NZ753456B2 - Non-human animals having an engineered immunoglobulin lambda light chain locus - Google Patents

Abstract

Non-human animals (and/or non-human cells) and methods of using and making the same are provided, which non-human animals (and/or non-human cells) have a genome comprising human antibody-encoding sequences (i.e., immunoglobulin genes). Non-human animals described herein express antibodies that contain human Ig? light chains, in whole or in part. In particular, non-human animals provided herein are, in some embodiments, characterized by expression of antibodies that contain human Ig? light chains, in whole or in part, that are encoded by human Ig? light chain-encoding sequences inserted into an endogenous Ig? light chain locus of said non-human animals. Methods for producing antibodies from non-human animals are also provided. in human Ig? light chains, in whole or in part. In particular, non-human animals provided herein are, in some embodiments, characterized by expression of antibodies that contain human Ig? light chains, in whole or in part, that are encoded by human Ig? light chain-encoding sequences inserted into an endogenous Ig? light chain locus of said non-human animals. Methods for producing antibodies from non-human animals are also provided.

Description

(12) Granted patent specificaon (19) NZ (11) 753456 (13) B2 (47) Publicaon date: 2021.12.24 (54) MAN S HAVING AN ENGINEERED IMMUNOGLOBULIN LAMBDA LIGHT CHAIN LOCUS (51) Internaonal Patent Classificaon(s): A01K 67/027 C12N 15/90 C12N 15/85 C07K 16/00 (22) Filing date: (73) Owner(s): 2017.11.03 REGENERON PHARMACEUTICALS, INC. (23) Complete specificaon filing date: (74) Contact: 2017.11.03 PHILLIPS ORMONDE TRICK (30) Internaonal Priority Data: (72) Inventor(s): US 62/567,932 2017.10.04 MCWHIRTER, John US 62/417,845 2016.11.04 MACDONALD, Lynn MURPHY, Andrew J. (86) Internaonal aon No.: TU, Naxin VORONINA, Vera GUO, Chunguang (87) aonal Publicaon number: LEVENKOVA, Natasha WO/2018/128691 HARRIS, Faith (57) Abstract: Non-human animals r non-human cells) and methods of using and making the same are provided, which non-human animals (and/or non-human cells) have a genome comprising human anbody-encoding sequences (i.e., globulin genes). Non-human animals described herein express anbodies that contain human Igλ light chains, in whole or in part. In parcular, man animals provided herein are, in some embodiments, characterized by expression of anbodies that contain human Igλ light chains, in whole or in part, that are encoded by human Igλ light chain-encoding sequences inserted into an endogenous Igλ light chain locus of said nonhuman animals. Methods for producing anbodies from non-human animals are also provided.

NZ 753456 B2 NON-HUMAN ANIMALS HAVING AN ENGINEERED IMMUNOGLOBULIN LAMBDA LIGHT CHAIN LOCUS RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application serial number 62/417,845, filed November 4, 2016, and U.S. Provisional Patent Application serial number 62/567,932, filed October 10, 2017, each of which is hereby incorporated by reference in its ty.

BACKGROUND Human antibodies are the most rapidly growing class of therapeutics. Of the technologies that are currently used for their production, the development of transgenic animals (e.g., s) engineered with genetic al encoding human dies, in whole or in part, has revolutionized the field of human therapeutic monoclonal antibodies for the treatment of various es. Still, development of improved in vivo systems for generating human monoclonal antibodies that maximize human antibody repertoires in host transgenic animals is needed.

SUMMARY In certain aspects, provided herein are improved in vivo systems for identifying and developing new antibody and antibody-based therapeutics that can be used for the treatment of a variety of diseases that affect humans. As disclosed herein, in certain embodiments the nonhuman animals (e.g., rodents) provided herein, having engineered globulin loci, in particular, engineered immunoglobulin (Ig)  light chain loci and/or otherwise expressing, producing or ning antibody repertoires characterized by light chains having human V regions, are useful, for example, for ting the diversity of human V sequences in the identification and development of new antibody-based eutics. In some embodiments, nonhuman animals described herein provide improved in vivo systems for development of antibodies and/or antibody-based therapeutics for administration to humans. In some embodiments, nonhuman animals described herein provide improved in vivo systems for development of dies and/or antibody-based therapeutics that contain human V domains characterized by ed performance as compared to antibodies and/or antibody-based therapeutics obtained from existing in viva systems that contain human V7» region sequences.

In certain aspects, provided herein is a non-human animal having an Ig7t light chain locus that contains ered immunoglobulin le and constant regions; in some certain embodiments, further comprises engineered regulatory region (or ce). As described herein, in certain embodiments the provided non-human animals, contain in their germline genome an Ig7t light chain locus comprising an engineered 1g?» light chain variable region characterized by the presence of one or more human V?» gene segments, one or more human I?» gene segments, one or more human C?» region genes and a rodent C?» region gene, which human V2», J7» and C7» gene segments are operably linked to each other and operably linked to said rodent Ck region gene.

In some embodiments, provided non-human animals comprise an ng light chain locus that comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 or at least 25 human V?» gene segments.

In some embodiments, provided non-human animals comprise an ng light chain locus that comprises 5 to 25, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5to 16, 5to 15, 5to 14, 5to 13, 5to 12, 5to 11, 5to 10, 5to9, 5to8,5to7, or5to6 human V7t gene segments. In some embodiments, ed man animals comprise an Ig7t light chain locus that comprises 10 to 70, 10 to 69, 10 to 68, 10 to 67, 10 to 66, 10 to 65, to 64, 10 to 63, 10 to 62, 10 to 61, 10 to 60, 10 to 59, 10 to 58,10 to 57, 10 to 56,10 to 55, 10 to 54, 10 to 53, 10 to 52, 10 to 51, 10 to 50,10 to 49, 10 to 48, 10 to 47, 10 to 46,10 to 45, 10 to 44, 10 to 43,10 to 42, 10 to 41,10 to 40, 10 to 39,10 to 38, 10 to 37, 10 to 36, to 35, 10 to 34, 10 to 33, 10 to 32, 10 to 31, 10 to 32, 10 to 31,10 to 30, 10 to 29,10 to 28, 10 to 27,10 to 26, 10 to 25,10 to 24, 10 to 23,10 to 22, 10 to 21, 10 to 20, 10 to 19, 10 t018, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 10 to 12, or 10 toll human V76 gene segments.

In some embodiments, provided man animals comprise an 1g?» light chain locus that comprises 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 11 to 25, 12 to 25, 13 to 25, 14 to 25, 15 to 25, 16 to 25, 17 to 25, 18 to 25, 19 to 25, 20 to 25,21 to 25, 22 to 25,23 to , or 24 to 25 human V?» gene ts. In some embodiments, provided non-human animals comprise an 1g?» light chain locus that comprises 11 to 70, 12 to 70, 13 to 70, 14 to 70, 15 to 70,16 to 70, 17 to 70, 18 to 70, 19 to 70, 20 to 70, 21 to 70, 22 to 70, 23 to 70,24 to 70, 25 to 70, 26 to 70, 27 to 70, 28 to 70, 29 to 70, 30 to 70, 31 to 70, 32 to 70, 33 to 70, 34 to 70, 35 to 70, 36 to 70, 37 to 70, 38 to 70, 39 to 70, 40 to 70, 41 to 70, 42 to 70, 43 to 70, 44 to 70, 45 to 70, 46 to 70, 47 to 70, 48 to 70, 49 to 70, 50 to 70, 51 to 70, 52 to 70,53 to 70, 54 to 70, 55 to 70, 56 to 70, 57 to 70, 58 to 70, 59 to 70, 60 to 70, 61 to 70, 62 to 70, 63 to 70, 64 to 70, 65 to 70, 66 to 70, 67 to 70, 68 to 70, or 69 to 70 human V?» gene segments.

In some embodiments, provided non-human animals se an 1g?» light chain locus that comprises 6 to 24, 7 to 23, 8 to 22, 9 to 21, 10 to 20, 11 to 19, 12 to 18, 13 to 17, 14 to 16, or 15 to 16 human V?» gene ts. In some ments, provided non-human animals comprise an 1g?» light chain locus that comprises 11 to 69, 12 to 68, 13 to 67, 14 to 66, 15 to 65,16 to 64, 17 to 63, 18 to 62, 19 to 61, 20 to 60, 21 to 59, 22 to 58, 23 to 57, 24 to 56, 25 to 55, 26 to 54, 27 to 53, 28 to 52, 29 to 51, 30 to 50, 31 to 49, 32 to 48, 33 to 47, 34 to 48,35 to 47, 36 to 46, 37 to 45, 38 to 44, 39 to 43, 40 to 42, or 41 to 42 human v2 gene segments.

In certain embodiments, provided non-human animals comprise an ng light chain locus that ses 5, 16 or 25 functional human Vl gene segments. In certain embodiments, provided non-human animals comprise an 1g?» light chain locus that comprises , 27 or 40 human V7t gene segments. In n embodiments, human V?» gene segments include consecutive human V?» gene segments as said human V?» gene segments appear in a human Ig7t light chain locus of a human cell.

In some embodiments, provided non-human animals comprise an 1g?» light chain locus that ses at least 5 human I?» gene segments (e.g, but not limited to, 5 human J?» gene segments, 6 human J71. gene segments, 7 human J71, gene segments, 8 human J71. gene segments, etc). In some embodiments, provided non-human animals se an ng light chain locus that comprises at least 4 human C?» region genes (e. g., but not limited to, 4 human C?» region genes, 5 human C?» region genes, 6 human C?» region genes, 7 human C7t region genes, 8 human C?» region genes, etc). In certain embodiments, provided non—human animals comprise an 1g?» light chain locus that comprises at least 25 human V?» gene segments, at least 5 human J7t gene segments and at least 4 human C?» region genes at an endogenous 1g?» light chain allele. In some embodiments, provided non-human animals comprise only one murine (e.g., mouse or rat) C?» region gene (e.g., a mouse CM region gene or a mouse CM gene segment) at an endogenous non-human 1g?» light chain locus. In some embodiments, said 1g?» light chain locus further comprises a human EX region (or sequence) that is characterized by three sequence elements.

In some ments, provided non—human animals contain human V7», J7» and Ck gene segments at an endogenous man ng light chain locus in natural or germline configuration. In some embodiments, provided non-human animals contain human V)», J?» and C?» gene ts at an endogenous non-human Igl light chain locus in a configuration that does not naturally appear in a human immunoglobulin 2 light chain locus of the germline genome of a human cell.

In some embodiments, provided non-human animals contain a DNA sequence at an endogenous non-human 1g?» light chain locus that includes a plurality of human V)», J?» and C?» coding sequences interspersed (or juxtaposed, associated, etc.) with non-coding human immunoglobulin 7t light chain sequence. In some embodiments, provided non-human animals contain a DNA sequence at an nous non-human Ig7t light chain locus that includes a ity of human V)», J?» and Cl coding sequences interspersed with non—coding non-human (e.g, murine) immunoglobulin 7t light chain ce.

In some embodiments, provided non-human animals are terized by expression of antibodies from endogenous non-human 1g?» light chain loci in the germline genome of said non-human animals, which antibodies contain human V2 domains and human or non-human CA domains. In some embodiments, provided non-human animals are characterized by an increased usage of human V7t regions from engineered immunoglobulin A light chain loci (e.g, a 60:40 1C). ratio) as compared to one or more nce engineered or Wild-type man animals (e. g., but not limited to, a 95:5 K27» ratio), In some embodiments, a non—human , non—human cell or non—human tissue is provided Whose genome comprises an endogenous immunoglobulin 9» light chain locus comprising insertion of one or more human V?» gene segments, one or more human J7» gene segments and one or more human C7» gene segments, Which human V)», J7» and CK gene segments are operably linked to a non-human Ck gene segment, and which endogenous immunoglobulin 1 light chain locus r comprises one or more non—human immunoglobulin 7t light chain enhancers (El) and one or more human immunoglobulin 9» light chain enhancers (EA).

In some embodiments, a man animal, non-human cell or man tissue is provided whose ine genome ses an endogenous immunoglobulin 7t light chain locus sing: (a) one or more human V?» gene segments, (b) one or more human J7» gene segments, and (c) one or more human C?» gene segments, wherein (a) and (b) are operably linked to (c) and a non-human Ck gene t, and wherein the endogenous immunoglobulin 7t light chain locus further comprises: one or more non-human immunoglobulin 1 light chain enhancers (El), and one or more human immunoglobulin 7» light chain enhancers (Bk).

In some embodiments, an endogenous immunoglobulin 7» light chain locus provided herein further comprises three human Eks. In some ments, an endogenous immunoglobulin 7t light chain locus further comprises one human E?» characterized by the presence of three sequence elements. In some certain embodiments, an endogenous immunoglobulin A light chain locus further comprises one human B?» characterized by the presence of three sequence elements that act (or function) in modular fashion.

In some embodiments, an endogenous immunoglobulin 7» light chain locus ed herein comprises two non-human Eks. In some certain ments, an endogenous immunoglobulin A light chain locus comprises two rodent Eks. In some certain embodiments, an endogenous immunoglobulin 1 light chain locus provided herein comprises two mouse Eks. In some certain embodiments, an endogenous immunoglobulin 7» light chain locus comprises a mouse E?» and a mouse Ek3-l. In some certain embodiments, an endogenous immunoglobulin 7t light chain locus provided herein does not contain (or lacks) a mouse E124. In some certain embodiments, an endogenous globulin A, light chain locus comprises two rat EXS.

In some embodiments, an endogenous immunoglobulin 7L light chain locus provided herein comprises a on of endogenous V?» and J?» gene ts, in whole or in part. In some certain embodiments, an endogenous immunoglobulin 9» light chain locus provided herein comprises a deletion of VXZ—VM —J?tZ—C7tZ gene segments and VM—J7t3— Ck3-J7&1 gene segments. In some certain embodiments, an endogenous immunoglobulin 7» light chain locus comprises a deletion of VXZ-VM-J?»2—C?»2—JMP-CMP gene segments and VM-J?t3-J?t3P-C?t3 -JM gene segments. In some embodiments, an endogenous immunoglobulin Might chain locus provided herein comprises a deletion of a non—human E?t2—4. In some certain embodiments, an endogenous immunoglobulin Might chain locus provided herein comprises a deletion of V?t2, V?t3, J?»2, CM, J?»4P, C?»4P, E?t2-4, VM, J?t3, J?t3P, C?»3 and JM. In some certain embodiments, an endogenous immunoglobulin ?» light chain locus provided herein comprises CM, E?» and E?t3-1 as the only non-human gene ts or sequence elements present.

In some embodiments, an endogenous immunoglobulin ?t light chain locus provided herein comprises insertion of the human V?» gene segments V?t4-69 to V?t3-l, at least the human t gene segment pairs J?»l—CM, J?t2—C?»2, J?t3—C?t3, J?t6—C?»6, the human J?t gene segment DJ and a rodent CM gene segment. In some embodiments, an endogenous immunoglobulin Might chain locus provided herein comprises insertion of the human V?» gene segments 2 to V?t3-l, at least the human J?t-C?t gene segment pairs JM-CM, J?t2-C?t2, J?t3-C?»3, J?»6-C?t6, the human J?» gene segment JM and a rodent CM gene segment, In some embodiments, an nous immunoglobulin Might chain locus provided herein comprises insertion of the human V?t gene segments V?t5-52 to VM-40 and Vk3-27 to V?t3-l, at least the human J?t-C?t gene segment pairs JM-CM, J?»2-C?t2, J?t3-C?t3, J?t6-C?t6, the human J?» gene segment JM and a rodent CM gene segment. In some n ments, the insertion includes human non-coding DNA that naturally appears between human 2 to VM-40 and V?t3 -27 to , human non-coding DNA that naturally appears between human t gene segment pairs JM—CM, J?t2—C?t2, J?»3-C?t3 and J?t6— C?t6, and human non—coding DNA that naturally appears upstream (or 5’) of human J?t gene segment J?»7.

In some embodiments, a non-human C?» gene segment is or comprises a rodent C?» gene segment. In some embodiments, a rodent C?» gene segment is or ses a murine (e.g, mouse or rat) C?» gene segmentIn some embodiments, a rodent C?» gene segment is or comprises a rat C?» gene segment. In some embodiments, a rodent C?» gene segment is or comprises a mouse C?t gene segment. In some certain ments, a rodent C?t gene segment is a mouse CM gene t.

In some embodiments, a mouse C?» gene (or gene segment) comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical or 100% identical to a mouse C2 gene selected from the group consisting of a mouse C21, mouse C22 and a mouse C23. In some embodiments, a mouse C2 gene comprises a sequence that is substantially identical or identical to a mouse C2 gene ed from the group consisting of a mouse C21, mouse C22 and a mouse C23. In some certain embodiments, a mouse C21 gene is or comprises SEQ ID NO:1. In some certain embodiments, a mouse C22 gene is or comprises SEQ ID NO:3, In some certain embodiments, a mouse C23 gene is or comprises SEQ ID NO:5. In some certain embodiments, a mouse C2 gene comprises a sequence that is cal to a mouse CM gene.

In some embodiments, a mouse C2 gene (or gene segment) comprises a sequence that is 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% identical to a mouse C2 gene selected from the group consisting of a mouse CM, mouse C22 and a mouse C23.

In some embodiments, a mouse C2 gene (or gene segment) comprises a sequence that is 50% to 98%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, or 50% to 55% identical to a mouse C2 gene selected from the group consisting of a mouse CM, mouse C22 and a mouse C23.

In some embodiments, a mouse C2 gene (or gene segment) comprises a sequence that is 55% to 98%, 60% to 95%, 65% to 90%, 70% to 85%, or 75% to 80%, identical to a mouse C2 gene selected from the group consisting of a mouse CM, mouse C22 and a mouse C23.

In some embodiments, a rat C2 gene (or gene t) comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical, or 100% identical to a rat C2 gene selected from the group consisting of a rat CM, rat C22, rat C23 and a rat C24 gene. In some embodiments, a rat C2 gene comprises a sequence that is ntially identical or identical to a rat C2 gene selected from the group consisting of a rat CM, rat C22, rat C23 and a rat C24 gene. In some certain embodiments, a rat CM gene is or comprises SEQ ID NO:7, In some certain embodiments, a rat C22 gene is or ses SEQ ID NO:9. In some certain embodiments, a rat C23 gene is or comprises SEQ ID N011 1. In some n embodiments, a rat C24 gene is or comprises SEQ ID NO: 13.

In some embodiments, a rat C7L gene (or gene segment) comprises a sequence that is 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% identical to a rat C?» gene selected from the group consisting of a rat CM, rat C7t2, rat CM and a rat CM gene.

In some ments, a rat C)» gene (or gene t) comprises a sequence that is 50% to 98%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, or 50% to 55% identical to a rat Ck gene ed from the group consisting of a rat CM, rat Ck2, rat C13 and a rat Ck4 gene, In some embodiments, a rat C7» gene (or gene segment) comprises a sequence that is 55% to 98%, 60% to 95%, 65% to 90%, 70% to 85%, or 75% to 80%, identical to a rat Ck gene selected from the group consisting of a rat CM, rat CM, rat CM and a rat CM gene.

In some ments of a provided non-human , non-human cell or non- human tissue, the germline genome or genome of said non-human animal, non-human cell or non-human tissue further comprises (i) an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a man immunoglobulin heavy chain constant region; or (ii) an endogenous immunoglobulin heavy chain locus sing ion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene ts are operably linked to a non-human immunoglobulin heavy chain constant region, and an endogenous immunoglobulin K light chain locus comprising insertion of one or more human VK gene segments and one or more human JK gene segments, which human VK and J14 gene segments are operably linked to a non-human immunoglobulin CK region.

In some embodiments, the inserted one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments replace non- human VH, DH gene segments. In certain embodiments, the insertion includes human non- coding DNA that naturally appears between human VH, DH and JH ts, and combinations thereof. In some embodiments, a non-human immunoglobulin heavy chain constant region is an endogenous non-human immunoglobulin heavy chain constant region, In some embodiments, an immunoglobulin heavy chain locus comprises insertion of the 2017/060006 human VH gene segments from VH3-74 to VH6-l, the human DH gene segments from DHl-l to DH7-27, and the human JH gene segments JHl-JH6. In certain embodiments, the insertion includes human non-coding DNA that naturally appears (occurs) between human VH3-74 to VH6-1, human non-coding DNA that naturally appears (occurs) between human DHl-l to DH7-27, and human non-coding DNA that naturally appears (occurs) between human JHl- JH6. In some embodiments, an globulin heavy chain locus ses insertion of all functional human VH gene ts, all functional human DH gene segments, and all functional human JH gene segments.

In some embodiments, an immunoglobulin heavy chain locus lacks an endogenous man Adam6 gene. In some embodiments, an immunoglobulin heavy chain locus further comprises an insertion of one or more nucleotide ces encoding one or more non-human Adam6 polypeptides. In some embodiments, one or more nucleotide ces encoding one or more rodent Adam6 polypeptides are inserted between a first and a second human VH gene segment, In some embodiments, a first human VH gene segment is human VH1-2 and a second human VH gene segment is human VH6—1. In some embodiments, one or more nucleotide sequences ng one or more rodent Adam6 polypeptides are inserted between a human VH gene segment and a human DH gene segment. In some embodiments, one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides are inserted in the place of a human Adam6 pseudogene.

In some embodiments, the inserted one or more human VK gene segments and one or more human JK gene segments replace non-human VK and JK gene ts. In some certain embodiments, the insertion includes human non-coding DNA that naturally appears n human VK and JK gene ts, and combinations thereof. In some embodiments, a non-human immunoglobulin CK region is an endogenous non-human CK region. In some embodiments, an immunoglobulin K light chain locus comprises insertion of the proximal VK duplication, in whole or in part, of a human immunoglobulin K light chain locus. In some embodiments, an immunoglobulin K light chain locus comprises insertion of the human VK gene segments from VK2-4O to VK4-l and the human JK gene segments from JKl-JKS. In some certain embodiments, the insertion includes human non-coding DNA that naturally appears between human VK2-40 to VK4-l, and human non-coding DNA that lly appears between human JKl-JKS.

In some ments of a non—human animal, non-human cell or non-human tissue provided herein, the non-human animal, non-human cell or non-human tissue is heterozygous or homozygous for an immunoglobulin heavy chain locus as described herein (e. g., an endogenous globulin heavy chain locus as described herein).

In some embodiments of a non—human animal, man cell or non-human tissue provided herein, the non-human animal, man cell or non-human tissue is heterozygous or homozygous for an immunoglobulin K light chain locus as described herein (e. g., an nous immunoglobulin K light chain locus as described herein).

In some embodiments of a non-human animal, non-human cell or non-human tissue provided herein, the non—human animal, non—human cell or non—human tissue is zygous or homozygous for an immunoglobulin X light chain locus as described herein (e.g, an endogenous immunoglobulin 7t light chain locus as described herein).

In some embodiments of a non-human animal, non-human cell or non-human tissue provided herein, the gerrnline genome of said non—human animal, non—human cell or non-human tissue further comprises insertion of one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides, and the animal is heterozygous or homozygous for said insertion.

In some embodiments, a non-human cell is a man lymphocyte. In some embodiments, a non-human cell is selected from a B cell, dendritic cell, macrophage, monocyte and a T cell.

In some embodiments, a non-human cell is a non-human embryonic stem (ES) cell. In some embodiments, a non-human ES cell is a rodent ES cell. In certain embodiments, a rodent ES cell is a mouse ES cell (e. g., from a 129 strain, C57BL strain, BALB/c or a mixture thereof). In some certain embodiments, a rodent embryonic stem cell is a mouse embryonic stem cell and is a mixture of 129 and C57BL strains. In some certain embodiments, a rodent embryonic stem cell is a mouse embryonic stem cell and is a e of 129, C57BL and BALB/c strains.

In some embodiments, use of a man ES cell described herein to make a non-human animal is provided. In certain embodiments, a non-human ES cell is a mouse ES cell and is used to make a mouse comprising engineered immunoglobulin 7t light chain locus as described herein. In certain embodiments, a man ES cell is a rat ES cell and is used to make a rat sing engineered immunoglobulin 9t light chain locus as described herein.

In some ments, a non-human tissue is selected from e, bladder, brain, , bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, , , ovum, and a combination thereof.

In some embodiments, an immortalized cell made, generated, produced or obtained from an isolated man cell or tissue as described herein is provided.

In some embodiments, a man embryo made, generated, produced, or ed from a man ES cell as described herein is provided. In some certain embodiments, a non—human embryo is a rodent embryo; in some embodiments, a mouse embryo, in some embodiments, a rat embryo.

In some embodiments, a kit comprising a non-human animal, non-human cell, non-human tissue, immortalized cell, non-human ES cell, or non-human embryo as described herein is provided, In some embodiments, a kit as described herein for use in the manufacture and/or development of a drug (e. g, an antibody or fragment thereof) for y or diagnosis is provided.

In some embodiments, a kit as described herein for use in the manufacture and/or development of a drug (e.g., an antibody or fragment thereof) for the treatment, prevention or amelioration of a disease, disorder or condition is provided.

In some embodiments, a method of making a non-human animal whose germline genome comprises an engineered endogenous immunoglobulin 9» light chain locus is provided, the method comprising (a) introducing a DNA fragment into a non-human nic stem cell, said DNA fragment comprising a nucleotide sequence that includes (i) one or more human V?» gene segments, (ii) one or more human Dc gene segments, and (iii) one or more human C?» gene segments, n (i)-(iii) are operably linked to a non-human Cl gene segment, and wherein the nucleotide sequence further comprises one or more human immunoglobulin 9» light chain enhancers (EX); (b) obtaining the non—human embryonic stem cell generated in (a); and (c) creating a non-human animal using the non- human embryonic stem cell of (b).

In some embodiments, a method of making a non-human animal whose germline genome comprises an ered endogenous immunoglobulin 9» light chain locus is provided, which engineered endogenous immunoglobulin 9» light chain locus comprises insertion of one or more human V?» gene segments, one or more human I?» gene segments and one or more human C?» gene segments, which human V?» and J?» gene segments are operably linked to a non—human and/or human C?» gene segment, and which endogenous immunoglobulin 7» light chain locus further comprises one or more non—human immunoglobulin 7t light chain enhancers (EX), and one or more human immunoglobulin 7» light chain enhancers (ER), is provided, the method comprising modifying the germline genome of a non-human animal so that it ses an engineered immunoglobulin 7t light chain locus that includes insertion of one or more human Vl gene segments, one or more human J7» gene segments and one or more human C?» gene segments, which human V?» and J7L gene segments are operably linked to a non-human and/or human Ck gene segment, and which endogenous immunoglobulin 7t light chain locus further comprises one or more rodent globulin 7t light chain enhancers (EA), and one or more human immunoglobulin 7» light chain ers (El), thereby making said non-human animal.

In some embodiments of a method of making a non-human animal ed herein, one or more human V?» gene segments include Vk4-69 to Vk3-1, VXS-SZ to Vk3—1 or Vk3—27 to Vk3—l. In some embodiments of a method of making a non—human animal, one or more human V?» gene segments include VXS-SZ to VM-4O and/or V96 -27 to VM-l. In some certain embodiments of a method of making a non—human animal, the one or more human Vk gene segments include human ding DNA that naturally appears between human VlS-SZ to VM-40 and/or Vk3-27 to Vk3-l. In some embodiments of a method of making a non-human , one or more human J?» gene segments and one or more human Ck gene segments include the human JNC?» gene segment pairs J7t1-C7t1, JkZ-CM, M3- C7t3, Jk6—C7t6 and the human R7 gene segment. In some certain embodiments of a method of making a non-human animal, the human JX-C?» gene t pairs Ill-CM, JkZ-CXZ, JX3-CK3 and t6 include human non—coding DNA that naturally appears between the human I)» and Ck gene t pairs, and the human M7 gene segment includes human ding DNA that naturally appears upstream (or 5’) of human J17.

In some certain embodiments of a method of making a non-human animal provided herein, insertion of the human V?» gene segments Vk5-52 to VM-40 and Vk3-27 to Vk3-1 includes human non—coding DNA that naturally appears between the human V?» gene segments, insertion of human INC)» gene segment pairs JM-CM, JKZ-CXZ, J7t3-C7t3 and JX6-C9t6 includes human ding DNA that naturally s between the human Jk-C?» gene segment pairs, and insertion of the human J27 gene segment includes human non- coding DNA that naturally appears upstream (or 5’) of human J27.

In some embodiments of a method of making a non-human animal provided herein, a non-human Ck gene segment is a rodent C?» gene segment; in some certain embodiments, a mouse CM gene segment.

In some ments of a method of making a non-human animal ed , a DNA fragment further comprises one or more selection markers. In some embodiments of a method of making a non—human animal, a DNA fragment r comprises one or more site-specific recombination sites. In some certain embodiments of a method of making a non-human animal provided herein, a DNA fragment further comprises one or more sets of site—specific recombination sites that recombine with the same recombinase. In some certain embodiments of a method of making a non-human animal, a DNA fragment r comprises one or more sets of site-specific recombination sites that recombine with different recombinases.

In some ments of a method of making a non-human animal provided herein, a DNA fragment is introduced into a man embryonic stem cell whose germline genome comprises (i) an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a non-human immunoglobulin heavy chain nt region; or (ii) an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are ly linked to a non-human immunoglobulin heavy chain constant region, and an endogenous immunoglobulin K light chain locus comprising insertion of one or more human VK gene segments and one or more human JK gene segments, which human VK and JK gene segments are operably linked to a non—human globulin CK region.

In some embodiments of a method of making a man animal provided herein, a DNA fragment is introduced into a non-human embryonic stem cell whose germline genome comprises (i) a wild—type endogenous immunoglobulin heavy chain locus; or (ii) a Wild-type endogenous immunoglobulin heavy chain locus and a wild-type endogenous immunoglobulin K light chain locus; and wherein the method further comprises WO 28691 a step of breeding a mouse made, generated, produced or obtained from said non-human embryonic stem cell with a second mouse.

In some embodiments of a method of making a non-human animal provided herein, modifying the germline genome of a non-human animal so that it comprises an engineered globulin 7» light chain locus is carried out in a non-human embryonic stem cell whose germline genome r comprises (i) an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene ts are operably linked to a non-human immunoglobulin heavy chain constant region; or (ii) an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a non—human immunoglobulin heavy chain constant region, and an endogenous immunoglobulin K light chain locus comprising ion of one or more human VK gene segments and one or more human .IK gene segments, which human VK and JK gene segments are ly linked to a non-human immunoglobulin CK region.

In some certain embodiments of a method of making a non-human animal provided herein, the insertion of one or more human VH gene ts, one or more human DH gene segments and one or more human JH gene segments includes human non-coding DNA that naturally appears between the one or more human VH gene segments, human non- coding DNA that naturally appears between the one or more human DH gene segments and human ding DNA that naturally appears between the one or more human JH gene segments. In some certain embodiments of a method of making a non-human animal provided herein, the insertion of one or more human VK gene segments and one or more human JK gene segments includes human ding DNA that naturally s between the one or more human VK gene segments and human non-coding DNA that lly appears between the one or more human JK gene segments.

In some embodiments of a method of making a non-human animal provided herein, modifying the germline genome of a non-human animal so that it comprises an engineered globulin 9t light chain locus is carried out in a non-human embryonic stem cell whose germline genome comprises (i) a wild-type endogenous immunoglobulin heavy chain locus; or (ii) a wild-type endogenous immunoglobulin heavy chain locus and a ype endogenous immunoglobulin K light chain locus; and wherein the method further ses a step of breeding a mouse made, generated, produced or ed from said non- human embryonic stem cell with a second mouse.

In some embodiments, a mouse as described herein has a germline genome comprising wild-type IgH and IgK loci, homozygous or heterozygous humanized IgH and IgK loci, which homozygous or heterozygous humanized IgH locus ns an inserted rodent Adamo-encoding sequence, or a homozygous or heterozygous humanized IgH locus (with or without insertion of Adam6—encoding sequence) and a gous or heterozygous inactivated IgK locus.

In some embodiments, a non—human animal made, generated, produced, obtained or obtainable from a method as described herein is provided.

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

In some embodiments, a method of producing a nucleic acid encoding a human lambda light chain variable domain in a non-human animal is provided, the method comprising the steps of (a) zing a non-human animal as described herein with an antigen of interest, (b) maintaining the non-human animal under conditions sufficient that the rodent produces an immune response to the antigen of interest, and (c) recovering a nucleic acid encoding a human lambda light chain variable domain from the non-human animal, or a non-human cell. In some embodiments, the method further comprises ring a nucleic acid ng a human heavy chain variable domain from the non-human animal, or a non- human cell.

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

In some embodiments of a method of producing an dy in a non-human animal, an antibody recovered from a rodent, or a rodent cell, that binds the antigen of interest ses a human heavy chain variable domain and a human lambda light chain variable domain.

In some embodiments a of a method of ing an antibody or a nucleic acid in a non-human animal, a human heavy chain variable domain es a rearranged human VH gene segment selected from the group consisting of VH3-74, VH3-73, VH3-72, VH2—70, VH1- 69, VH3-66, , VH4-61, VH4-59, VH1-58, VH3-53, VHS-51, VH3-49, VH3-48, VH1-46, VH1-45, VH3-43, , VH4-34, VH3-33, VH4-31, VH3—30, VH4—28, VH2-26, VH1-24, VH3-23,VH3-21,VH3-20,VHl-18,VH3-15,VH3-13,VH3-ll, VH3-9, vHi—s, VH3-7, sz—s, VH7l, VH4-4, VHl-3, VHl-Z and VH6-1.

In some embodiments a of a method of producing an antibody or a nucleic acid in a non-human animal, a human lambda light chain variable domain includes a rearranged human V?» gene segment selected from the group consisting of Vk4-69, Vk8-6l, VK4-60, Vk6-57, VMO—54, Wis—52, var—51, VA9-49, val—47, vx7—46, Wes—45, var—44, vm—43, val—40, Wis—39, vx5—37,vx1—36, vx3—27, vx3—25, va2—23, va3—22, V7L3-21, v>t3—19, VIZ-18, VK3-l6, VIZ-14, Vk3-12, VKZ-l l, Vk3-10, VX3-9, VXZ-S, Vk4—3 and Vk3-l.

In some embodiments, a method of inducing an antigen-specific immune response in a non-human animal is provided, the method comprising the steps of (a) immunizing a non—human animal as described herein with an antigen of interest, (b) maintaining the non-human animal under conditions sufficient that the rodent produces an immune se to the antigen of interest, In some embodiments, a non-human animal is provided whose germline genome comprises a homozygous endogenous immunoglobulin 7L light chain locus comprising insertion of (i) human V7t gene ts Vk4-69 to VM-l, Vh5-52 to VK3-l, Vk3-27 to Vk3—l, or VlS—SZ to O and VX3—27 to Vk3—l, (ii) human Jh—C?» gene segment pairs , JM-CXZ, JX3-C7t3 and 6, (iii) human 17L gene segment DJ, and (iv) three human immunoglobulin h light chain enhancers (or a human immunoglobulin k light chain enhancer having three sequence elements); n (i)-(iv) are operably linked to each other and the insertion is upstream of a non-human C7» gene segment, and wherein the endogenous immunoglobulin 7L light chain locus lacks an endogenous non-human globulin BAZ- In some embodiments, a non—human animal is ed whose germline genome comprises a homozygous endogenous immunoglobulin 7» light chain locus comprising: (i) human V7t gene segments VKS-SZ to W140 and VK3-27 to Vk3-1, (ii) human Dt-C?» gene segment pairs Ill-CM, JM-CXZ, Jl3-C7t3 and Jk6-C9t6, (iii) human IX gene segment M7, and (iv) three human immunoglobulin 7t light chain ers (or a human immunoglobulin 7» light chain enhancer having three sequence elements); wherein (i)-(iv) are operably linked to each other and (i)—(iii) are upstream (or 5’) of a non—human C?» gene segment, and wherein the endogenous immunoglobulin 9» light chain locus lacks an endogenous non-human immunoglobulin Ek2-4, the human Vk gene segments Vk5-52 to VM-40 and V76 -27 to Vk3—1 includes human non—coding DNA that naturally appears between the human V?» gene segments, the human Jl-Ck gene segments pairs Ill-CM, DtZ-CXZ, H3 -C}.3 and J7t6-C9t6 includes human non-coding DNA that naturally appears between the human Jk-Ck gene segments pairs, and the human J?» gene segment M7 es human non-coding DNA that lly appears am (or 5’) of human DJ.

In some certain embodiments of a provided non-human animal, a non—human C7» gene (or gene t) is a mouse CM gene (or gene segment). In some certain ments of a provided non-human animal, an nous immunoglobulin k light chain locus r ses endogenous non-human immunoglobulin A light chain enhancers E?» and Ek3-l. In some certain embodiments of 21 provided non-human animal, an endogenous immunoglobulin 9» light chain locus comprises a deletion of endogenous non- human VIZ-V13-J?t2-C7t2-J7t4P-C?t4P gene segments and VM-J7t3-J9t3P-C7t3 -J7tl gene segments.

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

In some embodiments, a non-human animal, non-human cell or non-human tissue as bed herein is provided for use in the manufacture of a medicament for the treatment, prevention or amelioration of a disease, disorder or condition.

In some embodiments, use of a non-human , non-human cell or non-human tissue as described herein in the manufacture and/or pment of a drug or e for use in medicine, such as use as a medicament, is provided.

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

In various embodiments, a provided non-human animal, man cell or non- human tissue as described herein is a rodent, rodent cell or rodent tissue; in some embodiments, a mouse, mouse cell or mouse tissue; in some embodiments, a rat, rat cell or rat tissue. In some embodiments, a mouse, mouse cell or mouse tissue as described herein ses a c background that includes a 129 strain, a BALE/c , a C57BL/6 strain, a mixed 129xC57BL/6 strain or combinations thereof.

As used in this application, the terms "about" and "approximately" are used as equivalents. Any numerals used in this application with or without about or approximately are meant to cover any normal fluctuations appreciated by one of ry skill in the relevant art.

BRIEF DESCRIPTION OF THE G The Drawing included herein, which is composed of the following Figures, is for illustration purposes only and not for limitation.

Figure 1 shows a schematic illustration, not to scale, of an exemplary strategy for uction of an engineered endogenous ng light chain locus in a rodent terized by the ce of a plurality of human V)», J9» and C?» coding sequences that are operably linked to each other and operably linked to a rodent C?» region (or rodent C7» gene). As depicted, five separate ing vectors (6286, 6571, 6596, 6597 and 6680) are shown with various amounts of genetic material from a human 1g?» light chain locus and are sequentially inserted into an endogenous rodent (e.g., mouse) ng light chain locus (shown at the top). A first targeting vector (6286) was inserted downstream of a rodent CM region and constructed to contain a modular human 1g?» enhancer (EX) region (or sequence) characterized by three sequence elements. A second targeting vector (6571) was inserted upstream of a rodent CM region and was engineered to contain five functional human V?» gene segments, four functional human Jk-C?» gene segment pairs and a human M7 gene segment (Human IM- Cll-JKZ-ClZ-JX3-Ck3 -J7t4-C7t4-J7t5-C?t5-J7t6-Cl6-J7t7). Third (6596) and fourth (6597) targeting vectors included further sets of additional human V?» gene ts (eleven and nine, respectively) that sequentially add to the total human V7t gene segment content of the endogenous mouse 1g?» light chain locus after successful targeting of the first targeting vector. Both ing vectors included regions of overlap (striped filled rectangles) on the 3’ ends to facilitate homologous recombination with the 5’ end of the preceding targeting vector once ated into the endogenous mouse 1g?» light chain locus. An alternative fifth targeting vector (6680) is also shown that has the same genetic material as the 6597 targeting vector except that this alternative targeting vector included a 5’ homology arm having a sequence that is identical to the sequence 5’ (or am) of a rodent V22 gene segment, thereby facilitating deletion of endogenous VIZ-V23—J?tZ-Ck2-Jl4P-Ck4P-E12V9tl-J9»3— J7t3P-C7t3-J7tl gene segments upon gous recombination with the targeting vector.

Unless otherwise indicated, closed symbols indicate rodent gene segments and/or sequences, while open s indicate human gene segments and/or sequences. Site—specific recombination recognition sites (eg, loxP, Frt) flanking ion cassettes (HYG: Hygromycin resistance gene [HYGR] under transcriptional control of a ubiquitin promoter; NEO: Neomycin resistance gene [NEOR] under transcriptional control of a ubiquitin promoter) are also shown, Selected nucleotide junction locations are marked with a line below each junction and each indicated by SEQ lD NO.

Figure 2 shows a schematic illustration, not to scale, of exemplary rodent 1g?» light chain alleles after tial insertion of targeting vectors described in Example 1. 6597 allele: an ng light chain allele that contains 25 functional human V?» gene segments, four functional human Dt-Ck gene t pairs and a human J27 gene segment operably linked to a rodent Ck region (e.g., a mouse CM region), and which lg?» light chain locus further includes endogenous VA-JK—C?» gene segments, three (i.e., E24, E and E3. 1) endogenous ng enhancer regions (or sequences) and a r human 1g?» enhancer region (or sequence) characterized by three ce elements. 6680 allele: an 1g?» light chain allele after site-specific on of endogenous Vl—JK—Ck gene segments and Ig7t enhancer E22-4, which 1g?» light chain allele contains 25 functional human V?» gene segments, four functional human Jl—C?» gene segment pairs and a human J27 gene segment operably linked to a rodent Cl region (e.g., a mouse CM region), which Ig7t light chain locus further includes two (i.e., E and E3. 1) endogenous 1g?» enhancer regions (or sequences) and a modular human 1g?» enhancer region (or sequence, see above). Unless otherwise indicated, closed symbols te rodent gene segments and/or sequences, while open symbols indicate human gene segments and/or sequences. Site-specific recombination recognition sites (e.g., Frt) flanking selection cassettes (HYG: Hygromycin resistance gene [HYGR] under transcriptional control of a ubiquitin er) are shown Dashed lines indicate deleted region between two illustrated 1g?» alleles. Selected nucleotide junction locations are marked with a line below each junction and each indicated by SEQ ID NO.

Figure 3 shows a schematic ration, not to scale, of an alternative exemplary gy for construction of an engineered endogenous 1g?» light chain locus in a rodent characterized by the presence of a plurality of human V7t, J7t and C7» coding sequences that are operably linked to each other and operably linked to a rodent Ck . As depicted, two different targeting vectors are shown with s amounts of genetic material from a human 1g?» light chain locus and are simultaneously inserted into an engineered rodent (e.g., mouse) 1g?» light chain locus (shown at the top) that contains five human V?» gene segments, a human JX—Ck cluster, and a mouse CM gene. The 6596 targeting vector is modified to remove a Neomycin selection te and incorporate overlapping sequences (striped filled rectangles) at the 5’and 3’ ends to provide regions of homology to facilitate recombination with a corresponding human ce. A second targeting vector is designed to contain an overlap region on the 3’end of the construct (striped filled gles) that shares sequence homology with the modified 6596 targeting vector (trimmed-6596 targeting vector), which facilitates homologous recombination with the 5’ end of the trimmed-6596 targeting vector.

These two targeting vectors e further sets of onal human V?» gene segments (eleven and nine, respectively) that sequentially add to the total human V1 gene segment content of the endogenous mouse ng light chain locus after successful targeting of a first targeting vector. The second targeting vector included a 5’ gy arm having a sequence that is identical to the ce 5’ (or upstream) of a rodent VXZ gene segment, thereby facilitating deletion of endogenous Viz-VB-J?t2-C?t2-J7t4P-C?t4P-E?t2-4—V?t1-J7t3 -J?t3P- Ck3-J7&1 gene segments upon homologous recombination with the targeting . The two targeting vectors are co-electroporated with guide RNAs (gRNA) to tate integration in the engineered ng light chain locus, which are marked with an arrow near each ce location and each indicated by SEQ ID NO. Unless otherwise indicated, closed symbols indicate rodent gene segments and/or sequences, while open symbols indicate human gene segments and/or sequences. Site-specific recombination recognition sites (e.g., loxP, Frt) flanking selection cassettes (HYG: Hygromycin resistance gene [HYGR] under transcriptional control of a tin promoter, NEO: Neomycin resistance gene [NEOR] under transcriptional control of a ubiquitin promoter) are also shown. Selected nucleotide junction locations are marked with a line below each junction and each indicated by SEQ ID Figure 4 shows a schematic illustration, not to scale, of wild-type and exemplary ered rodent 1g?» light chain alleles of rodents employed in the experiments described in Example 3. Wild-type allele: a wild—type mouse ng light chain locus (see also, e.g., Figure 2 of US. Patent No. 9,006,511); 6571 allele: an 1g?» light chain allele that contains 5 functional human V?» gene segments, four functional human Jk—C?» gene segment pairs and a human M7 gene segment operably linked to a rodent Ck region (e.g., a mouse CM region), and which Ig7t light chain locus further includes nous Vl—JK—Ck gene segments, three endogenous 1g?» enhancer regions (or sequences) and a modular human 1g?» enhancer region (or sequence, see above). 6597 : see above; 6680 allele: see above. Selected nucleotide junction locations are marked with a line below each junction and each indicated by SEQ ID Figures 5A and 5B show representative contour plots indicating single ated splenocytes (A) showing sion of CD19 (y-axis) and CD3 (x-axis), and absolute cell number per spleen (B) ted from mice gous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT).

Figures 6A and 6B show representative contour plots indicating mature and transitional B cells in splenocytes gated on CD19+ (A) showing expression of IgD (y-axis) and IgM (x-axis), and absolute cell number per spleen (B) harvested from mice homozygous for insertion of the 6680 targeting vector O) and wild-type littermates (WT). Specific B cell subpopulations are noted on each dot plot (e.g., , transitional).

Figures 7A and 7B show representative r plots indicating mouse 1g?» (mng, y-axis), mouse IgK (mIgK, x-axis) or human ng (h1g2, y-axis) expression in CD19+— gated splenocytes harvested from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT).

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

Figures 9A and 9B show representative r plots indicating gM1°WB220mt-gated bone marrow (A) showing expression of c-kit (y-axis) and CD43 s), and absolute cell number per femur (B) harvested from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT). Specific B cell subpopulations are noted on each dot plot (e.g, pro-B, . s 10A and 10B show representative contour plots indicating CD19+-gated bone marrow (A) showing expression of IgM (y-axis) and B220 (x-axis), and absolute cell number per femur (B) harvested from mice homozygous for insertion of the 6680 ing vector (6680HO) and wild-type littermates (WT). Specific B cell subpopulations are noted on each dot plot (eg, re, mature, pre- and pro-B).

Figures 11A and 11B show representative contour plots indicating immature bone marrow (CD19+IgM+B220m‘-gated) showing expression of mouse Ig2 (mIg2, y-axis), mouse IgK (mIgK, x-axis) or human Ig2 (h1g2, ) from mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild—type littermates (WT). s 12A and 12B show representative contour plots indicating mature bone marrow (CD19+IgM+B220+-gated) showing expression of mouse Ig2 (mlg2, ), mouse IgK (mIgK, x-axis) or human Ig2 (h1g2, y-axis) from mice homozygous for insertion of the 6680 targeting vector O) and wild-type mates (WT).

Figure 13 shows representative mean percent of IgK-expressing (% K C) and human pressing (% hum 2 C) B cells in spleen, immature bone marrow (immature BM) and mature bone marrow (mature BM) from selected engineered mouse strains as described herein. Data is presented as mean values with standard deviation also indicated. 6680HO/VI HO/Adam6 HO: an engineered mouse strain ning a gous engineered Ig2 light chain locus designed to contain 25 functional human V2 gene segments, four functional human J2—C2 gene segment pairs and a human J27 gene segment operably linked to a rodent C2 region (e.g., a mouse C2l region), which Ig2 light chain locus further includes two nous Ig2 enhancer regions (or sequences) and a modular human Ig2 enhancer region (or sequence, see above); and homozygous humanized IgH and IgK loci, which homozygous humanized IgH locus contained an inserted rodent Adam6-encoding sequence (see, e.g., US. Patent Nos. 8,642,835 and 8,697,940; hereby incorporated by reference in their entireties), 6889HO/VI HO/Adam6 HO: an engineered mouse strain containing a gous engineered Ig2 light chain locus containing 25 functional human V2 gene segments, four functional human J2—C2 gene segment pairs and a human J27 gene segment operably linked to a rodent C2 region (e.g., a mouse C21 region), which Ig2 light chain locus further includes two endogenous Ig2 enhancer regions (or sequences) and a modular human 1g?» enhancer region (or ce, see above); and homozygous humanized IgH and IgK loci, which homozygous humanized IgH locus contained an inserted rodent Adam6-encoding sequence (see, e.g., US. Patent Nos. 8,642,835 and 8,697,940, hereby incorporated by reference in their entireties). Number of mice for each genotype cohort shown included at least three and up to eight animals per group. s 14A and 14B show representative immunoblots (Western blots) of SDS- PAGE under ducing conditions using serum isolated from engineered mice homozygous for insertion of the 6680 targeting vector (6680HO) and wild-type littermates (WT) indicating expression of mouse (B, right image) or human (A, left image) 7» light chains, each sample was loaded into lanes at a volume of 1,5ul serum. PHS: pooled human serum at a volume of 0.25ul (Labquip Ltd Cat#9lOlA). Molecular weights in Kd are indicated on the right of each gel image.

Figure 15A shows representative human V7» (top) and human J9» (bottom) gene segment usage in human Ck—pn’med sequences amplified from RNA isolated from splenocytes harvested from T mice (n=5).

Figure 15B shows representative human V?» gene segment usage in mouse C?»- primed sequences ed from RNA isolated from splenocytes harvested from 6889HET mice (n=5).

Figure 15C shows representative human V?» (top) and human J2» (bottom) gene segment usage in human Cit-primed sequences amplified from RNA isolated from splenocytes harvested from 6889HO/VI HO/Adam6 HO mice (n=6).

Figure 15D shows representative human Vk gene t usage in mouse Ck- primed sequences amplified from RNA isolated from splenocytes ted from 6889HO/VI m6 HO mice (n=6).

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

Figures 17A—C show representative human 7» light chain (h1g2, left), mouse A light chain (mIgX, middle) and mouse K light chain (mIgK, right) titers in antigen-specific IgG in serum at days 0 and 22 collected from immunized mice heterozygous for insertion of the 6597 ET, n=6) or 6680 (6680HET, n=6) targeting vectors and immunized wild- type controls (WT, n=6).

Figures 18A and 18B show representative contour plots indicating single cell- gated splenocytes (left) showing expression of CD19 (y-axis) and CD3 (x-axis), and total B cells per spleen (right) harvested from mice homozygous for insertion of the 6889 targeting vector (6889HO VI HO Adam6 HO) and reference engineered mice (VI). 6889HO/VI HO/Adam6 HO: see above; VI: an engineered mouse strain containing homozygous zed IgH and IgK loci, which gous zed IgH locus contained an inserted rodent Adam6-encoding sequence (see, e.g., US. Patent Nos. 8,642,835 and 8,697,940; hereby incorporated by reference in their entireties). Live single-cell splenocytes were defined by viability staining (Thermo Fisher).

Figure 19 shows representative contour plots indicating human ng (h1g2, y-axis) and mouse IgK (mIgK, ) expression in CD19+-gated cytes harvested from mice homozygous for insertion of the 6889 targeting vector (6889HO VI HO Adam6 HO) and reference engineered mice (VI). 6889HO/VI HO/Adam6 HO: see above; VI: see above.

Figure 20 shows entative contour plots indicating single cell-gated lymphocytes from bone marrow g expression of IgM (y-axis) and B220 s) harvested from femurs of mice homozygous for insertion of the 6889 targeting vector (6889HO VI HO Adam6 HO) and reference engineered mice (VI). 6889HO/VI HO/Adam6 HO: see above; VI: see above. Immature and mature B cell subpopulations are noted on each contour plot.

Figure 21 shows representative contour plots indicating immature IgM+B220m‘-gated, left column) and mature (CD19+IgM+B220+-gated, right column) bone marrow showing expression of human Ig7t (hlgk, y-axis) and mouse IgK (mIgK, x-axis) from mice homozygous for insertion of the 6889 targeting vector (6889HO VI HO Adam6 HO) and reference engineered mice (VI). 6889HO/VI HO/Adam6 HO: see above; VI: see above.

BRIEF DESCRIPTION OF SELECTED SEQUENCES IN THE SEQUENCE LISTING Mouse CM DNA (SEQ ID NO:1): GCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCTCTGAAGAGCT CGAGACTAACAAGGCCACACTGGTGTGTACGATCACTGATTTCTACCCAGGTGT AGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGTATGGAGAC AACCCAGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGAC CCTGACAGCAAGAGCATGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCA TGAAGGTCACACTGTGGAGAAGAGTTTGTCCCGTGCTGACTGTTCC ] Mouse CM amino acid (SEQ ID NO:2): GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQ PSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS Mouse CAZ DNA (SEQ ID NO:3): GTCAGCCCAAGTCCACTCCCACTCTCACCGTGTTTCCACCTTCCTCTGAGGAGCT CAAGGAAAACAAAGCCACACTGGTGTGTCTGATTTCCAACTTTTCCCCGAGTGG TGTGACAGTGGCCTGGAAGGCAAATGGTACACCTATCACCCAGGGTGTGGACAC TTCAAATCCCACCAAAGAGGGCAACAAGTTCATGGCCAGCAGCTTCCTACATTT GACATCGGACCAGTGGAGATCTCACAACAGTTTTACCTGTCAAGTTACACATGA AGGGGACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTCTC Mouse CKZ amino acid (SEQ ID NO:4): GQPKSTPTLTVFPPSSEELKENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNP FMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL Mouse C23 DNA (SEQ ID NO:5): GTCAGCCCAAGTCCACTCCCACACTCACCATGTTTCCACCTTCCCCTGAGGAGCT CCAGGAAAACAAAGCCACACTCGTGTGTCTGATTTCCAATTTTTCCCCAAGTGGT GTGACAGTGGCCTGGAAGGCAAATGGTACACCTATCACCCAGGGTGTGGACACT TCAAATCCCACCAAAGAGGACAACAAGTACATGGCCAGCAGCTTCTTACATTTG GACCAGTGGAGATCTCACAACAGTTTTACCTGCCAAGTTACACATGAA GGGGACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTCTC Mouse Ck3 amino acid (SEQ ID NO:6): GQPKSTPTLTMFPPSPEELQENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNP TKEDNKYMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL Rat CM DNA (SEQ ID NO:7): GTCAGCCCAAGTCCACTCCCACACTCACAGTATTTCCACCTTCAACTGAGGAGCT CCAGGGAAACAAAGCCACACTGGTGTGTCTGATTTCTGATTTCTACCCGAGTGAT GTGGAAGTGGCCTGGAAGGCAAATGGTGCACCTATCTCCCAGGGTGTGGACACT GCAAATCCCACCAAACAGGGCAACAAATACATCGCCAGCAGCTTCTTACGTTTG ACAGCAGAACAGTGGAGATCTCGCAACAGTTTTACCTGCCAAGTTACACATGAA GGGAACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTGTC Rat CM amino acid (SEQ ID NO:8): GQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKANGAPISQGVDTAN PTKQGNKYIASSFLRLTAEQWRSRNSFTCQVTHEGNTVEKSLSPAECV Rat CA2 DNA (SEQ ID NO:9): ACCAACCCAAGGCTACGCCCTCAGTCACCCTGTTCCCACCTTCCTCTGAAGAGCT CAAGACTGACAAGGCTACACTGGTGTGTATGGTGACAGATTTCTACCCTGGTGTT ATGACAGTGGTCTGGAAGGCAGATGGTACCCCTATCACTCAGGGTGTGGAGACT ACCCAGCCTTTCAAACAGAACAACAAGTACATGGCTACCAGCTACCTGCTTTTG ACAGCAAAAGCATGGGAGACTCATAGCAATTACAGCTGCCAGGTCACTCACGAA GAGAACACTGTGGAGAAGAGTTTGTCCCGTGCTGAGTGTTCC ] Rat CKZ amino acid (SEQ ID NO:10): DQPKATPSVTLFPPSSEELKTDKATLVCMVTDFYPGVMTVVWKADGTPITQGVETT NKYMATSYLLLTAKAWETHSNYSCQVTHEENTVEKSLSRAECS Rat C2t3 DNA (SEQ ID NO:11): GTCAGCCCAAGTCCACTCCCACACTCACAGTATTTCCACCTTCAACTGAGGAGCT CCAGGGAAACAAAGCCACACTGGTGTGTCTGATTTCTGATTTCTACCCGAGTGAT GTGGAAGTGGCCTGGAAGGCAAATGGTGCACCTATCTCCCAGGGTGTGGACACT GCAAATCCCACCAAACAGGGCAACAAATACATCGCCAGCAGCTTCTTACGTTTG ACAGCAGAACAGTGGAGATCTCGCAACAGTTTTACCTGCCAAGTTACACATGAA GGGAACACTGTGGAAAAGAGTCTGTCTCCTGCAGAGTGTGTC Rat CA3 amino acid (SEQ ID : GQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKANGAPISQGVDTAN KYIASSFLRLTAEQWRSRNSFTCQVTHEGNTVEKSLSPAECV Rat CM DNA (SEQ ID NO:13): ACCAACCCAAGGCTACGCCCTCAGTCACCCTGTTCCCACCTTCCTCTGAAGAGCT CAAGACTGACAAGGCTACACTGGTGTGTATGGTGACAGATTTCTACCCTGGTGTT ATGACAGTGGTCTGGAAGGCAGATGGTACCCCTATCACTCAGGGTGTGGAGACT ACCCAGCCTTTCAAACAGAACAACAAGTACATGGCTACCAGCTACCTGCTTTTG ACAGCAAAAGCATGGGAGACTCATAGCAATTACAGCTGCCAGGTCACTCACGAA GAGAACACTGTGGAGAAGAGTTTGTCCCGTGCTGAGTGTTCC ] Rat CIA amino acid (SEQ ID NO:14): DQPKATPSVTLFPPSSEELKTDKATLVCMVTDFYPGVMTVVWKADGTPITQGVETT QPFKQNNKYMATSYLLLTAKAWETHSNYSCQVTHEENTVEKSLSRAECS DEFINITIONS The scope of the present invention is defined by the claims appended hereto and is not limited by certain embodiments described herein; those skilled in the art, reading the present specification, will be aware of various modifications that may be equivalent to such bed embodiments, or otherwise within the scope of the claims.

In general, terms used herein are in accordance with their understood meaning in the art, unless clearly indicated otherwise. it definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context. Additional definitions for the following and other terms are set forth throughout the specification. Patent and non- patent literature references cited within this cation, or relevant portions thereof, are incorporated herein by reference in their entireties.

Administration: as used herein, includes the administration of a composition to a subject or system (e. g., to a cell, organ, tissue, organism, or relevant component or set of components thereof). The skilled artisan will appreciate that route of administration may vary depending, for example, on the subject or system to which the composition is being administered, the nature of the ition, the purpose of the administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., to a human or a rodent) may be bronchial (including by bronchial lation), buccal, enteral, interdermal, intra-arterial, ermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and/or vitreal. In some embodiments, stration may involve intermittent dosing. In some ments, administration may involve continuous dosing (e.g., perfusion) for at least a ed period of time.

Amelioration: as used herein, includes the prevention, reduction or palliation of a state, or improvement of the state of a subject. Amelioration includes but does not require complete ry or complete prevention of a disease, er or condition.

Approximately: as applied to one or more values of interest, includes to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Biologically active: as used herein, refers to a characteristic of any agent that has activity in a biological system, in vitro or in vivo (e.g., in an sm). For instance, an agent that, when present in an sm, has a biological effect within that organism is considered to be biologically active. In particular ments, where a protein or polypeptide is biologically active, a n of that n or polypeptide that shares at least one ical activity of the protein or polypeptide is typically referred to as a "biologically active" portion.

] Comparable: as used herein, refers to two or more agents, entities, situations, sets of conditions, etc. that may not be identical to one another but that are sufficiently similar to permit comparison there between so that conclusions may reasonably be drawn based on differences or similarities ed. Persons of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, es, situations, sets of conditions, etc. to be considered comparable.

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

Control: as used herein, refers to the art—understood meaning of a "control" being a standard against which results are compared. Typically, controls are used to augment ity in experiments by isolating variables in order to make a conclusion about such variables. In some embodiments, a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparator. A "control" also includes a "control animal." A "control animal" may have a modification as described herein, a modification that is ent as described , or no ation (i.e., a wild- type animal). In one experiment, a "test" (i.e., a variable being tested) is applied. In a second experiment, the "control," the variable being tested is not applied. In some ments, a control is a historical control (i.e., of a test or assay performed previously, or an amount or result that is previously known). In some embodiments, a control is or comprises a printed or otherwise saved record. A control may be a positive control or a negative control.

Derivedfrom: when used ning a rearranged variable region gene or a variable domain "derived from" an unrearranged variable region and/or unrearranged variable region gene segments refers to the ability to trace the sequence of the rearranged variable region gene or le domain back to a set of unrearranged variable region gene segments that were rearranged to form the rearranged variable region gene that expresses the le domain (accounting for, where applicable, splice differences and somatic mutations). For e, a nged variable region gene that has undergone somatic mutation does not change the fact that it is d from the unrearranged variable region gene segments.

Disruption: as used , refers to the result of a gous recombination event with a DNA molecule (e.g., with an endogenous homologous sequence such as a gene or gene locus). In some embodiments, a disruption may e or represent an insertion, deletion, substitution, replacement, missense mutation, or a frame-shift of a DNA ce(s), or any combination thereof. Insertions may include the insertion of entire genes or gene fragments, e. g., exons, which may be of an origin other than the endogenous sequence (e.g., a heterologous sequence). In some embodiments, a tion may increase expression and/or activity of a gene or gene product (e.g., of a polypeptide encoded by a gene). In some embodiments, a disruption may decrease expression and/or activity of a gene or gene product. In some embodiments, a disruption may alter sequence of a gene or an encoded gene product (e. g., an encoded polypeptide). In some embodiments, a disruption may truncate or fragment a gene or an encoded gene product (e.g, an encoded polypeptide).

In some ments, a disruption may extend a gene or an encoded gene product. In some such embodiments, a disruption may achieve assembly of a fusion polypeptide. In some embodiments, a disruption may affect level, but not activity, of a gene or gene product. In some embodiments, a disruption may affect activity, but not level, of a gene or gene product.

In some embodiments, a disruption may have no cant effect on level of a gene or gene product. In some embodiments, a disruption may have no significant effect on activity of a gene or gene product. In some ments, a disruption may have no significant effect on either level or activity of a gene or gene product.

Determining, measuring, evaluating, ing, ng and analyzing: are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations, ng may be relative or absolute. "Assayingfor the presence of’ can be determining the amount of something present and/or determining whether or not it is t or absent.

Endogenous locus or endogenous gene: as used herein, refers to a genetic locus found in a parent or reference organism prior to uction of a disruption, deletion, replacement, alteration, or modification as described herein. In some embodiments, an endogenous locus has a sequence found in nature. In some ments, an endogenous locus is a ype locus. In some embodiments, an endogenous locus is an engineered locus. In some embodiments, a reference organism is a wild-type organism. In some embodiments, a reference organism is an engineered organism. In some embodiments, a nce sm is a laboratory-bred organism (whether wild-type or engineered). nous promoter: as used herein, refers to a promoter that is naturally associated, e.g., in a wild-type organism, with an endogenous gene.

Engineered: as used herein refers, in general, to the aspect of having been manipulated by the hand of man. For example, in some embodiments, a polynucleotide may be considered to be "‘engineerecl’ when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide. In some particular such embodiments, an engineered polynucleotide may comprise a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Alternatively, or additionally, in some embodiments, first and second nucleic acid sequences that each encode polypeptide elements or domains that in nature are not linked to one another may be linked to one another in a single engineered polynucleotide, Comparably, in some ments, a cell or sm may be considered to be "engineered" if it has been manipulated so that its genetic information is altered (e. g., new genetic material not previously present has been uced, or previously present genetic material has been altered or removed). As is common practice and is understood by persons of skill in the art, progeny of an engineered polynucleotide or cell are typically still ed to as "engineered" even though the actual manipulation was performed on a prior entity. Furthermore, as will be appreciated by persons of skill in the art, a variety of ologies are available through which "engineering" as described herein may be achieved. For e, in some embodiments, eering" may involve selection or design (e.g., of nucleic acid sequences, polypeptide sequences, cells, tissues, and/or organisms) through use of computer systems mmed to perform analysis or comparison, or otherwise to analyze, recommend, and/or select sequences, alterations, etc).

Alternatively, or onally, in some embodiments, "engineering" may involve use of in vitro chemical synthesis methodologies and/or recombinant nucleic acid technologies such as, for example, nucleic acid amplification (e. g., via the polymerase chain reaction) hybridization, mutation, transformation, transfection, etc, and/or any of a variety of controlled mating methodologies, As will be appreciated by those skilled in the art, a variety of established such techniques (eg., for recombinant DNA, oligonucleotide synthesis, and tissue e and transformation (e.g., electroporation, lipofection, etc.) are well known in the art and descn'bed in various general and more specific references that are cited and/or discussed hout the present specification. See e.g., ok et al., Molecular Cloning: A tory Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring , NY, 1989 and Principles of Gene Manipulation: An Introduction to Genetic Manipulation, 5th Ed, ed. By Old, R.W. and SB. Primrose, Blackwell Science, Inc, 1994.

Functional: as used herein, refers to a form or fragment of an entity (e.g., a gene or gene segment) that exhibits a particular property (e.g., forms part of a coding sequence) and/or activity. For e, in the context of immunoglobulins, variable domains are encoded by unique gene segments (i.e., V, D and/or J) that are assembled (or recombined) to form functional coding ces. When present in the genome, gene segments are organized in clusters, although variations do occur. A ional" gene segment is a gene segment represented in an expressed ce (i.e., a variable domain) for which the corresponding genomic DNA has been isolated (i.e., ) and identified by sequence.

Some immunoglobulin gene segment sequences contain open reading frames and are considered functional although not represented in an expressed repertoire, while other WO 28691 immunoglobulin gene segment ces contain mutations (e.g., point mutations, insertions, deletions, etc.) ing in a stop codon and/or truncated sequence which subsequently render(s) such gene segment sequences unable to perform the property/ies and/or activity/ies associated with a non-mutated sequence(s). Such sequences are not represented in expressed sequences and, therefore, categorized as pseudogenes.

Gene: as used herein, refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and/or a polypeptide product). In some embodiments, a gene includes coding sequence (i.e., ce that encodes a particular product). In some embodiments, a gene includes non—coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequence. In some embodiments, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences that, for example, may l or impact one or more aspects of gene sion (e,g, cell-type-specific sion, inducible expression, etc.) For the purpose of y, we note that, as used in the present disclosure, the term "gene" generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof, the term may optionally encompass regulatory sequences, as will be clear from context to those of ordinary skill in the art. This definition is not intended to exclude application of the term "gene" to non-protein-coding expression units but rather to clarify that, in most cases, the term as used in this document refers to a polypeptide-coding c acid.

Heterologous: as used herein, refers to an agent or entity from a different source.

For example, when used in reference to a polypeptide, gene, or gene product present in a ular cell or organism, the term clarifies that the relevant polypeptide, gene, or gene product: 1) was engineered by the hand of man; 2) was introduced into the cell or organism (or a precursor thereof) through the hand of man (e.g., via genetic engineering), and/or 3) is not naturally ed by or present in the relevant cell or organism (e.g., the relevant cell type or organism type). "Heterologous" also es a polypeptide, gene or gene product that is normally present in a particular native cell or organism, but has been altered or modified, for example, by on or placement under the control of non-naturally associated and, in some embodiments, non-endogenous regulatory elements (e.g, a promoter).

Host cell: as used herein, refers to a cell into which a nucleic acid or protein has been introduced. s of skill upon g this disclosure will understand that such terms refer not only to the particular subject cell, but also is used to refer to the progeny of such a cell. Because certain ations may occur in succeeding generations due to either mutation or environmental ces, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the phrase "host cell." In some embodiments, a host cell is or comprises a prokaryotic or eukaryotic cell. In general, a host cell is any cell that is suitable for receiving and/or producing a heterologous nucleic acid or n, less of the Kingdom of life to which the cell is designated. ary cells include those of prokaryotes and otes (single-cell or multiple-cell), bacterial cells (e.g., strains ofEscherichia coli, us Spp., Streptomyces Spp., etc), mycobacteria cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, Pichia methanolica, etc), plant cells, insect cells (cg, SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc), non—human animal cells, human cells, or cell fusions such as, for example, omas or quadromas, In some embodiments, a cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, a cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO Kl, DXB-ll CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, W138, MRC 5, C010205,HB 8065, HL—60, (e.g., BPH<21), Jurkat, Daudi, A431 (epidermal), CV-l, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell. In some embodiments, a cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e. g., a PER.C6® cell). In some ments, a host cell is or comprises an isolated cell. In some ments, a host cell is part of a tissue. In some embodiments, a host cell is part of an organism.

Identity: as used herein in connection with a comparison of sequences, refers to identity as determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity. In some embodiments, identities as described herein are determined using a ClustalW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnet similarity matrix (MACVECTORTM 10.0.2, MacVector Inc, 2008).

In vitro: as used herein refers to events that occur in an ial environment, e. g., in a test tube or reaction vessel, in cell culture, etc, rather than within a multi-cellular organism.

In viva: as used herein refers to events that occur within a multi-cellular organism, such as a human and/or a non—human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

Isolated: as used herein, refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, ed, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other ents with which they were initially associated. In some embodiments, isolated agents are separated from 10% to 100%, 15%-100%, 20%-100%, 25%-100%, 30%- 100%, 35%-100%, 40%-100%, 45%-100%, 50%-100%, 55%-100%, 60%—100%, 65%- 100%, 70%-100%, 75%—100%, 80%-100%, 85%—100%, 0%, 95%—100%, 96%- 100%, 97%-100%, 98%-100%, or 99%-100% of the other ents with which they were initially associated. In some embodiments, isolated agents are separated from 10% to 100%, %-99%, 10%-98%, 10%-97%, 10%-96%, 10%-95%, 10%-90%, %, 10%-80%, %-75%, 10%-70%, 10%-65%, 10%-60%, 10%-55%, 10%-50%, 10%-45%, %, %-3 5%, 10%-30%, 10%-25%, 10%-20%, or % of the other components with which they were initially associated. In some ments, isolated agents are separated from 11% to 99%, 12%-98%, 13%-97%, 14%-96%, 15%-95%, 20%-90%, 25%-85%, 30%— 80%, 35%-75%, 40%-70%, 45%-65%, 50%-60%, or 55%—60% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. In some embodiments, isolated agents are 80%—99%, %, 90%—99%, 95%—99%, 96%—99%, 97%-99%, or 98%-99% pure. In some ments, isolated agents are 80%-99%, 80%— 98%, 80%-97%, 80%-96%, 80%-95%, %, or 80%—85% pure. In some embodiments, isolated agents are 85%—98%, 90%—97%, or 95%—96% pure. In some embodiments, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be tood by those skilled in the art, a substance may still be considered "isolated" or even , after having been combined with n other components such as, for example, one or more carriers or excipients (e.g., , solvent, water, etc), in such embodiments, percent ion or purity of the substance is ated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polynucleotide that occurs in nature is ered to be "isolated" when: a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in ; or c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide that is ally synthesized, or is synthesized in a cellular system ent from that which produces it in nature, is considered to be an ted" polypeptide. Alternatively, or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components: a) with which it is associated in nature; and/or b) with which it was associated when initially produced.

Locus or loci: as used , refers to a specific location(s) of a gene (or significant sequence), DNA sequence, polypeptide-encoding sequence, or position on a some of the genome of an organism. For example, an "immunoglobulin locus" may refer to the specific location of an immunoglobulin gene segment (e.g., V, D, J or C), immunoglobulin gene segment DNA sequence, immunoglobulin gene segment-encoding sequence, or immunoglobulin gene t position on a chromosome of the genome of an organism that has been identified as to where such a ce resides. An "immunoglobulin locus" may comprise a regulatory element of an immunoglobulin gene segment, including, but not limited to, an enhancer, a promoter, 5’ and/or 3’ regulatory sequence or region, or a combination thereof. An "immunoglobulin locus" may comprise DNA that normally resides between gene segments in a wild—type locus, but the DNA itself lacks an immunoglobulin gene segment (e.g, an immunoglobulin DNA sequence that naturally s between a group of V gene segments and a group of J gene segments, an immunoglobulin DNA sequence that naturally resides between a group of I gene segments and a constant region gene, or an immunoglobulin DNA sequence that naturally resides 3’ of a constant region gene). Persons of ordinary skill in the art will appreciate that somes may, in some embodiments, contain hundreds or even thousands of genes and demonstrate physical oo— localization of similar genetic loci when comparing between different species. Such genetic loci can be described as having shared synteny.

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

Nucleic acid: as used herein, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain, In some embodiments, a "nucleic acid" is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e,g, nucleotides and/or nucleosides), in some embodiments, ic acid’ refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a "nucleic acid’ is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a "nucleic acid" is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a ic acid’ in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a "nucleic acid" is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone. Alternatively, or additionally, in some embodiments, a "nucleic acid" has one or more orothioate and/or 5’-N-phosphorarnidite linkages rather than phosphodiester bonds. In some embodiments, a ic acid" is, comprises, or ts of one or more natural nucleosides (e.g., adenosine, ine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a "nucleic acid" is, comprises, or consists of one or more side analogs (e.g, 2-aminoadenosine, 2—thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 yl-uridine, 2-aminoadenosine, CS—bromouridine, CS—fluorouridine, C5— idine, pynyl—uridine, C5-propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, denosine, 8-oxoguanosine, O(6)—methylguanine, 2—thiocytidine, methylated bases, intercalated bases, and combinations f). In some embodiments, a "nucleic acid" comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, ose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a "nucleic acid" has a nucleotide sequence that s a functional gene t such as an RNA or ptide. In some embodiments, a "nucleic acid’ includes one or more introns. In some embodiments, a "nucleic acid" es one or more exons. In some embodiments, a ic acid" is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in Vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a "nucleic acid’ is at least, e.g., but not limited to, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long, In some embodiments, a "nucleic acid" is single stranded, in some embodiments, a "nucleic acid" is double stranded. In some embodiments, a "nucleic acid" has a nucleotide sequence comprising at least one element that encodes, or is the ment of a sequence that encodes, a polypeptide. In some embodiments, a ic acid’ has enzymatic activity.

Operably linked: as used herein, refers to a juxtaposition wherein the components described are in a relationship ting them to function in their intended manner For example, unrearranged variable region gene segments are "operably ’ to a contiguous constant region gene if the unrearranged variable region gene segments are e of rearranging to form a rearranged variable region gene that is expressed in conjunction with the constant region gene as a polypeptide chain of an antigen binding protein. A control sequence "operably " to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

"Operably linked" ces include both expression control sequences that are contiguous with a gene of interest and expression l sequences that act in trans or at a distance to control a gene of interest (or sequence of interest). The term "expression control sequence" includes polynucleotide sequences, which are necessary to affect the expression and processing of coding ces to which they are ligated, "Expression control sequences" include: appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance polypeptide stability; and when desired, sequences that enhance polypeptide secretion. The nature of such control sequences differs ing upon the host organism. For example, in prokaryotes, such control sequences generally include promoter, ribosomal binding site and transcription ation sequence, while in eukaryotes typically such control sequences include promoters and transcription termination sequence. The term "control sequences" is ed to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

Physiological conditions: as used herein, refers to its art-understood meaning referencing conditions under which cells or organisms live and/or reproduce. In some embodiments, the term includes conditions of the external or internal milieu that may occur in nature for an organism or cell system. In some embodiments, physiological ions are those ions present within the body of a human or non-human animal, especially those ions present at and/or within a surgical site. logical conditions typically include, e.g., a temperature range of 20-400C, atmospheric pressure of 1, pH of 6-8, glucose tration of 1-20mM, oxygen concentration at atmospheric levels, and gravity as it is encountered on earth. In some embodiments, conditions in a laboratory are manipulated and/or maintained at physiologic conditions. In some embodiments, physiological conditions are tered in an organism.

Polypeptide: as used herein, refers to any polymeric chain of amino acids. In some ments, a polypeptide has an amino acid ce that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in . In some embodiments, a polypeptide has an amino acid sequence that contains ns that occur in nature separately from one another (ie., from two or more different organisms, for example, human and non-human portions). In some embodiments, a ptide has an amino acid sequence that is engineered in that it is designed and/or ed through action of the hand of man. In some embodiments, a polypeptide has an amino acid ce encoded by a sequence that does not occur in nature (e.g., a sequence that is engineered in that it is designed and/or produced through action of the hand of man to encode said polypeptide).

Recombinant: as used herein, is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell, polypeptides ed from a recombinant, combinatorial human polypeptide library nboom, H. R., 1997, TIB Tech. 15:62-70, Azzazy, H. and WE. Highsmith, 2002, Clin. m. 35:425-45; Gavilondo, J. V. and J.W. Larrick, 2002, BioTechniques 29:128- 45; Hoogenboom H., and P. Chames, 2000, Immunol. Today 21:371-8), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D, et a1, 1992, Nucl. Acids Res. 20:6287-95, Kellermann, S-A. and LL. Green, 2002, Curr. Opin. Biotechnol. 13:593-7, , M. et al., 2000, Immunol. Today 21:364-70; Osborn, MJ. et al., 2013, J. Immunol. 190:1481-90; Lee, E—C. et al., 2014, Nat. Biotech. 356-63, Macdonald, L.E. et al., 2014, Proc. Natl. Acad. Sci. USA, 111(14):5147-52, , A]. et a1., 2014, Proc. Natl. Acad. Sci. USA. 111(14):5153-8) or polypeptides prepared, sed, created or isolated by any other means that involves splicing selected sequence ts to one r. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in Silica. In some embodiments, one or more such selected sequence elements result from mutagenesis (eg, in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source. For example, in some embodiments, a inant polypeptide is comprised of sequences found in the genome of a source organism of interest (e.g., human, mouse, etc). In some embodiments, a recombinant polypeptide has an amino acid sequence that resulted from mutagenesis (e.g., in vitro or in vivo, for example, in a non-human animal), so that the amino acid sequences of the inant ptides are sequences that, while originating from and related to polypeptides sequences, may not naturally exist within the genome of a non-human animal in vivo.

Reference: as used herein, refers to a standard or control agent, animal, cohort, individual, population, sample, sequence or value t which an agent, animal, cohort, individual, population, sample, sequence or value of interest is compared. In some embodiments, a reference agent, animal, cohort, individual, population, sample, sequence or value is tested and/or determined substantially simultaneously with the testing or determination of an agent, animal, cohort, individual, population, sample, sequence or value of interest. In some embodiments, a reference agent, animal, cohort, individual, population, , sequence or value is a historical reference, optionally embodied in a tangible . In some embodiments, a reference may refer to a control. A "reference" also es a "reference animal." A "reference " may have a modification as bed herein, a modification that is different as described herein or no modification (i.e., a wild- type animal). Typically, as would be understood by persons of skill in the art, a reference agent, animal, cohort, individual, population, sample, sequence or value is determined or charactenzed under conditions comparable to those utilized to determine or characterize an agent, animal (e.g., a mammal), cohort, individual, tion, sample, sequence or value of interest.

Replacement: as used herein, refers to a process through which a "replaced" nucleic acid sequence (e.g., a gene) found in a host locus (e.g., in a genome) is d from that locus, and a different, "replacement" nucleic acid is d in its place. In some embodiments, the replaced nucleic acid sequence and the replacement nucleic acid sequences are comparable to one another in that, for example, they are homologous to one another and/or contain corresponding elements (eg, protein-coding elements, regulatory elements, etc). In some embodiments, a replaced nucleic acid sequence includes one or more of a promoter, an enhancer, a splice donor site, a splice acceptor site, an intron, an exon, an untranslated region (UTR), in some embodiments, a replacement nucleic acid ce includes one or more coding sequences. In some embodiments, a replacement nucleic acid sequence is a g or t (e. g., mutant) of the replaced nucleic acid sequence. In some embodiments, a replacement nucleic acid sequence is an ortholog or homolog of the replaced sequence. In some embodiments, a replacement nucleic acid sequence is or comprises a human nucleic acid sequence, In some embodiments, including where the replacement nucleic acid ce is or comprises a human nucleic acid sequence, the replaced nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse or rat sequence). In some embodiments, ing where the replacement nucleic acid sequence is or comprises a human nucleic acid sequence, the ed nucleic acid ce is or comprises a human sequence. In some embodiments, a replacement nucleic acid sequence is a variant or mutant (i.e., a sequence that contains one or more sequence differences, e.g, substitutions, as compared to the replaced sequence) of the replaced ce. The c acid sequence so placed may include one or more tory sequences that are part of source nucleic acid sequence used to obtain the sequence so placed (e.g., promoters, enhancers, 5'— or 3'-untranslated regions, etc). For example, in various embodiments, a ement is a substitution of an endogenous sequence with a heterologous sequence that results in the production of a gene product from the nucleic acid sequence so placed (comprising the heterologous sequence), but not expression of the endogenous sequence; a replacement is of an endogenous genomic sequence with a nucleic acid sequence that encodes a ptide that has a similar function as a polypeptide d by the endogenous sequence (e.g., the endogenous genomic sequence encodes a non-human variable domain polypeptide, in whole or in part, and the DNA fragment encodes one or more human variable domain polypeptides, in whole or in part). In s embodiments, an endogenous non-human immunoglobulin gene segment or fragment thereof is replaced with a human immunoglobulin gene segment or fragment f. ] ntially: as used herein, refers to the ative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest, One of ry skill in the biological alts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of teness inherent in many biological and chemical phenomena.

Substantial gy: as used herein, refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues in corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues with appropriately similar structural and/or functional teristics. For e, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or as having "polar" or "non-polar" side . Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution. Typical amino acid categorizations are summarized in the table below. e Ala A ar Neutral 1 .8 Arginine Arg R Polar Positive -4.5 Asparagine Asn N Polar Neutral -3.5 Aspartic acid Asp D Polar Negative -3.5 Cysteine Cys C Nonpolar Neutral 2.5 Glutamlc Glu E Polar Negative —3 ac1d . 5 Glutamine Gln Q Polar Neutral -3.5 Glycine Gly G Nonpolar Neutral -O.4 Histidine His H Polar Positive -3.2 Isoleucine Ile I ar l 4.5 Leucine Leu L Nonpolar \eutral 3.8 Lysine Lys K Polar Positive -3 .9 Methionine Met M Nonpolar \eutral 1.9 lghenylalamn Phe F Nonpolar \'eutral 2. 8 Proline Pro P Nonpolar _\eutral -1.6 Serine Ser S Polar \'eutral —0.8 Threonine Thr T Polar \eutral —0.7 Tryptophan Trp W Nonpolar l —0.9 Tyrosine Tyr Y Polar \ eutral -1.3 Valine Val V Nonpolar \eutral 4.2 Ambiguous Amino Acids 3-Letter er gine or aspartic acid Asx B Glutamine or ic acid GlX Z Leucine or Isoleucine Xle J Unspecified or unknown amino acid Xaa X As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial er programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, S. F. et al., 1990, J. Mol. Biol, 215(3): 403-10, Altschul, S.F. et al., 1996, Meth.

Enzymol. 266:460-80; Altschul, S.F. et al., 1997, Nucleic Acids Res, 25:3389-402; nis, AD. and B.F.F. Ouellette (eds) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds) Bioinformatics s and Protocols, Methods in Molecular Biology, Vol. 132, Humana Press, 1998. In addition to identifying homologous sequences, the programs mentioned above typically provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least, e.g., but not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding es are homologous over a relevant stretch of residues.

In some ments, the relevant stretch is a complete sequence. In some embodiments, the relevant h is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues. In some embodiments, the relevant stretch es contiguous residues along a complete sequence.

In some embodiments, the relevant stretch includes discontinuous residues along a complete sequence, for example, noncontiguous residues brought together by the folded conformation of a polypeptide or a portion thereof. In some embodiments, the relevant stretch is at least, e.g., but not limited to, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.

Substantial identity: as used herein, refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be "substantially identical" if they n identical residues in corresponding positions. As is well known in this art, amino acid or c acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, S. F. et al., 1990, J. Mol. Biol, 215(3): 403-10, Altschul, S.F. et al., 1996, Meth. l. 266:460-80; Altschul, SF. et al., 1997, Nucleic Acids Res, 2513389- 402, Baxevanis, AD. and REP Ouellette (eds) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998, and Misener et al. (eds) ormatjcs Methods and ols, s in lar Biology, Vol. 132, Humana Press, 1998. In addition to identifying identical sequences, the programs mentioned above typically provide an indication of the degree of identity. In some embodiments, two sequences are ered to be substantially cal if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding es are identical over a relevant stretch of residues. In some embodiments, a relevant stretch of es is a complete sequence. In some embodiments, a relevant stretch of residues is, e.g., but not limited to, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.

Targeting construct or targeting : as used herein, refers to a cleotide molecule that comprises a targeting region. A targeting region comprises a sequence that is identical or substantially identical to a sequence in a target cell, tissue or animal and provides for integration of the targeting construct into a position within the genome of the cell, tissue or animal via homologous ination. Targeting regions that target using pecific recombinase recognition sites (e.g., loxP or Frt sites) are also included and described herein.

In some embodiments, a targeting uct as described herein further comprises a nucleic acid sequence or gene of particular interest, a selectable marker, control and/or regulatory sequences, and other nucleic acid sequences that allow for recombination mediated through exogenous addition of proteins that aid in or facilitate recombination involving such sequences. In some embodiments, a targeting construct as described herein further comprises a gene of interest in whole or in part, wherein the gene of interest is a heterologous gene that encodes a polypeptide, in whole or in part, that has a similar function as a protein encoded by an nous ce. In some embodiments, a ing uct as described herein further comprises a humanized gene of interest, in whole or in part, wherein the humanized gene of interest encodes a polypeptide, in whole or in part, that has a r function as a polypeptide d by an endogenous sequence. In some ments, a targeting construct (or targeting vector) may comprise a nucleic acid sequence manipulated by the hand of man. For example, in some embodiments, a ing construct (or targeting vector) may be constructed to contain an engineered or recombinant cleotide that contains two or more sequences that are not linked together in that order in nature yet manipulated by the hand of man to be directly linked to one another in the engineered or recombinant polynucleotide.

Transgene or transgene construct: as used herein, refers to a nucleic acid sequence (encoding e.g, a ptide of interest, in whole or in part) that has been introduced into a cell by the hand of man such as by the methods described herein. A transgene could be partly or entirely logous, i.e., foreign, to the transgenic animal or cell into which it is introduced. A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns or promoters, which may be necessary for expression of a selected nucleic acid sequence.

Transgenic animal, transgenic non-human animal or Tg+z are used interchangeably herein and refer to any non-naturally occurring man animal in which one or more of the cells of the non-human animal contain heterologous nucleic acid and/or gene encoding a polypeptide of interest, in whole or in part. For example, in some embodiments, a "transgenic animal" or "transgenic non-human animal" refers to an animal or man animal that contains a transgene or transgene construct as described herein. In some embodiments, a heterologous c acid and/or gene is introduced into the cell, directly or ctly by introduction into a precursor cell, by way of rate genetic manipulation, such as by microinjection or by infection with a recombinant Virus. The term genetic manipulation does not include classic breeding techniques, but rather is directed to introduction of recombinant DNA molecule(s). This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. The term " Tg" includes animals that are heterozygous or homozygous for a heterologous nucleic acid and/or gene, and/or animals that have single or multi-copies of a heterologous nucleic acid and/or gene. t: as used herein, refers to an entity that shows significant structural identity with a reference entity, but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a "variant" also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a "variant" of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by persons of skill in the art, any biological or al reference entity has n teristic structural elements. A "variant", by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. To give but a couple examples, a polypeptide may have a characteristic ce t comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular biological function, or a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three—dimensional space. In another example, a "variantpolypeptide" may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e. g., ydrates, lipids, etc.) covalently attached to the polypeptide backbone.

In some embodiments, a "variantpolypeptide" shows an overall sequence identity with a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. Alternatively, or additionally, in some embodiments, a "variantpolypeptide" does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, a reference polypeptide has one or more ical activities. In some embodiments, a "variant polypeptide" shares one or more of the biological activities of the nce ptide. In some embodiments, a "variant polypeptide" lacks one or more of the biological ties of the reference polypeptide. In some embodiments, a "variantpolypeptide" shows a reduced level of one or more biological activities as ed with the reference polypeptide. In many embodiments, a polypeptide of interest is considered to be a "variant" of a parent or reference polypeptide if the polypeptide of st has an amino acid sequence that is identical to that of the parent but for a small number of sequence alterations at particular positions. Typically, fewer than 20%, %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the residues in the variant are tuted as ed with the parent. In some embodiments, a "variant" has, e.g, but not limited to,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue(s) as compared with a parent.

WO 28691 Often, a "variant" has a very small number (e. g., fewer than 5, 4, 3, 2, or 1) number of substituted functional residues (i.e., residues that ipate in a particular biological activity). Furthermore, a "variant" typically has not more than, e. g., but not limited to, 5, 4, 3, 2, or 1 ons or ons, and often has no additions or deletions, as compared with the parent. Moreover, any additions or deletions are typically fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues. In some ments, a parent or reference polypeptide is one found in . As will be understood by s of ordinary skill in the art, a plurality of variants of a particular polypeptide of interest may commonly be found in nature, particularly when the polypeptide of interest is an infectious agent polypeptide, Vector: as used herein, refers to a nucleic acid molecule capable of orting another nucleic acid to which it is associated, In some embodiment, vectors are capable of extra-chromosomal replication and/or expression of nucleic acids to which they are linked in a host cell such as a eukaryotic and/or prokaryotic cell. Vectors capable of directing the expression of operably linked genes are referred to herein as "expression vectors," Wild-type: as used herein, refers to an entity having a structure and/or activity as found in nature in a l" (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ry skill in the art will appreciate that wild-type genes and polypeptides often exist in multiple different forms (e.g, alleles).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS In certain aspects, provided herein, among other things, are engineered non- human animals having heterologous genetic material ng human valiable domains and, in some embodiments, human constant domains, which heterologous genetic material comprises human V)», J7» and C?» gene sequences (i.e., gene segments) and other human sequences that provide for proper rearrangement and expression of antibodies having a human portion and a non-human portion or antibodies having a sequence that is substantially or substantially all human. In various embodiments, ed engineered man s contain heterologous genetic material that is inserted in such a way so that antibodies containing light chains that have a human V?» domain and a human or non-human Ck domain are expressed in the antibody repertoire of the non-human animal. Further, provided engineered non-human animals contain logous genetic material that is inserted in such a way so that antibodies containing light chains that have a human V?» domain and a human or non-human Ck domain are expressed from engineered Ig7t light chain loci that e human and non—human 1g?» enhancer regions (or sequences) in the germline genome of the non-human animal.

] Without wishing to be bound by any particular theory, it is contemplated that embodiments of the man animals as described herein provide an improved in vivo system that exploits the expression of antibodies containing human V?» domains for the production of therapeutic antibodies. It is also contemplated that embodiments of the non- human animals as described herein, in some embodiments, provide alternative engineered forms of Ig9t light chain loci that contain heterologous genetic material for the development of human dy-based therapeutics (e.g., human monoclonal antibodies, multi—specific binding agents, scFvs, fusion ptides, etc.) to disease targets that are associated with biased antibody responses (e.g., antibody responses characterized by an elming proportion of either K or 7» light chains). Thus, embodiments of the non—human animals as described herein are particularly useful for the development of human antibodies against targets ated with poor immunogenicity (e. g., viruses) due, in part, to skewed antibody oires and/or responses.

In particular, in n aspects the present disclosure describes the production of a non-human animal (e.g, a rodent, such as a rat or mouse) having a ne genome that contains an engineered lg?» light chain locus that is, in some embodiments, characterized by the introduction of a plurality of human V7t, J7» and Cl gene sequences in operable linkage to a non-human C?» region resulting in the expression of antibodies that contain light chains that include a human V?» domain and a human or man C?» domain. As described herein, the tion of such an engineered Iglt light chain locus results in the expression of antibodies that contain light chains that include a human V?» domain and a human or non- human C?» domain from said engineered 1g?» light chain locus in the germline genome of the non-human animal. The germline genome of provided non-human animals, in some embodiments, further comprises (1) humanized IgH and IgK loci or (2) a humanized IgH locus and functionally silenced or otherwise rendered non-functional Ig1< light chain loci.

Provided man s, as described herein, express antibody repertoires that contain Ig7t light chains that include human Wt domains.

In some embodiments, non-human s as described herein n human and non—human 1g?» light chain sequences within a single 1g?» light chain locus. In some embodiments, non-human animals as described herein contain human Ig7t and murine (e,g, mouse or rat) 1g?» light chain sequences within an ng light chain locus. In many embodiments of non-human s as described herein, non-human 1th light chain sequences are or comprise murine sequences (e. g, mouse or rat).

In some ments, 1g?» light chain sequences include intergenic DNA that is of human and/or murine (e.g, mouse or rat) origin. In some embodiments, ng light chain sequences include intergenic DNA that is synthetic and based on a source sequence that is of human or murine (e.g, mouse or rat) origin. In some embodiments, said intergenic DNA is of the same immunoglobulin locus in which the intergenic DNA is so placed, inserted, positioned or engineered (e.g, Ig7t intergenic DNA in an ng light chain locus). In some certain embodiments, non-human animals as described herein contain an ered 1g?» light chain locus that contains intergenic DNA that includes 1g?» light chain sequence(s) of non-human origin (e.g., mouse or rat 1g?» light chain sequence).

In various embodiments, a humanized IgH locus contains a plurality of human VH, DH and JH gene segments operably linked to a non-human IgH constant region (e.g, an endogenous non—human IgH constant region that includes one or more IgH constant region genes such as, for example, IgM, IgG, etc). In various embodiments, a humanized IgK light chain locus contains a plurality of human VK and IK gene segments operably linked to a non- human IgK constant region. In some embodiments, provided non-human animals have a ne genome that es the immunoglobulin loci (or alleles) depicted in a g provided herein (e.g., see Figure 1, 2, 3 and/or 4). Such engineered non-human animals provide a source of human antibodies and human antibody fragments, and/or nucleic acids ng such human antibodies and human antibody fragments, and provide an improved in vivo system suitable for exploiting human V)» sequences for the production of human therapeutic antibodies.

] As described herein, in certain embodiments non-human animals are ed having a genome that contains a ity of human 7» light chain gene segments (e.g., V7», J?» and CA) in the place of non-human immunoglobulin X light chain gene ts at endogenous immunoglobulin 2 light chain loci, and e human non-coding intergenic DNA between the human variable region gene segments. In some embodiments, non—human animals provided herein have a genome that further ses human heavy (i.e., VH, DH and JH) and K light chain (e.g, VK and JK) variable region gene segments in the place of non- human heavy (i.e., VH, DH and JH) and K light chain (e. g., VK and JK) variable region gene segments at endogenous globulin heavy and K light chain loci, respectively. In many embodiments, human immunoglobulin gene segments (heavy and/or light) are engineered with human intergenic DNA (i.e., human non—coding immunoglobulin intergenic DNA) that is naturally associated with said gene ts (i.e., non-coding genomic DNA associated with said gene segments that naturally appears in a human immunoglobulin locus of a human cell). Such intergenic DNA es, for example, ers, leader sequences and recombination signal sequences that allow for proper recombination and expression of the human gene segments in the context of variable domains of antibodies. Persons of skill understand that non—human immunoglobulin loci also contain such non—coding intergenic DNA and that, upon reading this disclosure, other human or non-human intergenic DNA can be employed in ucting such engineered immunoglobulin loci resulting in the same expression of human variable domains in the context of antibodies in the non—human animal.

Such similar engineered immunoglobulin loci need only contain the human coding sequences (i.e., exons) of the desired human gene segments, or combination of human gene segments, to achieve sion of antibodies that contain human variable s.

Various aspects of certain embodiments are described in detail in the following sections, each of which can apply to any aspect or embodiment as described herein. The use of sections is not for limitation, and the use of "or" means "and/0r" unless stated otherwise.

Antibody repertoires in non-human animals ] Immunoglobulins (also called antibodies) are large (~150 kD), Y—shaped glycoproteins that are produced by B cells of a host immune system to lize foreign antigens (e.g., viruses, bacteria, etc). Each immunoglobulin (1g) is ed of two identical heavy chains and two identical light chains, each of which has two structural components: a variable domain and a constant domain. The heavy and light chain variable domains differ in antibodies produced by different B cells, but are the same for all antibodies produced by a single B cell or B cell clone. The heavy and light chain variable domains of each antibody together se the n—binding region (or antigen-binding site). globulins can exist in different varieties that are referred to as isotypes or classes based on the heavy chain constant regions (or domains) that they contain. The heavy chain constant domain is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The table below summarizes the nine antibody isotypes in mouse and man (human).

Mouse Human IgM IgM IgD IgD IgGl IgGl IgGZa IgG2 IgG2b IgG3 IgGZc IgG4 IgG3 IgE IgE IgAl IgA IgAZ onal isotypes have been identified in other s. Isotypes confer specialized biological ties on the antibody due to the different structural characteristics among the ent isotypes and are found in different locations (cells, tissues, etc.) within an animal body. Initially, B cells produce IgM and IgD with identical antigen-binding regions. Upon activation, B cells switch to different isotypes by a process referred to as class switching, which involves a change of the constant domain of the antibody produced by the B cell while the variable domains remain the same, y preserving antigen specificity of the original antibody (B cell).

Two separate loci (IgK and Igl) contain the gene segments that encode the light chains of dies, and exhibit both allelic and isotypic exclusion. The expression ratios of K+ to W B cells vary among species. For example, humans demonstrate a ratio of about 60:40 (K270. In mice and rats, a ratio of 95:5 (EA) is observed. Interestingly, the Kit ratio ed in cats (5:95) is opposite of mice and rats. Several studies have been conducted to elucidate the possible reasons behind these ed ratios and have proposed that both the complexity of the locus (i.e., number of gene segments, in particular, V gene segments) and the efficiency of gene segment ngement as rationale. The human immunoglobulin 7t light chain locus extends over 1,000kb and contains approximately 70 V?» gene segments (29 to 33 functional) and seven Jk-CX gene segment pairs (four to five functional) organized into three clusters (see, e.g., Fig. 1 of US. Patent No. 9,006,511). The majority of the observed V?» regions in the expressed dy repertoire are encoded by gene segments contained within the most proximal cluster (i.e., cluster A). The mouse immunoglobulin 7» light chain 2017/060006 locus is strikingly different than the human locus and, depending on the strain, contains only a few V?» and J7» gene segments organized in two distinct gene clusters (see, e.g, Fig. 2 of US. Patent No. 9,006,511).

Development of therapeutic antibodies for the treatment of various human diseases has y been centered on the creation of engineered non—human animal lines, in particular, engineered rodent lines, harboring varying amounts of genetic material in their genomes corresponding to human immunoglobulin genes (reviewed in, e.g., Bn‘jggemann, M. et al., 2015, Arch. Immunol. Ther. Exp. 63: 101-8). Initial efforts in creating such transgenic rodent lines focused on ation of portions of human immunoglobulin loci that could, by themselves, support recombination of gene segments and production of heavy and/or light chains that were entirely human while having endogenous immunoglobulin loci inactivated (see e.g., Bruggemann, M. et al., 1989, Proc. Nat. Acad. Sci. USA. 86:6713; Brfiggemann, M. et al., 1991, Eur. J. Immunol. 21 : 1323-6; Taylor, L.D. et al., 1992, Nucl.

Acids Res. 20:6287-6295, , N.P. et al., 1993, Biotechnol. 11:911-4, Green, L.L. et al., 1994, Nat. Genet. 1; Lonberg, N. et al., 1994, Nature 6—9, Taylor, L.D. et al., 1994, Int, Immunol. 91, Wagner, SD. et al., 1994, Eur. J. Immunol. 24:2672—81, Fishwild, D.M. et al., 1996, Nat. Biotechnol. 141845—51, Wagner, SD. et al., 1996, Genomics -14, Mendez, M.J. et al., 1997, Nat. Genet. 152146-56; Green, L.L. et al., 1998, J. Exp. Med. 188:483-95, Xian, J. et al., 1998, Transgenics 2:333-43; Little, M. et al., 2000, Immunol. Today 21:364-70, Kellerrnann, SA. and LL. Green, 2002, Cur. Opin. hnol. 13 1593-7). In particular, some efforts have included integration of human Ig9t light chain sequences (see, e.g., US. Patent Application ation Nos. 2002/0088016 A1, 2003/0217373 Aland 2011/0236378 A1; US. Patent Nos. 6,998,514 and 7,435,871; Nicholson, I.C. et al., 1999, J. Immunol. 163:6898-906, Popov, A.V et al., 1999, J. Exp.

Med. 189(10): 161 1—19). Such efforts have focused on the random integration of yeast artificial chromosomes containing human V1, Jk and Cl sequences thereby creating mouse strains that s fully human A light chains (i.e., human variable and human constant).

More recent s have employed similar strategies using constructs that also contain human V7», JR and CK sequences (Osborn, M.J. et al., 2013, J. Immunol. 190: 0; Lee, E-C. et al., 2014, Nat. Biotech. 32(4):356-63).

Yet other efforts have included the specific insertion of human V7» and J 7i. gene segments into endogenous rodent Ig light chain loci (K and I) so that said human V?» and J2 gene ts are operably linked to endogenous 1g light chain constant regions (see, e. g., US. Patent Nos. 511, 717, 9,029,628, 128, 9,066,502, 9,150,662 and 9,163,092; all of which are incorporated herein by reference in their entireties). In such animals, all of the human V?» gene segments from clusters A and B and either one or four human I?» gene ts were inserted into endogenous IgK and 1g?» light chain loci. As a result, several ent human V?» and J9» gene segments demonstrated proper rearrangement at both engineered rodent Ig light chain loci to form functional human V?» domains that were sed in the context of both CK and C7» regions in light chains of the rodent antibody repertoire (see, e.g., Table 7 and Figures ll-l3 ofU.S. Patent No. 9,006,511). In particular, mice having engineered IgK light chain loci harboring human VA and JR gene segments demonstrated a K27» ratio of about 1:1 in the splenic compartment (see, e.g., Table 4 of US.

Patent No. 9,006,511). Indeed, both engineered mouse strains (i.e., engineered IgK or engineered 1g?» light chain loci) demonstrated that human VQL domains could be expressed from endogenous Ig light chain loci in rodents, which normally display a large bias in light chain sion (see above). The present invention is based on the ition that other engineered Ig light chain locus structures can be produced to maximize usage of human V)» and J7L gene segments in antibody repertoires to therapeutic targets in non-human animals, in particular, as compared to non-human animals that contain an 1g?» light chain locus that lacks the complexity and robust quality (e. g., mice and rats) normally associated with a human 1g?» light chain locus (i.e., that appears in a human cell). Such alternative engineered Ig light chain locus structures e the capacity for unique antibody repertoires resulting from their design.

The present disclosure describes, among other , the successful production of a non-human animal whose germline genome contains an engineered endogenous 1g?» light chain locus sing a plurality of human V)», I?» and Cl gene ts in le linkage to a non-human ng light chain constant region. In particular, the present disclosure specifically demonstrates the successful production of an engineered non-human animal that expresses antibodies having human variable domains and non-human constant domains, which antibodies include light chains that contain a human V7» domain. As described herein, expression of such light chains is achieved by ion of said plurality of human V7t, J7» and C?» gene segments into an endogenous 1g?» light chain locus (or allele). Also, as described herein, provided non-human animals are, in some ments, engineered so that expression of light chains that contain endogenous V7t domains is inactivated (e.g, by gene deletion). Thus, the present disclosure, in at least some embodiments, embraces the development of an improved in viva system for the production of human antibodies by providing an engineered man animal containing an alternatively engineered 1g?» light chain locus that results in an expressed antibody repertoire containing human V7» domains.

DNA inserts Typically, a polynucleotide molecule containing human lg?» light chain sequences (e.g., V)», J)», C?» and 1g?» enhancers) or portion(s) thereof is inserted into a vector, preferably a DNA vector, in order to replicate the polynucleotide molecule in a host cell.

] Human 1g?» light chain sequences can be cloned ly from known sequences or sources (e.g., libraries) or synthesized from ne ces designed in silico based on published sequences available from GenBank or other publically available databases (e.g., IMGT). Alternatively, bacterial artificial chromosome (BAC) libraries can provide immunoglobulin DNA sequences of interest (e.g., human Vic gene segments, human lit-Ck gene segment pairs, human EX regions or sequences, and combinations thereof). BAC libraries can contain an insert size of lOO-lSOkb and are capable of harboring inserts as large as 300kb (Shizuya, et al., 1992, Proc. Natl. Acad. Sci, USA 4-8797; Swiatek, et al., 1993, Genes and Development 7:2071-2084; Kim, et al., 1996, Genomics 34 213-218; incorporated herein by reference in their entireties). For example, a human BAC library harboring average insert sizes of 164-196kb has been described (Osoegawa, K. et al., 2001, Genome Res. 483-96; Osoegawa, K, et al., 1998, Genomics 52: 1-8, Article No.

GE985423). Human and mouse genomic BAC libraries have been constructed and are commercially available (e. g., Fisher). Genomic BAC libraries can also serve as a source of immunoglobulin DNA sequences as well as transcriptional control regions. atively, globulin DNA sequences may be isolated, cloned and/or transferred from yeast artificial chromosomes (YACs). For example, the nucleotide sequence of the human ng light chain locus has been determined (see, e.g., Dunham, I. et al., 1999, Nature 402:489-95). r, YACs have previously been employed to assemble a human Ig7t light chain locus transgene (see, e.g., Popov, A.V. et al., 1996, Gene 177:195-201; Popov, A.V. et al., 1999, J. Exp. Med. 189(10):]611-19). An entire ng light chain locus (human or rodent) can be cloned and contained Within l YACs. If multiple YACs are ed and contain regions of overlapping homology, they can be recombined Within yeast host strains to e a single construct representing the entire locus or desired portions of the locus (e. g., a region to targeted with a ing vector). YAC arms can be additionally modified with mammalian selection cassettes by retrofitting to assist in introducing the constructs into embryonic stems cells or embryos by methods known in the art and/or described herein DNA and amino acid sequences of human Igh light chain gene segments for use in constructing an engineered 1g?» light chain locus as described herein may be obtained from published databases (e.g., GenBank, IMGT, etc.) and/or published antibody sequences. DNA inserts ning human 1g?» light chain gene segments, in some ments, comprise one or more human ng light chain enhancer sequences (or regions). DNA s, in some embodiments, comprise a human 1g?» light chain enhancer sequence (or region) that includes one or more sequence elements, e. g., one, two, three, etc. In some certain embodiments, DNA inserts comprise a human 1g?» light chain enhancer ce (or ), referred to as a human B?» having three distinct sequence elements. Thus, in some embodiments, a human EX as described herein is modular and one or more sequence elements function together as an enhancer sequence (or region). In some certain embodiments, DNA inserts containing human 1g?» light chain enhancer sequences comprise human 1g?» light chain enhancer sequences operably linked to a non-human 1g?» light chain sequence (e.g., a man 1g?» light chain constant region sequence). In some certain embodiments, DNA inserts containing human ng light chain enhancer sequences comprise human ng light chain enhancer sequences operably linked to a non-human ng light chain sequence (e.g., a non-human 1g?» light chain constant region sequence) and operably linked to one or more human V?» gene segments, one or more human JK-C?» gene segment pairs and/or one or more human I?» gene segments. In some n embodiments, DNA inserts containing human ng light chain enhancer sequences comprise human ng light chain enhancer sequences operably linked to a non-human 1g?» light chain sequence (e.g., a non-human ng light chain constant region sequence), one or more human V?» gene segments, one or more human J7» gene segments and one or more human C?» gene segments.

] DNA inserts can be prepared using methods known in the art. For e, a DNA insert can be prepared as part of a larger plasmid. Such ation allows the cloning and selection of the correct uctions in an efficient manner as is known in the art. DNA inserts containing human 1g?» light chain sequences, in whole or in part, as described herein can be d between convenient restriction sites on the plasmid so that they can be easily isolated from the remaining plasmid sequences for oration into a desired non-human animal.

Various methods employed in preparation of plasmids and transformation of host organisms are known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Principles of Gene Manipulation: An Introduction to Genetic Manipulation, 5th Ed, ed, By Old, R.W. and SB.

Primrose, Blackwell Science, Inc, 1994 and Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al., Cold Spring Harbor Laboratory Press: 1989.

Targeting Vectors Targeting vectors can be employed to introduce a DNA insert into a genomic target locus and comprise a DNA insert and homology arms that flank said DNA insert.

Targeting vectors can be in linear form or in circular form, and they can be -stranded or double-stranded. Targeting vectors can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). For ease of reference, homology arms are referred to herein as 5’ and 3’ (ie., upstream and downstream) homology arms. This terminology relates to the relative on of the homology arms to a DNA insert within a targeting vector. 5’ and 3’ homology arms correspond to regions within a targeted locus or to a region within another targeting vector, which are referred to herein as "5 ’ target sequence" and "3 ’ target sequence," respectively.

In some embodiments, homology arms can also function as a 5’ or a 3’ target sequence.

In some embodiments, methods described herein employ two, three or more ing vectors that are capable of recombining with each other. In various embodiments, targeting vectors are large targeting vectors (LTVEC) as bed elsewhere herein. In such embodiments, first, second, and third targeting s each comprise a 5’ and a 3’ homology arm. The 3’ gy arm of the first targeting vector comprises a sequence that overlaps with the 5’ gy arm of the second targeting vector (i.e., overlapping sequences), which allows for homologous recombination between first and second LTVECs.

In the case of double targeting methods, a 5’ homology arm of a first ing vector and a 3’ homology arm of a second ing vector are homologous to corresponding segments within a target genomic locus (i.e., a target sequence) which promotes homologous ination of the first and the second targeting vectors with ponding c ts and modifies the target genomic locus.

In the case of triple targeting methods, a 3’ homology arm of a second targeting vector comprises a sequence that overlaps with a 5’ homology arm of a third targeting vector (i.e., overlapping sequences), which allows for gous recombination between the second and the third LTVEC. The 5’ homology arm of the first targeting vector and the 3’ homology arm of the third targeting vector are homologous to ponding segments within the target genomic locus (i.e., the target sequence), which promotes homologous recombination of the first and the third targeting vectors with the corresponding genomic segments and s the target genomic locus.

A homology arm and a target sequence or two homology arms "correspond" or are "corresponding" to one r when the two s share a sufficient level of sequence identity to one another to act as substrates for a homologous recombination on. The term ogy" includes DNA sequences that are either identical or share sequence identity to a corresponding sequences The sequence identity between a given target sequence and the corresponding homology arm found on a targeting vector (i.e., overlapping sequence) or between two homology arms can be any degree of sequence identity that allows for homologous recombination to occur. To give but one example, an amount of sequence identity shared by a homology arm of a targeting vector (or a fragment thereof) and a target sequence of r targeting vector or a target sequence of a target genomic locus (or a fragment thereof) can be, e. g., but not limited to, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo homologous recombination.

Moreover, a corresponding region of gy between a homology arm and a corresponding target sequence can be of any length that is sufficient to promote homologous recombination at the target genomic locus. For example, a given homology arm and/or ponding target sequence can comprise corresponding regions of homology that are, e.g., but not limited to, at least about 5—10kb, 5—15kb, 5—20kb, , 5—30kb, 5—35kb, 5— 40kb, 5-45kb, 5-50kb, 5-55kb, 5-60kb, 5-65kb, 5-70kb, 5-75kb, 5-80kb, 5-85kb, 5-90kb, 5- 95kb, 5-100kb, lOO-ZOOkb, or 200-300kb in length or more (such as described elsewhere herein) such that a homology arm has sufficient homology to undergo homologous recombination with a corresponding target sequence(s) within a target c locus of the cell or within another targeting . In some embodiments, a given gy arm and/or corresponding target sequence comprise ponding regions of homology that are, e.g, WO 28691 but not limited to, at least about lO-lOOkb, 15-lOOkb, 20-100kb, 25-100kb, kb, 35- 100kb, 40-100kb, 45-100kb, 50-100kb, 55-100kb, 60-100kb, 65-100kb, 70-100kb, 75— lOOkb, 80—100kb, 85-100kb, 90-100kb, or kb in length or more (such as described elsewhere herein) such that a homology arm has sufficient homology to undergo homologous recombination with a corresponding target sequence(s) within a target genomic locus of the cell or within another targeting vector.

Overlapping sequences of a 3’ homology arm of a first targeting vector and a 5’ homology arm of a second targeting vector or of a 3’ homology arm of a second targeting vector and a 5’ homology arm of a third targeting vector can be of any length that is ent to promote homologous recombination between said targeting vectors. For example, a given overlapping sequence of a gy arm can comprise corresponding overlapping regions that are at least about 1-5kb, 5-lOkb, 5-15kb, 5—20kb, 5-25kb, 5-30kb, 5- 35kb, , 5-45kb, 5-50kb, , 5-60kb, 5-65kb, 5-70kb, 5-75kb, 5-80kb, , 5— 90kb, 5-95kb, 5-100kb, lOO-ZOOkb, or 200-300kb in length or more such that an overlapping sequence of a homology arm has sufficient homology to undergo gous recombination with a corresponding overlapping sequence within another targeting vector. In some embodiments, a given overlapping sequence of a gy arm comprises an pping region that is at least about l-lOOkb, 5-100kb, lO-lOOkb, 15-100kb, 20-100kb, 25-100kb, 30— lOOkb, 35-100kb, 40-100kb, kb, 50—100kb, 55-100kb, 60-100kb, 65-100kb, 70- lOOkb, 75-100kb, 80-100kb, 85-100kb, 90—100kb, or 95-100kb in length or more such that an overlapping sequence of a homology arm has sufficient homology to undergo homologous recombination with a corresponding overlapping sequence within another targeting . In some embodiments, an overlapping sequence is from l-5kb, inclusive. In some embodiments, an overlapping sequence is from about lkb to about 70kb, ive. In some embodiments, an overlapping sequence is from about lOkb to about 70kb, ive. In some embodiments, an overlapping sequence is from about lOkb to about 50kb, inclusive. In some embodiments, an overlapping sequence is at least lOkb. In some embodiments, an overlapping sequence is at least 20kb. For example, an overlapping sequence can be from about lkb to about 5kb, inclusive, from about 5kb to about 10kb, inclusive, from about lOkb to about 15kb, inclusive, from about 15kb to about 20kb, inclusive, from about 20kb to about 25kb, inclusive, from about 25kb to about 30 kb, ive, from about 30kb to about 35kb, inclusive, from about 35kb to about 40kb, inclusive, from about 40kb to about 45kb, inclusive, from about 45kb to about 50kb, inclusive, from about 50kb to about 60kb, WO 28691 inclusive, from about 60kb to about 7Okb, inclusive, from about 70kb to about 80kb, ive, from about 80kb to about 90kb, inclusive, from about 90kb to about 100kb, inclusive, from about 100kb to about 120kb, inclusive, from about 120kb to about 140kb, inclusive, from about 140kb to about 160kb, inclusive, from about 160kb to about 180kb, inclusive, from about 180kb to about 200kb, inclusive, from about 200kb to about 220kb, inclusive, from about 220kb to about 240kb, inclusive, from about 240kb to about 260kb, inclusive, from about 260kb to about 280kb, inclusive, or about 280kb to about 300 kb, inclusive. To give but one example, an overlapping sequence can be from about 20kb to about 60kb, inclusive. Alternatively, an overlapping sequence can be at least lkb, at least 5kb, at least 10kb, at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 35kb, at least 40kb, at least 45kb, at least 50kb, at least 60kb, at least 70kb, at least 80kb, at least 90kb, at least 100kb, at least 120kb, at least 140kb, at least 160kb, at least 180kb, at least 200kb, at least 220kb, at least 240kb, at least 260kb, at least 280kb, or at least 300ka Homology arms can, in some embodiments, correspond to a locus that is native to a cell (e. g, a targeted locus), or alternatively they can pond to a region of a heterologous or exogenous segment ofDNA that was integrated into the genome of the cell, including, for example, transgenes, expression cassettes, or heterologous or exogenous regions of DNA. Alternatively, homology arms can, in some embodiments, correspond to a region on a targeting vector in a cell. In some embodiments, homology arms of a targeting vector may correspond to a region of a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a human artificial chromosome, or any other engineered region contained in an appropriate host cell. Still further, homology arms of a ing vector may correspond to or be derived from a region of a BAC library, a cosmid y, or a P1 phage library. In some certain ments, gy arms of a targeting vector correspond to a locus that is native, heterologous, or exogenous to a prokaryote, a yeast, a bird (e.g., chicken), a non-human mammal, a rodent, a human, a rat, a mouse, a r a rabbit, a pig, a bovine, a deer, a sheep, a goat, a cat, a dog, a ferret, a primate (e. g., marmoset, rhesus ), a domesticated mammal, an agricultural mammal, or any other organism of interest. In some embodiments, homology arms correspond to a locus of the cell that is not targetable using a conventional method or that can be targeted only ectly or only with significantly low efficiency in the absence of a nick or -strand break induced by a nuclease agent (e.g., a Cas protein). In some embodiments, homology arms are derived from synthetic DNA.

In some embodiments, one of the 5’ or 3’ homology arms of a targeting vector(s) corresponds to a targeted genomic locus while the other of the 5’ or 3’ homology arms ponds to a region on another targeting vector.

In some embodiments, 5’ and 3’ homology arms of a targeting vector(s) correspond to a targeted genome. atively, gy arms can be from a related genome. For e, a ed genome is a mouse genome of a first strain, and targeting arms are from a mouse genome of a second strain, wherein the first strain and the second strain are different. In certain embodiments, homology arms are from the genome of the same animal or are from the genome of the same strain, e.g., the targeted genome is a mouse genome of a first strain, and the targeting arms are from a mouse genome from the same mouse or from the same .

A homology arm of a targeting vector can be of any length that is ent to promote a homologous recombination event with a corresponding target sequence, including, for example, at least l-5kb, inclusive, 5-10kb, inclusive, 5-15kb, inclusive, 5-20kb, inclusive, 5-25kb, inclusive, 5-30kb, inclusive, 5-35kb, inclusive, 5-40kb, inclusive, 5-45kb, inclusive, 5-50kb, inclusive, , inclusive, 5-60kb, inclusive, 5—65kb, inclusive, 5-70kb, inclusive, , inclusive, 5-80kb, inclusive, 5-85kb, inclusive, 5-90kb, inclusive, 5-95kb, inclusive, 5-100kb, inclusive, lOO-200kb, inclusive, or 200-300kb, inclusive, in length or greater, In some embodiments, a homology arm of a targeting vector has a length that is sufficient to promote a homologous recombination event with a ponding target sequence that is at least l-100kb, inclusive, 5-lOOkb, ive, lO-lOOkb, inclusive, 15- lOOkb, inclusive, 20—100kb, inclusive, 25-100kb, inclusive, 30-100kb, inclusive, 35-100kb, inclusive, 40-lOOkb, inclusive, 45-100kb, inclusive, 50-100kb, inclusive, 55-100kb, inclusive, 60-100kb, inclusive, 65-100kb, inclusive, 70-100kb, inclusive, 75-100kb, inclusive, 80-lOOkb, inclusive, 85-100kb, inclusive, 90-100kb, inclusive, or 95-lOOkb, inclusive, in length or greater. As described herein, large targeting vectors can employ targeting arms of greater length.

Nuclease agents (e.g., CRISPR/Cas systems) can be ed in combination with targeting vectors to facilitate the modification of a target locus (e.g., an 1g?» light chain locus). Such nuclease agents may promote homologous recombination n a targeting vector and a target locus. When se agents are ed in ation with a targeting vector, the ing vector can comprise 5’ and 3’ homology arms corresponding to 5’ and 3’ target sequences located in sufficient proximity to a nuclease cleavage site so as to promote the occurrence of a homologous recombination event between target sequences and homology arms upon a nick or double-strand break at the nuclease cleavage site. The term "nuclease cleavage site" includes a DNA sequence at which a nick or double—strand break is created by a nuclease agent (e.g., a Cas9 cleavage site). Target sequences within a targeted locus that correspond to 5’ and 3’ homology arms of a targeting vector are "located in suflicientproximity" to a nuclease cleavage site if the ce is such as to promote the occurrence of a homologous recombination event between 5’ and 3’ target sequences and homology arms upon a nick or double-strand break at the recognition site. Thus, in certain embodiments, target sequences corresponding to 5’ and/or 3’ homology arms of a targeting vector are within one nucleotide of a given ition site or are within at least 10 nucleotides to about 14kb of a given recognition site. In some embodiments, a nuclease cleavage site is immediately adjacent to at least one or both of the target sequences, The spatial relationship of target sequences that correspond to homology arms of a targeting vector and a nuclease ge site can vary. For example, target sequences can be located 5’ to a nuclease cleavage site, target sequences can be located 3’ to a ition site, or target sequences can flank a se cleavage site.

Combined use of a targeting vector (including, for example, a large ing vector) with a nuclease agent can result in an increased targeting efficiency compared to use of a targeting vector alone. For example, when a targeting vector is used in conjunction with a nuclease agent, targeting efficiency of a targeting vector can be increased by at least two— fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least sevenfold , at least fold, at least nine—fold, at least ld or within a range formed from these integers, such as 2fold when compared to use of a ing vector alone.

Some targeting vectors are "large targeting vectors" or S," which includes targeting vectors that se homology arms that correspond to and are d from nucleic acid sequences larger than those typically used by other approaches intended to perform homologous recombination in cells. A LTVEC can be, for example, at least lOkb in length, or the sum total of a 5’ gy arm and a 3’ homology arm can be, for example, at least lOkb. LTVECs also e targeting vectors comprising DNA inserts larger than those typically used by other approaches intended to perform homologous recombination in cells.

For example, LTVECs make possible the modification of large loci that cannot be accommodated by traditional plasmid-based targeting vectors because of their size limitations. For example, a targeted locus can be Ge, 5’ and 3’ homology arms can WO 28691 correspond to) a locus of a cell that is not targetable using a conventional method or that can be targeted only incorrectly or only with significantly low efficiency in the absence of a nick or double-strand break d by a se agent (e. g., a Cas protein).

In some embodiments, methods bed herein employ two or three LTVECs that are capable of recombining with each other and with a target genomic locus in a three- way or a four-way recombination event. Such methods make le the modification of large loci that cannot be achieved using a single LTVEC.

Examples of LTVECs include vectors derived from a bacterial artificial chromosome (BAC), a human artificial chromosome, or a yeast artificial chromosome (YAC). Examples of LTVECs and methods for making them are described, e. g, in US.

Patent Nos, 6,586,251, 6,596,541 and No. 7,105,348; and International Patent ation Publication No. , each h is incorporated herein by nce in its entirety. LTVECs can be in linear form or in circular form, LTVECs can be of any length, including, for example, from about 20kb to about 300kb, inclusive, from about 20kb to about 50 kb, inclusive, from about 50kb to about 75kb, inclusive, from about 75kb to about 100kb, inclusive, from about lOOkb to 125kb, inclusive, from about 125kb to about 150kb, inclusive, from about 150kb to about 175kb, inclusive, from about l75kb to about 200kb, inclusive, from about 200kb to about 225kb, inclusive, from about 225kb to about 250kb, inclusive, from about 250kb to about 275kb, inclusive, or from about 275kb to about 300kb, inclusive. Alternatively, a LTVEC can be at least 10kb, at least 15kb, at least 20kb, at least 30kb, at least 40kb, at least 50kb, at least 60kb, at least 70kb, at least 80kb, at least 90kb, at least 100kb, at least 150kb, at least 200kb, at least 250kb, at least 300kb, at least 350kb, at least 400kb, at least 450kb, or at least 500kb or greater, The size of a LTVEC can, in some ments, be too large to enable screening of targeting events by conventional assays, e.g., southern blotting and long-range (eg, lkb to 5kb) PCR.

In some embodiments, a LTVEC comprises a DNA insert ranging from about 5kb to about 200kb, inclusive, from about 5kb to about 10kb, inclusive, from about lOkb to about 20kb, inclusive, from about 20kb to about 30kb, inclusive, from about 30kb to about 40kb, inclusive, from about 40kb to about 50kb, inclusive, from about 60kb to about 70kb, inclusive, from about 80kb to about 90kb, inclusive, from about 90kb to about 100kb, inclusive, from about lOOkb to about 110kb, inclusive, from about 120kb to about 130kb, inclusive, from about 130kb to about 140kb, inclusive, from about 140kb to about 150kb, inclusive, from about 150kb to about 160kb, inclusive, from about 160kb to about 170kb, inclusive, from about 170kb to about 180kb, inclusive, from about 180kb to about 190kb, inclusive, or from about 190kb to about 200kb, inclusive. In some embodiments, a DNA insert can range from about 5kb to about 10kb, inclusive, from about 10kb to about 20kb, inclusive, from about 20kb to about 40kb, inclusive, from about 40kb to about 60kb, inclusive, from about 60kb to about 80kb, ive, from about 80kb to about 100kb, inclusive, from about 100kb to about 150kb, inclusive, from about 150kb to about 200kb, inclusive, from about 200kb to about 250kb, ive, from about 250kb to about 300kb, inclusive, from about 300kb to about 350kb, inclusive, or from about 350kb to about 400kb, inclusive. In some embodiments, a LTVEC comprises a DNA insert ranging from about 400kb to about 450kb, inclusive, from about 450kb to about 500kb, inclusive, from about 500kb to about 550kb, inclusive, from about 550kb to about 600kb, inclusive, about 600kb to about 650kb, inclusive, from about 650kb to about 700kb, inclusive, from about 700kb to about 750kb, inclusive, or from about 750kb to about 800kb, inclusive.

In some embodiments, the sum total of a 5’ gy arm and a 3’ homology arm of a LTVEC is at least 10kb, In some ments, a 5’ homology arm of a s) ranges from about lkb to about 100kb, inclusive, and/or a 3’ homology arm of a LTVEC(s) ranges from about lkb to about 100kb, inclusive. The sum total of 5’ and 3’ homology arms can be, for e, from about lkb to about 5kb, inclusive, from about 5kb to about 10kb, inclusive, from about 10kb to about 20kb, inclusive, from about 20kb to about 30kb, inclusive, from about 30kb to about 40kb, inclusive, from about 40kb to about 50kb, inclusive, from about 50kb to about 60kb, inclusive, from about 60kb to about 70kb, ive, from about 70kb to about 80kb, inclusive, from about 80kb to about 90kb, inclusive, from about 90kb to about 100kb, inclusive, from about lOOkb to about 110kb, inclusive, from about 110kb to about 120kb, inclusive, from about 120kb to about 130kb, inclusive, from about 130kb to about 140kb, inclusive, from about 140kb to about 150kb, inclusive, from about 150kb to about 160kb, inclusive, from about 160kb to about 170kb, inclusive, from about 170kb to about 180kb, inclusive, from about 180kb to about 190kb, inclusive, or from about 190kb to about 200kb, inclusive. Alternatively, each homology arm can, in some embodiments, be at least 5kb, at least 10kb, at least 15kb, at least 20kb, at least 30kb, at least 40kb, at least 50kb, at least 60kb, at least 70kb, at least 80 kb, at least 90kb, at least 100kb, at least 110kb, at least 120kb, at least 130kb, at least 140kb, at least 150kb, at least 160kb, at least 170kb, at least 180kb, at least 190kb, or at least 200kb. Likewise, the sum total of the 5’ and 3’ homology arms can, in some embodiments, be at least 5kb, at least 10kb, at least 15kb, at least 20kb, at least 30kb, at least 40kb, at least 50kb, at least 60kb, at least 70kb, at least 80kb, at least 90kb, at least 100kb, at least 110kb, at least 120kb, at least 130kb, at least 140kb, at least 150kb, at least 160kb, at least 170kb, at least 180kb, at least 190kb, or at least 200kb.

In some embodiments, a LTVEC and DNA insert are designed to allow for a deletion of an endogenous sequence at a target locus from about 5kb to about 10kb, inclusive, from about 10kb to about 20kb, inclusive, from about 20kb to about 40kb, inclusive, from about 40kb to about 60kb, inclusive, from about 60kb to about 80kb, inclusive, from about 80kb to about 100kb, inclusive, or from about lOOkb to about 150kb, inclusive, from about 150kb to about 200kb, inclusive, from about 200kb to about 300kb, inclusive, from about 300kb to about 400kb, inclusive, from about 400kb to about 500kb, inclusive, from about 500kb to about 600kb, inclusive, from about 600kb to about 700kb, inclusive, from about 700kb to about 800kb, inclusive, or from about 500kb to about le, ive, from about le to about 1.5Mb, inclusive, from about 1.5Mb to about 2Mb, inclusive, from about 2Mb to about 2.5Mb, inclusive, or from about 2.5Mb to about 3Mb, inclusive. Alternatively, a deletion can be from about 3Mb to about 4Mb, inclusive, from about 4Mb to about 5Mb, inclusive, from about 5Mb to about lOMb, inclusive, from about 10Mb to about 20Mb, inclusive, from about 20Mb to about 30Mb, inclusive, from about 30Mb to about 40Mb, inclusive, from about 40Mb to about 50Mb, inclusive, from about 50Mb to about 60Mb, inclusive, from about 60Mb to about 70Mb, ive, from about 70Mb to about 80Mb, ive, from about 80Mb to about 90Mb, inclusive, or from about 90Mb to about lOOMb, inclusive. Alternatively, a deletion can be at least 10kb, at least 20kb, at least 30kb, at least 40kb, at least 50kb, at least 60kb, at least 70kb, at least 80kb, at least 90kb, at least 100kb, at least 150kb, at least 200kb, at least 250kb, at least 300kb, at least 350kb, at least 400kb, at least 450kb, or at least 500kb or greater.

In some ments, a LTVEC and DNA insert are designed to allow for an insertion into a target locus of an ous nucleic acid sequence ranging from about 5kb to about 10kb, inclusive, from about 10kb to about 20kb, inclusive, from about 20kb to about 40kb, inclusive, from about 40kb to about 60kb, inclusive, from about 60kb to about 80kb, inclusive, from about 80kb to about 100kb, inclusive, from about 100kb to about 150kb, inclusive, from about 150kb to about 200kb, inclusive, from about 200kb to about 250kb, ive, from about 250kb to about 300kb, inclusive, from about 300kb to about 350kb, inclusive, or from about 350kb to about 400kb, inclusive. Alternatively, an insertion can, in some embodiments, be from about 400kb to about 450kb, inclusive, from about 450kb to about 500kb, inclusive, from about 500kb to about 550kb, ive, from about 550kb to about 600kb, inclusive, about 600kb to about 650kb, inclusive, from about 650kb to about 700kb, inclusive, from about 700kb to about 750kb, inclusive, or from about 750kb to about 800kb, inclusive. Alternatively, an insertion can be, in some embodiments, at least 10kb, at least 20kb, at least 30kb, at least 40kb, at least 50kb, at least 60kb, at least 70kb, at least 80kb, at least 90kb, at least 100kb, at least 150kb, at least 200kb, at least 250kb, at least 300kb, at least 350kb, at least 400kb, at least 450kb, or at least 500kb or greater, In yet other cases, a DNA insert and/or a region of an endogenous locus being altered, deleted, targeted, modified, engineered, etc, is at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides or at least lkb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, llkb, 12kb, 13kb, 14kb, 15kb, 16kb, 17kb, 18kb, 19kb, 20kb or r. In some ments, a DNA insert and/or a region of an endogenous locus being altered, deleted, targeted, modified, engineered, etc, is nucleotides to 20kb, 200 nucleotides to 20kb, 300 tides to 20kb, 400 nucleotides to 20kb, 500 nucleotides to 20kb, 600 nucleotides to 20kb, 700 nucleotides to 20kb, 800 nucleotides to 20kb, 900 nucleotides to 20kb, lkb to 20kb, 2kb to 20kb, 3kb to 20kb, 4kb to 20kb, 5kb to 20kb, 6kb to 20kb, 7kb to 20kb, 8kb to 20kb, 9kb to 20kb, lOkb to 20kb, llkb to 20kb, 12kb to 20kb, 13kb to 20kb, 14kb to 20kb, 15kb to 20kb, l6kb to 20kb, 17kb to 20kb, 18kb to 20kb, or l9kb to 20kb. In some embodiments, a DNA insert and/or a region of an endogenous locus being altered, deleted, targeted, modified, engineered, etc, is 100 nucleotides to 19kb, 100 nucleotides to 18kb, 100 nucleotides to 17kb, 100 nucleotides to 16kb, 100 nucleotides to 15kb, 100 nucleotides to 14kb, 100 nucleotides to 13kb, 100 nucleotides to 12kb, 100 nucleotides to llkb, 100 tides to 10kb, 100 nucleotides to 9kb, 100 nucleotides to 8kb, 100 nucleotides to 7kb, 100 nucleotides to 6kb, 100 tides to 5kb, 100 nucleotides to 4kb, 100 nucleotides to 3kb, 100 nucleotides to 2kb, 100 nucleotides to lkb, 100 tides to 900 nucleotides, 100 nucleotides to 800 nucleotides, 100 nucleotides to 700 nucleotides, 100 nucleotides to 600 nucleotides, 100 nucleotides to 500 nucleotides, 100 nucleotides to 400 nucleotides, 100 nucleotides to 300 nucleotides, or 100 nucleotides to 200 nucleotides. In some embodiments, a DNA insert and/or a region of an endogenous locus being altered, deleted, targeted, modified, engineered, etc. is 200 nucleotides to 19kb, 300 nucleotides to 18kb, 400 nucleotides to 17kb, 500 nucleotides to 16kb, 600 nucleotides to 15kb, 700 nucleotides to 14kb, 800 nucleotides to 13kb, 900 nucleotides to 12kb, lkb to llkb, Zkb to 10kb, 3kb to 9kb, 4kb to 8kb, Skb to 7kb, or Skb to 6kb.

Provided non-human animals In certain aspects, non-human s are provided that express dies that contain light chains that include a human lg?» light chain sequence, in whole or in part, resulting from integration of genetic material that corresponds to at least a n of a human Ig7t light chain locus, and which encodes at least a human V?» domain (i.e., a rearranged human Vl—Jk sequence), in the place of corresponding non-human 1g?» light chain sequences in the germline genome of the non—human animal. Suitable es described herein include, but are not limited to, rodents, in particular, rats or mice.

A human 1g?» light chain sequence, in some embodiments, comprises c material from a human Igl light chain locus, wherein the human Ig7t light chain sequence s an immunoglobulin light chain that comprises the encoded n of the genetic material from the human ng light chain locus. In some embodiments, a human 1g?» light chain sequence as described herein comprises at least one human Vl gene segment and at least one human J)» gene t, and one or more sequences necessary to promote rearrangement (e, g, recombination signal sequence[s]) of said at least one human V?» gene segment with said at least one human J?» gene segment to form a functional rearranged human Vl—J?» sequence that encodes a human V7» domain. In many embodiments, a human Ig7t light chain sequence comprises a plurality of human V?» gene segments and one or more sequences necessary to promote rearrangement of said human V?» gene segments with at least one human I). gene segment. In many embodiments, a human 1g?» light chain sequence as described herein is a genomic ce of a human 1g?» light chain locus (cg, isolated and/or cloned from a bacterial artificial chromosome) and contains a plurality of human VA gene segments in ne ration. In some embodiments, a human 1g?» light chain sequence comprises human V)», J9» and C7» sequences in germline configuration (i.e., as said human V7», J?» and Ck sequences appear in an 1g?» light chain locus in a human cell). In some embodiments, a human 1g?» light chain sequence is or comprises a human sequence that appears in the Drawing (e.g., see Figures 1-4). In some embodiments, a human Ig7t light chain sequence encodes an ng light chain polypeptide, in whole or in part, which ng light chain polypeptide appears in an immunoglobulin, in particular, an immunoglobulin that is expressed by a human B cell. Non-human animals, embryos, cells and targeting constructs for making non-human animals, non-human s, and cells ning said human 1g?» light chain sequence in the place of a corresponding non-human 1g?» light chain sequence (e. g., an nous rodent 1g?» light chain locus) are also provided.

In some embodiments, a human Ig?» light chain sequence is inserted in the place of a corresponding non-human lg?» light chain sequence within the germline genome of a non-human animal, In some embodiments, a human Ig?» light chain sequence is inserted am of a non-human 1g?» light chain sequence (e.g., a non-human 1g?» light chain nt region sequence). In some embodiments, a human ng light chain sequence is inserted in the midst of one or more non—human 1g?» light chain sequences so that said human 1g?» light chain sequence is juxtaposed by non-human 1g?» light chain sequences (e.g., see s 1, 2, 3 and/or 4).

In some embodiments, one or more non-human ng light chain sequences (or portion thereof) of non-human ng light chain locus are not d. In some embodiments, one or more non-human ng light chain sequences (e.g., V7», I?» and/or C?») of a non-human lg?» light chain locus are altered, displaced, disrupted, deleted or replaced with, among other , a human 1g?» light chain sequence as described herein (e. g, a sequence that includes one or more human V% gene segments, one or more human 1% gene segments, one or more human C7» gene segments, or combinations thereof) operably linked to a non—human 1g?» light chain constant region, and one or more enhancer and/or regulatory element(s) of a non- human Ig?» light chain locus. In some embodiments, all or substantially all of a man 1g?» light chain locus is replaced with one or more human lg?» light chain sequences (as described herein) that is operably linked to a man lg?» light chain constant region and one or more non-human 1g?» light chain enhancer and/or regulatory element(s) of a non— human Ig?» light chain locus. In some certain embodiments, one or more non-human ng light chain constant region genes are not deleted or replaced in a non—human animal that includes a human ng light chain sequence as described herein. To give but one non-limiting example, in the instance of an insertion of a human 1g?» light chain sequence that is inserted into a non- human ng light chain locus, said insertion is made in manner to maintain the integrity of non-human 1g?» light chain sequences near the insertion point (e.g., a non—human lg?» light chain constant region and/or a non—human 1g?» light chain enhancer region or sequence).

Thus, such non-human animals have a wild—type 1g?» light chain constant region. In some embodiments, a non-human 1g?» light chain locus that is d, displaced, disrupted, deleted, replaced or engineered with one or more human 1g?» light chain sequences as described herein is a murine (e. g., mouse or rat) 1g?» light chain locus. In some embodiments, a human 1g?» light chain sequence is inserted into one copy (i.e., ) of a non—human 1g?» light chain locus of the two copies of said man ng light chain locus, giving rise to a non-human animal that is heterozygous with respect to the human 1g?» light chain sequence.

In some embodiments, a non-human animal is provided that is homozygous for an ng light chain locus that includes a human ng light chain sequence as described .

In some embodiments, an engineered non—human 1g?» light chain locus as described herein comprises human V)», I?» and Cl gene ts operably linked to a non- human Ig7t light chain nt region and one or more non—human ng light chain enhancers and/or regulatory ts. In some embodiments, an engineered non-human ng light chain locus as described herein comprises human V)», J?» and C7» gene segments operably linked to a non—human Ig7t light chain constant region, one or more non-human ng light chain enhancers and/or tory elements and one or more human ng light chain enhancers and/or regulatory ts.

In some embodiments, a non-human animal contains an engineered ng light chain locus as described herein that is ly integrated into its genome (e. g., as part of a randomly integrated human ng light chain sequence). Thus, such non-human animals can be described as having a human 1g?» light chain transgene ning a plurality of human V7», I?» and/or Ck gene segments configured such that said human V2, J1 and/or C?» gene segments are capable of rearrangement and encoding an Iglt light chain, in whole or in part, of an antibody in the expressed repertoire of the non—human animal. An engineered Ig7t light chain locus or transgene as described herein can be detected using a variety of methods including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism , or a gain or loss of allele assay. In some embodiments, a non—human animal as described herein is heterozygous with respect to an engineered 1g?» light chain locus as described herein. In some embodiments, a non—human animal as bed herein is hemizygous with respect to an engineered ng light chain locus as described herein. In some embodiments, a non-human animal as described herein contains one or more copies of an engineered 1g?» light chain locus or transgene as described herein. In some embodiments, a non-human animal as described herein contains an 1g?» light chain locus as depicted in the Drawing (e.g., see Figure l, 2, 3 and/or 4).

In some embodiments, compositions and methods for making non-human animals whose gerrnline genome comprises an engineered 1g?» light chain locus that includes one or more human Ig7t light chain sequences (e.g., human V2, J7t and/or Ck gene segments) in the place of non-human Ig7t light chain ces, including human 1g?» light chain encoding sequences that include specific polymorphic forms of human V7t, I?» and/or Ck segments (e.g, specific V and/or J alleles or variants) are provided, including compositions and methods for making non-human animals that express antibodies comprising 1g?» light chains that contain human variable domains and human or non-human constant domains, assembled from an 1g?» light chain locus that ns human V2, Pt and Ch segments operably linked to a non-human 1g?» light chain constant region. In some embodiments, compositions and s for making non-human animals that s such antibodies under the control of an endogenous enhancer(s) and/or an endogenous regulatory sequence(s) are also provided. In some ments, compositions and methods for making non-human animals that express such antibodies under the control of a heterologous enhancer(s) and/or a heterologous regulatory sequence(s) are also provided.

In certain embodiments, methods described herein include ing a sequence encoding a human 1g?» light chain, in whole or in part, upstream of a non-human 1g?» light chain nt region (e. g, a murine C?» region) so that an antibody is expressed, which antibody is characterized by the presence of a light chain that contains at least a human V7» domain and, in some embodiments, a human VA and C?» domain, and is expressed both on the e ofB cells and in the blood serum of a non-human animal.

In some embodiments, methods include serial insertion of genetic material corresponding to a human 1g?» light chain locus. In some embodiments, genetic material corresponding to a human 1g?» light chain locus can be synthetic or genomic (e.g, cloned from a bacterial artificial chromosome). In some embodiments, genetic material corresponding to a human ng light chain locus can be designed from hed sources and/or bacterial ial chromosomes so that said genetic material contains human V7», DL and/or C7t segments in an ation that is different from that which appears in a human 1g?» light chain locus yet said c material still contains sequences to support ngement of said human V)», J?» and/or C7» segments to encode a functional 1g?» light chain. To give but one example, genetic material corresponding to a human 1g?» light chain locus can be ed using the guidance provided herein to construct a human 1g?» light chain sequence that ns human V7», J7» and/or C?» segments in an order and/or arrangement that is different than that which appears in a human 1g?» light chain locus of a human cell. In such an example, content of human V)», J7t and/or C7» segments would be equivalent to the corresponding segments in a human cell, however, the order and arrangement would be different. When constructing a human Ig?» light chain locus for generation of a non-human animal as described herein the ite recombination signal sequences can be configured so that the human segments can correctly rearrange and form a functional 1g?» light chain. Guidance for ne configuration of human 1g?» light chain segments and sequences necessary for proper recombination can be found in Molecular Biology ofB Cells, London: Elsevier Academic Press, 2004, Ed. Honjo, T., Alt, F.W., Neuberger, M, Chapters 4 (pp, 37-59) and 5 (61-82), incorporated herein by reference in their entireties.

In some embodiments, serial insertion es multiple insertions of portions of heterologous genetic material in a single ES cell clone. In some embodiments, serial ion includes sequential insertions of portions of logous genetic material in successive ES cell clones.

In some embodiments, methods include ion of about 11,822bp ofDNA downstream of a murine (e.g., mouse or rat) CM region so that said DNA is ly linked to said murine (e. g., mouse or rat) CM region, which DNA includes one or more human 1g?» light chain enhancer regions (or sequences). In some certain embodiments, methods include insertion of about 11,822bp of DNA that comprises three human ng light chain enhancer regions (or sequences), which said three human 1g?» light chain enhancer regions (or sequences) are inserted downstream of said murine (e.g., mouse or rat) CM region.

] In some embodiments, methods include insertion of about 3bp of DNA upstream of a murine (e.g., mouse or rat) CM region so that said DNA is ly linked to said murine (e. g, mouse or rat) CM region, which DNA includes human V?» gene segments VX3-10, Vx3-9, VXZ-S, Vk4-3, VX3-l, human Jk-Ck segment pairs JM-CM, JkZ-CKZ, J23- CM, Jk6-C9t6 and human J7L gene segment M7. In some certain embodiments, methods include insertion of about 11,822bp of DNA that comprises one or more human Ig7t light chain enhancer regions (or sequences), which one or more human Igl light chain enhancer 2017/060006 regions (or sequences) are inserted downstream of said murine (e,g., mouse or rat) CM region.

In some embodiments, methods include insertion of about 171,45pr ofDNA upstream of a murine (e.g., mouse or rat) CM region so that said DNA is operably linked to said murine (e,g., mouse or rat) CM region, which DNA includes human Vk gene segments V7tZ-1 1, Vk3-12, V7tZ-14, VM-16, VM-19, VM-Zl, VM-22, V2223, VM-ZS and Vk3-27.

In some certain embodiments, methods include insertion of about 171,458bp of DNA upstream of a human VM-IO gene segment that is operably linked to a murine (e.g., mouse or rat) CM region, which DNA includes human V?» gene segments V7tZ-11, 2, V22- 14, Vk3-16, , VM—21, V7t3-22, VIZ-23, VK3-25 and V23-27.

In some embodiments, methods include insertion of about 121,188bp of DNA upstream of a murine (e.g., mouse or rat) CM region so that said DNA is operably linked to said murine (e.g., mouse or rat) CM region, which DNA includes human VA gene segments Vk3-27, VM-36, Vk5-37, , VM-40, VK7-43, VM-44, VKS-45, VK7-46, VM-47, Vk9-49, VM-Sl and VXS-SZ. In some certain embodiments, methods include ion of about 121,188bp ofDNA upstream of a human Vk3-27 gene segment that is ly linked to a murine (e.g., mouse or rat) CM region, which DNA includes human V9t gene segments Vk3—27, VM-36, VA5—37, V7t5—39, VM-40, VM-43, VM-44, Wis—45, V>t7—46, VM—47, Vk9-49, Vkl-Sl and VXS—SZ.

In some embodiments, methods include insertion of about 121,188bp ofDNA upstream of a murine (e.g., mouse or rat) CM region so that said DNA is ly linked to said murine (e.g., mouse or rat) CM region, which DNA includes human V?» gene segments Vk3-27, VM-36, vx5—37, V1569, VM-40, V7t7-43, VM-44, 5, van—46, VM-47, Vk9-49, VM-Sl and V2562, and which DNA includes a homology arm that includes a sequence that is 5’ of a mouse VKZ gene segment. In some n embodiments, methods include ion of about 121,188bp ofDNA upstream of a human V96 -27 gene segment that is operably linked to a murine (e.g., mouse or rat) CM region, which DNA includes human V7L gene segments Vl3-27, VM-36, V25-37, V25-39, VM-40, VM-43, VM-44, VAS-45, VM-46, VM-47, VA9-49, VM-51 and V2562, and which DNA includes a homology arm that es a mouse sequence that is 5’ of a mouse V22 gene segment to direct deletion of a mouse 1g?» genomic sequence (e.g., a 1g?» light chain locus) upon homologous recombination with said DNA nt.

Insertion of additional human V7», J?» and/or C?» gene segments may be achieved using methods described herein to further supplement the diversity of an engineered ng light chain locus. For example, in some embodiments, methods can include insertion of about 300kb ofDNA am of a murine (e.g., mouse or rat) CM region so that said DNA is operably linked to said murine (e.g., mouse or rat) CM region, which DNA includes human V7t gene segments , Vk6-57, Vk4-60, Vk8-6l and Vk4-69. In such embodiments, said DNA is ed upstream of a human Vk5—52 gene segment that is operably linked to a murine (e.g., mouse or rat) CM region, which DNA includes human Vk gene segments VMO-54, Vk6-57, Vk4-60, Vk8-6l and . In some certain embodiments, said DNA includes a human VpreB gene. Additional human V?» segments described above may be cloned ly from commercially available BAC clones and ed in smaller DNA fragment using recombinant techniques described herein or otherwise known in the art.

Alternatively, additional human V?» gene segments described above can be synthesized into a DNA fragment and added to an engineered 1g?» light chain locus as described above.

Likewise, additional human J7t and/or C?» gene segments may be obtained from commercially available BAC clones or synthesized directly from published sequences. Also, nous 1g?» light chain enhancer regions (or ces) may be deleted from an engineered 1g?» light chain locus as described herein. An exemplary illustration that shows an engineered 1g?» light chain locus of non-human animals as described herein is set forth in any one ofFigmres l, 2, 3 and 4.

Where appropriate, a human Igl light chain sequence (ie, a sequence containing human V7», J7» and/or Ck gene segments) ng an 1g?» light chain, in whole or in part, may separately be modified to include codons that are optimized for expression in a non- human animal (e.g., see US. Patent Nos. 5,670,356 and 5,874,304). Codon optimized sequences are synthetic sequences, and preferably encode the identical polypeptide (or a biologically active fragment of a full length polypeptide which has ntially the same activity as the full length polypeptide) d by the non—codon optimized parent polynucleotide. In some embodiments, a human 1g?» light chain sequence encoding an 1g?» light chain, in whole or in part, may separately include an altered sequence to optimize codon usage for a particular cell type (e.g., a rodent cell). For example, the codons of each nucleotide sequence to be inserted into the genome of a non-human animal (e.g., a rodent) may be optimized for expression in a cell of the non-human animal. Such a sequence may be described as a codon-optimized sequence.

In some embodiments, ion of a nucleotide sequence encoding a human Ig7t light chain, in whole or in part, employs a minimal modification of the germline genome of a non-human animal as described herein and results in expression of antibodies comprising light chains that are human, in whole or in part. Methods for generating engineered non- human animals, including knockouts and knock-ins, are known in the art (see, e.g., Gene Targeting: A Practical Approach, Joyner, ed., Oxford University Press, Inc., 2000). For example, generation of transgenic rodents may optionally involve disruption of the genetic loci of one or more endogenous rodent genes (or gene ts) and introduction of one or more heterologous genes (or gene segments or nucleotide sequences) into the rodent genome, in some ments, at the same location as an nous rodent gene (or gene segments). In some embodiments, a tide sequence encoding a human 1g?» light chain, in whole or in part, is uced upstream of a murine (e. g., mouse or rat) 1g?» light chain constant region gene of a randomly inserted ng light chain transgene in the germline genome of a rodent. In some embodiments, a nucleotide sequence encoding a human 1g?» light chain, in whole or in part, is introduced upstream of a murine (e.g., mouse or rat) Ig7t light chain nt region gene of an endogenous 1g?» light chain locus in the germline genome of a rodent; in some certain embodiments, an endogenous 1g?» light chain locus is altered, modified, or engineered to n human 1g?» gene segments (e.g., V)», I?» and/or C9») operably linked to a rodent CM region.

A schematic illustration (not to scale) of exemplary engineered 1g?» light chain loci is provided in Figures 1-4. In ular, s 1 and 3 sets forth exemplary strategies for uction of engineered 1g?» light chain loci terized by insertion of nucleotide sequences containing a plurality of human V)», IX and Cl segments. As illustrated in Figure 1, a DNA fragment containing a human EX ce (or region) is inserted downstream of a rodent C7t region via homologous recombination. This DNA fragment contains a Neomycin selection cassette (e.g., a Neomycin resistance gene [NEOR] flanked by loxP recombination recognition sites) positioned 3’ to the human EX sequence, which contains three human B?» elements engineered downstream (or 3 ’) of the rodent CM region. Also illustrated in Figure l is a DNA fragment containing a first portion of human V7» segments, a set of human JX-C?» segment pairs (e.g., human JM-CM, JkZ-CXZ, J7t3-Cl3, J7t6-Clt6) and a human M7 segment is inserted upstream of a rodent CM region via homologous recombination. As illustrated a Hygromycin selection cassette (e.g, a Hygromycin resistance gene [HYGR] flanked by Frt recombination recognition sites) is positioned on the 5’ end of the targeting vector and upstream of the human 1g?» light chain sequence contained in the targeting vector. The Hygromycin selection cassette is removed via homologous recombination with subsequent ing vectors described in the example n below. The targeting vector is then electroporated into rodent embryonic stem (ES) cells to create a rodent whose germline genome comprises the engineered lg?» light chain locus. Once a positive rodent ES cell clone is confirmed, the other depicted targeting s are electroporated in successive fashion and confirmed at each step to complete construction of the engineered lg?» light chain locus (see Figure 2). The final targeting vector may be designed with (6680 targeting vector) or without (6597 targeting vector) a homology arm that directs deletion of endogenous lg?» light chain segments via homologous recombination ing in two potential engineered 1g?» light chain alleles (Figure 2). Additionally, any remaining selection cassette may be deleted as d via recombinase-mediated deletion. An alternative strategy for inserting additional human V?» gene segments into an engineered 1g?» light chain locus using guide RNAs (gRNAs) is set forth in Figure 3.

Once a human ng light chain sequence is inserted upstream of a man 1g?» light chain constant region of a BAC clone, a targeting vector for integration into an Iglt light chain locus is created. The BAC clone ed with a human 1g?» light chain sequence for creating a targeting vector can contain 5’ and/or 3’ flanking c DNA of murine (e.g., mouse or rat) origin. Alternatively, or additionally, a BAC clone targeted with a human 1g?» light chain ce for creating a targeting vector can n 5’ and/or 3’ flanking genomic DNA of human origin so that a region of overlap with a human 1g}. light chain sequence is created. In this way, successive targeting of multiple engineered BAC clones is d (e.g., see Figure l). The final targeting vectors are incorporated into an 1g?» light chain locus in the genome of a non—human cell (e. g., a rodent embryonic stem cell). In some embodiments, targeting vectors as described herein are incorporated into an ng light chain locus in the germline genome of a non-human cell that further contains human VH, DH and JH genomic DNA (e.g, containing a plurality of human VH, DH and JH gene segments) operably linked with one or more IgH constant region genes and/or human VK and JK genomic DNA 2017/060006 (e.g, containing a plurality of human VK and JK gene segments) operably linked with an IgK constant region gene (e.g., see US. Patent Nos. 8,502,018, 8,642,835, 940 and 8,791,323, incorporated herein by reference in their entireties).

A targeting vector is introduced into rodent (e.g., mouse) embryonic stem cells by electroporation so that the sequence contained in the targeting vector is inserted into the genome of the rodent embryonic stem cells and results in the capacity of a non-human cell or non—human animal (e. g., a mouse) that expresses antibodies having human 1g?» light chains, in whole or in part. As described herein, a transgenic rodent is generated where an engineered 1g?» light chain locus has been created in the germline of the rodent genome (e.g., an endogenous ng light chain locus containing a human ng light chain sequence operably linked to an endogenous rodent Ck region as described herein). dies are expressed on the surface of rodent B cells and in the serum of said rodent, which antibodies are characterized by light chains having human V7» domains and, in some embodiments, human Vlt and C?» domains. When an endogenous 1g?» light chain locus in the germline of the rodent genome is not targeted by the targeting vector, an engineered 1g?» light chain locus is preferably inserted at a location other than that of an endogenous rodent 1g?» light chain locus (e.g, randomly inserted transgene).

Creation of an engineered 1g?» light chain locus in a non-human animal as described above provides an ered rodent strain that produces antibodies that e Ig?» light chains expressed from such an engineered Igl light chain locus having a human V)» , and in some embodiments, human Vk and Cl domains. Leveraged with the presence of an engineered IgH locus that includes a plurality of human VH, DH and JH gene segments operably linked to IgH constant region genes, an ered rodent strain that produces antibodies and antibody components for the development of human antibody-based eutics is created. Thus, a single engineered rodent strain is realized that has the capacity to provide an alternative in viva system for exploiting human V7t domains for the development of new antibody-based medicines to treat human disease.

In some embodiments, the genome of a man animal as described herein further comprises (e.g, via cross-breeding or multiple gene targeting strategies) one or more human immunoglobulin heavy and/or light chain variable regions as bed in US. Patent Nos. 8,502,018, 835, 8,697,940 and 8,791,323, all of which are incorporated herein by reference in their entireties. Alternatively, the engineered lg?» light chain locus as described herein can be engineered into an embryonic stem cell sing humanized IgH and/or IgK loci, or a non-human animal comprising engineered ng light chain locus described herein may be bred with another non—human animal sing humanized IgH and/or IgK loci.

Various such animals comprising humanized IgH and/or IgK loci are known, e.g, a VELOCIMMUNE® strain (see, e.g., US. Patent Nos. 8,502,018 and/or 8,642,835; incorporated herein by nce in their entireties), a XENOMOUSETM strain (see, e.g., Mendez, MJ. et al., 1997, Nat. Genetics 15(2):146-56 and Jakobovits, A. et al., 1995, Ann.

NY Acad, Sci. 764:525-3 5). Homozygosity of the engineered 1g?» light chain locus as described herein can subsequently be achieved by breeding. Alternatively, in the case of a randomly inserted engineered Ig7t light chain transgene (described above), rodent strains can be selected based on, among other things, expression of human V2 domains from the transgene.

Alternatively, and/or additionally, in some embodiments, the germline genome of a non-human animal as described herein further comprises a deleted, vated, functionally silenced or otherwise non-functional nous IgK light chain locus. c modifications to delete or render non-functional a gene or genetic locus may be achieved using methods described herein and/or methods known in the art.

A transgenic founder non-human animal can be identified based upon the ce of an engineered ng light chain locus in its germline genome and/or expression of antibodies having a human 1g?» light chain sequence, in whole or in part, in tissues or cells of the non-human animal. A transgenic founder non-human animal can then be used to breed additional man animals carrying the engineered 1g?» light chain locus thereby creating a cohort of man animals each carrying one or more copies of an engineered ng light chain locus. er, transgenic non-human animals carrying an engineered ng light chain locus as described herein can further be bred to other transgenic non-human animals carrying other transgenes (e.g., human immunoglobulin genes) as d.

In some ments, transgenic non—human animals may also be produced to contain selected systems that allow for regulated, directed, inducible and/or cell-type c expression of the transgene or integrated sequence(s). For example, non—human animals as described herein may be engineered to contain a sequence encoding a human ng light chain, in whole or in part, of an antibody that is/are conditionally expressed (e. g., ed in Rajewski, K. et al., 1996, J. Clin. Invest. 98(3):600-3). Exemplary s include the WO 28691 Cre/loxP recombinase system of bacteriophage P1 (see, e.g., Lakso, M. et al., 1992, Proc.

Natl. Acad. Sci. USA. 89:6232—6) and the FLP/Frt recombinase system of S. cerevisiae (O’Gorman, S. et a1, 1991, Science 251 :1351-5). Such animals can be provided h the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene comprising a selected modification (e.g., an engineered 1g?» light chain locus as described herein) and the other containing a transgene encoding a recombinase (e.g., a Cre recombinase).

Non-human animals as described herein may be prepared as described above, or using methods known in the art, to comprise onal human, humanized or otherwise engineered genes, oftentimes depending on the intended use of the man animal.

Genetic material of such human, humanized or otherwise engineered genes may be introduced through the further alteration of the genome of cells (e.g., embryonic stem cells) having the genetic modifications or tions as described above or through breeding techniques known in the art with other genetically d or engineered s as desired.

In some embodiments, non-human animals as described herein are prepared to r se transgenic human IgH and/or IgK light chain genes or gene segments (see e.g, Murphy, A.J. et al., (2014) Proc. Natl. Acad. Sci. USA. 111(14):5153-5158; US, Patent No. 018, US. Patent No. 8,642,835; US. Patent No. 8,697,940, US. Patent No: 8,791,323; and US. Patent ation Publication No. 2013/0096287 A1; incorporated herein by reference in their entireties).

In some embodiments, non-human animals as described herein may be prepared by introducing a targeting vector described herein into a cell from a modified strain. To give but one e, a targeting vector, as described above, may be introduced into a VELOCIMMUNE® mouse. VELOCIMMUNE® mice express antibodies that have fully human variable domains and mouse constant domains. In some embodiments, non-human animals as described herein are prepared to further comprise human immunoglobulin genes ble and/or constant region genes). In some embodiments, non-human animals as described herein comprise an engineered Ig7t light chain locus as described herein and genetic material from a heterologous species (e. g., humans), wherein the genetic material encodes, in whole or in part, one or more human heavy and/or IgK light chain variable domains.

For e, as described , non-human animals comprising an engineered 1g?» light chain locus as described herein may further comprise (e. g., via cross-breeding or 2017/060006 multiple gene targeting strategies) one or more modifications as described in Murphy, AJ. et al., (2014) Proc. Natl. Acad, Sci. USA. 111(14):5153-8; Macdonald, LE, et a1., 2014, Proc.

Natl. Acad. Sci. USA. ):5147-52, US. Patent Nos. 8,502,018, 8,642,835, 940 and 8,791,323, all of which are incorporated herein by reference in their entirety. In some embodiments, a rodent comprising an engineered ng light chain locus as bed herein is crossed to a rodent comprising a humanized IgH and/or IgK light chain variable region locus (see, e.g., US. Patent Nos. 8,502,018, 8,642,835, 8,697,940 and/or 8,791,323; incorporated herein by reference in their entireties). In some embodiments, a rodent comprising an engineered ng light chain locus as described herein is d to a rodent comprising a humanized IgH variable region locus (see, e.g., US. Patent Nos. 8,502,018, 8,642,835, 8,697,940 and/or 8,791,323; incorporated herein by reference in their entireties) and an inactivated endogenous IgK light chain locus (see, e.g., US. Patent Nos. 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662 and 9,163,092, incorporated herein by reference in their entireties).

Although embodiments describing the construction of an engineered ng light chain locus in a mouse (i.e., a mouse with an engineered Ig7t light chain locus characterized by the presence of a plurality of human V9t, J?» and Cl gene ts operably linked with a mouse C?» region so that antibodies containing human Ig?» light chains, in whole or in part, are sed) are extensively discussed herein, other non-human animals that comprise an engineered 1g?» light chain locus are also provided. Such non-human animals include any of those which can be cally modified to express dies as described herein, including, e.g., mammals, e.g., mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey), etc. For e, for those non-human animals for which suitable genetically modifiable ES cells are not readily available, other s are employed to make a non—human animal comprising the genetic modification. Such methods include, e.g, modifying a non-ES cell genome (e.g, a fibroblast or an induced pluripotent cell) and employing somatic cell nuclear transfer (SCNT) to transfer the genetically modified genome to a suitable cell, e.g., an enucleated oocyte, and ing the modified cell (e.g., the modified oocyte) in a non-human animal under le conditions to form an embryo.

Methods for modifying the germline genome of a non-human animal (e.g., a pig, cow, , chicken, etc. genome) include, e. g, employing a zinc finger se (ZFN), a transcription activator-like effector nuclease (TALEN), or a Cas protein (i.e., a CRISPR/Cas system) to include engineered Ig7t light chain locus as described . ce for s for modifying the ine genome of a non-human animal can be found in, e.g., US. Patent Application Nos. 14/747,461 (filed June 23, 2015), ,221 (filed November , 2015) and 14/974,623 (filed December 18, 2015), in which all three applications are hereby incorporated herein by reference in their entireties.

In some embodiments, a non-human animal as described herein is a mammal. In some embodiments, a non—human animal as described herein is a small mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, a genetically modified animal as described herein is a rodent. In some embodiments, a rodent as described herein is selected from a mouse, a rat, and a hamster. In some embodiments, a rodent as described herein is selected from the amily Muroidea. In some embodiments, a genetically modified animal as described herein is from a family selected from Calomyscidae (e.g., mouse-like rs), Cricetidae (e. g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), idae (climbing mice, rock mice, white-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g, spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In some certain ments, a genetically modified rodent as described herein is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a crested rat. In some n embodiments, a genetically modified mouse as described herein is from a member of the family e. In some embodiment, a non—human animal as described herein is a rodent. In some certain embodiments, a rodent as described herein is selected from a mouse and a rat. In some embodiments, a non-human animal as described herein is a mouse.

In some embodiments, a non—human animal as described herein is a rodent that is a mouse of a C57BL strain ed from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLWN, C57BL/6, C57BL/6J, C57BL/6By], C57BL/6NJ, C57BL/10, C57BL/1OScSn, C57BL/10Cr, and C57BL/Ola. In some certain embodiments, a mouse as bed herein is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, l29P3, 129X1, l29Sl (e.g., l29Sl/SV, l29Sl/SVIm), 12982, 12984, l29S5, 129S9/SVEVH, 129/SVJae, 12986 (l29/SVEVTac), 129S7, 12988, 129T], 129T2 (see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach, W. et al., 2000, Biotechniques 29(5): 1024-1028, 1030, 1032). In some certain embodiments, a genetically modified mouse as described herein is a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain. In some certain embodiments, a mouse as described herein is a mix of aforementioned 129 strains, or a mix of aforementioned BL/6 strains. In some certain embodiments, a 129 strain of the mix as described herein is a 12986 (129/SvaTac) strain.

In some embodiments, a mouse as described herein is a BALB strain, e.g, BALB/c strain. In some embodiments, a mouse as described herein is a mix of a BALB strain and r aforementioned strain.

In some embodiments, a non-human animal as described herein is a rat. In some certain embodiments, a rat as described herein is ed from a Wistar rat, an LEA strain, a Sprague Dawley , a Fischer strain, F344, F6, and Dark Agouti. In some certain embodiments, a rat strain as bed herein is a mix of two or more s selected from the group consisting of Wistar, LEA, Sprague Dawley, r, F344, F6, and Dark Agouti, A rat pluripotent and/or totipotent cell can be from any rat strain, including, for example, an ACI rat strain, a Dark Agouti (DA) rat strain, a Wistar rat strain, a LEA rat strain, a Sprague Dawley (SD) rat strain, or a Fischer rat strain such as Fisher F344 or Fisher F6. Rat pluripotent and/or totipotent cells can also be obtained from a strain derived from a mix of two or more strains recited above. For example, the rat pluripotent and/or totipotent cell can be from a DA strain or an ACI strain. The ACI rat strain is characterized as having black agouti, with white belly and feet and an RTlavl haplotype. Such strains are available from a variety of sources ing Harlan Laboratories. An example of a rat ES cell line from an ACI rat is an ACIGl rat ES cell. The Dark Agouti (DA) rat strain is characterized as having an agouti coat and an RTlavl haplotype. Such rats are available from a variety of sources including Charles River and Harlan Laboratories. Examples of a rat ES cell line from a DA rat are the DA.2B rat ES cell line and the DA.2C rat ES cell line. In some embodiments, the rat pluripotent and/or totipotent cells are from an inbred rat strain (see, e.g., US. Patent Application Publication No. 2014-0235933 A1, published August 21, 2014, incorporated herein by reference in its entirety).

Specific Exemplary Embodiments — Engineered IgH loci In some embodiments, provided non-human animals comprise an engineered 1g?» light chain locus as described herein and further comprise engineered IgH loci (or alleles) terized by the presence of a plurality of human VH, DH and JH gene segments arranged in ine configuration and ly linked to non-human IgH constant regions, enhancers and regulatory regions. In some embodiments, an engineered IgH locus (or ) as described herein comprises one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments operably linked to a non-human IgH constant region.

In some embodiments, an engineered IgH locus (or allele) comprises 5, 10, 15, , 25, 30, 35, 40 or more (e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, etc.) human vH gene segments. In some n embodiments, an ered IgH locus (or allele) comprises all or substantially all the onal human VH gene segments found between human VH3-74 and human VH6-1 gene segments, inclusive, of a human IgH locus that appears in nature. In some certain ments, an engineered IgH locus (or allele) ses at least human VH gene segments VH3-74, VH3-73, , VH2-70, VH1-69, VH3-66, VH3-64, VH4-6l, VH4— 59, vH1—58, vH3—53, VH5—51, VH3—49, vH3—48, VH1—46, VH1—45, VH3—43, VH4—39, VH4-34, VH3-33, VH4-31, VH3-30, VH4-28, VH2-26, VH1-24, VH3-23, VH3-21, VH3-20, VHl-18, VH3-15, VH3-l3, VH3-l l, VH3-9, VHl-8, VH3-7, VH2-5, VH7l, VH4-4, VHl-3, VHl-Z and VH6-1.

In some embodiments, an engineered IgH locus (or ) comprises 5, 10, 15, , 25 or more (e.g., 26, 27, etc.) human DH gene segments. In some n embodiments, an engineered IgH locus (or allele) comprises all or substantially all of the functional human DH gene segment found between a human DHl-l and human DH7-27 gene segment, inclusive, of a human IgH locus that appears in nature. In some certain embodiments, an engineered IgH locus (or allele) comprises at least human DH gene ts DHl-l, DH2-2, DH3—3, DH4-4, DH5—5, DH6—6, DH1—7, DH2—8, DH3-9, DH3—10, DH5—12, DH6—l3, DH2—15, DH3-l6, DH4-l7, DH6-l9, DHl-20, DH2-21, , DH6—25, DH1-26 and DH7-27.

In some embodiments, an engineered IgH locus (or allele) comprises 1, 2, 3, 4, 5, 6 or more functional human JH gene segments. In some certain embodiments, an engineered IgH locus (or allele) comprises all or substantially all the functional human JH gene segments found between human JHl and human JH6 gene segments, inclusive, of a human IgH locus that appears in nature. In some certain embodiments, an engineered IgH locus (or allele) ses at least human JH gene segments JHl, JH2, JH3, JH4, JHS and JH6.

In some ments, a non-human IgH constant region includes one or more non-human IgH constant region genes such as, for e, immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin E (IgE) and immunoglobulin A (IgA). In some certain ments, a non-human IgH constant region includes a rodent IgM, rodent IgD, rodent IgG3, rodent IgGl, rodent IgG2b, rodent IgG2a, rodent IgE and rodent IgA constant region genes. In some embodiments, said human VH, DH WO 28691 and JH gene segments are operably linked to one or more man IgH enhancers (i.e., enhancer sequences or enhancer regions), In some embodiments, said human VH, DH and JH gene segments are operably linked to one or more non-human IgH regulatory regions (or regulatory sequences). In some embodiments, said human VH, DH and JH gene segments are operably linked to one or more man IgH enhancers (or enhancer sequence) and one or more non—human IgH regulatory regions (or regulatory sequence).

In some embodiments, an engineered IgH locus as described herein does not contain an endogenous Adam6 gene. In some embodiments, an engineered IgH locus as described herein does not contain an endogenous Adam6 gene (or Adam6-encoding sequence) in the same germline c position as found in a germline genome of a wild- type non-human animal of the same species. In some embodiments, an engineered IgH locus as bed herein does not contain a human Adam6 pseudogene. In some embodiments, an engineered IgH locus as described herein comprises insertion of at least one nucleotide sequence that encodes one or more non-human (e. g., rodent) Adam6 polypeptides. Said insertion may be outside of an engineered immunoglobulin heavy chain locus as described herein (e.g., upstream of a 5’ most VH gene segment), within an engineered IgH locus or elsewhere in the germline genome of a non-human animal (e.g., a ly uced non- human Adam6-encoding sequence), cell or tissue, In various embodiments, a provided non-human animal, non-human cell or non- human tissue as described herein does not detectably s, in whole or in part, an endogenous non-human VH region in an antibody molecule. In various embodiments, a provided non-human animal, non-human cell or non—human tissue as described herein does not contain (or lacks, or ns a deletion of) one or more nucleotide sequences that encode, in whole or in part, an endogenous non-human VH region (e.g., VH, DH and/or JH) in an antibody molecule. In various embodiments, a ed non-human animal, non-human cell or non-human tissue as described herein has a germline genome that includes a deletion of endogenous non—human VH, DH and JH gene segments, in whole or in part. In various embodiments, a provided non-human animal is fertile.

] Guidance for the creation of targeting vectors, non-human cells and animals harboring such engineered IgH loci (or alleles) can be found in, e. g, US. Patent Nos. 8,642,835 and 8,697,940, which are incorporated by reference in their ties. Persons of skill in the art are aware of a variety of technologies, known in the art, for lishing such genetic engineering and/or lation of non—human (e.g, ian) genomes or for otherwise preparing, providing, or manufacturing such sequences for introducing into the germline genome of non-human animals.

Specific Exemplary Embodiments — Engineered Iglclight chain loci In some embodiments, provided non-human animals comprise an engineered 1g?» light chain locus as described herein and further comprise engineered IgK light chain loci (or alleles) characterized by the presence of a plurality of human VK and JK gene segments ed in germline configuration and operably linked to a non-human IgK light chain constant region, IgK enhancers and regulatory regions. In some embodiments, an engineered IgK light chain locus (or allele) comprises one or more human VK gene segments and one or more human JK gene segments operably linked to a man IgK constant region (CK).

In some certain embodiments, an engineered IgK light chain locus (or allele) comprises at least human VK gene segments that appear in the distal variable cluster (or distal arm, or distal duplication) of a human IgK light chain locus that appears in nature. In some certain ments, an engineered IgK light chain locus (or allele) comprises at least human VK gene ts that appear in the proximal variable cluster (or proximal arm, or proximal duplication) of a human IgK light chain locus that appears in nature. In some n ments, an engineered IgK light chain locus (or allele) comprises human VK gene segments that appear in the distal and proximal le clusters of a human IgK light chain locus that s in nature. In some certain embodiments, an engineered ng light chain locus (or allele) comprises all or substantially all the functional human VK gene segments found between human VK2-40 (or VK3D-7) and human VK4-l gene segments, inclusive, of a human IgK light chain locus that appears in nature.

In some n embodiments, an engineered IgK light chain locus (or allele) comprises 5, 10, 15, 20, 25, 30, 35 or more (e.g., 36, 37, 38, 39, 40 etc.) human VK gene segments. In some certain embodiments, an engineered IgK light chain locus (or allele) comprises human VK gene segments VK3D-7, VKlD-S, VKlD-43, VK3D-l l, VKlD-lZ, VKlD-l3, VK3D-15, VKlD-l6, 7, VK3D-20, VK6D-21, VK2D-26, VKZD-28, VKZD—29, szD—30, 3, VKlD—39, szD—40, , VKl—39, VKl—33, , VK2-28, VKl-27, VK2-24, VK6-21, vK3—20, , vK1—16, W345, W142, VK3-l 1, VKl-9, VKl-8, VKl-6, VKl-S, VKS-Z and VK4-l. In some certain embodiments, an engineered IgK light chain locus (or allele) ses at least human VK gene segments VK3D-7, VKlD-S, VKlD-43, VK3D-ll, VKlD-12, VKlD-l3, VK3D-15, VKlD-l6, VKlD- l7, VK3D—20, VK6D—21, VK2D—26, VKZD—28, VKZD—29, VK2D—30, VKlD-33, 9 and VK2D-4O. In some certain embodiments, an engineered IgK light chain locus (or allele) comprises at least human VK gene segments VK2-40, VKl-39, VKl-33, VK2-30, VK2—28, VKl-27, VK2-24, VK6-21, VK3-20, , VKl-16, VK3-15, VKl-IZ, VK3-l 1, VKl-9, VKl-8, VKl-6, VKl-S, VK5-2 and VK4-1, In some embodiments, an engineered IgK light chain locus (or allele) comprises 1, 2, 3, 4, 5 or more functional human JK gene segments. In some certain embodiments, an ered IgK light chain locus (or allele) comprises all or substantially all the functional human JK gene segments found between human JKl and human JKS gene segments, ive, of a human IgK light chain locus that appears in nature. In some certain embodiments, an engineered IgK light chain locus (or allele) comprises at least human JK gene segments JKl, JK2, JK3, JK4 and JKS.

In some embodiments, said human VK and JK gene ts are operably linked to one or more non-human IgK light chain enhancers (i.e., enhancer ces or enhancer regions). In some ments, said human VIC and JK gene segments are operably linked to one or more non-human IgK light chain regulatory regions (or regulatory sequences). In some embodiments, said human VK and J14 gene segments are operably linked to one or more non-human IgK light chain enhancers (or enhancer sequences or enhancer s) and one or more non—human IgK light chain regulatory regions (or regulatory sequences).

In some embodiments, a man CK region of an engineered IgK light chain locus (or allele) includes a rodent CK region such as, for example, a mouse CK region or a rat CK region. In some certain embodiments, a non-human CK region of an engineered IgK light chain locus (or allele) is or comprises a mouse CK region from a genetic background that includes a 129 strain, a BALB/c strain, a C57BL/6 strain, a mixed 129xC57BL/6 strain or combinations thereof.

In some embodiments, provided non-human animals comprise an ered 1g?» light chain locus as bed herein and further comprise an inactivated IgK light chain loci (or alleles).

In various ments, a ed non-human animal, non-human cell or non- human tissue as described herein does not detectably express, in whole or in part, an endogenous non-human VK region in an antibody molecule. In various embodiments, a provided non-human animal, non—human cell or non-human tissue as described herein does not contain (or lacks, or contains a deletion of) one or more nucleotide sequences that encode, in whole or in part, an endogenous non-human VK region in an antibody molecule.

In various embodiments, a provided non-human animal, non—human cell or non-human tissue as bed herein has a germline genome that includes a deletion of endogenous non- human VK and JK gene segments, in whole or in part.

Guidance for the creation of targeting s, non-human cells and animals harboring such engineered IgK light chain loci (or alleles) can be found in, e. g, US. Patent Nos. 8,642,835 and 8,697,940, which are hereby orated by reference in their entireties.

Persons of skill in the art are aware of a variety of technologies, known in the art, for accomplishing such genetic engineering and/or manipulation of non—human (e.g., mammalian) genomes or for otherwise ing, providing, or cturing such sequences for ucing into the germline genome of non-human s.

Specific Exemplary Embodiments — Engineered lg]. light chain loci In some embodiments, provided non—human animals comprise an ered 1g?» light chain locus characterized by the presence of a plurality of human V)», J7» and Cl gene segments arranged in ne configuration and inserted upstream of, and operably linked to, a non-human C)» gene segment (or C?» region gene). As described herein, such engineered lg?» light chain locus further includes one or more human lg?» light chain enhancer regions (or enhancer ces). In some embodiments, an engineered 1g)» light chain locus comprises one or more human V?» gene segments and one or more human J?» gene segments operably linked to a non—human lg?» light chain constant (CK) region. In some certain embodiments, an engineered ng light chain locus (or allele) comprises human V?» gene segments that appear in at least cluster A of a human 1g?» light chain locus; in some embodiments, cluster A and cluster B of a human ng light chain locus; in some certain embodiments, r A, cluster B and cluster C of a human ng light chain locus.

In some ments, an engineered 1g?» light chain locus (or allele) comprises , 10, 15, 20, 25, 30 or more (e.g., 31, 32, 33, 34, 35, etc.) human V?» gene segments. In some certain embodiments, an engineered 1g?» light chain locus (or ) comprises all or substantially all the functional human V% gene segments found between human Vk4-69 and human Vh3—l gene ts, inclusive, of a human 1g?» light chain locus that appears in . In some certain embodiments, an engineered 1g?» light chain locus (or allele) comprises all or substantially all the functional human V?» gene segments found between human VKS-SZ and human Vk3-l gene segments, inclusive, of a human 1g?» light chain locus that appears in nature. In some certain embodiments, an engineered Ig7t light chain locus (or allele) comprises all or ntially all the functional human V?» gene ts found between human V76 -27 and human Vk3-l gene segments, ive, of a human ng light chain locus that appears in nature. In some certain embodiments, an engineered Ig7t light chain locus (or allele) comprises human V7t gene segments Vk4-69, , Vk4-60, V76- 57, VMO-54, Vk5—52, VM—Sl, VA9—49, V7tl—47, Vk7-46, Vk5-45, VM-44, Vk7-43, VA]- 40, VX5—39, VX5—37, VKl—36, VK3—27, VX3—25, VX2—23, VK3—22, , Vk3—l9, VX2—18, Vl3-16, vxz—14, Vk3-12, mm 1, W340, vx3—9, vxz—s, va4-3 and Vk3-1. In some n embodiments, an engineered ng light chain locus (or allele) comprises at least the functional human V)» gene ts from VX5152 to V7tl-4O and from Vk3-27 to Vk3-l.

In some embodiments, an engineered 1g?» light chain locus (or allele) comprises 1, 2, 3, 4, 5, 6, 7 or more functional human IX gene segments. In some certain embodiments, an engineered 1g?» light chain locus (or allele) comprises all or substantially all the functional human IX gene segments found between human JM and human M7 gene segments, inclusive, of a human 1g?» light chain locus that appears in nature. In some certain embodiments, an ered Ig7t light chain locus (or allele) comprises at least human IX gene segments JM, 1X2, J76, JM and H7.

In some embodiments, an engineered 1g?» light chain locus (or allele) comprises 1, 2, 3, 4, 5, 6, 7 or more functional human C?» gene segments. In some certain embodiments, an engineered 1g?» light chain locus (or allele) comprises all or substantially all the functional human C?» gene ts found between human CM and human C7t7 gene segments, inclusive, of a human 1g?» light chain locus that appears in nature. In some certain embodiments, an engineered Ig7t light chain locus (or allele) comprises at least human Ck gene segments CM, CM, CM and CM.

In some embodiments, an engineered 1g?» light chain locus (or allele) does not contain the same non-human Ig?t light chain er s (or enhancer sequences) that appear in a wild—type 1g?» light chain locus (or allele). In some embodiments, an ered Ig?» light chain locus (or allele) lacks at least one non-human Ig?» light chain enhancer region (or enhancer sequence), in whole or in part (e. g., an 1g?» enhancer 2—4 or E?»2-4).

In some ments, said human V?» and J?» gene segments are operably linked to one or more non-human Ig?» light chain enhancers (ie, enhancer sequences or enhancer s) and one or more human 1g?» light chain enhancers (i.e., enhancer sequences or enhancer regions). In some embodiments, said human V?L and J?» gene segments are operably linked to one or more non-human Ig?t light chain regulatory regions (or regulatory sequences). In some embodiments, said human V?» and J?» gene segments are operably linked to one or more non—human Ig?» light chain enhancers (or enhancer sequences or enhancer regions), one or more human Ig?» light chain enhancers (i.e., enhancer sequences or enhancer regions) and one or more non-human Ig?» light chain regulatory s (or regulatory sequences).

In some embodiments, an engineered Ig?» light chain locus (or allele) as bed herein does not contain a human VpreB gene (or human VpreB gene—encoding sequence).

] In some embodiments, a non-human C?» region of an engineered lg?» light chain locus (or allele) includes a rodent C?» region such as, for example, a mouse C?» region or a rat C?» region. In some certain embodiments, a non-human C?L region of an engineered Ig?t light chain locus (or allele) is or comprises a mouse C?» region from a genetic background that includes a 129 strain, a BALB/c strain, a C57BL/6 strain, a mixed 129xC57BL/6 strain or combinations thereof.

In some embodiments, a non-human C?» region of an engineered Ig?t light chain locus (or allele) as described herein ses a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID N021 (mouse C?tl), SEQ 1]) N03 (mouse C?»2) or SEQ ID N015 (mouse C?»3). In some certain embodiments, a man C?» region of an engineered 1g?» light chain locus (or allele) as bed herein comprises a sequence that is substantially identical or identical to SEQ ID N021 (mouse C?»1), SEQ ID NO:3 (mouse C?»2) or SEQ ID N025 (mouse C?t3). In some certain embodiments, a non- W0 20181128691 human C2 region of an engineered Ig2 light chain locus (or allele) as described herein is or ses the sequence of a mouse CM region.

In some embodiments, a non—human C2 region d by a sequence positioned at an engineered Ig2 light chain locus (or allele) as described herein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:2 (mouse C21), SEQ ID NO:4 (mouse C22) or SEQ ID NO:6 (mouse C23). In some certain ments, a non-human C2 region encoded by a sequence oned at an engineered Ig2 light chain locus (or allele) as described herein comprises a sequence that is ntially identical or identical to SEQ ID N0:2 (mouse C21), SEQ ID NO:4 (mouse C22) or SEQ ID NO:6 (mouse C23). In some certain embodiments, a non-human C2 region encoded by a sequence positioned at an engineered Ig2 light chain locus (or allele) as described herein is or comprises a mouse CM region polypeptide.

In some ments, a non-human C2 region of an engineered Ig2 light chain locus (or allele) as described herein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:7 (rat CM), SEQ ID N019 (rat C22), SEQ ID NO:11 (rat C23) or SEQ ID NO: 13 (rat C24). In some certain ments, a non-human C2 region of an engineered Ig2 light chain locus (or allele) as described herein comprises a sequence that is substantially identical or identical to SEQ ID NO:7 (rat C21), SEQ ID N019 (rat C22), SEQ ID NO:11 (rat C23) or SEQ ID NO: 13 (rat C24). In some certain embodiments, a non-human C2 region of an engineered Ig2 light chain locus (or allele) as described herein is or comprises the sequence of a rat C21 region.

In some embodiments, a non-human C2 region encoded by a sequence positioned at an engineered Ig2 light chain locus (or allele) as described herein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:8 (rat C21), SEQ ID NO: 10 (rat C22), SEQ ID NO: 12 (rat C23) or SEQ ID NO:14 (rat C24).

In some n embodiments, a non-human C2 region d by a ce positioned at an engineered Ig2 light chain locus (or allele) as described herein comprises a sequence that is substantially identical or identical to SEQ ID NO:8 (rat C21), SEQ ID NO: 10 (rat C22), SEQ ID NO: 12 (rat C23) or SEQ ID NO: 14 (rat CM). In some certain embodiments, a non- human C?» region encoded by a sequence positioned at an engineered Ig?» light chain locus (or allele) as described herein is or comprises a rat CM region polypeptide.

In some embodiments, an ered Ig7t light chain locus (or allele) as described herein is characterized by the ce of one or more unique nucleotide sequence junctions (or combinations of unique sequence junctions) resulting from the insertion of human genetic material corresponding to a human 1g?» light chain sequence (genomic or synthetic) in the place of or within a non-human ng light chain sequence at an endogenous locus, Exemplary nucleotide sequence junctions are set forth in SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129.

In some ments, an engineered Ig2t light chain locus (or allele) as described herein comprises one or more of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID , SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128 and SEQ ID NO:129.

In some embodiments, an engineered 1g?» light chain locus (or allele) as described herein comprises SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO:123.

] In some embodiments, an engineered 1g?» light chain locus (or allele) as described herein comprises SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID , SEQ ID NO:121, SEQ IDNO:122 and SEQ ID NO:123.

In some ments, an engineered 1g?» light chain locus (or allele) as described herein comprises SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:128 and SEQ ID NO:129.

In some embodiments, an engineered 1g?» light chain locus (or ) as described herein comprises SEQ ID NO: 120, SEQ ID , SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126 and SEQ ID NO: 127.

] In some embodiments, an engineered Ig7t light chain locus (or ) as described herein comprises SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124 and SEQ ID NO:125.

Guidance on human V1, 17» and C2 gene segments can be found in, e.g., Lefranc, M.P., 2000, Nomenclature of the human immunoglobulin lambda (IGL) genes, Current Protocols in Immunology, No. Supplement, 40:A. 1p. 1-A. lp.37. Among other , the present disclosure demonstrates that the presence of human V?» and IX gene segments at 1g?» light chain loci (or alleles) increases the diversity of the light chain repertoire of a provided man animal as compared to the ity of the light chains in the expressed antibody repertoire of a non-human animal that does not comprise such engineered 1g?» light chain alleles.

Methods In certain aspects, non-human s as described herein may be employed for making a human antibody and/or nucleic acid sequencse encoding human antibodies, which human antibody comprises variable domains derived from nucleic acid sequences encoded by genetic material of a cell of a non-human animal as described herein. For example, a non- human animal as bed herein is immunized with an antigen of interest under conditions and for a time sufficient that the non-human animal develops an immune response to said antigen of interest. Antibodies are isolated from the non-human animal (or one or more cells, for example, one or more B cells) so immunized and characterized using various assays measuring, for example, affinity, specificity, epitope g, ability for blocking ligand- receptor interaction, inhibition receptor activation, etc. In various ments, antibodies produced by non—human animals as described herein comprise one or more human variable s that are derived from one or more human variable region tide ces isolated from the non-human animal. In some embodiments, anti-drug antibodies (e.g., anti- idiotype antibody) may be raised in non-human animals as described herein.

In some embodiments, non-human s as described herein provide an improved in viva system and source of biological materials (e. g, cells) for producing human antibodies that are useful for a variety of assays. In various embodiments, non—human animals as described herein are used to develop therapeutics that target a polypeptide of interest (e.g., a transmembrane or secreted ptide) and/or te one or more activities associated with said polypeptide of interest and/or te interactions of said polypeptide of interest with other binding partners (e. g., a ligand or or polypeptide).

For example, in various embodiments, non-human s as described herein are used to develop therapeutics that target one or more receptor ptides, modulate receptor polypeptide activity and/or modulate receptor polypeptide interactions with other binding partners, In various embodiments, non-human animals as described herein are used to identify, screen and/or p candidate therapeutics (e.g., antibodies, siRNA, etc.) that bind one or more polypeptides of interest. In various embodiments, non-human animals as described herein are used to screen and develop candidate therapeutics (e.g., antibodies, siRNA, etc.) that block activity of one or more polypeptides of interest or that block the activity of one or more receptor polypeptides of interest. In various embodiments, non- human animals as described herein are used to determine the g profile of antagonists and/or agonists of one or more polypeptides of st. In some embodiments, man animals as described herein are used to determine the epitope or es of one or more candidate therapeutic antibodies that bind one or more polypeptides of interest.

In s embodiments, non-human animals as described herein are used to determine the pharmacokinetic profiles of one or more human antibody candidates. In various embodiments, one or more non-human animals as described herein and one or more l or reference non—human s are each exposed to one or more human antibody candidates at various doses (e.g., 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/mg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg or more). Candidate therapeutic antibodies may be dosed via any desired route of administration including parenteral and non-parenteral routes of administration. Parenteral routes e, e. g., intravenous, intraarterial, intraportal, intramuscular, subcutaneous, eritoneal, intraspinal, intrathecal, intracerebroventricular, intracranial, intrapleural or other routes of injection. Non-parenteral routes include, e. g., oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, ocular. Administration may also be by continuous infusion, local administration, sustained release from implants (gels, membranes or the like), and/or enous injection. Blood is isolated from non-human animals (humanized and control) at various time points (e.g., 0 hr, 6 hr, 1 day, 2 days, 3 days, 4 days, days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 or more days). Various assays may be performed to determine the pharmacokinetic profiles of administered candidate therapeutic antibodies using samples ed from non—human animals as described herein including, but not limited to, total IgG, anti-therapeutic antibody se, agglutination, etc.

] In various embodiments, non—human animals as described herein are used to measure the therapeutic effect of blocking or modulating the activity of a polypeptide of st and the effect on gene expression as a result of cellular changes or, in the context of a receptor polypeptide, the density of a receptor polypeptide on the surface of cells in the non-human animals. In various ments, a non-human animal as described herein or cells isolated therefrom are exposed to a candidate therapeutic that binds a polypeptide of interest and, after a subsequent period of time, analyzed for effects on specific cellular processes that are associated with said polypeptide of interest, for example, ligand-receptor interactions or signal transduction.

In certain s, man s as described herein express human antibody variable domains, thus cells, cell lines, and cell cultures can be generated to serve as a source of human antibody variable domains for use in binding and functional assays, e. g., to assay for binding or function of an antagonist or t, particularly where the antagonist or agonist is specific for a human antigen of interest or specific for an epitope that functions in -receptor interaction (binding). In various embodiments, epitopes bound by candidate therapeutic antibodies or siRNAs can be determined using cells ed from non-human animals as described .

Cells from ed man animals can be ed and used on an ad hoc basis, or can be maintained in culture for many generations. In various embodiments, cells from a provided non-human animal are immortalized (e.g, via use of a virus) and maintained in culture indefinitely (e.g., in serial cultures).

In some embodiments, non-human s as described herein provide an in vivo system for the generation of ts of human antibody variable domains that binds a polypeptide of interest (e.g., human V?» domain variants). Such variants include human antibody variable domains having a desired functionality, specificity, low cross-reactivity to a common epitope shared by two or more variants of a polypeptide of interest. In some embodiments, non—human animals as described herein are employed to generate panels of human antibody variable domains that contain a series of variant le domains that are screened for a desired or improved functionality.

In certain aspects, non—human animals as described herein provide an in viva system for generating human antibody variable region libraries (e.g., a human V?» domain library). Such libraries e a source for heavy and/or light chain variable region sequences that may be grafted onto different Fc regions based on a desired effector function, used as a source for affinity maturation of the variable region sequence using ques known in the art (e.g., site-directed mutagenesis, error-prone PCR, etc.) and/or used as a source of antibody components for the generation of antibody-based therapeutic molecules such as, for example, chimeric antigen receptors (i.e., a molecule ered using antibody components, e.g., an scFv), specifrc binding agents (e. g., bi-specifrc g ) and fusion proteins (e. g., single domain antibodies, scFvs, etc.) In some aspects, non-human animals as described herein provide an in vivo system for the analysis and testing of a drug or vaccine. In various embodiments, a candidate drug or vaccine may be delivered to one or more non-human animals as described herein, followed by monitoring of the non-human animals to determine one or more of the immune response to the drug or vaccine, the safety profile of the drug or vaccine, or the effect on a disease or condition and/or one or more symptoms of a disease or condition. Exemplary methods used to determine the safety profile include measurements of toxicity, optimal dose concentration, antibody (ile, rug) response, cy of the drug or vaccine and possible risk factors. Such drugs or vaccines may be ed and/or developed in such non- human animals.

Vaccine efficacy may be determined in a number of ways, Briefly, non—human animals as bed herein are vaccinated using methods known in the art and then challenged with a e or a vaccine is administered to already-infected non-human animals. The response of a non-human animal(s) to a vaccine may be measured by ring of, and/or performing one or more assays on, the non-human animal(s) (or cells isolated therefrom) to determine the efficacy of the vaccine. The response of a non-human animal(s) to the vaccine is then compared with control animals, using one or more measures known in the art and/or described herein.

Vaccine efficacy may further be determined by viral neutralization assays.

Briefly, non-human animals as described herein are immunized and serum is collected on various days post-immunization. Serial dilutions of serum are pre-incubated with a virus during which time dies in the serum that are specific for the Virus will bind to it. The virus/serum mixture is then added to permissive cells to determine infectivity by a plaque assay or microneutralization assay. If antibodies in the serum neutralize the virus, there are fewer plaques or lower relative luciferase units compared to a control group.

] In some embodiments, non-human animals as described herein produce human antibody variable domains and, therefore, provide an in viva system for the production of human dies for use in stic applications (e. g, immunology, serology, iology, cellular pathology, etc.) In various embodiments, non-human animals as described herein may be used to produce human antibody variable domains that bind relevant antigenic sites for fication of cellular changes such as, for e, expression of specific cell surface markers indicative of pathological changes. Such dies can be conjugated to various chemical es (e.g., a radioactive tracer) and be employed in various in vivo and/or in vitro assays as desired.

In some embodiments, non-human animals as described herein provide an improved in vivo system for development and selection of human antibodies for use in oncology and/or infectious diseases. In various embodiments, non-human animals as described herein and control non-human animals (e.g, having a genetic modification that is ent than as described herein or no genetic modification, i.e., wild-type) may be implanted with a tumor (or tumor cells) or infected with a virus (e.g., influenza, HIV, HCV, HPV, etc). Following tation or infection, non-human animals may be administered a candidate therapeutic. The tumor or virus may be allowed sufficient time to be established in one or more locations within the non-human animals prior to administration of a candidate eutic, atively, and/or onally, the immune response may be monitored in such non-human animals so as to characterize and select potential human antibodies that may be developed as a therapeutic.

Kits In some aspects, the present disclosure further provides a pack or kit comprising one or more ners filled with at least one non—human animal, man cell, DNA fragment, targeting vector, or any combination thereof, as described herein. Kits may be used in any applicable method (e.g, a research method). ally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) directions for use, and/or (0) a contract that governs the transfer of materials and/or biological products (e.g., a non-human animal or non—human cell as described herein) between two or more entities and combinations thereof.

Other features of n embodiments will become nt in the course of the following descriptions of exemplary embodiments, which are given for illustration and are not ed to be limiting thereof.

WO 28691 Additional Exemplary Embodiments In exemplary embodiment 1, provided herein is a rodent whose germline genome comprises an endogenous immunoglobulin it light chain locus comprising (a) one or more human V2 gene segments, (b) one or more human J?» gene segments, and (c) one or more human C7» gene segments, wherein (a) and (b) are operably linked to (c) and a rodent Cl gene segment, and wherein the endogenous immunoglobulin 9» light chain locus further comprises: one or more rodent immunoglobulin A light chain enhancers (El), and one or more human immunoglobulin 2 light chain enhancers (EA).

In exemplary embodiment 2, provided herein is the rodent of embodiment 1, wherein the endogenous globulin 7» light chain locus comprises two rodent Eks.

In exemplary embodiment 3, provided herein is the rodent of embodiment 2, wherein the two rodent Eks are a mouse E?» and a mouse Ek3-l.

In exemplary embodiment 4, provided herein is the rodent of any one of embodiments 1-3, wherein the endogenous immunoglobulin it light chain locus comprises three human Eks.

In ary embodiment 5, provided herein is the rodent of any one of embodiments 1-4, n the germline genome further comprises (i) an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant region, or (ii) an endogenous immunoglobulin heavy chain locus sing insertion of one or more human VH gene ts, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene ts are operably linked to a rodent immunoglobulin heavy chain nt region, and an endogenous immunoglobulin K light chain locus comprising insertion of one or more human VK gene segments and one or more human JK gene segments, which human VK and JK gene segments are ly linked to a rodent immunoglobulin CK region.

In exemplary embodiment 6, provided herein is the rodent of embodiment 5, n the insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments replace rodent VH, DH gene segments.

In exemplary embodiment 7, provided herein is the rodent of embodiments 6, wherein the insertion includes human non-coding DNA that lly appears n human VH, DH, and JH gene segments, and combinations thereof.

In exemplary embodiment 8, provided herein is the rodent of embodiments 5 or 6, wherein the insertion of one or more human VK gene segments and one or more human JK gene segments replace rodent VK and JK gene segments.

In exemplary embodiment 9, ed herein is the rodent of embodiment 8, wherein the insertion includes human ding DNA that naturally appears between human VK and JK gene segments, and ations thereof.

In exemplary embodiment 10, provided herein is the rodent of any one of embodiments 5-8, wherein the rodent globulin heavy chain nt region is an endogenous rodent globulin heavy chain constant region.

In exemplary embodiment 11, provided herein is the rodent of any one of embodiments 5-10, wherein the rodent CK region is an endogenous rodent CK region, In exemplary embodiment 12, provided herein is the rodent of any one of embodiments 1-9, wherein the endogenous immunoglobulin 9» light chain locus comprises a deletion of endogenous V7t and Dr gene segments, in whole or in part.

In exemplary embodiment 13, provided herein is the rodent of embodiment 12, wherein the endogenous immunoglobulin ?» light chain locus comprises a deletion of V22- V7t3—J7tZ—C22 gene segments and VM—J7t3—C7t3 —J?tl gene segments.

In exemplary ment 14, provided herein is the rodent of embodiment 12, wherein the endogenous immunoglobulin ?» light chain locus ses a on of V22- Vk3-DtZ-C7tZ-J9t4P-CX4P gene segments and VM-JM-JMP-CM-JM gene segments, In exemplary embodiment 15, provided herein is the rodent of any one of embodiments 1-14, n the rodent C?» gene segment is a mouse CM gene segment, In exemplary embodiment 16, provided herein is the rodent of any one of ments 1-13, wherein the endogenous immunoglobulin 7» light chain locus comprises a deletion of a rodent E224.

In exemplary embodiment 17, provided herein is the rodent of any one of embodiments 1-16, wherein the rodent does not detectably express endogenous immunoglobulin 2» light chains.

In exemplary ment 18, provided herein is the rodent of any one of embodiments 5-17, wherein the immunoglobulin heavy chain locus comprises insertion of the human VH gene segments from VH3-74 to VH6-l, the human DH gene segments from DHl-l to DH7-27, and the human In gene ts JHl-JH6.

In exemplary embodiment 19, provided herein is the rodent of ment 18, wherein the insertion includes human non-coding DNA that naturally appears between human VH3-74 to VH6-1, human non-coding DNA that naturally appears between human DHl-l to DH7-27, and human non-coding DNA that naturally appears between human JHl- JH6.

In exemplary ment 20, provided herein is the rodent of any one of embodiments 5-19, wherein the immunoglobulin K light chain locus ses ion of the proximal VK duplication, in whole or in part, of a human immunoglobulin K light chain locus.

In exemplary embodiment 21, provided herein is the rodent of embodiment 20, wherein the immunoglobulin K light chain locus comprises insertion of the human VK gene segments from VK2-4O to VK4-l and the human JK gene segments from JKl-JKS, In exemplary embodiment 22, provided herein is the rodent of embodiment 21, n the insertion includes human non-coding DNA that naturally appears between human VK2-40 to VK4-1, and human non-coding DNA that naturally appears between human JK l-JKS.

] In exemplary ment 23, provided herein is the rodent of any one of embodiments 1—22, wherein the endogenous globulin 7t light chain locus comprises insertion of the human V?» gene segments VKS-SZ to VM—40 and VK3-27 to VX3-1, at least the human INC?» gene segment pairs Ill-CM, JX2-C9t2, Jk3-C2t3, JA6-CX6, the human J?» gene segment J7L7 and a rodent CM gene segment.

In exemplary embodiment 24, provided herein is the rodent of embodiments 23, wherein the insertion includes human non-coding DNA that naturally appears between human V1562 to VM-40 and V13 -27 to Vk3-l, human non-coding DNA that lly appears n human Jk—C?» gene segment pairs Ill—CM, J7t2—CKZ, J7t3-C7t3 and M6— C16, and human non-coding DNA that naturally appears upstream (or 5’) of human I?» gene segment 1M.

In ary embodiment 25, provided herein is the rodent of any one of embodiments 5-24, wherein the immunoglobulin heavy chain locus lacks an endogenous rodent Adam6 gene.

In exemplary embodiment 26, provided herein is the rodent of any one of embodiments 5-25, n the immunoglobulin heavy chain locus further comprises insertion of one or more nucleotide ces encoding one or more rodent Adam6 polypeptides In exemplary ment 27, provided herein is the rodent of embodiment 26, wherein the one or more nucleotide sequences are inserted n a first and a second human VH gene segment.

In exemplary ment 28, provided herein is the rodent of embodiment 26, wherein the one or more nucleotide sequences are inserted in the place of a human Adam6 gene, In exemplary embodiment 29, provided herein is the rodent of embodiment 27, wherein the first human VH gene segment is human VHl-2 and the second human VH gene segment is human VH6-l.

] In exemplary embodiment 30, provided herein is the rodent of embodiment 26, wherein the one or more nucleotide sequences are inserted between a human VH gene segment and a human DH gene segment.

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

In exemplary embodiment 32, provided herein is the rodent of any one of embodiments 5-3], wherein the rodent is heterozygous or homozygous for the endogenous immunoglobulin K light chain locus.

In exemplary ment 33, provided herein is the rodent of any one of embodiments 1-32, wherein the rodent is heterozygous or homozygous for the endogenous immunoglobulin 7t light chain locus.

In exemplary ment 34, provided herein is the rodent of any one of embodiments 1-33, wherein the rodent is a rat or a mouse.

In exemplary embodiment 35, provided herein is an isolated rodent cell whose germline genome comprises an endogenous immunoglobulin 9t light chain locus comprising: (a) one or more human V?» gene segments, (b) one or more human JA. gene segments, and (c) WO 28691 one or more human C?» gene segments, (i) wherein (a) and (b) are operably linked to (c) and a rodent C?» gene segment, and (ii) wherein the endogenous globulin 2 light chain locus further comprises: one or more rodent immunoglobulin 2 light chain enhancers (E2) and one or more human immunoglobulin 7t light chain enhancers (EX).

In exemplary embodiment 36, provided herein is an immortalized cell made from the rodent cell of embodiment 35.

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

In exemplary embodiment 38, provided herein is a rodent embryo generated from the rodent embryonic stem cell of embodiment 35.

In exemplary embodiment 39, ed herein is a method of making a rodent whose germline genome comprises an engineered endogenous immunoglobulin 2 light chain locus, the method comprising (a) introducing a DNA fragment into a rodent embryonic stem cell, said DNA fragment comprising a nucleotide sequence that includes (i) one or more human V7L gene segments, (ii) one or more human I?» gene ts, and (iii) one or more human C7» gene segments, wherein (i)—(iii) are operably linked to a rodent C7» gene segment, and wherein the tide sequence further comprises one or more human immunoglobulin A light chain enhancers (El); (b) obtaining the rodent embryonic stem cell generated in (a); and (c) creating a rodent using the rodent embryonic stem cell of (b).

In ary embodiment 40, provided herein is the method of embodiment 39, wherein the nucleotide sequence further includes and one or more human immunoglobulin 2 light chain enhancers (EX).

In exemplary ment 41, provided herein is a method of making a rodent whose germline genome comprises an engineered endogenous immunoglobulin 7t light chain locus, which engineered endogenous immunoglobulin 9» light chain locus comprises insertion of one or more human V?» gene segments, one or more human IX gene ts and one or more human C7t gene ts, which human V?» and J?» gene segments are operably linked to a rodent or a human CA gene segment, and which endogenous immunoglobulin 7t light chain locus r comprises one or more rodent immunoglobulin 7t light chain enhancers (El), and one or more human immunoglobulin 2» light chain enhancers (Eh), the method comprising modifying the germline genome of a rodent so that it comprises an engineered immunoglobulin 2 light chain locus that includes insertion of one or more human V2 gene segments, one or more human J2 gene segments and one or more human C2 gene segments, which human V2 and J2 gene segments are operably linked to a rodent or a human C2 gene segment, and which endogenous immunoglobulin 2 light chain locus r comprises one or more rodent immunoglobulin 2 light chain enhancers (E2), and one or more human immunoglobulin 2 light chain enhancers (E2), y making said rodent.

In exemplary embodiment 42, ed herein is the method of embodiment 39 or 41, wherein the one or more human V2 gene ts include V25-52 to V2l-40 and/or V23-27 to V23-l.

In ary ment 43, provided herein is the method of embodiment 42, wherein the one or more human V2 gene segments include human non-coding DNA that naturally appears between human V25-52 to V21-40 and/or V23 -27 to V23-l, In exemplary embodiment 44, provided herein is the method of any one of embodiments 39-43, wherein the one or more human J2 gene segments and the one or more human C2 gene segments include the human J2-C2 gene segment pairs J2l-C2l, J22-C22, J23-C23, J26—C26 and the human J27 gene segment.

In exemplary embodiment 45, provided herein is the method of embodiment 44, wherein the human J2—C2 gene segment pairs J2l—C2l, J22-C22, J23 —C23 and J26—C26 include human non-coding DNA that naturally appears between the human J2 and C2 gene segment pairs, and the human J27 gene segment includes human non-coding DNA that naturally appears upstream (or 5’) of human J27.

In exemplary embodiment 46, provided herein is the method of any one of embodiments 39-45, wherein the rodent C2 gene segment is a mouse C21 gene segment.

In exemplary embodiment 47, ed herein is the method of any one of ments 39-46, wherein the endogenous immunoglobulin 2 light chain locus comprises three human E2s.

In exemplary embodiment 48, provided herein is the method of any one of embodiments 39-46, wherein endogenous immunoglobulin 2 light chain locus comprises two rodent E2s.

] In exemplary embodiment 49, provided herein is the method of embodiment 48, wherein the two rodent E2s are a mouse E2 and a mouse E23-l. 2017/060006 In exemplary embodiment 50, provided herein is the method of any one of embodiments 38 and 42-49, n the DNA fragment further comprises one or more selection markers.

] In exemplary embodiment 51, provided herein is the method of any one of embodiments 39 and 42-50, wherein the DNA fragment further ses one or more site- specific ination sites.

In ary embodiment 52, provided herein is the method of any one of embodiments 39 and 42-51, wherein the DNA fragment of (a) is introduced into a rodent embryonic stem cell Whose germline genome ses an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant region, or an endogenous immunoglobulin heavy chain locus sing insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant region, and an endogenous immunoglobulin K light chain locus comprising insertion of one or more human VK gene segments and one or more human JK gene ts, which human VK and JK gene segments are operably linked to a rodent immunoglobulin CK region.

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

] In exemplary embodiment 54, provided herein is the method of any one of embodiments 47-49, wherein the modifying the germline genome of a rodent so that it comprises an engineered immunoglobulin h light chain locus is carried out in a rodent embryonic stem cell whose ne genome further comprises an endogenous immunoglobulin heavy chain locus sing insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin 2017/060006 heavy chain constant region; or an nous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant , and an endogenous immunoglobulin K light chain locus comprising insertion of one or more human VK gene segments and one or more human JK gene segments, which human VK and JK gene segments are operably linked to a rodent immunoglobulin CK region.

In exemplary embodiment 55, provided herein is the method of embodiment 52 or 54, wherein the insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments es human non-coding DNA that naturally appears between the one or more human VH gene segments, human non- coding DNA that naturally appears between the one or more human DH gene segments and human non-coding DNA that lly appears between the one or more human JH gene segments.

In exemplary embodiment 56, provided herein is the method of embodiment 52 or 54, wherein the insertion of one or more human VK gene segments and one or more human JK gene segments includes human ding DNA that naturally appears between the one or more human VK gene segments and human non-coding DNA that naturally appears between the one or more human JK gene segments.

In exemplary embodiment 57, provided herein is the method of any one of embodiments 41-49, n the modifying the germline genome of a non-human animal so that it comprises an ered immunoglobulin X light chain locus is d out in a non- human embryonic stem cell whose germline genome comprises a wild—type nous globulin heavy chain locus; or a wild-type endogenous immunoglobulin heavy chain locus and a wild-type endogenous immunoglobulin K light chain locus, and wherein the method further comprises a step of breeding a mouse produced from said non-human embryonic stem cell with a second mouse.

In exemplary embodiment 58, provided herein is the method of embodiment 53 or 57, n the second mouse has a germline genome comprising wild-type IgH and IgK loci.

In exemplary embodiment 59, provided herein is the method of embodiment 53 or 57, wherein the second mouse has a germline genome comprising homozygous or heterozygous humanized IgH and IgK loci, which homozygous or heterozygous humanized IgH locus contains an inserted rodent Adam6-encoding sequence.

In exemplary embodiment 60, provided herein is the method of embodiment 53 or 57, n the second mouse has a germline genome sing a homozygous or heterozygous humanized IgH locus and a homozygous or heterozygous inactivated IgK locus.

In exemplary embodiment 61, provided herein is a method of producing an antibody in a rodent, the method comprising the steps of (l) immunizing a rodent with an antigen of interest, which rodent has a germline genome comprising an endogenous immunoglobulin 7t light chain locus comprising (ai) one or more human V?» gene segments, (b) one or more human J3’» gene segments, and (c) one or more human C?» gene segments, wherein (a) and (b) are operably linked to (c) and a rodent Ck gene segment, and wherein the endogenous immunoglobulin 7» light chain locus further comprises: one or more rodent immunoglobulin A light chain enhancers (El) and one or more human immunoglobulin 7t light chain enhancers (El); (2) maintaining the rodent under conditions sufficient that the rodent produces an immune se to the antigen of interest, and (3) recovering an antibody from the rodent, or a rodent cell, that binds the antigen of st.

In exemplary embodiment 62, provided herein is the method of embodiment 61, n the rodent has a germline genome further comprising: an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene ts and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant region, or an endogenous immunoglobulin heavy chain locus comprising ion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant region, and an endogenous immunoglobulin K light chain locus compiising insertion of one or more human VK gene segments and one or more human JK gene segments, which human VK and JK gene segments are ly linked to a rodent immunoglobulin CK region.

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

In exemplary embodiment 64, provided herein is the method of embodiment 61 or 62, wherein the rodent cell is a oma.

In exemplary embodiment 65, provided herein is the method of any one of embodiments 61-64, wherein the endogenous globulin 2 light chain locus comprises insertion of the human V7t gene segments VKS-SZ to VM-40 and Vl3-27 to VK3-1, the human t gene segment pairs JM—CM, JXZ—CKZ, JK3—C7v3, JK6-Ck6 and the human I?» gene segment H7.

In exemplary ment 66, provided herein is the method of embodiment 65, wherein the insertion includes human non—coding DNA that naturally appears between human V7t V2562 to VM-40 and Vk3-27 to Vk3-1, human non-coding DNA that naturally appears between human Jk-Ck gene segment pairs Ill-CM, JAZ-CXZ, JR3-C9t3 and M6- C9t6, and human non—coding DNA that naturally appears upstream (or 5’) of human J7» gene segment J17.

In exemplary embodiment 67, provided herein is the method of any one of embodiments 61-66, wherein the rodent C?» gene t is a mouse CM gene segment.

In exemplary ment 68, provided herein is the method of any one of embodiments 62-67, wherein the immunoglobulin heavy chain locus comprises insertion of the human VH gene segments from VH3-74 to VH6-l, the human DH gene segments from DH1—1 to DH7—27 and the human JH gene segments JHl—JH6, and which human VH, DH and JH gene segments are ly linked to an nous rodent immunoglobulin heavy chain constant region.

In exemplary embodiment 69, provided herein is the method of embodiment 68, wherein the insertion includes human ding DNA that naturally appears between human VH3-74 to VH6-1, human non-coding DNA that naturally appears between human DHl—l to DH7—27, and human non-coding DNA that naturally appears between human JHl- JH6.

In exemplary embodiment 70, provided herein is the method of embodiment 68, n the human VH, DH and JH gene segments replace rodent VH, DH and JH gene In exemplary embodiment 71, provided herein is the method of any one of embodiments 62-70, wherein the immunoglobulin K light chain locus comprises insertion of the human VK gene segments from VK2-40 to VK4-1 and the human JK gene segments from JKl-JKS, and which human VK and JK gene segments are operably linked to an endogenous rodent immunoglobulin CK region, In exemplary embodiment 72, provided herein is the method of embodiment 71, wherein the insertion includes human non-coding DNA that naturally appears between human VK2-40 to VK4-l, and human non-coding DNA that naturally appears between human JKl-JKS.

In exemplary embodiment 73, provided herein is the method of embodiment 71, wherein the human VK and JK gene segments replace rodent VK and JK gene segments.

In exemplary embodiment 74, ed herein is the method of any one of embodiments 61-73, wherein the germline genome of the rodent further comprises insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides.

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

In exemplary embodiment 76, ed herein is the method of embodiment 75, wherein the immunoglobulin heavy chain locus further ses insertion of one or more tide sequences encoding one or more rodent Adam6 polypeptides.

In exemplary embodiment 77, provided herein is the method of embodiment 76, wherein the one or more nucleotide ces ng one or more rodent Adam6 polypeptides are inserted between a first and a second human VH gene segment.

In exemplary embodiment 78, ed herein is the method of embodiment 77, wherein the first human VH gene segment is human VH1-2 and the second human VH gene segment is human VH6-1.

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

In ary embodiment 80, provided herein is the method of embodiment 76, wherein the one or more nucleotide ces encoding one or more rodent Adam6 polypeptides are ed between a human VH gene segment and a human DH gene segment.

] In exemplary embodiment 81, provided herein is the method of any one of embodiments 61-80, wherein the antibody recovered from the rodent, or a rodent cell, that binds the antigen of interest comprises a human heavy chain variable domain and a human lambda light chain variable domain, WO 28691 In exemplary embodiment 82, provided herein is the method of embodiment 81, wherein the human heavy chain variable domain includes a rearranged human VH gene segment selected from the group consisting of VH3-74, , VH3-72, VH2-70, VH1-69, VH3-66, VH3-64, VH4-61, VH4-59, VH1-58, VH3-53, VHS—51, VH3—49, VH3-48, , VH1-45, VH3-43, VH4-39, VH4-34, VH3—33, VH4-31, VH3—30, VH4-28, VH2-26, VH1-24, VH3-23, VH3-21, , VH1-18, VH3-15, VH3-13, , VH3-9, VH1-8, VH3-7, VHZ-S, VH71, VH4-4, VHl-3, VHl-Z and VH6-l.

In exemplary embodiment 83, provided herein is the method of embodiment 81 or 82, n the human lambda light chain variable domain includes a rearranged human V?» gene segment selected from the group consisting of Vk4-69, V18-61, VK4-60, V1667, V7t10-54, vxs-sz, VM-Sl, V1949, V7t1—47, V7t7-46, VX5-45, VM-44, V7t7-43, VM-40, W639, W537, VM—36, Vk3—27, VX3—25, V12—23, VX3—22, VX3—21, W349, W248, V9346, V1244, vm—iz, mm 1, WHO, vxs—9, vxz—s, vx4-3 and vx3-1.

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

In exemplary embodiment 85, provided herein is a rodent whose germline genome comprises a homozygous endogenous globulin 7» light chain locus comprising: (i) human VA gene segments VXS-SZ to VM-40 and V7L3 -27 to Vl3-l, (ii) human INC?» gene segment pairs JM-CM, JM-CM, JK3-C9t3 and t6, (iii) human J?» gene segment J27, and (iv) three human immunoglobulin A light chain enhancers; wherein (i)—(iv) are operably linked to each other and (i)-(iii) are upstream of a rodent C?» gene segment, and wherein the endogenous immunoglobulin 7» light chain locus lacks an endogenous rodent immunoglobulin E22-4, the human V7t gene segments VXS-SZ to VM-4O and V23 -27 to Vk3-1 includes human non—coding DNA that naturally s between the human V?» gene segments, the human INC?» gene segments pairs Jll-CM, JlZ-CKZ, J76- CB and Jlé-CM includes human non-coding DNA that naturally appears between the human Jl—C?» gene ts pairs, and the human IX gene segment M7 includes human non-coding DNA that naturally s am (or 5’) of human J27.

In exemplary embodiment 86, provided herein is the rodent of embodiment 85, wherein the rodent C?» gene segment is a mouse CM gene segment.

In exemplary embodiment 87, provided herein is the rodent of embodiment 85 or 86, wherein the nous immunoglobulin 7» light chain locus further comprises endogenous rodent immunoglobulin 9» light chain enhancers El and EA3-l.

In ary embodiment 88, provided herein is the rodent of any one of embodiments 85-87, wherein the endogenous globulin 9» light chain locus comprises a deletion of endogenous rodent VX2—V7t3 -J7t2-C7t2—J7t4P-C7L4P gene segments and VM- J7t3-J7t3P-C9t3-J7tl gene segments.

In exemplary embodiment 89, provided herein is the rodent of any one of embodiments 85-88, wherein the rodent is a rat or a mouse, In some embodiments, provided herein is a rodent whose germline genome comprises an endogenous immunoglobulin )L light chain locus sing (a) one or more human V?» gene segments, (b) one or more human J7» gene segments, and (c) one or more human Ck gene ts, wherein (a) and (b) are operably linked to (c) and a rodent Ck gene segment, and wherein the endogenous immunoglobulin 7» light chain locus further comprises: one or more rodent immunoglobulin 9» light chain enhancers (EX), and one or more human immunoglobulin A light chain enhancers (EA), In some embodiments, the endogenous immunoglobulin 7» light chain locus comprises two rodent Eks.

In some embodiments, the two rodent Eks are a mouse El and a mouse EM-l.

In some embodiments, the endogenous immunoglobulin 7» light chain locus comprises three human Els, ] In some embodiments, the ne genome further comprises (i) an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant region; or (ii) an nous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are ly linked to a rodent immunoglobulin heavy chain nt region, and an endogenous immunoglobulin K light chain locus comprising insertion of one or more human VK gene ts and one or more human JK gene segments, which human VK and JK gene segments are operably linked to a rodent immunoglobulin CK region.

WO 28691 In some embodiments, the insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments replace rodent VH, DH gene segments.

In some embodiments, the insertion includes human non-coding DNA that naturally appears between human VH, DH, and JH gene segments, and ations thereof.

In some embodiments, the insertion of one or more human VK gene segments and one or more human JK gene segments replace rodent VK and JK gene segments.

In some embodiments, the insertion includes human non-coding DNA that naturally appears between human VK and JK gene segments, and combinations thereof.

In some embodiments, the rodent immunoglobulin heavy chain constant region is an nous rodent immunoglobulin heavy chain constant .

In some embodiments, wherein the rodent CK region is an endogenous rodent CK region.

In some embodiments, the endogenous immunoglobulin 7» light chain locus comprises a deletion of endogenous V?» and J7» gene segments, in whole or in part.

In some embodiments, the endogenous immunoglobulin 7» light chain locus comprises a deletion of VX2—V7t3 —J7t2—C7tZ gene segments and VM—J7t3—C7t3—J7tl gene segments.

In some embodiments, the endogenous immunoglobulin X light chain locus comprises a deletion of vaz—vm -J?tZ-Ck2-J?t4P-CX4P gene segments and VM-JB-JMP— Cl3-J7tl gene segments.

In some embodiments, the rodent C?» gene segment is a mouse CM gene segment.

In some embodiments, the endogenous globulin 1 light chain locus ses a deletion of a rodent El2-4.

In some embodiments, the rodent does not detectably express endogenous globulin 7» light .

In some embodiments, the globulin heavy chain locus comprises insertion of the human VH gene segments from VH3-74 to VH6—l, the human DH gene segments from DH1-l to DH7-27, and the human JH gene segments JHl-JH6.

In some embodiments, the insertion includes human non-coding DNA that lly appears between human VH3—74 to VH6—l, human non—coding DNA that naturally appears between human DH1-1 to DH7-27, and human non-coding DNA that naturally appears between human 6.

In some embodiments, the immunoglobulin K light chain locus comprises insertion of the proximal VK duplication, in whole or in part, of a human globulin K light chain locus.

In some embodiments, the immunoglobulin K light chain locus comprises insertion of the human VK gene segments from VK2-4O to VK4-1 and the human JK gene segments from S.

In some embodiments, the insertion includes human non-coding DNA that naturally appears between human VK2-40 to VK4—l, and human non-coding DNA that lly appears between human JKl-JKS.

In some embodiments, the nous immunoglobulin 7» light chain locus comprises insertion of the human V?» gene segments VXS—SZ to VM—4O and Vk3—27 to VX3— l, at least the human Jl-Ck gene segment pairs Ill-CM, JM-CM, , , the human I?» gene segment J27 and a rodent CM gene segment.

In some embodiments, the insertion includes human non-coding DNA that naturally appears between human VXS-SZ to VM—40 and Vk3-27 to Vk3-1, human non- coding DNA that lly appears n human Dt-CX gene segment pairs JM-CM, J22- CM, JM—CM and JK6-C7t6, and human non-coding DNA that naturally appears upstream (or 5’) of human J7» gene segment J27.

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

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

In some embodiments, the one or more nucleotide sequences are inserted between a first and a second human VH gene segment.

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

In some embodiments, the first human VH gene segment is human VHl—2 and the second human VH gene segment is human VH6-l.

In some embodiments, the one or more nucleotide sequences are inserted between a human VH gene segment and a human DH gene t.

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

In some ments, the rodent is heterozygous or homozygous for the endogenous immunoglobulin K light chain locus.

In some embodiments, the rodent is heterozygous or gous for the endogenous immunoglobulin h light chain locus.

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

In some embodiments, provided herein is an isolated rodent cell whose germline genome comprises an endogenous immunoglobulin 7» light chain locus sing: (a) one or more human V?» gene segments, (b) one or more human J7» gene segments, and (0) one or more human C7» gene segments, (i) wherein (a) and (b) are operably linked to (c) and a rodent Ck gene segment, and (ii) wherein the endogenous immunoglobulin 9» light chain locus r comprises: one or more rodent immunoglobulin 7» light chain enhancers (EX) and one or more human immunoglobulin 9» light chain enhancers (EX).

In some embodiments, provided herein is an immortalized cell made from a rodent cell provided herein.

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

In some embodiments, provided herein is a rodent embryo generated from a rodent embryonic stem cell provided .

In some embodiments, ed herein is a method of making a rodent whose ne genome comprises an engineered endogenous immunoglobulin 9» light chain locus, the method comprising (a) introducing a DNA fragment into a rodent embryonic stem cell, said DNA fragment comprising a nucleotide sequence that es (i) one or more human V?» gene segments, (ii) one or more human J9t gene ts, and (iii) one or more human Cl gene segments, wherein (i)-(iii) are operably linked to a rodent C7» gene segment, and wherein the nucleotide sequence further comprises one or more human immunoglobulin 1 light chain enhancers (EX); (b) obtaining the rodent embryonic stem cell generated in (a); and (c) creating a rodent using the rodent embryonic stem cell of (b).

In some embodiments, the tide sequence further includes and one or more human immunoglobulin 9» light chain enhancers (ER).

In some embodiments, provided herein is a method of making a rodent whose germline genome comprises an ered endogenous immunoglobulin 7» light chain locus, which ered endogenous immunoglobulin 9» light chain locus comprises inseItion of one or more human V?» gene segments, one or more human J?» gene segments and one or more human C7» gene segments, which human V?» and J?» gene segments are operably linked to a rodent or a human C?» gene segment, and which endogenous globulin 7» light chain locus further comprises one or more rodent immunoglobulin A light chain enhancers (El), and one or more human immunoglobulin 7» light chain enhancers (El), the method comprising modifying the germline genome of a rodent so that it comprises an ered immunoglobulin 7t light chain locus that includes insertion of one or more human V?» gene segments, one or more human J7» gene segments and one or more human C?» gene segments, which human V?» and J7» gene segments are operably linked to a rodent or a human Ck gene segment, and which endogenous globulin 7t light chain locus further comprises one or more rodent immunoglobulin A light chain enhancers (El), and one or more human immunoglobulin 7t light chain enhancers (El), thereby making said rodent.

In some embodiments, the one or more human V?» gene segments include VXS—SZ to VM—40 and/or V23 —27 to Vk3—1.

In some embodiments, the one or more human Vl gene segments include human non-coding DNA that naturally appears between human VlS-SZ to VM-40 and/or V23 -27 to Vk3-1.

In some embodiments, the one or more human I?» gene ts and the one or more human C?» gene segments e the human Jl—C?» gene segment pairs Ill—CM, JAZ— CXZ, J7L3—CK3, J7L6-C?»6 and the human J7L7 gene t.

In some ments, the human INC?» gene segment pairs JM-CM, JkZ-CXZ, Jk3-C2t3 and Jk6—Cké include human non-coding DNA that naturally appears between the human J?» and Cl gene segment pairs, and the human J27 gene segment includes human non—coding DNA that naturally appears upstream (or 5’) of human J17.

In some embodiments, the rodent C7» gene segment is a mouse CM gene segment.

In some embodiments, the endogenous globulin A light chain locus comprises three human E15.

In some embodiments, the nous immunoglobulin 9» light chain locus comprises two rodent Eks.

In some embodiments, the two rodent Eks are a mouse E7» and a mouse Ek3—l.

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

] In some ments, the DNA fragment further comprises one or more site— c recombination sites In some embodiments, the DNA fragment of (a) is introduced into a rodent embryonic stem cell whose germline genome ses an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene ts, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene ts are operably linked to a rodent immunoglobulin heavy chain constant region; or an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant region, and an endogenous immunoglobulin K light chain locus comprising insertion of one or more human VK gene ts and one or more human JK gene ts, which human VK and JK gene segments are operably linked to a rodent immunoglobulin CK region.

In some embodiments, the DNA fragment of (a) is introduced into a rodent embryonic stem cell whose germline genome comprises a wild-type endogenous immunoglobulin heavy chain locus; or a wild-type endogenous immunoglobulin heavy chain locus and a ype endogenous immunoglobulin K light chain locus, and n the method further comprises a step of breeding a mouse produced from said non—human embryonic stem cell with a second mouse.

In some embodiments, the modifying the germline genome of a rodent so that it comprises an engineered immunoglobulin 7t light chain locus is carried out in a rodent embryonic stem cell whose germline genome further comprises an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant region, or an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent globulin heavy chain constant region, and an nous immunoglobulin K light chain locus sing insertion of one or more human VK gene segments and one or more human JK gene segments, which human VK and JK gene segments are operably linked to a rodent immunoglobulin CK region.

In some embodiments, the ion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments includes human non-coding DNA that naturally s between the one or more human VI-l gene segments, human non—coding DNA that naturally appears between the one or more human DH gene segments and human non-coding DNA that naturally appears between the one or more human JH gene segments.

In some embodiments, the insertion of one or more human VK gene segments and one or more human JK gene ts includes human non-coding DNA that naturally appears between the one or more human VK gene segments and human non—coding DNA that naturally appears between the one or more human JK gene segments.

In some embodiments, the modifying the germline genome of a non-human animal so that it comprises an engineered immunoglobulin 7» light chain locus is carried out in a non—human embryonic stem cell whose germline genome comprises a wild-type endogenous immunoglobulin heavy chain locus, or a wild-type endogenous immunoglobulin heavy chain locus and a wild-type endogenous immunoglobulin K light chain locus; and wherein the method further comprises a step of breeding a mouse produced from said non- human embryonic stem cell with a second mouse.

] In some embodiments, the second mouse has a germline genome comprising wild-type IgH and IgK loci.

] In some embodiments, the second mouse has a ne genome comprising homozygous or heterozygous humanized IgH and IgK loci, which homozygous or heterozygous humanized IgH locus contains an inserted rodent Adam6-encoding ce.

In some embodiments, the second mouse has a germline genome comprising a homozygous or heterozygous humanized IgH locus and a homozygous or heterozygous inactivated IgK locus.

In some embodiments, provided herein is a method of ing an antibody in a , the method comprising the steps of (l) immunizing a rodent with an antigen of interest, which rodent has a germline genome comprising an endogenous globulin A light chain locus comprising (ai) one or more human V?» gene segments, (b) one or more human J1 gene ts, and (0) one or more human Ck gene ts, wherein (a) and (b) are operably linked to (c) and a rodent C?» gene segment, and wherein the endogenous immunoglobulin 7» light chain locus further comprises: one or more rodent immunoglobulin 9» light chain enhancers (El) and one or more human globulin 7t light chain enhancers (EX); (2) maintaining the rodent under conditions sufficient that the rodent produces an immune response to the antigen of interest, and (3) recovering an antibody from the rodent, or a rodent cell, that binds the antigen of interest.

] In some embodiments, the rodent has a germline genome further comprising: an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene ts, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant region; or an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a rodent immunoglobulin heavy chain constant region, and an endogenous immunoglobulin K light chain locus comprising insertion of one or more human VK gene segments and one or more human JK gene segments, which human VK and JK gene segments are operably linked to a rodent immunoglobulin CK region, In some embodiments, the rodent cell is a B cell.

In some embodiments, the rodent cell is a hybridoma.

] In some ments, the nous immunoglobulin k light chain locus comprises insertion of the human V?» gene segments Vk5-52 to VM-40 and V76 -27 to VX3- l, the human LIX-Cl gene segment pairs JM-CM, JKZ-CKZ, Jk3-C7t3, Dt6-C7t6 and the human J?t gene segment M7.

In some embodiments, the insertion includes human ding DNA that naturally appears between human Wt Vk5-52 to VM-4O and V7t3 -27 to Vk3-l, human non- coding DNA that naturally appears between human INC?» gene segment pairs Ill-CM, J12- CXZ, M3 -C?t3 and JX6-C9t6, and human non-coding DNA that naturally appears upstream (or 57) of human J?» gene segment JM.

In some embodiments, the rodent C?» gene segment is a mouse CM gene segment.

In some embodiments, the immunoglobulin heavy chain locus comprises insertion of the human VH gene segments from VH3 -74 to VH6-1, the human DH gene segments from DHl-l to DH7-27 and the human JH gene segments JHl-JH6, and which human VH, DH and JH gene segments are operably linked to an endogenous rodent immunoglobulin heavy chain constant region.

In some embodiments, the insertion includes human non-coding DNA that naturally appears between human VH3-74 to VH6-l, human ding DNA that naturally appears between human DH1-1 to DH7-27, and human non-coding DNA that naturally appears between human JHl-JH6.

In some embodiments, the human VH, DH and JH gene segments replace rodent VH, DH and JH gene segments.

In some embodiments, the immunoglobulin K light chain locus ses insertion of the human VK gene segments from VK2-40 to VK4-l and the human JK gene segments from S, and which human VK and JK gene ts are operably linked to an endogenous rodent immunoglobulin CK region.

] In some ments, the insertion includes human non-coding DNA that naturally appears between human VK2—40 to VK4-l, and human non—coding DNA that naturally appears between human JKl-JKS.

In some ments, the human VK and JK gene segments replace rodent VK and JK gene segments.

In some embodiments, the germline genome of the rodent further comprises insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides.

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

In some embodiments, the immunoglobulin heavy chain locus further comprises insertion of one or more nucleotide ces encoding one or more rodent Adam6 polypeptides.

In some embodiments, the one or more nucleotide sequences ng one or more rodent Adam6 polypeptides are inserted between a first and a second human VH gene segment.

In some embodiments, the first human VH gene segment is human VHI-Z and the second human VH gene segment is human VH6-1, ] In some embodiments, the one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides are inserted in the place of a human Adam6 pseudogene.

In some embodiments, the one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides are inserted between a human VH gene segment and a human DH gene t.

In some embodiments, the antibody recovered from the , or a rodent cell, that binds the antigen of interest comprises a human heavy chain variable domain and a human lambda light chain le domain.

] In some embodiments, the human heavy chain variable domain includes a rearranged human VH gene segment selected from the group consisting of VH3 -74, VH3 -73, , VH2—70, vHi—69, VH3-66, VH3—64, VH4—61, VH4—59, VH1—58, VH3-53, VHS—51, VH3-49, VH3-48, VHl-46, VHl-45, VH3—43, VH4—39, VH4-34, vH3—33, VH4-31, VH3-30, VH4-28, VH2-26, Val-24, VH3-23, , VH3-20, VH1—18, VH3-15, VH3-13, VH3-11, VH3-9, VHl-8, VH3-7, VH2-5, VH7l, VH4-4, VHl-3, VHl—2 and VH6-l.

In some embodiments, the human lambda light chain variable domain includes a rearranged human V?» gene segment selected from the group consisting of Vk4-69, Vl8-61, v>t4—60, vm—57, VMO—54, vxs—sz, vx1—51, VX9—49, vm—47, vm—46, , VM—44, , Vltl—40, , , VX1-36, VX3-27, VK3-25, VK2—23, , vx3—21, VX3-19, V1248, Vx3-16, V1244, vx3—12, vx2—1 1, vx3—1o, vx3—9, VIZ-8, Vk4-3 and vx3—1.

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

In some embodiments, provided herein is a rodent whose germline genome comprises a homozygous nous immunoglobulin 7» light chain locus comprising: (i) human V?» gene segments VKS-SZ to VM-4O and Vk3-27 to Vk3-1, (ii) human INC?» gene segment pairs Ill-CM, J7t2—C9t2, JK3-C7t3 and J7L6—C9t6, (iii) human J7» gene segment DJ, and (iv) three human immunoglobulin 1 light chain enhancers; wherein (i)-(iv) are operably linked to each other and (i)-(iii) are upstream of a rodent Ck gene segment, and wherein the endogenous immunoglobulin 9» light chain locus lacks an endogenous rodent immunoglobulin E124, the human V?» gene segments Vk5-52 to VM-4O and V13 -27 to Vk3-l includes human non—coding DNA that naturally appears between the human V?» gene segments, the human INC?» gene segments pairs Del-CM, JM-CM, Jk3-Ck3 and JK6-C9t6 includes human non-coding DNA that naturally appears between the human INC?» gene segments pairs, and the human J7» gene segment J7t7 includes human non—coding DNA that naturally appears upstream (or 5’) of human R7.

In some embodiments, the rodent C?» gene segment is a mouse CM gene segment.

] In some ments, the endogenous immunoglobulin A light chain locus further comprises endogenous rodent immunoglobulin A light chain enhancers El and E23- In some embodiments, the endogenous immunoglobulin A light chain locus ses a on of endogenous rodent VlZ-VM-J?t2-C?tZ-J?t4P-C?t4P gene segments and VM -J?t3-J?»3P-C7t3-J?tl gene segments, ] In some embodiments, the rodent is a rat or a mouse.

EXAMPLES The following es are provided so as to describe to the skilled artisan how to make and use methods and compositions described , and are not intended to limit the scope of what the inventors of the present disclosure regard as their invention. Unless indicated otherwise, temperature is indicated in Celsius and pressure is at or near atmospheric.

Example 1. Construction ofa targeting vectorfor engineering a rodent Igz'l. light chain locus This example rates exemplary methods of constructing a targeting vector for insertion into the genome of a non—human animal such as a rodent (e. g., a mouse). The methods described in this example trate the production of a man animal whose germline genome comprises an engineered 1g?» light chain locus. In particular, this e demonstrates the construction of a series of targeting vectors for engineering an endogenous lg?» light chain locus in a non-human animal so that the non—human animal expresses and/or produces antibodies that include 1g?» light chains having human variable domains and non—human or, in some embodiments, human constant domains from said endogenous lg?» light chain locus in the germline genome of the non-human animal. As described below, a series of targeting vectors containing varying amounts of genetic material corresponding to a human 1g?» light chain locus (i.e., human V7», J9», C7» and Igl er sequences) are inserted into an endogenous rodent 1g?» light chain locus. In particular, said genetic material is inserted upstream of a rodent C?» gene (or gene segment) so that human V)», J?» and C?» gene segments are operably linked to said rodent C7» gene. The methods described in this example provide for retention and/or deletion of endogenous rodent ng gene segments (or sequences). An exemplary schematic illustration of a series of targeting vectors for constructing an engineered endogenous ng light chain locus is set forth in s 1—4.

A series of targeting vectors containing various amounts of human V7», J)L, C?» and 1g?» enhancer ces (or regions) for insertion into a rodent 1g?» light chain locus were created using VELOCIGENE® technology (see, e.g., US. Patent No. 6,586,251 and uela et al., 2003, Nature Biotech. 652-9, incorporated herein by reference in their entireties) and molecular biology techniques known in the art. The methods described in this example can be employed to utilize any human V)», J)», C?» and 1g?» enhancer ces, or combination of sequences (or sequence fragments) as desired. Table 1 sets forth brief descriptions of each targeting vector illustrated in Figure 1.

Briefly, about 12kb (11,822bp) of human 1g?» genomic ce from human bacterial artificial chromosome (BAC) clone CTD-2502ml6 was ligated into mouse BAC clone RP23-60e14. This mouse BAC clone was ered to shorten the BAC clone by about 90kb, insert unique AsiSI and PI—SceI restriction enzyme recognition sites downstream of a mouse C?»l gene and replace the original Chloramphenicol resistance (CMR) gene with a Spectinomycin resistance (SpecR) gene and unique I—CeuI restriction site by two consecutive bacterial homologous recombination (BHR) steps prior to ligation with the human 1g?» genomic sequence. The human BAC clone CTD-2502m16 was also modified by two consecutive BHR steps to trim about 53kb of human sequence from the 3 ’ end with a Neomycin selection cassette and a unique PI-SceI restriction site, and trim a CMR gene and about 101.5kb of human ce from the 5’ end with a ycin cassette and a unique AsiSI ction site, thereby placing the AsiSI site and the Neomycin selection cassette about 2885bp upstream and about 1418bp downstream, tively, of the modular human enhancer region (see, e.g., Asenbauer, H. and HG. k, 1996, Eur. J. l. 26(1): 142-50, which is hereby incorporated by reference in its entirety). The human 1g?» genomic sequence ned about 7.5kb corresponding to a human 1g?» enhancer (EX) region (or sequence), which is modular and contains three sequence elements (Figure 1, Asenbauer, H. and HG, Klobeck, 1996, Eur. J , Immunol, ]42-50), and 2,9kb and 1.4kb of 5’ and 3’ flanking sequence, respectively, as well as a Neomycin selection cassette (i.e., a Neomycin resistance gene [NEOR] under transcriptional control of a ubiquitin promoter and flanked by loxP sites). The modified human and mouse BAC clones were digested with AsiSI and PI—SceI sites and ligated together. After ligation to the engineered mouse BAC clone, the resulting targeting vector ned about 39,166bp of mouse ce as a 5’ gy arm and included mouse 1g?» gene segments VM, J13, IMP, C7t3, JM, and CM (6286 targeting vector, Figure 1). The 3’ homology arm (about 30,395bp) included a mouse 1g?» er (mEk). For simplicity, in the depiction of 6286 targeting vector in Figure 1, the mouse homology arms are not shown. Homologous ination with this ing vector resulted in the insertion of the three human 1g?» enhancer ces as well as the 5’ and 3’ flanking sequences without any deletion of mouse sequence.

Recombinase-mediated deletion of the in selection cassette was achieved in ES cells by transient expression of Cre recombinase (see e.g., Lakso, M. et al., 1992, Proc. Natl.

Acad. Sci. USA 89: 6232-6, Orban, P.C. et al., 1992, Proc. Natl. Acad. Sci. USA. 896861- ; Gu, H. et al., 1993, Cell 73(6):]155-64; Araki, K. et al., 1995, Proc. Natl. Acad. Sci.

USA. 92:160-4; i, S.M., 1996, Proc. Natl. Acad. Sci. USA. 93(12):6191-6, all of which are incorporated herein by reference in their entireties).

A second construct (6571 targeting vector) was engineered to include a group of five functional human V?» gene segments and a substantially all of a human Jk—CA cluster (i.e., human JM-CM-Jk2-C7t2-J7t3-C2t3 -JM-CM-JXS -C?»5 -Jk6-C7t6-hJk7) spanning about 125,473bp, which was obtained from human BAC clone CTD—2079i4. To construct the targeting vector, a human CM gene in human BAC clone CTD-2079i4 was first replaced by BHR with a mouse CM gene and about 1588bp of flanking sequence, which was amplified by PCR using mouse BAC clone RP23-60e14 as a template. A 5’ homology arm containing about 37,161bp of mouse sequence corresponding to sequence 5’ of a mouse CM gene in mouse BAC clone RP23-60e14 was then ligated to the modified human BAC clone CTD- 2079i4 containing the synthetic mouse CM gene using unique I—CeuI and PI—SceI restriction enzyme recognition sites tely introduced into both mouse and human BAC clones by BHR. This 5’ homology arm contained mouse VM, J23, JX3P, C23, and JM gene segments (Figure 1). The 3’ homology arm ned about p of human sequence corresponding to two of the human E7ts from the 6286 targeting vector (Figure 1).

A third uct (6596 targeting vector) was engineered to contain an additional eleven functional human V?» gene segments. This targeting vector contained about 171,458bp of human sequence from BAC clone RPl l-761Ll3. By design, the three human V)» gene segments were included to provide 3’ overlap homology (of about 33,469bp) with the 6571 targeting vector, As bed above, a 5’ homology arm containing about 37,161bp of mouse sequence was ligated to the 5’ end of the DNA fragment containing the human V?» gene segments using unique I-CeuI and A501 restriction enzyme recognition sites separately introduced into both mouse and human BAC clones by BHR.

A fourth construct (6597 targeting vector) was engineered to contain an additional nine functional human Vl gene segments. This targeting vector contained about 121,188bp of human sequence from two BAC clones, RP11-22L18 and RP11-761L13. As described above, the 3’ end of this human sequence contained onal human Vl gene segments that provided 3’ overlap homology with the 6596 targeting vector (about 27,468bp). As described above for the 6571 and 6596 targeting vectors, about 37,16lbp 5’ homology arm containing mouse sequence from BAC clone 0el4 was ligated to the ’ end of the human sequence using unique I-CeuI and A501 restriction enzyme recognition sites separately introduced into both mouse and human BAC clones by BHR.

In a similar manner, a fifth construct (6680 targeting vector) was engineered to n the same additional nine onal human V?» gene ts as the 6597 targeting , except that the 5’ homology arm was changed to allow for deletion of the mouse 1g?» light chain locus via homologous recombination. This 5’ gy arm contained about 22,298bp from mouse BAC clone RP23-15m16 and was ligated to the 5’ end of the human sequence (~121,188bp fragment, supra) using the unique I-CeuI and AscI ction enzyme recognition sites separately introduced into both mouse and human BAC clones by BHR.

This 5’ homology arm contains mouse sequence 5’ of a mouse VXZ gene segment, which, upon homologous recombination, effectively deletes the mouse 1g?» light chain locus. This targeting vector contained the same 3’ overlap homology as the 6597 targeting vector (described above). Figure 2 illustrates the ent alleles that result from insertion of the 6597 or 6680 targeting vectors.

In a similar , an additional engineered mouse strain was created via co- electroporation of two different targeting s into ES cells aided by the use of guide RNAs (gRNAs) using a CRISPR/Cas9 system (Figure 3), see, e.g., US. Patent No. 208 (granted January 5, 2016) and US. Patent Application Publication Nos. US. 2015-0159174 A1 (filed October 15, 2014), US. 2015-0376650 Al (filed June 5, 2015), US. 2015-0376628 A1 (filed June 23, 2015), US. 2016-0060657 A1 (filed October 30, 2015), US. 2016—0145646 A1 (filed er 20, 2015), and US. 177339 Al (filed December 18, 2015), all of which are incorporated herein by reference in their entireties. The ES cells had a genome heterozygous for insertion of the 6571 targeting vector construct.

Briefly, as shown in Figure 3, a trimmed 6596 targeting vector (i.e., without a 5’ homology arm and cassette as described above) was designed to contain about 33kb 3’ homology arm that includes overlap sequence corresponding to three human V9t gene segments in the 6571 targeting vector, about 111kb sequence that ses 11 additional human V?» gene segments, and about 27kb sequence that contains a single human V7t gene segment and serves as an overlap region with the second targeting vector. The second targeting vector (6680 targeting vector) comprises the same about 27kb overlap region sequence positioned on the 3’ end of the targeting , about 94kb sequence sing an additional nine human V)» gene segments, a Neomycin selection cassette (e.g., a Neomycin resistance gene [NEOR] under riptional control of a ubiquitin er flanked by Fit recombination recognition sites) and about 22kb 5’ mouse 2’» homology arm.

The ES cells employed in the electroporation of these two targeting vectors had a genome heterozygous for insertion of the 6571 targeting vector (Figure 3). These ES cells were co- electroporated with the two targeting vectors described above along with a guide RNA (gRNA) that s the Hygromycin resistance gene from the 6571 targeting vector at nucleotide sequence CGACCTGATG CAGCTCTCGG (SEQ ID NO: 130) and two gRNAs that target a region upstream of a mouse V22 gene segment (i.e., 3’ of a mouse V22 gene segment on the minus strand; gRNAl: GTACATCTTG ACGT, SEQ ID NO: 139, about 1000bp upstream of mouse V22, gRNA2: GTCCATAATT AATGTAGTTA C, SEQ ID NO: 140, about 380bp upstream of mouse VXZ) and promote double ed breaks at these sequences. The two co-electroporated targeting vectors were inserted by homologous recombination into the genome of the ES cells at the Hygromycin sequence, replacing the region containing and surrounding the Hygromycin selection cassette. The resulting ES cells contained an engineered endogenous 1g?» locus that included a human immunoglobulin WO 28691 variable region comprising 25 functional human V?» gene segments operably linked to a human INC?» cluster, a human JM gene t and operably linked to a mouse CM gene (Figure 3).

The targeting vectors described above were introduced into mouse embryonic stem (ES) cells to build the ered Ig7t light chain locus. Positive ES cell clones were confirmed after insertion of each targeting vector into the genome of ES cells (see below) prior to insertion of the next targeting vector. In some instances, intermediate strains were created for phenotypic analysis.

Table 1. Summary of Targeting Vectors Name Approximate Description h1g7» seguence 6286 1L822bp Insertion of human Eks into mouse Ig7t locus 6571 121473bp Insertion of five functional human V?» gene segments and portion of human Ik-Cl gene cluster 6596 17L458bp Insertion of additional eleven functional human V1 gene segments 6597 121,188bp Insertion of additional nine functional human V?» gene segments 6680 121,188bp Insertion of additional nine functional human V?» gene segments and deletion of mouse 1g?» gene segments 6889 121,188bp Insertion of additional nine onal human V?» gene segments and deletion of mouse 1g?» gene segments via simultaneous ion of two targeting vectors and guide RNAs The nucleotide sequence across selected junction points after insertion of the targeting vectors bed above was confirmed by cing. Selected on points indicated in Figures 1-4 are provided below.

SEQID CCCTATTCACTGAGTTCTGGAAGCTCTGCTATTTCCATGATCG NOIll7 TTCACACTGACCCCTGTTGATCTTACCGGTACCGAAGTTCCTA TTCCGAAGTTCCTA SEQID AGAAAGTATAGGAACTTCCTAGGGTTTCACCGGTGGC N02118 GCGCCGATGTACATCAGTTCAGTCTGGAAAGGTGGAACAGCT CCAGGTGAAGGCAGG SEQID CTCTACGGGTGATGTTCATCTAAGGTGACAGGAGTCAGTGAG NO:ll9 GGCTTCTCAAGCTTTATCTATGTCGGGTGCGGAGAAAGAGGT AATGAAATGGCACTCGAGCCCTGCTGGTGCCTTCTGTTGTATC CACGCCTTCAGTAGATTTGATGA SEQ ID GAGTTTTTCCCTTTCCTGTCTGTCGAAGGCTAAGGTCTAAGCC NO: 120 TGTCTGGTCACACTAGGTAAAGAATTTCTTTCTTCTCTAGATG CTTTGTCTCATTTC SEQ ID TATGTCACTGGAATTTAGAGTAGTGTGTGGAATGTCTTGGCAA N01121 CCTGGACACGCGTCCTGGCACCCAGTGAGAAAGTGGCCCTGA GGCTCATAG SEQ ID AGCAGCCGACATTTAGCAAAGAGGATTGGAAAATGAACCCCC NO: 122 CCTTAAAATACAGTTAAACACAGAGGAGGGAGCAAACCGGTA TAACTTCGTATAATGT SEQ ID TACGAAGTTATGTCGACCTCGAGGGGGGGCCCGGTA NO: 123 CCATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGTCT CATTCCTTCCCTGTCTCAAGGCATAATGGTTCAATATGCACCT SEQ ID TTCTCTCCAAGACTTGAGGTGCTTTTTGTTGTATACTTTCCCTT NO: 124 TCTGTATTCTGCTTCATACCTATACTGGTACCGAAGTTCCTATT CCGAAGTTCCTA SEQ ID TTCTCTAGAAAGTATAGGAACTTCCTAGGGTTTCACCGGTGGC NO: 125 GCGCCTGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGA TAAGCTTTGGATTGGATTCAGTGAGCAAGAATTCACAAACAC AATGGACTTATC SEQ ID TTCTCTCCAAGACTTGAGGTGCTTTTTGTTGTATACTTTCCCTT NO: 126 TCTGTATTCTGCTTCATACCTATACTGGTACCGAAGTTCCTATT TTCCTA SEQ ID TTCTCTAGAAAGTATAGGAACTTCCTAGGGTTTCACCGGTGGC NO: 127 CCCTGCTGGTGCCTTTTGTTGTATCCACGCCTTCAGT AGATTTGATGATGC SEQ ID TTCTCTCCAAGACTTGAGGTGCTTTTTGTTGTATACTTTCCCTT NO: 128 TCTGTATTCTGCTTCATACCTATACTGGTACCGAAGTTCCTATT CCGAAGTTCCTA SEQ ID TTCTCTAGAAAGTATAGGAACTTCCTAGGGTTTCACCGGTGGC NO: 129 GCGCCGATGTACATCAGTTCAGTCTGGAAAGGTGGAACAGCT CCAGGTGAAGGCAGG In the construction of engineered loci, in particular, engineered immunoglobulin loci, the inventors recognized that some human V?t gene segments may be missing from certain haplotypes and, therefore, not represented in selected BAC clones spanning a human ng light chain locus. To give but one example, one report has provided evidence that more recently discovered alleles contain insertion/deletion of one or more human V?» gene segments in cluster B of the human 1g?» light chain locus as compared to previously ed alleles (e.g., human V7tl-50, Vk5-48, V25-45 and V7tS-39; see Moraes, IC. and GA.

Passos, 2003, genetics 55(1): 10-5). Thus, the inventors have designed a strategy to include human V7t gene segments that are missing in a particular BAC clone used for targeting vector design and construction.

Briefly, human BAC clones are mapped to the human 1g?» light chain locus by end sequencing. In particular, using the GRCh3 7/hgl9 Assembly (UCSC Genome Browser, Human Feb. 2009) human BAC clones RPl l-346I4, CTD-2523F21 and CTD-2523E22 are identified to span a region of cluster B of the human 1g?» light chain locus that includes human Vk7-46 to VM-3 6. For example, one or more g human V}. gene segments (e.g., VX5139, Vh5-37 and/or VM-3 6) can be inserted into a targeting vector bed above (6680 or 6889) using any one of these BAC clones identified to contain one or more human V?» gene ts that are desired for insertion. For example, BAC clone CTD- 2523F21 is modified by replacing the 3’ with a ion cassette (e.g., Hygromycin resistance gene [HYGR] under transcriptional control of a ubiquitin promoter) flanked by recombinase recognition sites (e. g., [0x23 72) and a ~27kb 3’ homology arm having overlapping sequence with the 6597 targeting vector (see above). The 5’ end of human BAC clone serves as a 5’ homology arm having overlapping ce with the 6680 or 6889 targeting vector thereby tating homologous recombination and insertion of any missing human V?» gene segments along with the selection cassette. An optional last step of transient expression of a recombinase (e.g., Cre) may be employed to remove the selection cassette.

Example 2. Generation ofrodents having an engineered 1g). light chain locus This example demonstrates the tion of a non-human animal (e.g., rodent) whose germline genome ses an endogenous Igh light chain locus comprising insertion of a plurality of human V7t, Di and Ch sequences, which human Vh, Dt and Ch sequences are operably linked to a rodent C7» gene (or gene segment), and which endogenous 1g?» light chain locus further includes one or more human Ig7t enhancers (Els). In some embodiments, said endogenous 1g?» light chain locus es a deletion of one or more endogenous Ig7t light chain enhancer regions (or sequences). Such non-human animals are characterized, in some embodiments, by expression of Igh light chains that are fully human (i.e., human variable and constant domains).

Targeted ion of ing vectors described in Example 1 was confirmed by polymerase chain reaction. Targeted BAC DNA, confirmed by rase chain reaction, was then introduced into Fl hybrid (l29S6SvaTac/C57BL6NTac) mouse embryonic stem (ES) cells via electroporation followed by culturing in selection medium. In some embodiments, the ES cells used for electroporation of the series of targeting vectors may have a germline genome that includes ype IgH and IgK loci, homozygous humanized IgH and IgK loci, which gous zed IgH locus contained an inserted rodent Adam6-encoding sequence (see, e.g., US. Patent Nos. 8,642,835 and 8,697,940; hereby incorporated by reference in its entirety), or a homozygous humanized IgH locus (see, e. g., US, Patent Nos. 8,642,835 and 8,697,940, supra) and a homozygous inactivated IgK locus.

In other embodiments, after targeted ES cells as described herein are used to generate mice (see below), resultant mice comprising an engineered human 1g?» locus as described herein are used to breed with mice comprising humanized IgH and IgK loci, which humanized IgH locus contains an inserted rodent Adam6-encoding sequence (see, e.g., US. Patent Nos. 8,642,835 and 940, , or a humanized IgH locus (see, e.g., US. Patent Nos. 8,642,835 and 8,697,940, supra) and an inactivated IgK locus. Drug-resistant colonies were picked 10 days after electroporation and screened by TAQMANTM and karyotyping for correct targeting as previously described (Valenzuela et al., supra; Frendewey, D. et al., 2010, Methods Enzymol. 476:295—307). Table 2 sets forth exemplary primers/probes sets used for screening positive ES cell clones (F: forward , R: reverse primer; P: .

The VELOCIMOUSE® method (DeChiara, TM. et al., 2010, Methods Enzymol. 476:285-294, DeChiara, T.M., 2009, Methods Mol. Biol. 530:311-324, Poueymirou et al., 2007, Nat. Biotechnol. 25:91-99) was used, in which targeted ES cells were injected into uncompacted 8-cell stage Swiss Webster embryos, to produce healthy fully ES cell-derived F0 generation mice zygous for the engineered 1g?» light chain allele. F0 generation heterozygous mice were crossed with /NTac mice to generate F1 heterozygotes that were intercrossed to produce F2 tion s for phenotypic analyses.

Taken together, this example illustrates the generation of a rodent (e.g., a mouse) whose gerrnline genome comprises an engineered lg?» light chain locus terized by the presence of a plurality of human V7», J9» and C7» sequences ly linked to a rodent Ck gene, which rodent engineered lg?» light chain locus includes endogenous rodent and human 1g?» light chain enhancer sequences (or regions). The strategy described herein for inserting human V2, 19» and C?» sequences into an endogenous rodent 1g?» light chain locus enables the construction of a rodent that expresses antibodies that contain human V9» domains fused to W0 20181128691 either a human or rodent Ck domain. As described herein, such human V?» domains are expressed from endogenous 1g?» light chain loci in the germline rodent genome.

Table 2. Representative primer/probe sets for screening positive ES cell clones Name Sequence (5’-3’) GCATGGCCTAGAGATAACAAGAC (SEQ ID NO: 15) mIgLC lp3 ween-J GGCCTTGGATAACCTCAGGATAC (SEQ ID N0: 16) TCCATCCCAATAGATCTCATTCCTTCCC (SEQ ID N0: 17) www CCCTGTCAAGTCTCCAAGGTTG (SEQ ID NO: 18) HSSl-l CACTGTGGCCCAAGGATCAC (SEQ ID NO: 19) CACTCTGCCCAGGGAGTGTCTGG (SEQ ID NO:20) wwm GCATGGCCTAGAGATAACAAGACTG (SEQ ID NO:21) LoLjxnl GTGCTCTTCCCTTGGGAGA (SEQ ID N0z22) TCCATCCCAATAGAGCGATCGCA (SEQ ID NO:23) GAGGCTATTCGGC (SEQ ID N0z24) Neo www GAACACGGCGGCATCAG (SEQ ID NO:25) TGGGCACAACAGACAATCGGCTG (SEQ ID NO:26) AGCTGAATGGAAACAAGGCAA (SEQ ID N0:27) hIgLZ www GGAGACAATGCCCCAGTGA (SEQ ID NO:28) TGACATGAACCATCTGTTTCTCTCTCGACAA (SEQ ID NO:29) CCACCGCCAAGTTGACCTC (SEQ ID N030) hIgL4 "dpd’Tl TGAAGGACTAAGGCCCAGGATAG (SEQ ID N031) GCAAGGGCCCAGCCT (SEQ ID N032) TGGCTCAGTGACAAGAGTC (SEQ ID N033) hIgLS wwwwww CCAGGGACACAGCCTTTGC (SEQ ID N034) TGCATTGCAGAGACCAGGGACC (SEQ ID N035) TGCGGCCGATCTTAGCC (SEQ ID N036) GGGTTCGGCCCATTC (SEQ ID N037) TTGACCGATTCCTTGCGG (SEQ ID N038) ween-J TGTCGGGCGTACACAAATCG (SEQ ID N039) Hyg D GGGCGTCGGTTTCCACTATC (SEQ ID N0:40) CCGTCTGGACCGATGGCTGTGT (SEQ ID N0:41) CGACGTCTGTCGAGAAGTTTCTG (SEQ ID N0:42) Hyg U CACGCCCTCCTACATCGAA (SEQ ID N0:43) AGTTCGACAGCGTGTCCGACCTGA (SEQ ID N0:44) CGAGCTCCAGGTGT (SEQ ID N0:45) mlng wwwwpum AGGGCAGCCTTGTCTCCAA (SEQ ID N0:46) CCTGCCAGATTCTCAGGCTCCCTG (SEQ ID N0:47) mIgL6 '11 GGAGGTCAGGAATGAGGGAC (SEQ ID N0:48) CACTTGCTCACTGCAAAAGCA (SEQ ID N049) TGTGGGATTTTGGAATTCTATCTCACTGATAGGAAAG (SEQ ID N050) GCAGAGAGGATTCAAGAGCTGG (SEQ ID N051) mIgL 1 0 www TTTTTGCAATGCTTCACCTGA (SEQ ID N052) CAGGTGTCTGTATTGGAGGTCAATGGCA (SEQ ID N053) "UW’TJ GATTTGCTGAGGGCAGGGT (SEQ ID N054) mIng 1 CCCCAAGTCTGATCCTTCCTT (SEQ ID N055) CCTTCATACTCTTGCATCCTCCCTTCTCCA (SEQ ID N056) pun-1 GCTGACCAACGATCGCCTAA (SEQ ID N057) TAAGCGCCACACTGCACCT (SEQ ID NO:58) mIgL12 CTCTTCTGTGACTCAATTATTTGTGGACA (SEQ ID N059) wwu-j AACTGCTGATGCACTGGGC (SEQ ID N060) mIgL13 TGAATGCATGGAGTTGGCC (SEQ ID N061) TCTCCTTTGCAGTGGCTTAATTAGCTGAGTCA (SEQ ID N062) CCCTGGTGAAGCATGTTTGC (SEQ ID N063) 1467hTIl wwm TGTGGCCTGTCTGCCTTACG (SEQ ID N064) CCAAGCAGGAGGTGCTCAGTTCCCAA (SEQ ID N065) GGGACAGGTGAAGGGCCTATC (SEQ ID N066) 1467hT12 wwwj ACAGGATGCAGTTG (SEQ ID N067) CGCACCTGTATCTAACCAGTCCCAGCATC (SEQ ID N068) TAGACCCCGGAAGTC (SEQ ID N069) l467hTI3 wwu-j TCGCTTTGCCAGTTGATTCTC (SEQ ID N070) TCCACACTGTCGGCTGGGAGCTCA (SEQ ID N071) CGCTTCAATGACCCAACCA (SEQ ID N072) 1468h1 wwm TGTTGAAACGTAATCCCCAATG (SEQ ID N073) CTCCCACCAGGTGCCACATGCA (SEQ ID N074) www GGGCTACTTGAGGACCTTGCT (SEQ ID N075) 1468h2 GACAGCCCTTACAGAGTTTGGAA (SEQ ID NO:76) CAGGGCCTCCATCCCAGGCA (SEQ ID N077) www AGTGCAAACAGCAAGATGAGATCT (SEQ ID N078) 1468h3 GGCGCTGAGCAGAAAACAA (SEQ ID N079) AGACCACCAAGAAGGCCCAGAGTGACC (SEQ ID N080) wwm AAGACCAGGAGCTCTGCCTAAGT (SEQ ID N081) 1468h5 CCCATCACGAACTGAAGTTGAG (SEQ ID N082) TGTGTGAATCACTCTACCCTCC (SEQ ID N083) CCCTTCATGATGCTTTGTCATC (SEQ ID N084) 1468h6 wwu-J GTAGTGGCAAAGGCAGATTCCT (SEQ ID N085) CCTTCACTCCCCGAATGCCCTCC (SEQ ID N086) 6596V31 ’11 GCCCTGCTCCAGTCTTATTCC (SEQ ID N087) Pepe CTGCGTCTGGGCTTTGCT (SEQ ID NO:88) CCACAGATCCCAAGTTGAGCCTGC (SEQ ID NO:89) GTGAGCGGTACCCTGGAATC (SEQ ID NO:90) 6596V3-22—l www AGCCTCGTCTTCGGTCAGGAC (SEQ ID NO:91) TGAACGATTCTCTGGGTCCACC (SEQ ID NO:92) CCAGGATGGAATGAAG (SEQ ID NO:93) 6596V3-2l-1 wwr-n GATTTAAGAGGTTGTTAG (SEQ ID NO:94) GACCCCAGATAATTCCCCTG (SEQ ID NO:95) GAGTGCAGTGGCAGAATCTTG (SEQ ID NO:96) 6596VLdetect GGCAGGGAGCATTGGTAGA (SEQ ID NO:97) -1 Fawn-1 TACTGAAATCTCAGCCTCCCAGGC (SEQ ID NO:98) "UPU’fi TGGCTCCAGCTCAGGAAAV (SEQ ID NO:99) 6596V3-l9—1 CCCGGGAGTTACAGTAATAGTCA (SEQ ID NO: 100) CACAGCTTCCTTGACCATCACTGGG (SEQ ID NO: 101) CCAGCCCACCCAATTATGCTA (SEQ ID NO:102) 6597_h3'arm1 Pawn-r GCGTTTAGGGCCAGGTACAAAT (SEQ ID NO: 103) TGTCAAACACTTTCAGAGCA (SEQ ID NO: 104) wwm GAGGCTGCAGGGATGTAAC (SEQ ID NO: 105) 6597_h3'arm2 CCCATTCCAGGTCCAATTCTCA (SEQ ID NO: 106) TTTGTAAAGTGCATAACACAGACCCTGA (SEQ ID NO: 107) GGGTACAATGAGACAAGAATCAGA (SEQ ID NO: 108) 66805'Arm1 wwm GAAAGGCAAACACAAGTCACAGATG (SEQ ID NO: 109) TCAGCCCTCTGGAATGTAAGGATCA (SEQ ID N01110) GCTGCATCTTCTCAAGTCTTTAAGT (SEQ ID NO:1 l l) 66805'Arm2 "dW'fi GGGAACCAGTCAGGAACTCATAC (SEQ ID NO:112) TAAGCAGACCTATGCATCGCTCA (SEQ ID NO:113) GTGCTCCTTGTTCCCTTCACAG (SEQ ID NO:114) hIgLVpre2-8 wwm CTGAAGCATCTGCACCATCAAATC (SEQ ID NO:115) CCACCCACATGTGCCCGTGTG (SEQ ID N01 16) Example 3. Phenotypic assessment ofrodents having an engineered lg], light chain locus This example demonstrates the characterization of various immune cell populations in s (e.g., mice) engineered to contain an nous 1g?» light chain locus as described above. In ular, this example specifically demonstrates that rodents having an engineered endogenous 1g?» light chain locus as described herein display similar B cell development as compared to wild-type litterrnates. In particular, several engineered rodents harboring different amounts of genetic material corresponding to a human 1g)» light chain locus each detectably express 1g?» light chains having human variable and human or rodent constant domains on the surface of rodent B cells.

Briefly, spleens and femurs were harvested from ed engineered mouse s homozygous or heterozygous for the 1g?» light chain alleles depicted in Figure 4 and wild-type littermates. Bone marrow was collected from femurs by flushing with 1x phosphate buffered saline (PBS, Gibco) with 2.0% fetal bovine serum (FBS). Red blood cells from spleen and bone marrow preparations were lysed with ACK lysis buffer ) followed by washing with 1XPBS with 2.0% FBS. Isolated cells (1X106) were incubated with selected antibody ils for 30 min at +4°C (see Table 3).

Table 3. Antibodies for cell staining analyzed by flow try Antibody Label Vendor Clone Bone Marrow anti-mouse CD43 FITC BioLegend 1B1 1 anti—mouse c—Kit PE BioLegend 2B8 anti—mouse IgM PeCy7 eBiosciences 11/41 anti-mouse IgD PerCP-CyS .5 BioLegend 11-26c.2a anti—mouse CD3 PB end 17—A2 anti-mouse/—human B220 APC eBiosciences RA3-6B2 anti-mouse CD19 APC-H7 BD 1D3 Bone Marrow anti-mouse IgK FITC ED 187 .1 anti—mouse 1g?» PE BioLegend RML-42 anti-mouse 1gM PeCy7 eBiosciences 11/41 anti-mouse/—human B220 Cy5.5 BD RA3-6B2 anti-mouse CD3 PB BioLegend 17-A2 anti—human lg?» APC Biolegend MHL-3 8 anti-mouse CD19 APC-H7 BD 1D3 Spleen anti—mouse IgK FITC ED 187 .1 anti—mouse 1g?» PE BioLegend RML-42 anti-mouse IgM PeCy7 eBiosciences 11/41 anti-mouse IgD Cy5.5 BioLegend 11-26c.2a anti—mouse CD3 PB BioLegend 17—A2 anti-human 1g?» APC Biolegend MHL-3 8 anti-mouse CD19 APC-H7 BD 1D3 Following staining, cells were washed and fixed in 2% formaldehyde. Data acquisition was med on a BD FORTESSATM flow cytometer and analyzed with FLOWJOTM software. Representative results are set forth in Figures 5—13 and 18—21. Similar data was obtained for other strains depicted in Figure 4, but only selected ted strains are shown.

The results demonstrated that each strain harboring different amounts of genetic material corresponding to the human 1g?» light chain locus demonstrated similar immune cell tion s in the splenic and bone marrow compartments. In particular, as evident from the data shown in Figures 5-7, engineered mice demonstrated r number of CD194r splenic B cells, similar populations of mature and transitional B cell in the spleen, similar kappa usage in the spleen, and similar marginal zone and follicular B cell populations as their wild type littermate controls. Further, mice containing an engineered Igl light chain locus as described herein in the ce of additional humanized IgH and humanized IgK loci demonstrated no major differences in B cell development as ed with mice containing humanized IgH and zed IgK loci alone (e.g., see Figure 18A and 18B).

Also, as shown in mice represented in Figures 8-12, engineered mice had similar CD19+, pro-, pre-, immature and mature B cell number and similar kappa usage in bone marrow as their wild type littermate controls. A summary of the light chain expression in selected engineered s (homozygous — HO; heterozygous — I-IET) compared to their wild-type littermate ls is provided in Figure 13. Mice homozygous for an engineered humanized ng locus as described herein, and also homozygous for humanized IgH and IgK loci and homozygous for rodent Adam6-encoding sequence demonstrated increased utilization of lambda locus (about 40%) compared to the typical peripheral utilization (e.g., 5% in spleen) of lambda known for wild-type mice (see columns for 6680HO/VI HO/Adam6 HO and 6689HO/VI HO/Adam6 HO mice, Figure 13). Also, a small proportion mouse IgX-positive B cells was detected (~3-5%) in these mice, which confirms that the mouse C7» gene within the engineered 7» light chains locus is also expressed in the context of onal 7» light chains in these mice.

Example 4. Antibody sion in rodents having an engineered lg), light chain locus This example demonstrates the expression of antibodies from non-human animals, which antibodies contain light chains characterized by the presence of human V7» regions and human or rodent C?» regions, and which light chains are expressed from an engineered endogenous rodent 1g?» light chain locus. In particular, this example specifically demonstrates the expression of antibodies in the serum of non-human animals (e.g., rodents) whose gerrnline genome comprises an endogenous Iglt light chain locus comprising insertion of one or more human V?» gene segments, one or more human JA gene segments and one or more human Cit gene ts, which human Vlt, J)L and Ch gene segments are ly linked to a rodent Ck gene, and which endogenous immunoglobulin 9» light chain locus r comprises one or more rodent 1g?» light chain enhancers (El) and one or more human lg?» light chain enhancers (El).

Briefly, blood was drawn from selected engineered mouse strains and wild-type littermates (see Example 3). Serum was ted from blood using Eppendorf tubes centrifuged at 9000 rcf for five minutes at 4°C. Collected serum was used for immunoblotting to identify 1g light chain expression of antibodies. Mouse sera were diluted 1.5110 in PBS (without Ca2+ and Mg2+) and run on 4-20% Novex Tris-Glycine gels under reducing and non-reducing conditions. Gels were transferred to Polyvinylidene difluoride (PVDF) membranes according to manufacturer’s specifications. Blots were blocked overnight with 5% nonfat milk in Tris—Buffered Saline with 0.05% 20 (TBST, . PVDF membranes were d to different primary antibodies (mouse anti-h1g9» conjugated to HRP (Southern h); goat anti-mlglt conjugated to HRP, Southern Biotech) diluted 0 in 0.1% nonfat milk in TB ST for one hour at room temperature.

Blots were washed four times for ten minutes per wash and developed for one minute with Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences) according to manufacturer’s specifications. Blots were then imaged using GE Healthcare ImageQuant LAS-4000 Cooled CCD Camera Gel ntation System. Images were captured at 15 second intervals until 20 images were captured or images were fully exposed, whichever came first. Representative results are set forth in Figure 14A and 14B. The results demonstrated that all engineered strains expressed detectable levels of human Ig7t light chain containing antibodies in their sera (Figure 14 and data not shown).

Example 5. Human gene segment usage in s having an engineered Ig/‘l light chain locus ] This example demonstrates the human gene usage in light chains of antibodies expressed in rodents (e.g,, mice) engineered to contain an endogenous Ig7t light chain locus 2017/060006 as described herein. Usage of human 1g?» gene segments in selected engineered rodent strains described above was determined by Next tion Sequencing antibody repertoire analysis. , splenocytes were harvested from mice heterozygous for insertion of 25 functional human Vk gene segments, 4 functional human Jk—C?» clusters, and a human JM gene segment upstream of a mouse CM gene, and a human Ig?» enhancer inserted between the mouse CM gene and an endogenous mouse lg?» enhancer 3.1 (6889 heterozygous mice as in Figure 4). B cells were positively enriched from total splenocytes by anti—mouse CD19 magnetic beads and MACS columns (Miltenyi Biotech). Total RNA was isolated from splenic B cells using the RNeasy Plus kit n).

] Reverse transcription with an oligo—dT primer followed by gene specific PCR was performed to generate cDNA containing human ng constant region ces (CM, C12, C23 and C26), as well as cDNA containing mouse CM sequence, using SMARTerTM RACE cDNA Amplification Kit (Clontech). During reverse transcription, a specific DNA sequence (PIIA: 5’-CCCATGTACT CTGCGTTGAT ACCACTGCTT-3’, SEQ ID NO: 133) was attached to the 3’ end of the newly synthesized cDNAs. The cDNAs were purified by the Spin Gel and PCR Clean-Up Kit (Clontech), then further amplified using a primer reverse compliment to PIIA (5’ — AAGCAGTGGT ATCAACGCAG AGTACAT — 3 ’) paired with human C?» specific primer (5’-CACYAGTGTG GCCTTGTTGG CTTG—3’, SEQ ID ) and mouse CM specific primer (5’—CACCAGTGTG GCCTTGTTAG TCTC— 3’, SEQ ID NOzl32). d amplicons were then amplified by PCR using a PIIA specific primer (5’- GTGACTGGAG TTCAGACGTG TGCTCTTCCG ATCTAAGCAG TGGTATCAAC GCAGAGT-3’, SEQ ID NO: 134) and a nested human Ck specific primer (5’- ACACTCTTTC CCTACACGAC GCTCTTCCGA TCTCAGAGGA GAAC AGAGTG-3’, SEQ ID NO: 135) or a nested mouse CM specific primer (5’-ACACTCTTTC CCTACACGAC GCTCTTCCGA GTGG AAACAGGGTG ACTGATG-3’, SEQ ID NO: 136). PCR products between 450-690bp were isolated and collected by Pippin Prep (SAGE Science). These fragments were further amplified by PCR using following primers: GATACGG CCGA GATCTACACX XXXXXACACT CTTTCCCTAC ACGACGCTCT TCCGATC-3’, SEQ ID NO:137, and 5’-CAAGCAGAAG ACGGCATACG AGATXXXXXG TGACTGGAGT TCAGACGTGT GCTCTTCCGA TCT-3’, SEQ ID NO: 138 ("XXXXXX" is a 6bp index sequences to enable multiplexing samples for cing). PCR products between 490bp-710bp were isolated and collected by Pippin Prep, then quantified by qPCR using a KAPA Library Quantification Kit (KAPA Biosystems) before loading onto Miseq sequencer (Illumina) for sequencing (v3, 600- cycles), For bioinformatic analysis, the resulting Illumina ces were demultiplexed and trimmed for quality. Overlapping paired-end reads were then assembled and ted using local installation of igblast (NCBI, v2.2.25+). Reads were aligned to both human and mouse germline V7» and J7» segments se and sorted for the best hit. A sequence was marked as ambiguous and removed from analysis when multiple best hits with identical score were detected. A set of per] scripts was developed to analyze results and store data in mysql database. Representative results are set forth in Figures 15A (human Cl—primed) and 15B (mouse CX—primed).

In another experiment, splenic B cells harvested from mice (n=3) homozygous for insertion of the 6889 targeting vector (6889HO/VI m6 HO, see above) were analyzed for usage of human Ig7t gene segments by Next Generation Sequencing antibody repertoire analysis (described above). mRNA isolated from splenic B cells was amplified by ’RACE using primers to mouse mC7t (n=3) and human hC?» (n=3) constant regions and ced using MiSeq. Representative s are set forth in Figures 15C (human Ck— primed) and 15D (mouse CK-primed).

Mice generated using the 6889 targeting vector (i.e., co—electroporation of two targeting s and gRNAs) demonstrated expression of all 25 functional human V?» gene segments. Further, expression of human V?» gene segments from B cells of these mice demonstrated similar frequencies in isolated B cells as compared to human Vl gene segments observed in human cord blood. Taken together, this example demonstrates that rodents containing engineered Ig7t light chain loci as described herein in their germline genomes express 1g?» light chains containing human 1g?» ces in B cells. Further, such human 1g?» sequences can be readily distinguished in light chains containing a mouse or human Ck domain.

Example 6. Production ofantibodies in ered rodents This example demonstrates production of antibodies in a rodent that ses an engineered endogenous 1g?» light chain locus as described above using an n of interest WO 28691 (e.g., a single-pass or multi-pass membrane protein, etc). The methods described in this example, or immunization methods well known in the art, can be used to immunize rodents containing an ered endogenous ng light chain locus as described herein with polypeptides or fragments thereof (e.g, peptides derived from a desired epitope), or combination of polypeptides or fragments thereof, as desired. s of mice having an engineered lg?» light chain locus as described herein and a humanized IgH locus (described above), or an engineered Ig7t light chain locus as described herein and humanized IgH and IgK light chain loci (described above), are challenged with an antigen of interest using immunization methods known in the art. The antibody immune response is monitored by an ELISA immunoassay (i.e., serum titer). When a d immune response is ed, splenocytes (and/or other lymphatic tissue) are harvested and fused with mouse myeloma cells to preserve their viability and form immortal oma cell lines. The hybridoma cell lines are screened (e. g, by an ELISA assay) and selected to fy hybridoma cell lines that produce antigen—specific antibodies. omas may be further characterized for relative binding affinity and isotype as desired.

Using this technique, and the immunogen described above, several antigen-specific chimeric antibodies (i.e., antibodies possessing human variable domains and rodent constant domains) are obtained.

DNA encoding the variable domains of heavy chain and light chains may be isolated and linked to desirable isotypes (constant domains) of the heavy chain and light chain for the preparation of fully-human antibodies. Such an dy protein may be ed in a cell, such as a CHO cell. Fully human antibodies are then characterized for relative binding affinity and/or neutralizing activity of the antigen of interest.

DNA encoding the antigen-specific chimeric antibodies or the variable domains of light and heavy chains may be isolated directly from antigen-specific lymphocytes.

Initially, high affinity chimeric antibodies are isolated having a human le domain and a rodent constant domain and are characterized and selected for desirable characteristics, ing affinity, selectivity, epitope, etc. Rodent nt domains are ed with a desired human constant domain to generate fully-human dies. While the nt domain selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable domain. Antigen-specific antibodies are also isolated directly from antigen-positive B cells (from immunized mice) without fusion to myeloma cells, as described in, e,g., US. Patent No. 7,582,298, incorporated herein by reference in its entirety. Using this method, several fully human antigen-specific antibodies (i.e., antibodies possessing human variable domains and human constant domains) are made.

In one experiment, 6597het (n=6) and 6680het (n=6) mice were immunized via footpad administration with an ellular domain (ECD) of receptor polypeptide to determine the immune response in engineered mice.

Briefly, mice were primed with 2.35ug of antigen (receptor ptide ECD) plus 10ug of CpG adjuvant (Invivogen ODN1826), Mice were boosted seven times with 2.35ug of antigen (receptor polypeptide ECD), 10ug of CpG adjuvant and 25 ug of Adju- Phos (Brenntag). Two days after the final injection, blood was drawn from selected engineered mouse strains and controls. Serum was separated from blood using tainer capillary blood collector tubes (BD cat#365967) with centrifugation at 9000 rcf for five minutes at 4°C. Serum ELISA assays were performed to determine total IgG (Figure 16A), antigen-specific IgG (Figure 16B), mIgK (Figure 17C) mlgk (Figure 17B) and hng (Figure 17A) titers.

For a total IgG ELISA assay, Maxisorp plates (Nunc) were coated with 1 ug/mL goat anti-mouse IgG+IgM+IgA H&L (Abcam) in DPBS (with Ca and Mg) per well and incubated overnight at 4°C. The following day, plates were washed four times in PB S-T (PBS without Ca or Mg plus 0.1%Tween-20) and d in PB S-T with 1% BSA for one hour at room ature. Serum was diluted ten-fold (starting at 1:100 and ending at 1:109) in PB S-T with 0.1% BSA and mouse IgG standard (Sigma) was diluted three-fold (starting at lug/mL and ending at 0.05ng/mL) in PBS-T with 0.1% BSA. lOOul of each standard and sample dilution were added to the plate and incubated at room temperature for one hour, ed by washing four times in PBS-T. 100ul of goat anti-mouse IgG human ads-HRP (Southern h) diluted 122500 was added to each well, and plates were incubated an hour. Plates were washed four times in PBS-T, and 100111 of TMB substrate reagent (BD Biosciences) was added to each well for ten minutes. The reaction was stopped with 100 pl of 1N Sulfuric Acid per well, and tion was ed at 450 nm. Data were ed in GraphPad Prism and fit to a four-parameter curve.

] For an antigen-specific ELISA, rp plates (Nunc) were coated with 1 ug/mL antigen in DPBS (with Ca and Mg) per well and incubated overnight at 4°C. The following day, plates were washed four times in PBS-T and blocked in Sea Block (ThermoFisher) diluted 1:2 in PBS-T for one hour at room temperature. Serum was diluted (as above) in Sea Block diluted 1:5 in PB S-T. Each sample dilution was added to each plate and incubated at room temperature for one hour. Then plates were washed four times in PBS-T. lOOul of either goat anti-mouse IgG human P (Southern Biotech) diluted 1:2500, goat anti- mIgK-HRP (Southern Biotech) diluted 1:4000, goat anti-mlgk-HRP (Southern Biotech) diluted 1:4000, or goat anti-h1g7» mouse P (Southern Biotech) diluted 1:4000 were added to each well, and plates were incubated for one hour. Plates were washed four times in PBS-T, and ped as described above. Representative results are set forth in Figure 16 and 17‘ Taken together, this example specifically demonstrates that rodents engineered to contain Ig7t light chain loci as described herein generate strong antibody responses to immunization with an antigen of interest. Further, such engineered rodents demonstrate total and antigen-specific IgG levels comparable to wild-type controls, which confirms the capacity for a robust immune response in these ered animals. Indeed, h1g9» titers were stronger than mIg7t titers upon immunization (Figure 17A and 17B). Thus, engineered s as described herein provide an ed in vivo system for the generation of antibodies for the development of human antibody-based therapeutics, in particular, human antibody—based therapeutics that utilize human 1g?» light chain sequences.

EQUIVALENTS It is to be appreciated by those d in the art that s alterations, modifications, and improvements to the present disclosure will readily occur to those d in the art, Such alterations, modifications, and improvements are intended to be part of the present disclosure, and are intended to be within the spirit and scope of the invention.

Accordingly, the foregoing description and g are by way of example only and any invention described in the present sure if further described in detail by the claims that follow.

Use of ordinal terms such as "first," "second," "third," etc, in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to guish one claim element having a certain name from another t having a same name (but for use of the ordinal term) to distinguish the claim elements.

The articles "a" and "an" in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are t in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or s.

The invention also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim ent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g, in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in l, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, es, etc, certain ments of the invention or aspects of the invention consist, or consist essentially of, such elements, es, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of r the specific exclusion is recited in the specification.

Those skilled in the art will appreciate l standards of ion or error attributable to values obtained in assays or other processes as described herein. The publications, websites and other reference materials nced herein to describe the ound of the invention and to provide additional detail regarding its practice are hereby orated by reference in their entireties.

Claims (21)

1. A mouse whose germline genome comprises an endogenous immunoglobulin  light chain locus comprising: (a) one or more human V gene segments, (b) one or more human J gene segments, and (c) one or more human C gene segments, wherein (a) and (b) are operably linked to (c) and a mouse C gene segment, and wherein the endogenous immunoglobulin  light chain locus further comprises one or more mouse immunoglobulin  light chain enhancers (E), and one or more human immunoglobulin  light chain enhancers (E), and wherein the mouse expresses an immunoglobulin λ light chain sing a human λ le domain and either a human λ constant domain or a mouse λ constant domain.

2. The mouse of claim 1, wherein the endogenous immunoglobulin  light chain locus comprises two mouse Es.

3. The mouse of claim 2, wherein the two mouse Es are a mouse E and a mouse E3-1.

4. The mouse of claim 1, wherein the endogenous immunoglobulin  light chain locus comprises three human Es.

5. The mouse of claim 1, n the germline genome further comprises (i) an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene ts, which human VH, DH and JH gene segments are operably linked to a mouse immunoglobulin heavy chain constant region; or (ii) an endogenous immunoglobulin heavy chain locus comprising insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments, which human VH, DH and JH gene segments are operably linked to a mouse immunoglobulin heavy chain constant region, and an endogenous immunoglobulin  light chain locus comprising insertion of one or more human V gene segments and one or more human J gene segments, which human V and J gene segments are operably linked to a mouse immunoglobulin C .

6. The mouse of claim 5, wherein the insertion of one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments replace mouse VH, DH gene segments.

7. The mouse of claim 6, wherein the insertion includes human non-coding DNA that lly appears between human VH, DH, and JH gene segments, and combinations thereof.

8. The mouse of claim 5, wherein the insertion of one or more human V gene segments and one or more human J gene ts replace mouse V and J gene segments.

9. The mouse of claim 8, n the insertion includes human non-coding DNA that naturally appears between human V and J gene segments, and combinations thereof.

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

11. The mouse of claim 5, wherein the mouse C region is an endogenous mouse C region.

12. The mouse of claim 1, wherein the endogenous immunoglobulin  light chain locus comprises a deletion of endogenous V and J gene segments, in whole or in part.

13. The mouse of claim 1, wherein the mouse C gene segment is a mouse C gene segment.

14. The mouse of claim 5, wherein the immunoglobulin  light chain locus comprises ion of the proximal V duplication, in whole or in part, of a human immunoglobulin  light chain locus.

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

16. The mouse of claim 15, wherein the immunoglobulin heavy chain locus further comprises insertion of one or more nucleotide ces encoding one or more mouse Adam6 polypeptides.

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

18. The mouse of claim 5, wherein the mouse is gous for the endogenous globulin  light chain locus.

19. The mouse of claim 1, wherein the mouse is homozygous for the endogenous immunoglobulin  light chain locus.

20. A method of making a genetically modified mouse comprising: modifying the germline genome of the mouse so that it comprises an engineered immunoglobulin  light chain locus that includes: (a) one or more human V gene ts, (b) one or more human J gene segments, and (c) one or more human C gene segments, wherein (a) and (b) are operably linked to (c) and a mouse C gene segment, and wherein the endogenous immunoglobulin  light chain locus further comprises one or more mouse immunoglobulin  light chain enhancers (E), and one or more human globulin  light chain enhancers (E), thereby making said genetically modified mouse.

21. The method according to claim 20, comprising (i) introducing one or more DNA fragments into a mouse embryonic stem cell, said one or more DNA fragments together comprise: (a) one or more human V gene segments, (b) one or more human J gene segments, and (c) one or more human C gene segments, wherein ) are operably linked to (c) and a mouse C gene segment, and n the one or more DNA fragments further comprise one or more human immunoglobulin  light chain enhancers (E); (ii) obtaining the mouse embryonic stem cell generated in (i); and (iii) creating the genetically modified mouse using the mouse embryonic stem cell of (ii).