non-human animal, as used herein, refers to any vertebrate organism that is not a human. In some embodiments, a non-human animal is a cyclostomes, a bony fish, a cartilaginous fish (e.g., a shark or ray), an amphibian, a reptile, a mammal, and a bird. In some embodiments, a non-human animal is a mammal. In some embodiments, a non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, a non-human animal is a rodent, such as a rat or a mouse.

[00140] A nucleic acid, as used herein, refers to any compound and / or substance that is or can be included in an oligonucleotide chain.In some embodiments, a "nucleic acid" is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be apparent from the context, in some embodiments, a "nucleic acid" refers to individual nucleic acid units (e.g., nucleotides and / or nucleosides); in some embodiments, a "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid units. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a "nucleic acid" is, comprises, or consists of one or more naturally occurring nucleic acid units.In some embodiments, the "nucleic acid" is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a "nucleic acid" in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the "nucleic acid" is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds. Alternatively, or additionally, in some embodiments, the "nucleic acid" has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages instead of phosphodiester linkages.In some embodiments, the "nucleic acid" is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the "nucleic acid" is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C5-propynylcytidine, C5-propynyluridine, 2-aminoadenosine, C5-bromuridine, C5-fluorouridine, C5-ioduridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof.In some embodiments, the "nucleic acid" comprises one or more modified sugar residues (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In some embodiments, the "nucleic acid" has a nucleotide sequence that encodes a functional gene product, such as RNA or a polypeptide. In some embodiments, the "nucleic acid" comprises one or more introns. In some embodiments, the "nucleic acid" comprises one or more exons. In some embodiments, the "nucleic acid" is produced by one or more of isolation from a natural source, enzymatic synthesis by complementary template-based polymerization (in vivo or in vivo), propagation in a recombinant cell or system, and chemical synthesis.In some embodiments, the length of the “nucleic acid” is at least, such as without limitation, 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 some embodiments, the "nucleic acid" is single-stranded; in some embodiments, the "nucleic acid" is double-stranded. In some embodiments, the "nucleic acid" has a nucleotide sequence that contains at least one element that encodes a polypeptide or is complementary to a sequence that encodes a polypeptide. In some embodiments, the "nucleic acid" has enzymatic activity.

[00141] Operably linked, as used herein, refers to a juxtaposition in which the components described are in a relationship that allows them to function properly. For example, segments of an unrearranged variable region gene are "operably linked" to an adjacent constant region gene if the segments of the unrearranged variable region gene are capable of rearranging to form a rearranged variable region gene that is expressed in combination with the constant region gene as the polypeptide chain of an antigen-binding protein. A control sequence "operably linked" to a coding sequence is ligated thereto such that expression of the coding sequence is achieved under conditions compatible with the function of the control sequences."Operably linked" sequences include both expression control sequences adjacent to the gene of interest and expression control sequences acting in trans or at a distance to exert control over the gene of interest (or sequence of interest). The term "expression control sequence" includes polynucleotide sequences necessary to influence the expression and processing of coding sequences to which they are ligated. "Expression control sequences" include appropriate sequences for transcription initiation, termination, promoter, and enhancer sequences; signals for efficient RNA processing, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e.,Kozak consensus sequence); sequences that enhance polypeptide stability; and, if necessary, sequences that enhance polypeptide secretion. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence, whereas in eukaryotes, such control sequences typically include promoters and a transcription termination sequence. The term "control sequences" is intended to include components whose presence is essential for expression and processing and may also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences.

[00142] Physiological conditions, as used herein, refer to standard conditions understood in the art under which cells or organisms live and / or reproduce. In some embodiments, the term includes external or internal environmental conditions that may exist naturally relative to an organism or cellular system. In some embodiments, physiological conditions are conditions present within the body of a human or non-human animal, in particular those conditions present at the site and / or within the area of ​​surgical intervention. Physiological conditions typically include, for example, a temperature range of 20-40°C, an atmospheric pressure of 1, a pH of 6-8, a glucose concentration of 1-20 mM, an oxygen concentration at atmospheric levels, and gravity that is found on planet Earth.In some embodiments, laboratory conditions are controlled and / or maintained at physiological conditions. In some embodiments, the physiological conditions occur in an organism.

[00143] A polypeptide, as used herein, refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that is found in nature. In some embodiments, a polypeptide has an amino acid sequence that is not found in nature. In some embodiments, a polypeptide has an amino acid sequence that contains portions that are found in nature separately from each other (i.e., from two or more different organisms, such as portions belonging to a human organism and a non-human organism).In some embodiments, the polypeptide has an amino acid sequence that is engineered in the sense that it is designed and / or produced by human action. In some embodiments, the polypeptide has an amino acid sequence encoded by a sequence that is not naturally occurring (e.g., a sequence that is engineered in the sense that it is designed and / or produced by human action to encode said polypeptide).

[00144] Recombinant, as used herein, is intended to refer to polypeptides that are designed, constructed, produced, expressed, created, or isolated by recombinant methods, such as polypeptides expressed by a recombinant expression vector introduced by transfection into a host cell, polypeptides isolated from a combinatorial library of recombinant human polypeptides (Hoogenboom, H. R., 1997, TIB Tech. 15:62-70; Azzazy, H. and W. E. Highsmith, 2002, Clin. Biochem. 35:425-45; Gavilondo, JV and JW Larrick, 2002, BioTechniques 29:128-45; Hoogenboom H., and P. Chames, 2000, Immunol. Today 21:371-8), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor, LD et al., 1992, Nucl. Acids Res. 20:6287-95; Kellermann, SA and LL Green, 2002, Curr. Opin. Biotechnol. 13:593-7; Little, M et al., 2000, Immunol. Today 21:364-70; Osborn, MJ et al., 2013, J. Immunol. 190:1481-90; Lee, EC et al., 2014, Nat. Biotech. 32(4):356-63; Macdonald, LE et al., 2014, Proc. Natl. Acad. Sci. USA 111(14):5147-52; Murphy, AJ et al., 2014, Proc. Natl. Acad. Sci. USA 111(14):5153-8), or polypeptides produced, expressed, created, or isolated by any other means that involve linking selected sequence elements together.In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements are obtained by mutagenesis (e.g., in vivo or in vitro) of an element of a known sequence, e.g., from a natural or synthetic source. For example, in some embodiments, the recombinant polypeptide consists of sequences found in the genome of the source organism of interest (e.g., human, mouse, etc.).In some embodiments, the recombinant polypeptide has an amino acid sequence obtained by mutagenesis (e.g., in vitro or in vivo, such as in a non-human animal), such that the amino acid sequences of the recombinant polypeptides are sequences that, although derived from and related to the polypeptide sequences, may not naturally exist in vivo within the genome of a non-human animal.

[00145] A reference, as used herein, refers to a standard or control agent, animal, cohort, individual, population, sample, sequence, or value to which the agent, animal, cohort, individual, population, sample, sequence, or value of interest 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 agent, animal, cohort, individual, population, sample, sequence, or value of interest. In some embodiments, the reference agent, animal, cohort, individual, population, sample, sequence, or value is a historical reference, which does not necessarily exist in tangible form on a tangible carrier. In some embodiments, the reference may relate to a control. "Reference" also includes a "reference animal." A "reference animal" may have a modification as described herein, a modification different from that described herein, or no modification (i.e., be a wild-type animal).As will be understood by those skilled in the art, a reference agent, animal, cohort, individual, population, sample, sequence, or value is typically determined or characterized under conditions comparable to the conditions used to determine or characterize the agent, animal (e.g., mammal), cohort, individual, population, sample, sequence, or value of interest.

[00146] Substitution, as used herein, refers to the process by which a "substitute" nucleic acid sequence (e.g., a gene) found at a host locus (e.g., a genome) is removed from that locus and another, "replacement" nucleic acid is placed in its place.In some embodiments, the nucleic acid sequence to be replaced and the replacement nucleic acid sequences are comparable to each other in that, for example, they are homologous to each other and / or comprise corresponding elements (e.g., protein coding elements, regulatory elements, etc.). In some embodiments, the nucleic acid sequence to be replaced comprises one or more of a promoter, an enhancer, a splice donor site, a splice acceptor site, an intron, an exon, an untranslated region (UTR); in some embodiments, the replacement nucleic acid sequence comprises one or more coding sequences. In some embodiments, the replacement nucleic acid sequence is a homologue or a variant (e.g., a mutant) of the nucleic acid sequence to be replaced.In some embodiments, the replacement nucleic acid sequence is an ortholog or homolog of the sequence being replaced. In some embodiments, the replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, including those in which the replacement nucleic acid sequence is or comprises a human nucleic acid sequence, the replaced nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse or rat sequence). In some embodiments, including those in which the replacement nucleic acid sequence is or comprises a human nucleic acid sequence, the replaced nucleic acid sequence is or comprises a human sequence.In some embodiments, the replacement nucleic acid sequence is a variant or mutant (i.e., a sequence containing one or more sequence differences, such as substitutions, compared to the sequence being replaced) of the sequence being replaced. The nucleic acid sequence positioned in this manner may contain one or more regulatory sequences that are part of the primary nucleic acid sequence used to produce the sequence positioned in this manner (e.g., promoters, enhancers, 5'- or 3'-untranslated regions, etc.).For example, in various embodiments, the substitution is the replacement of an endogenous sequence with a heterologous sequence, resulting in the production of a gene product with a nucleic acid sequence so positioned (containing the heterologous sequence), but not the expression of the endogenous sequence; wherein the endogenous genomic sequence is replaced with a nucleic acid sequence that encodes a polypeptide that has a function similar to that of the polypeptide encoded by the endogenous sequence (e.g., the endogenous genomic sequence encodes a complete or partial non-human variable domain polypeptide, and the DNA fragment encodes one or more human complete or partial variable domain polypeptides).In various embodiments, an endogenous segment of a non-human immunoglobulin gene or a fragment thereof is replaced with a segment of a human immunoglobulin gene or a fragment thereof.

[00147] Substantially, as used herein, refers to the qualitative factor of exhibiting a full or substantially full measure or degree of expression of a characteristic or property of interest. One skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, continue to completion and / or proceed to completion or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to encompass the potential lack of completeness inherent in many biological and chemical phenomena.

[00148] Substantially homologous, as used herein, refers to a comparison between amino acid sequences or nucleic acid sequences.As those skilled in the art will understand, two sequences are generally considered "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 correspondingly similar structural and / or functional characteristics. For example, as is well known to those skilled in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid for another of the same type can often be considered a "homologous" substitution. A typical categorization of amino acids is summarized in the table below.

[00149] As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Such exemplary programs are described in Altschul, SF et al., 1990, J. Mol. Biol., 215(3): 403-10; Altschul, SF et al., 1996, Meth. Enzymol. 266:460-80; Altschul, SF et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, A.D. and B.F.F. Ouellette (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 mentioned above typically provide an indication of the degree of homology.In some embodiments, two sequences are considered to be substantially homologous if at least, such as without limitation, 50%, 55%, 60%, 65%, 70%, 75%), 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their respective residues are homologous over a given fragment of residues. In some embodiments, the given fragment is an entire sequence. In some embodiments, the given fragment comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues. In some embodiments, the given fragment comprises contiguous residues along the entire sequence. In some embodiments, the fragment in question comprises interspaced residues along the entire sequence, such as interspaced residues brought together by a folded conformation of the polypeptide or a portion thereof.In some embodiments, the fragment in question is at least, for example, without limitation, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[00150] Substantially identical, 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 "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Such exemplary programs are described in Altschul, SF et al., 1990, J. Mol. Biol., 215(3): 403–10; Altschul, SF et al., 1996, Meth. Enzymol. 266:460–80; Altschul, SF et al., 1997, Nucleic Acids Res., 25:3389–402; Baxevanis, AD and BFF Ouellette (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 identical sequences, the programs mentioned above typically 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 a given fragment of residues. In some embodiments, the given fragment of residues is the entire sequence.In some embodiments, the moiety of residues in question is, for example, but not limited to, at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[00151] A 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 allows for the integration of the targeting construct into a location in the genome of the cell, tissue, or animal through homologous recombination. Also included and described herein are targeting regions that are targeted by recognition sites for a site-specific recombinase (e.g., loxP and / or Frt sites).In some embodiments, the targeting construct described herein further comprises a nucleic acid sequence or gene of particular interest, a selectable marker, control and / or regulatory sequences, as well as other nucleic acid sequences that allow for recombination mediated by the addition of exogenous proteins that promote or facilitate recombination involving such sequences. In some embodiments, the targeting construct described herein further comprises a complete or partial gene of interest, wherein the gene of interest is a heterologous gene that encodes a complete or partial polypeptide having a function similar to that of the protein encoded by the endogenous sequence.In some embodiments, the targeting construct described herein further comprises a humanized complete or partial gene of interest, wherein the humanized gene of interest encodes a complete or partial polypeptide having a function similar to that of a polypeptide encoded by an endogenous sequence. In some embodiments, the targeting construct (or targeting vector) may comprise a nucleic acid sequence that has been manipulated by humans. For example, in some embodiments, the targeting construct (or targeting vector) may be constructed to comprise an engineered or recombinant polynucleotide that comprises two or more sequences that are not naturally associated with each other in this order until they are manipulated by humans to link directly to each other in the engineered or recombinant polynucleotide.

[00152] A transgene or transgene construct, as used herein, refers to a nucleic acid sequence (encoding, for example, a complete or partial polypeptide of interest) that has been introduced into a human cell using methods such as those described herein. The transgene may be partially or completely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced. The transgene may contain one or more transcriptional regulatory sequences and any other nucleic acid, such as introns or promoters, that may be necessary for the expression of the selected nucleic acid sequence.

[00153] A transgenic animal, a transgenic non-human animal, or a T8. +The term "transgenic animal" is used interchangeably herein and refers to any non-naturally occurring non-human animal in which one or more cells of the non-human animal contain a heterologous nucleic acid and / or gene encoding a complete or partial polypeptide of interest. For example, in some embodiments, a "transgenic animal" or "transgenic non-human animal" refers to an animal or non-human animal that contains a transgene or transgene construct described herein. In some embodiments, the heterologous nucleic acid and / or gene is introduced into a cell directly or indirectly, by introduction into a precursor cell, by deliberate genetic manipulation, such as by microinjection, or by infection with a recombinant virus.The term "genetic manipulation" does not include classical crossbreeding techniques, but rather refers to the introduction of a recombinant DNA molecule(s). This molecule may be integrated into a chromosome, or it may be extrachromosomally replicating DNA. The term "Tg." +" includes animals that are heterozygous or homozygous for a heterologous nucleic acid and / or gene, and / or animals that have one or multiple copies of a heterologous nucleic acid and / or gene.

[00154] A variant, as used herein, refers to an entity that exhibits significant structural identity to a reference entity, but structurally differs from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, a "variant" also differs functionally from its reference entity. Generally, whether a particular entity is appropriate to be considered a "variant" of a reference entity depends on the degree of its structural identity to the reference entity. As will be understood by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements.A "variant" is defined as a distinct chemical entity that shares one or more such characteristic structural elements. As just a couple of examples, a polypeptide may have a characteristic sequence element consisting of a plurality of amino acids characterized by specific positions relative to one another in linear or three-dimensional space and / or providing a specific biological function, or a nucleic acid may have a characteristic sequence element consisting of a plurality of nucleotide residues characterized by specific positions relative to one another in linear or three-dimensional space. In another example, a "variant polypeptide" may differ from a reference polypeptide due to 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 an overall sequence identity with a reference polypeptide of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. Alternatively, or additionally, in some embodiments, a "variant polypeptide" lacks at least one characteristic sequence element in common with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a "variant polypeptide" has one or more biological activities in common with the reference polypeptide. In some embodiments, a "variant polypeptide" lacks one or more biological activities of the 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 polypeptide of interest is considered a "variant" of a parent or reference polypeptide if the polypeptide of interest has an amino acid sequence identical to that of the parent molecule, except for a small number of changes at specific positions in the sequence. Typically, a variant has less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues substituted compared to the parent molecule. In some embodiments, a "variant" has, for example, but not limited to, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue(s) substituted compared to the parent molecule. Often a "variant" has a very small number (e.g. less than 5, 4, 3, 2 or 1) of functional residues replaced (i.e.residues involved in a particular biological activity). Furthermore, a "variant" typically has no more than, for example, without limitation, 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, compared to the parent molecule. Moreover, any additions or deletions typically involve 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 often involve less than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is found in nature.As will be appreciated by those skilled in the art, multiple variants of a particular polypeptide of interest can often be found in nature, particularly if the polypeptide of interest is a polypeptide of an infectious agent.

[00155] A vector, as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, vectors are capable of extrachromosomal replication and / or expression of nucleic acids to which they are linked in a host cell, such as a eukaryotic and / or prokaryotic cell. Vectors capable of directing the expression of operably linked genes are referred to herein as "expression vectors."

[00156] Wild type, as used herein, refers to an entity having a structure and / or activity found in nature in a "normal" (as opposed to mutant, diseased, altered, etc.)) state or environment. Those skilled in the art will appreciate that wild-type genes and polypeptides often exist in several different forms (e.g., alleles). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[00157] In certain aspects, provided herein are, inter alia, engineered non-human animals having heterologous genetic material encoding human variable domains and, in some embodiments, human constant domains, wherein the heterologous genetic material comprises human gene sequences (i.e., segments of human genes) Vλ, Jλ, and Cλ and other human sequences that allow for the suitable rearrangement and expression of antibodies having a human portion and a non-human portion, or antibodies having a sequence that is substantially or substantially entirely human.In various embodiments, the provided engineered non-human animals comprise heterologous genetic material that is inserted such that antibodies comprising light chains that have a human Vλ domain and a human or non-human Cλ domain are expressed in the antibody repertoire of the non-human animal. Furthermore, the provided engineered non-human animals comprise heterologous genetic material that is inserted such that antibodies comprising light chains that have a human Vλ domain and a human or non-human Cλ domain are expressed from engineered Ig λ light chain loci that comprise human and non-human enhancer regions (or sequences) of the Ig λ chain in the germline genome of the non-human animal.

[00158] Without wishing to be bound by any particular theory, it is contemplated that the non-human animal embodiments described herein provide an improved in vivo system that utilizes the expression of antibodies comprising human Vλ domains to produce therapeutic antibodies. It is also contemplated that the non-human animal embodiments described herein, in some embodiments, provide alternative engineered forms of Ig λ light chain loci that comprise heterologous genetic material to develop human antibody-based therapeutics (e.g., monoclonal human antibodies, multispecific binders, scFvs, fusion polypeptides, etc.) directed to disease-causing targets associated with biased antibody responses (e.g., antibody responses characterized by a predominant proportion of either κ or λ light chains).Thus, the non-human animal embodiments described herein are particularly useful for developing human antibodies directed to targets associated with low immunogenicity (e.g., viruses) due, in part, to aberrant antibody repertoires and / or responses.

[00159] In particular, in certain aspects, the present invention describes producing a non-human animal (e.g., a rodent such as a rat or mouse) having a germline genome that comprises an engineered Ig λ light chain locus that, in some embodiments, is characterized by the introduction of a plurality of human Vλ, Jλ, and Cλ gene sequences operably linked to a non-human Cλ region, resulting in the expression of antibodies that comprise light chains comprising a human Vλ domain and a human or non-human Cλ domain.As described herein, obtaining such an engineered Ig λ light chain locus results in the expression of antibodies that comprise light chains comprising a human Vλ domain and a human or non-human Cλ domain from said engineered Ig λ light chain locus in the germline genome of a non-human animal. In some embodiments, the germline genome of the provided non-human animals further comprises (1) humanized Ig H chain and Ig κ chain loci, or (2) a humanized Ig H chain locus and functionally suppressed or otherwise deficient Ig κ light chain loci. The provided non-human animals described herein express antibody repertoires that comprise Ig λ light chains comprising human Vλ domains.

[00160] In some embodiments, the non-human animals described herein comprise human and non-human Ig λ light chain sequences at a single Ig λ light chain locus. In some embodiments, the non-human animals described herein comprise human Ig λ light chain sequences and murine (e.g., mouse or rat) Ig λ light chain sequences at the Ig λ light chain locus. In many embodiments of the non-human animals described herein, the non-human Ig λ light chain sequences are or comprise murine (e.g., mouse or rat) sequences.

[00161] In some embodiments, the Ig λ light chain sequences comprise intergenic DNA that is human and / or murine (e.g., mouse or rat) derived.In some embodiments, the Ig λ light chain sequences comprise intergenic DNA that is synthetic and based on a primary sequence that is of human and / or murine (e.g., mouse or rat) origin. In some embodiments, the intergenic DNA is from the same immunoglobulin locus into which the intergenic DNA is thus placed, inserted, located, or integrated (e.g., Ig λ chain intergenic DNA at an Ig λ light chain locus). In some specific embodiments, the non-human animals described herein comprise an engineered Ig λ light chain locus that comprises intergenic DNA comprising Ig λ light chain sequence(s) of non-human origin (e.g., a mouse or rat Ig λ light chain sequence).

[00162] In various embodiments, the humanized Ig H chain locus comprises a plurality of human V gene segments. H , DH and J H, operably linked to a non-human Ig H chain constant region (e.g., an endogenous non-human Ig H chain constant region that contains one or more Ig H chain constant region genes, such as IgM, IgG, etc.). In various embodiments, the humanized Ig κ light chain locus comprises a plurality of human Vκ and Jλ gene segments operably linked to a non-human Ig κ chain constant region. In some embodiments, the provided non-human animals have a germline genome that comprises the immunoglobulin loci (or alleles) depicted in the drawings provided herein (e.g., see 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 and provide an improved in vivo system suitable for using human Vκ sequences designed to produce human therapeutic antibodies.

[00163] As described herein, certain embodiments provide non-human animals that have a genome that contains multiple segments of human λ light chain genes (e.g., Vλ, Jλ, and Cλ) in place of segments of non-human immunoglobulin λ light chain genes at endogenous immunoglobulin λ light chain loci and contain non-coding human intergenic DNA between the segments of human variable region genes.In some embodiments, the non-human animals provided herein have a genome that further comprises segments of human heavy variable region genes (i.e., V. H , D H and J H ) and light κ chains (e.g., Vκ and Jλ) instead of segments of non-human heavy variable region genes (i.e., V H , D H and J H) and κ-light chains (e.g., Vκ and Jλ) at the endogenous immunoglobulin heavy and light κ-chain loci, respectively. In many embodiments, the human immunoglobulin gene segments (heavy and / or light chains) are constructed using human intergenic DNA (i.e., non-coding human immunoglobulin intergenic DNA) that is naturally associated with said gene segments (i.e., non-coding genomic DNA associated with said gene segments that occurs naturally at the human immunoglobulin locus of a human cell). Such intergenic DNA includes, for example, promoters, leader sequences, and recombination signal sequences that provide for the appropriate recombination and expression of the human gene segments in the case of antibody variable domains.Those skilled in the art will appreciate that non-human immunoglobulin loci also contain such non-coding intergenic DNA, and that, given the present invention, other human or non-human intergenic DNA can be used to create such engineered immunoglobulin loci that result in the same level of expression of human variable domains in antibodies in a non-human animal. Such similar engineered immunoglobulin loci need only contain human coding sequences (i.e., exons) from the desired human gene segments or combinations of human gene segments to achieve expression of antibodies that contain human variable domains.

[00164] Various aspects of certain embodiments are described in detail in the following sections, each of which can be applied 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 indicated. Non-human Animal Antibody Repertoires

[00165] Immunoglobulins (also called antibodies) are large (~150 kDa), Y-shaped glycoproteins that are 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 of which has two structural components: a variable domain and a constant domain. The variable domains of the heavy and light chains differ among antibodies produced by different B cells, but are identical among all antibodies produced by a single B cell or B cell clone. The variable domains of the heavy and light chains of each antibody together constitute the antigen-binding region (or antigen-binding site).Immunoglobulins can exist in different varieties, called isotypes or classes, based on the constant regions (or domains) of their heavy chains. The heavy chain constant domain is identical in all antibodies of the same isotype but differs in antibodies of different isotypes. The table below briefly describes the nine isotypes of mouse and human antibodies.

[00166] Additional isotypes have been identified in other species. Isotypes confer specialized biological properties on an antibody due to different structural characteristics between the different isotypes, and they are found in different locations (cells, tissues, etc.) within an animal. Initially, B cells produce IgM and IgD with identical antigen-binding regions. Upon activation, B cells switch to different isotypes through a process called class switching, which involves a change in the constant domain of the antibody produced by the B cell while the variable domains remain the same, thereby maintaining the antigen specificity of the original antibody (B cell).

[00167] Two distinct loci (Ig κ chains and Ig λ chains) contain gene segments that encode the light chains of antibodies and exhibit both allelic and isotypic exclusion. κ expression ratios + and λ +B cell ratios vary among species. For example, humans exhibit a ratio of approximately 60:40 (κ:λ). Mice and rats exhibit a ratio of 95:5 (κ:λ). Interestingly, the κ:λ ratio observed in cats (5:95) is the opposite of that observed in mice and rats. Several studies have been conducted to elucidate possible reasons underlying these observed ratios, and both locus complexity (i.e., the number of gene segments, particularly V gene segments) and the efficiency of gene segment rearrangement have been proposed as explanations. The human immunoglobulin λ light chain locus is over 1,000 kb in length. and contains approximately 70 Vλ gene segments (29 to 33 functional) and seven pairs of Jλ-Cλ gene segments (four to five functional), organized into three clusters (see, for example, Fig. 1 of U.S. Patent No. 9,006,511). Most of the observed Vλ regions in the expressed antibody repertoire are encoded by gene segments,contained in the most proximal cluster (i.e., cluster A). The mouse immunoglobulin λ light chain locus is strikingly different from the human locus and, depending on the strain, contains only a few Vλ and Jλ gene segments organized into two distinct gene clusters (see, e.g., Fig. 2 of U.S. Patent No. 9,006,511).

[00168] The development of therapeutic antibodies for the treatment of various human diseases has primarily focused on the creation of engineered strains of non-human animals, in particular engineered strains of rodents, carrying varying amounts of genetic material in their genomes corresponding to human immunoglobulin genes (for a brief review, see, e.g., Bruggemann, M. et al., 2015, Arch. Immunol. Ther. Exp. 63:101-8). Initial attempts to create such transgenic rodent lines focused on integrating portions of human immunoglobulin loci that could, on their own,allow recombination of gene segments to produce heavy and / or light chains that were fully human but had inactivated endogenous immunoglobulin loci (see, 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, LD et al., 1992, Nucl. Acids Res. 20:6287-6295; Davies, NP et al., 1993, Biotechnol. 11:911-4; Green, LL et al., 1994, Nat. Genet. 7:13-21; Lonberg, N. et al., 1994, Nature 368:856-9; Taylor, L. D. et al., 1994, Int. Immunol. 6:579-91; Wagner, SD et al., 1994, Eur. J. Immunol. 24:2672-81; Fishwild, D. M. et al., 1996, Nat. Biotechnol. 14:845-51; Wagner, SD et al., 1996, Genomics 35:405-14; Mendez, MJ et al., 1997, Nat. Genet. 15:146-56; Green, L.L. et al., 1998, J. Exp. Med. 188:483-95; Xian, J. et al., 1998, Transgenics 2:333-43; Little, M. et al. 2000, Immunol. Today 21:364-70; Kellermann, S. A. and L. L. Green, 2002,Cur. Opin. Biotechnol. 13:593-7). In particular, some attempts have involved the integration of human Ig λ light chain sequences (see, e.g., U.S. Patent Application Publications Nos. 2002 / 0088016 A1, 2003 / 0217373 A1, and 2011 / 0236378 A1; U.S. Patent Publications Nos. 6,998,514 and 7,435,871; Nicholson, I.C. et al., 1999, J. Immunol. 163:6898-906; Popov, A.V. et al., 1999, J. Exp. Med. 189(10): 1611-19). Such efforts have focused on the random integration of yeast artificial chromosomes containing human Vλ, Jλ, and Cλ sequences, thereby generating mouse strains that express fully human λ light chains (i.e., the human variable and human constant domains). More recent efforts have employed similar strategies using constructs that also contain human Vλ, Jλ, and Cλ sequences (Osborn, MJ et al., 2013, J. Immunol. 190:1481–90; Lee, EC et al., 2014,Nat. Biotech. 32(4):356-63).

[00169] Still other attempts have involved the specific insertion of human Vλ and Jλ gene segments into endogenous rodent Ig light chain loci (κ chain and λ chain) such that said human Vλ and Jλ gene segments are operably linked to endogenous Ig light chain constant regions (see, e.g., U.S. Pat. Nos. 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662, and 9,163,092; all of which are incorporated herein by reference in their entireties). In these animals, all of the human Vλ gene segments from clusters A and B and either one or four human Jλ gene segments were inserted into the endogenous Ig κ and Ig λ light chain loci. As a result, several different human Vλ and Jλ gene segments were properly rearranged in both engineered rodent Ig light chain loci to form functional human Vλ domains that were expressed in close association with both the Cκ regions,as well as with the Cλ regions in the light chains of the rodent antibody repertoire (see, e.g., Table 7 and Figures 11-13 of U.S. Patent No. 9,006,511). In particular, mice carrying engineered Ig κ light chain loci carrying segments of the human Vλ and Jλ genes exhibited a κ:λ ratio of approximately 1:1 in the spleen compartment (see, e.g., Table 4 of U.S. Patent No. 9,006,511). Indeed, both engineered mouse strains (i.e., engineered Ig κ light chain or Ig λ light chain loci) demonstrated that human Vλ domains can be expressed from endogenous Ig light chain loci in rodents, which typically exhibit a large bias in light chain expression (see above). The present invention is based on the recognition thatthat other engineered Ig light chain locus structures can be produced to maximize the utilization of human Vλ and Jλ gene segments in antibody repertoires for therapeutic targets in non-human animals, particularly as compared to non-human animals that contain an Ig light chain locus that lacks the complexity and high quality (e.g., mice and rats) typically associated with a human Ig light chain locus (i.e., that resides in a human cell). Such alternative engineered Ig light chain locus structures provide the ability to produce unique antibody repertoires based on their structure.

[00170] The present invention describes, inter alia, the successful production of a non-human animal whose germline genome comprises an engineered endogenous Ig light chain locus comprising a plurality of human Vλ, Jλ, and Cλ gene segments,operably linked to a non-human constant region of an Ig light λ chain. In particular, the present invention specifically demonstrates the successful production of an engineered non-human animal that expresses antibodies comprising human variable domains and non-human constant domains, wherein the antibodies comprise light chains that comprise a human Vλ domain. As described herein, the expression of such light chains is achieved by inserting said plurality of human Vλ, Jλ, and Cλ gene segments into an endogenous Ig light λ chain locus (or allele). Also, as described herein, the provided non-human animals are, in some embodiments, engineered such that expression of light chains that comprise endogenous Vλ domains is inactivated (e.g., by gene deletion). Thus, in the present invention,In at least some embodiments, the development of an improved in vivo system for producing human antibodies is encompassed by providing an engineered non-human animal comprising an alternatively engineered Ig λ light chain locus that results in an expressible antibody repertoire comprising human Vλ domains. DNA Inserts

[00171] Typically, a polynucleotide molecule comprising human Ig λ light chain sequences (e.g., Vλ, Jλ, Cλ, and Ig λ chain enhancers) or portion(s) thereof is inserted into a vector, preferably a DNA vector, for the purpose of replicating the polynucleotide molecule in a host cell.

[00172] Human Ig λ light chain sequences can be cloned directly from known sequences or sources (e.g., libraries) or synthesized from germline sequences designed in silico based on published sequences,available from GenBank or other publicly accessible databases (e.g., IMGT). Alternatively, bacterial artificial chromosome (BAC) libraries can 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 inserts of 100-150 kbp in size, and they are capable of carrying inserts up to 300 kbp in size. (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; incorporated herein by reference in their entireties.) For example, a human BAC library carrying inserts with an average size of 164–196 kb has been described (Osoegawa, K. et al., 2001, Genome Res. 11(3):483–96; Osoegawa, K. et al., 1998, Genomics 52:1–8,Article number GE985423). Human and mouse BAC genomic libraries have been constructed and are commercially available (e.g., ThermoFisher). BAC genomic libraries can also serve as a source of immunoglobulin DNA sequences, as well as transcriptional control regions.

[00173] Alternatively, immunoglobulin DNA sequences can be isolated, cloned, and / or transferred from yeast artificial chromosomes (YACs). For example, the nucleotide sequence of the human Ig light chain λ locus has been determined (see, e.g., Dunham, I. et al., 1999, Nature 402:489–95). In addition, YACs have previously been used to assemble a transgene for the human Ig λ light chain locus (see, e.g., Popov, AV et al., 1996, Gene 177:195–201; Popov, AV et al., 1999, J. Exp. Med. 189(10): 1611–19). A complete Ig λ light chain locus (human or rodent) can be cloned and contained within multiple YACs. If multiple YACs are used,and they contain overlapping homologous regions, they can be recombined in yeast host strains to produce a single construct representing the entire locus or the necessary portions of the locus (e.g., the region to be targeted by a targeting vector). The YAC arms can be further modified using mammalian cell selection cassettes by enhancing them in a manner that facilitates the introduction of the constructs into embryonic stem cells or embryos using methods known in the art and / or described herein.

[00174] DNA and amino acid sequences of human Ig λ light chain gene segments for use in constructing the engineered Ig λ light chain locus described herein can be obtained from published databases (e.g., GenBank, IMGT, etc.) and / or published antibody sequences. DNA inserts containing human Ig λ light chain gene segments,in some embodiments, comprise one or more human Ig λ light chain enhancer sequences (or regions). In some embodiments, the DNA inserts comprise a human Ig λ light chain enhancer sequence (or region) that comprises one or more sequence elements, such as one, two, three, etc. In some specific embodiments, the DNA inserts comprise a human Ig λ light chain enhancer sequence (or region), referred to as human Eλ, which has three different sequence elements. Thus, in some embodiments, the human Eλ described herein is modular, and the one or more sequence elements function together as an enhancer sequence (or region). In some specific embodiments, DNA inserts comprising human Ig λ light chain enhancer sequences,contain human Ig λ light chain enhancer sequences operably linked to a non-human Ig λ light chain sequence (e.g., a non-human Ig λ light chain constant region sequence). In some specific embodiments, DNA inserts containing human Ig λ light chain enhancer sequences contain human Ig λ light chain enhancer sequences operably linked to a non-human Ig λ light chain sequence (e.g., a non-human Ig λ light chain constant region sequence) and operably linked to one or more human Vλ gene segments, one or more pairs of human Jλ-Cλ gene segments, and / or one or more human Jλ gene segments. In some specific embodiments, DNA inserts containing human Ig λ light chain enhancer sequences,comprise human Ig λ light chain enhancer sequences 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 segments of a human Vλ gene, one or more segments of a human Jλ gene, and one or more segments of a human Cλ gene.

[00175] DNA inserts can be produced using methods known in the art. For example, a DNA insert can be produced as part of a larger plasmid. Such production allows for efficient cloning and selection of the correct constructs, as is known in the art. DNA inserts containing complete or partial human Ig λ light chain sequences described herein can be positioned between suitable restriction sites on the plasmid such thatthat they can be readily isolated from the remaining plasmid sequences for insertion into the desired non-human animal.

[00176] Various methods used in producing plasmids and transforming host organisms are known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombination procedures, see Principles of Gene Manipulation: An Introduction to Genetic Manipulation, 5th Ed., ed. By Old, R.W. and S.B. Primrose, Blackwell Science, Inc., 1994, and Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al., Cold Spring Harbor Laboratory Press: 1989.Targeting Vectors

[00177] Targeting vectors can be used to introduce a DNA insert into a genomic target locus and contain the DNA insert and homology arms that flank the DNA insert. Targeting vectors can be in linear form or in circular form,and they can be single-stranded or double-stranded. Targeting vectors can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). For ease of reference, the homology arms are referred to herein as the 5' and 3' (i.e., upstream and downstream) homology arms. This terminology refers to the relative position of the homology arms relative to the DNA insert in the targeting vector. The 5' and 3' homology arms correspond to regions in the locus to be targeted or a region in another targeting vector, which are respectively referred to herein as the "5' gap sequence" and the "3' target sequence." In some embodiments, the homology arms can also function as the 5' or 3' target sequence.

[00178] In some embodiments, the methods described herein provide two, three or more targeting vectors,which are capable of recombination with each other. In various embodiments, the targeting vectors are large targeting vectors (LTVECs) as described elsewhere herein. In such embodiments, each of the first, second, and third targeting vectors comprises a 5' and a 3' homology arm. The 3' homology arm of the first targeting vector comprises a sequence that overlaps with the 5' homology arm of the second targeting vector (i.e., overlapping sequences) that allow for homologous recombination between the first and second LTVECs.

[00179] In the case of dual targeting methods, the 5' homology arm of the first targeting vector and the 3' homology arm of the second targeting vector are homologous to corresponding segments within the target genomic locus (i.e., the target sequence),which facilitates homologous recombination of the first and second targeting vectors with the corresponding genomic segments and provides for modification of the target genomic locus.

[00180] In the case of triple targeting methods, the 3' homology arm of the second targeting vector comprises a sequence that overlaps with the 5' homology arm of the third targeting vector (i.e., overlapping sequences), which provide for homologous recombination between the second and third LTVEC. The 5' homology arm of the first targeting vector and the 3' homology arm of the third targeting vector are homologous to the corresponding segments in the target genomic locus (i.e., target sequence),which facilitates homologous recombination of the first and third targeting vectors with the corresponding genomic segments and provides for modification of the target genomic locus.

[00181] A homology arm and a target sequence, or two homology arms, "correspond" or "are correspondent" to each other if the two regions have a sufficient level of sequence identity with each other to act as substrates for the homologous recombination reaction. The term "homology" encompasses DNA sequences that are either identical to or share sequence identity with a corresponding sequence. The sequence identity between a specified target sequence and the corresponding homology arm revealed in a targeting vector (i.e., an overlapping sequence), or between two homology arms, may represent any degree of sequence identity,which ensures the occurrence of homologous recombination. As one example, the degree of sequence identity between the homology arm of a targeting vector (or fragment thereof) and the target sequence of another targeting vector or the target sequence of a target genomic locus (or fragment thereof) can be, for example, 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, as a result of which the sequences undergo homologous recombination.

[00182] Furthermore, the corresponding region of homology between the homology arm and the corresponding target sequence may be of any length sufficient to activate homologous recombination at the target genomic locus. For example, a given homology arm and / or the corresponding target sequence may contain corresponding regions of homology,which have a length of, for example, without limitation, at least about 5-10 tons, 5-15 tons, 5-20 tons, 5-25 tons, 5-30 tons, 5-35 tons, 5-40 tons, 5-45 tons, 5-50 tons, 5-55 tons, 5-60 tons, 5-65 tons, 5-70 tons, 5-75 tons, 5-80 tons, 5-85 tons, 5-90 tons, 5-95 tons, 5-100 tons, 100-200 tons, or 200-300 tons. or more (as described elsewhere herein), whereby the homology arm has sufficient homology to undergo homologous recombination with the corresponding target sequence(s) in the target genomic locus of the cell or in another targeting vector. In some embodiments, the homology arm and / or the corresponding target sequence comprise corresponding regions of homology that are, for example, without limitation, 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 or greater (as described elsewhere herein), whereby the homology arm has sufficient homology to undergo homologous recombination with the corresponding target sequence(s) in the target genomic locus of the cell or in another targeting vector.

[00183] The overlapping sequences of the 3' homology arm of the first targeting vector and the 5' homology arm of the second targeting vector or the 3' homology arm of the second targeting vector and the 5' homology arm of the third targeting vector may be of any length that is sufficient to ensure homologous recombination between said targeting vectors. For example, a given overlapping homology arm sequence may comprise corresponding overlapping regions that have a length of at least approximately 1-5 kb, 5-10 kb,5-15 t.n., 5-20 t.n., 5-25 t.n., 5-30 t.n., 5-35 t.n., 5-40 t.n., 5-45 t.n., 5-50 t.n., 5-55 t.n., 5-60 t.n., 5-50 t.n., 5-70 t.n. t.n., 5-75 t.n., 5-80 t.n., 5-85 t.n., 5-90 t.n., 5-95 t.n., 5-100 t.n., 100-200 t.n. or 200-300 t.n. or greater, whereby the overlapping homology arm sequence is characterized by sufficient homology to undergo homologous recombination with the corresponding overlapping sequence in another targeting vector. In some embodiments, said overlapping homology shoulder sequence comprises an overlapping region that is at least about 1-100 bp, 5-100 bp, 10-100 bp, 15-10, 0, 20-100 bp, 20-100 bp in length. 25-100 t.n., 30-100 t.n., 35-100 t.n., 40-100 t.n., 45-100 t.n., 50-100 t.n., 55-100 t.n., 60-100 t.n., 0.05-100 t.0.00 t.n. t.n., 75-100 t.n., 80-100 t.n., 85-100 t.n., 90-100 t.n. or 95-100 t.n. or more,as a result of which the overlapping sequence of the homology arm is characterized by sufficient homology,to undergo homologous recombination with a corresponding overlapping sequence in another targeting vector. In some embodiments, the overlapping sequence is 1-5 kb, inclusive. In some embodiments, the overlapping sequence is from about 1 kb to about 70 kb, inclusive. In some embodiments, the overlapping sequence is from about 10 kb to about 70 kb, inclusive. In some embodiments, the overlapping sequence is from about 10 kb to about 50 kb, inclusive. In some embodiments, the overlapping sequence is at least 10 kb. In some embodiments, the overlapping sequence is at least 20 kb. For example,The overlapping sequence may be from about 1 kb to about 5 kb inclusive, from about 5 kb to about 10 kb inclusive, from about 10 kb to about 15 kb inclusive, from about 15 kb to about 20 kb inclusive, from about 20 kb to about 25 kb inclusive, from about 25 kb to about 30 kb inclusive, from about 30 kb to about 35 kb inclusive, from about 35 kb to about 40 kb inclusive, from about 40 kb to about 45 kb inclusive, from about 45 kb to about 50 kb inclusive, from about 50 kb to about 60 kb. inclusive, from approximately 60 t. to approximately 70 t. inclusive, from approximately 70 t. to approximately 80 t. inclusive, from approximately 80 t. to approximately 90 t. inclusive,from about 90 tbsp to about 100 tbsp inclusive, from about 100 tbsp to about 120 tbsp inclusive, from about 120 tbsp to about 140 tbsp inclusive, from about 140 tbsp to about 160 tbsp inclusive, from about 160 tbsp to about 180 tbsp inclusive, from about 180 tbsp to about 200 tbsp inclusive, from about 200 tbsp to about 220 tbsp inclusive, from about 220 tbsp to about 240 tbsp inclusive, from about 240 tbsp to about 260 tbsp inclusive, from about 260 tbsp to about 280 tbsp inclusive, or from about 280 tbsp up to and including approximately 300 kb. As one example, the overlapping sequence may be from approximately 20 kb to and including approximately 60 kb. Alternatively, the overlapping sequence may be at least 1 kb.at least 5 t, at least 10 t, at least 15 t, at least 20 t, at least 25 t, at least 30 t, at least 35 t, at least 40 t, at least 45 t, at least 50 t, at least 60 t, at least 70 t, at least 80 t, at least 90 t, at least 100 t, at least 120 t, at least 140 t, at least 160 t, at least 180 t, at least 200 t, at least 220 t, at least 240 kb, at least 260 kb, at least 280 kb, or at least 300 kb.

[00184] The homology arms, in some embodiments, may correspond to a locus that is native to the cell (e.g., the locus to be targeted), or, alternatively, they may correspond to a region of a heterologous or exogenous DNA segment that has been integrated into the genome of the cell, including, for example,transgenes, expression cassettes, or heterologous or exogenous regions of DNA. Alternatively, the homology arms, in some embodiments, may correspond to a region of the targeting vector in the cell. In some embodiments, the homology arms of the targeting vector may correspond to a region of a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a human artificial chromosome, or any other engineered region contained in the appropriate host cell. Furthermore, the homology arms of the targeting vector may correspond to or be derived from a region of a BAC library, a cosmid library, or a P1 phage library. In some specific embodiments, the homology arms of the targeting vector correspond to a locus that is native, heterologous, or exogenous to a prokaryote, a yeast cell, an avian (e.g., chicken), a non-human mammal, a rodent, a human, a rat, a mouse,a hamster, a rabbit, a pig, a bovine, a deer, a sheep, a goat, a cat, a dog, a ferret, a primate (e.g., a marmoset, a rhesus macaque), a domesticated mammal, a farm mammal, or any other organism of interest. In some embodiments, the homology arms correspond to a cellular locus that cannot be targeted using a conventional method, or that can be targeted only incorrectly or only with significantly low efficiency in the absence of a single-strand break or a double-strand break induced by a nuclease-based agent (e.g., a Cas protein). In some embodiments, the homology arms are derived from synthetic DNA.

[00185] In some embodiments, one of the 5' or 3' homology arms of the targeting vector(s) corresponds to a genomic locus to be targeted,wherein the other of the 5' or 3' homology arms corresponds to a region of another targeting vector.

[00186] In some embodiments, the 5' and 3' homology arms of the targeting vector(s) correspond to the genome to be targeted. Alternatively, the homology arms may be from a related genome. For example, the genome to be targeted is the genome of a mouse from a first lineage, and the targeting arms are from the genome of a mouse from a second lineage, wherein the first lineage and the second lineage are different. In certain embodiments, the homology arms are from the genome of the same animal or from the genome of the same lineage, such as the genome to be targeted is the genome of a mouse from a first lineage, and the targeting arms are from the genome of a mouse of the same mouse or from the same lineage.

[00187] The homology arm of the targeting vector can be of any length,which is sufficient to ensure a homologous recombination event with the corresponding target sequence, including, for example, a length of at least 1-5 kb inclusive, 5-10 kb inclusive, 5-15 kb inclusive, 5-20 kb inclusive, 5-25 kb inclusive, 5-30 kb inclusive, 5-35 kb inclusive, 5-40 kb inclusive, 5-45 kb inclusive, 5-50 kb inclusive, 5-55 kb inclusive, 5-60 kb inclusive, 5-65 kb inclusive, 5-70 kb inclusive, 5-75 kb inclusive, 5-80 kb inclusive, 5-85 kb inclusive. inclusive, 5-90 kb inclusive, 5-95 kb inclusive, 5-100 kb inclusive, 100-200 kb inclusive, or 200-300 kb inclusive or more. In some embodiments, the homology arm of the targeting vector has a length that is sufficient to ensure a homologous recombination event with the corresponding target sequence,which is at least 1-100 tons inclusive, 5-100 tons inclusive, 10-100 tons inclusive, 15-100 tons inclusive, 20-100 tons inclusive, 25-100 tons inclusive, 30-100 tons inclusive, 35-100 tons inclusive, 40-100 tons inclusive, 45-100 tons inclusive, 50-100 tons inclusive, 55-100 tons inclusive, 60-100 tons inclusive, 65-100 tons inclusive, 70-100 tons inclusive, 75-100 tons inclusive, 80-100 tons inclusive, 85-100 kb inclusive, 90-100 kb inclusive, or 95-100 kb inclusive or longer. As described herein, longer targeting arms may be used in larger targeting vectors.

[00188] Nuclease-based tools (e.g., CRISPR / Cas systems) may be used in combination with targeting vectors to facilitate modification of the target locus (e.g.,Ig light chain locus). Such nuclease-based agents can promote homologous recombination between a targeting vector and a target locus. If nuclease-based agents are used in combination with a targeting vector, the targeting vector may contain 5' and 3' homology arms corresponding to the target 5' and 3' sequences located close enough to the nuclease cleavage site to promote the fidelity of the homologous recombination event between the target sequences and the homology arms following a single-strand break or double-strand break at the nuclease cleavage site. The term "nuclease cleavage site" refers to a DNA sequence in which a single-strand break or double-strand break is made by the nuclease-based agent (e.g., the cleavage site for Cas9). Target sequences at the locus to be targeted,that correspond to the 5' and 3' homology arms of the targeting vector are "located sufficiently close" to a nuclease cleavage site if the distance is such as to promote the fidelity of a homologous recombination event between the target 5' and 3' sequences and the homology arms following a single-strand or double-strand break in the recognition site. Thus, in certain embodiments, the target sequences corresponding to the 5' and / or 3' homology arms of the targeting vector are one nucleotide away from the recognition site or are at least 10 to about 14 kb away from the recognition site. In some embodiments, the nuclease cleavage site is immediately adjacent to at least one or both of the target sequences.

[00189] The spatial relationship of the target sequences that correspond to the homology arms of the targeting vector,and the nuclease cleavage site may vary. For example, the target sequences may be located 5' to the nuclease cleavage site, the target sequences may be located 3' to the recognition site, or the target sequences may flank the nuclease cleavage site.

[00190] Combined use of a targeting vector (including, for example, a large targeting vector) with a nuclease-based agent may result in increased targeting efficiency compared to use of the targeting vector alone. For example, when a targeting vector is used in combination with a nuclease-based agent, the targeting efficiency of the targeting vector may be increased by at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold, at least nine-fold,at least ten times or a range formed by such integers, such as 2-10 times, compared to using the targeting vector alone.

[00191] Some targeting vectors are "large targeting vectors" or "LTVECs," which include targeting vectors containing homology arms that correspond to and are derived from nucleic acid sequences that are larger than those typically used in other approaches designed to achieve homologous recombination in cells. An LTVEC may be, for example, at least 10 kb in length, or the total sum of the lengths of the 5' homology arm and the 3' homology arm may be, for example, at least 10 kb. LTVECs also include targeting vectors containing DNA inserts that are larger in size than those typically used in other approaches designed to achieve homologous recombination in cells. For example, LTVECs make it possible to modify large loci,which cannot be targeted using traditional plasmid-based targeting vectors due to their size limitations. For example, the locus to be targeted may be (i.e., the 5' and 3' arms of homology may correspond to the locus) a cellular locus that cannot be targeted using traditional methods, or that can only be targeted incorrectly or only with significantly low efficiency in the absence of a single-strand break or a double-strand break induced by a nuclease-based agent (e.g., a Cas protein).

[00192] In some embodiments, the methods described herein utilize two or three LTVECs that are capable of recombination with each other and with the target genomic locus in a three-way or four-way recombination event. Such methods enable the modification of large loci,which cannot be achieved using a single LTVEC.

[00193] Examples of LTVECs include vectors derived from a bacterial artificial chromosome (BAC), a human artificial chromosome, or a yeast artificial chromosome (YAC). Examples of LTVECs and methods for making them are described, for example, in U.S. Patent Nos. 6,586,251, 6,596,541, and 7,105,348 and International Patent Application Publication No. WO 2002 / 036789, each of which is incorporated herein by reference in its entirety. LTVECs may be in linear form or circular form.

[00194] LTVECs may be of any length, including, for example, from about 20 kb to about 300 kb, inclusive, from about 20 kb to about 50 kb, inclusive, from about 50 kb to about 50 kb, inclusive, up to and including approximately 75 tons, from approximately 75 tons to and including approximately 100 tons, from approximately 100 tons to and including 125 tons, from approximately 125 tons to and including approximately 150 tons,from about 150 tbsp to about 175 tbsp inclusive, from about 175 tbsp to about 200 tbsp inclusive, from about 200 tbsp to about 225 tbsp inclusive, from about 225 tbsp to about 250 tbsp inclusive, from about 250 tbsp to about 275 tbsp inclusive, or from about 275 tbsp to about 300 tbsp inclusive. Alternatively, the LTVEC may be at least 10 t, at least 15 t, at least 20 t, at least 30 t, at least 40 t, at least 50 t, at least 60 t, at least 70 t, at least 80 t, at least 90 t, at least 100 t, at least 150 t, at least 200 t, at least 250 t, at least 300 t, at least 350 t, at least 400 t,at least 450 kb, or at least 500 kb, or more. In some embodiments, the size of the LTVEC may be too large to allow screening for targeting events using traditional assays, such as Southern blotting and long fragment PCR (e.g., 1 kb to 5 kb).

[00195] In some embodiments, the LTVEC comprises a DNA insert having a length in the range of from about 5 kb to about 200 kb, inclusive, from about 5 kb to about 10 kb, inclusive, from about 10 kb to about 20 kb, inclusive, from about 20 kb to about 30 kb, inclusive, from about 30 kb to about 40 kb, inclusive, from about 40 kb to about 50 kb. inclusive, from approximately 60 t. to approximately 70 t. inclusive, from approximately 80 t. to approximately 90 t. inclusive,from about 90 tbsp to about 100 tbsp inclusive, from about 100 tbsp to about 110 tbsp inclusive, from about 120 tbsp to about 130 tbsp inclusive, from about 130 tbsp to about 140 tbsp inclusive, from about 140 tbsp to about 150 tbsp inclusive, from about 150 tbsp to about 160 tbsp inclusive, from about 160 tbsp to about 170 tbsp inclusive, from about 170 tbsp to about 180 tbsp inclusive, from about 180 tbsp to about 190 tbsp inclusive, or from about 190 tbsp to about 200 tbsp inclusive. In some embodiments, the length of the DNA insert may be in the range of from about 5 kb to about 10 kb, inclusive, from about 10 kb to about 20 kb, inclusive, from about 20 kb to about 40 kb, inclusive,from about 40 kb to about 60 kb inclusive, from about 60 kb to about 80 kb inclusive, from about 80 kb to about 100 kb inclusive, from about 100 kb to about 150 kb inclusive, from about 150 kb to about 200 kb inclusive, from about 200 kb to about 250 kb inclusive, from about 250 kb to about 300 kb inclusive, from about 300 kb to about 350 kb inclusive, or from about 350 kb to about 400 kb inclusive. In some embodiments, the LTVEC comprises a DNA insert having a length in the range of from about 400 kb to about 450 kb. inclusive, from approximately 450 tons to approximately 500 tons inclusive, from approximately 500 tons to approximately 550 tons inclusive, from approximately 550 tons to approximately 600 tons inclusive,from about 600 kb to about 650 kb, inclusive, from about 650 kb to about 700 kb, inclusive, from about 700 kb to about 750 kb, inclusive, or from about 750 kb to about 800 kb, inclusive.

[00196] In some embodiments, the total sum of the lengths of the 5' homology arm and the 3' homology arm of LTVEC is at least 10 kb. In some embodiments, the length of the 5' homology arm of LTVEC is in the range of about 1 kb to about 100 kb, inclusive, and / or the length of the 3' homology arm of LTVEC is in the range of about 1 kb to about 100 kb, inclusive. The total sum of the lengths of the 5' and 3' arms of homology may be, for example, from about 1 kb to about 5 kb inclusive, from about 5 kb to about 10 kb inclusive, from about 10 kb to about 20 kb inclusive,from about 20 t to about 30 t inclusive, from about 30 t to about 40 t inclusive, from about 40 t to about 50 t inclusive, from about 50 t to about 60 t inclusive, from about 60 t to about 70 t inclusive, from about 70 t to about 80 t inclusive, from about 80 t to about 90 t inclusive, from about 90 t to about 100 t inclusive, from about 100 t to about 110 t inclusive, from about 110 t to about 120 t inclusive, from about 120 t to about 130 t inclusive, from approximately 130 t. to approximately 140 t. inclusive, from approximately 140 t. to approximately 150 t. inclusive, from approximately 150 t. to approximately 160 t. inclusive,from about 160 tn to about 170 tn inclusive, from about 170 tn to about 180 tn inclusive, from about 180 tn to about 190 tn inclusive, or from about 190 tn to about 200 tn inclusive. Alternatively, the length of each homology arm, in some embodiments, may 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 so-called, at least 190 kb or at least 200 kb. Similarly, the total sum of the lengths of the 5'- and 3'-arms of homology,in some embodiments, may be at least 5 tbsp, at least 10 tbsp, at least 15 tbsp, at least 20 tbsp, at least 30 tbsp, at least 40 tbsp, at least 50 tbsp, at least 60 tbsp, at least 70 tbsp, at least 80 tbsp, at least 90 tbsp, at least 100 tbsp, at least 110 tbsp, at least 120 tbsp, at least 130 tbsp, at least 140 tbsp, at least 150 tbsp, at least 160 tbsp, at least 170 tbsp, at least 180 tbsp, at least 190 kb or at least 200 kb.

[00197] In some embodiments, the LTVEC and DNA insert are designed to allow for a deletion of endogenous sequence at the target locus of from about 5 kb to about 10 kb, inclusive, from about 10 kb to about 20 kb, inclusive, from about 20 kb to about 40 kb, inclusive,from about 40 tons to about 60 tons inclusive, from about 60 tons to about 80 tons inclusive, from about 80 tons to about 100 tons inclusive, or from about 100 tons to about 150 tons inclusive, from about 150 tons to about 200 tons inclusive, from about 200 tons to about 300 tons inclusive, from about 300 tons to about 400 tons inclusive, from about 400 tons to about 500 tons inclusive, from about 500 tons to about 600 tons inclusive, from about 600 tons to about 700 tons inclusive, from about 700 tons up to and including approximately 800 thou. N. or from approximately 500 thou. N. to and including approximately 1 million N., from approximately 1 million N. to and including approximately 1.5 million N., from approximately 1.5 million N. to and including approximately 2 million N.,from about 2 million bp to about 2.5 million bp, inclusive, or from about 2.5 million bp to about 3 million bp, inclusive. Alternatively, the deletion size may be from about 3 million bp to about 4 million bp, inclusive, from about 4 million bp to about 5 million bp, inclusive, from about 5 million bp to about 10 million bp, inclusive, from about 10 million bp to about 20 million bp, inclusive, from about 20 million bp to about 30 million bp, inclusive, from about 30 million bp to about 40 million bp, inclusive, from about 40 million bp to about 50 million bp, inclusive, from about 50 million bp to about 60 million bp, inclusive, from about 60 million bp to about 70 million bp. inclusive, from approximately 70 million N. to approximately 80 million N. inclusive,from approximately 80 million N to approximately 90 million N, inclusive, or from approximately 90 million N to approximately 100 million N, inclusive. Alternatively, the size of 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.

[00198] In some embodiments, the LTVEC and DNA insert are designed to allow insertion into the target locus of an exogenous nucleic acid sequence having a size in the range of about 5 kb to about 10 kb, inclusive,from about 10 tons to about 20 tons inclusive, from about 20 tons to about 40 tons inclusive, from about 40 tons to about 60 tons inclusive, from about 60 tons to about 80 tons inclusive, from about 80 tons to about 100 tons inclusive, from about 100 tons to about 150 tons inclusive, from about 150 tons to about 200 tons inclusive, from about 200 tons to about 250 tons inclusive, from about 250 tons to about 300 tons inclusive, from about 300 tons to about 350 tons inclusive, or from about 350 tons to about 400 tons inclusive. Alternatively, the insert size, in some embodiments, may be from about 400 tbsp to about 450 tbsp, inclusive, from about 450 tbsp to about 500 tbsp, inclusive,from about 500 tons to about 550 tons inclusive, from about 550 tons to about 600 tons inclusive, from about 600 tons to about 650 tons inclusive, from about 650 tons to about 700 tons inclusive, from about 700 tons to about 750 tons inclusive, or from about 750 tons to about 800 tons inclusive. Alternatively, the insert size may be, in some embodiments, at least 10 kt, at least 20 kt, at least 30 kt, at least 40 kt, at least 50 kt, at least 60 kt, at least 70 kt, at least 80 kt, at least 90 kt, at least 100 kt, at least 150 kt, at least 200 kt, at least 250 kt, at least 300 kt, at least 350 kt, at least 400 kt,at least 450 tons or at least 500 tons or more.

[00199] In still other cases, the size of the DNA insert and / or region of the endogenous locus altered, 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 tn, 20 kb or more. In some embodiments, the size of the DNA insert and / or the region of the endogenous locus subject to alteration, deletion, targeting, modification, engineering, etc. is from nucleotides to 20 kb, from 200 nucleotides to 20 kb, from 300 nucleotides to 20 kb, from 400 nucleotides to 20 kb, from 500 nucleotides to 20 kb, from 600 nucleotides to 20 kb, from 700 nucleotides to 20 kb, from 800 nucleotides to 20 kb, from 900 nucleotides to 20 kb,from 1 t to 20 t, from 2 t to 20 t, from 3 t to 20 t, from 4 t to 20 t, from 5 t to 20 t, from 6 t to 20 t, from 7 t to 20 t, from 8 t to 20 t, from 9 t to 20 t, from 10 t to 20 t, from 11 t to 20 t, from 12 t to 20 t, from 13 t to 20 t, from 14 t to 20 t, from 15 t up to 20 tons, from 16 tons to 20 tons, from 17 tons to 20 tons, from 18 tons to 20 tons or from 19 tons to 20 tons. In some embodiments, the size of the DNA insert and / or region of the endogenous locus that is altered, deleted, targeted, modified, engineered, etc. is from 100 nucleotides to 19 kb, from 100 nucleotides to 18 kb, from 100 nucleotides to 17 kb, from 100 nucleotides to 16 kb, from 100 nucleotides to 15 kb, from 100 nucleotides to 14 kb, from 100 nucleotides to 13 kb, from 100 nucleotides to 12 kb, from 100 nucleotides to 11 kb, from 100 nucleotides to 10 kb, from 100 nucleotides to 9 kb, from 100 nucleotides to 8 so-called,from 100 nucleotides to 7 bp, from 100 nucleotides to 6 bp, from 100 nucleotides to 5 bp, from 100 nucleotides to 4 bp, from 100 nucleotides to 2 bp, from 100 nucleotides to 2 bp. 100 nucleotides to 1 bn, from 100 nucleotides to 900 nucleotides, from 100 nucleotides to 800 nucleotides, from 100 nucleotides to 700 nucleotides, from 100 nucleotides to 600 nucleotides, from 1000 nucleotides nucleotides to 500 nucleotides, from 100 nucleotides to 400 nucleotides, from 100 nucleotides to 300 nucleotides, or from 100 nucleotides to 200 nucleotides. In some embodiments, the size of the DNA insertion and / or the region of the endogenous locus subject to alteration, deletion, targeting, modification, design, etc. is from 200 nucleotides to 19 bp, from 300 nucleotides to 18 to 400 bp. nucleotides to 17 bp, from 500 nucleotides to 16 bp, from 600 nucleotides to 15 bp, from 700 nucleotides to 14 bp, from 800 nucleotides to 13 bp, from 900 nucleotides to 11 bp. t.n. to 11 tons,2 kb to 10 kb, 3 kb to 9 kb, 4 kb to 8 kb, 5 kb to 7 kb, or 5 kb to 6 kb. Non-human Animals Provided

[00200] In certain aspects, non-human animals are provided that express antibodies comprising light chains that comprise a complete or partial sequence of a human Ig λ light chain, resulting from the integration of genetic material that corresponds to at least a portion of a human Ig λ light chain locus and that encodes at least a human Vλ domain (i.e., a rearranged human Vλ-Jλ sequence), in place of the corresponding sequences of the non-human Ig λ light chains in the germline genome of the non-human animal. Suitable examples described herein include, but are not limited to, rodents, in particular rats or mice.

[00201] The sequence of a human Ig light λ chain, in some embodiments,comprises genetic material from a human Ig λ light chain locus, wherein the human Ig λ light chain sequence encodes an immunoglobulin light chain that comprises an encoded portion of genetic material from a human Ig λ light chain locus. In some embodiments, the human Ig λ light chain sequence described herein comprises at least one human Vλ gene segment and at least one human Jλ gene segment, and one or more sequences necessary to provide for the rearrangement (e.g., recombination signal sequence(s)) of at least one said human Vλ gene segment with at least one said human Jλ gene segment to form a functional, rearranged human Vλ-Jλ sequence,which encodes a human Vλ domain. In many embodiments, the human Ig λ light chain sequence comprises a plurality of human Vλ gene segments and one or more sequences necessary to provide for the rearrangement of said human Vλ gene segments with at least one human Jλ gene segment. In many embodiments, the human Ig λ light chain sequence described herein is the genomic sequence of a human Ig λ light chain locus (e.g., isolated and / or cloned from a bacterial artificial chromosome) and comprises a plurality of human Vλ gene segments in germline configuration. In some embodiments, the human Ig λ light chain sequence comprises human Vλ, Jλ, and Cλ sequences in germline configuration (i.e., as indicated, human Vλ,Jλ and Cλ are located in the Ig λ light chain locus in a human cell). In some embodiments, the human Ig λ light chain sequence is or comprises a human sequence as depicted in the drawings (e.g., see Figures 1-4). In some embodiments, the human Ig λ light chain sequence encodes a complete or partial Ig λ light chain polypeptide, wherein the Ig λ light chain polypeptide is located in an immunoglobulin, particularly an immunoglobulin that is expressed by a human B cell. Also provided are non-human animals, non-human embryos, cells, and targeting constructs for producing non-human animals, non-human embryos, and cells comprising said human Ig λ light chain sequence in place of the corresponding non-human Ig λ light chain sequence (e.g.,

[00202] In some embodiments, a human Ig λ light chain sequence is inserted in place of a corresponding non-human Ig λ light chain sequence into the germline genome of a non-human animal. In some embodiments, the human Ig λ light chain sequence is inserted upstream of the non-human Ig λ light chain sequence (e.g., the non-human Ig λ light chain constant region sequence). In some embodiments, the human Ig λ light chain sequence is inserted among one or more non-human Ig λ light chain sequences such that the human Ig λ light chain sequence is located adjacent to the non-human Ig λ light chain sequences (e.g., see Figures 1, 2,3 and / or 4).

[00203] In some embodiments, one or more sequences (or a portion thereof) of the non-human Ig λ light chain are not removed from the non-human Ig λ light chain locus. In some embodiments, one or more sequences of the non-human Ig λ light chains (e.g., Vλ, Jλ, and / or Cλ) from the non-human Ig λ light chain locus are altered, substituted, disrupted, deleted, or replaced with, among other things, a human Ig λ light chain sequence described herein (e.g., a sequence that comprises one or more human Vλ gene segments, one or more human Jλ gene segments, one or more human Cλ gene segments, or combinations thereof),operably linked to a non-human Ig λ light chain constant region and one or more enhancers and / or regulatory elements of a non-human Ig λ light chain locus. In some embodiments, all or substantially all of the non-human Ig λ light chain locus is replaced with one or more human Ig λ light chain sequences (as described herein) that are operably linked to a non-human Ig λ light chain constant region and one or more enhancers of a non-human Ig λ light chain and / or regulatory elements of a non-human Ig λ light chain locus. In some specific embodiments, the one or more non-human Ig λ light chain constant region genes are not deleted or replaced in the non-human animal that comprises the human Ig light chain X sequence,described herein. As just one non-limiting example, in the case of an insertion of a human Ig λ light chain sequence that is inserted into a non-human Ig λ light chain locus, said insertion is made in a manner that maintains the integrity of the non-human Ig λ light chain sequences near the insertion site (e.g., a non-human Ig λ light chain constant region and / or enhancer region or a non-human Ig λ light chain sequence). Thus, such non-human animals have a wild-type Ig λ light chain constant region. In some embodiments, the non-human Ig λ light chain locus that is altered, substituted, disrupted, deleted, replaced, or engineered with one or more of the human Ig λ light chain sequences described herein is murine (e.g.,mouse or rat) Ig λ light chain locus. In some embodiments, a human Ig λ light chain sequence is inserted into one copy (i.e., an allele) of a non-human Ig λ light chain locus from two copies of said non-human Ig λ light chain locus to produce a non-human animal that is heterozygous for the human Ig λ light chain sequence. In some embodiments, a non-human animal is provided that is homozygous for an Ig λ light chain locus that comprises a human Ig λ light chain sequence described herein.

[00204] In some embodiments, the engineered non-human Ig λ light chain locus described herein comprises segments of human Vλ, Jλ, and Cλ genes,operably linked to a non-human Ig λ light chain constant region and one or more enhancers and / or regulatory elements of a non-human Ig λ light chain. In some embodiments, the engineered non-human Ig λ light chain locus described herein comprises segments of the human Vλ, Jλ, and Cλ genes operably linked to a non-human Ig λ light chain constant region, one or more enhancers and / or regulatory elements of a non-human Ig λ light chain, and one or more enhancers and / or regulatory elements of a human Ig λ light chain.

[00205] In some embodiments, the non-human animal comprises the engineered Ig λ light chain locus described herein that is randomly integrated into its genome (e.g., as part of a randomly integrated human Ig λ light chain sequence). Thus,Such non-human animals may be described as having a human Ig λ light chain transgene comprising a plurality of human Vλ, Jλ, and / or Cλ gene segments configured such that the human Vλ, Jλ, and / or Cλ gene segments are capable of rearranging and encoding a complete or partial Ig λ light chain from an antibody in the expressible repertoire of the non-human animal. The engineered Ig λ light chain locus or transgene described herein can be detected using a variety of methods, including, for example, PCR, Western blotting, Southern blotting, restriction fragment length polymorphism (RFLP), or allele gain or loss analysis. In some embodiments, the non-human animal described herein is heterozygous for the engineered Ig λ light chain locus,described herein. In some embodiments, a non-human animal described herein is hemizygous for an engineered Ig λ light chain locus described herein. In some embodiments, a non-human animal described herein comprises one or more copies of an engineered Ig λ light chain locus or transgene described herein. In some embodiments, a non-human animal described herein comprises an Ig λ light chain locus depicted in the drawings (e.g., see Figures 1, 2, 3, and / or 4).

[00206] In some embodiments, compositions and methods are provided for producing non-human animals whose germline genome comprises an engineered Ig λ light chain locus that comprises one or more human Ig λ light chain sequences (e.g., human Vλ gene segments,Jλ and / or Cλ) instead of sequences of non-human Ig λ light chains, including sequences encoding human Ig X light chains that include specific polymorphic forms of human Vλ, Jλ and / or Cλ segments (e.g., specific V and / or J alleles or variants), including compositions and methods for producing non-human animals that express antibodies comprising Ig λ light chains that comprise human variable domains and human or non-human constant domains assembled from an Ig λ light chain locus that comprises human Vλ, Jλ, and Cλ segments operably linked to a non-human Ig λ light chain constant region. In some embodiments, compositions and methods for producing non-human animals are also provided,which express such antibodies under the control of endogenous enhancer(s) and / or endogenous regulatory sequence(s). In some embodiments, compositions and methods for producing non-human animals that express such antibodies under the control of heterologous enhancer(s) and / or heterologous regulatory sequence(s) are also provided.

[00207] In certain embodiments, the methods described herein comprise inserting a sequence encoding a complete or partial human Ig λ light chain upstream of a non-human Ig λ light chain constant region (e.g., a murine Cλ region) such that an antibody is expressed, wherein the antibody is characterized by having a light chain that comprises at least a human Vλ domain and, in some embodiments, human Vλ and Cλ domains,and is expressed on both the surface of B cells and in the serum of a non-human animal.

[00208] In some embodiments, the methods comprise making a series of insertions of genetic material corresponding to a human Ig λ light chain locus. In some embodiments, the genetic material corresponding to a human Ig λ light chain locus may be synthetic or genomic (e.g., cloned from a bacterial artificial chromosome). In some embodiments, the genetic material corresponding to a human Ig λ light chain locus may be constructed based on published sources and / or bacterial artificial chromosomes such that the genetic material comprises human Vλ, Jλ, and / or Cλ segments in an orientation different from that in which they are found in the human Ig λ light chain locus,however, said genetic material still contains sequences to provide for the rearrangement of said human Vλ, Jλ, and / or Cλ segments to encode a functional Ig λ light chain. As just one example, genetic material corresponding to a human Ig λ light chain locus can be designed using the guidance provided herein to construct a human Ig λ light chain sequence that contains human Vλ, Jλ, and / or Cλ segments in an order and / or grouping that differs from those found in the human Ig λ light chain locus of a human cell. In such an example, the content of human Vλ, Jλ, and / or Cλ segments will be equivalent to the corresponding segments in a human cell, however, the order and grouping will be different. When constructing a human Ig λ light chain locus to create a non-human animal as described herein,The necessary recombination signal sequences can be configured so that the human-specific segments can rearrange correctly and form a functional Ig λ light chain. Guidance for the germline configuration of the human Ig λ light chain segments and sequences required for proper recombination can be found in Molecular Biology of B Cells, London: Elsevier Academic Press, 2004, Ed. Honjo, T., Alt, F.W., Neuberger,M. Chapters 4 (pp. 37-59) and 5 (61-82); incorporated herein by reference in their entireties.

[00209] In some embodiments, making a series of insertions comprises making multiple insertions of portions of heterologous genetic material into a single ES cell clone. In some embodiments, making a series of insertions comprises making sequential insertions of portions of heterologous genetic material into successful ES cell clones.

[00210] In some embodiments, the methods comprise making an insertion of approximately 11,822 bp of DNA downstream of a murine (e.g., mouse or rat) Cλ1 region such that the DNA is operably linked to the murine (e.g., mouse or rat) Cλ1 region,wherein the DNA comprises one or more human Ig λ light chain enhancer regions (or sequences). In some specific embodiments, the methods comprise inserting approximately 11,822 bp of DNA that comprises three human Ig λ light chain enhancer regions (or sequences), wherein said three human Ig λ light chain enhancer regions (or sequences) are inserted downstream of said murine (e.g., mouse or rat) Cλ1 region.

[00211] In some embodiments, the methods comprise inserting approximately 125,473 bp of DNA that comprises three human Ig λ light chain enhancer regions (or sequences), wherein said three human Ig λ light chain enhancer regions (or sequences) are inserted downstream of said murine (e.g., mouse or rat) Cλ1 region. DNA upstream of a murine (e.g., mouse or rat) Cλ1 region such that said DNA is operably linked to said murine (e.g., mouse or rat) Cλ1 region, wherein the DNA comprises 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,Kλ6-Cλ6, and a segment of the human Jλ gene, Jλ7. In some specific embodiments, the methods comprise inserting approximately 11,822 bp of DNA that comprises one or more human Ig λ light chain enhancer regions (or sequences), wherein the one or more human Ig λ light chain enhancer regions (or sequences) are inserted downstream of said murine (e.g., mouse or rat) Cλ1 region.

[00212] In some embodiments, the methods comprise inserting approximately 171,458 bp of DNA that comprises one or more human Ig λ light chain enhancer regions (or sequences), wherein the one or more human Ig λ light chain enhancer regions (or sequences) are inserted downstream of said murine (e.g., mouse or rat) Cλ1 region. DNA upstream of a murine (e.g., mouse or rat) Cλ1 region such that said DNA is operably linked to said murine (e.g., mouse or rat) Cλ1 region, wherein the DNA comprises segments of the human Vλ gene, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ3-19, VL3-21, Vλ3-22, Vλ2-23,Vλ3-25 and Vλ3-27. In some particular embodiments, the methods comprise inserting approximately 171,458 bp of DNA upstream of a human Vλ3-10 gene segment that is operably linked to a murine (e.g., mouse or rat) Cλ1 region, wherein the DNA comprises 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.

[00213] In some embodiments, the methods comprise inserting approximately 121,18 ... DNA upstream of a murine (e.g., mouse or rat) Cλ1 region such that said DNA is operably linked to said murine (e.g., mouse or rat) Cλ1 region, wherein the DNA comprises segments of the human Vλ gene, Vλ3-27, Vλ1-36, Vλ5-31, Vλ5-39, Vλ1-40, Vλ1-43, Vλ1-44, Vλ5-45, Vλ1-46, Vλ1-41, Vλ9-49,Vλ1-51 and Vλ5-52. In some particular embodiments, the methods comprise inserting approximately 121,188 bp of DNA upstream of a human Vλ3-21 gene segment that is operably linked to a murine (e.g., mouse or rat) Cλ1 region, wherein the DNA comprises 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.

[00214] In some ... DNA upstream of a murine (e.g., mouse or rat) Cλ1 region such that said DNA is operably linked to said murine (e.g., mouse or rat) Cλ1 region, wherein the DNA comprises segments of the human Vλ gene, 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 wherein the DNA comprises an arm of homology,which comprises a sequence located 5' relative to the mouse Vλ2 gene segment. In some specific embodiments, the methods comprise inserting approximately 121,188 bp. DNA upstream of a human Vλ3-27 gene segment that is operably linked to a murine (e.g., mouse or rat) Cλ1 region, wherein the DNA comprises 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 wherein the DNA comprises a homology arm that comprises a mouse sequence located 5'-direction relative to the mouse Vλ2 gene segment to drive deletion of the mouse Ig λ chain genomic sequence (e.g., Ig λ light chain locus) after homologous recombination with said fragment DNA.

[00215] Insertions of additional segments of human Vλ, Jλ and / or Cλ genes can be achieved using methodsdescribed herein, to further expand the divergence of the engineered Ig λ light chain locus. For example, in some embodiments, the methods may comprise inserting approximately 300 kb of DNA upstream of a murine (e.g., mouse or rat) Cλ1 region such that said DNA is operably linked to said murine (e.g., mouse or rat) Cλ1 region, wherein the DNA comprises segments of the human Vλ gene, 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 that is operably linked to a murine (e.g., mouse or rat) Cλ1 region, wherein the DNA comprises human Vλ gene segments, Vλ10-54, Vλ6-57, Vλ4-60, Vλ8-61, and Vλ4-69. In some specific embodiments, the DNA comprises a human VpreB gene. The additional human Vλ segments described abovecan be cloned directly from commercially available BAC clones and assembled into a smaller DNA fragment using recombinant techniques described herein or otherwise known in the art. Alternatively, additional human Vλ gene segments described above can be synthesized into a DNA fragment and added to the engineered Ig light X chain locus described above. Similarly, additional human Jλ and / or Cλ gene segments can be obtained from commercially available BAC clones or synthesized directly from published sequences. Also, endogenous Ig light λ chain enhancer regions (or sequences) can be removed from the engineered Ig light λ chain locus described herein. An illustrative image showing the engineered Ig light λ chain locus in non-human animals described herein is shown in any of Figures 1, 2,3 and 4.

[00216] If desired, a human Ig λ light chain sequence (i.e., a sequence comprising segments of the human Vλ, Jλ, and / or Cλ genes) encoding a complete or partial Ig λ light chain can be separately modified to include codons optimized for expression in a non-human animal (e.g., see U.S. Patent Nos. 5,670,356 and 5,874,304). Codon-optimized sequences are synthetic sequences and preferably encode a polypeptide (or a biologically active fragment of a full-length polypeptide having substantially the same activity as the full-length polypeptide) identical to that encoded by the original polynucleotide that is not codon-optimized. In some embodiments, a human Ig λ light chain sequence encoding a complete or partial Ig λ light chain,may separately include an altered sequence to optimize codon usage for a particular cell type (e.g., a rodent cell). For example, the codons of each nucleotide sequence to be inserted into the genome of a non-human animal (e.g., a rodent) may be optimized for expression in a cell of the non-human animal. Such a sequence may be described as a codon-optimized sequence.

[00217] In some embodiments, inserting a nucleotide sequence encoding a complete or partial human Ig λ light chain involves minimal modification of the germline genome of the non-human animal described herein and results in the expression of antibodies comprising light chains that are fully or partially human. Methods for creating engineered non-human animals, including using knockouts and knockins,are known in the art (see, for example, Gene Targeting: A Practical Approach, Joyner, ed., Oxford University Press, Inc. 2000). For example, the creation of transgenic rodents may optionally involve disrupting the genetic loci of one or more endogenous genes (or gene segments) of the rodent and introducing one or more heterologous genes (or gene segments, or nucleotide sequences) into the genome of the rodent, in some embodiments, at the same location as the endogenous gene (or gene segments) of the rodent. In some embodiments, a nucleotide sequence encoding a complete or partial human Ig λ light chain is introduced upstream of a murine (e.g., mouse or rat) Ig λ light chain constant region gene of a randomly inserted Ig λ light chain transgene into the rodent germline genome. In some embodiments, the nucleotide sequence,encoding a complete or partial human Ig λ light chain, is introduced upstream of a murine (e.g., mouse or rat) Ig λ light chain constant region gene of an endogenous Ig λ light chain locus into the germline genome of a rodent; in some specific embodiments, the endogenous Ig λ light chain locus is altered, modified, or engineered to contain segments of human Ig λ chain genes (e.g., Vλ, Jλ, and / or Cλ) operably linked to a rodent Cλ1 region.

[00218] A schematic representation (not to scale) of exemplary engineered Ig λ light chain loci is provided in Figures 1-4. In particular, Figures 1 and 3 show illustrative strategies for constructing engineered Ig λ light chain loci characterized by the insertion of nucleotide sequences containing multiple segments of human Vλ, Jλ, and Cλ. As illustrated in Figure 1, the DNA fragment,containing the human Eλ sequence (or -region), is inserted downstream of the rodent Cλ region by homologous recombination. This DNA fragment contains a cassette for selection for neomycin resistance (e.g., the neomycin resistance gene [NEO, R] flanked by loxP recombination recognition sites) located 3' to the human Eλ sequence that contains three human EX elements constructed downstream (or 3') of the rodent Cλ1 region. Also illustrated in Figure 1 is a DNA fragment containing the first portion of human Vλ segments, a collection of human Jλ-CX segment pairs (e.g., human Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6), and the human Jλ7 segment inserted upstream of the rodent Cλ1 region by homologous recombination. As illustrated, the hygromycin resistance selection cassette (e.g., the hygromycin resistance gene [HYG R] flanked by Frt recombination recognition sites) is located at the 5' end of the targeting vector and upstream of the human Ig λ light chain sequence contained in the targeting vector. The hygromycin resistance selection cassette is removed by homologous recombination with subsequent targeting vectors described in the examples section below. The targeting vector is then electroporated into rodent embryonic stem (ES) cells to generate a rodent whose germline genome contains the engineered Ig λ light chain locus. Once the positivity of the rodent ES cell clone is confirmed, the other depicted targeting vectors are electroporated in a sequential manner and confirmation is performed at each step to complete the construction of the engineered Ig λ light chain locus (see Figure 2).The final targeting vector can be engineered with or without a homology arm (targeting vector 6680) or without (targeting vector 6597) that directs the deletion of endogenous Ig λ light chain segments via homologous recombination, resulting in two potential engineered Ig λ light chain alleles (Figure 2). In addition, any remaining selection cassette can be removed, if desired, via recombinase-mediated deletion. An alternative strategy for implementing the insertion of additional human Vλ gene segments into the engineered Ig λ light chain locus using guide RNAs (gRNAs) is shown in Figure 3.

[00219] Once the human Ig λ light chain sequence is inserted upstream of the non-human Ig λ light chain constant region from the BAC clone, a targeting vector is generated for integration into the Ig λ light chain locus.A BAC clone to be targeted with a human Ig λ light chain sequence to produce a targeting vector may comprise 5'- and / or 3'-flanking genomic DNA of murine (e.g., mouse or rat) origin. Alternatively or additionally, a BAC clone to be targeted with a human Ig λ light chain sequence to produce a targeting vector may comprise 5'- and / or 3'-flanking genomic DNA of human origin such that a region of overlapping human Ig λ light chain sequence is formed. This allows for the successful targeting of multiple engineered BAC clones (e.g., see Figure 1). The final targeting vectors are introduced into the Ig λ 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 introduced into the Ig light chain locus of the germline genome of a non-human cell that further comprises human V. H , D H and J H (for example, containing multiple segments of human V genes H , D H and J H), operably linked to one or more Ig H chain constant region genes, and / or human Vκ and Jκ genomic DNA (e.g., comprising multiple segments of human Vκ and J genes), operably linked to an Ig κ chain constant region gene (e.g., see U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, and 8,791,323, incorporated herein by reference in their entireties).

[00220] The targeting vector is introduced into rodent embryonic stem cells (e.g., a mouse) by electroporation such that the sequence contained in the targeting vector is inserted into the genome of the rodent embryonic stem cells, resulting in the ability of a non-human cell or non-human animal (e.g., a mouse) to express antibodies with complete or incomplete human Ig λ light chains.As described herein, a transgenic rodent is created in which an engineered Ig λ light chain locus (e.g., an endogenous Ig λ light chain locus comprising a human Ig λ light chain sequence operably linked to an endogenous rodent Cλ region described herein) has been engineered into the germline genome of the rodent. Antibodies are expressed on the surface of rodent B cells and in the serum of said rodent, wherein the antibodies are characterized by light chains with human Vλ domains and, in some embodiments, human Vλ and Cλ domains. If the endogenous Ig light λ-chain locus in the germline of the rodent genome is not to be targeted by the targeting vector, then the engineered Ig light λ-chain locus is preferably inserted in a location different from that of the endogenous rodent Ig light λ-chain locus (e.g., a randomly inserted transgene).

[00221] Obtaining an engineered Ig light chain λ locus in a non-human animal as described above provides for the production of an engineered rodent line that produces antibodies that comprise Ig light chains expressed from such an engineered Ig light chain λ locus with a human Vλ domain, and in some embodiments, human Vλ and Cλ domains. By effectively utilizing the presence of an engineered Ig H chain locus that contains multiple segments of human V genes. H , D Hand JH, operably linked to Ig H chain constant region genes, an engineered rodent line has been generated that produces antibodies and antibody components for the development of human antibody therapeutics. Thus, a single engineered rodent line has been practically generated that has the potential to provide an alternative in vivo system for utilizing human Vλ domains to develop novel antibody therapeutics for the treatment of human disease.

[00222] In some embodiments, the genome of the non-human animal described herein further comprises (e.g., through crossbreeding or multiple gene targeting strategies) one or more human immunoglobulin heavy and / or light chain variable regions, as described in U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, and 8,791,323; all of which are incorporated herein by reference in their entireties.Alternatively, the engineered Ig λ light chain locus described herein may be inserted into an embryonic stem cell containing humanized Ig H chain and / or Ig κ chain loci, or a non-human animal containing the engineered Ig λ light chain locus described herein may be crossed with another non-human animal containing humanized Ig H chain and / or Ig κ chain loci. Various such animals containing humanized Ig H chain and / or Ig κ chain loci are known, such as the VELOCIMMUNE® line (see, e.g., U.S. Patent Nos. 8,502,018 and / or 8,642,835; incorporated herein by reference in their entireties), the XENOMOUSE™ line (see, e.g., Mendez, MJ et al., 1997, Nat. Genetics 15(2): 146-56, and Jakobovits, A. et al., 1995, Ann. NY Acad. Sci. 764:525-35). Homozygosity of the engineered Ig λ light chain locus described herein can subsequently be achieved by crossing.Alternatively, in the case of a randomly inserted engineered Ig λ light chain transgene (described above), rodent strains can be selected based on, among other things, the level of expression of human Vλ domains from the transgene.

[00223] Alternatively and / or additionally, in some embodiments, the germline genome of the non-human animal described herein further comprises a deleted, inactivated, functionally suppressed, or otherwise non-functional endogenous Ig κ light chain locus. Genetic modifications to remove or render functional a gene or genetic locus can be achieved using the methods described herein and / or methods known in the art.

[00224] A transgenic non-human founder animal can be identified based on the presence of an engineered Ig light X chain locus in its germline genome and / or the expression of antibodies with a complete or partial human Ig λ light chain sequence in tissues or cells of the non-human animal. The transgenic non-human founder animal can then be used to breed additional non-human animals carrying the engineered Ig λ light chain locus, thereby producing a cohort of non-human animals, each carrying one or more copies of the engineered Ig λ light chain locus. Moreover, transgenic non-human animals carrying the engineered Ig λ light chain locus described herein can be further crossed, if desired, with other transgenic non-human animals carrying other transgenes (e.g., human immunoglobulin genes).

[00225] In some embodiments, transgenic non-human animals can also be generated to contain selected systems that provide controlled, manipulated, inducible, and / or cell type-specific expression of the transgene or integrated sequence(s). For example, the non-human animals described herein can be engineered to contain a sequence encoding a complete or partial human Ig light X chain from an antibody that is expressed under certain conditions (e.g., summarized in Rajewski, K. et al., 1996, J. Clin. Invest. 98(3):600-3). Illustrative systems include the Cre / loxP recombinase system from bacteriophage P1 (see, for example, Lakso, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6) and the FLP / Frt recombinase system from S. cerevisiae (O'Gorman, S. et al, 1991, Science 251:1351-5).Such animals can be produced by creating "doubly" transgenic animals, such as by mating two transgenic animals, one containing a transgene containing a selected modification (e.g., the engineered Ig light chain λ locus described herein) and the other containing a transgene encoding a recombinase (e.g., Cre recombinase).

[00226] The non-human animals described herein can be produced as described above or using methods known in the art so as to contain additional human, humanized, or otherwise engineered genes, often depending on the intended use of the non-human animal.If desired, the genetic material of such human, humanized, or otherwise engineered genes may be introduced by further altering the genome of cells (e.g., embryonic stem cells) having the genetic modifications or alterations described above, or by using crossbreeding techniques known in the art with other genetically modified or engineered lines. In some embodiments, the non-human animals described herein are produced such that they further comprise transgenic human Ig H chain and / or Ig κ 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 No. 8502018; U.S. Patent No. 8642835; U.S. Patent No. 8697940; U.S. Patent No. 8791323 and U.S. Patent Application Publication No. 2013 / 0096287 A1; incorporated herein by reference in their entireties).

[00227] In some embodiments, the non-human animals described herein can be produced by introducing the targeting vector described herein into a cell from a modified cell line. As just one example, the targeting vector described above can be introduced into a VELOCIMMUNE® mouse. VELOCIMMUNE® mice express antibodies that have fully human variable domains and mouse constant domains. In some embodiments, the non-human animals described herein are produced such that they additionally contain human immunoglobulin genes (variable and / or constant region genes).In some embodiments, the non-human animals described herein comprise an engineered Ig λ light chain locus as described herein and genetic material from a heterologous species (e.g., humans), wherein the genetic material encodes complete or partial one or more human Ig heavy chain and / or κ light chain variable domains.

[00228] For example, the non-human animals described herein that comprise an engineered Ig λ light chain locus as described herein can further comprise (e.g., via crossbreeding or multiple gene targeting strategies) one or more modifications described in 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. 8502018, 8642835, 8697940, and 8791323; all of which are incorporated herein by reference in their entireties.In some embodiments, a rodent comprising an engineered Ig λ light chain locus described herein is crossed with a rodent comprising a humanized Ig H chain variable region locus and / or Ig κ light chain variable region locus (see, e.g., U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, and / or 8,791,323; incorporated herein by reference in their entireties). In some embodiments, a rodent comprising an engineered Ig λ light chain locus described herein is crossed with a rodent comprising a humanized Ig H chain variable region locus (see, e.g., U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, and / or 8,791,323; incorporated herein by reference in their entireties) and an inactivated endogenous Ig κ light chain locus (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, incorporated herein by reference in their entireties).

[00229] While embodiments that describe the construction of an engineered Ig λ light chain locus in a mouse (i.e., a mouse with an engineered Ig λ light chain locus characterized by having multiple segments of human Vλ, Jλ, and Cλ genes operably linked to a mouse Cλ region such that antibodies containing complete or partial human Ig λ light chains are expressed) are actively contemplated herein, other non-human animals that contain an engineered Ig λ light chain locus are also contemplated. Such non-human animals include any of those that can be genetically modified to express the antibodies described herein, including, for example, mammals such as a mouse, rat, rabbit, pig, bovine (e.g., cow, ox, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus macaque), etc.For example, for those non-human animals for which suitable genetically modified ES cells are not readily available, other methods are used to produce a non-human animal containing a genetic modification. Such methods include, for example, modifying the genome of cells other than ES cells (e.g., a fibroblast or an induced pluripotent cell) and using somatic cell nuclear transfer (SCNT) to transfer the genetically modified genome into a suitable cell, such as an anucleated oocyte, and gestating the modified cell (e.g., the modified oocyte) in the non-human animal under suitable conditions to form an embryo.

[00230] Methods for modifying the germline genome of a non-human animal (e.g., the genome of a pig, a cow, a rodent, a chicken, etc.)) include, for example, the use of a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a Cas protein (i.e., a CRISPR / Cas system) to incorporate the engineered Ig light chain λ locus described herein. Guidance for methods of modifying the germline genome of a non-human animal 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 hereby incorporated by reference in their entireties.

[00231] In some embodiments, the non-human animal described herein is a mammal. In some embodiments, the non-human animal described herein is a small mammal, such as a member of the superfamily Dipodoidea or Muroidea.In some embodiments, the genetically modified animal described herein is a rodent. In some embodiments, the rodent described herein is selected from a mouse, a rat, and a hamster. In some embodiments, the rodent described herein is selected from the superfamily Muroidea. In some embodiments, the genetically modified animal described herein belongs to a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, dwarf gerbils, spiny mice, woolly hamsters), Nesomyidae (climbing mice, rock mice, white-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats, bamboo rats, and zokors).In certain specific embodiments, the genetically modified rodent described herein is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a woolly hamster. In certain specific embodiments, the genetically modified mouse described herein is a member of the family Muridae. In certain specific embodiments, the non-human animal described herein is a rodent. In certain specific embodiments, the rodent described herein is selected from a mouse and a rat. In certain specific embodiments, the non-human animal described herein is a mouse.

[00232] In some embodiments, the non-human animal described herein is a rodent that is a C57BL mouse 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 specific embodiments, the mouse described herein is a 129 strain selected from the group consisting of 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 specific embodiments, the genetically modified mouse described herein is a cross between the aforementioned 129 strain and the aforementioned C57BL / 6 strain.In some specific embodiments, the mouse described herein is a cross between the aforementioned 129 strains or a cross between the aforementioned BL / 6 strains. In some specific embodiments, the 129 strain in the cross described herein is the 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse described herein is of the BALB strain, such as the BALB / c strain. In some embodiments, the mouse described herein is a cross between the BALB strain and another of the aforementioned strains.

[00233] In some embodiments, the non-human animal described herein is a rat. In some specific embodiments, the rat described herein is selected from the Wistar strain, the LEA strain, the Sprague Dawley strain, the Fischer strain, the F344, the F6, and the Dark Agouti strain.In some specific embodiments, the rat strain described herein is a cross between two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[00234] The pluripotent and / or totipotent rat cell can 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. The pluripotent and / or totipotent rat cells can also be derived from a strain derived from a cross between two or more strains listed above. For example, the pluripotent and / or totipotent rat cell can be derived from the DA strain or the ACI strain. The ACI rat strain is characterized by a black agouti coloration with a white belly and paws and the RT1av1 haplotype. These strains are available from a variety of sources, including Harlan Laboratories.An example of a rat ES cell line from an ACI rat is the ACI.G1 rat ES cell. The Dark Agouti (DA) rat line is characterized by the agouti coloration and the RT1av1 haplotype. Such rats are available from a variety of sources, including Charles River and Harlan Laboratories. Examples of a rat ES cell line from a DA rat 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 line (see, for example, U.S. Patent Application Publication No. 2014-0235933 A1, published August 21, 2014, incorporated herein by reference in its entirety).Specific illustrative embodiments of engineered Ig H chain loci

[00235] In some embodiments, the provided non-human animals comprise an engineered Ig λ light chain locus as described herein and further comprise engineered Ig H chain loci (or alleles) characterized by the presence of multiple segments of human V genes. H , D H and J H , grouped in a germline configuration and operably linked to non-human Ig H chain constant regions, enhancers, and regulatory regions of the human Ig H chain. In some embodiments, the engineered Ig H chain locus (or allele) described herein comprises one or more segments of the human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H, operably linked to a non-human Ig H chain constant region.

[00236] In some embodiments, the engineered Ig H chain 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 gene segments H In certain specific embodiments, the engineered Ig H chain locus (or allele) comprises all or substantially all of the functional segments of the human V gene. H , which are located between human V gene segments H 3-74 and V H 6-1 inclusive, from a human Ig H chain locus that occurs naturally. In some specific embodiments, the engineered Ig H chain locus (or allele) comprises at least segments of the human V gene H , 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 H3-64, 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, W7-4-1, VH4-4, V H 1–3, V H 1-2 and VH6-1.

[00237] In some embodiments, the engineered locus (or allele) of the H-chain Ig comprises 5, 10, 15, 20, 25 or more (e.g., 26, 27, etc.) segments of the human gene D HIn certain specific embodiments, the engineered Ig H chain locus (or allele) comprises all or substantially all of the functional segments of the human D gene H , which are located between human D gene segments H 1-1 and D H 7-27 inclusive, from a human Ig H chain locus that occurs naturally. In some specific embodiments, the engineered Ig H chain locus (or allele) comprises at least segments of the human D gene H , D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26 and D H7-27.

[00238] In some embodiments, the engineered Ig H chain locus (or allele) comprises 1, 2, 3, 4, 5, 6 or more functional segments of the human J gene H In certain specific embodiments, the engineered Ig H chain locus (or allele) comprises all or substantially all of the functional segments of the human J gene H , which are located between human J gene segments H 1 and J H 6 inclusive, from a human Ig H chain locus that occurs naturally. In some specific embodiments, the engineered Ig H chain locus (or allele) comprises at least segments of the human J gene H , J H 1, J H 2, J H 3, J H 4, J H 5 and J H6.

[00239] In some embodiments, the non-human Ig H chain constant region comprises one or more non-human Ig H chain constant region genes, such as, for example, an immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin E (IgE), and immunoglobulin A (IgA) gene. In some specific embodiments, the non-human Ig H chain constant region comprises rodent IgM, rodent IgD, rodent IgG3, rodent IgG1, rodent IgG2b, rodent IgG2a, rodent IgE, and rodent IgA constant region genes. In some embodiments, said human V gene segments H , D H and J H operably linked to one or more enhancers (i.e., enhancer sequences or enhancer regions) of a non-human Ig H chain. In some embodiments, said human V gene segments H , D H and J Hoperably linked to one or more regulatory regions (or regulatory sequences) of a non-human Ig H chain. In some embodiments, said segments of human V genes H , D H and J Hoperably linked to one or more enhancers (or enhancer sequences) of a non-human Ig H chain and to one or more regulatory regions (or regulatory sequences) of a non-human Ig H chain.

[00240] In some embodiments, the engineered Ig H chain locus described herein does not comprise an endogenous Adam6 gene. In some embodiments, the engineered Ig H chain locus described herein does not comprise an endogenous Adam6 gene (or an Adam6 coding sequence) at the same position in the germline genome as it is found in the germline genome of a non-human wild-type animal of the same species. In some embodiments, the engineered Ig H chain locus described herein does not comprise a human Adam6 pseudogene.In some embodiments, the engineered Ig H chain locus described herein comprises an insertion of at least one nucleotide sequence that encodes one or more non-human (e.g., rodent) Adam6 polypeptides. The insertion may be located outside the engineered immunoglobulin heavy chain locus described herein (e.g., upstream of the closest 5' segment of the V gene). H), within an engineered Ig H chain locus or elsewhere in the germline genome of a non-human animal (e.g., a randomly introduced non-human Adam6 coding sequence), a non-human cell or tissue.

[00241] In various embodiments, the provided non-human animal, non-human cell, or non-human tissue described herein does not express, at a detectable level, a complete or partial endogenous non-human V H -region in an antibody molecule. In various embodiments, the provided non-human animal, non-human cell, or non-human tissue described herein does not contain (or is characterized by the absence or deletion of) one or more nucleotide sequences that encode a complete or partial endogenous non-human V H -area (for example, VH , D H and / or J H ) in an antibody molecule. In various embodiments, the provided non-human animal, non-human cell, or non-human tissue described herein is characterized by a germline genome that includes a deletion of segments of the endogenous non-human V genes. H , D H and J H, in whole or in part. In various embodiments, the provided non-human animal is fertile.

[00242] Guidance for producing targeting vectors for non-human cells and non-human animals carrying such engineered Ig H chain loci (or alleles) can be found, for example, in U.S. Patent Nos. 8,642,835 and 8,697,940, which are incorporated by reference in their entireties. Those skilled in the art are aware of a number of technologies known in the art for performing such genetic engineering and / or manipulation of non-human (e.g., mammalian) genomes or for obtaining, providing, or otherwise making such sequences for introduction into the germline genome of non-human animals.Specific Illustrative Embodiments of Engineered Ig κ Light Chain Loci

[00243] In some embodiments, the provided non-human animals comprise an engineered Ig λ light chain locus as described herein and further comprise engineered Ig κ light chain loci (or alleles) characterized by having a plurality of human Vκ and Jκ gene segments arranged in a germline configuration and operably linked to a non-human Ig κ light chain constant region, enhancers, and Ig κ chain regulatory regions. In some embodiments, the engineered Ig κ light chain locus (or allele) comprises one or more human Vκ gene segments and one or more human Jκ gene segments operably linked to a non-human Ig κ chain (Cκ) constant region.

[00244] In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises at least segments of the human Vκ gene that are in the distal variable cluster (or distal arm, or distal duplication) of a naturally occurring human Ig κ light chain locus. In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises at least segments of the human Vκ gene that are in the proximal variable cluster (or proximal arm, or proximal duplication) of a naturally occurring human Ig κ light chain locus. In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises segments of the human Vκ gene that are located in the distal and proximal variable clusters of the naturally occurring human Ig κ light chain locus.In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises all or substantially all functional human Vκ gene segments that are between and including the human Vκ2-40 (or Vκ3D-7) and Vκ4-1 gene segments, from a human Ig κ light chain locus that occurs in nature.

[00245] In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises 5, 10, 15, 20, 25, 30, 35, or more (e.g., 36, 37, 38, 39, 40, etc.) human Vκ gene segments.In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises human Vκ gene segments, Vκ3D-7, Vκ1D-8, VκID-43, Vκ3D-1 1, Vκ1D-12, VκID-13, Vκ3D-15, Vκ1D-16, Vκ1D-17, Vκ3D-20, Vκ6D-21, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, VκID-33, VκID-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 specific embodiments, the engineered Ig κ light chain locus (or allele) comprises at least segments of the human Vκ gene, 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 specific embodiments, the engineered Ig κ light chain locus (or allele) comprises at least segments of the human Vκ gene, 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, Vκ5-2h, Vκ4-1.

[00246] In some embodiments, the engineered Ig κ light chain locus (or allele) comprises 1, 2, 3, 4, 5, or more functional segments of the human Jκ gene. In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises all or substantially all functional segments of the human Jκ gene that are located between and including the human Jκ1 and Jκ5 gene segments, from a naturally occurring human Ig κ light chain locus.In certain specific embodiments, the engineered Ig κ light chain locus (or allele) comprises at least segments of a human Jκ, Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5 gene.

[00247] In some embodiments, the human Vκ and Jκ gene segments are operably linked to one or more enhancers (i.e., enhancer sequences or enhancer regions) of a non-human Ig κ light chain. In some embodiments, the human Vκ and Jκ gene segments are operably linked to one or more regulatory regions (or regulatory sequences) of a non-human Ig κ light chain.In some embodiments, the human Vκ and Jκ gene segments are operably linked to one or more enhancers (or enhancer sequences, or enhancer regions) of a non-human Ig κ light chain and one or more regulatory regions (or regulatory sequences) of a non-human Ig κ light chain.

[00248] In some embodiments, the non-human CK region of the engineered Ig κ light chain locus (or allele) comprises a rodent CK region, such as, for example, a mouse CK region or a rat CK region. In some specific embodiments, the non-human Cκ region of the engineered Ig κ light chain locus (or allele) is or comprises a mouse Cκ region from a genetic background that includes the 129 strain, the BALB / c strain, the C57BL / 6 strain, the 129xC57BL / 6 crossbreed, or combinations thereof.

[00249] In some embodiments, the provided non-human animals comprise the engineered Ig λ light chain locus described herein and further comprise inactivated Ig κ light chain loci (or alleles).

[00250] In various embodiments, the provided non-human animal, non-human cell, or non-human tissue described herein does not express at a detectable level a complete or partial endogenous non-human Vκ region in an antibody molecule. In various embodiments, the provided non-human animal, non-human cell, or non-human tissue described herein does not comprise (or is characterized by the absence or deletion of) one or more nucleotide sequences that encode a complete or partial endogenous non-human Vκ region in an antibody molecule.In various embodiments, the provided non-human animal, non-human cell, or non-human tissue described herein is characterized by a germline genome that includes a deletion of segments of the endogenous non-human Vκ and Jκ genes, in whole or in part.

[00251] Guidance for producing targeting vectors for non-human cells and non-human animals carrying such engineered Ig κ light chain loci (or alleles) can be found, for example, in U.S. Patent Nos. 8,642,835 and 8,697,940, which are hereby incorporated by reference in their entireties.Those skilled in the art are aware of a number of technologies known in the art for performing such genetic engineering and / or manipulation of non-human (e.g., mammalian) genomes or for obtaining, providing, or otherwise making such sequences for introduction into the germline genome of non-human animals. Specific illustrative embodiments - engineered Ig λ light chain loci

[00252] In some embodiments, the provided non-human animals comprise an engineered Ig λ light chain locus characterized by having a plurality of 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 Ig λ light chain locus further comprises one or more enhancer regions (or enhancer sequences) of a human Ig λ light chain. In some embodiments, the engineered Ig λ light chain locus comprises one or more human Vλ gene segments and one or more human Jλ gene segments operably linked to a non-human Ig λ light chain constant region (Cλ). In some specific embodiments, the engineered Ig λ light chain locus (or allele) comprises human Vλ gene segments that are in at least cluster A of the human Ig λ light chain locus; in some embodiments, cluster A and cluster B of the human Ig λ light chain locus; in some specific embodiments, cluster A, cluster B, and cluster C of the human Ig λ light chain locus.

[00253] In some embodiments, the engineered Ig λ 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 specific embodiments, the engineered Ig λ light chain locus (or allele) comprises all or substantially all functional human Vλ gene segments that are between and including human gene segments Vλ4-69 and Vλ3-1, from a naturally occurring human Ig λ light chain locus. In some specific embodiments, the engineered Ig λ light chain locus (or allele) comprises all or substantially all functional human Vλ gene segments that are between and including the human Vλ5-52 and Vλ3-1 gene segments, from a naturally occurring human Ig λ light chain locus.In some specific embodiments, the engineered Ig λ light chain locus (or allele) comprises all or substantially all functional human Vλ gene segments that are between and including the human Vλ3-27 and Vλ3-1 gene segments, from a naturally occurring human Ig λ light chain locus. In some specific embodiments, the engineered Ig λ light chain locus (or allele) comprises 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, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some specific embodiments, the engineered Ig λ light chain locus (or allele) comprises at least functional segments of the human Vλ gene from Vλ5-52 to Vλ1-40 and from Vλ3-27 to Vλ3-1.

[00254] In some embodiments, the engineered Ig λ light chain locus (or allele) comprises 1, 2, 3, 4, 5, 6, 7, or more functional segments of a human Jλ gene. In some specific embodiments, the engineered Ig λ light chain locus (or allele) comprises all or substantially all functional segments of a human Jλ gene that are between and including the human Jλ1 and Jλ7 gene segments, from a naturally occurring human Ig λ light chain locus. In some specific embodiments, the engineered Ig λ light chain locus (or allele) comprises at least the human Jλ, Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7 gene segments.

[00255] In some embodiments, the engineered Ig λ light chain locus (or allele) comprises 1, 2, 3, 4, 5, 6, 7 or more functional segments of the human Cλ gene.In some specific embodiments, the engineered Ig λ light chain locus (or allele) comprises all or substantially all functional segments of the human Cλ gene that are between and including the human Cλ1 and Cλ7 gene segments from a naturally occurring human Ig λ light chain locus. In some specific embodiments, the engineered Ig λ light chain locus (or allele) comprises at least the human Cλ, Cλ1, Cλ2, Cλ3, and Cλ6 gene segments.

[00256] In some embodiments, the engineered Ig λ light chain locus (or allele) does not comprise the same enhancer regions (or enhancer sequences) of a non-human Ig λ light chain as are found in a wild-type Ig λ light chain locus (or allele).In some embodiments, the engineered Ig λ light chain locus (or allele) completely or partially lacks at least one enhancer region (or enhancer sequence) of a non-human Ig λ light chain (e.g., Ig λ enhancer 2-4 or Eλ2-4).

[00257] In some embodiments, the human Vλ and Jλ gene segments are operably linked to one or more enhancers (i.e., enhancer sequences or enhancer regions) of a non-human Ig λ light chain and one or more enhancers (i.e., enhancer sequences or enhancer regions) of a human Ig λ light chain. In some embodiments, said human Vλ and Jλ gene segments are operably linked to one or more regulatory regions (or regulatory sequences) of a non-human Ig λ light chain.In some embodiments, the human Vλ and Jλ gene segments are operably linked to one or more enhancers (or enhancer sequences, or enhancer regions) of a non-human Ig λ light chain, one or more enhancers (i.e., enhancer sequences or enhancer regions) of a human Ig λ light chain, and one or more regulatory regions (or regulatory sequences) of a non-human Ig λ light chain.

[00258] In some embodiments, the engineered Ig λ light chain locus (or allele) described herein does not comprise a human VpreB gene (or a sequence encoding a human VpreB gene).

[00259] In some embodiments, the non-human Cλ region of the engineered Ig light chain locus (or allele) comprises a rodent Cλ region, such as, for example, a mouse Cλ region or a rat Cλ region.In some specific embodiments, the non-human Cλ region of the engineered Ig light chain locus (or allele) is or comprises a mouse Cλ region from a genetic background that includes the 129 strain, the BALB / c strain, the C57BL / 6 strain, the 129xC57BL / 6 crossbreed, or combinations thereof.

[00260] In some embodiments, the non-human Cλ region of the engineered Ig λ 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 certain specific embodiments, the non-human Cλ region of the engineered Ig λ light chain locus (or allele) described herein comprises a sequence that is substantially identical to or identical to SEQ ID NO: 1 (mouse Cλ1), SEQ ID NO: 3 (mouse Cλ2), or SEQ ID NO: 5 (mouse Cλ3). In certain specific embodiments, the non-human Cλ region of the engineered Ig λ light chain locus (or allele) described herein is or comprises a mouse Cλ1 region sequence.

[00261] In some embodiments, the non-human Cλ region that is encoded by a sequence located within the engineered Ig λ 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 specific embodiments, the non-human Cλ region that is encoded by a sequence located within the engineered Ig light chain λ 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 certain specific embodiments, the non-human Cλ region that is encoded by a sequence located within the engineered Ig light chain λ locus (or allele) described herein is or comprises a mouse Cλ1 region polypeptide.

[00262] In some embodiments, the non-human Cλ region of the engineered Ig λ 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: 7 (rat Cλ1), SEQ ID NO: 9 (rat Cλ2), SEQ ID NO: 11 (rat Cλ3), or SEQ ID NO: 13 (rat Cλ4).In some specific embodiments, the non-human Cλ region of the engineered Ig λ light chain locus (or allele) described herein comprises a sequence that is substantially identical to 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 specific embodiments, the non-human Cλ region of the engineered Ig λ light chain locus (or allele) described herein is or comprises the sequence of a rat Cλ1 region.

[00263] In some embodiments, the non-human Cλ region that is encoded by a sequence located within the engineered Ig λ 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 specific embodiments, the non-human Cλ region encoded by a sequence located within the engineered Ig λ light chain locus (or allele) described herein comprises a sequence that is substantially identical to 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 specific embodiments, the non-human Cλ region encoded by a sequence located within the engineered Ig λ light chain locus (or allele) described herein is or comprises a rat Cλ1 region polypeptide.

[00264] In some embodiments, the engineered Ig λ light chain locus (or allele) described herein is characterized by having one or more unique nucleotide sequence junctions (or combinations of unique sequence junctions) resulting from the insertion of human genetic material corresponding to a (genomic or synthetic) human Ig λ light chain sequence in place of or within a non-human Ig λ light chain sequence at an endogenous locus. Exemplary nucleotide sequence compounds are provided under 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.

[00265] In some embodiments, the 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.

[00266] In some embodiments, the engineered Ig λ light chain locus (or allele) described herein comprises SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123.

[00267] In some embodiments, the engineered Ig λ light chain locus (or allele) described herein comprises 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.

[00268] In some embodiments, the engineered Ig λ light chain locus (or allele) described herein comprises 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.

[00269] In some embodiments, the engineered Ig λ light chain locus (or allele) described herein comprises 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.

[00270] In some embodiments, the engineered Ig λ light chain locus (or allele) described herein comprises 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.

[00271] Methodology regarding human Vλ, Jλ, and Cλ gene segments can be found, for example, in Lefranc, M.P., 2000, Nomenclature of the human immunoglobulin lambda (IGL) genes, Current Protocols in Immunology, No. Supplement, 40:A.1p.1-A.1p.37.Among other things, the present invention demonstrates that the presence of human Vλ and Jλ gene segments in the Ig λ light chain loci (or alleles) provides for an increase in the diversity of the light chain repertoire in the provided non-human animal, compared to the diversity of light chains in the expressed antibody repertoire of a non-human animal that does not contain such engineered Ig λ light chain alleles. Methods

[00272] In certain aspects, the non-human animals described herein can be used to produce a human antibody and / or nucleic acid sequences encoding human antibodies, wherein the human antibody comprises variable domains derived from nucleic acid sequences encoded by the genetic material of a cell of a non-human animal described herein.For example, a non-human animal described herein is immunized with an antigen of interest under conditions and for a time sufficient to cause the non-human animal to mount an immune response to said antigen of interest. Antibodies are isolated from the non-human animal (or one or more cells, such as one or more B cells) so immunized and characterized using various assays, such as affinity and specificity measurements, epitope mapping, ligand-receptor blocking, and receptor activation inhibition.In various embodiments, the antibodies produced by the non-human animals described herein comprise one or more human variable domains that are derived from one or more human variable region nucleotide sequences isolated from a non-human animal. In some embodiments, the non-human animals described herein can be induced to produce antibodies to drugs (e.g., an anti-idiotypic antibody).

[00273] In some embodiments, the non-human animals described herein provide an improved in vivo system and source of biological materials (e.g., cells) for the production of human antibodies that are useful in a variety of 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 or secreted polypeptide) and / or modulate one or more activities associated with said polypeptide of interest and / or modulate interactions of said polypeptide of interest with other binding partners (e.g., a ligand or a receptor polypeptide). 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 the activity of a receptor polypeptide, and / or modulate interactions of a receptor polypeptide 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, siRNA, etc.) that bind 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, siRNA, etc.) that block the activity of one or more polypeptides of interest or that block 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 polypeptides of interest.In some embodiments, the non-human animals described herein are used to determine the epitope or epitopes of one or more candidate therapeutic antibodies that bind one or more polypeptides of interest.

[00274] In various embodiments, the non-human animals described herein are used to determine the pharmacokinetic profiles of one or more human antibody candidates. In various embodiments, each of the one or more non-human animals described herein and one or more control or reference non-human animals is exposed to one or more human antibody candidates at various doses (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).The candidate therapeutic antibodies can be administered in doses via any desired route of administration, including parenteral and non-parenteral routes. Parenteral routes include, for example, intravenous, intra-arterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intracerebroventricular, intracranial, intrapleural and other injection routes. Non-parenteral routes include, for example, oral, intranasal, transdermal, intrapulmonary, rectal, buccal, vaginal, intraocular. Administration can also be accomplished by continuous infusion, topical administration, sustained release from implants (gels, membranes, etc.) and / or intravenous injection. From non-human animals (humanized and control), blood is collected at various time points (e.g., 0 h, 6 h)., 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 can be performed to determine the pharmacokinetic profiles of the administered candidate therapeutic antibodies using samples obtained from the non-human animals described herein, including, but not limited to, assays for total IgG levels, humoral response to therapeutic agents, agglutination, etc.

[00275] In various embodiments, the non-human animals described herein are used to measure the therapeutic effect of blocking or modulating the activity of a polypeptide of interest and the effect on gene expression as a result of cellular changes or, in the case of a receptor polypeptide, the number of receptor polypeptide molecules on the surface of cells of non-human animals.In various embodiments, a non-human animal described herein, or cells isolated therefrom, is exposed to a candidate therapeutic agent that binds a polypeptide of interest and, after an additional period of time, is analyzed for effects on specific cellular processes that are associated with said polypeptide of interest, such as ligand-receptor interactions or signal transduction.

[00276] In certain aspects, the non-human animals described herein express human variable domains of antibodies, thus providing cells, cell lines, and cell cultures that serve as a source of human variable domains of antibodies for use in binding assays and functional assays, such as for assaying antagonist or agonist binding or function, particularly where the antagonist or agonist is specific for a human antigen of interest or specific for an epitope that functions in ligand-receptor interaction (binding). In various embodiments, the epitopes to which the candidate therapeutic antibodies or siRNAs bind can be determined using cells isolated from the non-human animals described herein.

[00277] Cells from the provided non-human animals can be isolated and used as needed, or they can be maintained in culture for many generations. In various embodiments, cells from the provided non-human animal are immortalized (e.g., using a virus) and maintained in culture for an indefinite period of time (e.g., in serial cultures).

[00278] In some embodiments, the non-human animals described herein provide an in vivo system for producing variants of human variable domains of antibodies that bind a polypeptide of interest (e.g., variants of human Vλ domains). Such variants provide human variable domains of antibodies with the desired functionality, specificity, and low cross-reactivity with a common epitope shared by two or more variants of the polypeptide of interest.In some embodiments, the non-human animals described herein are used to produce panels of human antibody variable domains comprising series of variant variable domains that are screened for desired or improved functionality.

[00279] In certain aspects, the non-human animals described herein provide an in vivo system for producing libraries of human antibody variable regions (e.g., a library of human Vλ domains). Such libraries provide a source of heavy and / or light chain variable region sequences that can be grafted onto various Fc regions based on the desired effector function and can be used as a source for "affinity maturation" of the variable region sequence using techniques known in the art (e.g., site-directed mutagenesis, error-prone PCR, etc.).), and / or can be used as a source of antibody components for the production of antibody-based therapeutic molecules, such as, for example, chimeric antigen receptors (i.e., a molecule engineered using antibody components, such as scFv), polyspecific binders (e.g., bispecific binders), and fusion proteins (e.g., single-domain antibodies, scFv, etc.).

[00280] In some aspects, the non-human animals described herein provide an in vivo system for assaying and testing a drug or vaccine.In various embodiments, the candidate drug or vaccine may be delivered to one or more non-human animals described herein, followed by observation of the non-human animals to determine one or more immune responses to the drug or vaccine, the safety profile of the drug or vaccine, or the effect on the disease or condition and / or one or more symptoms of the disease or condition. Illustrative methods used to determine the safety profile include measurements of toxicity, optimal dose concentration, antibody response (i.e., antibody to the drug), drug or vaccine efficacy, and potential risk factors. Such drugs or vaccines may be improved and / or developed using such non-human animals.

[00281] Vaccine efficacy may be determined using a number of methods.Briefly, the non-human animals described herein are vaccinated using methods known in the art and then challenged with the vaccine, or the vaccine is administered to an already infected non-human animal. The response of the non-human animal(s) to the vaccine can be measured by monitoring the non-human animal(s) (or cells isolated from them) and / or conducting one or more assays on them to determine the efficacy of the vaccine. The response of the non-human animal(s) to the vaccine is then compared with the response of control animals using one or more measures known in the art and / or described herein.

[00282] Vaccine efficacy can be further determined using virus neutralization assays. Briefly, the non-human animals described herein are immunized and blood serum is collected on various days after immunization.Serial dilutions of blood serum are pre-incubated with the virus, during which time antibodies in the serum specific for the virus bind to it. The virus / serum mixture is then added to permissive cells to determine infectivity using a plaque assay or a microneutralization assay. If the antibodies in the serum neutralize the virus, a lower number of plaques or a lower number of relative luciferase units is observed compared to a control group.

[00283] In some embodiments, the non-human animals described herein produce human variable domains of antibodies and, therefore, provide an in vivo system for the production of human antibodies intended for use in diagnostic applications (e.g., immunology, serology, microbiology, cellular pathology, etc.).In various embodiments, the non-human animals described herein can be used to produce human variable domains of antibodies that bind corresponding antigenic sites to identify cellular changes, such as, for example, the expression of specific cell surface markers indicative of pathological changes. Such antibodies can be conjugated to various chemical moieties (e.g., a radioactive label) and used, as needed, in various in vivo and / or in vitro assays.

[00284] In some embodiments, the non-human animals described herein provide an improved in vivo system for the development and selection of human antibodies intended for use in oncological and / or infectious diseases.In various embodiments, non-human animals described herein and control non-human animals (e.g., characterized by a genetic modification that differs from that described herein, or without a genetic modification, i.e., wild-type) can be implanted with a tumor (or tumor cells) or infected with a virus (e.g., influenza virus, HIV, HCV, HPV, etc.). Following implantation or infection, the non-human animals can be administered a candidate therapeutic agent. Prior to administration of the candidate therapeutic agent, the tumor or virus can be allowed a sufficient amount of time to develop in one or more locations within the body of the non-human animal.Alternatively and / or additionally, the immune response in such non-human animals can be monitored to characterize and select potential human antibodies that can be developed as a therapeutic agent. Kits

[00285] In some aspects, the present invention further provides a package or kit comprising one or more containers filled with at least one non-human animal, non-human cell, DNA fragment, targeting vector, or any combination thereof described herein. The kits can be used according to any applicable method (e.g., a research method).Optionally associated with such container(s) may be a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, the notice reflecting (a) approval by the agency regulating the manufacture, use, or sale for administration to humans, (b) directions for use, and / or (c) an agreement that governs the transfer of materials and / or biological products (e.g., a non-human animal or a non-human cell described herein) between two or more entities, and combinations thereof.

[00286] Other features of certain embodiments will become apparent from reading the following descriptions of exemplary embodiments, which are provided for illustrative purposes and are not intended to be limiting.Additional Exemplary Embodiments

[00287] In exemplary embodiment 1, provided herein is a rodent 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) and a rodent Cλ gene segment, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers.

[00288] 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.

[00289] In exemplary embodiment 3, provided herein is a rodent according to embodiment 2, wherein the two rodent Eλs are mouse Eλs and mouse Eλ3-1.

[00290] 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 Eλs.

[00291] In exemplary embodiment 5, provided herein is a rodent according to any one of embodiments 1-4, wherein the germline genome further comprises (i) an endogenous immunoglobulin heavy chain locus comprising an insertion of one or more human V gene segments. H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J Hfunctionally linked to a rodent immunoglobulin heavy chain constant region; or (ii) an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J H operably linked to a constant region of a rodent immunoglobulin heavy chain, 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 Cκ region of a rodent immunoglobulin.

[00292] In exemplary embodiment 6, provided herein is a rodent according to embodiment 5, wherein the insertion of one or more human Vκ gene segments H, one or more segments of the human D gene H and one or more segments of the human J gene H provides replacement of V gene segments H , D H rodent.

[00293] In exemplary embodiment 7, provided herein is a rodent according to embodiment 6, wherein the insert comprises non-coding human DNA that occurs naturally between segments of human V genes. H , D H and J Hand combinations thereof.

[00294] 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 provides for replacement of the rodent Vκ and Jκ gene segments.

[00295] In exemplary embodiment 9, provided herein is a rodent according to embodiment 8, wherein the insert comprises non-coding human DNA that occurs naturally between the human Vκ and Jκ gene segments and combinations thereof.

[00296] In exemplary embodiment 10, provided herein is a rodent according to any of embodiments 5-8, wherein the rodent immunoglobulin heavy chain constant region is an endogenous rodent immunoglobulin heavy chain constant region.

[00297] 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.

[00298] In exemplary embodiment 12, provided herein is a rodent according to any one of embodiments 1-9, wherein the endogenous immunoglobulin λ light chain locus comprises a deletion of the endogenous Vλ and Jλ gene segments, in whole or in part.

[00299] In exemplary embodiment 13, provided herein is a rodent according to 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.

[00300] In exemplary embodiment 14, provided herein is a rodent according to 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.

[00301] 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.

[00302] 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.

[00303] In exemplary embodiment 17, provided herein is a rodent according to any one of embodiments 1-16, wherein the rodent does not express detectable levels of endogenous immunoglobulin λ light chains.

[00304] In exemplary embodiment 18, provided herein is a rodent according to any one of embodiments 5-17, wherein the immunoglobulin heavy chain locus comprises an insertion of human V gene segments. H from V H 3-74 to V H 6-1, segments of the human D gene H from D H 1-1 to D H 7-27 and segments of the human J gene H , J H IJ H 6.

[00305] In exemplary embodiment 19, provided herein is a rodent according to embodiment 18, wherein the insert comprises non-coding human DNA that occurs naturally between human V H 3-74 and V H 6-1, human noncoding DNA that occurs naturally between human D H 1-1 and D H 7-27, and human noncoding DNA, which occurs naturally between human J H I and J H6.

[00306] In an exemplary embodiment 20, provided herein is a rodent according to any one of embodiments 5-19, wherein the immunoglobulin κ light chain locus comprises an insertion of a proximal duplication of Vκ, in whole or in part, of a human immunoglobulin κ light chain locus.

[00307] In an exemplary embodiment 21, provided herein is a rodent according to embodiment 20, wherein the immunoglobulin κ light chain locus comprises an insertion of the human Vκ gene segments Vκ2-40 through Vκ4-1 and the human Jκ gene segments JκI through Jκ5.

[00308] In exemplary embodiment 22, provided herein is a rodent according to embodiment 21, wherein the insert comprises human non-coding DNA that occurs naturally between human Vκ2-40 and Vκ4-1, and human non-coding DNA that occurs naturally between human JκI and Jκ5.

[00309] In an exemplary embodiment 23, provided herein is a rodent according to any one of embodiments 1-22, wherein the endogenous immunoglobulin λ light chain locus comprises an insertion of human Vλ gene segments from Vλ5-52 to Vλ1-40 and from Vλ3-27 to Vλ3-1, at least pairs of human Jλ-Cλ, Jλ1-Cλ1, Jλ2-CX2, Jλ3-Cλ3, Jλ6-Cλ6 gene segments, a human Jλ gene segment, Jλ7, and a rodent Cλ1 gene segment.

[00310] In an exemplary embodiment 24, provided herein is a rodent according to embodiments 23, wherein the insert comprises human non-coding DNA that occurs naturally between human Vλ5-52 and Vλ1-40 and Vλ3-27 and Vλ3-1, human non-coding DNA that occurs naturally between pairs of human gene segments Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3 and Jλ6-Cλ6, and human non-coding DNA that occurs naturally upstream (or 5'-direction) of a human gene segment Jλ, Jλ7.

[00311] 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.

[00312] In exemplary embodiment 26, provided herein is a rodent according to any one of embodiments 5-25, wherein the immunoglobulin heavy chain locus further comprises an insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides.

[00313] In exemplary embodiment 27, provided herein is a rodent according to embodiment 26, wherein the one or more nucleotide sequences are inserted between the first and second segments of the human V gene. H.

[00314] In exemplary embodiment 28, provided herein is a rodent according to embodiment 26, wherein one or more nucleotide sequences are inserted in place of the human Adam6 pseudogene.

[00315] In exemplary embodiment 29, provided herein is a rodent according to embodiment 27, wherein the first segment of the human V gene H represents the human V H 1-2, and the second segment of the human V gene H represents the human V H 6- 1.

[00316] In exemplary embodiment 30, provided herein is a rodent according to embodiment 26, wherein one or more nucleotide sequences are inserted between a segment of the human V gene H and a segment of the human D gene H.

[00317] 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.

[00318] 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.

[00319] 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 λ light chain locus.

[00320] In an illustrative embodiment 34, provided herein is a rodent according to any one of embodiments 1-33,wherein the rodent is a rat or a mouse.

[00321] In an exemplary embodiment 35, provided herein is an isolated rodent cell, the germline genome of which 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 to a rodent Cλ gene segment, and (ii) wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers.

[00322] In an illustrative embodiment 36, an immortalized cell is provided herein,

[00323] In exemplary embodiment 37, provided herein is an isolated rodent cell of embodiment 35, wherein the rodent cell is a rodent embryonic stem cell.

[00324] In exemplary embodiment 38, provided herein is a rodent embryo produced using the rodent embryonic stem cell of embodiment 35.

[00325] In exemplary embodiment 39, provided herein is a method of 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 that comprises (i) one or more segments of a human Vλ gene,(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 λ light chain (Eλ) immunoglobulin enhancers; (b) obtaining a rodent embryonic stem cell generated from (a); and (c) obtaining a rodent using a rodent embryonic stem cell of (b).

[00326] In exemplary embodiment 40, provided herein is the method of embodiment 39, wherein the nucleotide sequence further comprises one or more human λ light chain (Eλ) immunoglobulin enhancers.

[00327] In an exemplary embodiment 41, this document provides a method for 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 segments of a human Vλ gene, one or more segments of a human Jλ gene, and one or more segments of a human Cλ gene, wherein the segments of the human Vλ and Jλ genes are operably linked to a segment of a rodent or human Cλ gene, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more enhancers of a rodent immunoglobulin λ light chain (Eλ) and one or more enhancers of a human immunoglobulin λ light chain (Eλ); wherein the method comprises modifying the germline genome of a rodent such that it contains an engineered immunoglobulin λ light chain locus that contains an insertion of one or more segments of a human Vλ gene, one or more segments of a human Jλ gene, and one or more segments of a human Cλ gene,wherein the human Vλ and Jλ gene segments are operably linked to a rodent or human Cλ gene segment, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers, thereby producing said rodent.

[00328] In an exemplary embodiment 42, provided herein is a method according to 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.

[00329] In exemplary embodiment 43, the present document provides a method according to embodiment 42, wherein one or more segments of the human Vλ gene comprise non-coding human DNA,which occurs naturally between human Vλ5-52 and Vλ1-40 and / or Vλ3-27 and Vλ3-1.

[00330] In an exemplary embodiment 44, provided herein is a method according to any one of embodiments 39 43, wherein the one or more human Jλ gene segments and the one or more human Cλ gene segments comprise pairs of human gene segments Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6, and a human gene segment Jλ7.

[00331] In an exemplary embodiment 45, provided herein is the method of embodiment 44, wherein the pairs of human gene segments Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6 comprise human non-coding DNA that occurs naturally between the pairs of human gene segments Jλ and Cλ, and the human gene segment Jλ7 comprises human non-coding DNA,which occurs naturally upstream (or 5') of human Jλ7.

[00332] 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.

[00333] In exemplary embodiment 47, provided herein is a method according to any one of embodiments 39-46, wherein the endogenous immunoglobulin λ light chain locus comprises three human Eλ.

[00334] In exemplary embodiment 48, provided herein is a method according to any one of embodiments 39-46, wherein the endogenous immunoglobulin λ light chain locus comprises two rodent Eλ.

[00335] In exemplary embodiment 49, provided herein is a method according to embodiment 48,wherein the two rodent Eλ are mouse Eλ and mouse Eλ3-1.

[00336] In an 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 selection markers.

[00337] In an 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.

[00338] In an exemplary embodiment 52, provided herein is a method according to any one of embodiments 39 and 42-51, wherein the DNA fragment of (a) is introduced into a rodent embryonic stem cell whose germline genome comprises an endogenous immunoglobulin heavy chain locus comprising an insertion of one or more human V gene segments, H , one or more segments of the human D gene Hand one or more segments of the human J gene H , while segments of human V genes H , D H and J H functionally linked to a rodent immunoglobulin heavy chain constant region; or an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J Hoperably linked to a constant region of a rodent immunoglobulin heavy chain, 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.

[00339] In an exemplary embodiment 53, provided herein is a method according to any one of embodiments 39 and 42-51, wherein the DNA fragment of (a) is introduced into a rodent embryonic stem cell whose germline genome comprises an endogenous wild-type immunoglobulin heavy chain locus or an endogenous wild-type immunoglobulin heavy chain locus and an endogenous wild-type immunoglobulin κ light chain locus; and wherein the method further comprises the step of crossing the mouse obtained using said non-human embryonic stem cell with a second mouse.

[00340] In exemplary embodiment 54, provided herein is a method according to any one of embodiments 47-49, wherein the modification of the rodent germline genome to comprise an engineered immunoglobulin λ light chain locus is performed in a rodent embryonic stem cell, the germline genome of which further comprises an endogenous immunoglobulin heavy chain locus comprising an insertion of one or more human V gene segments. H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J H functionally linked to a rodent immunoglobulin heavy chain constant region; or an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H , one or more segments of the human D gene Hand one or more segments of the human J gene H , while segments of human V genes H , D H and J H 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.

[00341] In exemplary embodiment 55, provided herein is a method according to embodiment 52 or 54, wherein the insertion of one or more human Vκ gene segments H , one or more segments of the human D gene H and one or more segments of the human J gene H contains non-coding human DNA that occurs naturally between one or more segments of the human V gene H, human noncoding DNA that occurs naturally between one or more segments of the human D gene H , and human noncoding DNA that occurs naturally between one or more segments of the human J gene H.

[00342] 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 non-coding human DNA that occurs naturally between the one or more human Vκ gene segments and non-coding human DNA that occurs naturally between the one or more human Jκ gene segments.

[00343] In exemplary embodiment 57, provided herein is a method according to any of embodiments 41-49, wherein the modification of the germline genome of a non-human animal to comprise an engineered immunoglobulin λ light chain locus is performed in a non-human embryonic stem cell,the germline genome of which comprises an endogenous wild-type immunoglobulin heavy chain locus or an endogenous wild-type immunoglobulin heavy chain locus and an endogenous wild-type immunoglobulin κ light chain locus; and wherein the method further comprises the step of crossing the mouse produced by said non-human embryonic stem cell with a second mouse.

[00344] In exemplary embodiment 58, provided herein is the method of embodiment 53 or 57, wherein the second mouse is characterized by a germline genome comprising wild-type Ig H chain and Ig κ chain loci.

[00345] In exemplary embodiment 59, provided herein is the method of embodiment 53 or 57, wherein the second mouse has a germline genome comprising homozygous or heterozygous humanized Ig H chain and Ig κ chain loci,wherein the homozygous or heterozygous humanized Ig H chain locus comprises an inserted sequence encoding rodent Adam6.

[00346] In an exemplary embodiment 60, provided herein is a method according to embodiment 53 or 57, wherein the second mouse is characterized by a germline genome comprising a homozygous or heterozygous humanized Ig H chain locus and a homozygous or heterozygous inactivated Ig κ chain locus.

[00347] In an exemplary embodiment 61, provided herein is a method for producing an antibody in a rodent, the method comprising the steps of (1) immunizing the rodent with an antigen of interest, wherein the rodent is characterized by a germline genome comprising an endogenous immunoglobulin λ light chain locus that comprises (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 operably linked to (c) and to a rodent Cλ gene segment, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers; (2) maintaining the rodent under conditions sufficient for the rodent to develop an immune response to an antigen of interest; and (3) isolating an antibody from the rodent or a rodent cell that binds the antigen of interest.

[00348] In exemplary embodiment 62, provided herein is the method of embodiment 61, wherein the rodent is characterized by a germline genome that further comprises an endogenous immunoglobulin heavy chain locus comprising an insertion of one or more human V gene segments, H , one or more segments of the human D gene H and one or more segments of the human J gene H, while segments of human V genes H , D H and J H functionally linked to a rodent immunoglobulin heavy chain constant region; or an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J Hoperably linked to a constant region of a rodent immunoglobulin heavy chain, 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.

[00349] In exemplary embodiment 63, provided herein is a method of embodiment 61 or 62, wherein the rodent cell is a B cell.

[00350] In exemplary embodiment 64, provided herein is a method of embodiment 61 or 62, wherein the rodent cell is a hybridoma.

[00351] In an exemplary embodiment 65, provided herein is a method according to any one of embodiments 61-64, wherein the endogenous immunoglobulin λ light chain locus comprises an insertion of human Vλ gene segments from Vλ5-52 to Vλ1-40 and from Vλ3-27 to Vλ3-1, human Jλ-Cλ gene segment pairs, Jλ-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6, human Jλ gene segment, Jλ7.

[00352] In an exemplary embodiment 66, provided herein is a method according to embodiment 65, wherein the insert comprises human non-coding DNA that occurs naturally between human Vλ from Vλ5-52 to Vλ1-40 and from Vλ3-27 to Vλ3-1, human non-coding DNA that occurs naturally between the pairs of human Jλ-Cλ gene segments Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3 and Jλ6-Cλ6, and human non-coding DNA that occurs naturally upstream (or 5'-direction) of the human Jλ gene segment, Jλ7.

[00353] 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.

[00354] In exemplary embodiment 68, provided herein is a method according to any one of embodiments 62-67, wherein the immunoglobulin heavy chain locus comprises an insertion of human V gene segments. H from V H 3-74 to V H 6-1, segments of the human D gene H from D H 1-1 to D H 7-27 and segments of the human J gene H , J H 1-J H 6, and at the same time segments of human V genes H , D H and J Hoperably linked to an endogenous constant region of a rodent immunoglobulin heavy chain.

[00355] In an exemplary embodiment 69, provided herein is a method according to embodiment 68, wherein the insert comprises non-coding human DNA that occurs naturally between human V H 3-74 and V H 6-1, human noncoding DNA that occurs naturally between human D H 1-1 and D H 7-27, and human noncoding DNA, which occurs naturally between human J H 1 and J H 6.

[00356] In exemplary embodiment 70, provided herein is a method according to embodiment 68, wherein the human V gene segments H , D H and J H provide replacement of V gene segments H , D H and J H

[00357] In an exemplary embodiment 71, provided herein is a method according to any one of embodiments 62-70, wherein 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 through Jκ5, and wherein the human Vκ and Jκ gene segments are operably linked to an endogenous rodent immunoglobulin Cκ region.

[00358] In an exemplary embodiment 72, provided herein is a method according to embodiment 71, wherein the insert comprises human non-coding DNA that occurs naturally between human Vκ2-40 and Vκ4-1 and human non-coding DNA that occurs naturally between human Jκ1 and Jκ5.

[00359] In exemplary embodiment 73, provided herein is the method of embodiment 71, wherein the human Vκ and Jκ gene segments provide a replacement for the rodent Vκ and Jκ gene segments.

[00360] In exemplary embodiment 74, provided herein is a method according to any of embodiments 61-73, wherein the rodent germline genome further comprises an insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides.

[00361] In exemplary embodiment 75, provided herein is a method according to any of embodiments 62-74, wherein the immunoglobulin heavy chain locus lacks an endogenous rodent Adam6 gene.

[00362] In exemplary embodiment 76, provided herein is a method according to embodiment 75, wherein the immunoglobulin heavy chain locus further comprises an insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides.

[00363] In exemplary embodiment 77, provided herein is the method of embodiment 76, wherein one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides are inserted between the first and second segments of the human V gene. H .

[00364] In exemplary embodiment 78, provided herein is a method according to embodiment 77, wherein the first segment of the human V gene H represents the human V H 1-2, and the second segment of the human V gene H represents the human V H6-1.

[00365] In exemplary embodiment 79, provided herein is the method of embodiment 76, wherein one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides are inserted in place of the human Adam6 pseudogene.

[00366] In exemplary embodiment 80, provided herein is the method of embodiment 76, wherein one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides are inserted between a segment of the human V gene H and a segment of the human D gene H.

[00367] In exemplary embodiment 81, provided herein is the method of any one of embodiments 61-80, wherein the antibody obtained from a rodent or rodent cell that binds the antigen of interest comprises a human heavy chain variable domain and a human lambda light chain variable domain.

[00368] In exemplary embodiment 82, provided herein is the method of embodiment 81, wherein the human heavy chain variable domain comprises a rearranged segment of the human V gene H , selected from the group consisting of 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, VH 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 H6-1.

[00369] In an exemplary embodiment 83, provided herein is the method of embodiment 81 or 82, wherein the human variable domain of a lambda light chain comprises a rearranged segment of a human Vλ gene selected from the group consisting of Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ-51, Vλ9-49, Vλ-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1.

[00370] In exemplary embodiment 84, provided herein is a method according to any one of embodiments 39-83, wherein the rodent is a mouse or a rat.

[00371] In exemplary embodiment 85, provided herein is a rodent whose germline genome comprises a homozygous endogenous immunoglobulin λ light chain locus,which comprises (i) the human Vλ gene segments Vλ5-52 to Vλ1-40 and Vλ3-27 to Vλ3-1, (ii) pairs of human Jλ-Cλ gene segments, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, (iii) a human Jλ gene segment, Jλ7, and (iv) three enhancers of the human λ light chain of immunoglobulin; wherein (i)-(iv) are operably linked to one another and (i)-(iii) are located upstream of the rodent Cλ gene segment, and 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 human noncoding DNA that occurs naturally between the human Vλ gene segments, the human Jλ-Cλ gene segment pairs, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6, comprise human noncoding DNA that occurs naturally between the human Jλ-Cλ gene segment pairs, and the human Jλ gene segment, Jλ7, comprises human noncoding DNA,which occurs naturally upstream (or 5') of human Jλ7.

[00372] In exemplary embodiment 86, provided herein is a rodent of embodiment 85, wherein the rodent Cλ gene segment is a mouse Cλ1 gene segment.

[00373] 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.

[00374] In exemplary embodiment 88, provided herein is a rodent of any of embodiments 85-87,wherein the endogenous immunoglobulin λ light chain locus comprises a deletion of the endogenous Vλ2-Vλ3-Jλ2-Cλ2-Jλ4P-Cλ4P gene segments and the Vλ1-Jλ3-Jλ3P-Cλ3-Jλ1 gene segments of the rodent.

[00375] In an 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.

[00376] In some embodiments, provided herein is a rodent 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) and to a rodent Cλ gene segment,and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers.

[00377] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises two rodent Eλ.

[00378] In some embodiments, the two rodent Eλ are mouse Eλ and mouse Eλ3-1.

[00379] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises three human Eλ.

[00380] In some embodiments, the germline genome further comprises (i) an endogenous immunoglobulin heavy chain locus comprising an insertion of one or more human V gene segments, H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J Hfunctionally linked to a rodent immunoglobulin heavy chain constant region; or (ii) an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J H 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.

[00381] In some embodiments, the insertion of one or more human Vκ gene segments H , one or more segments of the human D gene Hand one or more segments of the human J gene H provides replacement of V gene segments H , D H rodent.

[00382] In some embodiments, the insert comprises non-coding human DNA that occurs naturally between segments of human V genes. H , D H and J Hand combinations thereof.

[00383] In some embodiments, the insertion of one or more human Vκ gene segments and one or more human Jκ gene segments provides for the replacement of rodent Vκ and Jκ gene segments.

[00384] In some embodiments, the insertion comprises human non-coding DNA that occurs naturally between the human Vκ and Jκ gene segments and combinations thereof.

[00385] In some embodiments, the rodent immunoglobulin heavy chain constant region is an endogenous rodent immunoglobulin heavy chain constant region.

[00386] In some embodiments, wherein the rodent Cκ region is an endogenous rodent Cκ region.

[00387] In some embodiments, the endogenous immunoglobulin λ light chain locus provides for the deletion of endogenous Vλ and Jλ gene segments, in whole or in part.

[00388] 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.

[00389] 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.

[00390] In some embodiments, the rodent Cλ gene segment is a mouse Cλ1 gene segment.

[00391] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises a deletion of rodent Eλ2-4.

[00392] In some embodiments, the rodent does not express detectable levels of endogenous immunoglobulin λ light chains.

[00393] In some embodiments, the immunoglobulin heavy chain locus comprises an insertion of segments of the human V gene. H from V H 3-74 to V H 6-1, segments of the human D gene H from D H1-1 to D H 7-27 and segments of the human J gene H , J H 1-J H 6.

[00394] In some embodiments, the insert comprises non-coding human DNA that occurs naturally between human V H 3-74 and V H 6-1, human noncoding DNA that occurs naturally between human D H 1-1 and D H 7-27, and human noncoding DNA, which occurs naturally between human J H 1-J H6.

[00395] In some embodiments, the immunoglobulin κ light chain locus comprises an insertion of a proximal duplication of Vκ, in whole or in part, of a human immunoglobulin κ light chain locus.

[00396] 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 through Jκ5.

[00397] In some embodiments, the insertion comprises human non-coding DNA that occurs naturally between human Vκ2-40 and Vκ4-1 and human non-coding DNA that occurs naturally between human Jκ1 through Jκ5.

[00398] 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 pairs of human Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6 gene segments, a human Jλ gene segment, Jλ7, and a rodent Cλ1 gene segment.

[00399] In some embodiments, the insert comprises human non-coding DNA that naturally occurs between human Vλ5-52 and Vλ1-40 and Vλ3-27 and Vλ3-1, human non-coding DNA that naturally occurs between pairs of human gene segments Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3 and Jλ6-Cλ6, and human non-coding DNA that naturally occurs upstream (or 5') of the human Jλ gene segment, Jλ7.

[00400] In some embodiments, the immunoglobulin heavy chain locus lacks the endogenous rodent Adam6 gene.

[00401] 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.

[00402] In some embodiments, the one or more nucleotide sequences are inserted between the first and second segments of the human V gene. H.

[00403] In some embodiments, one or more nucleotide sequences are inserted in place of the human Adam6 pseudogene.

[00404] In some embodiments, the first segment of the human V gene H represents the human V H 1-2, and the second segment of the human V gene H represents the human V H 6-1.

[00405] In some embodiments, one or more nucleotide sequences are inserted between a segment of the human V gene H and a segment of the human D gene H.

[00406] In some embodiments, the rodent is heterozygous or homozygous for an endogenous immunoglobulin heavy chain locus.

[00407] In some embodiments, the rodent is heterozygous or homozygous for an endogenous immunoglobulin κ light chain locus.

[00408] In some embodiments, the rodent is heterozygous or homozygous for an endogenous immunoglobulin λ light chain locus.

[00409] In some embodiments, the rodent is a rat or a mouse.

[00410] 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) and to a rodent Cλ gene segment,and (ii) wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers.

[00411] In some embodiments, provided herein is an immortalized cell produced by a rodent cell provided herein.

[00412] In some embodiments, the rodent cell is a rodent embryonic stem cell.

[00413] In some embodiments, provided herein is a rodent embryo produced by a rodent embryonic stem cell provided herein.

[00414] In some embodiments, provided herein is a method of producing a rodent whose germline genome comprises an engineered endogenous immunoglobulin λ light chain locus,wherein the method comprises (a) introducing a DNA fragment into a rodent embryonic stem cell, wherein the DNA fragment comprises a nucleotide sequence that comprises (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 λ light chain (Eλ) immunoglobulin enhancers; (b) obtaining a rodent embryonic stem cell generated from (a); and (c) producing a rodent using the rodent embryonic stem cell of (b).

[00415] In some embodiments, the nucleotide sequence further comprises one or more human λ light chain (Eλ) immunoglobulin enhancers.

[00416] In some embodiments, this document provides a method for producing a rodent,the germline genome of which 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 a rodent or human Cλ gene segment, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers; wherein the method comprises modifying the germline genome of a rodent such that it comprises an engineered immunoglobulin λ light chain locus that comprises an insertion of one or more segments of a human Vλ gene,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 a rodent or human Cλ gene segment, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers, thereby producing said rodent.

[00417] In some embodiments, the one or more human Vλ gene segments comprise Vλ5-52 to Vλ1-40 and / or Vλ3-27 to Vλ3-1.

[00418] In some embodiments, the one or more human Vλ gene segments comprise human non-coding DNA,which occurs naturally between human Vλ5-52 and Vλ1-40 and / or Vλ3-27 and Vλ3-1.

[00419] In some embodiments, the one or more human Jλ gene segments and the one or more human Cλ gene segments comprise pairs of human gene segments Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6, and a human Jλ7 gene segment.

[00420] In some embodiments, the human Jλ-Cλ, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, and Jλ6-Cλ6 gene segment pairs comprise human noncoding DNA that occurs naturally between the human Jλ and Cλ gene segment pairs, and the human Jλ7 gene segment comprises human noncoding DNA,which occurs naturally upstream (or 5') of human Jλ7.

[00421] In some embodiments, the rodent Cλ gene segment is a mouse Cλ1 gene segment.

[00422] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises three human Eλ.

[00423] In some embodiments, the endogenous immunoglobulin λ light chain locus comprises two rodent Eλ.

[00424] In some embodiments, the two rodent Eλ are mouse Eλ and mouse Eλ3-1.

[00425] In some embodiments, the DNA fragment further comprises one or more selection markers.

[00426] In some embodiments, the DNA fragment further comprises one or more site-specific recombination sites.

[00427] In some embodiments, the DNA fragment from (a) is introduced into a rodent embryonic stem cell whose germline genome contains an endogenous immunoglobulin heavy chain locus,containing an insertion of one or more segments of the human V gene, H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J H functionally linked to a rodent immunoglobulin heavy chain constant region; or an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J Hoperably 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.

[00428] In some embodiments, the DNA fragment of (a) is introduced into a rodent embryonic stem cell whose germline genome comprises an endogenous wild-type immunoglobulin heavy chain locus or an endogenous wild-type immunoglobulin heavy chain locus and an endogenous wild-type immunoglobulin κ light chain locus; and wherein the method further comprises the step of crossing a mouse produced by said non-human embryonic stem cell with a second mouse.

[00429] In some embodiments, the modification of the germline genome of a rodent to contain an engineered immunoglobulin λ light chain locus is performed in a rodent embryonic stem cell whose germline genome further comprises an endogenous immunoglobulin heavy chain locus comprising an insertion of one or more segments of a human V gene. H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J H functionally linked to a rodent immunoglobulin heavy chain constant region; or an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genesH , D H and J H 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.

[00430] In some embodiments, the insertion of one or more human Vκ gene segments H , one or more segments of the human D gene H and one or more segments of the human J gene H contains non-coding human DNA that occurs naturally between one or more segments of the human V gene H , human noncoding DNA that occurs naturally between one or more segments of the human D gene H, and human noncoding DNA that occurs naturally between one or more segments of the human J gene H.

[00431] In some embodiments, the insertion of one or more human Vκ gene segments and one or more human Jκ gene segments comprises human non-coding DNA that occurs naturally between the one or more human Vκ gene segments, and human non-coding DNA that occurs naturally between the one or more human Jκ gene segments.

[00432] In some embodiments, modifying the germline genome of a non-human animal to comprise an engineered immunoglobulin λ light chain locus is performed in a non-human embryonic stem cell whose germline genome comprises an endogenous wild-type immunoglobulin heavy chain locus or an endogenous wild-type immunoglobulin heavy chain locus and an endogenous wild-type immunoglobulin κ light chain locus; and wherein the method further comprises the step of crossing a mouse,obtained using said non-human embryonic stem cell, with a second mouse.

[00433] In some embodiments, the second mouse is characterized by a germline genome comprising wild-type Ig H chain and Ig κ chain loci.

[00434] In some embodiments, the second mouse is characterized by a germline genome comprising homozygous or heterozygous humanized Ig H chain and Ig κ chain loci, wherein the homozygous or heterozygous humanized Ig H chain locus comprises an inserted sequence encoding rodent Adam6.

[00435] In some embodiments, the second mouse is characterized by a germline genome comprising a homozygous or heterozygous humanized Ig H chain locus and a homozygous or heterozygous inactivated Ig κ chain locus.

[00436] In some embodiments, a method for producing an antibody in a rodent is provided,wherein the method comprises the steps of (1) immunizing a rodent with an antigen of interest, wherein the rodent is characterized by a germline genome comprising an endogenous immunoglobulin λ light chain locus that comprises (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 operably linked to (c) and to 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 (Eλ) enhancers; (2) maintaining the rodent under conditions sufficient for the rodent to develop an immune response to the antigen of interest; and (3) isolating antibodies from a rodent or rodent cell,that binds an antigen of interest.

[00437] In some embodiments, the rodent is characterized by a germline genome that further comprises an endogenous immunoglobulin heavy chain locus comprising an insertion of one or more human V gene segments, H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J H functionally linked to a rodent immunoglobulin heavy chain constant region; or an endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene H , one or more segments of the human D gene H and one or more segments of the human J gene H , while segments of human V genes H , D H and J Hoperably 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.

[00438] In some embodiments, the rodent cell is a B cell.

[00439] In some embodiments, the rodent cell is a hybridoma.

[00440] In some embodiments, the endogenous immunoglobulin λ light chain loc...

Claims

1. A method for producing an antibody, wherein the method comprises: (a) immunizing a genetically modified mouse with an antigen of interest, wherein the genetically modified mouse is characterized by a germline genome comprising an engineered endogenous immunoglobulin λ light chain locus that comprises (i) one or more segments of the human Vλ gene, (ii) one or more segments of the human Jλ gene and (iii) one or more segments of the human Cλ gene, wherein (i) and (ii) are operably linked to (iii) and to a mouse Cλ gene segment, and wherein the engineered endogenous immunoglobulin λ light chain locus further comprises one or more mouse immunoglobulin λ light chain (Eλ) enhancers and one or more human immunoglobulin λ light chain (Eλ) enhancers; (b) maintaining the genetically modified mouse under conditions sufficient to cause the genetically modified mouse to develop an immune response to the antigen of interest; and (c) isolating an antibody from the genetically modified mouse or cell thereof, wherein the antibody: (i) is capable of binding the antigen of interest and (ii) comprises an immunoglobulin λ light chain comprising a human variable λ domain and either a human constant λ domain or a mouse constant λ domain.

2. The method of claim 1, wherein the engineered endogenous immunoglobulin λ light chain locus comprises two mouse Eλ.

3. The method according to claim 2, wherein the two Eλ mice are Eλ mice and Eλ3-1 mice.

4. The method of claim 1, wherein the engineered endogenous immunoglobulin λ light chain locus comprises three human Eλ.

5. The method according to claim 1, wherein the germline genome of the genetically modified mouse further comprises: (i) an engineered endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene Н , one or more segments of the human D gene Н and one or more segments of the human J gene Н , while segments of human V genes Н , D Н and J Н functionally linked to the constant region of a mouse immunoglobulin heavy chain; or (ii) an engineered endogenous immunoglobulin heavy chain locus containing an insertion of one or more segments of the human V gene Н , one or more segments of the human D gene Н and one or more segments of the human J gene Н , while segments of human V genes Н , D Н and J Нoperably linked to a mouse immunoglobulin heavy chain constant region, and an engineered 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 the Cκ region of a mouse immunoglobulin.

6. The method according to claim 5, wherein the insertion of one or more segments of the human V gene Н , one or more segments of the human D gene Н and one or more segments of the human J gene Н provides replacement of endogenous V gene segments Н , D Н and J Н mice.

7. The method of claim 6, wherein the insert comprises non-coding human DNA that occurs naturally between segments of the human V gene Н , segments of the human D gene Н , segments of the human J gene Н or combinations thereof.

8. The method of claim 5, wherein the insertion of one or more segments of a human Vκ gene and one or more segments of a human Jκ gene provides a replacement for the endogenous segments of the mouse Vκ and Jκ genes.

9. The method of claim 8, wherein the insert comprises non-coding human DNA that occurs naturally between segments of the human Vκ gene, segments of the human Jκ gene, or combinations thereof.

10. The method of claim 5, wherein the mouse immunoglobulin heavy chain constant region is an endogenous mouse immunoglobulin heavy chain constant region.

11. The method according to claim 5, wherein the Cκ region of the mouse immunoglobulin is an endogenous mouse Cκ region.

12. The method of claim 1, wherein the engineered endogenous immunoglobulin λ light chain locus comprises deletion of endogenous segments of the Vλ and Jλ genes, in whole or in part.

13. The method according to claim 1, wherein the mouse Cλ gene segment is a mouse Cλ1 gene segment.

14. The method of claim 5, wherein the engineered endogenous immunoglobulin κ light chain locus comprises an insertion of a proximal duplication of Vκ, in whole or in part, of a human immunoglobulin κ light chain locus.

15. The method of claim 5, wherein the engineered endogenous immunoglobulin heavy chain locus lacks the endogenous mouse Adam6 gene.

16. The method of claim 15, wherein the engineered endogenous immunoglobulin heavy chain locus further comprises an insertion of one or more nucleotide sequences encoding one or more murine Adam6 polypeptides.

17. The method of claim 5, wherein the genetically modified mouse is homozygous for the engineered endogenous immunoglobulin heavy chain locus.

18. The method of claim 5, wherein the genetically modified mouse is homozygous for the engineered endogenous immunoglobulin κ light chain locus.

19. The method of claim 1, wherein the genetically modified mouse is homozygous for the engineered endogenous immunoglobulin λ light chain locus.

20. The method according to claim 1, wherein the cell of the genetically modified mouse is a B cell.

21. The method of claim 20, further comprising obtaining a hybridoma from a B cell.

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