Hybrid light chain mouse

Abstract

To provide a hybrid light chain mouse.SOLUTION: There is provided a mouse genetically modified to appear a Homo sapiens λ variable (hVλ) sequence (containing a mouse for appearing the hVλ sequence from an endogenous mouse λ light chain gene locus, a mouse for appearing an hVλ sequence from an endogenous mouse κ light chain gene locus, and a mouse for appearing hVλ sequence from a transducing gene or an episome). Here, the hVλ sequence is connected to a mouse steady sequence. There is provided a mouse for becoming an origin of the Homo sapiens λ variable sequence which has been varied into a body cell variation useful for preparing an antigen binding protein. There are provided a composition and a method for preparing an antigen binding protein containing a human λ variable sequence containing a human antibody.SELECTED DRAWING: Figure 7B

Description

[Technical field]

[0001] Field A genetically modified mouse comprising a mouse or human lambda variable (Vλ) light chain sequence operably linked to a mouse or human light chain constant region (λ or kappa (κ)). The mouse comprises a human lambda variable (hVλ) gene segment, a variable domain derived from a human lambda J (hJλ) gene segment, and a mouse light chain constant (C L A genetically modified mouse that expresses an epitope-binding protein comprising an immunoglobulin light chain that comprises an HV domain. A genetically modified mouse that comprises an unrearranged immunoglobulin lambda (λ) light chain variable nucleic acid sequence at an endogenous mouse light chain locus. A genetically modified mouse that expresses a chimeric human λ / mouse C from an endogenous light chain locus that comprises replacement of all endogenous mouse light chain variable region gene segments with one or more hVλ gene segments and one or more hJλ gene segments. L A mouse capable of rearranging and expressing light chains. hVλ domains and mouse C L Somatically mutated antibodies containing the domain. [Background technology]

[0002] background Mice that express fully human antibodies or antibodies that are partly human and partly mouse antibodies are known in the art.For example, transgenic mice that express fully human antibodies from transgenes that contain human light and heavy chain immunoglobulin variable region genes have been reported.Similarly, genetically modified mice that contain human heavy chain variable region (HCVR) and light chain variable region (LCVR) gene segments to replace endogenous mouse HCVR gene segments and kappa (κ) LCVR gene segments and produce chimeric antibodies with chimeric human / mouse kappa chains are also known.

[0003] Antibody light chains are encoded by one of two separate genetic loci: kappa (κ) and lambda (λ). Mouse antibody light chains are predominantly of the κ type. The ratio of κ to λ light chain usage in humans is approximately 60:40, whereas in mice it is approximately 95:5. The biased κ light chain usage in mice is reportedly maintained in genetically modified mice capable of expressing fully or partially human antibodies. Thus, mice expressing fully or partially human antibodies appear to have suppressed lambda variable usage. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need in the art to generate lambda variable regions, whether murine or human, for use in generating epitope-binding proteins. There is a need in the art for mice that express fully or partially human antibodies that exhibit high lambda variable (Vλ) usage.

[0005] There is a need in the art for mice that express fully or partially human antibodies that exhibit high lambda variable (Vλ) usage. [Means for solving the problem]

[0006] Abstract Genetically modified mice, embryos, cells, tissues, and nucleic acid constructs for modifying mice, as well as methods and compositions for making and using same are provided. Mice and cells that produce lambda (λ) variable regions (human or non-human) in the context of kappa (κ) light chains are provided. Mice and cells that produce human λ variable regions in the context of κ or λ light chains, e.g., from endogenous mouse light chain loci, are also provided. Methods for making antibodies that include lambda variable regions are also provided. Methods for selecting heavy chains that express with cognate lambda variable regions are also provided.

[0007] Chimeric and human antigen binding proteins (e.g., antibodies) comprising somatically mutated variable regions (including antibodies having light chains comprising variable domains derived from human Vλ and human Jλ gene segments fused to mouse light chain constant domains) and nucleic acids encoding them are provided.

[0008] In one embodiment, a mouse is provided that expresses a human λ variable region sequence in a light chain that comprises a mouse constant region. In one embodiment, a mouse is provided that expresses a human λ variable region sequence in a light chain that comprises a κ constant region. In one embodiment, a mouse is provided that expresses a light chain that comprises a human λ variable region sequence from an endogenous mouse light chain locus. In one embodiment, a mouse is provided that comprises a rearranged light chain gene that comprises a human λ variable sequence connected to a mouse constant region sequence; in one embodiment, the mouse constant region sequence is a λ constant sequence; in one embodiment, the mouse constant region sequence is a κ constant sequence.

[0009] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises an unrearranged human λ light chain variable gene segment (hVλ) and a human λ joining gene segment (hJλ). In one embodiment, the unrearranged hVλ and hJλ are present at a mouse light chain locus. In one embodiment, the unrearranged hVλ and unrearranged hJλ are present on a transgene and operably linked to human or mouse constant region sequences. In one embodiment, the unrearranged hVλ and unrearranged hJλ are present on an episome. In one embodiment, the mouse comprises unrearranged hVλ and hJλ sequences and a mouse light chain constant region (C L The present invention provides a method for producing an immunoglobulin comprising the steps of: (a) producing an immunoglobulin having a light chain derived from a nucleic acid sequence comprising: (a) a human heavy chain variable domain (hV) fused to a mouse heavy chain constant region; H ), and (b) mouse C LAntibodies are provided that comprise a human Vλ fused to a domain, including those in which one or more of the variable domains have been somatically mutated, for example, during selection of antibodies or immune cells in a mouse of the invention. In one embodiment, the unrearranged hVλ and the unrearranged hJλ are operably linked to a human or mouse κ constant region (Cκ). In one embodiment, the unrearranged hVλ and the unrearranged hJλ are operably linked to a human or mouse λ constant region (Cλ).

[0010] In one embodiment, a mouse is provided that comprises a human λ light chain variable region sequence at an endogenous mouse light chain locus in the germline, wherein the human lambda variable region sequence is expressed in a light chain that comprises a mouse immunoglobulin constant region gene sequence.

[0011] In one embodiment, the endogenous mouse light chain locus is the λ locus. In one embodiment, the endogenous mouse light chain locus is the κ locus.

[0012] In one embodiment, the mouse lacks an endogenous light chain variable sequence at an endogenous mouse light chain locus.

[0013] In one embodiment, all or substantially all of the endogenous mouse light chain variable region gene segments are replaced with one or more human λ variable region gene segments.

[0014] In one embodiment, the human λ light chain variable region sequence comprises a human Jλ sequence, hi one embodiment, the human Jλ sequence is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and a combination thereof.

[0015] In one embodiment, the human lambda light chain variable region sequence comprises a fragment of cluster A of the human light chain locus. In a specific embodiment, the fragment of cluster A of the human lambda light chain locus spans from hVλ3-27 to hVλ3-1.

[0016] In one embodiment, the human λ light chain variable region sequence comprises a fragment of cluster B of the human light chain locus. In a specific embodiment, the fragment of cluster B of the human λ light chain locus spans hVλ5-52 to hVλ1-40.

[0017] In one embodiment, the human lambda light chain variable region sequence comprises a genomic fragment of cluster A and a genomic fragment of cluster B. In one embodiment, the human lambda light chain variable region sequence comprises at least one gene segment of cluster A and at least one gene segment of cluster B.

[0018] In one embodiment, greater than 10% of the mouse's light chain naive repertoire is derived from at least two hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49. In one embodiment, greater than 20% of the mouse's light chain naive repertoire is derived from at least three hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49. In one embodiment, greater than 30% of the mouse's light chain naive repertoire is derived from at least four hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49.

[0019] In one embodiment, a mouse is provided that expresses an immunoglobulin light chain comprising a human λ variable sequence fused to a mouse constant region, wherein the mouse exhibits a ratio of κ to λ usage of about 1:1.

[0020] In one embodiment, the immunoglobulin light chain is expressed from an endogenous mouse light chain locus.

[0021] In one embodiment, a mouse is provided that comprises a λ light chain variable region sequence (Vλ) and at least one J sequence (J) contiguous with a mouse κ light chain constant region sequence.

[0022] In one embodiment, the mouse lacks functional mouse Vκ and / or mouse Jκ gene segments.

[0023] In one embodiment, the Vλ is a human Vλ (hVλ) and the J is a human Jλ (hJλ). In one embodiment, the hVλ and hJλ are unrearranged gene segments.

[0024] In one embodiment, the mouse comprises a plurality of unrearranged hVλ gene segments and at least one hJλ gene segment, hi a specific embodiment, the plurality of unrearranged hVλ gene segments is at least 12 gene segments, at least 28 gene segments, or at least 40 gene segments.

[0025] In one embodiment, the at least one hJλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof.

[0026] In one embodiment, the endogenous mouse λ light chain locus is deleted in whole or in part.

[0027] In one embodiment, the mouse κ light chain constant region sequence is present at an endogenous mouse κ light chain locus.

[0028] In one embodiment, about 10% to about 45% of B cells of the mouse express an antibody comprising a light chain comprising a human λ light chain variable (Vλ) domain and a mouse κ light chain constant (Cκ) domain.

[0029] In one embodiment, the human lambda variable domains are derived from a rearranged hVlambda / hJlambda sequence selected from the group consisting of 3-1 / 1, 3-1 / 7, 4-3 / 1, 4-3 / 7, 2-8 / 1, 3-9 / 1, 3-10 / 1, 3-10 / 3, 3-10 / 7, 2-14 / 1, 3-19 / 1, 2-23 / 1, 3-25 / 1, 1-40 / 1, 1-40 / 2, 1-40 / 3, 1-40 / 7, 7-43 / 1, 7-43 / 3, 1-44 / 1, 1-44 / 7, 5-45 / 1, 5-45 / 2, 5-45 / 7, 7-46 / 1, 7-46 / 2, 7-46 / 7, 9-49 / 1, 9-49 / 2, 9-49 / 7, and 1-51 / 1.

[0030] In one embodiment, the mouse further comprises a human VK-JK intergenic region from a human κ light chain locus, where the human VK-JK intergenic region is contiguous with a Vλ sequence and a J sequence. In a specific embodiment, the human VK-JK intergenic region is disposed between the Vλ sequence and the J sequence.

[0031] In one embodiment, a mouse is provided that comprises: (a) at an endogenous mouse light chain locus, at least 12, up to at least 40 unrearranged human λ light chain variable region gene segments and at least one human Jλ gene segment; (b) a human Vκ-Jκ intergenic sequence disposed between the at least 12, up to at least 40 human light chain variable region gene segments and the at least one human Jλ sequence; wherein the mouse expresses an antibody comprising a light chain comprising a human Vλ domain and a mouse Cκ domain.

[0032] In one embodiment, a mouse is provided that expresses an antibody comprising a light chain comprising a λ variable sequence and a κ constant sequence.

[0033] In one embodiment, the mice exhibit a ratio of κ to λ usage of about 1:1.

[0034] In one embodiment, the population of immature B cells obtained from the bone marrow of the mice exhibits a ratio of κ to λ usage of about 1:1.

[0035] In one embodiment, a genetically modified mouse is provided, wherein the mouse is a mouse C L The mouse light chain locus comprises unrearranged immunoglobulin Vλ and Jλ gene segments operably linked to a mouse light chain locus comprising a gene.

[0036] In one embodiment, the V and / or J gene segments are human gene segments. In one embodiment, the V and / or J gene segments are mouse gene segments and are L is mouse Cκ.

[0037] In one embodiment, the endogenous mouse light chain locus is a κ light chain locus. In one embodiment, the endogenous mouse light chain locus is a λ light chain locus.

[0038] In one embodiment, the unrearranged Vλ and Jλ gene segments are present at the endogenous mouse light chain locus.

[0039] In one embodiment, unrearranged immunoglobulin Vλ and Jλ gene segments are present on a transgene.

[0040] In one embodiment, the mouse further comprises a replacement of one or more heavy chain V, D and / or J gene segments at an endogenous mouse heavy chain immunoglobulin locus with one or more human V, D and / or J gene segments.

[0041] In one embodiment, the mouse comprises unrearranged immunoglobulin Vλ and Jλ gene segments at an endogenous mouse κ light chain locus that comprises a mouse Cκ gene.

[0042] In one embodiment, the mouse comprises an unrearranged human immunoglobulin λ light chain variable gene segment (Vλ) and a λ joining gene segment (Jλ) at an endogenous mouse λ light chain locus that comprises a mouse Cλ gene.

[0043] In one embodiment, the locus of the light chain variable gene ("V L In one embodiment, the V locus ("V locus") comprises at least one human Vλ (hVλ) gene segment. L The locus comprises at least one human Jλ (hJλ) gene segment. L The locus comprises up to four hJλ gene segments. In one embodiment, the V L The locus comprises a contiguous sequence that includes human λ and human κ genomic sequences.

[0044] In one embodiment, the κ light chain variable gene locus ("κ locus") comprises at least one human Vλ (hVλ) gene segment. In one embodiment, the κ locus comprises at least one human Jλ (hJλ) gene segment. In one embodiment, the κ locus comprises up to four hJλ gene segments. In one embodiment, the κ locus comprises at least one hVλ and at least one hJλ, lacks or substantially lacks a functional Vκ region gene segment, and lacks or substantially lacks a functional Jκ region gene segment. In one embodiment, the mouse does not comprise a functional Vκ region gene segment. In one embodiment, the mouse does not comprise a functional Jκ region gene segment.

[0045] In one embodiment, the λ light chain variable gene locus ("λ locus") comprises at least one hVλ gene segment. In one embodiment, the λ locus comprises at least one human Jλ (hJλ) gene segment. In another embodiment, the λ locus comprises up to four hJλ gene segments.

[0046] In one embodiment, V L The locus comprises a plurality of hVλs. In one embodiment, the plurality of hVλs is selected to provide for expression of a λ light chain variable region repertoire that reflects about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of the Vλ usage observed in humans. L The locus includes gene segments hVλ1-40, 1-44, 2-8, 2-14, 3-21 and combinations thereof.

[0047] In one embodiment, the hVλ comprises 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, and 3-12. L The locus comprises a contiguous sequence of the human λ light chain locus spanning from Vλ3-12 to Vλ3-1. L The locus comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hVλ. In certain embodiments, the hVλ comprises 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, and 3-12. L The locus comprises a contiguous sequence of the human λ locus spanning from Vλ3-12 to Vλ3-1. L The locus is present at the endogenous κ locus. L The locus is present at the endogenous κ locus and the endogenous λ light chain locus is partially or completely deleted. L The locus is present at the endogenous λ locus. L The locus is present at the endogenous λ locus and the endogenous κ locus is partially or completely deleted.

[0048] In one embodiment, V LThe locus comprises 13-28 or more hVλs. In certain embodiments, the hVλs include 2-14, 3-16, 2-18, 3-19, 3-21, 3-22, 2-23, 3-25, and 3-27. In certain embodiments, the κ locus comprises a contiguous sequence of the human λ locus spanning Vλ3-27 to Vλ3-1. In one embodiment, V L The locus is present at the endogenous κ locus. L In another embodiment, the locus is present at the endogenous κ locus and the endogenous λ light chain locus is partially or completely deleted. L The locus is present at the endogenous λ locus. L The locus is present at the endogenous λ locus and the endogenous κ locus is partially or completely deleted.

[0049] In one embodiment, V L The locus comprises 29-40 hVλ. In certain embodiments, the κ locus comprises a contiguous sequence of the human λ locus spanning from Vλ3-29 to Vλ3-1 and a contiguous sequence of the human λ locus spanning from Vλ5-52 to Vλ1-40. In certain embodiments, all or substantially all of the sequence between hVλ1-40 and hVλ3-29 in the genetically modified mouse consists essentially of approximately 959 bp of human λ sequence found naturally (e.g., in the human population) downstream of the hVλ1-40 gene segment (downstream of the 3' untranslated portion), a restriction enzyme site (e.g., PI-SceI), followed by approximately 3,431 bp of human λ sequence upstream of the naturally found hVλ3-29 gene segment. In one embodiment, the V L The locus is present in the endogenous mouse κ locus. L The locus is present at the endogenous mouse κ locus and the endogenous mouse λ light chain locus is partially or completely deleted. L The locus is present in the endogenous mouse λ locus. LThe locus is present at the endogenous mouse λ locus, and the endogenous mouse κ locus is partially or completely deleted.

[0050] In one embodiment, V L The locus comprises at least one hJλ. L The locus comprises multiple hJλ. In one embodiment, L The locus comprises at least 2, 3, 4, 5, 6 or 7 hJλ. L The locus comprises four hJλ. In a specific embodiment, the four hJλ are hJλ1, hJλ2, hJλ3 and hJλ7. L In certain embodiments, the locus is the κ locus. L The locus is present at the endogenous κ locus and the endogenous λ light chain locus is partially or completely deleted. L The locus comprises one hJλ. In a specific embodiment, the one hJλ is hJλ1. In one embodiment, L The locus is present at the endogenous κ locus. L In another embodiment, the locus is present at the endogenous κ locus and the endogenous λ light chain locus is partially or completely deleted. L The locus is present at the endogenous λ locus. L The locus is present at the endogenous λ locus and the endogenous κ locus is partially or completely deleted.

[0051] In one embodiment, V L The locus comprises at least one hVλ, at least one hJλ and a mouse Cκ gene. LThe locus comprises at least one hVλ, at least one hJλ, and a mouse Cλ gene. In certain embodiments, the mouse Cλ gene is Cλ2. In certain embodiments, the mouse Cλ gene is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, 96%, 97%, 98% or at least 99% identical to mouse Cλ2.

[0052] In one embodiment, the mouse comprises a replacement of an endogenous mouse VK gene segment with one or more hVλ gene segments at the endogenous mouse κ locus, where the hVλ gene segments are operably connected to an endogenous mouse Cκ region gene, and the mouse rearranges the human Vλ gene segments and expresses a reverse chimeric immunoglobulin light chain comprising a human Vλ domain and a mouse Cκ. In one embodiment, 90-100% of the unrearranged mouse VK gene segments are replaced with at least one unrearranged hVλ gene segment. In a particular embodiment, all or substantially all of the endogenous mouse VK gene segments are replaced with at least one unrearranged hVλ gene segment. In one embodiment, the replacement is with at least 12, at least 28, or at least 40 unrearranged hVλ gene segments. In one embodiment, the replacement is with at least 7 functional unrearranged hVλ gene segments, at least 16 functional unrearranged hVλ gene segments, or at least 27 functional unrearranged hVλ gene segments. In one embodiment, the mouse comprises a replacement of all mouse Jκ gene segments with at least one unrearranged hJλ gene segment. In one embodiment, the at least one unrearranged hJλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ4, Jλ5, Jλ6, Jλ7, and a combination thereof. In certain embodiments, the one or more hVλ gene segments are selected from a 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, 3-12, 2-14, 3-16, 2-18, 3-19, 3-21, 3-22, 2-23, 3-25, 3-27, 1-40, 7-43, 1-44, 5-45, 7-46, 1-47, 5-48, 9-49, 1-50, 1-51, 5-52 hVλ gene segments, and combinations thereof.In a specific embodiment, the at least one unrearranged hJλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof.

[0053] In one embodiment, the mouse comprises a replacement of an endogenous mouse Vλ gene segment with one or more human Vλ gene segments at the endogenous mouse λ locus, where the hVλ gene segments are operably linked to a mouse Cλ region gene, and the mouse rearranges the hVλ gene segments and expresses a reverse chimeric immunoglobulin light chain comprising an hVλ domain and a mouse Cλ. In a specific embodiment, the mouse Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2. In one embodiment, 90-100% of the unrearranged mouse Vλ gene segments are replaced with at least one unrearranged hVλ gene segment. In a specific embodiment, all or substantially all of the endogenous mouse Vλ gene segments are replaced with at least one unrearranged hVλ gene segment. In one embodiment, the replacement is with at least 12, at least 28, or at least 40 unrearranged hVλ gene segments. In one embodiment, the replacement is with at least 7 functional unrearranged hVλ gene segments, at least 16 functional unrearranged hVλ gene segments, or at least 27 functional unrearranged hVλ gene segments. In one embodiment, the mouse comprises a replacement of all mouse Jλ gene segments with at least one unrearranged hJλ gene segment. In one embodiment, the at least one unrearranged hJλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ4, Jλ5, Jλ6, Jλ7, and a combination thereof.In certain embodiments, the one or more hVλ gene segments are selected from a 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, 3-12, 2-14, 3-16, 2-18, 3-19, 3-21, 3-22, 2-23, 3-25, 3-27, 1-40, 7-43, 1-44, 5-45, 7-46, 1-47, 5-48, 9-49, 1-50, 1-51, 5-52 hVλ gene segment, and combinations thereof. In certain embodiments, the at least one unrearranged hJλ gene segment is selected from a Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof.

[0054] In one embodiment, a genetically modified mouse is provided that comprises a human Vκ-Jκ intergenic region sequence located at the endogenous mouse κ light chain locus.

[0055] In one embodiment, the human VK-JK intergenic region sequence is present in an endogenous κ light chain locus of a mouse that comprises hVλ and hJλ gene segments, and the human VK-JK intergenic region sequence is disposed between an hVλ gene segment and an hJλ gene segment. In a specific embodiment, the hVλ and hJλ gene segments are capable of recombining to form a functional human λ light chain variable domain in the mouse.

[0056] In one embodiment, a mouse is provided that comprises multiple hVλs and one or more hJλs, where, from a transcriptional standpoint, the human Vκ-Jκ intergenic region sequence is positioned downstream of the proximal or 3'-most hVλ sequence and upstream or 5' of the first hJλ sequence.

[0057] In one embodiment, the human Vκ-Jκ intergenic region is a region located about 130 bp downstream or 3' of the human Vκ4-1 gene segment, i.e., about 130 bp downstream of the 3' untranslated region of the human Vκ4-1 gene segment, and extends to about 600 bp upstream or 5' of the human Jκ1 gene segment. In a particular embodiment, the human Vκ-Jκ intergenic region is about 22.8 kb in size. In one embodiment, the Vκ-Jκ intergenic region is about 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or about 95% or more identical to the human Vκ-Jκ intergenic region that extends from the end of the 3' untranslated region of the human Vκ4-1 gene segment to about 600 bp upstream of the human Jκ1 gene segment. In one embodiment, the Vκ-Jκ intergenic region comprises SEQ ID NO:100. In a particular embodiment, the Vκ-Jκ intergenic region comprises a functional fragment of SEQ ID NO:100. In a specific embodiment, the Vκ-Jκ intergenic region is SEQ ID NO:100.

[0058] In one aspect, mice, mouse cells (e.g., mouse embryonic stem cells), mouse embryos and mouse tissues are provided that comprise a recited human VK-JK intergenic region sequence, wherein the intergenic region sequence is ectopic. In a specific embodiment, the ectopic sequence is located in a humanized endogenous mouse immunoglobulin locus.

[0059] In one aspect, an isolated nucleic acid construct is provided that includes the recited human VK-JK intergenic region sequence. In one embodiment, the nucleic acid construct includes a targeting arm that targets the human VK-JK intergenic region sequence to a mouse light chain locus. In a specific embodiment, the mouse light chain locus is a κ locus. In a specific embodiment, the targeting arm targets the human VK-JK intergenic region to a modified endogenous mouse κ locus, where the targeting is to a position between the hVλ sequence and the hJλ sequence.

[0060] In one embodiment, a genetically modified mouse is provided, wherein the mouse comprises no more than two light chain alleles, wherein the light chain alleles are: (a) a mouse C L (b) an endogenous mouse light chain locus containing unrearranged immunoglobulin human Vλ and Jλ gene segments; and (b) a mouse C L The endogenous mouse light chain locus contains the gene, which is unrearranged immunoglobulin V L and J. L Contains gene segments.

[0061] In one embodiment, the endogenous mouse light chain locus is the κ locus. In another embodiment, the endogenous mouse light chain locus is the λ locus.

[0062] In one embodiment, the two or less light chain alleles are selected from a κ allele and a λ allele, two κ alleles and two λ alleles, hi a specific embodiment, one of the two light chain alleles is a λ allele that comprises a Cλ2 gene.

[0063] In one embodiment, the mouse comprises one functional immunoglobulin light chain locus and one non-functional light chain locus, where the functional light chain locus comprises unrearranged immunoglobulin human Vλ and Jλ gene segments at an endogenous mouse κ light chain locus that comprises a mouse Cκ gene.

[0064] In one embodiment, the mouse comprises one functional immunoglobulin light chain locus and one non-functional light chain locus, where the functional light chain locus comprises unrearranged immunoglobulin human Vλ and Jλ gene segments at an endogenous mouse λ light chain locus that comprises a mouse Cλ gene. In one embodiment, the Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2.

[0065] In one embodiment, the mouse further comprises at least one immunoglobulin heavy chain allele, which comprises a human VV ... H Gene segment, human D H Gene segments and human J H In a specific embodiment, the mouse comprises two immunoglobulin heavy chain alleles and expresses a human / mouse heavy chain.

[0066] In one embodiment, the mouse comprises a first light chain allele at the endogenous mouse κ locus that comprises the endogenous Cκ gene, comprising an unrearranged hVκ and an unrearranged hJκ; and a second light chain allele at the endogenous mouse κ locus that comprises the endogenous Cκ gene, comprising an unrearranged hVλ and an unrearranged hJλ. In a particular embodiment, the first and second light chain alleles are the only functional light chain alleles in the genetically modified mouse. In a particular embodiment, the mouse comprises a non-functional λ locus. In one embodiment, the genetically modified mouse does not express a light chain comprising a λ constant region.

[0067] In one embodiment, the mouse comprises a first light chain allele comprising an unrearranged hVκ and an unrearranged hJκ at the endogenous mouse κ locus comprising the endogenous Cκ gene; and a second light chain allele comprising an unrearranged hVλ and an unrearranged hJλ at the endogenous mouse λ locus comprising the endogenous Cλ gene. In a specific embodiment, the first and second light chain alleles are the only functional light chain alleles in the genetically modified mouse. In one embodiment, the endogenous Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2.

[0068] In one embodiment, the mouse comprises six immunoglobulin alleles, wherein a first allele comprises unrearranged immunoglobulin Vλ and Jλ gene segments at an endogenous mouse κ light chain locus comprising a mouse Cκ gene, a second allele comprises unrearranged immunoglobulin Vκ and Jκ gene segments at an endogenous mouse κ light chain locus comprising a mouse Cκ gene, a third allele comprises unrearranged immunoglobulin Vλ and Jλ gene segments at an endogenous mouse λ light chain locus comprising a mouse Cλ gene, and a fourth and fifth allele each independently comprises an unrearranged Vλ and Jλ gene segments at an endogenous mouse heavy chain locus comprising a mouse heavy chain gene. H and D. H and J. H and the sixth allele comprises unrearranged immunoglobulin Vλ and Jλ gene segments at an endogenous mouse λ light chain locus that contains a mouse Cλ gene, (b) comprises a non-functional λ locus, or (c) comprises a deletion of the λ locus in whole or in part.

[0069] In one embodiment, the first allele comprises unrearranged hVλ and hJλ. In one embodiment, the second allele comprises unrearranged hVκ and hJκ. In one embodiment, the third allele comprises unrearranged hVλ and hJλ. In one embodiment, the fourth and fifth alleles each independently comprise unrearranged hV. H and hD H and hJ H In one embodiment, the sixth allele comprises a deleted or partially deleted endogenous mouse λ locus.

[0070] In one embodiment, the mouse comprises six immunoglobulin alleles, wherein a first allele comprises unrearranged immunoglobulin Vλ and Jλ gene segments at an endogenous mouse λ light chain locus comprising a mouse Cλ gene, a second allele comprises unrearranged immunoglobulin Vλ and Jλ gene segments at an endogenous mouse λ light chain locus comprising a mouse Cλ gene, a third allele comprises unrearranged immunoglobulin Vκ and Jκ gene segments at an endogenous mouse κ light chain locus comprising a mouse Cκ gene, and a fourth and fifth allele each independently comprises an unrearranged Vκ and Jκ gene segments at an endogenous mouse heavy chain locus comprising a mouse heavy chain gene. H and D. H and J. H and the sixth allele comprises gene segments that are unrearranged immunoglobulin Vκ and Jκ gene segments at an endogenous mouse κ light chain locus that contains the mouse Cκ gene, (b) comprises a non-functional κ locus, or (c) comprises a deletion of one or more elements of the κ locus.

[0071] In one embodiment, the first allele comprises unrearranged hVλ and hJλ gene segments. In one embodiment, the second allele comprises unrearranged hVλ and hJλ gene segments. In one embodiment, the third allele comprises unrearranged hVκ and hJκ gene segments. In one embodiment, the fourth and fifth alleles each independently comprise unrearranged hV H and hD H and hJ H In one embodiment, the sixth allele comprises a functionally silenced endogenous mouse κ locus.

[0072] In one embodiment, the genetically modified mouse is a mouse C L In one embodiment, the mouse C20 comprises a B cell comprising a rearranged antibody gene comprising a rearranged hVλ domain operably linked to a rearranged hVλ domain. L The domain is selected from a mouse Cκ and a mouse Cλ domain. In certain embodiments, the mouse Cλ domain is derived from a Cλ2 gene. In certain embodiments, the mouse Cλ domain is derived from a Cλ domain that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to mouse Cλ2.

[0073] In one embodiment, the C is C L In one embodiment, a genetically modified mouse is provided that expresses a Vλ region in a C selected from human Cκ, human Cλ, or mouse Cκ. L In one embodiment, a genetically modified mouse is provided that expresses an hVλ region in a mouse Cκ.

[0074] In one embodiment, about 10-50% of the splenocytes of the mouse are B cells (i.e., CD19 positive) or about 9-28% of them express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.

[0075] In certain embodiments, about 23-34% of the splenocytes of the mouse are B cells (i.e., CD19 positive) or about 9-11% of them express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.

[0076] In certain embodiments, about 19-31% of the splenocytes of the mouse are B cells (i.e., CD19 positive) or about 9-17% of them express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.

[0077] In certain embodiments, about 21-38% of the splenocytes of the mouse are B cells (i.e., CD19 positive) or about 24-27% of them express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.

[0078] In certain embodiments, about 10-14% of the splenocytes of the mouse are B cells (i.e., CD19 positive) or about 9-13% of them express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.

[0079] In certain embodiments, about 31-48% of the splenocytes of the mouse are B cells (i.e., CD19 positive) or about 15-21% of them express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain. In certain embodiments, about 30-38% of the splenocytes of the mouse are B cells (i.e., CD19 positive) and about 33-48% of them express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.

[0080] In one embodiment, about 52-70% of the bone marrow of the mouse are B cells (i.e., CD19 positive) or about 31-47% of the immature B cells (i.e., CD19 positive / B220 intermediate positive / IgM positive) express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.

[0081] In one embodiment, about 60% of the bone marrow of the mouse are B cells (i.e., CD19 positive) or about 38.3% of the immature B cells (i.e., CD19 positive / B220 intermediate positive / IgM positive) express an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.

[0082] In one embodiment, the mouse expresses an antibody comprising a light chain comprising variable domains derived from human V and human J gene segments and a constant domain derived from a mouse constant region gene. In one embodiment, the mouse constant region gene is a Cκ gene. In another embodiment, the mouse constant region gene is a Cλ gene. In a particular embodiment, the Cλ region is Cλ2. In a particular embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2. In a particular embodiment, the antibody further comprises a heavy chain comprising variable domains derived from human V, human D and human J gene segments, and a heavy chain constant domain derived from a mouse heavy chain constant region gene. In one embodiment, the mouse heavy chain constant region gene comprises a hinge-CH of the heavy chain constant domain. 2 -CH 3 In another embodiment, the mouse heavy chain constant region gene comprises the sequence 1 - Hinge-CH 2 -CH 3 In another embodiment, the mouse heavy chain constant region gene comprises the sequence 1 -CH 2 -CH3 -CH 4 In another embodiment, the mouse heavy chain constant region gene comprises the sequence 2 -CH 3 -CH 4 Contains arrays.

[0083] In one embodiment, the mouse expresses an antibody comprising a light chain comprising a rearranged human Vλ-Jλ sequence and a mouse Cκ sequence. In one embodiment, the rearranged human Vλ-Jλ sequence is derived from a rearrangement of hVλ gene segments selected from the 3-1, 4-3, 2-8, 3-9, 3-10, 2-14, 3-19, 2-23, 3-25, 1-40, 7-43, 1-44, 5-45, 7-46, 1-47, 9-49, and 1-51 gene segments. In one embodiment, the rearranged human Vλ-Jλ sequence is derived from a rearrangement of hJλ gene segments selected from the Jλ1, Jλ2, Jλ3, and Jλ7 gene segments.

[0084] In one embodiment, the mouse is 3-1 / 1, 3-1 / 7, 4-3 / 1, 4-3 / 7, 2-8 / 1, 3-9 / 1, 3-10 / 1, 3-10 / 3, 3-10 / 7, 2-14 / 1, 3-19 / 1, 2-23 / 1, 3-25 / 1, 1-40 / 1, 1-40 / 2, 1-40 / 3, 1-40 / 7, 7-43 / 1, 7-43 / 2, 7-43 / 1, 7-43 / 3, 7-43 / 4, 7-43 / 5, 7-43 / 6, 7-43 / 7, 7-43 / 8, 7-43 / 9, 7-43 / 1, 7-43 / 2, 7-43 / 3, 7-43 / 4, 7-43 / 5, 7-43 / 6, 7-43 / 7, 7-43 / 8, 7-43 / 9, 7-43 / 1 ...1, 7-43 / 1, 7-43 / 2, 7-43 / 3, 7-43 / 4, 7-43 / 5, 7-43 / 6, 7-43 / 1, 7-43 / In certain embodiments, the B cell expresses an antibody comprising a light chain comprising a rearranged immunoglobulin lambda light chain variable region comprising a human Vλ / Jλ sequence selected from among Vλ / Jλ / 3, 1-44 / 1, 1-44 / 7, 5-45 / 1, 5-45 / 2, 5-45 / 7, 7-46 / 1, 7-46 / 2, 7-46 / 7, 9-49 / 1, 9-49 / 2, 9-49 / 7, and 1-51 / 1. In certain embodiments, the B cell expresses an antibody comprising a human immunoglobulin heavy chain variable domain fused to a mouse heavy chain constant domain and a human immunoglobulin lambda light chain variable domain fused to a mouse kappa light chain constant domain.

[0085] In one embodiment, (a) a heavy chain comprising a heavy chain variable domain derived from an unrearranged human heavy chain variable region gene segment, wherein the heavy chain variable domain is a mouse heavy chain constant (CH and (b) a light chain comprising a light chain variable domain derived from an unrearranged hVλ and hJλ, wherein the light chain variable domain is fused to a mouse C L In one embodiment, a mouse is provided that expresses an antibody comprising the nucleotide sequence fused to the nucleotide sequence of the target gene (e.g., nucleotide sequence fused to the target gene) and a region fused to the target gene (e.g., nucleotide sequence fused to the target gene).

[0086] In one embodiment, the mouse comprises: (i) a replacement of all or substantially all functional endogenous mouse V, D, and J gene segments with all or substantially all functional human V, D, and J gene segments; H The mouse comprises: (i) a heavy chain locus comprising a gene; (ii) a first κ light chain locus comprising a mouse Cκ gene; (iii) a second κ light chain locus comprising a mouse Cκ gene; and (iv) a heavy chain locus comprising a mouse Cκ gene; (v) a heavy chain locus comprising a gene comprising a gene; (vi) a first κ light chain locus comprising a mouse Cκ gene; (vi) a first κ light chain locus comprising a mouse Cκ gene; (vi) a second κ light chain locus comprising a mouse Cκ gene; and (vi) a first κ light chain locus comprising a mouse Cκ gene; and ...

[0087] In one embodiment, the mouse comprises: (i) a replacement of all or substantially all functional endogenous mouse V, D, and J gene segments with all or substantially all functional human V, D, and J gene segments; HThe invention further comprises a heavy chain locus comprising a mouse Cλ gene, (ii) a first λ light chain locus comprising a replacement of all or substantially all of the functional endogenous mouse Vλ and Jλ gene segments with all, substantially all or a plurality of functional hVλ and hJλ gene segments, and a mouse Cλ gene, (iii) a second λ light chain locus comprising a replacement of all or substantially all of the functional endogenous mouse Vλ and Jλ gene segments with all, substantially all or a plurality of functional hVλ and hJλ gene segments, and a mouse Cλ gene. In certain embodiments, the mouse Cλ gene is Cλ2. In certain embodiments, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to mouse Cλ2.

[0088] In one embodiment, the mouse comprises a deletion of a Cκ gene and / or a Vκ and / or a Jκ gene segment, hi one embodiment, the mouse comprises a non-functional κ light chain locus.

[0089] In one aspect, a genetically modified mouse is provided that expresses an antibody, wherein more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 60%, more than 70%, more than 80%, or more than 90% of total IgG antibodies produced by the mouse comprise a lambda-derived variable domain, and the mouse expresses an antibody comprising a Kappa-derived variable domain fused to a mouse C Kappa region. In certain embodiments, about 15-40%, 20-40%, 25-40%, 30-40%, or 35-40% of total antibodies produced by the mouse comprise a lambda-derived variable domain.

[0090] In one embodiment, the λ-derived variable domain is derived from hVλ and hJλ. In one embodiment, the λ-derived variable domain is present in a light chain that comprises a mouse Cκ region. In a particular embodiment, the λ-derived variable domain is present in a light chain that comprises a mouse Cλ region. In another particular embodiment, the Cλ region is a Cλ2 region. In one embodiment, the κ-derived variable domain is derived from hVκ and hJκ, and in a particular embodiment, is present in a light chain that comprises a mouse Cκ region.

[0091] In one embodiment, an isolated DNA construct is provided that includes an upstream homology arm and a downstream homology arm, where the upstream and downstream homology arms target the construct to the mouse κ locus, and the construct includes a functional unrearranged hVλ segment and a functional unrearranged hJλ segment and a selection or marker sequence.

[0092] In one embodiment, an isolated DNA construct is provided that includes, in a 5' to 3' direction of transcription, a targeting arm for targeting a mouse λ sequence upstream of mouse Vλ2, a selection cassette flanked on the 5' and 3' sides by recombinase recognition sites, and a targeting arm for targeting a mouse λ sequence 3' to mouse Jλ2. In one embodiment, the selection cassette is a Frt'ed Hyg-TK cassette. In one embodiment, the 3' targeting arm includes mouse Cλ2, Jλ4, Cλ4, and mouse enhancer 2.4.

[0093] In one embodiment, an isolated DNA construct is provided that includes, in a 5' to 3' direction of transcription, a targeting arm for targeting the mouse λ locus 5' to Vλ1, a selection cassette flanked on the 5' and 3' sides by recombinase recognition sites, and a 3' targeting arm for targeting the mouse λ sequence 3' to mouse Cλ1. In one embodiment, the selection cassette is a loxed neomycin cassette. In one embodiment, the 3' targeting arm includes the mouse λ3' enhancer and the mouse λ3' enhancer 3.1.

[0094] In one embodiment, an isolated DNA construct is provided that includes, in a 5' to 3' direction of transcription, a targeting arm for targeting the mouse λ locus 5' to Vλ2, a selection cassette flanked on the 5' and 3' sides by recombinase recognition sites, and a 3' targeting arm for targeting the mouse λ sequence 3' to mouse Jλ2 and 5' to mouse Cλ2. In one embodiment, the selection cassette is a hygromycin-TK cassette flanked by Frt. In one embodiment, the 3' targeting arm includes the mouse Cλ2-Jλ4-Cλ4 gene segment and mouse λ enhancer 2.4.

[0095] In one embodiment, an isolated DNA construct is provided that includes, in a 5' to 3' direction of transcription, a targeting arm for targeting the mouse λ locus 5' to Vλ2, a selection cassette flanked on the 5' and 3' sides by recombinase recognition sites, a human genomic fragment that includes a contiguous region of the human λ light chain locus from hVλ3-12 downstream to the end of hJλ1, and a 3' targeting arm for targeting the mouse λ sequence 3' to mouse Jλ2. In one embodiment, the selection cassette is a neomycin cassette flanked by Frt. In one embodiment, the 3' targeting arm includes the mouse Cλ2-Jλ4-Cλ4 gene segment and the mouse λ enhancer 2.4.

[0096] In one embodiment, an isolated DNA construct is provided that includes a contiguous region of the human λ light chain locus from hVλ3-12 downstream to the end of hJλ1.

[0097] In one embodiment, an isolated DNA construct is provided that includes, in a 5' to 3' direction of transcription, a targeting arm for targeting the mouse λ locus 5' to Vλ2, a selection cassette flanked on the 5' and 3' sides by recombinase recognition sites, and a human genomic fragment that includes a contiguous region of the human λ light chain locus from hVλ3-27 downstream to the end of hVλ2-8. In one embodiment, the selection cassette is a hygromycin cassette flanked by Frts. In one embodiment, the human genomic fragment constitutes the 3' targeting arm. In a particular embodiment, the 3' targeting arm includes approximately 53 kb of the human λ light chain locus from hVλ3-12 downstream to the end of hVλ2-8.

[0098] In one embodiment, an isolated DNA construct is provided that includes a contiguous region of the human λ light chain locus from hVλ3-27 downstream to the end of hVλ3-12.

[0099] In one embodiment, an isolated DNA construct is provided, comprising, in a 5' to 3' direction of transcription, a targeting arm for targeting the mouse λ locus 5' to Vλ2, a selection cassette flanked on the 5' and 3' sides by recombinase recognition sites, a first human genomic fragment comprising a contiguous region of the human λ light chain locus from hVλ5-52 downstream of the end of hVλ1-40, a restriction enzyme site, and a second human genomic fragment comprising a contiguous region of the human λ light chain locus from hVλ3-29 downstream of the end of hVλ82K. In one embodiment, the selection cassette is a neomycin cassette flanked by Frt. In one embodiment, the restriction enzyme site is a site for a homing endonuclease. In a particular embodiment, the homing endonuclease is PI-SceI. In one embodiment, the second human genomic fragment is a 3' targeting arm. In a specific embodiment, the 3' targeting arm comprises approximately 27 kb of the human λ light chain locus from hVλ3-29 downstream to the end of hVλ82K.

[0100] In one embodiment, an isolated DNA construct is provided that includes a contiguous region of the human λ light chain locus from hVλ5-52 downstream to the end of hVλ1-40.

[0101] In one embodiment, an isolated DNA construct is provided, comprising, in a 5' to 3' direction of transcription, a targeting arm for targeting the mouse kappa locus 5' to the endogenous Vkappa gene segment, two juxtaposed recombinase recognition sites, a selection cassette 3' to the juxtaposed recombinase recognition sites, and a 3' targeting arm for targeting the mouse kappa sequence 5' to the kappa light chain variable gene segment. In one embodiment, the juxtaposed recombinase recognition sites are in opposite orientation to each other. In a particular embodiment, the recombinase recognition sites are different. In another particular embodiment, the recombinase recognition sites are loxP and lox511 sites. In one embodiment, the selection cassette is a neomycin cassette.

[0102] In one embodiment, an isolated DNA construct is provided, comprising, in the 5' to 3' direction of transcription, a targeting arm for targeting the mouse kappa locus 5' to the mouse Jkappa gene segment, a selection cassette, a recombinase recognition site 3' to the selection cassette, and a 3' targeting arm for targeting the mouse kappa sequence 3' to the mouse Jkappa gene segment and 5' to the mouse kappa intronic enhancer. In one embodiment, the selection cassette is a hygromycin-TK cassette. In one embodiment, the recombinase recognition site is in the same orientation as the selection cassette with respect to transcription. In a particular embodiment, the recombinase recognition site is a loxP site.

[0103] In one embodiment, an isolated DNA construct is provided that includes, in a 5' to 3' direction of transcription, a first mouse genomic fragment that includes a sequence 5' of an endogenous mouse Vκ gene segment, a first recombinase recognition site, a second recombinase recognition site, and a second mouse genomic fragment that includes a sequence 3' of the endogenous mouse Jκ gene segment and 5' of a mouse κ intronic enhancer.

[0104] In one embodiment, a genetically modified mouse is provided, wherein the genetic modification comprises modification with one or more of the DNA constructs described above or herein.

[0105] In one embodiment, there is provided a use of an isolated DNA construct as described herein for making a mouse as described herein.In one embodiment, there is provided a use of an isolated DNA construct as described herein in a method for making an antigen binding protein.

[0106] In one embodiment, a non-human stem cell is provided that comprises a targeting vector that comprises a DNA construct as described above and herein.In one embodiment, a non-human stem cell is provided, wherein the non-human stem cell is derived from a mouse as described herein.

[0107] In one embodiment, the non-human stem cells are embryonic stem (ES) cells. In a specific embodiment, the ES cells are mouse ES cells.

[0108] In one embodiment, there is provided a use of non-human stem cells as described herein to generate a mouse as described herein. In one embodiment, there is provided a use of non-human stem cells as described herein to generate an antigen binding protein.

[0109] In one aspect, a mouse embryo is provided, wherein the mouse embryo comprises a genetic modification as provided herein. In one embodiment, a host mouse embryo is provided that comprises a donor ES cell, wherein the donor ES cell comprises a genetic modification as described herein. In one embodiment, the mouse embryo is a pre-morula stage embryo. In a specific embodiment, the pre-morula stage embryo is a 4-cell stage embryo or an 8-cell stage embryo. In another specific embodiment, the mouse embryo is a blastocyst.

[0110] In one embodiment, there is provided a use of a mouse embryo as described herein to generate a mouse as described herein. In one embodiment, there is provided a use of a mouse embryo as described herein to generate an antigen binding protein.

[0111] In one embodiment, a non-human cell is provided, wherein the non-human cell comprises a rearranged immunoglobulin light chain gene sequence derived from a genetically modified mouse as described herein. In one embodiment, the cell is a B cell. In one embodiment, the cell is a hybridoma. In one embodiment, the cell encodes a somatically mutated immunoglobulin light chain variable domain and / or an immunoglobulin heavy chain variable domain.

[0112] In one embodiment, a non-human cell is provided, wherein the non-human cell comprises a rearranged immunoglobulin light chain gene sequence derived from a genetically modified mouse as described herein. In one embodiment, the cell is a B cell. In one embodiment, the cell is a hybridoma. In one embodiment, the cell encodes a somatically mutated immunoglobulin light chain variable domain and / or an immunoglobulin heavy chain variable domain.

[0113] In one embodiment, there is provided a use of a non-human cell as described herein to generate a mouse as described herein. In one embodiment, there is provided a use of a non-human cell as described herein to generate an antigen binding protein.

[0114] In one embodiment, (a) variable regions derived from hVλ and hJλ gene segments; and (b) mouse C L A mouse B cell is provided that expresses an immunoglobulin light chain comprising the gene. LThe gene is selected from a Cκ and a Cλ gene. In a specific embodiment, the Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to mouse Cλ2. In one embodiment, the mouse B cell further comprises (c) an hV H , hD H and (d) a variable region derived from hJ H In one embodiment, the B cells express a cognate heavy chain comprising a rearranged λ gene. In another embodiment, the B cells do not comprise a rearranged κ gene.

[0115] In one embodiment, a method for producing an antibody in a genetically modified mouse is provided, the method comprising: (a) exposing a genetically modified mouse to an antigen, wherein the mouse has a genome that includes at least one hVλ and at least one hJλ at an endogenous light chain locus, the endogenous light chain locus being a mouse C L (b) causing the genetically modified mouse to generate an immune response against the antigen; and (c) isolating from the mouse of (b) an antibody that specifically recognizes the antigen, or isolating from the mouse of (b) a cell that contains an immunoglobulin domain that specifically recognizes the antigen, wherein the antibody is selected from the hVλ, hJλ and mouse C L In a specific embodiment, the mouse C L The gene is a mouse Cκ gene.

[0116] In one embodiment, a method for producing antibodies in a genetically modified mouse is provided, the method comprising: (a) exposing a genetically modified mouse to an antigen, wherein the mouse has a genome comprising at least one hVλ at an endogenous κ locus and at least one hJλ at its κ locus, wherein the κ locus comprises a mouse Cκ gene; (b) causing the genetically modified mouse to generate an immune response to the antigen; and (c) isolating from the mouse of (b) an antibody that specifically recognizes the antigen, or isolating from the mouse of (b) a cell that comprises an immunoglobulin domain that specifically recognizes the antigen, wherein the antibody comprises a light chain derived from an hVλ, an hJλ, and a mouse Cκ gene.

[0117] In one embodiment, the κ light chain constant gene is selected from a human Cκ gene and a mouse Cκ gene.

[0118] In one embodiment, a method for producing an antibody in a genetically modified mouse is provided, the method comprising: (a) exposing a genetically modified mouse to an antigen, wherein the mouse has a genome comprising at least one hVλ at a λ light chain locus and at least one Jλ at its λ light chain locus, wherein the λ light chain locus comprises a mouse Cλ gene; (b) causing the genetically modified mouse to generate an immune response to the antigen; and (c) isolating from the mouse of (b) an antibody that specifically recognizes the antigen, or isolating from the mouse of (b) a cell that comprises an immunoglobulin domain that specifically recognizes the antigen, or identifying in the mouse of B a nucleic acid sequence encoding a heavy and / or light chain variable domain that binds to the antigen, wherein the antibody comprises a light chain derived from an hVλ, an hJλ, and a mouse Cλ gene.

[0119] In one embodiment, the λ light chain constant gene is selected from a human Cλ gene and a mouse Cλ gene. In one embodiment, the λ light chain constant gene is a human Cλ gene. In a specific embodiment, the human Cλ gene is selected from Cλ1, Cλ2, Cλ3 and Cλ7. In one embodiment, the λ light chain constant gene is a mouse Cλ gene. In a specific embodiment, the mouse Cλ gene is selected from Cλ1, Cλ2 and Cλ3. In a more specific embodiment, the mouse Cλ gene is Cλ2. In another specific embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2.

[0120] In one embodiment, a method is provided for generating rearranged antibody genes in a genetically modified mouse, the method comprising: (a) exposing the genetically modified mouse to an antigen, wherein the genetic modification comprises hVλ and hJλ at an endogenous light chain locus, the endogenous light chain locus being a mouse C L and (b) identifying a rearranged immunoglobulin gene in said mouse, said rearranged immunoglobulin gene comprising a λ light chain variable region gene segment and a C light chain variable region gene segment or a functional fragment thereof. L The term includes a gene or a functional fragment thereof.

[0121] In one embodiment, the method further comprises cloning nucleic acid sequences encoding heavy and / or light chain variable regions from the mouse, wherein the heavy and / or light chain variable regions are selected from human Vλ and mouse Cλ. L The antibody is derived from an antibody comprising

[0122] In one embodiment, the mouse C L The gene or a functional fragment thereof is L Genes and mouse C Lgenes or functional fragments thereof.

[0123] In one embodiment, a method is provided for making rearranged antibody genes in a genetically modified mouse comprising: (a) exposing a genetically modified mouse to an antigen, wherein the genetic modification comprises an hVλ and an hJλ at a κ light chain locus, wherein the κ light chain locus comprises a mouse Cκ gene, or a functional fragment thereof; and (b) identifying rearranged immunoglobulin genes in the mouse, wherein the rearranged immunoglobulin genes comprise a λ light chain variable region gene segment and a Cκ gene, or a functional fragment thereof.

[0124] In one embodiment, the kappa light chain constant gene or a functional fragment thereof is selected from a human Cκ gene and a mouse Cκ gene or a functional fragment thereof.

[0125] In one embodiment, the method further comprises cloning a nucleic acid sequence encoding a heavy and / or light chain variable region from the mouse, wherein the heavy and / or light chain variable region is derived from an antibody comprising a human Vλ and a mouse Cκ.

[0126] In one embodiment, a method is provided for making rearranged antibody genes in a genetically modified mouse comprising: (a) exposing a genetically modified mouse to an antigen, wherein the genetic modification comprises an hVλ and an hJλ at a mouse λ light chain locus, wherein the λ light chain locus comprises a mouse Cλ gene, or a functional fragment thereof; and (b) identifying rearranged immunoglobulin genes in the mouse, wherein the rearranged immunoglobulin genes comprise a λ light chain variable region gene segment and a Cλ gene, or a functional fragment thereof.

[0127] In one embodiment, the lambda light chain constant gene or a functional fragment thereof is selected from a human Cλ gene and a mouse Cλ gene or a functional fragment thereof, in a specific embodiment, the lambda light chain constant gene is a mouse Cλ gene or a functional fragment thereof.

[0128] In one embodiment, the method further comprises cloning a nucleic acid sequence encoding a heavy and / or light chain variable region from the mouse, wherein the heavy and / or light chain variable region is derived from an antibody comprising a human Vλ and a mouse Cλ.

[0129] In one embodiment, a method for producing antibodies is provided, comprising exposing a mouse as described herein to an antigen, causing the mouse to mount an immune response including production of an antibody that specifically binds to the antigen, identifying a rearranged nucleic acid sequence encoding a heavy chain in the mouse and a rearranged nucleic acid sequence encoding a cognate light chain variable domain sequence of an antibody in the mouse, wherein the antibody specifically binds to the antigen, and using the nucleic acid sequences of the heavy and light chain variable domains fused to a human constant domain to produce the desired antibody, wherein the desired antibody is a C L In one embodiment, the V domain is a human domain and is a light chain comprising a Vλ domain fused to a C L In one embodiment, the V domain is a human or mouse C domain. In one embodiment, the V domain is a mouse domain and is L The domain is a human or mouse Cκ domain.

[0130] In one embodiment, a method for making antibodies is provided comprising exposing a mouse as described herein to an antigen, causing the mouse to mount an immune response comprising production of an antibody that specifically binds to the antigen, identifying a rearranged nucleic acid sequence encoding a heavy chain in the mouse and a rearranged nucleic acid sequence encoding a cognate light chain variable domain sequence of an antibody in the mouse, where the antibody specifically binds to the antigen, and using the nucleic acid sequences of the heavy and light chain variable domains fused to a nucleic acid sequence of a human constant domain to make the desired antibody, where the desired antibody comprises a light chain comprising a Vλ domain fused to a Cκ domain.

[0131] In one embodiment, a method for making antibodies is provided that includes exposing a mouse as described herein to an antigen, causing the mouse to mount an immune response including production of an antibody that specifically binds to the antigen, identifying in the mouse a rearranged nucleic acid sequence encoding a heavy chain variable domain and a rearranged nucleic acid sequence encoding a cognate light chain variable domain sequence of an antibody, which specifically binds to the antigen, and using the nucleic acid sequence fused to a nucleic acid sequence encoding a human heavy chain constant domain and a human light chain constant domain to make an antibody derived from the human sequences, where the antibody that specifically binds to the antigen comprises a light chain comprising a human Vλ domain fused to a mouse Cλ region.

[0132] In one embodiment, the mouse Cλ region is selected from Cλ1, Cλ2 and Cλ3. In a specific embodiment, the mouse Cλ region is Cλ2.

[0133] In one embodiment, a method for generating a rearranged antibody light chain variable region gene sequence is provided, the method comprising: (a) exposing a mouse to an antigen as described herein; (b) mounting an immune response in the mouse; (c) subjecting the mouse to an antigen-binding domain; Lidentifying a cell in the mouse that comprises a nucleic acid sequence encoding a rearranged human Vλ domain sequence fused to a human V domain, H Domain and Mouse C H (d) extracting from the cell a nucleic acid sequence encoding a human Vλ domain and a cognate human Vλ domain, the cell also encoding a cognate heavy chain comprising the human Vλ domain, the cell expressing an antibody that binds to the antigen; H (e) cloning a nucleic acid sequence encoding a human Vλ domain and a cognate human V H The process includes generating fully human antibodies using cloned nucleic acid sequences encoding the domains.

[0134] In one embodiment, a method for generating a rearranged antibody light chain variable region gene sequence is provided, the method comprising: (a) exposing a mouse to an antigen as described in this disclosure; (b) mounting an immune response in the mouse; and (c) identifying a cell in the mouse that comprises a nucleic acid sequence encoding a mouse Cκ domain and a nucleic acid sequence encoding a rearranged human Vλ domain sequence contiguous on the same nucleic acid molecule, wherein the cell is a human V H Domain and Mouse C H (d) extracting from the cell a nucleic acid sequence encoding a human Vλ domain and a cognate human Vλ domain, the cell also encoding a cognate heavy chain comprising the human Vλ domain, the cell expressing an antibody that binds to the antigen; H (e) cloning a nucleic acid sequence encoding a human Vλ domain and a cognate human V H The process includes generating fully human antibodies using cloned nucleic acid sequences encoding the domains.

[0135] In one embodiment, a method for generating a rearranged antibody light chain variable region gene sequence is provided, the method comprising: (a) exposing a mouse as described herein to an antigen; (b) causing the mouse to mount an immune response to the antigen; (c) identifying a cell in the mouse that comprises DNA encoding a rearranged human Vλ domain sequence fused to a mouse Cλ domain, wherein the cell is a human Vλ domain. H Domain and Mouse C H (d) extracting from the cell a nucleic acid sequence encoding a rearranged human Vλ domain and a cognate human Vλ domain, the cell also encoding a cognate heavy chain comprising the rearranged human Vλ domain, the cell expressing an antibody that binds to the antigen; H (e) cloning a nucleic acid sequence encoding a human Vλ domain and a cognate human V H The method includes generating a fully human antibody using a cloned nucleic acid sequence encoding the mouse Cλ domain. In one embodiment, the mouse Cλ domain is mouse Cλ2. In a specific embodiment, the mouse Cλ domain is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to mouse Cλ2.

[0136] In one embodiment, the endogenous light chain constant region (C L A genetically modified mouse is provided that expresses a human λ-derived light chain fused to a mouse C L In one embodiment, the mouse C L In one embodiment, the mouse C domain is selected from a Cκ domain and a Cλ domain. L In one embodiment, the mouse C LThe domain is a Cλ domain. In certain embodiments, the Cλ domain is Cλ2. In certain embodiments, the mouse Cλ domain is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2.

[0137] In one aspect, a genetically modified mouse is provided that comprises a modified endogenous κ or λ light chain locus as described herein that expresses multiple immunoglobulin λ light chains associated with multiple immunoglobulin heavy chains. In one embodiment, the heavy chain comprises a human sequence. In various embodiments, the human sequence comprises a variable sequence, CH 1 , Hinge, C.H. 2 , C.H. 3 and combinations thereof. In one embodiment, the plurality of immunoglobulin lambda light chains comprises human sequences. In various embodiments, the human sequences are selected from variable sequences, constant sequences, and combinations thereof. In one embodiment, the mouse comprises a disabled endogenous immunoglobulin locus and expresses heavy chains and / or lambda light chains from a transgene or an extrachromosomal episome. In one embodiment, the mouse expresses some or all of the endogenous mouse heavy chain gene segments (i.e., V, D, J), and / or some or all of the endogenous mouse heavy chain constant sequences (e.g., CH, 1 , Hinge, C.H. 2 , C.H. 3 or combinations thereof) and / or replacement at the endogenous mouse locus of some or all of the endogenous mouse light chain sequences (e.g., V, J, constant or combinations thereof) with one or more human immunoglobulin sequences.

[0138] In one aspect, a mouse suitable for producing antibodies having a human λ-derived light chain is provided, wherein all or substantially all antibodies produced in the mouse are expressed with a human λ-derived light chain. In one embodiment, the human λ-derived light chain is expressed from an endogenous light chain locus. In one embodiment, the endogenous light chain locus is a κ light chain locus. In a specific embodiment, the κ light chain locus is a mouse κ light chain locus.

[0139] In one embodiment, a method for generating a λ-derived light chain for a human antibody is provided, the method comprising obtaining light and heavy chain sequences from a mouse as described herein and using the light and heavy chain sequences in generating a human antibody.

[0140] In one embodiment, a method for making an antigen binding protein is provided, the method comprising exposing a mouse to an antigen as described herein; mounting an immune response in the mouse; and obtaining from the mouse an antigen binding protein that binds to the antigen, or obtaining from the mouse a sequence for use in making an antigen binding protein that binds to the antigen.

[0141] In one aspect, a cell derived from a mouse as described herein is provided. In one embodiment, the cell is selected from an embryonic stem cell, a pluripotent cell, an induced pluripotent cell, a B cell, and a hybridoma.

[0142] In one embodiment, a cell is provided that comprises a genetic modification as described herein.In one embodiment, the cell is a mouse cell.In one embodiment, the cell is selected from a hybridoma and a quadroma.In one embodiment, the cell expresses an immunoglobulin light chain that comprises a human λ variable sequence fused with a mouse constant sequence.In a particular embodiment, the mouse constant sequence is a mouse κ constant sequence.

[0143] In one embodiment, tissue derived from a mouse as described herein is provided.

[0144] In one embodiment, the use of a mouse or cell as described herein to generate an antigen-binding protein is provided. In one embodiment, the antigen-binding protein is a human protein. In one embodiment, the human protein is a human antibody.

[0145] In one embodiment, an antigen-binding protein is provided that is produced by a mouse, cell, tissue, or method as described herein.In one embodiment, the antigen-binding protein is a human protein.In one embodiment, the human protein is a human antibody.

[0146] Any of the embodiments and aspects described herein can be used in combination with each other unless otherwise indicated or apparent from the context. Other embodiments will become apparent to those of skill in the art upon review of the following description. For example, the present invention provides the following: (Item A1) A mouse comprising a nucleic acid sequence of an immunoglobulin lambda light chain variable region (Vλ) contiguous with a nucleic acid sequence of a mouse immunoglobulin kappa light chain constant region and at least one nucleic acid sequence of a J (J). (Item A2) The mouse of item A1, wherein the mouse lacks a functional mouse Vκ gene segment and / or a functional mouse Jκ gene segment. (Item A3) The mouse of item A1, wherein the Vλ is a human Vλ (hVλ) and the J is a human Jλ (hJλ). (Item A4) The mouse of paragraph A3, wherein the hVλ and the hJλ are unrearranged gene segments. (Item A5) The mouse of paragraph A4, comprising a plurality of unrearranged hVλ gene segments and at least one hJλ gene segment. (Item A6) The mouse of item A5, wherein the plurality of unrearranged hVλ gene segments is at least 12 gene segments, at least 28 gene segments, or at least 40 gene segments. (Item A7) The mouse of item A3, wherein the at least one hJλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof. (Item A8) The mouse of paragraph A1, wherein the endogenous mouse immunoglobulin lambda light chain locus is deleted, in whole or in part. (Item A9) The mouse of paragraph A1, wherein the nucleic acid sequence of the mouse immunoglobulin κ light chain constant region is present in an endogenous mouse immunoglobulin κ light chain locus. (Item A10) The mouse according to item A1, wherein about 10% to about 45% of B cells of the mouse express an antibody comprising an immunoglobulin light chain comprising a human immunoglobulin λ light chain variable (Vλ) domain and a mouse immunoglobulin κ light chain constant (Cκ) domain. (Item A11) The human lambda variable domain comprises: (chemical 1) The mouse of item A10, wherein the nucleic acid sequence of a rearranged hVλ / hJλ is selected from the group consisting of: TIFF2025027148000002.tif41154. (Item A12) The mouse of paragraph A1, further comprising a human Vκ-Jκ intergenic region derived from a human immunoglobulin κ light chain locus, wherein the human Vκ-Jκ intergenic region is contiguous with a nucleic acid sequence of the Vλ and a nucleic acid sequence of the J. (Item A13) The mouse of item A12, wherein the human Vκ-Jκ intergenic region is located between a nucleic acid sequence of the Vλ and a nucleic acid sequence of the J. (Item A14) (a) at least 12 to at least 40 unrearranged human immunoglobulin λ light chain variable region gene segments and at least one human Jλ gene segment at an endogenous mouse immunoglobulin light chain locus; (b) a human Vκ-Jκ intergenic sequence disposed between the at least 12 to at least 40 human immunoglobulin light chain variable region gene segments and the at least one human Jλ nucleic acid sequence; 1. A mouse comprising: (Item A15) A mouse that expresses an antibody comprising an immunoglobulin light chain comprising an immunoglobulin λ variable sequence and an immunoglobulin κ light chain constant sequence. (Item A16) The mouse of item A15, wherein the mouse exhibits a ratio of κ usage to λ usage of about 1:1. (Item A17) The mouse of item A16, wherein a population of immature B cells obtained from bone marrow of said mouse exhibits a ratio of κ to λ usage of about 1:1. (Item A18) Use of a mouse according to paragraphs A1-17 to generate an antigen-binding protein. (Item A19) An antigen-binding protein produced using a mouse described in paragraphs A1-17. (Item A20) A cell or tissue derived from a mouse according to any one of paragraphs A1 to A17, the cell or tissue comprising a nucleic acid sequence of an immunoglobulin λ light chain variable region (Vλ) contiguous with a nucleic acid sequence of an endogenous immunoglobulin κ light chain constant region and at least one nucleic acid sequence of J (J). (Item A21) 1. A method for generating a somatically mutated antibody that binds to an antigen of interest, the method comprising: (a) exposing the mouse according to any one of items A1 to A17 to an antigen of interest; (b) obtaining one or more B lymphocytes of the mouse of (a), wherein the one or more B lymphocytes produce an antibody that binds to the antigen of interest; and (c) identifying a nucleic acid sequence encoding an immunoglobulin light chain of the antibody of (b), the immunoglobulin light chain comprising a human immunoglobulin lambda light chain variable domain and a mouse immunoglobulin kappa constant domain; and (d) using the nucleic acid sequence of a human immunoglobulin light chain constant region and the nucleic acid sequence of (c) to generate a human antibody that binds to the antigen of interest. The method includes: [Brief description of the drawings]

[0147] [Figure 1] FIG. 1 shows a detailed illustration, not to scale, of the human λ light chain locus including clusters of Vλ gene segments (A, B and C) and Jλ and Cλ region pairs (JC pairs). [Diagram 2] FIG. 2 shows, not to scale, a general illustration of the targeting strategy used to inactivate the endogenous mouse λ light chain locus. [Diagram 3] FIG. 3 shows a general illustration, not to scale, of the targeting strategy used to inactivate the endogenous mouse kappa light chain locus. [Figure 4A] FIG. 4A shows a general schematic, not to scale, of the initial targeting vector for targeting the endogenous mouse λ light chain locus with a human λ light chain sequence comprising 12 hVλ gene segments and an hJλ1 gene segment (12 / 1-λ targeting vector). [Figure 4B]FIG. 4B shows a general, not to scale, illustration of four initial targeting vectors for targeting an endogenous mouse κ light chain locus with a human λ light chain sequence comprising 12 hVλ gene segments and an hJλ1 gene segment (12 / 1-κ targeting vector), 12 hVλ gene segments and hJλ1, 2, 3, and 7 gene segments (12 / 4-κ targeting vector), 12 hVλ gene segments, a human Vκ-Jκ genomic sequence, and an hJλ1 gene segment (12(κ)1-κ targeting vector), and 12 hVλ gene segments, a human Vκ-Jκ genomic sequence, and hJλ1, 2, 3, and 7 gene segments (12(κ)4-κ targeting vector). [Figure 5A] FIG. 5A shows a general illustration, not to scale, of the targeting strategy for the progressive insertion of 40 hVλ gene segments and a single hJλ gene segment into the mouse λ light chain locus. [Figure 5B] FIG. 5B shows a general illustration, not to scale, of the targeting strategy for the progressive insertion of 40 hVλ gene segments and a single hJλ gene segment into the mouse κ locus. [Figure 6] FIG. 6 shows a general, not to scale, illustration of the targeting and molecular engineering steps used to generate unique human λ-κ hybrid targeting vectors for constructing hybrid light chain loci containing human κ intergenic sequences, multiple hJλ gene segments, or both. [Figure 7A] FIG. 7A shows a general illustration, not to scale, of the locus structure for a modified mouse λ light chain locus comprising 40 hVλ gene segments and a single hJλ gene segment operably linked to the endogenous Cλ2 gene. [Figure 7B]FIG. 7B shows a generalized, not to scale, representation of the locus structures for four independent modified mouse κ light chain loci comprising 40 hVλ gene segments and one or four hJλ gene segments, with or without contiguous human Vκ-Jκ genomic sequences operably linked to the endogenous Cκ gene. [Figure 8A] FIG. 8A shows contour plots of Igλ+ and Igκ+ splenocytes gated on CD19+ from a wild-type mouse (WT), a mouse homozygous for 12 hVλ gene segments and 4 hJλ gene segments comprising the human Vκ-Jκ genomic sequence (12hVλ-VκJκ-4hJλ), and a mouse homozygous for 40 hVλ gene segments and 1 hJλ gene segment (40hVλ-1hJλ). [Figure 8B] Figure 8B shows the total number of CD19+ B cells in spleens recovered from wild-type (WT), mice homozygous for 12 hVλ gene segments and 4 hJλ gene segments comprising the human Vκ-Jκ genomic sequence (12hVλ-VκJκ-4hJλ), and mice homozygous for 40 hVλ gene segments and 1 hJλ gene segment (40hVλ-1hJλ). [Figure 9A] The top panel of Figure 9A shows contour plots of splenocytes gated on singlets and stained for B and T cells (CD19+ and CD3+, respectively) from a wild-type mouse (WT) and a mouse homozygous for 40 hVλ and 4 Jλ gene segments comprising the human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ). The bottom panel shows contour plots of splenocytes gated on CD19+ and stained for Igλ+ and Igκ+ expression from a wild-type mouse (WT) and a mouse homozygous for 40 hVλ and 4 Jλ gene segments comprising the human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ). [Figure 9B]FIG. 9B shows the total numbers of CD19+, CD19+Igκ+, and CD19+Igλ+ B cells in spleens recovered from wild-type mice (WT) and mice homozygous for 40 hVλ and 4 Jλ gene segments comprising the human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ). [Figure 9C] Figure 9C shows contour plots of splenocytes gated for CD19+ and stained for immunoglobulin D (IgD) and immunoglobulin M (IgM) from wild-type mice (WT) and mice homozygous for 40 hVλ and 4 Jλ gene segments containing human Vκ-Jκ genomic sequences (40hVλ-VκJκ-4hJλ). In each of the contour plots, mature B cells (72 for WT, 51 for 40hVλ-VκJκ-4hJλ) and transitional B cells (13 for WT, 22 for 40hVλ-VκJκ-4hJλ) are displayed. [Figure 9D] FIG. 9D shows the total numbers of CD19+ B cells, transitional B cells (CD19+IgMhiIgDlo), and mature B cells (CD19+IgMloIgDhi) in spleens recovered from wild-type mice (WT) and mice homozygous for 40 hVλ and 4 Jλ gene segments comprising the human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ). [Figure 10A]The top panel of FIG. 10A shows contour plots of bone marrow stained for B and T cells (CD19+ and CD3+, respectively) from a wild-type mouse (WT) and a mouse homozygous for 40 hVλ and 4 Jλ gene segments containing human Vκ-Jκ genomic sequences (40hVλ-VκJκ-4hJλ). The bottom panel shows contour plots of bone marrow gated for CD19+ and stained for ckit+ and CD43+ from a wild-type mouse (WT) and a mouse homozygous for 40 hVλ and 4 Jλ gene segments containing human Vκ-Jκ genomic sequences (40hVλ-VκJκ-4hJλ). The contour plots in the bottom panel show pro B cells and pre B cells. [Figure 10B] FIG. 10B shows the numbers of pro-B cells (CD19+CD43+ckit+) and pre-B cells (CD19+CD43-ckit-) in bone marrow recovered from femurs of wild-type mice (WT) and mice homozygous for 40 hVλ and 4 Jλ gene segments comprising the human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ). [Figure 10C] Figure 10C shows contour plots of bone marrow gated on singlets stained for immunoglobulin M (IgM) and B220 from a wild-type mouse (WT) and a mouse homozygous for 40 hVλ and 4 Jλ gene segments containing the human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ). In each of the contour plots, immature, mature and pro / pre-B cells are displayed. [Figure 10D] FIG. 10D shows the total numbers of immature B cells (B220intIgM+) and mature B cells (B220hiIgM+) in bone marrow isolated from femurs of wild-type mice (WT) and mice homozygous for 40 hVλ and 4 Jλ gene segments comprising the human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ). [Figure 10E]FIG. 10E shows contour plots of bone marrow gated on immature B cells (B220intIgM+) and mature B cells (B220hiIgM+) stained for Igλ and Igκ expression isolated from femurs of wild-type mice (WT) and mice homozygous for 40 hVλ and 4 Jλ gene segments comprising the human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ). [Figure 11] Figure 11 shows the nucleotide sequence alignment of the Vλ-Jλ-Cκ junctions of 18 independent RT-PCR clones amplified from splenocyte RNA of mice carrying a human λ light chain gene sequence at the endogenous mouse κ light chain locus. A6 = SEQ ID NO: 57; B6 = SEQ ID NO: 58; F6 = SEQ ID NO: 59; B7 = SEQ ID NO: 60; E7 = SEQ ID NO: 61; F7 = SEQ ID NO: 62; C8 = SEQ ID NO: 63; E12 = SEQ ID NO: 64; 1-4 = SEQ ID NO: 65; 1-20 = SEQ ID NO: 66; 3B43 = SEQ ID NO: 67; 5-8 = SEQ ID NO: 68; 5-19 = SEQ ID NO: 69; 1010 = SEQ ID NO: 70; 11A1 = SEQ ID NO: 71; 7A8 = SEQ ID NO: 72; 3A3 = SEQ ID NO: 73; 2-7 = SEQ ID NO: 74. Lowercase bases indicate non-germline bases resulting from mutations and / or N additions during recombination. Consensus amino acids within the framework 4 region (FWR4) encoded by the nucleotide sequences of hJλ1 and mouse Cκ are indicated below the sequence alignment. [Figure 12]Figure 12 shows the nucleotide sequence alignment of the Vλ-Jλ-Cκ junctions of 12 independent RT-PCR clones amplified from splenocyte RNA of mice carrying human λ light chain gene sequences containing contiguous human Vκ-Jκ genomic sequences at the endogenous mouse κ light chain locus. 5-2=SEQ ID NO:87; 2-5=SEQ ID NO:88; 1-3=SEQ ID NO:89; 4B-1=SEQ ID NO:90; 3B-5=SEQ ID NO:91; 7A-1=SEQ ID NO:92; 5-1=SEQ ID NO:93; 4A-1=SEQ ID NO:94; 11A-1=SEQ ID NO:95; 5-7=SEQ ID NO:96; 5-4=SEQ ID NO:97; 2-3=SEQ ID NO:98. Lowercase bases indicate non-germline bases resulting from mutations and / or N additions during recombination. The consensus amino acids within the framework 4 region (FWR4) encoded by the nucleotide sequences of each of the human Jλ and mouse Cκ are displayed below the sequence alignment. [Figure 13] Figure 13 shows the nucleotide sequence alignment of the Vλ-Jλ-Cλ junctions of three independent RT-PCR clones amplified from splenocyte RNA of mice carrying human λ light chain gene sequences at the endogenous mouse λ light chain locus. 2D1 = SEQ ID NO: 101; 2D9 = SEQ ID NO: 102; 3E15 = SEQ ID NO: 103. Lowercase bases indicate non-germline bases resulting from mutations and / or N additions during recombination. Consensus amino acids within the framework 4 region (FWR4) encoded by the nucleotide sequences of hJλ1 and mouse Cλ2 are indicated below the sequence alignment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0148] Detailed Description Although specific features of various embodiments are disclosed in detail, the description of specific aspects, embodiments and examples does not limit the subject matter of the claims; the claims describe the scope of the invention. All terms and phrases used in this disclosure include the meanings normally given to them in the art.

[0149] The term "contiguous" includes reference to being on the same nucleic acid molecule, for example, two nucleic acid sequences are "contiguous" when they are on the same nucleic acid molecule but are interrupted by another nucleic acid sequence. For example, the last codon of a rearranged V(D)J sequence is not directly following the first codon of the constant region sequence, but the V(D)J sequence is "contiguous" with the constant region gene sequence. In another example, two V gene segment sequences are "contiguous" when they are on the same genomic fragment, but they may be separated by a sequence that does not code for the V region codons, for example, they may be separated by a regulatory sequence, such as a promoter or other non-coding sequence. In one embodiment, a contiguous sequence includes a genomic fragment that includes genomic sequences arranged as they are found in a wild-type genome.

[0150] The phrase "derived from," when used in reference to a variable region "derived from" a referenced gene or gene segment, includes tracing the sequence back to the particular unrearranged gene segment or to the gene segments that have been rearranged to form the gene expressing that variable domain (accounting for splice differences and somatic mutations, if applicable).

[0151] The term "functional" when used in reference to a variable region gene segment or joining gene segment refers to the use of an expressed antibody repertoire; for example, in humans, Vλ gene segments 3-1, 4-3, 2-8, etc. are functional, while Vλ gene segments 3-2, 3-4, 2-5, etc. are non-functional.

[0152] The "heavy chain locus" is a region of the heavy chain variable H ), heavy chain diversity (D H ), heavy chain linkage (J H ) and heavy chain constant (C H ) region's DNA sequence is found, including the location on a chromosome, for example, on a mouse chromosome.

[0153] A "κ locus" includes a chromosomal location, e.g., a location on a mouse chromosome, at which the DNA sequences for the κ variable (Vκ), κ joining (Jκ), and κ constant (Cκ) regions are found in wild-type mice.

[0154] A "lambda locus" includes a chromosomal location, eg, a location on a mouse chromosome, at which the DNA sequences of the lambda variable (Vlambda), lambda joining (Jlambda), and lambda constant (Clambda) regions are found in wild-type mice.

[0155] The term "unrearranged" includes the state of an immunoglobulin locus in which the V and J gene segments (as well as the D gene segments in the case of heavy chains) are maintained separately but can be linked to form rearranged V(D)J genes comprising a single V,(D),J of the V(D)J repertoire.

[0156] Mice expressing human lambda variable domains Mice expressing fully human or partially human and partially mouse antibodies have been previously reported. VELOCIMMUNE® engineered mice contain a replacement of unrearranged V(D)J gene segments with human V(D)J gene segments at the endogenous mouse locus. VELOCIMMUNE® mice express chimeric antibodies with human variable domains and mouse constant domains (see, e.g., U.S. Patent No. 7,605,237). Most other reports concern mice expressing fully human antibodies from fully human transgenes in mice with disabled endogenous immunoglobulin loci.

[0157] Antibody light chains are encoded by one of two separate loci: kappa (κ) and lambda (λ). Mouse antibody light chains are predominantly of the κ type. Mice producing mouse antibodies and modified mice producing fully human or chimeric human-mouse antibodies show a bias in light chain usage. Humans also show a light chain bias, but it is less pronounced than mice; the ratio of κ to λ light chains in mice is about 95:5, while in humans it is about 60:40. It is believed that the more pronounced bias in mice does not seriously affect antibody diversity, since the λ variable loci are less diverse in mice to begin with. This is not the case in humans. The human λ light chain loci are highly diverse.

[0158] The human λ light chain locus spans 1,000 kb and contains more than 80 genes encoding variable (V) or joining (J) segments (Figure 1). Within the human λ light chain locus, the majority of all observed Vλ domains are encoded by gene segments 1-40, 1-44, 2-8, 2-14, and 3-21. Overall, approximately 30 human Vλ gene segments are considered functional. There are seven Jλ gene segments, of which only four (Jλ1, Jλ2, Jλ3, and Jλ7) are generally considered to be functional Jλ gene segments.

[0159] The human λ light chain locus is similar in structure to both the mouse and human κ loci in that it has several variable region gene segments that can recombine to form functional light chain proteins. The human λ light chain locus contains approximately 70 V gene segments and seven Jλ-Cλ gene segment pairs. Only four of these Jλ-Cλ gene segment pairs appear to be functional. In some alleles, the fifth Jλ-Cλ gene segment pair is reportedly a pseudogene (Cλ6). The 70 Vλ gene segments appear to contain 38 functional gene segments. The 70 Vλ sequences are arranged into three clusters, all of which contain distinct members of different V gene family groups (clusters A, B, and C; FIG. 1). This represents a potentially rich source of relatively untapped diversity for generating antibodies with human V regions in non-human animals.

[0160] In stark contrast, the mouse λ light chain locus contains only two or three mouse Vλ region gene segments (depending on the strain) (Figure 2). At least for this reason, the large κ bias in mice is not thought to be particularly detrimental to overall antibody diversity.

[0161] According to the published map of the mouse λ light chain locus, the locus consists essentially of two clusters of gene segments within approximately 200 kb (Figure 2). The two clusters contain two sets of V, J, and C genes capable of independent rearrangements: Vλ2-Jλ2-Cλ2-Jλ4-Cλ4 and Vλ1-Jλ3-Cλ3-Jλ1-Cλ1. Vλ2 has been found to recombine with all Jλ gene segments, whereas Vλ1 appears to recombine exclusively with Cλ1. Cλ4 is thought to be a pseudogene lacking a splice site.

[0162] The mouse kappa light chain locus is significantly different. The structure and number of gene segments that participate in recombination events to yield functional light chain proteins from the mouse kappa locus is considerably more complex (Figure 3). Thus, mouse lambda light chains do not contribute significantly to the diversity of the antibody population in a typical mouse.

[0163] In particular, exploiting the rich diversity of human λ light chain loci in mice could provide a source for a more complete human repertoire of light chain V domains. Previous attempts to exploit this diversity have used human transgenes that contain chunks of human λ light chain loci randomly integrated into the mouse genome (see, for example, U.S. Patent Nos. 6,998,514 and 7,435,871). Mice containing these randomly integrated transgenes reportedly express complete human λ light chains, however, in some cases, one or both endogenous light chain loci remain intact. This situation is undesirable, since the human λ light chain sequences compete with mouse light chains (κ or λ) in the expressed antibody repertoire of the mouse.

[0164] In contrast, we describe genetically modified mice that can express one or more λ light chain nucleic acid sequences directly from a mouse light chain locus (including by replacement at the endogenous mouse light chain locus). The genetically modified mice that can express human λ light chain sequences from an endogenous locus can be further bred with mice that contain a human heavy chain locus and can therefore be used to express antibodies that contain V regions (heavy and light chains) that are fully human. In various embodiments, the V regions are expressed with mouse constant regions. In various embodiments, the endogenous mouse immunoglobulin gene segments are absent and the V regions are expressed with human constant regions. These antibodies will prove to be beneficial in numerous applications, both diagnostic and therapeutic.

[0165] A number of advantages can be realized for various embodiments in which binding proteins derived from human Vλ and Jλ gene segments are expressed in mice. Advantages can be realized by placing a human λ sequence at an endogenous light chain locus, e.g., the mouse κ or λ locus. Antibodies produced from such mice have the same structure as mouse C L The mouse may have a light chain that comprises a human Vλ domain fused to a human Cκ or Cλ region, specifically a mouse Cκ or Cλ region. L They may also express human Vλ domains suitable for identification and cloning for use with the regions (specifically, Cκ and / or Cλ regions). Because B cell development in such mice is otherwise normal, it is possible to generate compatible Vλ domains (including somatically mutated Vλ domains) in the context of a Cλ or Cκ region.

[0166] Genetically modified mice are described that contain unrearranged Vλ gene segments at the immunoglobulin κ or λ light chain loci. Mice are described that express antibodies that contain light chains with human Vλ domains fused to Cκ and / or Cλ regions.

[0167] Sterile transcripts at the immunoglobulin kappa light chain locus Variations on the theme of expressing human immunoglobulin λ sequences in mice are reflected in various embodiments of genetically modified mice capable of such expression.Thus, in some embodiments, the genetically modified mouse comprises certain non-coding sequences from human locus.In one embodiment, the genetically modified mouse comprises human Vλ and Jλ gene segments at the endogenous κ light chain locus, and further comprises a human κ light chain genomic fragment.In a particular embodiment, the human κ light chain genomic fragment is a non-coding sequence that is naturally found between the human Vκ gene segment and the human Jκ gene segment.

[0168] The human and mouse kappa light chain loci contain sequences that encode null transcripts lacking either an initiation codon or an open reading frame and that are identified as elements controlling the transcription of the kappa light chain locus. These null transcripts arise from intergenic sequences located downstream or 3' of the most proximal Vkappa gene segment and upstream or 5' of the kappa light chain intronic enhancer (Ekappa) that is located upstream of the kappa light chain constant region gene (Ckappa). The null transcripts arise from rearrangement of the intergenic sequences, resulting in the formation of a VkappaJkappa1 segment fused to Ckappa.

[0169] Replacement of the κ light chain locus upstream of the Cκ gene can remove the intergenic region that encodes an invalid transcript. Thus, in various embodiments, replacement of the mouse κ light chain sequence upstream of the mouse Cκ gene with a human λ light chain gene segment can result in a humanized mouse κ light chain locus that contains human Vλ and Jλ gene segments but does not contain the κ light chain intergenic region that encodes an invalid transcript.

[0170] As described herein, humanization of an endogenous mouse κ light chain locus with human λ light chain gene segments, where the humanization removes the intergenic region, results in a significant increase in λ light chain usage, as well as a significant decrease in κ light chain locus usage. Thus, humanized mice lacking the intergenic region are useful in that the mice can produce antibodies with human light chain variable domains (e.g., human λ or κ domains), but with reduced usage from the locus.

[0171] In addition to the insertion of a human κ intergenic region to create a Vλ locus that includes a κ intergenic region between the final human Vλ gene segment and the first human Jλ gene segment with respect to transcription, humanization of an endogenous mouse κ light chain locus with human Vλ and Jλ gene segments has also been described; it shows a higher expressing B cell population than a locus lacking the κ intergenic region. This finding is consistent with the hypothesis that the intergenic region suppresses the use of the endogenous λ light chain locus (directly or indirectly via abolished transcripts). Under such a hypothesis, the inclusion of the intergenic region may reduce the frequency of use of the endogenous λ light chain locus, thereby resulting in a mouse with limited options, but with antibodies produced using the modified locus (λ to κ).

[0172] In various embodiments, the replacement of the mouse κ light chain sequence upstream of the mouse Cκ gene with the human λ light chain sequence further comprises a human κ light chain intergenic region located between the 3' untranslated region of the 3' most Vλ gene segment from the point of transcription and 5' to the first human Jλ gene segment. Alternatively, such an intergenic region can be removed from the endogenous κ light chain locus being replaced (upstream of the mouse Cκ gene) by creating a deletion in the endogenous λ light chain locus. Similarly, in this embodiment, the mice produce antibodies from an endogenous κ light chain locus that includes a human λ light chain sequence.

[0173] Approaches to engineer mice to express human Vλ domains endogenous C LVarious approaches are described for generating genetically modified mice that produce antibodies that include light chains with human Vλ domains fused to (e.g., Cκ or Cλ) regions. In various embodiments, genetic modifications are described that include deletion of one or both endogenous light chain loci. For example, deletion of a first Vλ-Jλ-Cλ gene cluster and total or partial replacement of the Vλ-Jλ gene segments of the second gene cluster with human Vλ-Jλ gene segments can be performed to eliminate mouse λ light chains from the endogenous antibody repertoire. Genetically modified mouse embryos, cells, and targeting constructs for generating the mice, mouse embryos and cells are also provided.

[0174] Since the endogenous Cλ genes remain intact and therefore retain normal functionality and the ability to assemble with the constant regions of endogenous heavy chains, in various embodiments, the deletion of one endogenous Vλ-Jλ-Cλ gene cluster and the replacement of the Vλ-Jλ gene segments of another endogenous Vλ-Jλ-Cλ gene cluster employs relatively minimal disruption of the assembly and function of the native antibody constant regions in the animal. Thus, in such embodiments, the modification does not affect other endogenous heavy chain constant regions that depend on a functional light chain constant region for the assembly of a functional antibody molecule comprising two heavy chains and two light chains. Furthermore, in various embodiments, the modification does not affect the assembly of a functional membrane-bound antibody molecule comprising endogenous heavy and light chains, e.g., hVλ domains connected to mouse Cλ regions. Because at least one functional Cλ gene is retained at the endogenous locus, an animal comprising a replacement of a Vλ-Jλ gene segment of an endogenous Vλ-Jλ-Cλ gene cluster with a human Vλ-Jλ gene segment should be able to produce normal λ light chains capable of binding to antigens during an immune response, depending on the human Vλ-Jλ gene segments present in the animal's expressed antibody repertoire.

[0175] A schematic (not to scale) of the endogenous mouse Vλ-Jλ-Cλ gene cluster to be deleted is provided in FIG. 2. As shown, the mouse λ light chain locus is organized into two gene clusters, both of which contain functional gene segments that can recombine to form a functional mouse λ light chain. The endogenous mouse Vλ1-Jλ3-Cλ3-Jλ1-Cλ1 gene cluster is deleted by a targeting construct (targeting vector 1) with a neomycin cassette flanked by recombination sites. The other endogenous gene cluster (Vλ2-Vλ3-Jλ2-Cλ2-Jλ4-Cλ4) is partially deleted by a targeting construct (targeting vector 2) with a hygromycin-thymidine kinase cassette flanked by recombination sites. In this second targeting event, the Cλ2-Jλ4-Cλ4 endogenous gene segment is retained. The second targeting construct (targeting vector 2) is constructed with a recombination site different from that in the first targeting construct (targeting vector 1), which allows the selective deletion of the selection cassette after successful targeting. The resulting double-targeted locus is functionally silenced in that it cannot produce endogenous λ light chain. This modified locus can be used for the insertion of human Vλ and Jλ gene segments, thereby creating an endogenous mouse λ locus that contains human Vλ and Jλ gene segments (wherein, upon recombination at the modified locus, the animal produces λ light chains that contain rearranged human Vλ and Jλ gene segments connected to endogenous mouse Cλ gene segments).

[0176] Genetically modifying mice to render endogenous λ gene segments non-functional, in various embodiments, results in mice that display exclusively κ light chains in their antibody repertoires, making them useful for evaluating the role of λ light chains in their immune responses and for generating antibody repertoires that contain Vκ but not Vλ domains.

[0177] Genetically modified mice expressing hVλ linked to mouse Cλ gene recombined in endogenous mouse λ light chain locus can be generated by any method known in the art. A schematic (not to scale) of the replacement of endogenous mouse Vλ2-Vλ3-Jλ2 gene segments with human Vλ and Jλ gene segments is provided in FIG. 4A. As shown, the non-functionalized endogenous mouse λ light chain locus is replaced by a targeting construct (12 / 1-λ targeting vector) containing a neomycin cassette flanked by recombination sites. The Vλ2-Vλ3-Jλ2 gene segments are replaced with a genomic fragment containing a human λ sequence containing 12 hVλ gene segments and a single hJλ gene segment.

[0178] Thus, by this first approach, one or more hVλ gene segments are positioned at the endogenous λ light chain locus contiguous with a single hJλ gene segment (FIG. 4A).

[0179] Further modifications to the modified endogenous λ light chain locus can be achieved using a similar approach to insert more hVλ gene segments. For example, a schematic diagram of two additional targeting constructs (+16-λ and +12-λ targeting vectors) used for the incremental insertion of additional human hVλ gene segments is provided in FIG. 5A. As shown, additional genomic fragments containing specific human hVλ gene segments are inserted into the modified endogenous λ light chain locus in sequential steps, using the homology provided by the previous insertion of human λ light chain sequences. Upon recombination with each of the illustrated targeting constructs, 28 additional hVλ gene segments are inserted into the modified endogenous λ light chain locus in a sequential manner. This creates a chimeric locus that produces λ light chain proteins containing human Vλ-Jλ gene segments connected to mouse Cλ genes.

[0180] The above approach to insert human λ light chain gene segments into the mouse λ locus maintains enhancers located downstream of the Cλ2-Jλ4-Cλ4 gene segment (designated Enh2.4, Enh and Enh3.1, FIG. 4A and FIG. 5A). This approach results in a single modified allele at the endogenous mouse λ light chain locus (FIG. 7A).

[0181] Compositions and methods are provided for generating mice that express light chains comprising hVλ and Jλ gene segments operably linked to mouse Cλ gene segments, including for generating mice that express such genes from an endogenous mouse λ light chain locus, comprising selectively rendering one endogenous mouse Vλ-Jλ-Cλ gene cluster non-functional (e.g., by targeted deletion) and expressing hVλ domains in the mouse using hVλ and Jλ gene segments at the endogenous mouse λ light chain locus.

[0182] Alternatively, in the second approach, human λ light chain gene segments can be located at endogenous κ light chain locus. The genetic modification, in various embodiments, includes deletion of endogenous κ light chain locus. For example, mouse Vκ and Jκ gene segments can be deleted to eliminate mouse κ light chain from endogenous antibody repertoire. Also provided are genetically modified mouse embryos, cells, and targeting constructs for producing the mouse, mouse embryos and cells.

[0183] For the reasons stated above, relatively minimal disruption is used in the deletion of mouse Vκ and Jκ gene segments. A schematic (not to scale) of the mouse Vκ and Jκ gene segments to be deleted is provided in FIG. 3. Endogenous mouse Vκ and Jκ gene segments are deleted via recombinase-mediated deletion of mouse sequences located between two precisely positioned targeting vectors, each using a site-specific recombination site. A first targeting vector (Jκ targeting vector) is used in a first targeting event to delete mouse Jκ gene segments. A second targeting vector (Vκ targeting vector) is used in a second sequential targeting event to delete sequences located 5' of the most distal mouse Vκ gene segment. Both targeting vectors contain site-specific recombination sites, allowing selective deletion of both selection cassettes and all intervening mouse κ light chain sequences after successful targeting. The resulting deleted locus is functionally silenced in that it cannot produce endogenous κ light chain.This modified locus can be used for the insertion of hVλ and Jλ gene segments, thereby creating an endogenous mouse κ locus that contains hVλ and Jλ gene segments (wherein, when recombination occurs at the modified locus, the animal produces λ light chains that contain rearranged hVλ and Jλ gene segments operably connected to endogenous mouse Cκ gene segments).Various targeting vectors that contain human λ light chain sequences can be used in combination with this deleted mouse κ locus to create hybrid light chain loci that contain human λ gene segments operably connected to mouse Cκ region.

[0184] Thus, by the second approach, one or more human Vλ gene segments are placed into the mouse κ light chain locus contiguous with a single human Jλ gene segment (12 / 1-κ targeting vector, FIG. 4B).

[0185] In various embodiments, variations on this approach can be made by adding gene segments and / or regulatory sequences to optimize the usage of human λ light chain sequences from the mouse κ locus within the mouse antibody repertoire.

[0186] In the third approach, one or more hVλ gene segments are positioned at the mouse κ light chain locus contiguous with four hJλ gene sequences (12 / 4-κ targeting vector, FIG. 4B).

[0187] In the third approach, one or more hVλ gene segments are placed into the mouse κ light chain locus contiguous with human κ intergenic sequences and a single hJλ gene sequence (12(κ)1-κ targeting vector, FIG. 4B).

[0188] In the fourth approach, one or more hVλ gene segments are positioned at the mouse κ light chain locus contiguous with the human κ intergenic sequence, four hJλ gene sequences (12(κ)4-κ targeting vector FIG. 4B).

[0189] All of the above approaches to insert human lambda light chain gene segments into the mouse kappa locus maintain the kappa intronic enhancer element (designated Ekappa, Figures 4B and 5B) upstream of the Ckappa gene and the 3' kappa enhancer (designated Ekappa3', Figures 4B and 5B) downstream of the Ckappa gene. This approach results in four separate modified alleles at the endogenous mouse kappa light chain locus (Figure 7B).

[0190] In various embodiments, the genetically modified mouse comprises a knockout of the endogenous mouse λ light chain locus. In one embodiment, the λ light chain locus is knocked out by a strategy that deletes the region spanning Vλ2 to Jλ2 and the region spanning Vλ1 to Cλ1 (FIG. 2). Any strategy that reduces or eliminates the ability of the endogenous λ light chain locus to express an endogenous λ domain is suitable for use with the embodiments in this disclosure.

[0191] Genetically engineered mouse-derived lambda domain antibodies Mice containing human λ sequences at the mouse κ or λ light chain loci are L These include: (a) mice that contain a functionally silenced light chain locus (e.g., a knockout of an endogenous mouse κ or λ light chain locus); (b) mice that contain an endogenous mouse λ light chain locus that contains hV and hJ gene segments operably linked to an endogenous mouse Cλ gene; (c) mice that contain an endogenous mouse κ light chain locus that contains hVκ and hJκ gene segments operably linked to an endogenous mouse Cκ gene; and (d) mice in which one κ allele contains hVκ and hJκ; the other κ allele contains hVλ and hJλ; one λ allele contains hVλ and hJλ and one λ allele is silenced or knocked out, or both λ alleles contain hVλ and hJλ; and each contains hV H , hD H and hJ H The mouse is advantageously mated to a mouse containing two heavy chain alleles comprising

[0192] Antibodies containing hVλ domains expressed in the context of Cκ or Cλ are used to generate fully human antibodies by cloning the nucleic acid encoding the hVλ domain into an expression construct with a gene encoding a human Cλ The resulting expression construct is transfected into a host cell suitable for expression of antibodies displaying a complete hVλ domain fused to hCλ. EXAMPLES

[0193] The following examples are provided to illustrate how to make and use the methods and compositions of the invention and are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise indicated, temperatures are given in degrees Celsius and pressures are at or near atmospheric.

[0194] Example I Deletions in the mouse immunoglobulin light chain locus To engineer mouse genomic bacterial artificial chromosome (BAC) libraries to inactivate mouse kappa and lambda light chain loci, various targeting constructs were generated using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659).

[0195] Deletion of the Mouse λ Light Chain Locus. To inactivate the endogenous mouse λ light chain locus by targeted deletion of the Vλ-Jλ-Cλ gene cluster, DNA from the mouse BAC clone RP23-135k15 (Invitrogen) was modified by homologous recombination (FIG. 2).

[0196] Briefly, the entire proximal cluster containing the Vλ1-Jλ3-Cλ3-Jλ1-Cλ1 gene segments was deleted in a single targeting event using a targeting vector containing a neomycin cassette flanked by loxP sites with a 5' mouse homology arm containing sequences 5' of the Vλ1 gene segment and a 3' mouse homology arm containing sequences 3' of the Cλ1 gene segment (Figure 2, targeting vector 1).

[0197] To precisely delete the distal endogenous mouse λ gene cluster containing Vλ2-Jλ2-Cλ2-Jλ4-Cλ4, a second targeting construct was prepared, except that the second targeting construct contained a 5' mouse homology arm containing sequences 5' to the Vλ2 gene segment and a 3' mouse homology arm containing sequences 5' to the endogenous Cλ2 gene segment (FIG. 2, targeting vector 2). Thus, the second targeting construct precisely deleted Vλ2-Jλ2, leaving Cλ2-Jλ4-Cλ4 intact at the endogenous mouse λ locus. ES cells containing an inactivated endogenous λ locus (as described above) were confirmed by karyotype analysis and screening methods known in the art (e.g., TAQMAN®). DNA was then isolated from the modified ES cells and subjected to treatment with CRE recombinase to mediate deletion of the proximal targeting cassette containing the neomycin marker gene, leaving only a single loxP site in the deletion (Figure 2, bottom).

[0198] Deletion of the Mouse Kappa Light Chain Locus. Several targeting constructs were generated using methods similar to those described above to inactivate the mouse kappa light chain locus in a two-step process by modifying DNA from mouse BAC clones RP23-302g12 and RP23-254m04 (Invitrogen) by homologous recombination (FIG. 3).

[0199] Briefly, a targeting vector containing a hygromycin-thymidine kinase (hyg-TK) cassette containing a single loxP site 3' to the hyg-TK cassette was used to delete the Jκ gene segments (1-5) of the endogenous mouse kappa light chain locus in a single targeting event (Figure 3, Jκ targeting vector). The homology arms used to create this targeting vector contained mouse genomic sequence 5' and 3' to the endogenous mouse Jκ gene segment. In a second targeting event, a second targeting vector was prepared to delete a portion of the mouse genomic sequence upstream (5') to the most distal endogenous mouse Vκ gene segment (Figure 3, Vκ targeting vector). This targeting vector contained an inverted lox511 site, a loxP site, and a neomycin cassette. The homology arms used to create this targeting vector contained mouse genomic sequence upstream of the most distal mouse Vκ gene segment. The targeting vectors were used in a sequential manner (i.e., Jκ followed by Vκ) to target DNA in ES cells. ES with a double-targeted chromosome (i.e., a single endogenous mouse κ locus targeted with both targeting vectors) were confirmed by karyotyping and screening methods known in the art (e.g., Taqman™). DNA was then isolated from the modified ES cells and subjected to treatment with Cre recombinase to mediate the deletion of the endogenous mouse Vκ gene segment and both selection cassettes, with two lox sites juxtaposed in the opposite direction to each other (Figure 3, bottom; SEQ ID NO: 1).

[0200] In this way, two modified endogenous light chain loci (κ and λ) were created that contain intact enhancers and constant regions for the stepwise insertion of unrearranged human λ germline gene segments in a precise manner using the targeting vectors described below.

[0201] Example II Replacement of the mouse light chain locus with a human lambda light chain minilocus. Using methods similar to those described above, multiple targeting vectors were generated for the progressive insertion of human λ gene segments into the endogenous mouse κ and λ light chain loci. Multiple independent initial modifications were made to the endogenous light chain locus, each of which resulted in a chimeric light chain locus comprising hVλ and Jλ gene segments operably linked to mouse light chain constant genes and enhancers.

[0202] Human λ minilocus containing 12 human Vλ and 1 human Jλ gene segments. A series of initial targeting vectors were generated using a human BAC clone designated RP11-729g4 (Invitrogen) to contain the first 12 contiguous human Vλ gene segments and an hJλ1 gene segment or 4 hJλ gene segments from cluster A. Figures 4A and 4B show the targeting vectors constructed for the initial insertion of human λ light chain gene segments at the mouse λ and κ light chain loci, respectively.

[0203] For the first set of initial targeting vectors, a 124,125 bp DNA fragment from the 729g4 BAC clone containing 12 hVλ and hJλ1 gene segments was engineered to contain a PI-SceI site 996 bp downstream (3') of the hJλ1 gene segment for ligation of a 3' mouse homology arm. Two different sets of homology arms were used for ligation to the human fragment; one set of homology arms contained endogenous mouse λ sequences from the 135k15 BAC clone (Figure 4A), and the other set contained endogenous κ sequences 5' and 3' of the mouse Vκ and Jκ gene segments of mouse BAC clones RP23-302g12 and RP23-254m04, respectively (Figure 4B).

[0204] For the 12 / 1-λ targeting vector (FIG. 4A), a PI-SceI site was created at the 5′ end of a 27,847 bp DNA fragment containing mouse Cλ2-Jλ4-Cλ4 and enhancer 2.4 of the modified mouse λ locus described in Example 1. The approximately 28 kb mouse fragment was used as a 3′ homology arm by ligation to a approximately 124 kb human λ fragment to create a 3′ junction that included, from 5′ to 3′, the hJλ1 gene segment, 996 bp of human λ sequence 3′ to the hJλ1 gene segment, 1229 bp of mouse λ sequence 5′ to the mouse Cλ2 gene, the mouse Cλ2 gene, and the remainder of the approximately 28 kb mouse fragment. Upstream (5') of the human Vλ3-12 gene segment was an additional 1456 bp of human λ sequence, preceded by the start of a 5' mouse homology arm that contained 23,792 bp of mouse genomic DNA corresponding to sequences 5' from the endogenous mouse λ locus. Between the 5' homology arm and the start of the human λ sequence was a neomycin cassette flanked by Frt sites.

[0205] Thus, the 12 / 1-λ targeting vector contained, from 5' to 3', a 5' homology arm containing approximately 24 kb of mouse λ genomic sequence 5' to the endogenous λ locus, a 5' Frt site, a neomycin cassette, a 3' Frt site, approximately 123 kb of human genomic λ sequence including the first 12 contiguous hVλ and hJλ1 gene segments, a PI-SceI site, and approximately 28 kb of mouse genomic sequence including the endogenous Cλ2-Jλ4-Cλ4 gene segment, mouse enhancer 2.4 sequence, and additional mouse genomic sequence downstream (3') of enhancer 2.4 (Figure 4A).

[0206] In a similar manner, the 12 / 1-Kappa targeting vector (Figure 4B) used the same approximately 124 human λ fragment, except that it used a mouse homology arm containing mouse κ sequences so that targeting to the endogenous Kappa locus could be achieved by homologous recombination. Thus, the 12 / 1-Kappa targeting vector contained, from 5' to 3', a 5' homology arm containing approximately 23 kb of mouse genomic sequence 5' to the endogenous Kappa locus, an I-CeuI site, a 5' Frt site, a neomycin cassette, a 3' Frt site, approximately 124 kb of human genomic λ sequence including the first 12 consecutive hVλ and hJλ1 gene segments, a PI-SceI site, and approximately 28 kb of mouse genomic sequence including the endogenous mouse Cκ gene, Eκi and Eκ3', and additional mouse genomic sequence downstream (3') of the Eκ3' (Figure 4B, 12 / 1-Kappa targeting vector).

[0207] Homologous recombination with either of these two original targeting vectors generated a modified mouse light chain locus (κ or λ) containing 12 hVλ and hJλ1 gene segments operably linked to endogenous mouse light chain constant genes and enhancers (Cκ or Cλ2 and Eκi / Eκ3' or Enh2.4 / Enh3.1) genes, which upon recombination would result in the formation of a chimeric λ light chain.

[0208] Human λ minilocus with 12 human Vλ gene segments and 4 human Jλ gene segments. In another approach to add diversity to the chimeric λ light chain locus, a third initial targeting vector was created to insert the first 12 contiguous human Vλ gene segments from cluster A and the hJλ1, 2, 3 and 7 gene segments into the mouse κ light chain locus (FIG. 4B, 12 / 4-κ targeting vector). A DNA segment containing the hJλ1, Jλ2, Jλ3 and Jλ7 gene segments, including each Jλ gene segment and approximately 100 bp of human genomic sequence from both the 5′ and 3′ regions immediately adjacent to each Jλ gene segment, was generated by de novo DNA synthesis (Integrated DNA Technologies). A PI-SceI site was created at the 3′ end of this approximately 1 kb DNA fragment and ligated to a chloroamphenicol cassette. Homology arms were PCR amplified from human λ sequences at positions 5' and 3' to the hJλ1 gene segment of human BAC clone 729g4. Homologous recombination with this intermediate targeting vector was performed in a modified 729g4BAC clone that was previously targeted upstream (5') of the human Vλ3-12 gene segment with a neomycin cassette flanked by Frt sites that also contained an I-CeuI site 5' to the 5'Frt site. The doubly targeted 729g4BAC clone contained, from 5' to 3', an I-CeuI site, a 5'Frt site, a neomycin cassette, a 3'Frt site, an approximately 123 kb fragment containing the first 12 hVλ gene segments, an approximately 1 kb fragment containing the human Jλ1, 2, 3 and 7 gene segments, a PI-SceI site and a chloramphenicol cassette. The third targeting vector was generated by simultaneously digesting this intermediate targeting vector with I-CeuI and PI-SceI and then ligating it to the modified mouse BAC clone (described above).

[0209] This ligation resulted in a third targeting vector for insertion of human λ sequences into the endogenous κ light chain locus that contained, from 5′ to 3′, a 5′ mouse homology arm containing approximately 23 kb of genomic sequence 5′ to the endogenous mouse κ locus, an I-CeuI site, a 5′ Frt site, a neomycin cassette, a 3′ Frt site, an approximately 123 kb fragment containing the first 12 hVλ gene segments, an approximately 1 kb fragment containing the hJλ1, 2, 3, and 7 gene segments, a PI-SceI site, and a 3′ homology arm containing approximately 28 kb of mouse genomic sequence including the endogenous mouse Cκ gene, Eκi, and Eκ3′, as well as additional mouse genomic sequence downstream (3′) of the Eκ3′ ( FIG. 4B , 12 / 4-κ targeting vector). Homologous recombination with this third targeting vector created a modified mouse κ light chain locus containing 12 hVλ and 4 hJλ gene segments operably linked to the endogenous mouse Cκ gene, which upon recombination result in the formation of a chimeric human λ / mouse κ light chain.

[0210] Human λ minilocus with integrated human κ light chain sequence. In a similar manner, two additional targeting vectors similar to the targeting vectors created to perform the initial insertion of human λ gene segments into the endogenous κ light chain locus (FIG. 4B, 12 / 1-κ and 12 / 4-κ targeting vectors) were created to progressively insert human λ light chain gene segments using uniquely constructed targeting vectors containing contiguous human λ and κ genomic sequences. These targeting vectors were constructed to contain approximately 23 kb of human κ genomic sequence that is naturally located between the human Vκ4-1 and Jκ1 gene segments. This human κ genomic sequence was specifically positioned between the human Vλ and Jλ gene segments in these two additional targeting vectors (FIG. 4B, 12(κ)1-κ and 12(κ)4-κ targeting vectors).

[0211] Both targeting vectors containing the human kappa genomic sequence were generated using the modified RP11-729g4BAC clone described above (Figure 6). This modified BAC clone was targeted with a spectinomycin selection cassette flanked by NotI and AsiSI restriction enzyme recognition sites (Figure 6, top left). Homologous recombination with the spectinomycin cassette resulted in a double targeted 729g4BAC clone that contained, from 5' to 3', an I-CeuI site, a 5'Frt site, a neomycin cassette, a 3'Frt site, a .about.123 kb fragment containing the first 12 hVλ gene segments, a NotI site approximately 200 bp downstream (3') of the nonamer sequence of the hVλ3-1 gene segment, a spectinomycin cassette, and an AsiSI site. A separate human BAC clone (CTD-2366j12) containing human κ sequences was independently targeted twice to create a restriction enzyme recognition site located between the hVκ4-1 and hJκ1 gene segments to allow subsequent cloning of a ∼23 kb fragment for ligation with the hVλ gene segment contained in the doubly targeted modified 729g4 BAC clone (Figure 6, top right).

[0212] Briefly, the 2366j12 BAC clone is approximately 132 kb in size and contains hVκ gene segments 1-6, 1-5, 2-4, 7-3, 5-2, 4-1, human κ genomic sequences downstream of those Vκ gene segments, hJκ gene segments 1-5, hCκ, and approximately 20 kb of additional genomic sequences of the human κ locus. This clone was first targeted with a targeting vector containing a hygromycin cassette flanked by Frt sites and a NotI site downstream (3') of the 3'Frt site. The homology arms to this targeting vector contained human genomic sequences 5' and 3' of the Vκ gene segments in the BAC clone, such that upon homologous recombination with this targeting vector, the Vκ gene segments were deleted and a NotI site was created approximately 133 bp downstream of the hVκ4-1 gene segment (Figure 6, top right). This modified 2366j12BAC clone was independently targeted at the 3' end with two targeting vectors to delete the hJκ gene segment with a chloramphenicol cassette that also contained either the hJλ1 gene segment, PI-SceI and AsiSI sites, or a human λ genomic fragment containing four hJλ gene segments (supra), PI-SceI and AsiSI sites (Figure 6, top right). The homology arms for these two similar targeting vectors included sequences 5' and 3' to the hJκ gene segment. Homologous recombination with these second targeting vectors and the modified 2366j12BAC clones yielded doubly targeted 2366j12 clones that contained, from 5' to 3', a 5'Frt site, a hygromycin cassette, a 3'Frt site, a NotI site, a 22,800 bp genomic fragment of the human κ locus containing the intergenic region between the Vκ4-1 and Jκ1 gene segments, either the hJλ1 gene segment or a human λ genomic fragment containing hJλ1, Jλ2, Jλ3, and Jλ7, a PI-SceI site, and a chloramphenicol cassette (Figure 6, top right).Two ligation steps using the dual targeted 729g4 and 2366j12 clones yielded two final targeting vectors for making two additional modifications.

[0213] Digestion of the dual targeted 729g4 and 2366j12 clones with NotI and AsiSI, respectively, yielded one fragment containing the neomycin cassette and the hVλ gene segment, and another fragment containing a ∼23 kb genomic fragment of the human κ locus containing the intergenic region between the Vκ4-1 and Jκ1 gene segments, either the hJλ1 gene segment or a genomic fragment containing the hJλ1, Jλ2, Jλ3 and Jλ7 gene segments, a PI-SceI site and a chloramphenicol cassette. Ligation of these fragments generated two unique BAC clones that contained, from 5' to 3', the hVλ gene segment, the human κ genomic sequence between the Vκ4-1 and Jκ1 gene segments, either the hJλ1 gene segment or a genomic fragment containing the hJλ1, Jλ2, Jλ3 and Jλ7 gene segments, a PI-SceI site and a chloramphenicol cassette (Figure 6, bottom). These new BAC clones were then digested with I-CeuI and PI-SceI to release unique fragments containing the upstream neomycin cassette and the contiguous human λ and κ sequences, which were then sequenced from 5' to 3' to consist of mouse genomic sequence 5' to the endogenous κ locus, an I-CeuI site, a 5'Frt site, a neomycin cassette, a 3'Frt site, the hVλ gene segment (3-12 to 3-1), and a NotI site approximately 200 bp downstream of Vλ3-1. The approximately 23 kb human κ sequence naturally found between the human Vκ4-1 and Jκ1 gene segments, either the hJλ1 gene segment or a genomic fragment containing the hJλ1, Jλ2, Jλ3 and Jλ7 gene segments, was ligated into a modified mouse BAC clone 302g12 containing mouse Eκi, mouse Cκ gene and Eκ3' (Figure 4, 12hVλ-VκJκ-hJλ1 and 12hVλ-VκJκ-4hJλ targeting vectors).Homologous recombination with both of these targeting vectors generated two separate modified mouse κ light chain loci containing 12 hVλ gene segments, human κ genomic sequences and one or four hJλ gene segments operably linked to the endogenous mouse Cκ gene, which upon recombination result in the formation of a chimeric human λ / mouse κ light chain.

[0214] Example III Engineering additional human V.lambda. gene segments into the human .lambda. light chain minilocus. Using similar targeting vectors and methods (Figure 5A, +16-λ targeting vector and Figure 5B, +16-κ targeting vector), additional hVλ gene segments were added independently to each of the initial modifications described in Example 2.

[0215] Introduction of 16 additional human Vλ gene segments. The upstream (5') homology arm used in constructing the targeting vectors to add the 16 additional hVλ gene segments to the modified light chain locus described in Example 2 contained mouse genomic sequence 5' to the endogenous κ or λ light chain locus. The 3' homology arm was the same for all targeting vectors and contained human genomic sequence overlapping the 5' end of the human λ sequence of the modification as described in Example 2.

[0216] Briefly, two targeting vectors were generated to introduce 16 additional hVλ gene segments into the modified mouse light chain locus described in Example 2 (FIGS. 5A and 5B, +16-λ or +16-κ targeting vectors). A ∼172 kb DNA fragment from human BAC clone RP11-761113 (Invitrogen) containing 21 contiguous hVλ gene segments from cluster A was generated with a 5′ homology arm containing mouse genomic sequence 5′ to the endogenous κ or λ light chain locus and a 3′ homology arm containing human genomic λ sequence. The 5′ mouse κ or λ homology arm used in these targeting constructs was the same 5′ homology arm as described in Example 2 (FIGS. 5A and 5B). The 3′ homology arm contained a 53,057 bp overlap of human genomic λ sequence corresponding to the equivalent 5′ end of the ∼123 kb fragment of human genomic λ sequence described in Example 2. These two targeting vectors contained, from 5' to 3', a 5' mouse homology arm containing approximately 23 kb of genomic sequence 5' to the endogenous mouse κ light chain locus or approximately 24 kb of mouse genomic sequence 5' to the endogenous λ light chain locus, a 5' Frt site, a hygromycin cassette, a 3' Frt site, and 171,457 bp of human genomic λ sequence containing 21 contiguous hVλ gene segments (of which approximately 53 kb overlaps with the 5' end of the human λ sequence described in Example 3 and serves as the 3' homology arm for the targeting construct) (Figures 5A and 5B, +16-λ or +16-κ targeting vectors). Homologous recombination with these targeting vectors generated independently engineered mouse κ and λ light chain loci, each containing 28 hVλ and hJλ1 gene segments operably linked to endogenous mouse constant genes (Cκ or Cλ2), which upon recombination result in the formation of chimeric light chains.

[0217] In a similar manner, the +16-κ targeting vector was also used to introduce 16 additional hVλ gene segments into the other initial modification described in Example 2 that incorporates multiple hJλ gene segments with and without integrated human κ sequences ( FIG. 4B ). Homologous recombination with this targeting vector in the endogenous mouse κ locus containing the other initial modification generated a mouse κ light chain locus containing 28 hVλ gene segments with and without human Vκ-Jκ genomic sequences operably linked to the endogenous mouse Cκ gene, as well as hJλ1, 2, 3, and 7 gene segments, which upon recombination result in the formation of a chimeric λ-κ light chain.

[0218] Introduction of 12 additional human Vλ gene segments. Using similar targeting vectors and methods, additional hVλ gene segments were added independently to each of the modifications described above. The structure of the final locus resulting from homologous recombination with the targeting vector containing the additional hVλ gene segments is shown in Figures 7A and 7B.

[0219] Briefly, a targeting vector was created to introduce 12 additional hVλ gene segments into the modified mouse κ and λ light chain loci described above (Figures 5A and 5B, +12-λ or 12-κ targeting vectors). A 93,674 bp DNA fragment from human BAC clone RP11-22118 (Invitrogen) containing 12 consecutive hVλ gene segments from cluster B was engineered with a 5' homology arm containing mouse genomic sequences 5' to the endogenous mouse κ or λ light chain locus and a 3' homology arm containing human genomic λ sequences. The 5' homology arm used in this targeting construct was the same 5' homology arm used to add the 16 hVλ gene segments described above (Figures 5A and 5B). The 3' homology arm was generated by creating a PI-SceI site approximately 3431 bp 5' to the human Vλ3-29P gene segment contained in the 27,468 bp genomic fragment of human λ sequence from BAC clone RP11-761113. This PI-SceI site served as a ligation point to join an additional 94 kb fragment of human λ sequence with an approximately 27 kb fragment of human λ sequence that overlaps with the 5' end of the human λ sequence in the previous modification using the +16-λ or +16-κ targeting vectors (Figures 5A and 5B). These two targeting vectors contained, from 5' to 3', a 5' homology arm containing approximately 23 kb of mouse genomic sequence 5' to the endogenous κ light chain locus or approximately 24 kb of mouse genomic sequence 5' to the endogenous λ light chain locus, a 5' Frt site, a neomycin cassette, a 3' Frt site, and 121,188 bp of human genomic λ sequence containing 16 hVλ gene segments and a PI-SceI site (of which approximately 27 kb overlaps with the 5' end of the human λ sequence from the insertion of the 16 additional hVλ gene segments and serves as the 3' homology arm for the targeting construct) (Figures 5A and 5B, +12-λ or 12-κ targeting vectors).Homologous recombination with these targeting vectors independently generated modified mouse κ and λ light chain loci containing 40 hVλ gene segments operably linked to endogenous mouse constant genes (Cκ or Cλ2) and human Jλ1, which upon recombination result in the formation of chimeric light chains (Figures 5A and 5B, bottom).

[0220] In a similar manner, the +12-κ targeting vector was also used to introduce 12 additional hVλ gene segments into the other initial modification incorporating multiple hJλ gene segments with and without integrated human κ sequences ( FIG. 4B ). Homologous recombination with this targeting vector in the endogenous mouse κ locus containing the other modifications generated a mouse κ light chain locus containing 40 hVλ gene segments with and without human Vκ-Jκ genomic sequences operably linked to the endogenous mouse Cκ gene, as well as hJλ1, 2, 3, and 7 gene segments, which upon recombination result in the formation of a chimeric λ-κ light chain.

[0221] Example IV Identification of targeted ES cells carrying human λ light chain gene segments The targeted BAC DNA produced according to the previous examples was used to electroporate mouse ES cells to generate modified ES cells for the production of chimeric mice expressing human λ light chain gene segments. ES cells containing unrearranged human λ light chain gene segment insertions were identified by quantitative TAQMAN® assay. Specific primer sets and probes were designed for the insertion of human λ sequences and associated selection cassettes (gain of allele, GOA), loss of endogenous mouse sequences and any selection cassettes (loss of allele, LOA), and retention of adjacent mouse sequences (retention of allele, AR). For each additional insertion of human λ sequences, additional primer sets and probes were used to confirm the presence of the additional human λ sequences, and previous primer sets and probes were used to confirm the retention of the previously targeted human sequences. Table 1 shows the primers and associated probes used in the quantitative PCR assay. Table 2 shows the combinations used to confirm the insertion of each section of human λ light chain gene segments in ES cell clones.

[0222] To remove the Frt-flanked neomycin cassette introduced by insertion of the targeting construct containing the human Vλ5-52-Vλ1-40 gene segments, ES cells carrying the human λ light chain gene segments are optionally transfected with a construct expressing FLP (FIGS. 5A and 5B). The neomycin cassette can be optionally removed by breeding with mice expressing FLP recombinase (e.g., U.S. Patent No. 6,774,279). The neomycin cassette is optionally retained in the mice.

[0223] [Table 1]

[0224] [Table 2-1]

[0225] [Table 2-2] Example V Generation of mice expressing human lambda light chains from the endogenous light chain locus The targeted ES cells described above were used as donor ES cells and introduced into 8-cell stage mouse embryos by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses). (See Nature Biotech. 25(1):91-99.) VELOCIMICE® (F0 mice entirely derived from donor ES cells) independently carrying human λ gene segments were identified by genotyping using a modified allele assay (Valenzuela et al., supra) that detects the presence of unique human λ gene segments (supra).

[0226] Kappa:lambda light chain usage in mice carrying human lambda light chain gene segments. Mice homozygous for each of three consecutive insertions of hVlambda gene segments containing a single hJlambda gene segment (FIG. 5B), and mice homozygous for the first insertion of a hVlambda gene segment containing a single hJlambda gene segment or four human Jlambda gene segments (FIG. 4B), containing a human VK-JK genomic sequence, were analyzed for Kappa and lambda light chain expression in splenocytes using flow cytometry.

[0227] Briefly, spleens were collected from groups of mice (ranging from 3-7 animals per group) and crushed using a glass slide. After lysing red blood cells (RBCs) using ACK lysis buffer (Lonza Walkersville), splenocytes were stained with fluorochrome-conjugated antibodies specific for mouse CD19 (clone 1D3; BD Biosciences), mouse CD3 (17A2; Biolegend), mouse Igκ (187.1; BD Biosciences) and mouse Igλ (RML-42; Biolegend). Data were acquired using a BD™ LSR II flow cytometer (BD Biosciences) and analyzed using FLOWJO™ software (Tree Star, Inc.). Table 3 shows the observed B cell (CD19 + ), κ light chain (CD19 + Igκ + Igλ - ) and λ light chain (CD19 + Igκ - Igλ + ) Mean percentage values ​​for expression are shown.

[0228] In a similar experiment, the B cell content of the splenic compartment from mice homozygous for the first insertion of 12 hVλ gene segments and 4 hJλ gene segments comprising human Vκ-Jκ genomic sequences operably linked to the mouse Cκ gene (FIG. 4B, bottom) and mice homozygous for 40 hVλ gene segments and 1 hJλ gene segment (FIG. 5B, bottom or FIG. 7B, top) was analyzed for Igκ and Igλ expression using flow cytometry (as described above). FIG. 8A shows CD19 expression for representative mice from each group. + Igλ and Igκ expression in B cells is shown. CD19 per spleen + The number of B cells was also recorded for each mouse (Figure 8B).

[0229] In a separate experiment, the B cell content of the splenic and bone marrow compartments from mice homozygous for 40 hVλ and 4 hJλ gene segments containing human Vκ-Jκ genomic sequences operably linked to the mouse Cκ gene (Fig. 7B, bottom) was analyzed over the course of B cell development using flow cytometry of various cell surface markers.

[0230] Briefly, two groups of mice (N=3 each, 9-12 weeks old, male and female) homozygous for 40 hVλ gene segments and 4 hJλ gene segments containing wild type and human Vκ-Jκ genomic sequences operably connected to mouse Cκ genes were sacrificed and spleens and bone marrow were harvested. Bone marrow was harvested from femurs by flushing with complete RPMI medium (RPMI medium supplemented with fetal bovine serum, sodium pyruvate, Hepes, 2-mercaptoethanol, non-essential amino acids, and gentamicin). RBCs from spleen and bone marrow preparations were lysed with ACK lysis buffer (Lonza Walkersville) followed by washing with complete RPMI medium. 1×10 6 Cells were incubated with anti-mouse CD16 / CD32 (2.4G2, BD Biosciences) for 10 min on ice and then labeled with a selected panel of antibodies for 30 min on ice.

[0231] Bone marrow panel: anti-mouse FITC-CD43 (1B11, BioLegend), PE-ckit (2B8, BioLegend), PeCy7-IgM (II / 41, eBioscience), PerCP-Cy5.5-IgD (11-26c.2a, BioLegend), APC-B220 (RA3-6B2, eBioscience), APC-H7-CD19 (ID3, BD) and Pacific Blue-CD3 (17A2, BioLegend).

[0232] Bone marrow and spleen panel: anti-mouse FITC-Igκ (187.1, BD), PE-Igλ (RML-42, BioLegend), PeCy7-IgM (II / 41, ebioscience), PerCP-Cy5.5-IgD (11-26c.2a, BioLegend), Pacific Blue-CD3 (17A2, BioLegend), APC-B220 (RA3-6B2, eBioscience), APC-H7-CD19 (ID3, BD).

[0233] After staining, cells were washed and fixed in 2% formaldehyde. Data were acquired on a FACSCANTOII™ flow cytometer (BD Biosciences) and analyzed using FLOWJO™ software (Tree Star, Inc.). Figures 9A-9D show the results for the splenic compartment of one representative mouse from each group. Figures 10A-10E show the results for the bone marrow compartment of one representative mouse from each group. Table 4 shows the B cell (CD19) population observed in splenocytes from groups of animals with various genetic modifications. + ), κ light chain (CD19 + Igκ + Igλ - ) and λ light chain (CD19 + Igκ - Igλ + Table 5 shows the mean percentage values ​​for B cell (CD19) expression observed in the bone marrow of wild type and mice homozygous for 40 hVλ gene segments and 4 hJλ gene segments comprising human Vκ-Jκ genomic sequences operably linked to the mouse Cκ gene. + ), mature B cells (B220 hi IgM + ), immature B cells (B220 int IgM + ), immature B cells expressing κ light chains (B220 int IgM + Igκ + ) and immature B cells expressing lambda light chains (B220 int IgM + Igλ +) are shown. This experiment was repeated with additional groups of mice as described above, with similar results (data not shown).

[0234] [Table 3]

[0235] [Table 4]

[0236] [Table 5] Human Vλ gene usage in mice carrying human λ light chain gene segments. Mice heterozygous for the first insertion of human λ sequences (hVλ3-12-hVλ3-1 and hJλ1, FIG. 5B) and mice homozygous for the third insertion of human λ sequences (hVλ5-52-hVλ3-1 and hJλ1, FIG. 5B) were analyzed for human λ light chain gene usage by reverse transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from splenocytes.

[0237] Briefly, spleens were harvested and perfused with 10 mL of RPMI-1640 (Sigma) containing 5% HI-FBS in a sterile disposable bag. Each bag containing one spleen was then placed in a STOMACHER™ (Seward) and homogenized at medium setting for 30 seconds. The homogenized spleens were filtered through a 0.7 μm cell strainer and then pelleted in a centrifuge (1000 rpm for 10 minutes) and RBCs were lysed in BD PHARM LYSE™ (BD Biosciences) for 3 minutes. Splenocytes were diluted in RPMI-1640 and centrifuged again before being resuspended in 1 mL of PBS (Irvine Scientific). RNA was isolated from pelleted splenocytes using standard techniques known in the art.

[0238] RT-PCR was performed on splenocyte RNA using primers specific for human hVλ gene segments and mouse Cκ gene (Table 6). PCR products were gel purified, cloned into pCR2.1-TOPO TA vector (Invitrogen) and sequenced using primers M13Forward (GTAAAACGACGGCCAG; SEQ ID NO: 55) and M13Reverse (CAGGAAACAGCTATGAC; SEQ ID NO: 56) located adjacent to the cloning site in the vector. hVλ gene usage was determined by sequencing a total of 84 clones derived from the first and third insertions of human λ sequence (Table 7). Nucleotide sequences of the hVλ-hJλ1-mCκ junction for selected RT-PCR clones are shown in FIG. 11.

[0239] In a similar manner, mice homozygous for a third insertion of human λ light chain gene sequences operably linked to the endogenous mouse Cκ gene (i.e., 40 hVλ gene segments and 4 hJλ gene segments comprising human Vκ-Jκ genomic sequences, FIG. 7B, bottom) were analyzed for human λ light chain gene usage by RT-PCR using RNA isolated from splenocytes (as described above). The human λ light chain gene segment usage for the 26 selected RT-PCR clones is shown in Table 8. The nucleotide sequence of the hVλ-hJλ-mCκ junction for the selected RT-PCR clones is shown in FIG. 12.

[0240] In a similar manner, mice homozygous for the first insertion of human λ light chain gene segments (12 hVλ gene segments and hJλ1, FIG. 4A and FIG. 5A) operably linked to the endogenous mouse Cλ2 gene were analyzed for human λ light chain gene usage by RT-PCR using RNA isolated from splenocytes (as described above). Primers specific for the hVλ gene segments (Table 6) were paired with one of two primers specific for the mouse Cλ2 gene; Cλ2-1 (SEQ ID NO: 104) or Cλ2-2 (SEQ ID NO: 105).

[0241] Multiple hVλ gene segments rearranged into hλ1 were observed from RT-PCR clones derived from mice carrying human λ light chain gene segments at the endogenous mouse λ light chain locus. The nucleotide sequences of the hVλ-hJλ-mCλ2 junctions for selected RT-PCR clones are shown in FIG.

[0242] [Table 6]

[0243] [Table 7] FIG. 11 shows the sequence of the hVλ-hJλ1-mCκ junction for RT-PCR clones from mice carrying the first and third insertions of hVλ gene segments with a single hJλ gene segment. The sequences shown in FIG. 11 show unique rearrangements involving various hVλ gene segments with hJλ1 recombined into the mouse Cκ gene. Both heterozygous mice with a single modified endogenous κ locus containing 12 hVλ gene segments and hJλ1 and homozygous mice with two modified endogenous κ loci containing 40 hVλ gene segments and hJλ1 were able to generate human λ gene segments operably linked to the mouse Cκ gene and were able to produce B cells expressing human λ light chains. These rearrangements demonstrate that the chimeric loci were able to independently rearrange human λ gene segments in multiple independent B cells of these mice. Furthermore, as evidenced by the 16 distinct hVλ gene segments that were observed to rearrange with hJλ1 (Table 7), these modifications to the endogenous κ light chain locus did not render any hVλ gene segments inoperable, nor did the chimeric locus prevent recombination of multiple hVλ and hJλ (Jλ1) gene segments during B cell development. Moreover, these mice produced functional antibodies that contained rearranged human Vλ-Jλ gene segments operably linked to the mouse Cκ gene as part of their endogenous immunoglobulin light chain repertoire.

[0244] FIG. 12 shows the sequence of the hVλ-hJλ-mCκ junction for selected RT-PCR clones from mice homozygous for 40 hVλ gene segments and 4 hJλ gene segments that comprise the human Vκ-Jκ genomic sequence. The sequences shown in FIG. 12 show unique additional rearrangements involving multiple different hVλ gene segments throughout the chimeric locus (multiple different hJλ gene segments rearranged and operably connected to the mouse Cκ gene). Homozygous mice with modified endogenous κ loci containing 40 hVλ gene segments and 4 hJλ gene segments were also able to generate human λ gene segments operably connected to the mouse Cκ gene and produce B cells expressing human λ light chains. These rearrangements further demonstrate that all stages of the chimeric locus were able to independently rearrange human λ gene segments in multiple independent B cells of these mice. Furthermore, as evidenced by the 12 distinct hVλ gene segments that were observed to rearrange with all four hJλ gene segments from the 26 selected RT-PCR clones (Table 8), these additional modifications to the endogenous κ light chain locus demonstrate that each insertion of a human λ gene segment did not render any hVλ and / or Jλ gene segments inoperable, nor did the chimeric locus prevent recombination of hVλ and Jλ gene segments during B cell development. Moreover, these mice also produced functional antibodies that contained human Vλ-Jλ gene segments operably linked to mouse Cκ regions as part of their endogenous immunoglobulin light chain repertoire.

[0245] Figure 13 shows the sequences of the hVλ-hJλ-mCλ2 junction for 12 hVλ gene segments and 3 individual RT-PCR clones from mice homozygous for hJλ1. The sequences shown in Figure 13 show additional unique rearrangements involving different hVλ gene segments across the length of the first insertion (hJλ1 is rearranged and operably connected to the mouse Cλ2 gene) (2D1=Vλ2-8Jλ1; 2D9=Vλ3-10Jλ1; 3E15=Vλ3-1Jλ1). One clone showed a non-productive rearrangement due to the addition of an N at the hVλ-hJλ junction (2D1, Figure 13). This is not uncommon in V(D)J recombination, since the joining of gene segments during recombination has been shown to be imprecise. Although this clone represents a non-productive recombinant present in the light chain repertoire of these mice, it demonstrates that the genetic mechanisms contributing to the diversity of junctions between antibody genes are operating normally in these mice, thereby resulting in an antibody repertoire that contains light chains with greater diversity.

[0246] Homozygous mice with modified endogenous λ loci containing 12 hVλ gene segments and hJλ1 were also able to generate human λ gene segments operably connected to endogenous mouse Cλ genes and were able to produce B cells expressing reverse chimeric λ light chains containing hVλ regions connected to mouse Cλ regions. These rearrangements further demonstrate that human λ light chain gene segments placed at other light chain loci (i.e., λ loci) were able to independently rearrange human λ gene segments in multiple independent B cells of these mice. Furthermore, the modifications to the endogenous λ light chain locus demonstrate that the insertion of human λ gene segments did not render any of the hVλ gene segments and / or hJλ1 gene segments inoperable, and that the chimeric locus did not prevent recombination of hVλ and hJλ1 gene segments during B cell development. Furthermore, these mice also produced functional antibodies containing human Vλ-Jλ gene segments operably connected to mouse Cλ regions as part of their endogenous immunoglobulin light chain repertoire.

[0247] Since functional light chains are required at various checkpoints of B cell development in both the spleen and bone marrow, as shown in this example, mice carrying human λ light chain gene segments at endogenous κ and λ light chain loci are able to rearrange human λ light chain gene segments and express them in the context of mouse Cκ and / or Cλ regions as part of the mouse's normal antibody repertoire. Furthermore, early subsets of B cells (e.g., pre-, pro- and transitional B cells) display a normal phenotype in these mice compared to wild-type littermates (Figures 9D, 10A and 10B). Minor defects were observed in bone marrow and peripheral B cell populations, which may be due to the deletion of a subset of autoreactive immature B cells and / or suboptimal association of human λ light chains with mouse heavy chains. However, the Igκ / Igλ usage observed in these mice represents a more similar picture to human light chain expression than that observed in mice.

[0248] Example VI Breeding of mice expressing human lambda light chains from the endogenous light chain locus To optimize the frequency of human λ gene segment usage at the endogenous mouse light chain locus, a mouse with unrearranged human λ gene segments is bred with another mouse with a deletion at the opposing endogenous light chain locus (κ or λ). For example, the human λ gene segments located at the endogenous κ locus may be the only functional light chain gene segments present in a mouse that also has a deletion at the endogenous λ light chain locus. In this manner, the resulting offspring may express only human λ light chains as described in the preceding examples. Breeding is performed by standard techniques recognized in the art and alternatively by commercial companies, such as The Jackson Laboratory. Mouse strains with human λ light chain gene segments at the endogenous κ locus and deletions at the endogenous λ light chain locus are screened for the presence of unique reverse chimeric (human-mouse) λ light chains and the absence of endogenous mouse λ light chains.

[0249] Mice with unrearranged human λ light chain loci are also bred with mice that contain a replacement of the endogenous mouse heavy chain variable gene locus with a human heavy chain variable gene locus (see U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE® engineered mice). VELOCIMMUNE® mice include, in part, mice with genomes that contain a human heavy chain variable region operably linked to an endogenous mouse constant region locus (the mice produce antibodies that contain a human heavy chain variable region and a mouse heavy chain constant region in response to antigenic stimulation). DNA encoding the variable region of the heavy chain of those antibodies can be isolated and operably linked to DNA encoding a human heavy chain constant region. The DNA can then be expressed in a cell capable of expressing the complete human heavy chain of the antibody. When a suitable breeding schedule is performed, mice are obtained that have a replacement of the endogenous mouse heavy chain locus with a human heavy chain locus and have an unrearranged human λ light chain locus at the endogenous κ light chain locus. Upon immunization with an antigen of interest, antibodies can be isolated that contain a somatically mutated human heavy chain variable region and a human lambda light chain variable region.

[0250] Example VII Production of antibodies from mice expressing human heavy chains and human lambda light chains Mice containing an unrearranged human λ light chain locus are bred with various desired strains containing modifications and deletions of other endogenous Ig loci (as described above) and then the selected mice are immunized with the antigen of interest.

[0251] Typically, VELOCIMMUNE® mice containing one of the single rearranged human germline light chain regions are challenged with an antigen, and lymphocytes (e.g., B cells) are harvested from the animal's serum. Immortal hybridoma cell lines are prepared by fusing the lymphocytes with myeloma cell lines, and hybridoma cell lines that produce antibodies specific to the antigen used for immunization, including human heavy chains and human λ light chains, can be identified by screening and selecting such hybridoma cell lines. DNA encoding the variable regions of the heavy and λ light chains can be isolated and connected to constant regions of the desired isotypes of the heavy and light chains. Due to the presence of additional hVλ gene segments compared to the endogenous mouse λ locus, the diversity of the light chain repertoire is dramatically increased, conferring higher diversity to the antigen-specific repertoire upon immunization. The resulting cloned antibody sequences can then be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes (eg, B cells).

[0252] First, high affinity chimeric antibodies with human variable regions and mouse constant regions are isolated. As described above, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. By replacing the mouse constant regions with the desired human constant regions, fully human antibodies are generated that contain somatically mutated human heavy chains and human λ light chains derived from the unrearranged human λ light chain locus of the present invention. Suitable human constant regions include, for example, wild-type or modified IgG1, IgG2, IgG3, or IgG4. (Item 1) A mouse comprising a λ light chain variable region sequence (Vλ) and at least one J sequence (J) contiguous with a mouse κ light chain constant region sequence. (Item 2) The mouse of item 1, wherein the mouse lacks a functional mouse Vκ gene segment and / or a functional mouse Jκ gene segment. (Item 3) The mouse described in Item 1, wherein the Vλ is a human Vλ (hVλ) and the J is a human Jλ (hJλ). (Item 4) The mouse of item 3, wherein the hVλ and the hJλ are unrearranged gene segments. (Item 5) The mouse described in Item 4, comprising multiple unrearranged hVλ gene segments and at least one hJλ gene segment. (Item 6) The mouse described in Item 5, wherein the unrearranged hVλ gene segments are at least 12 gene segments, at least 28 gene segments, or at least 40 gene segments. (Item 7) The mouse described in Item 3, wherein the at least one hJλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof. (Item 8) The mouse described in Item 1, in which the endogenous mouse λ light chain locus is deleted, in whole or in part. (Item 9) The mouse described in Item 1, wherein the mouse κ light chain constant region sequence is present in an endogenous mouse κ light chain locus. (Item 10) The mouse according to Item 1, wherein about 10% to about 45% of B cells of the mouse express an antibody comprising a light chain comprising a human λ light chain variable (Vλ) domain and a mouse κ light chain constant (Cκ) domain. Item 11. The mouse of item 10, wherein the human lambda variable domain is derived from a rearranged hVlambda / hJlambda sequence selected from the group consisting of 3-1 / 1, 3-1 / 7, 4-3 / 1, 4-3 / 7, 2-8 / 1, 3-9 / 1, 3-10 / 1, 3-10 / 3, 3-10 / 7, 2-14 / 1, 3-19 / 1, 2-23 / 1, 3-25 / 1, 1-40 / 1, 1-40 / 2, 1-40 / 3, 1-40 / 7, 7-43 / 1, 7-43 / 3, 1-44 / 1, 1-44 / 7, 5-45 / 1, 5-45 / 2, 5-45 / 7, 7-46 / 1, 7-46 / 2, 7-46 / 7, 9-49 / 1, 9-49 / 2, 9-49 / 7 and 1-51 / 1. (Item 12) The mouse of item 1, further comprising a human Vκ-Jκ intergenic region derived from a human κ light chain locus, wherein the human Vκ-Jκ intergenic region is contiguous with the Vλ sequence and the J sequence. (Item 13) The mouse described in Item 12, wherein the human Vκ-Jκ intergenic region is positioned between the Vλ sequence and the J sequence. (Item 14) (a) at least 12 to at least 40 unrearranged human λ light chain variable region gene segments and at least one human Jλ gene segment in an endogenous mouse light chain locus; (b) a human Vκ-Jκ intergenic sequence disposed between the at least 12 to at least 40 human light chain variable region gene segments and the at least one human Jλ sequence; wherein the mouse expresses an antibody comprising a light chain comprising a human Vλ domain and a mouse Cκ domain. (Item 15) A mouse expressing an antibody comprising a light chain comprising a lambda variable sequence and a kappa constant sequence. (Item 16) The mouse of item 15, wherein the mouse exhibits a ratio of κ usage frequency to λ usage frequency of about 1:1. (Item 17) The mouse described in Item 16, wherein a population of immature B cells obtained from the bone marrow of the mouse exhibits a ratio of κ usage frequency to λ usage frequency of about 1:1. (Item 18) Use of a mouse according to any one of items 1 to 17 for producing an antigen-binding protein. (Item 19) An antigen-binding protein produced using a mouse according to Items 1 to 17. (Item 20) A cell or tissue derived from a mouse described in Items 1 to 17.

Claims

[Claim 1] The method, mouse or composition described in the specification.

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