WO2017214089A1

WO2017214089A1 - Non-human animals expressing antibodies with human lambda light chains

Info

Publication number: WO2017214089A1
Application number: PCT/US2017/036071
Authority: WO
Inventors: Lynn Macdonald; Vera VORONINA; Chunguang GUO; John Mcwhirter; Andrew J. Murphy
Original assignee: Regeneron Pharmaceuticals, Inc.
Priority date: 2016-06-06
Filing date: 2017-06-06
Publication date: 2017-12-14

Abstract

In certain aspects provided herein are non-human animals that express antibodies having human Vκ and νλ domains. In some embodiments, the ratio of antibodies containing human Vκ domains to antibodies containing human νλ domains is similar to that which occurs in humans.

Description

NON-HUMAN ANIMALS EXPRESSING ANTIBODIES WITH HUMAN LAMBDA

LIGHT CHAINS

RELATED APPLICATION

[001] This application claims the benefit of priority to U.S. Provisional Patent Application serial number 62/345,910, filed June 6, 2016, hereby incorporated by reference in its entirety.

BACKGROUND

[002] Human antibodies are a rapidly growing class of therapeutics. Of the technologies that are currently used for their production, the development of transgenic animals (e.g., rodents) engineered with genetic material encoding human antibodies, in whole or in part, has revolutionized the generation of human therapeutic monoclonal antibodies for the treatment of various diseases. Still, development of improved in vivo systems for generating human monoclonal antibodies would be desirable.

SUMMARY

[003] In some embodiments, provided herein are non-human animals that provide improved in vivo systems for development of antibodies and/or antibody-based therapeutics for administration to humans. In certain embodiments, provided herein are non-human animals that provide improved in vivo systems for development of antibodies and/or antibody-based therapeutics that contain human VK or νλ domains characterized by improved performance as compared to antibodies and/or antibody-based therapeutics obtained from in vivo systems that deviate from provided non-human animals.

[004] In certain aspects, provided herein is a non-human animal having immunoglobulin heavy and κ light chain loci, which immunoglobulin heavy and κ light chain loci each contain an engineered immunoglobulin variable region. As described herein, provided non-human animals, in some embodiments, contain in their germline genome (1) an immunoglobulin heavy chain locus comprising an engineered immunoglobulin heavy chain variable region characterized by the presence 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 (CH) and/or a CH gene (e.g., a rodent immunoglobulin heavy chain constant region gene such as, for example, a Cμ gene, a C5 gene, a Cy gene, a Ce gene and/or a Ca gene), (2) a first immunoglobulin light chain locus comprising an engineered immunoglobulin κ light chain variable region characterized by the presence 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 first non-human immunoglobulin κ light chain constant region (CK) and/or a CK gene (e.g., a rodent immunoglobulin CK gene), and (3) a second immunoglobulin light chain locus comprising an engineered immunoglobulin λ light chain variable region characterized by the presence of one or more human νλ gene segments and one or more human Ιλ gene segments, which human νλ and Ιλ gene segments are operably linked to a second non-human

immunoglobulin CK region and/or a CK gene (e.g., a rodent immunoglobulin CK gene). In many embodiments, the engineered immunoglobulin λ light chain variable region further comprises a human immunoglobulin κ light chain sequence between said one or more human νλ gene segments and one or more human Ιλ gene segments. In some certain embodiments, the human immunoglobulin κ light chain sequence is or comprises a genomic sequence that naturally appears between a human VK4- 1 gene segment and a human JKI gene segment of a human immunoglobulin κ light chain locus. In many embodiments, provided non-human animals further comprise functionally silenced endogenous immunoglobulin λ light chain loci. In some embodiments, provided non-human animals contain human V, D and/or J gene segments (i.e., heavy and/or light) in natural or germline configuration.

[005] In some embodiments, provided non-human animals are characterized by expression of antibodies containing engineered κ light chains that include either a human VK domain or a human νλ domain. In some embodiments, provided non-human animals are characterized by expression of immunoglobulin light chains from first and second κ light chain alleles in a proportion that is similar or substantially similar to the expression of κ and λ light chains observed in humans (e.g., 60:40). In some embodiments, provided non-human animals are characterized by expression of immunoglobulin light chains from first and second κ light chain alleles in a proportion that is different from the expression of κ and λ light chains observed in wild-type mice (e.g., 80:20, 70:30, 60:40, 50: 50, 40:60, and the like). [006] In some embodiments, a non-human animal, non-human cell or non-human tissue is provided whose germline genome comprises an immunoglobulin heavy chain locus comprising 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 CH region and/or a Qj gene; a first immunoglobulin κ light chain locus (or allele) comprising 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 CK region and/or a CK gene; a second immunoglobulin κ light chain locus (or allele) comprising one or more human νλ gene segments and one or more human Ιλ gene segments, which human νλ and Ιλ gene segments are operably linked to a non-human CK region and/or a CK gene; and an endogenous immunoglobulin λ light chain locus that is deleted in whole or in part.

[007] In some embodiments of a provided non-human animal, the germline genome of said non-human animal further comprises insertion of one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides. In some certain embodiments, the germline genome of said non-human animal comprises insertion of two nucleotide sequences, each of which encode a non-human Adam6 polypeptide (e.g., two copies of an Adam6- encoding nucleotide sequence or homozygous for an Adam6-encoding nucleotide sequence). In some embodiments, two nucleotide sequences include a mouse Adam6a-encoding sequence and a mouse Adam6b-encoding sequence.

[008] In some embodiments, an immunoglobulin heavy chain locus comprises a replacement of non-human VH, DH and JH gene segments with the one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments. In some embodiments, an immunoglobulin heavy chain locus comprises the human VH gene segments from VH3-74 to VH6- 1 , the human DH gene segments from DH1 - 1 to DH7-27, and the human JH gene segments from JHI to JH6.

[009] In some embodiments, a non-human immunoglobulin heavy chain constant region is an endogenous non-human immunoglobulin heavy chain constant region.

[0010] In some embodiments, an immunoglobulin heavy chain locus lacks an endogenous non-human Adam6 gene. In some embodiments, an immunoglobulin heavy chain locus further comprises insertion of one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides.

[0011] In some certain embodiments, one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides are inserted between a first and a second human VH gene segment. In some embodiments, one or more nucleotide sequences encoding one or more non-human 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 non-human Adam6 polypeptides are inserted between a first and a second human DH gene segment. In some embodiments, one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides are inserted between a human DH gene segment and a human JH gene segment. In some embodiments, one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides are inserted between a first and a second human JH gene segment. In some embodiments, one or more nucleotide sequences encoding one or more non- human Adam6 polypeptides are inserted so that said one or more nucleotide sequences are adjacent to at least one human VH gene segment.

[0012] In some embodiments, a first human VH gene segment is human VH3-74 and a second human VH gene segment is human VH6-1. 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 encoding one or more non-human Adam6 polypeptides are inserted in the place of a human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides are inserted at a location that resembles the approximate position of a non-human Adam6 gene or non-human Adam6-encoding sequence in a non-human animal genome; in some certain embodiments, a host non-human animal genome.

[0013] In some embodiments, a first immunoglobulin κ light chain locus (or allele) comprises a replacement of non-human VK and JK gene segments with the one or more human VK gene segments and one or more human JK gene segments.

[0014] In some embodiments, a first immunoglobulin κ light chain locus (or allele) comprises the proximal duplication (or proximal repeat, or proximal arm), in whole or in part, of a human immunoglobulin κ light chain locus. In some certain embodiments, a first immunoglobulin κ light chain locus (or allele) comprises the human VK gene segments from VK2-40 to VK4-1 and the human JK gene segments from JKI to JK5.

[0015] In some embodiments, a second immunoglobulin κ light chain locus (or allele) comprises a replacement of rodent VK and JK gene segments with the one or more human νλ gene segments and one or more human Ιλ gene segments. In some embodiments, a second immunoglobulin κ light chain locus (or allele) further comprises a human immunoglobulin κ light chain sequence between the one or more human νλ gene segments and the one or more human Ιλ gene segments. In some certain embodiments, a human immunoglobulin κ light chain sequence is or comprises a genomic sequence that naturally appears between a human VK4-1 gene segment and a human JKI gene segment of a human immunoglobulin κ light chain locus. In some embodiments, a human immunoglobulin κ light chain sequence is or comprises a sequence that is located about 130bp 3' of a 3'UTR of a human VK4-1 gene segment to about 600bp 5' of a human JKI gene segment. In some embodiments, a human immunoglobulin κ light chain sequence is about 22,800bp or about 23kb. In some embodiments, a second immunoglobulin κ light chain locus (or allele) comprises the human νλ gene segments from V 5-52 to νλ1-40 and/or V 3-27 to V 3-1, and the human Ιλ gene segments Ιλΐ, Ιλ2, Ιλ3 and .

[0016] In some embodiments, a first and/or second immunoglobulin κ light chain locus (or allele) is an endogenous non-human immunoglobulin κ light chain locus (or allele). In some embodiments, a non-human CK region and/or CK gene of a first immunoglobulin κ light chain locus (or allele) is an endogenous non-human CK region and/or CK gene. In some

embodiments, a non-human CK region and/or CK gene of a second immunoglobulin κ light chain locus (or allele) is an endogenous non-human CK region and/or CK gene. In some embodiments, a non-human CK region and/or CK gene of a first immunoglobulin κ light chain locus (or allele) is an endogenous non-human CK region and/or CK gene, and wherein a non- human CK region and/or CK gene of a second immunoglobulin κ light chain locus (or allele) is an endogenous non-human CK region and/or CK gene.

[0017] In some embodiments, an endogenous immunoglobulin λ light chain locus is deleted in part. In some certain embodiments, an endogenous immunoglobulin λ light chain locus comprises a deletion of νλ2-νλ3-Ιλ2 gene segments and/or V l-J 3-C 3-J l-Od gene segments. In some certain embodiments, an endogenous immunoglobulin λ light chain locus comprises a deletion of V 2-V 3-J 2-C 2-J 4P-C 4P gene segments and/or V 1-J 3-C 3- J l-C l gene segments. In some certain embodiments, an endogenous immunoglobulin λ light chain locus comprises a deletion of C 2-J 4P-C 4P gene segments and/or C 3-J l-Od gene segments. In some certain embodiments, an endogenous immunoglobulin λ light chain locus comprises a deletion of at least C 2, C 3, Ckl and combinations thereof.

[0018] In some embodiments, a provided non-human animal, non-human cell or non- human tissue is homozygous for an immunoglobulin heavy chain locus as described herein.

[0019] In some embodiments, a provided non-human animal, non-human cell or non- human tissue is homozygous for an endogenous immunoglobulin λ light chain locus that is deleted, in whole or in part.

[0020] In some embodiments, a cell is a lymphocyte. In some embodiments, a cell is selected from a B cell, dendritic cell, macrophage, monocyte, and a T cell.

[0021] In some embodiments, a cell is a non-human embryonic stem (ES) cell. In some embodiments, a non-human ES cell is a rodent ES cell. In some certain embodiments, a rodent ES cell is a mouse ES cell and is 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 mixture of 129, C57BL and BALB/c strains.

[0022] In some embodiments, use of a non-human ES cell as described herein to make a non-human animal is provided. In some certain embodiments, a non-human ES cell is a mouse ES cell and is used to make a mouse comprising engineered immunoglobulin heavy, κ light and λ light chain loci as described herein. In some certain embodiments, a non-human ES cell is a rat ES cell and is used to make a rat comprising engineered immunoglobulin heavy, κ light and λ light chain loci as described herein.

[0023] In some embodiments, a tissue is selected from adipose, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, ovum, and a combination thereof. [0024] In some embodiments, an immortalized cell made, generated, produced or obtained from an isolated non-human cell or tissue as described herein is provided.

[0025] In some embodiments, a non-human embryo made, generated, produced, or obtained from a non-human 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.

[0026] In some embodiments, use of a non-human embryo as described herein to make a non-human animal is provided. In some certain embodiments, a non-human embryo is a mouse embryo and is used to make a mouse comprising engineered immunoglobulin heavy, κ light and λ light chain loci as described herein. In some certain embodiments, a non-human embryo is a rat embryo and is used to make a rat comprising engineered immunoglobulin heavy, κ light and λ light chain loci as described herein.

[0027] In some embodiments, a kit comprising a non-human animal, an isolated non- human cell or tissue, an immortalized cell, a non-human ES cell, or a non-human embryo as described herein is provided.

[0028] In some embodiments, a kit as described herein for use in the manufacture and/or development of a drug (e.g., an antibody or antigen-binding fragment thereof) for therapy or diagnosis is provided.

[0029] In some embodiments, a kit as described herein for use in the manufacture and/or development of a drug (e.g., an antibody or antigen-binding fragment thereof) for the treatment, prevention or amelioration of a disease, disorder or condition is provided.

[0030] In some embodiments, a method of making a non-human animal is provided, the method comprising a step of generating a non-human animal from a non-human cell, non- human ES cell or non-human embryo as described herein, thereby making the non-human animal.

[0031] In some embodiments, a non-human animal made, generated, produced, obtained or obtainable from a method as described herein is provided.

[0032] 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 non-human animal produces an immune response to the antigen of interest; and (c) recovering an antibody from the non-human animal, or a non-human cell, that binds the antigen of interest.

[0033] In some embodiments of a method of producing an antibody in a non-human animal, a non-human cell is a B cell. In some embodiments of a method of producing an antibody in a non-human animal, a non-human cell is a hybridoma. In some embodiments of a method of producing an antibody in a non-human animal, human VH, DH and JH gene segments replace non-human VH, DH and JH gene segments.

[0034] In some embodiments, a non-human animal is provided whose germline genome comprises two endogenous immunoglobulin heavy chain alleles each comprising 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 an endogenous CH region and/or CH gene; a first

immunoglobulin κ light chain allele comprising one or more human VK gene segments and one or more human JK gene segments operably linked to an endogenous CK region and/or CK gene; a second immunoglobulin κ light chain allele comprising one or more human νλ gene segments and one or more human Ιλ gene segments operably linked to an endogenous CK region and/or CK gene; and two endogenous immunoglobulin λ light chain alleles each comprising a deletion of νλ2-νλ3-Ιλ2 gene segments and V l-J 3-C 3-J l-Od gene segments, wherein the non-human animal expresses antibodies comprising human VK domains generated from rearrangement of human VK and JK gene segments of the first immunoglobulin K light chain allele and νλ domains generated from rearrangement of human νλ and Ιλ gene segments of the second immunoglobulin κ light chain allele, and wherein said VK and νλ domains are represented in the expressed antibody repertoire of the non-human animal in a 4: 1 ratio (or 80:20 ratio), a 7:3 ratio (or 70:30 ratio), a 3:2 ratio (or 60:40 ratio), a 1 : 1 ratio (or 50:50 ratio), or a 2:3 ratio (or 40:60 ratio), preferably a 3:2 ratio (or 60:40 ratio).

[0035] In some embodiments of a provided non-human animal, two endogenous immunoglobulin heavy chain alleles each containing a deletion in whole or in part of the endogenous non-human immunoglobulin heavy chain variable region. In some embodiments of a provided non-human animal, one or both of two endogenous immunoglobulin heavy chain alleles further comprise insertion of one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides.

[0036] In some embodiments of a provided non-human animal, a first and/or second immunoglobulin κ light chain allele comprises a deletion in whole or in part of the endogenous non-human immunoglobulin κ light chain variable region. In some certain embodiments of a provided non-human animal, a first and second immunoglobulin κ light chain allele each comprises a deletion in whole or in part of the endogenous non-human immunoglobulin κ light chain variable region.

[0037] 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.

[0038] In some embodiments, a non-human animal, non-human cell or non-human tissue as described herein is provided for use in the manufacture of a medicament for the treatment, prevention or amelioration of a disease, disorder or condition.

[0039] In some embodiments, use of a non-human animal, non-human cell or non-human tissue as described herein in the manufacture and/or development of a drug or vaccine for use in medicine, such as use as a medicament, is provided.

[0040] 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.

[0041] In various embodiments, a provided non-human animal, non-human 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 comprises a genetic background that includes a 129 strain, a BALB/c strain, a C57BL/6 strain, a mixed 129xC57BL/6 strain or combinations thereof.

[0042] As used in this application, the terms "abo f 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 ordinary skill in the relevant art. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The Drawings included herein, which is composed of the following Figures, is for illustration purposes only and not for limitation.

[0044] Figure 1 shows representative illustrations, not to scale, of engineered

immunoglobulin heavy and light chain loci of various rodent strains described herein. For some strains, only a single immunoglobulin allele or locus (heavy or light) is shown.

Elsewhere in the present disclosure, homozygosity or heterozygosity for various engineered immunoglobulin loci is indicated. Open symbols indicate human gene segments and/or sequence, closed symbols indicate non-human (e.g., rodent) gene segments and/or sequence, and site-specific recombination recognition sites (e.g., Fit) are also indicated. Unless otherwise indicated, number of V, D and/or J gene segments depicted herein are illustrative. VI 3: a rodent strain having a genome comprising a homozygous immunoglobulin heavy chain locus containing a plurality of human VH, DH and JH gene segments operably linked to a rodent immunoglobulin heavy chain constant region including rodent heavy chain enhancers and regulatory regions, and containing a nucleotide sequence encoding one or more functional murine Adam6 genes (e.g., U.S. Patent Nos. 8,642,835 and 8,697,940); and a homozygous immunoglobulin κ light chain locus containing human VK and JK gene segments operably linked to a rodent immunoglobulin κ light chain constant region including rodent κ light chain enhancers. It is noted that in various embodiments in the present application, other

VELOCF MUNE® strains that may or may not comprise a nucleotide sequence encoding one or more functional Adam6 genes can also be used. Thus, in various embodiments, the heavy chain locus of the non-human animal described herein may comprise a plurality of human VH, DH and JH gene segments operably linked to a rodent immunoglobulin heavy chain constant region including rodent heavy chain enhancers and regulatory regions, without a nucleotide sequence encoding one or more functional murine Adam6 genes. LoK: a rodent strain having a genome comprising a homozygous immunoglobulin heavy chain locus containing a plurality of human VH, DH and JH gene segments operably linked to a rodent immunoglobulin heavy chain constant region including rodent heavy chain enhancers and regulatory regions, and containing a nucleotide sequence encoding one or more functional murine Adam6 genes (e.g., see U.S. Patent Nos. 8,642,835 and 8,697,940); and a homozygous immunoglobulin κ light chain locus containing human νλ and Ιλ gene segments operably linked to a rodent immunoglobulin κ light chain constant region including rodent κ light chain enhancers, and a human immunoglobulin κ light chain sequence positioned between the human νλ and Jl gene segments (e.g., see U.S. Patent Nos. 9,006,511, 9,029,628, 9,035,128, 9,066,502, 9, 150,662, 9, 163,092, 9,206,261, 9,206,262, 9,206,263, 9,226,484, and 9,334,333), a human

immunoglobulin κ light chain sequence between human νλ and Jl gene segments is indicated by an open bar filed with closed diamonds, mlg ^{' '}: a rodent strain having an inactivated endogenous immunoglobulin λ light chain locus resulting from a deletion of V 2-V 3-J 2 and V l-J13-C 3-Jll-OJ gene segments thereby inactivating endogenous λ light chain expression.

[0045] Figure 2 shows a detailed illustration, not to scale, of exemplary immunoglobulin loci (heavy and light) of a non-human animal as described herein (referred to herein as LoK/KoK, where the non-human animal is hemizygous at each of the LOK or KOK loci). Unless otherwise indicated, number of V, D and/or J gene segments depicted herein are illustrative. As depicted, an exemplary non-human animal comprises: two immunoglobulin heavy chain alleles each containing a plurality of human VH, DH and JH gene segments operably linked to a rodent immunoglobulin heavy chain constant region including rodent heavy chain enhancers and regulatory regions, and optionally containing a nucleotide sequence encoding one or more functional murine Adam6 genes in the place of a human Adam6 pseudogene; two immunoglobulin κ light chain alleles with a first allele containing a plurality of human VK and JK gene segments operably linked to a rodent immunoglobulin κ light chain constant region including rodent κ light chain enhancers, and a second allele containing a plurality of human νλ and Ιλ gene segments operably linked to a rodent immunoglobulin κ light chain constant region including rodent κ light chain enhancers, and a human

immunoglobulin κ light chain sequence (open bar filed with closed diamonds) positioned between the human νλ and Tk gene segments; and two immunoglobulin λ light chain alleles each containing a deletion of νλ2-νλ3-Ιλ2 and νλ1-Ιλ3-Ολ3-.Τλ1-Ολ1 gene segments thereby inactivating endogenous λ light chain expression. Open symbols indicate human gene segments and/or sequence, closed symbols indicate rodent gene segments and/or sequence, and site-specific recombination recognition sites (e.g., Fit, loxP) and hygromycin selection cassettes (pgk-hyg-tk) are also indicated. Ei: intronic heavy chain enhancer; Εκί: intronic κ light chain enhancer; 3 Έκ: 3 ' κ light chain enhancer.

[0046] Figures 3A to 3D show representative flow cytometry contour plots of immune cell populations from harvested spleens and femurs of various engineered rodent strains described herein. Percentages of cells within each quadrant of a contour plot were calculated from 20,000 events (i.e., cells). Figure 3A: splenocytes gated on singlets showing expression of CD19 (y- axis) and CD3 (x-axis). Figure 3B: splenocytes gated on CD19⁺ expression showing expression of mouse immunoglobulin λ (¾λ) or κ (IgK) light chain constant regions. Figure 3C: bone marrow gated on singlets showing expression of immunoglobulin M (IgM, y-axis) and B220 (x-axis) indicating immature (IgM⁺B220^low) and mature (IgM⁺B220^{hi h}) B cells. Figure 3D: bone marrow gated on immature (top row, IgM⁺B220^low) and mature (bottom row, ¾Μ⁺Β220^Μ^) B cells showing expression of mouse immunoglobulin λ (Igk) or κ (¾κ) light chain constant regions. Genotype of each engineered rodent strain is noted at the top of each contour plot (see Figure 1 and the examples section below for further description of immunoglobulin loci for each strain).

[0047] Figure 4 shows representative repertoire sequencing illustrating human VK and νλ gene usage from three LoK/KoK mice. Human VK (left graph) and human νλ (right graph) gene segments are noted below each graph. The x-axis of the left graph recites from left to right human VK gene segments ordered as most proximal human VK gene segment (i.e., 3 ' end of human VK region closest to human JK region of a human IgK light chain locus) to most distal human VK gene segment (i.e., 5' end of VK region farthest from JK region within a proximal V- cluster of a human IgK light chain locus). The x-axis of the right graph recites from left to right human νλ gene segments ordered as distal human νλ gene segment (i.e., most 5' human νλ gene segment of cluster B of a human Igk light chain locus) to most proximal human νλ gene segment (i.e., 3 ' end of human νλ region closets to human Jk-Ck pairs of a human Igk light chain locus).

DEFINITIONS

[0048] 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 described embodiments, or otherwise within the scope of the claims.

[0049] In general, terms used herein are in accordance with their understood meaning in the art, unless clearly indicated otherwise. Explicit 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. References cited within this specification, or relevant portions thereof, are incorporated herein by reference.

[0050] 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). Those of ordinary skill will appreciate that routes of administration may vary depending, for example, on the subject or system to which the composition is being administered, the nature of the composition, 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 instillation), buccal, enteral, interdermal, intra-arterial, intradermal, 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, administration may involve intermittent dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0051] 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 recovery or complete prevention of a disease, disorder or condition.

[0052] 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). [0053] 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 so that conclusions may reasonably be drawn based on differences or similarities observed. Those 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, entities, situations, sets of conditions, etc. to be considered comparable.

[0054] Control, as used herein, refers to the art-understood meaning of a "controF being a standard against which results are compared. Typically, controls are used to augment integrity 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 "controF also includes a "control animal." A "control animaF may have a modification as described herein, a modification that is different as described herein, or no modification (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 embodiments, 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.

[0055] Determining, measuring, evaluating, assessing, assaying 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. Assaying may be relative or absolute. "Assaying for the presence of can be determining the amount of something present and/or determining whether or not it is present or absent.

[0056] 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 particular 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 produced by or present in the relevant cell or organism (e.g., the relevant cell type or organism type). "Heterologous" also includes a polypeptide, gene or gene product that is normally present in a particular native cell or organism, but has been modified, for example, by mutation or placement under the control of non-naturally associated and, in some

embodiments, non-endogenous regulatory elements (e.g., a promoter).

[0057] Host cell, as used herein, refers to a cell into which a nucleic acid or protein has been introduced. Persons of skill upon reading 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 modifications may occur in succeeding generations due to either mutation or environmental influences, 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 protein, regardless of the

Kingdom of life to which the cell is designated. Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of Escherichia coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO Kl, DXB-11 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, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, 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, the 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 embodiments, a host cell is or comprises an isolated cell. In some embodiments, a host cell is part of a tissue. In some embodiments, a host cell is part of an organism. [0058] 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

(MACVECTOR™ 10.0.2, Mac Vector Inc., 2008).

[0059] In vitro, as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0060] In vivo, 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).

[0061] 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, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%), about 97%), about 98%, about 99%, or more than about 99% of the other components with which they were 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, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated' or even "pure", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated 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 considered 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 nature; 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 chemically synthesized, or is synthesized in a cellular system different from that which produces it in nature, is considered to be an "isolated" 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.

[0062] Locus or loci, as used herein, refers to a specific location(s) of a gene (or significant sequence), DNA sequence, polypeptide-encoding sequence, or position on a chromosome 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 or J), immunoglobulin gene segment DNA sequence, immunoglobulin gene segment-encoding sequence, or immunoglobulin gene segment position on a chromosome of the genome of an organism that has been identified as to where such a sequence 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. Those of ordinary skill in the art will appreciate that chromosomes may, in some embodiments, contain hundreds or even thousands of genes and demonstrate physical co-localization of similar genetic loci when comparing between different species. Such genetic loci can be described as having shared synteny.

[0063] Non-human animal, as used herein, refers to any animal that is not a human. In some embodiments, a non-human animal is a cyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian, a reptile, a mammal, and a bird. In some embodiments, a non- human animal is a mammal. In some embodiments, a non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, a non-human animal is a rodent such as a rat or a mouse. [0064] 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, "nucleic 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 "nucleic 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, are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a "nucleic acid' has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than

phosphodiester bonds. In some embodiments, a "nucleic acid' is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a "nucleic acid" is, comprises, or consists of one or more nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a "nucleic acid' comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a "nucleic acid' has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a "nucleic acid' includes one or more introns. In some embodiments, a "nucleic acid" includes one or more exons. In some embodiments, a "nucleic acid' is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a "nucleic acid' is at least 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 complement of a sequence that encodes, a polypeptide. In some embodiments, a "nucleic acid' has enzymatic activity.

[0065] Operably linked, as used herein, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. For example, unrearranged immunoglobulin variable region gene segments are operably linked to a constant region gene if, following V(D)J recombination (and, in some instances, class switch recombination) the resulting rearranged immunoglobulin variable region gene and the constant region gene are expressed together as an antibody heavy or light chain. A control sequence "operably linked" 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' sequences include both expression control sequences that are contiguous with a gene of interest and expression control sequences that act in trans or at a distance to control a gene of interest. The term "expression control sequence" includes polynucleotide sequences, which are necessary to affect the expression and processing of coding sequences 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 protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism. For example, in prokaryotes, such control sequences generally include promoter, ribosomal binding site and transcription termination sequence, while in eukaryotes typically such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended 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.

[0066] 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 isolated from a recombinant, combinatorial human polypeptide library

(Hoogenboom, H. R., 1997, TIB Tech. 15:62-70; Azzazy, H. and W.E. Highsmith, 2002, Clin. Biochem. 35:425-45; Gavilondo, J. V. and J.W. Larrick, 2002, BioTechniques 29: 128-45; Hoogenboom H., and P. Chames, 2000, Immunol. Today 21 :371-8), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D. et al., 1992, Nucl. Acids Res. 20:6287-95; Kellermann, S-A. and L.L. Green, 2002, Curr. Opin. Biotechnol. 13 :593-7; Little, M. et al., 2000, Immunol. Today 21 :364-70; Murphy, A.J. et al., 2014, Proc. Natl. Acad. Sci. U.S.A. 111(14):5153-8) or polypeptides prepared, expressed, created or isolated by any other means that involves splicing selected sequence elements to one another. 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 silico. In some embodiments, one or more such selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source. For example, in some embodiments, a recombinant 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 recombinant polypeptides 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.

[0067] Reference, as used herein, refers to a standard or control agent, animal, cohort, individual, population, sample, sequence or value against 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, sample, sequence or value is a historical reference, optionally embodied in a tangible medium. In some embodiments, a reference may refer to a control. A "reference" also includes a "reference animaF '. A "reference animaF may have a modification as described herein, a modification that is different as described herein or no modification (i.e., a wild-type animal). Typically, as would be understood by those skilled in the art, a reference agent, animal, cohort, individual, population, sample, sequence or value is determined or characterized under conditions comparable to those utilized to determine or characterize an agent, animal (e.g., a mammal), cohort, individual, population, sample, sequence or value of interest.

[0068] Substantially , as used herein, refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts 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 completeness inherent in many biological and chemical phenomena.

[0069] Substantial homology , 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 characteristics. For example, 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 chains. Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution. Typical amino acid categorizations are summarized below. Alanine Ala A Nonpolar 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

Glutamic acid Glu E Polar Negative -3.5

Glutamine Gin Q Polar Neutral -3.5

Glycine Gly G Nonpolar Neutral -0.4

Histidine His H Polar Positive -3.2

Isoleucine He I Nonpolar Neutral 4.5

Leucine Leu L Nonpolar Neutral 3.8

Lysine Lys K Polar Positive -3.9

Methionine Met M Nonpolar Neutral 1.9

Phenylalanine Phe F Nonpolar Neutral 2.8

Proline Pro P Nonpolar Neutral -1.6

Serine Ser S Polar Neutral -0.8

Threonine Thr T Polar Neutral -0.7

Tryptophan Tip w Nonpolar Neutral -0.9

Tyrosine Tyr Y Polar Neutral -1.3

Valine Val V Nonpolar Neutral 4.2

Ambiguous Amino Acids 3 -Letter 1 -Letter

Asparagine or aspartic acid Asx B

Glutamine or g lutamic acid Glx Z

Leucine or Isoleucine Xle J

Unspecified or unknown amino acid Xaa X

[0070] 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 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. Enzymol. 266:460- 80; Altschul, S.F. et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, A.D. and B.F.F.

Ouellette (eds.) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.) Bioinformatics Methods and Protocols, Methods in Molecular Biology, Vol. 132, Humana Press, 1998. In addition to identifying homologous sequences, the programs mentioned above typically provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%), 95%), 96%), 97%), 98%>, 99% or more of their corresponding residues are homologous over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues. In some embodiments, the relevant stretch includes contiguous residues along 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 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.

[0071] 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 contain identical residues in corresponding positions. 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 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. Enzymol. 266:460-80; Altschul, S.F. et al., 1997, Nucleic Acids Res., 25:3389- 402; Baxevanis, A.D. and B.F.F. Ouellette (eds.) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.) Bioinformatics Methods and Protocols, Methods in Molecular Biology, Vol. 132, Humana Press, 1998. In addition to identifying identical sequences, the programs mentioned above typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be

substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues. [0072] Wild-type: as used herein, refers to an entity having a structure and/or activity as found in nature in a "normal" (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

DETAILED DESCRIPTION

[0073] Provided herein are, among other things, engineered non-human animals having heterologous genetic material encoding human immunoglobulin heavy and light chain variable domains, which heterologous genetic material comprises human immunoglobulin V(D)J 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 (i.e., antibodies that contain a human variable domain and a non-human constant domain). In various embodiments, provided engineered non-human animals contain heterologous genetic material that is inserted in such a way so that antibodies containing light chains that have either a human VK or a human νλ domain are expressed in the antibody repertoire of the non-human animal. Further, provided engineered non-human animals contain heterologous genetic material that is inserted in such a way so that antibodies containing light chains are expressed exclusively from engineered κ light chain loci in the germline genome of the non-human animal.

[0074] Without wishing to be bound by any particular theory, it is contemplated that provided non-human animals provide an improved in vivo system that exploits the expression of antibodies containing human νλ domains for the production of therapeutic antibodies. It is also contemplated that non-human animals as described herein, in some embodiments, provide alternative engineered forms of immunoglobulin light chain loci for the development of antibody-based therapeutics to disease targets that are associated with biased antibody responses (e.g., antibody responses characterized by an overwhelming proportion of either κ or λ light chains). Thus, non-human animals as described herein are particularly useful for the development of human antibodies against targets that have proven to date to be difficult to generate neutralizing human antibodies against due, in part, to skewed antibody repertoires and/or responses. [0075] In particular, the present invention encompasses the production of a non-human animal (e.g., a rodent) having a germline genome that contains engineered immunoglobulin heavy chain and κ light chain loci, each of which are characterized by the introduction of human variable region gene sequences (i.e., human V, D and/or J gene segments) in operable linkage to non-human constant regions and/or constant region genes resulting in the expression of antibodies that contain heavy chains that include a human variable domain and light chains that include either a human VK or a νλ domain. The germline genome of provided non-human animals further comprises functionally silenced immunoglobulin λ light chain loci and, as described herein, the non-human animals express antibody repertoires that exclusively contain K light chains (i.e., light chains that include a non-human CK domain, or light chains expressed from an endogenous immunoglobulin κ light chain locus) that include human VL domains. In some embodiments, non-human animals as described herein contain human immunoglobulin κ and/or λ light chain sequences within a single immunoglobulin κ light chain locus. Exemplary immunoglobulin heavy and light chain loci, in particular, immunoglobulin heavy and light chain variable region loci, of a non-human animal as described herein are set forth in the Drawings (e.g., see Figures 1 and/or 2). Such engineered non-human animals provide a source of human antibodies and human antibody fragments, and provide an improved in vivo system suitable for exploiting human νλ sequences for the production of human therapeutic antibodies.

[0076] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of "or" means "and/or" unless stated otherwise.

Antibody repertoires in non-human animals

[0077] Development of therapeutic antibodies for the treatment of various human diseases has largely 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 (Ig) genes (reviewed in, e.g., Briiggemann, M. et al., 2015, Arch. Immunol. Ther. Exp. 63 : 101-8). Initial efforts in creating such transgenic rodent lines focused on integration 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., Briiggemann, M. et al., 1989, Proc. Nat. Acad. Sci. U. S.A. 86:67-09-13;

Bruggemann, M. et al., 1991, Eur. J. Immunol. 21 : 1323-6; Taylor, L.D. et al., 1992, Nucl. Acids Res. 20:6287-6295; Davies, N.P. et al., 1993, Biotechnol. 1 1 :91 1-4; Green, L.L. et al., 1994, Nat. Genet. 7: 13-21 ; Lonberg, N. et al., 1994, Nature 368:856-9; Taylor, L.D. et al., 1994, Int. Immunol. 6:579-91 ; Wagner, S.D. et al., 1994, Eur. J. Immunol. 24:2672-81 ;

Fishwild, D M. et al., 1996, Nat. Biotechnol. 14:845-51 ; Wagner, S.D. et al., 1996, Genomics 35 :405-14; Mendez, M.J. et al., 1997, Nat. Genet. 15 : 146-56; Green, L.L. et al., 1998, J. Exp. Med. 188:483-95; Xian, J. et al., 1998, Transgenics 2:333-43; Little, M. et al., 2000, Immunol. Today 21 :364-70; Kellermann, S.A. and L.L. Green, 2002, Cur. Opin. Biotechnol. 13 :593-7). In particular, some efforts have included integration of human λ light chain gene sequences (see, e.g., U.S. Patent Application Publication Nos. 2002/0088016 Al, 2003/0217373 Al and 201 1/0236378 Al ; U. S. 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 νλ, Ιλ and C sequences thereby creating mouse strains that express fully human λ light chains (i.e., human variable and human constant). More recent efforts have employed similar strategies using constructs that also contain human νλ, Τλ and C sequences (Osborn, M.J. et al., 2013, J. Immunol. 190: 1481-90; Lee, E-C. et al., 2014, Nat. Biotech. 32(4):356-63).

[0078] Two separate loci (κ and λ) contain the gene segments that encode the light chains of antibodies, and exhibit both allelic and isotypic exclusion. The expression ratios of κ⁺ to λ⁺ B cells vary among species. For example, in humans the ratio is about 60:40 (κ:λ). In mice and rats, the ratio is 95 :5 (κ:λ). Interestingly, the κ:λ ratio observed in cats (5 :95) is opposite of mice and rats. Several studies have been conducted to elucidate the possible reasons behind these observed 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

rearrangement as rationales. The human immunoglobulin λ light chain locus extends over l,000kb and contains approximately 70 νλ gene segments (29 to 33 functional) and seven Ιλ- C gene segment pairs (four to five functional) organized into three clusters (see, e.g., Fig. 1 of U.S. Patent No. 9,006,511). The majority of the observed νλ regions in the expressed antibody repertoire are encoded by gene segments contained within the most proximal cluster (i.e., cluster A). The mouse immunoglobulin λ light chain locus is strikingly different than the human locus and, depending on the strain, contains only a few νλ and Jl gene segments organized in two distinct gene clusters (see, e.g., Fig. 2 of U.S. Patent No. 9,006,511).

[0079] Demonstrated herein is the successful production of a non-human animal whose germline genome contains (1) an immunoglobulin heavy chain locus comprising a plurality of human VH, human DH and human JH gene segments in operable linkage to a non-human CH region and/or gene; (2) a first immunoglobulin κ light chain locus (or allele) comprising a plurality of human VK and human JK gene segments in operable linkage to a non-human CK region and/or gene (i.e., a first non-human CK region and/or gene); (3) a second

immunoglobulin κ light chain locus (or allele) comprising a plurality of human νλ and human Jl gene segments in operable linkage to a non-human CK region and/or gene (i.e., a second non-human CK region and/or gene); and (4) an endogenous immunoglobulin λ light chain locus that is deleted in whole or in part. 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 either a human VK or human νλ domain. As described herein, expression of such light chains is achieved by combination of first and second engineered immunoglobulin κ light chain loci (or alleles). Also, as described herein, provided non-human animals are engineered so that expression of endogenous immunoglobulin heavy, κ light and λ light chain variable regions is inactivated (e.g., by gene deletion). Thus, the present disclosure, in at least some embodiments, embraces the development of an improved in vivo system for the production of human antibodies by providing an engineered non-human animal containing alternatively engineered immunoglobulin loci that result in an expressed antibody repertoire characterized by a light chain population that is exclusively expressed from engineered κ light chain loci (or alleles). Specific Exemplary Embodiments— IgHLoci

[0080] In many embodiments, provided non-human animals comprise engineered immunoglobulin heavy chain loci (or alleles) characterized by the presence of a plurality of human VH, DH and JH gene segments arranged in germline configuration and operably linked to non-human CH regions, CH genes, enhancers and regulatory regions (described, e.g., in U.S. Patent Nos.9,353,394, 9,371,553 and 9,528,136). Exemplary engineered immunoglobulin heavy chain loci or alleles of provided non-human animals are depicted in Figure 1 or 2. In some embodiments, an engineered immunoglobulin heavy chain locus (or allele) 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 CH region and/or gene. In some certain embodiments, an engineered immunoglobulin heavy chain locus (or allele) comprises at least human VH gene segments VH3-74, VH3-73, VH3-72, VH2-70, VHI- 69, VH3-66, VH3-64, VH4-61, VH4-59, VH1-58, VH3-53, VH5-51, VH3-49, VH3-48, VH1-46, VH1-45, VH3-43, VH 4-39, VH4-34, VH3-33, VH4-31, VH3-30, VH4-28, VH2-26, VH1-24, VH3- 23, VH3-21, VH3-20, VH1-18, VH3-15, VH3-13, VH3-11, VH3-9, VH1-8, VH3-7, VH2-5, VH7-4- 1, VH4-4, VH1-3, VH1-2 and VH6-1. In some certain embodiments, an engineered

immunoglobulin heavy chain locus (or allele) comprises at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 of human VH gene segments selected from VH3-74, VH3-73, VH3-72, VH2-70, VHI- 69, VH3-66, VH3-64, VH4-61, VH4-59, VH1-58, VH3-53, VH5-51, VH3-49, VH3-48, VH1-46, VH1-45, VH3-43, VH 4-39, VH4-34, VH3-33, VH4-31, VH3-30, VH4-28, VH2-26, VH1-24, VH3- 23, VH3-21, VH3-20, VH1-18, VH3-15, VH3-13, VH3-11, VH3-9, VH1-8, VH3-7, VH2-5, VH7-4- 1, VH4-4, VH1-3, VH1-2 and VH6-1. In some certain embodiments, an engineered

immunoglobulin heavy chain locus (or allele) comprises at least human DH gene segments DH1-1, DH2-2, DH3-3, DH4-4, DH5-5, DH6-6, DH1-7, DH2-8, DH3-9, DH3-10, DH5-12, DH6-13, DH2-15, DH3-16, DH4-17, DH6-19, DH1-20, DH2-21, DH3-22, DH6-25, DH1-26 and DH7-27. In some certain embodiments, an engineered immunoglobulin heavy chain locus (or allele) comprises at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 human DH gene segments selected from DH1-1, DH2-2, DH3-3, DH4-4, DH5-5, DH6-6, DH1-7, DH2-8, DH3-9, DH3-10, DH5-12, DH6-13, DH2-15, DH3-16, DH4-17, DH6-19, DH1-20, DH2-21, DH3-22, DH6-25, DH1-26 and DH7-27. In some certain embodiments, an engineered immunoglobulin heavy chain locus (or allele) comprises at least human JH gene segments JHI , JH2, JH3, JH4, JH5 and JH6. In some certain embodiments, an engineered immunoglobulin heavy chain locus (or allele) comprises at least 1, 2, 3, 4, or 5 human JH gene segments selected from JHI , JH2, JH3, JH4, JH5 and JH6.

[0081] In some embodiments, a non-human immunoglobulin heavy chain constant region includes one or more non-human constant region genes such as, for example, genes that encode immunoglobulin M (IgM) ^μ), immunoglobulin D (IgD) (C5), immunoglobulin G (IgG) (Cy), immunoglobulin E (IgE) (Ce) and immunoglobulin A (IgA) (Ca). In some certain

embodiments, a non-human immunoglobulin heavy chain constant region includes a rodent C^ rodent C5, rodent Cy3, rodent Cyl, rodent Cyb, rodent Cy2a, rodent Ce and rodent Ca constant region genes. In many embodiments, said human VH, DH and JH gene segments are operably linked to one or more non-human immunoglobulin heavy chain enhancers (i.e., enhancer sequences or enhancer regions). In many embodiments, said human VH, DH and JH gene segments are operably linked to one or more non-human immunoglobulin heavy chain regulatory regions (or regulatory sequences). In many embodiments, said human VH, DH and JH gene segments are operably linked to one or more non-human immunoglobulin heavy chain enhancers (or enhancer sequence) and one or more non-human immunoglobulin heavy chain regulatory regions (or regulatory sequence).

[0082] In some embodiments, an engineered immunoglobulin heavy chain locus as described herein does not contain (e.g., lacks or has a deletion of) an endogenous Adam6 gene. In some embodiments, an engineered immunoglobulin heavy chain locus as described herein does not contain an endogenous Adam6 gene (or Adam6-encoding sequence) in the same germline genomic position as found in a germline genome of a wild-type non-human animal of the same species. In some embodiments, an engineered immunoglobulin heavy chain locus as described herein does not contain a human Adam6 pseudogene. In some embodiments, an engineered immunoglobulin heavy chain 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 immunoglobulin heavy chain locus (e.g., see Figures 1 and 2) or elsewhere in the germline genome of a non-human animal (e.g., a randomly introduced non-human Adam6-encoding sequence), cell or tissue.

[0083] In various embodiments, a provided 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 VH region in an antibody molecule.

[0084] 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 VH region (e.g., VH, DH and/or JH) in an antibody molecule.

[0085] In various embodiments, a provided 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.

[0086] Exemplary guidance for the creation of targeting vectors, non-human cells and animals harboring such engineered immunoglobulin heavy chain loci (or alleles) can be found in, for example, U.S. Patent Nos. 8,642,835 and 8,697,940. Those skilled 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 preparing, providing, or manufacturing such sequences for introducing into the germline genome of non-human animals.

Specific Exemplary Embodiments— Igic Light Chain Loci

[0087] In many embodiments, provided non-human animals comprise engineered immunoglobulin κ light chain loci (or alleles) characterized by separately distinct variable region structure. As described herein, engineered immunoglobulin κ light chain loci (or alleles) separately contain a plurality of human VL and JL gene segments arranged in germline configuration and operably linked to non-human immunoglobulin κ light chain constant regions and/or genes. Exemplary engineered immunoglobulin κ light chain loci (or alleles) of provided non-human animals are depicted in Figure 2. In many embodiments, provided non- human animals comprise first and second engineered immunoglobulin κ light chain loci (or alleles). [0088] In some embodiments, a first engineered immunoglobulin κ 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 non-human immunoglobulin κ light chain constant (CK) region and/or gene (i.e., a first non-human CK region and/or gene). In some certain embodiments, a first engineered immunoglobulin κ light chain locus (or allele) comprises at least human VK gene segments that appear in the proximal variable cluster (or proximal arm, or proximal duplication) of a human immunoglobulin κ light chain locus. In some certain embodiments, a first engineered immunoglobulin κ light chain locus (or allele) comprises at least human VK gene segments VK2-40, VK1-39, VK1-33, VK2-30, VK2-28, VK1-27, VK2-24, VK6-21, VK3-20, VK1-17, VK1-16, VK3-15, VK1-12, VK3-11, VK1-9, VK1-8, VK1-6, VK1-5, VK5-2 and VK4-1. In some certain embodiments, a first engineered immunoglobulin κ light chain locus (or allele) comprises at least 1, 2, 3, 4, 5, 10, or 15 human VK gene segments selected from VK2-40, VKI - 39, VK1-33, VK2-30, VK2-28, VK1-27, VK2-24, VK6-21, VK3-20, VK1-17, VK1-16, VK3-15, VK1-12, VK3-11, VK1-9, VK1-8, VK1-6, VK1-5, VK5-2 and VK4-1. In some certain

embodiments, a first engineered immunoglobulin κ light chain locus (or allele) comprises human JK gene segments JKI , JK2, JK3, JK4 and JK5. In some certain embodiments, a first engineered immunoglobulin κ light chain locus (or allele) comprises at least 1, 2, 3, or 4 human JK gene segments selected from JKI , JK2, JK3, JK4 and JK5.

[0089] In many embodiments, said human VK and JK gene segments are operably linked to one or more non-human immunoglobulin κ light chain enhancers (i.e., enhancer sequences or enhancer regions). In many embodiments, said human VK and JK gene segments are operably linked to one or more non-human immunoglobulin κ light chain regulatory regions (or regulatory sequences). In many embodiments, said human VK and JK gene segments are operably linked to one or more non-human immunoglobulin κ light chain enhancers (or enhancer sequences or enhancer regions) and one or more non-human immunoglobulin κ light chain regulatory regions (or regulatory sequences).

[0090] In some embodiments, a second engineered immunoglobulin κ light chain locus (or allele) comprises one or more human νλ gene segments and one or more human Ιλ gene segments operably linked to a non-human immunoglobulin κ light chain constant (CK) region and/or gene (i.e., a second non-human CK region and/or gene). In some certain embodiments, a second engineered immunoglobulin κ light chain locus (or allele) comprises human νλ gene segments that appear in at least cluster A of a human immunoglobulin λ light chain locus; in some embodiments, cluster A and cluster B of a human immunoglobulin λ light chain locus. In some certain embodiments, a second engineered immunoglobulin κ light chain locus (or allele) comprises at least human νλ gene segments νλ5-52, νλ1-51, V 9-49, νλ1-47, νλ7-46, νλ5- 45, νλ1-44, V 7-43, νλ1-40, V 3-27, νλ3-25, V 2-23, V 3-22, V 3-21, V 3-19, νλ2-18, νλ3-16, V 2-14, V 3-12, νλ2-11, νλ3-10, V 3-9, V 2-8, V 4-3 and νλ3-1. In some certain embodiments, a second engineered immunoglobulin κ light chain locus (or allele) comprises at least 1, 2, 3, 4, 5, 10, 15, or 20 human νλ gene segments selected from V 5-52, νλ1-51, νλ9- 49, νλ1-47, νλ7-46, V 5-45, νλ1-44, V 7-43, νλ1-40, V 3-27, V 3-25, V 2-23, V 3-22, V 3-21, V 3-19, νλ2-18, νλ3-16, V 2-14, V 3-12, νλ2-11, νλ3-10, V 3-9, V 2-8, V 4-3 and νλ3-1. In some certain embodiments, a second engineered immunoglobulin κ light chain locus (or allele) comprises at least human Ιλ gene segments Ιλΐ, Ιλ2, Ιλ3, Ιλ6 and Ιλ7. In some certain embodiments, a second engineered immunoglobulin κ light chain locus (or allele) comprises at least 1, 2, 3, or 4 human Ιλ gene segments selected from Ιλΐ, Ιλ2, Ιλ3, Ιλ6 and Ιλ7.

[0091] In many embodiments, a second engineered immunoglobulin κ light chain locus or allele does not contain a human immunoglobulin λ light chain constant region (or human immunoglobulin λ light chain constant region encoding sequence), in whole or in part. In many embodiments, a second engineered immunoglobulin κ light chain locus or allele does not contain a human immunoglobulin λ light chain enhancer (or human immunoglobulin λ light chain enhancer sequence), in whole or in part.

[0092] In many embodiments, said human νλ and Ιλ gene segments are operably linked to one or more non-human immunoglobulin κ light chain enhancers (i.e., enhancer sequences or enhancer regions). In many embodiments, said human νλ and Ιλ gene segments are operably linked to one or more non-human immunoglobulin κ light chain regulatory regions (or regulatory sequences). In many embodiments, said human νλ and Ιλ gene segments are operably linked to one or more non-human immunoglobulin κ light chain enhancers (or enhancer sequences or enhancer regions) and one or more non-human immunoglobulin κ light chain regulatory regions (or regulatory sequences). [0093] In many embodiments, a second engineered immunoglobulin κ light chain locus or allele as described herein further comprises a genomic sequence that appears in a human immunoglobulin κ light chain locus, in particular, between a human VK4-1 gene segment and a human JKI gene segment. As described herein, said genomic sequence as it appears in a human immunoglobulin κ light chain locus is positioned between a human νλ3-1 gene segment and a human Γλΐ gene segment. Thus, a second engineered immunoglobulin κ light chain locus or allele as described herein is characterized by the presence of a VK-JK intergenic sequence positioned between human νλ and Jl gene segments. In many embodiments, a second engineered immunoglobulin κ light chain locus or allele as described herein does not contain a human νλ-Ιλ intergenic region that naturally appears between a human νλ3-1 gene segment and a human Ιλΐ gene segment in a human immunoglobulin λ light chain locus. In some embodiments, a second engineered immunoglobulin κ light chain locus or allele as described herein does not contain a human VpreB gene (or human VpreB gene-encoding sequence).

[0094] In some embodiments, a non-human immunoglobulin CK region of a first and/or second engineered immunoglobulin κ light chain locus (or allele) includes a rodent

immunoglobulin CK region such as, for example, a mouse immunoglobulin CK region or a rat immunoglobulin CK region. In some certain embodiments, a non-human immunoglobulin CK region of a first and/or second engineered immunoglobulin κ light chain locus (or allele) is or comprises a mouse immunoglobulin 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.

[0095] Exemplary guidance for the creation of targeting vectors, non-human cells and animals harboring engineered immunoglobulin κ chain loci (or alleles) as described herein can be found in, for example, U.S. Patent Nos. 9,006,511, 9,029,628, 9,035, 128, 9,066,502, 9, 150,662, 9, 163,092, 9,206,261, 9,206,262, 9,026,263, 9,226,484, and 9,334,333. Those skilled 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 preparing, providing, or manufacturing such sequences for introducing into the germline genome of non-human animals. [0096] In various embodiments, a provided 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 domain in an antibody molecule.

[0097] 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 domain in an antibody molecule.

[0098] In various embodiments, a provided 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 VK and JK gene segments, in whole or in part.

[0099] Among other things, the present disclosure demonstrates that the presence of human VK and JK gene segments and human νλ and Ιλ gene segments at separate immunoglobulin κ light chain loci (or alleles, i.e., first and second alleles as described herein) increases the diversity of the light chains in the expressed antibody repertoire of a provided non-human animal as compared to the diversity of the light chains in the expressed antibody repertoire of a non-human animal that does not comprise such engineered immunoglobulin κ light chain alleles. That is, a non-human animal engineered to contain engineered immunoglobulin κ light chain loci (or alleles) as described herein results in the expression of immunoglobulin κ light chains in the antibody repertoire of the non-human animal that are characterized by a ratio of human VK to human νλ domains of about 4: 1 (or about 80:20), about 7:3 (or about 70:30), about 3:2 (or about 60:40), about 1 : 1 (or about 50: 50), or about 2:3 (or about 40:60), preferably about 3:2 (or about 60:40).

Specific Exemplary Embodiments— IgX Light Chain Loci

[00100] In many embodiments, provided non-human animals comprise immunoglobulin λ light chain loci (or alleles) that are functionally silenced. For example, as described herein, immunoglobulin λ light chain loci (or alleles) are functionally silenced by gene deletion of specific gene segments that include at least all the functional endogenous νλ and Ιλ gene segments (e.g., νλ2-νλ3-Γλ2 and V 1 -J 3-C 3-J 1 -C 1). Exemplary functionally silenced immunoglobulin λ light chain loci (or alleles) of provided non-human animals are depicted in Figure 1 or 2. In many embodiments, provided non-human animals comprise two functionally silenced immunoglobulin λ light chain loci (or alleles; i.e., homozygous for a functionally silenced immunoglobulin λ light chain locus).

[00101] In various embodiments, a provided 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 νλ region in an antibody molecule.

[00102] 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 νλ region in an antibody molecule.

[00103] In various embodiments, a provided 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 νλ and Ιλ gene segments, in whole or in part.

[00104] Exemplary guidance for the creation of targeting vectors, non-human cells and animals harboring silenced immunoglobulin λ chain loci (or alleles) can be found in, for example, U.S. Patent Nos. 9,006,511, 9,029,628, 9,035, 128, 9,066,502, 9, 150,662, 9, 163,092, 9,206,261, 9,206,262, 9,026,263, 9,226,484, and 9,334,333. Those skilled in the art upon reading this disclosure will be aware of other methods for functionally silencing endogenous immunoglobulin λ light chain loci (or alleles) including, but not limited to, gene deletion of gene segments different than as described herein (e.g., deletion of C 2 and C 3-J 1-C 1). Those skilled in the art are also 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 preparing, providing, or manufacturing such deletions for

introducing into the germline genome of non-human animals.

Methods

[00105] Non-human animals as described herein may be employed for making a human antibody, 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 described 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 mapping, ability for blocking ligand-receptor interaction, inhibition receptor activation, etc. In various embodiments, antibodies produced by non-human animals as described herein comprise one or more human variable regions that are derived from one or more human variable region nucleotide sequences isolated from the non-human animal. In some embodiments, anti-drug antibodies (e.g., antiidiotype antibody) may be raised in non-human animals as described herein.

[00106] Non-human animals as described herein provide an improved in vivo 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 interested (e.g., a transmembrane or secreted polypeptide) and/or modulate one or more activities associated with said polypeptide of interest and/or modulate interactions of said polypeptide of interest with other binding partners (e.g., a ligand or receptor polypeptide). For example, in various embodiments, non-human animals as described herein are used to develop therapeutics that target one or more receptor polypeptides and/or 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 develop 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 binding profile of antagonists and/or agonists of one or more polypeptides of interest. In some embodiments, non-human animals as described herein are used to determine the epitope or epitopes of one or more candidate therapeutic antibodies that bind one or more polypeptides of interest. [00107] In various 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 control or reference non-human animals 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 include, e.g., intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, 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 intravenous 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, 5 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 obtained from non-human animals as described herein including, but not limited to, total IgG, anti-therapeutic antibody response, agglutination, etc.

[00108] 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 interest 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 embodiments, 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.

[00109] Non-human animals 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 regions for use in binding and functional assays, e.g., to assay for binding or function of an antagonist or agonist, particularly where the antagonist or agonist is specific for a human antigen of interest or specific for an epitope that functions in ligand-receptor interaction (binding). In various embodiments, epitopes bound by candidate therapeutic antibodies or siRNAs can be determined using cells isolated from non-human animals as described herein.

[00110] Cells from provided non-human animals can be isolated 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).

[00111] Non-human animals as described herein provide an in vivo system for the generation of variants of human antibody variable domains that bind a polypeptide of interest. 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 variable regions that are screened for a desired or improved functionality.

[00112] Non-human animals as described herein provide an in vivo system for generating human antibody variable region libraries. Such libraries provide a source for heavy and 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 techniques known in the art (e.g., site-directed mutagenesis, error-prone PCR, etc.) and/or used as a source of antibody components for the generation of antibody-based therapeutic molecules such as, for example, chimeric antigen receptors (i.e., a molecule engineered using antibody components, e.g., an scFv), multi-specific binding agents (e.g., bi- specific binding agents) and fusion proteins (e.g., single domain antibodies, scFvs, etc.).

[00113] 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 (i.e., anti-drug) response, efficacy of the drug or vaccine and possible risk factors. Such drugs or vaccines may be improved and/or developed in such non-human animals.

[00114] Vaccine efficacy may be determined in a number of ways. Briefly, non-human animals as described herein are vaccinated using methods known in the art and then challenged with a vaccine 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 monitoring 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.

[00115] 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 antibodies 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.

[00116] Non-human animals as described herein produce human antibody variable domains and, therefore, provide an in vivo system for the production of human antibodies for use in diagnostic applications (e.g., immunology, serology, microbiology, 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 identification of cellular changes such as, for example, expression of specific cell surface markers indicative of pathological changes. Such antibodies can be conjugated to various chemical entities (e.g., a radioactive tracer) and be employed in various in vivo and/or in vitro assays as desired.

[00117] 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 different 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 implantation of 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 therapeutic. Alternatively and/or additionally, 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

[00118] Also provided herein is a pack, package or kit comprising one or more containers filled with at least one non-human animal or non-human cell as described herein. Kits may be used in any applicable method (e.g., a research method). Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of such products, which notice reflects (a) directions for use, (b) 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, or combinations thereof.

[00119] Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments, which are given for illustration and are not intended to be limiting thereof.

EXAMPLES

[00120] The following examples are provided so as to describe to those of ordinary skill in the art how to make and use methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Unless indicated otherwise, temperature is indicated in Celsius, and pressure is at or near atmospheric. Example 1. Generation of rodents containing multiple engineered immunoglobulin loci

[00121] This Example illustrates the generation of an engineered rodent (e.g., mouse) strain whose genome comprises immunoglobulin heavy and κ light chain loci designed with human variable region gene sequences operably linked to rodent constant region sequences, and λ light chain loci that are functionally silenced. In particular, this example demonstrates the creation of an engineered rodent (e.g., mouse) strain whose germline genome includes a homozygous immunoglobulin heavy chain locus containing a plurality of human VH, DH and JH gene segments operably linked to rodent immunoglobulin heavy chain constant and

enhancer/regulatory regions and also containing a functional mouse Adam6 gene, a first immunoglobulin κ light chain allele that contains human VK and JK gene segments operably linked to a rodent immunoglobulin CK region, a second immunoglobulin κ light chain allele that contains human νλ and Jl gene segments operably linked to a rodent immunoglobulin CK region, and a homozygous immunoglobulin λ light chain locus that is functionally silenced by gene deletion. Thus, in some embodiments, provided non-human animals are characterized as hemizygous for said first and second immunoglobulin κ light chain alleles. As described below, the engineered rodent strain was created by classical breeding methods. Figure 1 sets forth representative engineered immunoglobulin loci of individual engineered rodent strains as described herein. Alternatively, breeding of one or more engineered rodent strains as described herein may be performed by commercial services (e.g., Jackson Laboratory).

[00122] Briefly, individual mouse strains homozygous for the engineered immunoglobulin loci depicted in Figure 1 were bred together over multiple breeding' s to establish a mouse strain homozygous for the illustrated engineered immunoglobulin heavy chain locus, hemizygous for the illustrated engineered immunoglobulin κ light chain loci, and homozygous for the inactivated immunoglobulin λ light chain locus (referred to herein as "LoK/KoK mice", see Figure 2). LoK/KoK were produced by crossing LoK mice comprising λ light chain variable gene segments operably linked to κ light chain constant region at the endogenous κ locus as described previously (see, e.g., U. S. Patent Nos. 9,006,51 1, 9,035, 128, 9,066,502, 9, 150,662 and 9, 163,092) with VI3 mice which comprise human V(D)J heavy and κ light chain variable region gene segments operably linked to mouse heavy and κ light chain constant region sequences, respectively, and comprising a functional mouse Adam6 gene(s) (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940) and mice inactivated for endogenous immunoglobulin λ light chain expression (mIg ^"/_; see Fig. 2 of U.S. Patent No. 9,006,511). The final strain (52.566% BALB/cAnNTac 25.445% C57BL/6NTac 21.989%

129S6/SvEvTac) was maintained by intercrosses between homozygous animals for each of the engineered loci. All offspring were confirmed for the desired genotype by PCR analysis of tail tissue DNA, karyotyping using Giemsa staining and/or screening methods known in the art (e.g., see Valenzuela, D M. et al., 2003, Nat. Biotechnol. 21 :652-9). All experimental procedures were carried out in accordance with Regeneron Pharmaceuticals, Inc.'s Institutional Animal Care and Use Committee (IACUC) protocol at their facilities in Tarrytown, NY.

Cohorts of LoK/KoK mice were further analyzed for their immune cell compartments by flow cytometry (see below).

Example 2. Assessment of immune cell populations

[00123] This example demonstrates the characterization of various immune cell populations in rodents (e.g., mice) designed to contain multiple engineered immunoglobulin loci that contain heterologous (e.g., human) variable region sequences operably linked to rodent constant, enhancer and regulatory regions. In particular, this example specifically

demonstrates that rodents having engineered immunoglobulin heavy, κ light and λ light chain loci described above (see also Figure 2) display essentially normal B cell development as compared to other engineered and/or wild-type rodent strains. This example also demonstrates that engineered rodent strains described in Example 1 contain an expressed antibody repertoire that includes immunoglobulin light chains that utilize all human light chain variable region sequences (VK and νλ) contained within the engineered immunoglobulin κ light chain alleles.

[00124] Briefly, spleens and femurs were harvested from various groups of mice having genotypes set forth in Table 1 (wt = wild-type; "VI3" [n=3]; "LoK" [n=3]; "LoK/mlg^^""

[n=3]; "LoK/KoK/mlg ^{" "}" [n=4]). Bone marrow was collected from femurs by flushing with lx phosphate buffered saline (PBS, Gibco) with 2.5% fetal bovine serum (FBS). Red blood cells from spleen and bone marrow preparations were lysed with ACK lysis buffer (Gibco) followed by washing with lxPBS with 2.5% FBS. Isolated cells (lxlO⁶) were incubated with selected antibody cocktails for 30 min at +4°C: anti-mlgK-FITC (187.1, BD Biosciences), anti- mlg -PE (RML-42, BioLegend), anti-mouse IgM-PeCy7 (11/41, eBioscience), anti-mouse CD3 -Pacific Blue (17A2, BioLegend), anti-mouse B220-APC (RA3-6B2, eBioscience), anti- mouse CD19-APC-H7 (ID3, BD Biosciences). Following staining, cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on a BD LSRFORTESSA™ flow cytometer and analyzed with FLOWJO™ software. Representative results are set forth in Figures 3A-3D.

[00125] As shown in Figure 3 A, LoK and LoK/mlg ^{" "} mice have comparable splenic CD19/CD3 distribution (left two plots). In contrast, LoK/KoK mice demonstrate a splenic CD19/CD3 distribution that resembles that of the VI3 rodent strain containing humanized heavy and κ light chain loci (i.e., heavy and κ light chain loci with human variable regions operably linked to rodent constant regions, right two plots). Further, deletion of the endogenous immunoglobulin λ light chain locus led to exclusive expression from engineered κ light chain alleles (both LOK and KOK alleles, as detected by antibody against mouse κ constant region) in mIg ^_/" strains (Figure 3B). In the bone marrow, essentially normal immature/mature ratios and similar light chain expression profiles were observed among analyzed engineered strains (Figures 3C and 3D).

[00126] In another experiment, usage of human antibody genes (i.e., V(D)J gene segments) in engineered rodent strains was determined by Next Generation Sequencing antibody repertoire analysis. Briefly, splenic B cells were positively enriched from total splenocytes by magnetic cell sorting using mouse anti-CD 19 magnetic beads and MACS® columns (Miltenyi Biotech). Total RNA was isolated from purified splenic B cells using an RNeasy Plus RNA isolation kit (Qiagen) according to manufacturer's specifications. Reverse transcription was performed to generate cDNA containing IgK constant region sequence, using a SMARTer™ RACE cDNA Amplification Kit (Clontech) and an IgK specific primer. During this process, a DNA sequence, reverse complement to 3' of a template switching (TS) primer, was attached to the 3' end of newly synthesized cDNAs. Purified IgK-specific cDNAs were then amplified by the 1^st round PCR reaction using the TS specific primer and an IgK constant specific primer. PCR products ranging from ~450-700bp were isolated using Pippin Prep (SAGE Science) and then these fragments were further amplified by a 2^nd round PCR reaction. All primers used for repertoire library construction are set forth in Table 2 ("XXXXXX" represents a random 6bp index sequence (e.g., ATCACG) (SEQ ID NO: 1) to enable multiplexing samples for sequencing and sorting after the reactions are complete; for: forward; rev: reverse). PCR products ranging from ~400bp-700bp were isolated, purified, and quantified by qPCR using a KAPA Library Quantification Kit (KAPA Biosystems) before loading onto a Miseq sequencer (Illumina) for sequencing using Miseq Reagent Kits v3 (2x300 cycles).

[00127] For bioinformatic analysis, Raw Illumina sequences were de-muliplexed and filtered based on quality, length and match to corresponding constant region primer.

Overlapping paired-end reads were merged and analyzed using custom in-house pipeline. The pipeline used local installation of IgBLAST (NCBI, v2.2.25+) to align rearranged light chain sequences to human germline V and J gene segment database, and denoted productive and nonproductive joins along with the presence of stop codons. CDR3 sequences were extracted using boundaries as defined in International Immunogenetics Information System (FMGT). Representative results are set forth in Figure 4.

[00128] The results demonstrated that all of the human VK and νλ regions are represented in the expressed antibody repertoire of LoK/KoK/mlg ^{" "} mice. In particular, the usage ratio of human VK to human νλ regions within the population of light chain sequences was observed at 3 :2, which is similar to the κ:λ ratio reported in humans). A high percentage of V gene segments (i.e., VK or νλ) from distal portions of each engineered light chain loci was observed among selected rearrangements for both engineered light chain loci (Figure 4).

[00129] Taken together, this example specifically demonstrates that rodents engineered to contain two immunoglobulin heavy chain alleles containing a plurality of human VH, DH and JH gene segments operably linked to a rodent heavy chain constant region, a first

immunoglobulin κ light chain allele containing a plurality of human VK and JK gene segments operably linked to a rodent CK region, a second κ light chain allele containing a plurality of human νλ and Ιλ gene segments operably linked to a rodent CK region, and two

immunoglobulin λ light chain alleles that are functionally silenced by gene deletion display an essentially normal B cell development. Further, such engineered rodents demonstrate usage of all human VK and νλ gene segments in a proportion typically observed in humans. Thus, the present invention provides improved in vivo systems that exploit a unique combination of immunoglobulin light chain locus structures that optimize expression of human light chains engineered in the genome of non-human animals and highlight their use for the production of human therapeutic antibodies.

TABLE 1

Locus

Mouse strain IgH JgK_ L

VI3 hV_H, hD_H, hJ_H hVK, hJK wt LOK hV_H, hDH, hJ_H hV , hJ wt

LOK/mlg^^" hV_H, hDH, hJ_H hV , hJ deleted LOK/KOK/mlg -' hV_H, hDH, hJ_H hVK, hJK/hV , hJ deleted

TABLE 2

Primer Sequence (5 '-3')

TS primer CACCATCGAT GTCGACACGC CTAGGG (SEQ ID NO: l)

IgKC (RT primer) AAGAAGCACA CGACTGAGGC AC (SEQ ID NO:2)

ACACTCTTTC CCTACACGAC GCTCTTCCGA TCTGGAAGAT

IgKC (l^st PCR)

GGATACAGTT GGTGC (SEQ ID NO:3)

TS specific (1^st GTGAC TGGAG TTCAGACGTG TGCTCTTCCG ATCTCACCAT PCR) CGATGTCGAC ACGCCTA (SEQ ID NO:4)

AATGATACGG CGACCACCGA GATCTACAC XXXXXX

for (2^nd PCR) ACACTCTTTC CCTACACGAC GCTCTTCCGA TCT (SEQ ID

NO: 5)

CAAGCAGAAG ACGGCATACG AGAT XXXXXX

rev (2^nd PCR) GTGAC TGGAG TTCAGACGTG TGCTCTTCCG ATCT (SEQ ID

NO: 6)

Example 3. Production of antibodies in rodents

[00130] This example demonstrates production of antibodies in a rodent that comprises engineered immunoglobulin heavy, κ light and λ light chain loci as described above using an antigen of interest (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 engineered immunoglobulin loci 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. [00131] Cohorts of LoK/KoK/mlg ^"'^" mice 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 desired immune response is achieved, splenocytes (and/or other lymphatic tissue) are harvested and fused with mouse myeloma cells to preserve their viability and form immortal hybridoma cell lines. The hybridoma cell lines are screened (e.g., by an ELISA assay) and selected to identify hybridoma cell lines that produce antigen-specific antibodies. Hybridomas 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.

[00132] DNA encoding the variable regions of heavy chain and light chains may be isolated and linked to desirable isotypes (constant regions) of the heavy chain and light chain for the preparation of fully-human antibodies. Such an antibody protein may be produced 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.

[00133] 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 variable region and a mouse constant region and are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. Mouse constant regions are replaced with a desired human constant region to generate fully-human antibodies. As described herein, LoK mice produce antibodies having a unique light chain format (i.e., hV -mCK). Such light chains may be reformatted into fully human Υλ-C or V -CK as desired and/or depending on downstream use. While the constant region selected may vary according to specific use, high affinity antigen- binding and target specificity characteristics reside in the variable region. 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., U.S. Patent No. 7,582,298, specifically 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. EQUIVALENTS

[00134] It is to be appreciated by those skilled in the art that various alterations,

modifications, and improvements to the present disclosure will readily occur to those skilled 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 drawing are by way of example only and any invention described in the present disclosure if further described in detail by the claims that follow.

[00135] 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 distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[00136] 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 present 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 process. 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 dependent 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 general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, 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 whether the specific exclusion is recited in the specification.

[00137] Those skilled in the art will appreciate typical standards of deviation or error attributable to values obtained in assays or other processes as described herein. The publications, websites and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

Claims

1. A rodent comprising:

an immunoglobulin heavy chain locus comprising one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments and a rodent CH gene, wherein the human VH, DH and JH gene segments are operably linked to the rodent CH gene;

a first immunoglobulin light chain locus comprising one or more human VK gene segments, one or more human JK gene segments and a rodent CK gene, wherein the human VK and JK gene segments are operably linked to the rodent CK gene;

a second immunoglobulin light chain locus comprising one or more human νλ gene segments and one or more human J gene segments and a rodent CK gene, wherein the human νλ and J gene segments are operably linked to the rodent CK gene; and

an endogenous immunoglobulin λ light chain locus that is deleted in whole or in part.

2. The rodent of claim 1, wherein the immunoglobulin heavy chain locus is an

endogenous immunoglobulin heavy chain locus.

3. The rodent of claim 2, wherein the immunoglobulin heavy chain locus comprises a replacement of rodent VH, DH and JH gene segments with the human VH, DH and JH gene segments.

4. The rodent of any one of claims 1 to 3, wherein the first immunoglobulin light chain locus is an endogenous immunoglobulin κ light chain locus.

5. The rodent of claim 4, wherein the first immunoglobulin light chain locus comprises a replacement of rodent VK and JK gene segments with the human VK and JK gene segments gene segments.

6. The rodent of any one of claims 1 to 5, wherein the second immunoglobulin light chain locus is an endogenous immunoglobulin κ light chain locus.

7. The rodent of claim 6, wherein the second immunoglobulin light chain locus comprises a replacement of rodent VK and JK gene segments with the human νλ and Ιλ gene segments.

8. The rodent of claim 1 , wherein the first immunoglobulin light chain locus is at a first endogenous immunoglobulin κ light chain locus and the second immunoglobulin light chain locus is at a second endogenous immunoglobulin κ light chain locus such that the rodent is hemizygous for the first and the second immunoglobulin light chain loci.

9. The rodent of any one of the preceding claims, wherein the second immunoglobulin light chain locus further comprises a human immunoglobulin κ light chain sequence between the one or more human νλ gene segments and the one or more human Ιλ gene segments.

10. The rodent of claim 9, wherein the human immunoglobulin κ light chain sequence is or comprises a genomic sequence that naturally appears between a human VK4-1 gene segment and a human JKI gene segment of a human immunoglobulin κ light chain locus.

1 1. The rodent of any one of claims 1 - 10, wherein the rodent CK gene of the first immunoglobulin light chain locus is an endogenous rodent CK gene.

12. The rodent of any one of claims 1 - 1 1 , wherein the rodent CK gene of the second immunoglobulin light chain locus is an endogenous rodent CK gene.

13. The rodent of any one of the preceding claims, wherein the endogenous

immunoglobulin λ light chain locus is deleted in part.

14. The rodent of claim 13, wherein the endogenous immunoglobulin λ light chain locus comprises a deletion of V 2-V 3-J12 gene segments and/or V 1-J13-C 3-J J-C 1 gene segments.

15. The rodent of any one of the preceding claims, wherein the immunoglobulin heavy chain locus comprises the human VH gene segments from VH3-74 to VH6-1, the human DH gene segments from DH1-1 to DH7-27, and the human JH gene segments from JHI to JH6.

16. The rodent of any one of the preceding claims, wherein the first immunoglobulin light chain locus comprises the proximal duplication, in whole or in part, of a human

immunoglobulin κ light chain locus.

17. The rodent of claim 16, wherein the first immunoglobulin light chain locus comprises the human VK gene segments from VK2-40 to VK4-1 and the human JK gene segments from JKI to JK5.

18. The rodent of any one of the preceding claims, wherein the second immunoglobulin light chain locus comprises the human νλ gene segments from νλ5-52 to νλ1-40 and νλ3-27 to νλ3-1, and the human J gene segments J l, J 2, J 3 and J 7.

19. The rodent of any one of claims 1-18, wherein the immunoglobulin heavy chain locus lacks an endogenous rodent Adam6 gene.

20. The rodent of any one of claims 1-19, wherein the immunoglobulin heavy chain locus further comprises insertion of one or more nucleotide sequences encoding one or more functional rodent Adam6 polypeptides.

21. The rodent of claim 20, wherein the one or more nucleotide sequences are inserted between a first and a second human VH gene segment.

22. The method of claim 20, wherein the one or more nucleotide sequences are inserted in the place of a human Adam6 pseudogene.

23. The rodent of claim 21 or 22, wherein the first human VH gene segment is human VHI - 2 and the second human VH gene segment is human VH6- 1 .

24. The rodent of claim 19, wherein the one or more nucleotide sequences are inserted between a human VH gene segment and a human DH gene segment.

25. The rodent of any one of the preceding claims, wherein the rodent is homozygous for the immunoglobulin heavy chain locus.

26. The rodent of any one of the preceding claims, wherein the rodent is homozygous for the endogenous immunoglobulin λ light chain locus that is deleted, in whole or in part.

27. The rodent of any one claims 1 -26, wherein the rodent is a mouse or a rat.

28. An isolated rodent cell comprising:

29. An immortalized cell made from the rodent cell of claim 28.

30. The isolated rodent cell of claim 28, wherein the rodent cell is a rodent embryonic stem cell.

31. A rodent embryo generated from the rodent embryonic stem cell of claim 30.

32. A method of making a rodent, the method comprising a step of

generating a rodent from the rodent embryonic stem cell of claim 30 or the rodent embryo of claim 31, thereby making the rodent.

33. A method of producing an antibody in a rodent, the method comprising the steps of

(a) immunizing a rodent with an antigen of interest, wherein the rodent comprises:

(i) an immunoglobulin heavy chain locus comprising one or more human VH gene segments, one or more human DH gene segments and one or more human JH gene segments and a rodent CH gene, wherein the human VH, DH and JH gene segments are operably linked to the rodent CH gene;

(ii) a first immunoglobulin light chain locus comprising one or more human VK gene segments, one or more human JK gene segments and a rodent CK gene, wherein the human VK and JK gene segments are operably linked to the rodent CK gene;

(iii) a second immunoglobulin light chain locus comprising one or more human νλ gene segments and one or more human J gene segments and a rodent CK gene, wherein the human νλ and J gene segments are operably linked to the rodent CK gene; and

(iv) an endogenous immunoglobulin λ light chain locus that is deleted in whole or in part;

(b) maintaining the rodent under conditions sufficient that the rodent produces an immune response to the antigen of interest; and

(c) recovering from the rodent or a rodent cell an antibody that binds the antigen of interest or a nucleic acid encoding a variable region of such an antibody.

34. The method of claim 33, wherein the rodent cell is a B cell.

35. The method of claim 33, wherein the rodent cell is a hybridoma made from the B cell obtained from the rodent.

36. The method of any one of claims 33-35, wherein the immunoglobulin heavy chain locus comprises the human VH gene segments from VH3-74 to VH6-1, the human DH gene segments from DH1-1 to DH7-27, and the human JH gene segments from JHI to JH6.

37. The method of any one of claims 33-36, wherein the human VH, DH and JH gene segments replace rodent VH, DH and JH gene segments.

38. The method of any one of claims 37, wherein rodent further comprises insertion of one or more nucleotide sequences encoding one or more functional rodent Adam6 polypeptides.

39. The method of any one of claims 33-36, wherein the immunoglobulin heavy chain locus lacks an endogenous rodent Adam6 gene.

40. The method of claim 37, wherein the immunoglobulin heavy chain locus further comprises insertion of one or more nucleotide sequences encoding one or more functional rodent Adam6 polypeptides.

41. The method of claim 40, wherein the one or more nucleotide sequences are inserted between a first and a second human VH gene segment.

42. The method of claim 41, wherein the one or more nucleotide sequences are inserted in the place of a human Adam6 pseudogene.

43. The method of claim 41 or 42, wherein the first human VH gene segment is human VH1-2 and the second human VH gene segment is human VH6-1.

44. The method of claim 41 , wherein the one or more nucleotide sequences are inserted between a human VH gene segment and a human DH gene segment.

45. The method of any one of claims 33-44, wherein the first immunoglobulin light chain locus comprises the human VK gene segments from VK2-40 to VK4-1 and the human JK gene segments from JKI to JK5.

46. The method of any one of claims 33-45, wherein the second immunoglobulin light chain locus comprises the human νλ gene segments from νλ5-52 to νλ1-40 and/or νλ3-27 to νλ3-1, and the human Jl gene segments Γλΐ, Tk2, J13 and J17.

47. The method of any one of claims 33-46, wherein the first immunoglobulin light chain locus is at a first endogenous immunoglobulin κ light chain locus and the second

immunoglobulin light chain locus is at a second endogenous immunoglobulin κ light chain locus such that the rodent is hemizygous for the first and the second immunoglobulin light chain loci.

48. The method of any one of claims 33-47, wherein the rodent is a mouse or a rat.

49. The method of any one of claims 33-47, wherein the antibody comprises a λ light chain variable region.

50. A rodent comprising:

two endogenous immunoglobulin heavy chain alleles each comprising 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 an endogenous CH gene,

a first immunoglobulin light chain allele at an endogenous κ light chain locus, the first immunoglobulin light chain allele comprising one or more human VK gene segments and one or more human JK gene segments operably linked to an endogenous CK gene, a second immunoglobulin light chain allele at an endogenous κ light chain locus, the second immunoglobulin light chain allele comprising one or more human νλ gene segments and one or more human Ιλ gene segments operably linked to an endogenous CK gene, and a third and a fourth endogenous immunoglobulin light chain allele, each at an endogenous λ light chain locus and each comprising a deletion of νλ2-νλ3-Ιλ2 gene segments and V l-J13-C 3-Jll-OJ gene segments,

wherein the rodent expresses antibodies comprising human VK domains generated from rearrangement of human VK and JK gene segments of the first immunoglobulin κ light chain allele and νλ domains generated from rearrangement of human νλ and Jl gene segments of the second immunoglobulin κ light chain allele, and wherein said VK and νλ domains are represented in the expressed antibody repertoire of the rodent in about a 3 :2 ratio.

51. The rodent of claim 50, wherein the two endogenous immunoglobulin heavy chain alleles each contain a deletion in whole or in part of the endogenous rodent immunoglobulin heavy chain variable region.

52. The rodent of claim 50 or 51, wherein the first and/or second immunoglobulin light chain allele comprises a deletion in whole or in part of the endogenous rodent immunoglobulin K light chain variable region.

53. The rodent of any one of claims 50-52, wherein the rodent further comprises an insertion of one or more nucleotide sequences encoding one or more functional rodent Adam6 polypeptides.

54. The rodent of any one of claims 50-52, wherein one or both of the two endogenous immunoglobulin heavy chain alleles further comprise insertion of one or more nucleotide sequences encoding one or more functional rodent Adam6 polypeptides.

55. The rodent of claim 53 or 54, wherein the one or more nucleotide sequences are inserted in the place of a human Adam6 pseudogene.

56. The rodent of claim 53 or 54, wherein the one or more nucleotide sequences are inserted between a first and a second human VH gene segment.

57. The rodent of claim 56, wherein the first human VH gene segment is human VH1-2 and the second human VH gene segment is human VH6-1.

58. The rodent of claim 53 or 54, wherein the one or more nucleotide sequences are inserted between a human VH gene segment and a human DH gene segment.

59. The rodent of any one of claims 50-58, wherein the rodent is a mouse or a rat.