Mice expressing light chains containing the human λ variable region and the mouse constant region.

Abstract

To provide a mouse that expresses a light chain containing the human λ variable region and the mouse constant region. [Solution] A genetically modified mouse expressing a human λ variable (hVλ) sequence is provided (including mice expressing the hVλ sequence from the endogenous mouse λ light chain locus, mice expressing the hVλ sequence from the endogenous mouse κ light chain locus, and mice expressing the hVλ sequence from a transgene or episome), wherein the hVλ sequence is linked to a mouse constant sequence. A mouse is provided that serves as the source of somatically mutated human λ variable sequences useful for the production of antigen-binding proteins. A composition and method for producing an antigen-binding protein containing a human λ variable sequence containing a human antibody is also provided.

Description

[Technical Field]

[0001] field Genetically modified mice containing a mouse or human lambda variable (Vλ) light chain sequence operably connected to a mouse or human light chain constant region (λ or kappa (κ)). Variable domains derived from the human lambda variable (hVλ) gene segment, the human lambda J (hJλ) gene segment, and the mouse light chain constant (C L A genetically modified mouse expressing an epitope-binding protein containing an immunoglobulin light chain including the ) domain. A genetically modified mouse in which the endogenous mouse light chain locus contains an unrearranged immunoglobulin lambda (λ) light chain variable nucleic acid sequence. A chimeric human-λ / mouse C from an endogenous light chain locus, including substitution of all endogenous mouse light chain variable region gene segments by one or more hVλ gene segments and one or more hJλ gene segments. L A mouse capable of rearranging and expressing the light chain. hVλ domain and mouse C L Antibodies with somatic mutations that include a domain. [Background technology]

[0002] background Mice expressing antibodies that are either entirely human or partially human and partially mouse antibodies are known in this field. For example, transgenic mice expressing fully human antibodies from transgenes containing human light chain and heavy chain immunoglobulin variable region genes have been reported. Similarly, genetically modified mice are known that produce chimeric antibodies with chimeric human / mouse copper chains, including substitutions of endogenous mouse HCVR gene segments and copper (κ)LCVR gene segments with human heavy chain variable region (HCVR) and light chain variable region (LCVR) gene segments.

[0003] Antibody light chains are encoded by one of two distinct gene loci: kappa (κ) and lambda (λ). Mouse antibody light chains are predominantly of the κ type. The usage ratio of κ to λ light chains in humans is approximately 60:40, while in mice it is approximately 95:5. Reports indicate that the biased usage of κ light chains in mice is maintained even in genetically modified mice capable of expressing fully human or partially human antibodies. Therefore, it appears that mice expressing fully human or partially human antibodies exhibit suppressed use of lambda-variable light chains. [Overview of the project] [Problems that the invention aims to solve]

[0004] In this field, it is necessary to create lambda variable regions for use in the production of epitope-binding proteins, whether from mice or humans. This field requires mice that express fully human or partially human antibodies that exhibit a high frequency of lambda variable (Vλ) utilization.

[0005] In this field, there is a need for mice that express fully human or partially human antibodies that exhibit a high frequency of λ variable (Vλ) usage. [Means for solving the problem]

[0006] Abstract Genetically modified mice, embryos, cells, tissues, and nucleic acid constructs for modifying mice are provided, as well as methods and compositions for producing and using them. Mice and cells that produce lambda (λ) variable regions (human or non-human) in the background of a copper (κ) light chain are provided. For example, mice and cells that produce human λ variable regions in the background of a κ or λ light chain from an endogenous mouse light chain locus are also provided. Methods for producing antibodies containing lambda variable regions are also provided. Methods for selecting heavy chains to be expressed together with congeneral lambda variable regions are also provided.

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

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

[0009] In one embodiment, a genetically modified mouse is provided, wherein the mouse includes an unrearranged human λ light chain variable gene segment (hVλ) and a human λ linking gene segment (hJλ). In one embodiment, the unrearranged hVλ and hJλ reside at a mouse light chain locus. In one embodiment, the unrearranged hVλ and unrearranged hJλ reside on a transgene and are operably linked to a human or mouse constant region sequence. In one embodiment, the unrearranged hVλ and unrearranged hJλ reside on an episome. In one embodiment, the mouse includes the unrearranged hVλ and hJλ sequences as well as the mouse light chain constant region (C L ) It is possible to produce immunoglobulins containing light chains derived from nucleic acid sequences. Methods and compositions for creating and using genetically modified mice are also provided. (a) Human heavy chain variable domain (hV) fused to the mouse heavy chain constant region H ), and (b) Mouse C LAntibodies are provided that contain human Vλ fused to a domain, including, for example, antibodies in which one or more of their variable domains have undergone somatic mutation during the selection of antibodies or immune cells in mice according to the present invention. In one embodiment, the unarranged hVλ and unarranged hJλ are operably connected to a human or mouse κ constant region (Cκ). In one embodiment, the unarranged hVλ and unarranged hJλ are operably connected to a human or mouse λ constant region (Cλ).

[0010] In one embodiment, a mouse is provided in which a human lambda variable region sequence is present in an endogenous mouse light chain gene locus in the germline, wherein the human lambda variable region sequence is expressed in a light chain containing 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 the endogenous mouse light chain gene locus.

[0013] In one embodiment, all or substantially all 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 includes a human Jλ sequence. In one embodiment, the human Jλ sequence is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7 and combinations thereof.

[0015] In one embodiment, the human λ light chain variable region sequence includes a fragment of cluster A of the human light chain locus. In a particular embodiment, the fragment of cluster A of the human λ light chain locus extends from hVλ3-27 to hVλ3-1.

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

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

[0018] In one embodiment, more than 10% of the light chain naive repertoire of the mouse 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, more than 20% of the light chain naive repertoire of the mouse 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, more than 30% of the light chain naive repertoire of the mouse 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 containing a human λ variable sequence fused to a mouse constant region, wherein the mouse exhibits a κ-to-λ utilization ratio of approximately 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 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.

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

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

[0024] In one embodiment, the mouse comprises multiple unrearranged hVλ gene segments and at least one hJλ gene segment. In a particular embodiment, the multiple unrearranged hVλ gene segments are at least 12 gene segments, at least 28 gene segments, or at least 40 gene segments.

[0025] In one embodiment, 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 entirely or partially.

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

[0028] In one embodiment, approximately 10% to 45% of the mouse B cells express antibodies containing light chains that include human λ light chain variable (Vλ) domains and mouse κ light chain constant (Cκ) domains.

[0029] In one embodiment, the human λ variable domain is derived from a rearranged hVλ / hJλ 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 Vκ-Jκ intergene region derived from the human κ light chain locus, where the human Vκ-Jκ intergene region is contiguous with the Vλ and J sequences. In a particular embodiment, the human Vκ-Jκ intergene region is located between the Vλ and J sequences.

[0031] In one embodiment, a mouse is provided having (a) at an endogenous mouse light chain locus at least 12 to 40 unrearranged human λ light chain variable region gene segments and at least one human Jλ gene segment; and (b) a human Vκ-Jκ intergeneric sequence positioned between at least 12 to 40 human light chain variable region gene segments and at least one human Jλ sequence, wherein the mouse expresses an antibody comprising a light chain containing 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 containing a λ variable sequence and a κ constant sequence.

[0033] In one embodiment, the mouse exhibits a ratio of approximately 1:1 between the frequency of use of κ and the frequency of use of λ.

[0034] In one embodiment, a population of immature B cells obtained from the bone marrow of the mouse exhibits a ratio of approximately 1:1 between the frequency of κ utilization and the frequency of λ utilization.

[0035] In one embodiment, a genetically modified mouse is provided, where the mouse is mouse C L It includes unrearranged immunoglobulin Vλ and Jλ gene segments operably connected to mouse light chain loci containing the genes.

[0036] In one embodiment, the Vλ and / or Jλ gene segments are human gene segments. In one embodiment, the Vλ and / or Jλ gene segments are mouse gene segments, C L This is mouse Cκ.

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

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

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

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

[0041] In one embodiment, the mouse includes unrearranged immunoglobulin Vλ and Jλ gene segments in the endogenous mouse κ light chain locus containing the mouse Cκ gene.

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

[0043] In one embodiment, the locus of the light chain variable gene (the "V L locus") contains at least one human Vλ (hVλ) gene segment. In one embodiment, its V L locus contains at least one human Jλ (hJλ) gene segment. In another embodiment, the V L locus contains up to four hJλ gene segments. In one embodiment, its V L locus contains a continuous sequence containing human λ and human κ genomic sequences.

[0044] In one embodiment, the locus of the κ light chain variable gene (the "κ locus") contains at least one human Vλ (hVλ) gene segment. In one embodiment, its κ locus contains at least one human Jλ (hJλ) gene segment. In one embodiment, its κ locus contains up to four hJλ gene segments. In one embodiment, its κ locus contains 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 contain a functional Vκ region gene segment. In one embodiment, the mouse does not contain a functional Jκ region gene segment.

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

[0046] In one embodiment, V L The locus contains multiple hVλ. In one embodiment, multiple hVλ are selected so as to result in the expression of a λ light chain variable region repertoire that reflects the Vλ usage frequencies observed in humans at approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or higher. In one embodiment, V L The locus includes gene segments hVλ1-40, 1-44, 2-8, 2-14, 3-21, and combinations thereof.

[0047] In one embodiment, hVλ includes 3-1, 4-3, 2-8, 3-9, 3-10, 2-11 and 3-12. In a particular embodiment, V L The locus contains a contiguous sequence of the human λ light chain locus extending from Vλ3-12 to Vλ3-1. In one embodiment, V L The gene locus contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hVλ. In certain embodiments, hVλ include 3-1, 4-3, 2-8, 3-9, 3-10, 2-11 and 3-12. In certain embodiments, V L The locus contains a contiguous sequence of human lambda loci extending from Vλ3-12 to Vλ3-1. In one embodiment, V L The gene locus is located at the endogenous κ gene locus. In certain embodiments, V L The gene locus is located at the endogenous κ locus, and the endogenous λ light chain locus is partially or completely deleted. In one embodiment, V L The gene locus is located at the endogenous λ gene locus. In a particular embodiment, V L The gene locus is located at the endogenous λ locus, while the endogenous κ locus is partially or completely deleted.

[0048] In one embodiment, V LThe locus contains 13 to 28 or more hVλ. In certain embodiments, these hVλ 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 contains a contiguous sequence of human λ loci extending from Vλ3-27 to Vλ3-1. In one embodiment, V L The gene locus is located at the endogenous κ gene locus. In certain embodiments, V L The gene locus is located at the endogenous κ locus, and the endogenous λ light chain locus is partially or completely deleted. In another embodiment, V L The gene locus is located at the endogenous λ gene locus. In a particular embodiment, V L The gene locus is located at the endogenous λ locus, while the endogenous κ locus is partially or completely deleted.

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

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

[0051] In one embodiment, V L The gene locus includes at least one hVλ, at least one hJλ, and the mouse Cκ gene. In one embodiment, V LThe locus comprises at least one hVλ, at least one hJλ, and a mouse Cλ gene. In a particular embodiment, the mouse Cλ gene is Cλ2. In a particular embodiment, 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 substitution of an endogenous mouse Vκ gene segment at the endogenous mouse κ locus by one or more hVλ gene segments, wherein the hVλ gene segment is operably connected to an endogenous mouse Cκ region gene, and the mouse rearranges the human Vλ gene segment to express a reverse chimeric immunoglobulin light chain containing the human Vλ domain and mouse Cκ. In one embodiment, 90–100% of the unrearranged mouse Vκ gene segment is replaced by at least one unrearranged hVλ gene segment. In a particular embodiment, all or substantially all of the endogenous mouse Vκ gene segment is replaced by at least one unrearranged hVλ gene segment. In one embodiment, the substitution is by at least 12, at least 28, or at least 40 unrearranged hVλ gene segments. In one embodiment, the substitution is a substitution by at least seven functionally unrearranged hVλ gene segments, at least 16 functionally unrearranged hVλ gene segments, or at least 27 functionally unrearranged hVλ gene segments. In one embodiment, the mouse includes the substitution of all mouse Jκ gene segments by 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 combinations thereof. In certain embodiments, one or more hVλ gene segments are selected from the 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 particular 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 has an endogenous mouse λ locus containing a substitution of the endogenous mouse Vλ gene segment with one or more human Vλ gene segments, where the hVλ gene segment is operably connected to a mouse Cλ region gene, and the mouse rearranges the hVλ gene segment to express a reverse chimeric immunoglobulin light chain containing the hVλ domain and mouse Cλ. In a particular embodiment, the mouse Cλ gene is Cλ2. In a particular 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 segment is replaced with at least one unrearranged hVλ gene segment. In a particular embodiment, all or substantially all of the endogenous mouse Vλ gene segment is replaced with at least one unrearranged hVλ gene segment. In one embodiment, the substitution is a substitution by at least 12, at least 28, or at least 40 unrearranged hVλ gene segments. In one embodiment, the substitution is a substitution by at least 7 functionally unrearranged hVλ gene segments, at least 16 functionally unrearranged hVλ gene segments, or at least 27 functionally unrearranged hVλ gene segments. In one embodiment, the mouse includes the substitution of all mouse Jλ gene segments by 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 combinations thereof.In certain embodiments, one or more hVλ gene segments are selected from 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, 5-52hVλ gene segments and combinations thereof. In certain embodiments, at least one unrearranged hJλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ7 and combinations thereof.

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

[0055] In one embodiment, the human Vκ-Jκ intergenic region sequence is located at the endogenous κ light chain locus of a mouse containing the hVλ and hJλ gene segments, and the human Vκ-Jκ intergenic region sequence is positioned between the hVλ and hJλ gene segments. In a particular embodiment, the hVλ and hJλ gene segments can be recombined in the mouse to form a functional human λ light chain variable domain.

[0056] In one embodiment, a mouse comprising multiple hVλ and one or more hJλ is provided, and in terms of transcription, the human Vκ-Jκ intergeneric region sequence is located downstream of the proximal or most 3' hVλ sequence and upstream or 5' of the first hJλ sequence.

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

[0058] In one embodiment, mice, mouse cells (e.g., mouse embryonic stem cells), mouse embryos, and mouse tissues containing the enumerated human Vκ-Jκ intergenetic region sequence are provided, where the intergenetic region sequence is ectopic. In certain embodiments, the ectopic sequence is located at a humanized endogenous mouse immunoglobulin locus.

[0059] In one embodiment, an isolated nucleic acid construct is provided comprising an enumerated human Vκ-Jκ intergenetic region sequence. In one embodiment, the nucleic acid construct includes a targeting arm that targets the human Vκ-Jκ intergenetic region sequence to a mouse light chain locus. In a particular embodiment, the mouse light chain locus is a κ locus. In a particular embodiment, the targeting arm targets the human Vκ-Jκ intergenetic region to a modified endogenous mouse κ locus, where the targeting is relative to a position between the hVλ and hJλ sequences.

[0060] In one embodiment, a genetically modified mouse is provided, wherein the mouse comprises two or fewer light chain alleles, wherein the light chain alleles are (a) mouse C L (b) Unrearranged immunoglobulin human Vλ and Jλ gene segments at the endogenous mouse light chain locus containing the gene; and (b) mouse C L Unrearranged immunoglobulin V is present at the endogenous mouse light chain locus containing the gene. L and J L Includes 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 fewer light chain alleles are selected from a κ allele and a λ allele, two κ alleles and two λ alleles. In a particular embodiment, one of the two light chain alleles is a λ allele containing the Cλ2 gene.

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

[0064] In one embodiment, the mouse comprises one functional immunoglobulin light chain locus and one non-functional light chain locus, wherein the functional light chain locus comprises an endogenous mouse λ light chain locus containing the mouse Cλ gene, and unrearranged immunoglobulin human Vλ and Jλ gene segments. In one embodiment, the Cλ gene is Cλ2. In a particular 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. In one embodiment, the at least one immunoglobulin heavy chain allele is located at an endogenous mouse heavy chain locus containing a human heavy chain gene that expresses human / mouse heavy chain, and contains human V H Gene segment, Human D H Gene segments and human J H The gene segment is included. In a particular embodiment, the mouse contains two immunoglobulin heavy chain alleles, and the mouse expresses human / mouse heavy chains.

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

[0067] In one embodiment, the mouse has a first light chain allele containing unrearranged hVκ and unrearranged hJκ at the endogenous mouse κ locus containing the endogenous Cκ gene; and a second light chain allele containing unrearranged hVλ and unrearranged hJλ at the endogenous mouse λ locus containing the endogenous Cλ gene. In a particular embodiment, the only functional light chain alleles in the genetically modified mouse are its first and second light chain alleles. In one embodiment, the endogenous Cλ gene is Cλ2. In a particular 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, where the first allele comprises unrearranged immunoglobulin Vλ and Jλ gene segments in the endogenous mouse κ light chain locus containing the mouse Cκ gene, the second allele comprises unrearranged immunoglobulin Vκ and Jκ gene segments in the endogenous mouse κ light chain locus containing the mouse Cκ gene, the third allele comprises unrearranged immunoglobulin Vλ and Jλ gene segments in the endogenous mouse λ light chain locus containing the mouse Cλ gene, and the fourth and fifth alleles each independently comprise unrearranged V in the endogenous mouse heavy chain locus containing the mouse heavy chain gene H and D H and J H The gene segment comprises, and the sixth allele comprises (a) an endogenous mouse λ light chain locus containing the mouse Cλ gene, containing unrearranged immunoglobulin Vλ and Jλ gene segments, (b) a non-functional λ locus, or (c) a deletion of the λ locus entirely or partially.

[0069] In one embodiment, the first allele described above comprises unrearranged hVλ and hJλ. In one embodiment, the second allele described above comprises unrearranged hVκ and hJκ. In one embodiment, the third allele described above comprises unrearranged hVλ and hJλ. In one embodiment, the fourth and fifth alleles described above each independently comprise unrearranged hV H and hD H and hJ H This includes, in one embodiment, the sixth allele described above comprises an endogenous mouse λ locus that is deleted entirely or partially.

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

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

[0072] In one embodiment, the genetically modified mouse is mouse C L The invention comprises B cells containing a rearranged antibody gene containing a rearranged hVλ domain operably connected to the domain. In one embodiment, the mouse C L The domain is selected from mouse Cκ and mouse Cλ domains. In certain embodiments, the mouse Cλ domain is derived from the 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, Cκ is C L A genetically modified mouse is provided that expresses the Vλ region in [location]. In one embodiment, C selected from human Cκ, human Cλ, or mouse Cκ is provided. L A genetically modified mouse is provided that expresses the hVλ region in mouse Cκ. In one embodiment, a genetically modified mouse is provided that expresses the hVλ region in mouse Cκ.

[0074] In one embodiment, approximately 10–50% of the mouse splenocytes described above are B cells (i.e., CD19-positive), or approximately 9–28% express immunoglobulin light chains containing an hVλ domain fused to a mouse Cκ domain.

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

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

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

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

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

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

[0081] In one embodiment, approximately 60% of the bone marrow of the mouse described above consists of B cells (i.e., CD19-positive), or approximately 38.3% of its immature B cells (i.e., CD19-positive / moderately B220-positive / IgM-positive) express an immunoglobulin light chain containing an hVλ domain fused to a mouse Cκ domain.

[0082] In one embodiment, the mouse expresses an antibody comprising a light chain containing variable domains derived from human V and human J gene segments and a constant domain derived from a mouse constant domain gene. In one embodiment, the mouse constant domain gene is the Cκ gene. In another embodiment, the mouse constant domain gene is the 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 containing 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 domain gene. In one embodiment, the mouse heavy chain constant domain gene contains a hinge-CH2-CH3 sequence of the heavy chain constant domain. In another embodiment, the mouse heavy chain constant domain gene contains a CH1-hinge-CH2-CH3 sequence of the heavy chain constant domain. In another embodiment, the mouse heavy chain constant domain gene includes the CH1-CH2-CH3-CH4 sequence of the heavy chain constant domain.

[0083] In one embodiment, the mouse expresses an antibody comprising a light chain containing 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 an hVλ gene segment 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 an hJλ gene segment selected from the Jλ1, Jλ2, Jλ3, and Jλ7 gene segments.

[0084] In one embodiment, the mice are 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 The B cells express antibodies containing a light chain that includes a rearranged immunoglobulin λ light chain variable region containing a human Vλ / Jλ sequence selected from / 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 cells express antibodies containing a human immunoglobulin heavy chain variable domain fused with a mouse heavy chain constant domain and a human immunoglobulin λ light chain variable domain fused with a mouse κ light chain constant domain.

[0085] In one embodiment, (a) a heavy chain containing a heavy chain variable domain derived from an unrearranged human heavy chain variable region gene segment (where the heavy chain variable domain is mouse heavy chain constant (C) H (b) a light chain containing light chain variable domains derived from unrearranged hVλ and hJλ (where the light chain variable domains are mouse C L A mouse is provided that expresses an antibody containing (which is fused to the region).

[0086] In one embodiment, the mouse (i) replaces 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, mouse C H The mouse comprises a heavy chain locus containing the gene, (ii) substitution of all or substantially all functional endogenous mouse Vκ and Jκ gene segments with all, substantially all or more functional hVλ and hJλ gene segments, and a first κ light chain locus containing the mouse Cκ gene, and (iii) substitution of all or substantially all functional endogenous mouse Vκ and Jκ gene segments with all, substantially all or more functional hVκ and hJκ gene segments, and a second κ light chain locus containing the mouse Cκ gene. In one embodiment, the mouse does not express an antibody containing the Cλ region. In one embodiment, the mouse comprises a deletion of the Cλ gene and / or Vλ and / or Jλ gene segments. In one embodiment, the mouse comprises a non-functional λ light chain locus. In a particular embodiment, the λ light chain locus is deleted whole or partially.

[0087] In one embodiment, the mouse (i) replaces 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, mouse C HThe locus comprises (ii) a heavy chain locus containing the gene, (ii) a first λ light chain locus containing all or substantially all functional endogenous mouse Vλ and Jλ gene segments replaced by all, substantially all or more functional hVλ and hJλ gene segments and the mouse Cλ gene, and (iii) a second λ light chain locus containing all or substantially all functional endogenous mouse Vλ and Jλ gene segments replaced by all, substantially all or more functional hVλ and hJλ gene segments and the mouse Cλ gene. In a particular embodiment, the mouse Cλ gene 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.

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

[0089] In one embodiment, a genetically modified mouse expressing an antibody is provided, 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 the total IgG antibody produced by the mouse contains a λ-derived variable domain, and the mouse expresses an antibody containing a κ-derived variable domain fused to a mouse Cκ region. In a particular embodiment, about 15–40%, 20–40%, 25–40%, 30–40%, or 35–40% of the total antibody produced by the mouse contains a λ-derived variable domain.

[0090] In one embodiment, the λ-derived variable domains described above are derived from hVλ and hJλ. In one embodiment, the λ-derived variable domains described above are located in a light chain containing a mouse Cκ region. In a particular embodiment, the λ-derived variable domains described above are located in a light chain containing a mouse Cλ region. In another particular embodiment, the Cλ region is the Cλ2 region. In one embodiment, the κ-derived variable domains are derived from hVκ and hJκ, and in a particular embodiment, are located in a light chain containing a mouse Cκ region.

[0091] In one embodiment, an isolated DNA construct is provided comprising an upstream homology arm and a downstream homology arm, wherein the upstream and downstream homology arms target the construct to a mouse κ locus, and the construct comprises a functionally unrearranged hVλ segment and a functionally unrearranged hJλ segment, as well as a selection sequence or marker sequence.

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

[0093] In one embodiment, an isolated DNA construct is provided comprising a targeting arm for targeting a mouse λ locus 5' to Vλ1 with respect to the direction of transcription from 5' to 3', a recombinase recognition site and a selection cassette adjacent to the 5' and 3' sides, and a 3' targeting arm for targeting a 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 comprises a mouse λ3' enhancer and a mouse λ3' enhancer 3.1.

[0094] In one embodiment, an isolated DNA construct is provided comprising a targeting arm for targeting a mouse λ locus 5' to Vλ2 with respect to the direction of transcription from 5' to 3', a recombinase recognition site and a selection cassette adjacent on the 5' and 3' sides, and a 3' targeting arm for targeting a 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 comprises a mouse Cλ2-Jλ4-Cλ4 gene segment and a mouse λ enhancer 2.4.

[0095] In one embodiment, an isolated DNA construct is provided comprising a targeting arm for targeting a mouse λ locus 5' to Vλ2 with respect to the transcription direction from 5' to 3', a recombinase recognition site and a selection cassette adjacent at the 5' and 3' ends, a human genome fragment containing a contiguous region of human λ light chain loci derived from hVλ3-12 downstream of the end of hJλ1, and a 3' targeting arm for targeting a 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 comprises a mouse Cλ2-Jλ4-Cλ4 gene segment and a mouse λ enhancer 2.4.

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

[0097] In one embodiment, an isolated DNA construct is provided comprising a human genome fragment containing a targeting arm for targeting a mouse λ locus 5' to Vλ2 with respect to the transcription direction from 5' to 3', a recombinase recognition site and a selection cassette adjacent at the 5' and 3' ends, and a contiguous region of a human λ light chain locus derived from hVλ3-27 downstream of the ends of hVλ2-8. In one embodiment, the selection cassette is a hygromycin cassette flanked by Frt. In one embodiment, the human genome fragment constitutes a 3' targeting arm. In a particular embodiment, the 3' targeting arm contains a human λ light chain locus of approximately 53 kb derived from hVλ3-12 downstream of the ends of hVλ2-8.

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

[0099] In one embodiment, an isolated DNA construct is provided comprising a targeting arm for targeting the mouse λ locus 5' to Vλ2 with respect to the transcription direction from 5' to 3', a recombinase recognition site and a selection cassette adjacent on the 5' and 3' sides, a first human genome fragment containing a contiguous region of human λ light chain loci derived from hVλ5-52 downstream of the terminals of hVλ1-40, a restriction enzyme site, and a second human genome fragment containing a contiguous region of human λ light chain loci derived from hVλ3-29 downstream of the terminals 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 (on) embodiment, the second human genome fragment described above is a 3' targeting arm. In certain embodiments, the 3' targeting arm includes approximately 27 kb of human λ light chain locus derived from hVλ3-29 downstream of the terminal of hVλ82K.

[0100] In one embodiment, an isolated DNA construct is provided, comprising a contiguous region of human λ light chain loci derived from hVλ5-52 downstream of the ends of hVλ1-40.

[0101] In one embodiment, an isolated DNA construct is provided comprising, in the direction of transcription from 5' to 3', a targeting arm for targeting a mouse κ locus on the 5' side with respect to an endogenous Vκ gene segment, two juxtaposed recombinase recognition sites, a selection cassette on the 3' side with respect to the juxtaposed recombinase recognition sites, and a 3' targeting arm for targeting a mouse κ sequence on the 5' side with respect to a κ light chain variable gene segment. In one embodiment, the juxtaposed recombinase recognition sites are oriented opposite 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 a targeting arm for targeting a mouse κ locus 5' to the mouse Jκ gene segment with respect to the direction of transcription from 5' to 3', a selection cassette, a recombinase recognition site 3' to the selection cassette, and a 3' targeting arm for targeting a mouse κ sequence 3' to the mouse Jκ gene segment and 5' to the mouse κ intron enhancer. In one embodiment, the selection cassette is a hygromycin-TK cassette. In one embodiment, the recombinase recognition site is oriented in the same direction 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, comprising a first mouse genome fragment containing the 5' side sequence of an endogenous mouse Vκ gene segment, a first recombinase recognition site, a second recombinase recognition site, and a second mouse genome fragment containing the 3' side sequence of an endogenous mouse Jκ gene segment and the 5' side sequence of a mouse κ intron enhancer, oriented 5' to 3' with respect to the direction of transcription.

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

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

[0106] In one embodiment, non-human stem cells are provided, comprising a targeted vector containing a DNA construct as described above and herein. In one embodiment, non-human stem cells are provided, wherein the non-human stem cells are derived from a mouse as described herein.

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

[0108] In one embodiment, the use of non-human stem cells as described herein for producing mice as described herein is provided. In one embodiment, the use of non-human stem cells as described herein for producing antigen-binding proteins is provided.

[0109] In one embodiment, a mouse embryo is provided, wherein the mouse embryo includes genetic modifications as provided herein. In one embodiment, a host mouse embryo containing donor ES cells is provided, wherein the donor ES cells include genetic modifications as described herein. In one embodiment, the mouse embryo is an embryo at the pre-morula stage. In a particular embodiment, the pre-morula stage embryo is a 4-cell stage embryo or an 8-cell stage embryo. In another particular embodiment, the mouse embryo is a blastocyst.

[0110] In one embodiment, the use of a mouse embryo as described herein for producing a mouse as described herein is provided. In one embodiment, the use of a mouse embryo as described herein for producing an antigen-binding protein is provided.

[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 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 immunoglobulin heavy chain variable domain.

[0113] In one embodiment, the use of non-human cells as described herein for producing mice as described herein is provided. In one embodiment, the use of non-human cells as described herein for producing antigen-binding proteins is provided.

[0114] In one embodiment, (a) variable regions derived from the hVλ gene segment and the hJλ gene segment; and (b) mouse C L Mouse B cells expressing an immunoglobulin light chain containing a gene are provided. In one embodiment, the mouse C LThe gene is selected from the Cκ and Cλ genes. In certain embodiments, the 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. In one embodiment, the mouse B cells are further (c)hV H , hD H Variable region derived from (d)hJ H The cells express a congeneral heavy chain containing a segment. In one embodiment, the B cells do not contain a rearranged λ gene. In another embodiment, the B cells do not contain a rearranged κ gene.

[0115] In one embodiment, a method is provided for producing antibodies in a genetically modified mouse, the method comprising: (a) exposing a genetically modified mouse to an antigen (where the mouse has a genome containing at least one hVλ and at least one hJλ in its endogenous light chain locus, and its endogenous light chain locus is mouse C L (b) a step of inducing an immune response to the antigen in the genetically modified mouse; and (c) a step of isolating an antibody that specifically recognizes the antigen from the mouse in (b), or a step of isolating cells containing an immunoglobulin domain that specifically recognizes the antigen from the mouse in (b) (wherein the antibody is hVλ, hJλ and mouse C L Includes (including light chains derived from genes). In certain embodiments, the above mouse C L The gene in question is the mouse Cκ gene.

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

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

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

[0119] In one embodiment, the λ light chain constant gene is selected from the human Cλ gene and the mouse Cλ gene. In one embodiment, the λ light chain constant gene is the human Cλ gene. In a particular embodiment, the human Cλ gene is selected from Cλ1, Cλ2, Cλ3, and Cλ7. In one embodiment, the λ light chain constant gene is the mouse Cλ gene. In a particular embodiment, the mouse Cλ gene is selected from Cλ1, Cλ2, and Cλ3. In a more particular embodiment, the mouse Cλ gene is Cλ2. In another 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.

[0120] In one embodiment, a method is provided for producing a rearranged antibody gene in a genetically modified mouse, the method comprising: (a) exposing a genetically modified mouse to an antigen (where the genetic modification includes hVλ and hJλ in the endogenous light chain loci, and the endogenous light chain loci are in mouse C L (b) a step of identifying an immunoglobulin gene that has been rearranged in the mouse (wherein the rearranged immunoglobulin gene is the λ light chain variable region gene segment and C L Includes genes or their functional fragments.

[0121] In one embodiment, the method further comprises the step of cloning a nucleic acid sequence encoding a heavy chain and / or light chain variable region from the mouse, wherein the heavy chain and / or light chain variable region is human Vλ and mouse Cλ. L It is derived from antibodies containing this substance.

[0122] In one embodiment, the mouse C L The gene or its functional fragment is human C L Genes and mouse C LSelected from genes or their functional fragments.

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

[0124] In one embodiment, the κ light chain constant gene or its functional fragment is selected from the human Cκ gene and the mouse Cκ gene or their functional fragments.

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

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

[0127] In one embodiment, the λ light chain constant gene or its functional fragment is selected from the human Cλ gene and the mouse Cλ gene or their functional fragments. In a particular embodiment, the λ light chain constant gene is the mouse Cλ gene or its functional fragment.

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

[0129] In one embodiment, a method for producing an antibody is provided, the method comprising the steps of: exposing a mouse as described herein to an antigen; initiating an immune response in the mouse, including the production of an antibody that specifically binds to the antigen; identifying a rearranged nucleic acid sequence encoding a heavy chain within the mouse and a rearranged nucleic acid sequence encoding a homologous light chain variable domain sequence of the antibody within the mouse (wherein the antibody specifically binds to the antigen); and producing a desired antibody using the nucleic acid sequences of a heavy chain and a light chain variable domain fused to a human constant domain (wherein the desired antibody is C L It includes a light chain containing a Vλ domain fused to the domain. In one embodiment, the Vλ domain is a human domain, and C L The domain is a human or mouse Cλ domain. In one embodiment, the Vλ domain is a mouse domain, and C L The domain is the human or mouse Cκ domain.

[0130] In one embodiment, a method for producing an antibody is provided, comprising the steps of: exposing a mouse to an antigen as described herein; initiating an immune response in the mouse, including the production of an antibody that specifically binds to the antigen; identifying a rearranged nucleic acid sequence encoding a heavy chain and a rearranged nucleic acid sequence encoding a homologous light chain variable domain sequence of the antibody in the mouse (wherein the antibody specifically binds to the antigen); and producing a desired antibody using the nucleic acid sequences of a heavy chain and a light chain variable domain fused to a human constant domain nucleic acid sequence (wherein the desired antibody includes a light chain containing a Vλ domain fused to a Cκ domain).

[0131] In one embodiment, a method for producing an antibody is provided, the method comprising the steps of: exposing a mouse to an antigen as described herein; initiating an immune response in the mouse, including the production of an antibody that specifically binds to the antigen; identifying a rearranged nucleic acid sequence in the mouse that encodes a heavy chain variable domain and a rearranged nucleic acid sequence that encodes a homologous light chain variable domain sequence of the antibody (wherein the antibody specifically binds to the antigen); and producing an antibody derived from a human sequence using a nucleic acid sequence fused to a nucleic acid sequence encoding a human heavy chain constant domain and a human light chain constant domain (wherein the antibody that specifically binds to the antigen includes a light chain containing 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 particular embodiment, the mouse Cλ region is Cλ2.

[0133] In one embodiment, a method is provided for producing a rearranged antibody light chain variable region gene sequence, the method comprising: (a) exposing a mouse as described herein to an antigen; (b) initiating an immune response in the mouse; and (c) mouse C LA step of identifying, in the mouse, a cell comprising a nucleic acid sequence encoding a rearranged human Vλ domain sequence fused with a domain (wherein the cell also encodes a cognate heavy chain comprising a human V H domain and a mouse C H domain, and the cell expresses an antibody that binds to the antigen); (d) a step of cloning a nucleic acid sequence encoding a human Vλ domain and a nucleic acid sequence encoding a cognate human V H domain from the cell; and (e) a step of producing a fully human antibody using the cloned nucleic acid sequence encoding a human Vλ domain and the cloned nucleic acid sequence encoding a cognate human V H domain.

[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 as described in the present disclosure to an antigen; (b) initiating an immune response in the mouse; (c) identifying, in the mouse, a cell comprising 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 also encodes a cognate heavy chain comprising a human V H domain and a mouse C H domain, and the cell expresses an antibody that binds to the antigen); (d) a step of cloning a nucleic acid sequence encoding a human Vλ domain and a nucleic acid sequence encoding a cognate human V H domain from the cell; and (e) a step of producing a fully human antibody using the cloned nucleic acid sequence encoding a human Vλ domain and the cloned nucleic acid sequence encoding a cognate human V H domain.

[0135] In one embodiment, a method is provided for producing a rearranged antibody light chain variable region gene sequence, the method comprising: (a) exposing a mouse as described herein to an antigen; (b) initiating an immune response in the mouse to the antigen; and (c) identifying cells in the mouse that contain DNA encoding a rearranged human Vλ domain sequence fused to a mouse Cλ domain (wherein the cells are human Vλ H Domain and Mouse C H (d) The cell also encodes a congeneral heavy chain containing the domain, and the cell expresses an antibody that binds to the antigen; (d) From the cell, the nucleic acid sequence encoding the rearranged human Vλ domain and congeneral human V H (e) the process of cloning a nucleic acid sequence encoding the domain; and (e) the cloned nucleic acid sequence encoding the human Vλ domain and the congener human V H The process includes the step of producing a fully human antibody using a cloned nucleic acid sequence encoding the domain. In one embodiment, the mouse Cλ domain is mouse Cλ2. In a particular 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 ), where the mouse, when immunized with an antigen, becomes mouse C L It produces antibodies containing a human Vλ domain fused to the domain. In one embodiment, the mouse C L The domain is selected from the Cκ domain and the Cλ domain. In one embodiment, the mouse C L The domain is the Cκ domain. 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 embodiment, a genetically modified mouse is provided comprising a modified endogenous κ or λ light chain locus, as described herein, expressing multiple immunoglobulin λ light chains associated with multiple immunoglobulin heavy chains. In one embodiment, the heavy chains comprise human sequences. In various embodiments, the human sequences are selected from variable sequences, CH1, hinge, CH2, CH3, and combinations thereof. In one embodiment, the multiple immunoglobulin λ light chains comprise 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, expressing heavy chains and / or λ light chains from a transgene or extrachromosomal episome. In one embodiment, the mouse comprises substitutions of one or more human immunoglobulin sequences at endogenous mouse loci of 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., CH1, Hinge, CH2, CH3 or a combination thereof) and / or some or all of the endogenous mouse light chain sequences (e.g., V, J, constant or a combination thereof).

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

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

[0140] In one embodiment, a method for producing an antigen-binding protein is provided, the method comprising the steps of exposing a mouse to an antigen as described herein; initiating an immune response in the mouse; obtaining an antigen-binding protein from the mouse that binds to the antigen, or obtaining a sequence from the mouse that is used in producing an antigen-binding protein that binds to the antigen.

[0141] In one embodiment, cells derived from a mouse as described herein are provided. In one embodiment, the cells are selected from embryonic stem cells, pluripotent cells, induced pluripotent cells, B cells, and hybridomas.

[0142] In one embodiment, cells comprising genetic modifications as described herein are provided. In one embodiment, the cells are mouse cells. In one embodiment, the cells are selected from hybridomas and quadromas. In one embodiment, the cells express an immunoglobulin light chain comprising 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, a mouse-derived tissue as described herein is provided.

[0144] In one embodiment, the use of a mouse or cells as described herein for producing 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] Unless otherwise indicated or evident from the context, any embodiment and aspect described herein can be used in combination with one another. Other embodiments will become apparent to those skilled in the art by considering the following description. For example, the present invention provides the following items: (Item A1) A mouse in which the germline contains the nucleic acid sequence of the human immunoglobulin λ light chain variable region at the endogenous immunoglobulin light chain gene locus, wherein the nucleic acid sequence of the human immunoglobulin λ variable region is expressed in the immunoglobulin light chain containing the nucleic acid sequence of the mouse immunoglobulin constant region. (Item A2) The mouse described in item A1, wherein the endogenous immunoglobulin light chain gene locus is the immunoglobulin λ gene locus. (Item A3) The mouse described in item A1, wherein the endogenous immunoglobulin light chain gene locus is the immunoglobulin κ gene locus. (Item A4) The above mouse is the mouse described in item A1, which lacks the endogenous immunoglobulin light chain variable sequence at the above endogenous immunoglobulin light chain gene locus. (Item A5) A mouse as described in item A1, wherein all or substantially all endogenous immunoglobulin light chain variable region gene segments are replaced by one or more human immunoglobulin λ variable region gene segments. (Item A6) The mouse described in item A1, which contains the above human immunoglobulin λ light chain variable region sequence, specifically the human Jλ sequence. (Item A7) The mouse described in item A6, wherein the above human Jλ sequence is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7 and combinations thereof. (Item A8) The mouse described in item A1, wherein the above human immunoglobulin λ light chain variable region sequence contains a fragment of cluster A of the human immunoglobulin light chain locus. (Item A9) The mouse described in item A8, wherein the fragment of cluster A of the above human immunoglobulin λ light chain locus spans from hVλ3-27 to hVλ3-1. (Item A10) The mouse described in item A1, wherein the above human immunoglobulin λ light chain variable region sequence contains a fragment of cluster B of the human immunoglobulin light chain locus. (Item A11) The mouse described in item A10, wherein the fragment of cluster B of the human immunoglobulin λ light chain locus extends from hVλ5-52 to hVλ1-40. (Item A12) The mouse described in item A1, wherein the above human immunoglobulin λ light chain variable region sequence includes genomic fragments of cluster A and cluster B of the human immunoglobulin λ light chain locus. (Item A13) The mouse described in item A12, wherein the above-mentioned human immunoglobulin λ light chain variable region sequence includes at least one gene segment of cluster A and at least one gene segment of cluster B of the human immunoglobulin λ light chain locus. (Item A14) The mouse described in item A1, wherein more than 10% of the immunoglobulin light chain repertoire of the above mouse is derived from at least two hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49. (Item A15) The mouse described in item A14, wherein more than 20% of the immunoglobulin light chain repertoire of the above mouse is derived from at least three hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49. (Item A16) The mouse described in item A15, wherein more than 30% of the immunoglobulin light chain repertoire of the above mouse is derived from at least four hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49. (Item A17) A mouse expressing an immunoglobulin light chain containing a human lambda variable sequence fused to a mouse constant region, wherein the mouse exhibits a ratio of approximately 1:1 between the frequency of κ use and the frequency of λ use. (Item A18) The above-mentioned immunoglobulin light chain is expressed from the endogenous immunoglobulin light chain gene locus in the mouse described in item A17. (Item A19) Use of a mouse described in any one of the above items for the production of an antigen-binding protein. (Item A20) The above antigen-binding protein is a human antigen-binding protein, as described in item A19. (Item A21) Cells or tissues derived from a mouse described in any one of items A1 to A18, wherein the cells or tissues contain a nucleic acid sequence of the human immunoglobulin λ light chain variable region that is contiguous with the nucleic acid sequence of the endogenous immunoglobulin light chain constant region. (Item A22) A method for producing a somatically mutated antibody that binds to a target antigen, the method being as follows: (a) A step of exposing a mouse described in any one of items A1 to A18 to the target antigen; (b) A step of obtaining one or more B lymphocytes from the mouse described in (a), wherein the one or more B lymphocytes produce antibodies that bind to the target antigen; and A step of identifying a nucleic acid sequence encoding an immunoglobulin light chain of the antibody of (c)(b), wherein the immunoglobulin light chain comprises a human immunoglobulin λ light chain variable domain and a mouse immunoglobulin light chain constant domain; and (d) A step of producing a human antibody that binds to the target antigen using the nucleic acid sequence of the constant region of the human immunoglobulin light chain and the nucleic acid sequence of (c). A method that includes [Brief explanation of the drawing]

[0147] [Figure 1] Figure 1 shows a detailed, non-constantly magnified diagram of the human λ light chain locus, including clusters of Vλ gene segments (A, B, and C) and the region pair of Jλ and Cλ (JC pair). [Figure 2] Figure 2 shows an overall diagram, not a constant magnification ratio, of the targeting strategies used to inactivate the endogenous mouse λ light chain locus. [Figure 3] Figure 3 shows an overall diagram, not a constant magnification ratio, of the targeting strategies used to inactivate the endogenous mouse κ light chain locus. [Figure 4A] Figure 4A shows a general, non-constant scaling diagram of the initial targeting vector (12 / 1-λ targeting vector) for targeting the endogenous mouse λ light chain locus using a human λ light chain sequence containing 12 hVλ gene segments and hJλ1 gene segments. [Figure 4B]Figure 4B shows an overall diagram, not a constant magnification ratio, of four initial targeting vectors for targeting the endogenous mouse κ light chain locus using human λ light chain sequences, including 12 hVλ gene segments and 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, human Vκ-Jκ genome sequence and hJλ1 gene segment (12(κ)1-κ targeting vector), and 12 hVλ gene segments, human Vκ-Jκ genome sequence and hJλ1, 2, 3, and 7 gene segments (12(κ)4-κ targeting vector). [Figure 5A] Figure 5A shows an overall diagram illustrating the targeting strategy for progressively inserting 40 hVλ gene segments and a single hJλ gene segment into the mouse λ light chain locus, not a constant scaling ratio. [Figure 5B] Figure 5B shows an overall diagram illustrating the targeting strategy for progressively inserting 40 hVλ gene segments and a single hJλ gene segment into the mouse κ locus, not a constant scaling ratio. [Figure 6] Figure 6 shows an overall diagram, not a constant magnification ratio, of the targeting and molecular manipulation steps used to construct a unique human λ-κ hybrid targeting vector for constructing a hybrid light chain locus containing human κ inter-gene sequences, multiple hJλ gene segments, or both. [Figure 7A] Figure 7A shows an overall diagram, not a constant scaling ratio of the locus structure, for a modified mouse λ light chain locus containing 40 hVλ gene segments and a single hJλ gene segment operably connected to the endogenous Cλ2 gene. [Figure 7B]Figure 7B shows a general description, not a constant expansion ratio of locus structure, for four independent modified mouse κ light chain loci, each containing 40 hVλ gene segments and 1 or 4 hJλ gene segments, with or without a contiguous human Vκ-Jκ genomic sequence operably connected to the endogenous Cκ gene. [Figure 8A] Figure 8A shows contour plots of Igλ+ and Igκ+ splenocytes gated to CD19+ from wild-type mice (WT), mice homozygous for 12 hVλ gene segments and 4 hJλ gene segments containing human Vκ-Jκ genome sequences (12hVλ-VκJκ-4hJλ), and mice 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 the spleen recovered from wild-type (WT) mice, mice homozygous for 12 hVλ gene segments and 4 hJλ gene segments containing human Vκ-Jκ genome sequences (12hVλ-VκJκ-4hJλ), and mice homozygous for 40 hVλ gene segments and 1 hJλ gene segment (40hVλ-1hJλ). [Figure 9A] The upper panel of Figure 9A shows contour plots of splenocytes from wild-type (WT) mice and homozygous mice (40hVλ-VκJκ-4hJλ) containing 40 hVλ and 4 Jλ gene segments including human Vκ-Jκ genome sequences, gated for singlet cells and stained for B and T cells (CD19+ and CD3+, respectively). The lower panel shows contour plots of splenocytes from wild-type (WT) mice and homozygous mice (40hVλ-VκJκ-4hJλ) containing 40 hVλ and 4 Jλ gene segments including human Vκ-Jκ genome sequences, gated for CD19+ and stained for Igλ+ and Igκ+ expression. [Figure 9B]Figure 9B shows the total number of CD19+, CD19+Igκ+, and CD19+Igλ+ B cells in the spleen recovered from wild-type (WT) mice and homozygous mice (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments containing human Vκ-Jκ genome sequences. [Figure 9C] Figure 9C shows contour plots of splenocytes from wild-type mice (WT) and homozygous mice (40hVλ-VκJκ-4hJλ) containing 40 hVλ and 4 Jλ gene segments including human Vκ-Jκ genome sequences, gated for CD19+ and stained for immunoglobulin D (IgD) and immunoglobulin M (IgM). In each contour plot, mature B cells (72 for WT, 51 for 40hVλ-VκJκ-4hJλ) and transitional B cells (13 for WT, 22 for 40hVλ-VκJκ-4hJλ) are shown. [Figure 9D] Figure 9D shows the total number of CD19+ B cells, transitional B cells (CD19+IgMloIgDlo) and mature B cells (CD19+IgMloIgDlo) recovered from the spleen of mice homozygous for 40 hVλ and 4 Jλ gene segments, including wild-type (WT) and human Vκ-Jκ genome sequences (40hVλ-VκJκ-4hJλ). [Figure 10A]The upper panel of Figure 10A shows contour plots of bone marrow stained for B and T cells (CD19+ and CD3+, respectively) from wild-type (WT) mice and homozygous mice (40hVλ-VκJκ-4hJλ) containing 40 hVλ and 4 Jλ gene segments including human Vκ-Jκ genome sequences. The lower panel shows contour plots of bone marrow from wild-type (WT) mice and homozygous mice (40hVλ-VκJκ-4hJλ) containing 40 hVλ and 4 Jλ gene segments including human Vκ-Jκ genome sequences, gated for CD19+ and stained for ckit+ and CD43+. Pro B cells and pre B cells are shown in the contour plots of the lower panel. [Figure 10B] Figure 10B shows the number of pro-B cells (CD19+CD43+ckit+) and pre-B cells (CD19+CD43-ckit-) in the bone marrow recovered from the femurs of mice (40hVλ-VκJκ-4hJλ) that were homozygous for 40 hVλ and 4 Jλ gene segments, including wild-type (WT) and human Vκ-Jκ genome sequences. [Figure 10C] Figure 10C shows contour plots of bone marrow gated to singlets stained for immunoglobulin M (IgM) and B220, derived from wild-type (WT) mice and homozygous mice (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments containing human Vκ-Jκ genome sequences. Immature, mature, and pro / pre B cells are shown in each contour plot. [Figure 10D] Figure 10D shows the total number of immature B cells (B220intIgM+) and mature B cells (B220hiIgM+) isolated from the bone marrow of mice homozygous for 40 hVλ and 4 Jλ gene segments, including wild-type (WT) and human Vκ-Jκ genome sequences (40hVλ-VκJκ-4hJλ). [Figure 10E]Figure 10E shows a contour plot of bone marrow gated for immature B cells (B220intIgM+) and mature B cells (B220hiIgM+) stained for Igλ and Igκ expression, isolated from the femurs of wild-type (WT) mice and homozygous mice (40hVλ-VκJκ-4hJλ) containing 40 hVλ and 4 Jλ gene segments, including human Vκ-Jκ genome sequences. [Figure 11] Figure 11 shows the nucleotide sequence alignment of the Vλ-Jλ-Cκ junction of 18 independent RT-PCR clones amplified from splenocyte RNA of mice possessing the 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-germ cell lineage bases resulting from mutations and / or N addition during recombination. The consensus amino acids within the Framework 4 region (FWR4) encoded by the nucleotide sequences of hJλ1 and mouse Cκ are shown below the sequence alignment. [Figure 12]Figure 12 shows the nucleotide sequence alignment of the Vλ-Jλ-Cκ junction of 12 independent RT-PCR clones amplified from mouse splenocyte RNA having a human λ light chain gene sequence containing a consecutive human Vκ-Jκ genomic sequence 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-germ cell lineage bases resulting from mutations and / or N additions during recombination. The consensus amino acids within the Framework 4 region (FWR4) encoded by the respective nucleotide sequences of human Jλ and mouse Cκ are shown below the sequence alignment. [Figure 13] Figure 13 shows the nucleotide sequence alignment of the Vλ-Jλ-Cλ junction of three independent RT-PCR clones amplified from mouse splenocyte RNA carrying the human λ light chain gene sequence 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. The consensus amino acids within the Framework 4 region (FWR4) encoded by the nucleotide sequences of hJλ1 and mouse Cλ2 are shown below the sequence alignment. [Modes for carrying out the invention]

[0148] Detailed explanation While the specific features of various embodiments are described in detail, the descriptions of these specific aspects, embodiments, and examples do not limit the scope of the claims; the claims describe the scope of the invention. All terms and phrases used in this disclosure have the meanings commonly given to them in the art.

[0149] The term “contiguous” encompasses references to being located on the same nucleic acid molecule. For example, two nucleic acid sequences are “contiguous” if they are located on the same nucleic acid molecule but are interrupted by another nucleic acid sequence. For instance, the last codon of a rearranged V(D)J sequence does not directly follow the first codon of a 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 located on the same genomic fragment are “contiguous,” but they may be interrupted by sequences that do not code for codons in the V region, for example, they may be interrupted by regulatory sequences, such as promoters or other non-coding sequences. In one embodiment, contiguous sequences include a genomic fragment containing a genomic sequence arranged as found in a wild-type genome.

[0150] When the phrase "derived from" is used in reference to a variable region "derived from" a gene or gene segment being referred to, it includes the ability to trace its sequence back to a particular unrearranged gene segment or a gene segment rearranged to form a gene expressing that variable domain (including, where applicable, splice differences and somatic mutations).

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

[0152] The "heavy chain locus" is heavy chain variable in wild-type mice (V H ), heavy chain diversity (D H ), heavy chain linkage (J H ) and heavy chain steady state (C H This includes locations on chromosomes where the DNA sequence of the region can be observed, for example, locations on mouse chromosomes.

[0153] A "κ locus" refers to a chromosomal location in wild-type mice where the DNA sequences of the κ variable (Vκ), κ linked (Jκ), and κ constant (Cκ) regions are found, for example, a location on a mouse chromosome.

[0154] A "λ locus" refers to a chromosomal location in wild-type mice where the DNA sequences of the λ variable (Vλ), λ linked (Jλ), and λ constant (Cλ) regions are found, for example, a location on a mouse chromosome.

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

[0156] Mouse expressing the human λ variable domain Mice expressing antibodies that are either entirely human or partially human and partially mouse have been previously reported. VELOCIMMUNE® genetically engineered mice involve the substitution of an unrearranged V(D)J gene segment with a human V(D)J gene segment at an endogenous mouse locus. VELOCIMMUNE® mice express chimeric antibodies having a human variable domain and a mouse constant domain (see, for example, U.S. Patent No. 7,605,237). Most other reports concern mice expressing fully human antibodies from fully human transgenes in mice with a deactivated endogenous immunoglobulin locus.

[0157] Antibody light chains are encoded by one of two distinct gene loci: kappa (κ) and lambda (λ). Mouse antibody light chains are predominantly of the κ type. Mice that produce mouse antibodies, and modified mice that produce fully human antibodies or chimeric human-mouse antibodies, show a bias in the frequency of light chain use. Humans also show a light chain bias, but not as pronounced as in mice; the ratio of κ light chains to λ light chains in mice is approximately 95:5, while in humans it is approximately 60:40. In mice, the λ variable locus is not as diverse, so the more pronounced bias in mice is thought not to have a serious impact on antibody diversity. This is not the case in humans. The human λ light chain locus is highly diverse.

[0158] The human λ light chain locus extends to 1,000 kb and contains more than 80 genes encoding variable (V) or ligated (J) segments (Figure 1). Of 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 functional.

[0159] The human λ light chain locus is structurally similar to both the mouse and human κ loci in that it contains several variable region gene segments that can be recombined to form functional light chain proteins. The human λ light chain locus contains approximately 70 V gene segments and 7 Jλ-Cλ gene segment pairs. Only four of these Jλ-Cλ gene segment pairs appear to be functional. In some alleles, a 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 in three clusters, all of which contain different members of different V gene families (clusters A, B, and C; Figure 1). This represents a potentially rich source of relatively underutilized diversity for producing 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). For this reason, at least, the large bias towards κ in mice is not considered particularly detrimental to overall antibody diversity.

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

[0162] The mouse κ light chain locus is remarkably diverse. The structure and number of gene segments involved in recombination events, which yield functional light chain proteins from the mouse κ locus, are considerably complex (Figure 3). Consequently, the mouse λ light chain does not significantly contribute to the diversity of antibody populations in typical mice.

[0163] In particular, leveraging the rich diversity of the human λ light chain locus in mice could provide a source for a more complete human repertoire of light chain V domains. Previous attempts to utilize this diversity have used human transgenes containing clusters of human λ light chain loci randomly integrated into the mouse genome (see, e.g., U.S. Patents 6,998,514 and 7,435,871). Reportedly, mice containing these randomly integrated transgenes express a complete human λ light chain, however, in some cases, one or both endogenous light chain loci remain intact. This situation is undesirable because the human λ light chain sequence competes with mouse light chains (κ or λ) in the mouse antibody repertoire.

[0164] In contrast, we describe genetically modified mice capable of directly expressing one or more λ light chain nucleic acid sequences from a mouse light chain locus (including through substitutions at the endogenous mouse light chain locus). Genetically modified mice capable of expressing human λ light chain sequences from the endogenous locus can be further crossed with mice containing a human heavy chain locus and thus can be used to express antibodies containing V regions (heavy and light chains) that are entirely human. In various embodiments, these V regions are expressed together with the mouse constant region. In various embodiments, the endogenous mouse immunoglobulin gene segment is absent, and their V regions are expressed together with the human constant region. These antibodies will prove useful in a number of applications, both diagnostic and therapeutic.

[0165] Many advantages can be realized in various embodiments of expressing binding proteins derived from human Vλ and Jλ gene segments in mice. These advantages can be realized by positioning human λ sequences at endogenous light chain loci, for example, the mouse κ or λ loci. Antibodies produced from such mice are mouse C L The mouse may have a light chain containing a human Vλ domain fused to a region (specifically, a mouse Cκ or Cλ region). L Human Vλ domains suitable for identification and cloning can also be expressed for use in conjunction with the region (specifically, the Cκ and / or Cλ region). Since B cell development in such mice is otherwise normal, it is possible to generate compatible Vλ domains (including somatically mutated Vλ domains) in the background of the Cλ or Cκ region.

[0166] A genetically modified mouse is described that contains a Vλ gene segment that has not been rearranged at the immunoglobulin κ or λ light chain locus. A mouse expressing an antibody containing a light chain with a human Vλ domain fused to the Cκ and / or Cλ region is also described.

[0167] Sterile transcript of the immunoglobulin κ 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. Therefore, in some embodiments, the genetically modified mouse contains certain non-coding sequences derived from human loci. In one embodiment, the genetically modified mouse contains human Vλ and Jλ gene segments in the endogenous κ light chain locus, and further contains a human κ light chain genomic fragment. In certain embodiments, the human κ light chain genomic fragment is a non-coding sequence found naturally between the human Vκ gene segment and the human Jκ gene segment.

[0168] The human and mouse κ light chain loci described above contain sequences that encode invalid transcripts lacking either a start codon or an open reading frame, and which are considered to be elements that regulate transcription of the κ light chain locus. These invalid transcripts arise from intergenetic sequences located downstream or 3' of the most proximal Vκ gene segment and upstream or 5' of the κ light chain intron enhancer (Eκi), which is located upstream of the κ light chain constant region gene (Cκ). These invalid transcripts arise from rearrangements of these intergenetic sequences, thereby forming the VκJκ1 segment fused to Cκ.

[0169] Substitution of the κ light chain locus upstream of the Cκ gene can remove the intergeneric region encoding an invalid transcript. Therefore, in various embodiments, substitution of the mouse κ light chain sequence upstream of the mouse Cκ gene with a human λ light chain gene segment may result in a humanized mouse κ light chain locus that includes human Vλ and Jλ gene segments but does not contain the intergeneric region encoding an invalid transcript.

[0170] As described herein, humanization of the endogenous mouse κ light chain locus with a human λ light chain gene segment (where the intergenic region is removed during humanization) results in a significant increase in the frequency of λ light chain use, as well as a substantial decrease in the frequency of κ light chain locus use. Therefore, humanized mice lacking the intergenic region are useful in that they can produce antibodies containing human light chain variable domains (e.g., human λ or κ domains), but the frequency of use from that locus is reduced.

[0171] Regarding transcription, in addition to the insertion of a human κ intergenic region to create a Vλ locus containing a κ intergenic region between the final human Vλ gene segment and the first human Jλ gene segment, the humanization of the endogenous mouse κ light chain locus by human Vλ and Jλ gene segments is also described; it shows a higher expression of B cell populations than loci 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 invalid 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 making the mouse a limited choice, but antibodies are produced using the modified locus (λ to κ).

[0172] In various embodiments, the substitution of a mouse κ light chain sequence upstream of the mouse Cκ gene with a human λ light chain sequence further includes a human κ light chain intergeneric region located between the 3' untranslated region of the Vλ gene segment furthest 3' from transcriptional point of view and the 5' side of the first human Jλ gene segment. Alternatively, such an intergeneric region may be removed from the endogenous κ light chain locus to be substituted (upstream of the mouse Cκ gene) by creating a deletion at the endogenous λ light chain locus. Similarly, in this embodiment, these mice produce antibodies from the endogenous κ light chain locus containing the human λ light chain sequence.

[0173] An approach to manipulate mice to express the human Vλ domain. endogenous C LVarious approaches are described for creating genetically modified mice that produce antibodies containing light chains having human Vλ domains fused to (e.g., Cκ or Cλ) regions. In various embodiments, genetic modifications involving deletion of one or both endogenous light chain loci are described. For example, deletion of a first Vλ-Jλ-Cλ gene cluster and whole or partial replacement of the Vλ-Jλ gene segment of a second gene cluster with a human Vλ-Jλ gene segment may be performed to eliminate mouse λ light chains from the endogenous antibody repertoire. Genetically modified mouse embryos, cells, and targeted constructs for creating those mice, mouse embryos, and cells are also provided.

[0174] Since the endogenous Cλ gene remains intact and therefore retains normal functionality and the ability to associate with the constant region of the endogenous heavy chain, in various embodiments, deletion of one endogenous Vλ-Jλ-Cλ gene cluster and substitution of the Vλ-Jλ gene segment of another endogenous Vλ-Jλ-Cλ gene cluster result in relatively minimal disruption of the association and function of the native antibody constant region in that animal. Thus, in such embodiments, the modification does not affect other endogenous heavy chain constant regions that depend on the functional light chain constant region for the assembly of a functional antibody molecule containing two heavy chains and two light chains. Furthermore, in various embodiments, the modification also does not affect the assembly of a membrane-bound functional antibody molecule containing endogenous heavy and light chains, for example, an hVλ domain attached to the mouse Cλ region. Since at least one functional Cλ gene is held at the endogenous locus, an animal containing the substitution of the Vλ-Jλ gene segment in the endogenous Vλ-Jλ-Cλ gene cluster with the human Vλ-Jλ gene segment should be able to produce normal λ light chains that can bind to antigens during the immune response, due to the human Vλ-Jλ gene segment present in the animal's expressed antibody repertoire.

[0175] A schematic diagram (not at a constant magnification) of the deleted endogenous mouse Vλ-Jλ-Cλ gene cluster is provided in Figure 2. As illustrated, the mouse λ light chain locus is organized into two gene clusters, both containing functional gene segments that can be recombined to form a functional mouse λ light chain. The endogenous mouse Vλ1-Jλ3-Cλ3-Jλ1-Cλ1 gene cluster is deleted by a targeted construct (targeted vector 1) having a neomycin cassette adjacent to the recombination site. The other endogenous gene cluster (Vλ2-Vλ3-Jλ2-Cλ2-Jλ4-Cλ4) is partially deleted by a targeted construct (targeted vector 2) having a hygromycin-thymidine kinase cassette adjacent to the recombination site. In this second targeted event, the Cλ2-Jλ4-Cλ4 endogenous gene segment is retained. The second targeting construct (targeting vector 2) is constructed using a different recombination site than that in the first targeting construct (targeting vector 1), thereby enabling selective deletion of its selection cassette after successful targeting. The resulting double-targeted locus is functionally silenced in that it cannot produce endogenous λ light chains. This modified locus is used for insertion of human Vλ and Jλ gene segments, thereby creating an endogenous mouse λ locus containing human Vλ and Jλ gene segments (where, upon recombination at the modified locus, the animal produces λ light chains containing rearranged human Vλ and Jλ gene segments attached to an endogenous mouse Cλ gene segment).

[0176] Genetically modifying mice to render the endogenous λ gene segment nonfunctional, in various embodiments, results in mice whose antibody repertoire exclusively exhibits κ light chains. These mice are useful for evaluating the role of λ light chains in their immune response and for creating antibody repertoires that contain Vκ domains but not Vλ domains.

[0177] Genetically modified mice expressing hVλ linked to the mouse Cλ gene, which is recombinant at the endogenous mouse λ light chain locus, can be produced by any method known in the art. A schematic diagram (not at a constant magnification) of the substitution of the endogenous mouse Vλ2-Vλ3-Jλ2 gene segment with human Vλ and Jλ gene segments is provided in Figure 4A. As illustrated, the non-functional endogenous mouse λ light chain locus is replaced by a targeted construct (12 / 1-λ targeted vector) containing a neomycin cassette adjacent to the recombination site. The Vλ2-Vλ3-Jλ2 gene segment is replaced with a genomic fragment containing a human λ sequence containing 12 hVλ gene segments and a single hJλ gene segment.

[0178] Therefore, this first approach positions one or more hVλ gene segments at an endogenous λ light chain locus that is contiguous with a single hJλ gene segment (Figure 4A).

[0179] Further modifications to the modified endogenous λ light chain locus can be achieved using similar techniques to insert more hVλ gene segments. For example, schematic diagrams of two additional targeting constructs (+16-λ and +12-λ targeting vectors) used for the gradual insertion of additional human hVλ gene segments are provided in Figure 5A. As illustrated, additional genomic fragments containing specific human hVλ gene segments are inserted into the modified endogenous λ light chain locus in a sequential manner, using the homology provided by the previous insertion of the human λ light chain sequence. In recombination using each illustrated targeting construct, 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 a λ light chain protein containing a human Vλ-Jλ gene segment attached to the mouse Cλ gene.

[0180] The above approach, which involves inserting a human λ light chain gene segment into the mouse λ locus, preserves the enhancer located downstream of the Cλ2-Jλ4-Cλ4 gene segment (named Enh2.4, Enh, and Enh3.1, Figures 4A and 5A). This approach results in a single modified allele at the endogenous mouse λ light chain locus (Figure 7A).

[0181] Compositions and methods are provided for producing mice expressing a light chain comprising hVλ and Jλ gene segments operably attached to a mouse Cλ gene segment (including compositions and methods for producing mice expressing such genes from an endogenous mouse λ light chain locus). These methods include selectively deactivating one endogenous mouse Vλ-Jλ-Cλ gene cluster (e.g., by targeted deletion) and expressing the hVλ domain in a mouse using the hVλ and Jλ gene segments at that endogenous mouse λ light chain locus.

[0182] Alternatively, in a second approach, the human λ light chain gene segment may be positioned at the endogenous κ light chain locus. The genetic modification involves deletion of the endogenous κ light chain locus in various embodiments. For example, deletion of the mouse Vκ and Jκ gene segments may be performed to eliminate the mouse κ light chain from the endogenous antibody repertoire. Genetically modified mouse embryos, cells, and targeted constructs for producing such mice, mouse embryos, and cells are also provided.

[0183] For the reasons stated above, relatively minimal disruption is used in the deletion of the mouse Vκ and Jκ gene segments. A schematic diagram (not at a constant magnification) of the mouse Vκ and Jκ gene segments to be deleted is provided in Figure 3. The endogenous mouse Vκ and Jκ gene segments are deleted via a deletion mediated by a recombinase of a mouse sequence located between two precisely positioned targeting vectors (each using a site-specific recombination site). The first targeting vector (Jκ targeting vector) is used in the first targeting event to delete the mouse Jκ gene segment. The second targeting vector (Vκ targeting vector) is used in the second sequential targeting event to delete the sequence located at 5' of the most distal mouse Vκ gene segment. Both targeting vectors contain site-specific recombination sites, thereby allowing for selective deletion of both select 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 chains. This modified locus can be used for the insertion of hVλ and Jλ gene segments, thereby creating an endogenous mouse κ locus containing the hVλ and Jλ gene segments (where, upon recombination at the modified locus, the animal produces a λ light chain containing rearranged hVλ and Jλ gene segments operably linked to the endogenous mouse Cκ gene segment). Various targeting vectors containing human λ light chain sequences can be used in combination with this deleted mouse κ locus to create a hybrid light chain locus containing a human λ gene segment operably linked to the mouse Cκ region.

[0184] Therefore, the second approach positions one or more human Vλ gene segments at a mouse κ light chain locus that is contiguous with a single human Jλ gene segment (12 / 1-κ targeted vector, Figure 4B).

[0185] In various embodiments, modifications to this approach may optimize the frequency of use of human λ light chain sequences from the mouse κ locus within the mouse antibody repertoire by adding gene segments and / or regulatory sequences.

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

[0187] In the third approach, one or more hVλ gene segments are positioned at a mouse κ light chain locus that is contiguous with the human κ inter-gene sequence and a single hJλ gene sequence (12(κ)1-κ targeted vector, Figure 4B).

[0188] In the fourth approach, one or more hVλ gene segments are positioned at mouse κ light chain loci that are contiguous with human κ inter-gene sequences and four hJλ gene sequences (12(κ)4-κ targeted vector Figure 4B).

[0189] All of the above approaches, which involve inserting a human λ light chain gene segment into the mouse κ locus, maintain a κ intron enhancer element (named Eκi, Figures 4B and 5B) upstream of the Cκ gene and a 3' κ enhancer (named Eκ3', Figures 4B and 5B) downstream of the Cκ gene. This approach results in four distinct and modified alleles at the endogenous mouse κ light chain locus (Figure 7B).

[0190] In various embodiments, the genetically modified mice include 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 regions extending from Vλ2 to Jλ2 and from Vλ1 to Cλ1 (Figure 2). Any strategy that reduces or eliminates the endogenous λ light chain locus's ability to express the endogenous λ domain is suitable for use with the embodiments of this disclosure.

[0191] Genetically modified mouse-derived lambda domain antibody Mice containing a human λ sequence in the mouse κ or λ light chain locus are classified as mouse C L These mice express a light chain containing an hVλ region fused to a (Cκ or Cλ) region. These include (a) mice containing a functionally silenced light chain locus (e.g., knockout of an endogenous mouse κ or λ light chain locus); (b) mice containing an endogenous mouse λ light chain locus containing hV and hJ gene segments operably connected to the endogenous mouse Cλ gene; (c) mice containing an endogenous mouse κ light chain locus containing hVκ and hJκ gene segments operably connected to the 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 the other 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 It conveniently crosses with mice containing two heavy chain alleles, including the one mentioned above.

[0192] Antibodies containing the hVλ domain, expressed against a Cκ or Cλ background, are used to produce fully human antibodies by cloning the nucleic acid encoding the hVλ domain into an expression construct containing the gene encoding human Cλ. The resulting expression construct is then transfected into host cells suitable for expressing antibodies exhibiting the complete hVλ domain fused to hCλ. [Examples]

[0193] The following examples are provided to illustrate methods for preparing and using the methods and compositions of the present invention and are not intended to limit the scope that the inventors consider to be their invention. Unless otherwise indicated, temperatures are expressed in degrees Celsius and pressures are atmospheric pressure or near atmospheric pressure.

[0194] Example I Deletion of mouse immunoglobulin light chain gene locus To modify the mouse genomic bacterial artificial chromosome (BAC) library to inactivate the mouse κ and λ light chain loci, various targeted constructs were created using VELOCIGENE® technology (see, for example, U.S. Patent 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 mouse BAC clone RP23-135k15 (Invitrogen) was modified by homologous recombination (Figure 2).

[0196] In short, a targeting vector containing a neomycin cassette adjacent to the loxP site, along with a 5' mouse homology arm containing the 5' sequence of the Vλ1 gene segment and a 3' mouse homology arm containing the 3' sequence of the Cλ1 gene segment, was used to delete the entire proximal cluster containing the Vλ1-Jλ3-Cλ3-Jλ1-Cλ1 gene segment in a single targeting event (Figure 2, Targeting Vector 1).

[0197] A second targeting construct was prepared to precisely delete the distal endogenous mouse λ gene cluster containing Vλ2-Jλ2-Cλ2-Jλ4-Cλ4. However, this second targeting construct included a 5' mouse homology arm containing the 5' sequence of the Vλ2 gene segment and a 3' mouse homology arm containing the 5' sequence of the endogenous Cλ2 gene segment (Figure 2, Targeting Vector 2). Therefore, this second targeting construct precisely deleted Vλ2-Jλ2 while leaving Cλ2-Jλ4-Cλ4 intact at the endogenous mouse λ locus. ES cells containing the inactivated endogenous λ locus (as described above) were identified by karyotyping and screening methods known in the field (e.g., TAQMAN®). Next, DNA was isolated from the modified ES cells and subjected to treatment with CRE recombinase, which mediated the deletion of a proximal targeting cassette containing the neomycin marker gene, resulting in the deletion of only one loxP site (Figure 2, bottom).

[0198] Deletion of the mouse κ light chain locus. The mouse κ light chain locus was inactivated in a two-step process by modifying the DNA from mouse BAC clones RP23-302g12 and RP23-254m04 (Invitrogen) by homologous recombination using several targeted constructs similar to those described above (Figure 3).

[0199] In short, a targeting vector containing a hyg-TK cassette with a single loxP site at 3' relative to the hygromycin-thymidine kinase (hyg-TK) cassette was used to delete the Jκ gene segment (1-5) of the endogenous mouse κ light chain locus in a single targeting event (Figure 3, Jκ targeting vector). The homology arm used to construct this targeting vector contained mouse genome sequences on the 5' and 3' sides of the endogenous mouse Jκ gene segment. For a second targeting event, a second targeting vector was prepared to delete a portion of the upstream (5') mouse genome sequence of the most distal endogenous mouse Vκ gene segment (Figure 3, Vκ targeting vector). This targeting vector contained the reversed lox511 site, loxP site, and neomycin cassette. The homology arm used to construct this targeting vector contained mouse genome sequences upstream of the most distal mouse Vκ gene segment. These targeting vectors were used sequentially (i.e., Jκ followed by Vκ) to target DNA in ES cells. ES cells possessing a dual-targeting chromosome (i.e., a single endogenous mouse κ locus targeted by both targeting vectors) were identified by karyotyping and screening methods known in the art (e.g., Taqman®). DNA was then isolated from these modified ES cells and subjected to treatment with Cre recombinase, which mediated deletion of the endogenous mouse Vκ gene segment and both selective cassettes, with the two lox sites juxtaposed in opposite orientations to each other (Figure 3, bottom; Sequence ID 1).

[0200] In this way, two modified endogenous light chain loci (κ and λ) containing intact enhancers and constant regions were constructed for the precise and progressive insertion of unrearranged human λ germline gene segments using the targeted vectors described below.

[0201] Example II Replacement of mouse light chain loci by human lambda light chain miniloci Using a method similar to that described above, we created multiple targeted vectors for the gradual insertion of human λ gene segments into endogenous mouse κ and λ light chain loci. Multiple independent initial modifications were performed on the endogenous light chain loci, each resulting in a chimeric light chain locus containing hVλ and Jλ gene segments operably connected to mouse light chain constant genes and enhancers.

[0202] A human λ minilocentre containing 12 human Vλ and 1 human Jλ gene segment. Using a human BAC clone (Invitrogen) named RP11-729g4, a series of initial targeting vectors were constructed to contain the first 12 consecutive human Vλ gene segments and either the hJλ1 gene segment or 4 hJλ gene segments from cluster A. Figures 4A and 4B show the targeting vectors constructed to perform the initial insertion of human λ light chain gene segments in the mouse λ and κ light chain loci, respectively.

[0203] For the first set of the initial targeting vector, a 124,125 bp DNA fragment from a 729g4BAC clone containing 12 hVλ gene segments and an hJλ1 gene segment was created to include a PI-SceI site 996 bp downstream (3') of the hJλ1 gene segment for ligation of the 3' mouse homology arm. Two different sets of homology arms were used for ligation to this human fragment; one set of homology arms contained endogenous mouse λ sequences from a 135k15BAC clone (Figure 4A), and the other set contained endogenous κ sequences at the 5' and 3' ends of the mouse Vκ and Jκ gene segments from mouse BAC clones RP23-302g12 and RP23-254m04, respectively (Figure 4B).

[0204] In the case of the 12 / 1-λ targeting vector (Figure 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. By using this approximately 28 kb mouse fragment as a 3' homology arm by ligating it to an approximately 124 kb human λ fragment, a 3' junction was constructed from 5' to 3', containing the hJλ1 gene segment, a 996 bp human λ sequence on the 3' side of the hJλ1 gene segment, a 1229 bp mouse λ sequence on the 5' side relative to the mouse Cλ2 gene, the mouse Cλ2 gene, and the remaining portion of its approximately 28 kb mouse fragment. Upstream (5') of the human Vλ3-12 gene segment, an additional 1456 bp human λ sequence was present prior to the start of the 5' mouse homology arm, which contained 23,792 bp of mouse genomic DNA corresponding to the 5' sequence of the endogenous mouse λ locus. Between the 5' homology arm and the beginning of the human λ sequence, a neomycin cassette adjacent to the Frt region was present.

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

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

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

[0208] A human λ minilocentesis containing 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 consecutive human Vλ gene segments, as well as the hJλ1, 2, 3, and 7 gene segments, from cluster A into the mouse κ light chain locus (Figure 4B, 12 / 4-κ targeting vector). DNA segments containing the hJλ1, Jλ2, Jλ3, and Jλ7 gene segments, each containing approximately 100 bp of human genome sequence from both the 5' and 3' regions immediately adjacent to each Jλ gene segment, were constructed 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 the 5' and 3' positions of the hJλ1 gene segment of human BAC clone 729g4. Homological recombination with this intermediate-targeting vector was performed in a modified 729g4BAC clone that was pre-targeted upstream (5') of the human Vλ3-12 gene segment with a neomycin cassette adjacent to the Frt site, which also included an I-CeuI site on the 5' side of the 5'Frt site. The dual-targeted 729g4BAC clone contained, from 5' to 3', the I-CeuI site, the 5'Frt site, the neomycin cassette, the 3'Frt site, a fragment of approximately 123kb containing the first 12 hVλ gene segments, a fragment of approximately 1kb containing human Jλ1, 2, 3, and 7 gene segments, the PI-SceI site, and a chloramphenicol cassette. This intermediate targeting vector was simultaneously digested with I-CeuI and PI-SceI, and then ligated into a modified mouse BAC clone (described above) to produce a third targeting vector.

[0209] This ligation resulted in a third targeting vector for inserting the human λ sequences into the endogenous κ light chain locus, which, from 5' to 3', included a 5' mouse homology arm containing approximately 23 kb of genomic sequence on the 5' side of the endogenous mouse κ locus, an I-CeuI site, a 5' Frt site, a neomycin cassette, a 3' Frt site, a fragment of approximately 123 kb containing the first 12 hVλ gene segments, a fragment of approximately 1 kb 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' and additional mouse genomic sequence downstream of its Eκ3' (3') (Figure 4B, 12 / 4-κ targeting vector). Homologous recombination with this third targeting vector created a modified mouse κ light chain locus containing 12 hVλ gene segments and 4 hJλ gene segments operably linked to the endogenous mouse Cκ gene (which, upon recombination, results in the formation of chimeric human λ / mouse κ light chains).

[0210] A human λ minilocus having an integrated human κ light chain sequence. In a similar fashion, two additional targeting vectors were created, similar to the targeting vectors (Figure 4B, 12 / 1-κ and 12 / 4-κ targeting vectors) created for the initial insertion of human λ gene segments into the endogenous κ light chain locus, and the human λ light chain gene segments were inserted progressively 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 naturally located between the human Vκ4-1 gene segment and the Jκ1 gene segment. This human κ genomic sequence was specifically positioned between the human Vλ gene segment and the human Jλ gene segment in these two additional targeting vectors (Figure 4B, 12(κ)1-κ and 12(κ)4-κ targeting vectors).

[0211] Both targeting vectors containing the above human κ genome sequence were constructed using the modified RP11-729g4BAC clone described above (Figure 6). This modified BAC clone was targeted with a spectinomycin selector cassette adjacent to the NotI and AsiSI restriction enzyme recognition sites (Figure 6, top left). Homologous recombination with the spectinomycin cassette yielded a dual-targeted 729g4BAC clone, which contained, from 5' to 3', an I-CeuI site, a 5'Frt site, a neomycin cassette, a 3'Frt site, a fragment of approximately 123kb containing the first 12 hVλ gene segments, a NotI site approximately 200bp downstream (3') of the nonumeric sequence of the hVλ3-1 gene segment, a spectinomycin cassette, and an AsiSI site. To enable the subsequent cloning of an approximately 23kb fragment for ligation with the hVλ gene segment contained in a dual-targeted modified 729g4BAC clone, a separate human BAC clone (CTD-2366j12) containing a human κ sequence was independently targeted twice to create a restriction enzyme recognition site between the hVκ4-1 gene segment and the hJκ1 gene segment (Figure 6, upper right).

[0212] In short, the 2366j12BAC clone is approximately 132kb in size and contains hVκ gene segments 1-6, 1-5, 2-4, 7-3, 5-2, 4-1, human κ genome sequences downstream of those Vκ gene segments, hJκ gene segments 1-5, hCκ, and approximately 20kb of additional genome sequences of the human κ locus. First, this clone was targeted with a targeting vector containing a hygromycin cassette adjacent to the Frt site and a NotI site downstream (3') of the 3'Frt site. The homology arm for this targeting vector contained human genome sequences on the 5' and 3' sides of the Vκ gene segments within its BAC clone (therefore, upon homologous recombination by this targeting vector, the Vκ gene segments were deleted and a NotI site was created approximately 133bp downstream of the hVκ4-1 gene segment) (Figure 6, upper right). This modified 2366j12BAC clone was independently targeted at its 3' end with two targeting vectors, and the hJκ gene segment was deleted using a chloramphenicol cassette (this chloramphenicol cassette also contains either the hJλ1 gene segment, the PI-SceI site and the AsiSI site, or a human λ genome fragment containing four hJλ gene segments (as previously mentioned), the PI-SceI site and the AsiSI site) (Figure 6, upper right). The homology arms for these two similar targeting vectors contained the 5' and 3' sequences of the hJκ gene segment. Homologous recombination with these second targeting vectors and the modified 2366j12BAC clone yielded a double-targeted 2366j12 clone, which 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 including the intergenetic 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, upper right).Two final targeting vectors for making two additional modifications were obtained by two ligation steps using the dual-targeted 729g4 and 2366j12 clones.

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

[0214] Example III Manipulation of additional human Vλ gene segments to human λ light chain miniloci Additional hVλ gene segments were independently added to each of the initial modifiers described in Example 2 using similar targeting vectors and methods (Figure 5A, +16-λ targeting vector and Figure 5B, +16-κ targeting vector).

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

[0216] In short, two targeting vectors were created to introduce 16 additional hVλ gene segments into the modified mouse light chain locus described in Example 2 (Figures 5A and 5B, +16-λ or +16-κ targeting vectors). A 172kb DNA fragment from human BAC clone RP11-761l13 (Invitrogen) containing 21 consecutive hVλ gene segments from cluster A was constructed using a 5' homology arm containing the mouse genome sequence at the 5' end relative to the endogenous κ or λ light chain locus and a 3' homology arm containing the human genome 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 (Figures 5A and 5B). The 3' homology arm contained a 53,057 bp duplication of the human genome λ sequence corresponding to the equivalent 5' end of the approximately 123kb fragment of the human genome λ sequence described in Example 2. These two targeting vectors contained a 5' mouse homology arm containing approximately 23 kb of genomic sequence from the 5' side of the endogenous mouse κ light chain locus or approximately 24 kb of mouse genomic sequence from the 5' side of the endogenous λ light chain locus, a 5' Frt region, a hygromycin cassette, a 3' Frt region, and a 171,457 bp human genomic λ sequence containing 21 consecutive hVλ gene segments (of which approximately 53 kb overlaps with the 5' end of the human λ sequence described in Example 3 and acts as a 3' homology arm for this targeting construct) (Figures 5A and 5B, +16-λ or +16-κ targeting vectors). Homologous recombination using these targeted vectors independently produced modified 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)). (These result in the formation of chimeric light chains upon recombination.)

[0217] In a similar manner, 16 additional hVλ gene segments were introduced into the other initial modifier described in Example 2 (Figure 4B), which incorporates multiple hJλ gene segments with and without the incorporated human κ sequence, by using a +16-κ targeted vector. Homologous recombination by this targeted vector at the endogenous mouse κ locus, including the other initial modifier, created a mouse κ light chain locus containing 28 hVλ gene segments and hJλ1, 2, 3, and 7 gene segments with and without the human Vκ-Jκ genomic sequence operably attached to the endogenous mouse Cκ gene (these result in the formation of chimeric λ-κ light chains when recombination occurs).

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

[0219] In short, targeted vectors were 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 targeted vectors). A 93,674 bp DNA fragment from human BAC clone RP11-22l18 (Invitrogen) containing 12 consecutive hVλ gene segments from cluster B was manipulated using a 5' homology arm containing the mouse genome sequence on the 5' side and a 3' homology arm containing the human genome λ sequence relative to the endogenous mouse κ or λ light chain loci. The 5' homology arm used in this targeted construct was the same 5' homology arm used for the addition of the 16 hVλ gene segments described above (Figures 5A and 5B). A 3' homology arm was created by adding a PI-SceI site approximately 3431 bp to the 5' end of the human Vλ3-29P gene segment contained in a 27,468 bp genomic fragment of a human λ sequence derived from BAC clone RP11-761l13. This PI-SceI site acted as a ligation point, linking an additional approximately 94 kb fragment of the human λ sequence with an approximately 27 kb fragment of the human λ sequence that overlapped with the 5' end of the human λ sequence in the previous modification, using a +16-λ or +16-κ targeted vector (Figures 5A and 5B). These two targeting vectors contained a 5' homology arm containing approximately 23kb of mouse genome sequence on the 5' side of the endogenous κ light chain locus or approximately 24kb of mouse genome sequence on the 5' side of the endogenous λ light chain locus, a 5' Frt site, a neomycin cassette, a 3' Frt site, and a 121,188bp human genome λ sequence containing 16 hVλ gene segments and a PI-SceI site (of which approximately 27kb overlaps with the 5' end of the human λ sequence from the insertion of 16 additional hVλ gene segments, acting as a 3' homology arm for this targeting construct) (Figures 5A and 5B, +12-λ or 12-κ targeting vectors).Homologous recombination using these targeted 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 (these result in the formation of chimeric light chains upon recombination) (bottom of Figures 5A and 5B).

[0220] In a similar manner, 12 additional hVλ gene segments were introduced into the other initial modifier (Figure 4B) that incorporated multiple hJλ gene segments with and without the incorporated human κ sequence, by using the +12-κ targeted vector. Homologous recombination by this targeted vector at the endogenous mouse κ locus, including the other modifiers, created a mouse κ light chain locus containing 40 hVλ gene segments and hJλ1, 2, 3, and 7 gene segments with and without the human Vκ-Jκ genomic sequence operably attached to the endogenous mouse Cκ gene (these result in the formation of chimeric λ-κ light chains when recombination occurs).

[0221] Example IV Identification of targeted ES cells containing the human λ light chain gene segment Modified ES cells expressing human λ light chain gene segments were generated for creating chimeric mice by electroporating mouse ES cells using targeted BAC DNA prepared according to the previously described examples. ES cells containing insertions of unrearranged human λ light chain gene segments were identified by quantitative TAQMAN® assay. Specific primer sets and probes were designed for insertion of human λ sequences and associated selective cassettes (allele acquisition, GOA), loss of endogenous mouse sequences and any selective cassette (allele loss, LOA), and retention of adjacent mouse sequences (allele retention, AR). For each additional insertion of a human λ sequence, the presence of the additional human λ sequence was confirmed using additional primer sets and probes, and retention of the previously targeted human sequence was confirmed using the same primer sets and probes. Table 1 shows the primers and associated probes used in the quantitative PCR assay. Table 2 shows the combinations used to confirm insertion of each segment of the human λ light chain gene segment in ES cell clones.

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

[0223] [Table 1]

[0224] [Table 2-1]

[0225] [Table 2-2] Example V Creating mice that express human λ light chains from an endogenous light chain gene locus. The targeted ES cells described above were used as donor ES cells and introduced into 8-cell stage mouse embryos using the VELOCIMOUSE® method (e.g., U.S. Patent 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 possessing the human λ gene segment was identified by genotyping using a modified allele assay (Valenzuela et al., previously mentioned) that detects the presence of a unique human λ gene segment.

[0226] Frequency of κ:λ light chain use in mice possessing human λ light chain gene segments. κ and λ light chain expression in splenocytes was analyzed using flow cytometry in mice homozygous for each of three consecutive insertions of the hVλ gene segment (Figure 5B) containing a single hJλ gene segment, and in mice homozygous for the first insertion of the hVλ gene segment (Figure 4B) containing either a single hJλ gene segment or four human Jλ gene segments, both containing human Vκ-Jκ genome sequences.

[0227] Briefly, spleens were harvested from groups of mice (ranging from 3 to 7 animals per group) and disrupted using a slide glass. After lysing red blood cells (RBC) using ACK lysis buffer (Lonza Walkersville), splenocytes were stained with fluorescent dye-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 (trademark) LSR II flow cytometer (BD Biosciences) and analyzed using FLOWJO (trademark) software (Tree Star, Inc.). Table 3 shows the mean percent values observed for B cell (CD19 + ), κ light chain (CD19 + Igκ + Igλ - ), and λ light chain (CD19 + Igκ - Igλ + ) expression in splenocytes from groups of animals with each genetic modification.

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

[0229] In another experiment, the B cell content of spleen and bone marrow compartments from homozygous mice containing 40 hVλ gene segments and 4 hJλ gene segments (bottom of Figure 7B) operably linked to the mouse Cκ gene, along with the B cell development process, was analyzed using flow cytometry of various cell surface markers.

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

[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, the cells were washed and fixed in 2% formaldehyde. Data were acquired using a FACSCANTO II® flow cytometer (BD Biosciences) and analyzed using FLOWJO® software (Tree Star, Inc.). Figures 9A-9D show the results for the spleen compartment of a representative mouse from each group. Figures 10A-10E show the results for the bone marrow compartment of a representative mouse from each group. Table 4 shows the B cells (CD19) observed in spleen cells from animal groups with various genetic modifications. + ), κ light chain (CD19 + Igκ + Igλ - ) and λ light chain (CD19 + Igκ - Igλ + Table 5 shows the average percentage values ​​for expression. Table 5 shows the B cell (CD19) observed in the bone marrow of homozygous mice for 40 hVλ gene segments and 4 hJλ gene segments, including wild-type and human Vκ-Jκ genome 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 λ light chains (B220 int IgM + Igλ +The average percentage values ​​for ) are shown. This experiment was repeated using the additional mouse groups described above and yielded similar results (data not shown).

[0234] [Table 3]

[0235] [Table 4]

[0236] [Table 5] Frequency of use of the human Vλ gene in mice possessing the human λ light chain gene segment. The frequency of use of the human λ light chain gene was analyzed using reverse transcriptase polymerase chain reaction (RT-PCR) with RNA isolated from splenocytes in heterozygous mice for the first insertion of the human λ sequence (hVλ3-12-hVλ3-1 and hJλ1, Figure 5B) and homozygous mice for the third insertion of the human λ sequence (hVλ5-52-hVλ3-1 and hJλ1, Figure 5B).

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

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

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

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

[0241] Multiple hVλ gene segments rearranged at hλ1 were observed in RT-PCR clones derived from mice possessing human λ light chain gene segments at the endogenous mouse λ light chain locus. The nucleotide sequences of the hVλ-hJλ-mCλ2 junction for the selected RT-PCR clones are shown in Figure 13.

[0242] [Table 6]

[0243] [Table 7] Figure 11 shows the sequence of the hVλ-hJλ1-mCκ junction for mouse-derived RT-PCR clones having first and third insertions of the hVλ gene segment along with a single hJλ gene segment. The sequence shown in Figure 11 exhibits a unique rearrangement involving various hVλ gene segments along with hJλ1 recombinant to 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 produce B cells expressing human λ light chains. These rearrangements demonstrate that their 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 observed when rearranged with hJλ1 (Table 7), these modifications to the endogenous κ light chain locus did not render any hVλ gene segment operable, nor did the chimeric locus interfere with the recombination of multiple hVλ and hJλ(Jλ1) gene segments during B cell development. Moreover, these mice produced functional antibodies containing rearranged human Vλ-Jλ gene segments operably linked to the mouse Cκ gene as part of the endogenous immunoglobulin light chain repertoire.

[0244] Figure 12 shows the sequences of the hVλ-hJλ-mCκ junction for selected RT-PCR clones from homozygous mice containing 40 hVλ gene segments and 4 hJλ gene segments, each containing human Vκ-Jκ genome sequences. The sequences shown in Figure 12 exhibit unique additional rearrangements containing multiple different hVλ gene segments across the entire chimeric locus (multiple different hJλ gene segments are rearranged and operably connected to the mouse Cκ gene). Homozygous mice with the modified endogenous κ locus 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 the chimeric locus at all stages was able to independently rearrange human λ gene segments in multiple independent B cells of these mice. Furthermore, these additional modifications to the endogenous κ light chain locus demonstrate that each insertion of a human λ gene segment did not render any of the hVλ and / or Jλ gene segments inoperable, nor did the chimeric locus interfere with the recombination of the hVλ and Jλ gene segments during B cell development, as evidenced by the 12 distinct hVλ gene segments observed when rearranged with all four hJλ gene segments from 26 selected RT-PCR clones (Table 8).

[0245] Figure 13 shows the sequences of the hVλ-hJλ-mCλ2 junction for 12 hVλ gene segments and three distinct RT-PCR clones from homozygous mice for hJλ1. The sequences shown in Figure 13 show additional unique rearrangements containing different hVλ gene segments over 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 N at the hVλ-hJλ junction (2D1, Figure 13). This is not unusual in V(D)J recombination, as the ligation of gene segments during recombination has been shown to be inaccurate. This clone represents a non-productive recombinant present in the light chain repertoire of these mice, demonstrating that the genetic mechanisms contributing to the diversity of junctions between antibody genes are functioning correctly in these mice, resulting in an antibody repertoire containing light chains with greater diversity.

[0246] Homozygous mice possessing a modified endogenous λ locus containing 12 hVλ gene segments and hJλ1 were also able to generate human λ gene segments operably linked to the endogenous mouse Cλ gene, and produce B cells expressing a reverse chimeric λ light chain containing an hVλ region linked to the mouse Cλ region. 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 the human λ gene segments in multiple independent B cells of these mice. Furthermore, modifications to the endogenous λ light chain locus demonstrate that insertion of human λ gene segments did not render any hVλ gene segment and / or hJλ1 gene segment inoperable, and that the chimeric locus did not interfere with the recombination of hVλ and hJλ1 gene segments during B cell development. In addition, these mice also produced functional antibodies containing human Vλ-Jλ gene segments operably linked to the mouse Cλ region as part of the endogenous immunoglobulin light chain repertoire.

[0247] Since functional light chains are required at various checkpoints in B cell development in both the spleen and bone marrow, mice possessing human λ light chain gene segments at endogenous κ and λ light chain loci, as shown in this embodiment, can rearrange the human λ light chain gene segments and express them against the background 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) exhibit 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 (this 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 frequency observed in these mice is more similar to human light chain expression than to light chain expression observed in mice.

[0248] Example VI Crossbreeding mice that express human λ light chains from an endogenous light chain locus. To optimize the frequency of use of the human λ gene segment at the endogenous mouse light chain locus, mice possessing an unrearranged human λ gene segment are crossed with other mice containing deletions at the opposing endogenous light chain locus (κ or λ). For example, only the human λ gene segment located at the endogenous κ locus may be a functional light chain gene segment present in mice that also have a deletion at the endogenous λ light chain locus. In this configuration, the resulting offspring may express only the human λ light chain, as described in the examples above. The crosses are performed using standard methods accepted in the field, and / or by commercial companies, such as The Jackson Laboratory. Mouse lines possessing the human λ light chain gene segment at the endogenous κ locus and having a deletion at the endogenous λ light chain locus are screened for the presence of a unique reverse-skimer (human-mouse) λ light chain and the absence of the endogenous mouse λ light chain.

[0249] Mice possessing an unrearranged human λ light chain locus can also be crossed with mice that have a substitution of the endogenous mouse heavy chain variable gene locus by the human heavy chain variable gene locus (see U.S. Patent No. 6,596,541, Regeneron Pharmaceuticals; VELOCIMMUNE® genetically engineered mice). VELOCIMMUNE® mice partially include mice with a genome containing a human heavy chain variable region operably linked to an endogenous mouse constant region locus (these mice produce antibodies containing both the human heavy chain variable region and the mouse heavy chain constant region in response to antigenic stimulation). The DNA encoding the heavy chain variable region of these antibodies can be isolated and operably linked to the DNA encoding the human heavy chain constant region. This DNA can then be expressed in cells capable of expressing the complete human heavy chain of the antibody. A suitable crossing schedule yields mice that have a substitution of the endogenous mouse heavy chain locus by the human heavy chain locus and possess an unrearranged human λ light chain locus at the endogenous κ light chain locus. Upon immunization with the target antigen, antibodies containing somatically mutated human heavy chain variable regions and human λ light chain variable regions can be isolated.

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

[0251] Generally, VELOCIMMUNE® mice containing one of a single rearranged human germline light chain regions are challenged with an antigen, and lymphocytes (e.g., B cells) are recovered from the animal's serum. By fusing these lymphocytes with a myeloma cell line, immortal hybridoma cell lines are prepared, and by screening and selecting such hybridoma cell lines, 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. The DNA encoding the variable regions of the heavy chain and λ light chain can be isolated and ligated to the constant regions of the desired isotypes of the heavy chain and light chain. Thanks to the presence of an additional hVλ gene segment compared to the endogenous mouse λ locus, the diversity of the light chain repertoire is dramatically increased, conferring greater diversity to the antigen-specific repertoire when immunized. The resulting cloned antibody sequences can then be generated in cells such as CHO cells. Alternatively, antigen-specific chimeric antibodies or DNA encoding variable domains of the light and heavy chains can be directly isolated from antigen-specific lymphocytes (e.g., B cells).

[0252] First, high-affinity chimeric antibodies having a human variable region and a mouse constant region are isolated. As described above, these antibodies are characterized, and desirable characteristics such as affinity, selectivity, and epitope are selected. By substituting their mouse constant regions with a desired human constant region, a fully human antibody is generated containing a somatically mutated human heavy chain and a human λ light chain 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 in which the germline contains a human lambda variable region sequence at an endogenous mouse light chain gene locus, wherein the human lambda variable region sequence is expressed in a light chain containing a mouse immunoglobulin constant region gene sequence. (Item 2) The mouse described in Item 1, wherein the endogenous mouse light chain gene locus is the λ gene locus. (Item 3) The mouse described in Item 1, wherein the endogenous mouse light chain gene locus is the κ gene locus. (Item 4) The mouse described in Item 1, wherein the mouse lacks the endogenous light chain variable sequence at the endogenous mouse light chain gene locus described above. (Item 5) A mouse as described in Item 1, wherein all or substantially all of the endogenous mouse light chain variable region gene segments are replaced by one or more human λ variable region gene segments. (Item 6) The mouse described in Item 1, wherein the above human λ light chain variable region sequence contains the human Jλ sequence. (Item 7) The mouse described in Item 6, wherein the above human Jλ sequence is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7 and combinations thereof. (Item 8) The mouse described in Item 1, wherein the above human λ light chain variable region sequence contains a fragment of cluster A of the human light chain locus. (Item 9) The mouse described in Item 8, wherein the fragment of cluster A of the human λ light chain locus described above spans from hVλ3-27 to hVλ3-1. (Item 10) The mouse described in Item 1, wherein the above human λ light chain variable region sequence contains a fragment of cluster B of the human light chain locus. (Item 11) The mouse described in Item 10, wherein the fragment of cluster B of the human λ light chain locus described above spans from hVλ5-52 to hVλ1-40. (Item 12) The mouse described in Item 1, wherein the above human λ light chain variable region sequence includes the genomic fragment of cluster A and the genomic fragment of cluster B. (Item 13) The mouse described in Item 12, wherein the human λ light chain variable region sequence comprises at least one gene segment of cluster A and at least one gene segment of cluster B. (Item 14) The mouse described in Item 1, wherein more than 10% of the light chain repertoire of the mouse is derived from at least two hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45 and 9-49. (Item 15) The mouse described in Item 14, wherein more than 20% of the light chain repertoire of the mouse is derived from at least three hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45 and 9-49. (Item 16) The mouse described in Item 15, wherein more than 30% of the light chain repertoire of the mouse is derived from at least four hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45 and 9-49. (Item 17) A mouse expressing an immunoglobulin light chain containing a human lambda variable sequence fused to a mouse constant region, wherein the mouse exhibits a ratio of approximately 1:1 between the frequency of κ use and the frequency of λ use. (Item 18) The mouse described in Item 17, in which the above immunoglobulin light chain is expressed from the endogenous mouse light chain gene locus. (Item 19) Use of a mouse described in any one of the above items for the production of an antigen-binding protein. (Item 20) Use as described in Item 19, provided that the antigen-binding protein is a human antigen-binding protein. (Item 21) Cells or tissues derived from a mouse as described in any one of items 1 to 18.

Claims

[Claim 1] The cell or antibody or method described in the specification.

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