Non-human animals possessing engineered immunoglobulin λ light chains and the uses of said non-human animals.

Abstract

Non-human animals (and / or non-human cells) and methods for using said non-human animals (and / or non-human cells) are provided, said non-human animals (and / or non-human cells) having a genome containing sequences encoding human antibodies (i.e., immunoglobulin genes). The non-human animals described herein express antibodies containing immunoglobulin (Ig) light chains, said immunoglobulin light chains being characterized by the presence of a human Vλ domain. In some embodiments, the non-human animals provided herein are characterized by expressing antibodies containing a human Vλ light chain encoded by a sequence encoding a human Igλ light chain inserted into an endogenous Igκ light chain locus of said non-human animal. Methods for producing antibodies from non-human animals are also provided, said antibodies containing a human variable region and a mouse constant region.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201880078631.1, filed on December 4, 2018, entitled "Non-human animal having an engineered immunoglobulin λ light chain and use of said non-human animal". The original application was a national phase application of International Application No. PCT / US2018 / 063841, which claims priority to U.S. Provisional Application No. 62 / 594,944, filed December 5, 2017; U.S. Provisional Application No. 62 / 594,946, filed December 5, 2017; U.S. Provisional Application No. 62 / 609,241, filed December 21, 2017; and U.S. Provisional Application No. 62 / 609,251, filed December 21, 2017; each of which is incorporated herein by reference. Background Technology

[0002] Human antibodies represent the most advanced class of therapeutic agents. Among current antibody production technologies, the development of genetically engineered animals (e.g., rodents) using genetic material encoding human antibodies (in whole or in part) has revolutionized the field of human therapeutic monoclonal antibodies for treating a wide range of diseases. There remains a need to develop improved in vivo systems for producing human monoclonal antibodies that maximize the human antibody repertoire in genetically engineered host animals. Summary of the Invention

[0003] In some embodiments, this disclosure provides a rodent whose phylogenetic genome comprises:

[0004] An engineered endogenous immunoglobulin κ light chain locus, wherein the engineered endogenous immunoglobulin κ light chain locus comprises:

[0005] (a) One or more human Vλ gene segments

[0006] (b) One or more human Jλ gene segments, and

[0007] (c) One or more Cλ genes.

[0008] The one or more human Vλ gene segments and the one or more human Jλ gene segments are operatively linked to the one or more Cλ genes, and the rodents lack the rodent Cκ gene at the engineered endogenous immunoglobulin κ gene locus.

[0009] In some embodiments, the one or more Cλ genes are Cλ genes. In some embodiments, the Cλ gene is or includes a rodent Cλ gene. In some embodiments, the rodent Cλ gene has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the mouse Cλ1, mouse Cλ2, or mouse Cλ3 genes. In some embodiments, the rodent Cλ gene is or includes the mouse Cλ1 gene. In some embodiments, the rodent Cλ gene is or includes the rat Cλ gene. In some embodiments, the rat Cλ gene has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the rat Cλ1, rat Cλ2, rat Cλ3, or rat Cλ4 genes.

[0010] In some embodiments, one or more human Vλ gene segments and one or more human Jλ gene segments replace one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof. In some embodiments, one or more human Vλ gene segments and one or more human Jλ gene segments replace one or more rodent Vκ gene segments, one or more rodent Jκ gene segments, or any combination thereof. In some embodiments, one or more human Vλ gene segments and one or more human Jλ gene segments replace all functional rodent Vκ gene segments and / or all functional rodent Jκ gene segments.

[0011] In some embodiments, one or more human Vλ gene segments include Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof. In some embodiments, one or more human Vλ gene segments include Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof. In some implementations, one or more human Vλ gene segments include Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1.

[0012] In some embodiments, one or more human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any combination thereof. In some embodiments, one or more human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0013] In some embodiments, the engineered endogenous immunoglobulin κ light chain locus includes one or more human Vλ non-coding sequences, each of which is adjacent to at least one of one or more human Vλ gene segments, wherein the one or more human Vλ non-coding sequences naturally occur adjacent to the human Vλ gene segment in the endogenous human immunoglobulin κ light chain locus. For example, refer to Figure 20 The first exemplary endogenous human Vλ non-coding sequence naturally occurs adjacent to the Vλ3-12 gene segment in the endogenous human immunoglobulin λ light chain locus (and at the 3′ end of the Vλ3-12 gene segment). An engineered endogenous immunoglobulin κ light chain locus containing the first exemplary endogenous human Vλ non-coding sequence may include the non-coding sequence at a position adjacent to the Vλ3-12 gene segment in the engineered endogenous immunoglobulin κ light chain locus (and preferably at the 3′ end of the Vλ3-12 gene segment). An engineered endogenous immunoglobulin κ light chain locus containing the first exemplary endogenous human Vλ non-coding sequence may also include the non-coding sequence at a position adjacent to the Vλ2-11 gene segment in the engineered endogenous immunoglobulin κ light chain locus (and preferably at the 5′ end of the Vλ2-11 gene segment). In some embodiments, the engineered endogenous immunoglobulin κ light chain locus comprising the first exemplary endogenous human Vλ non-coding sequence may further include the non-coding sequence at a location adjacent to the Vλ3-12 gene segment (and preferably the 3′ end of the Vλ3-12 gene segment) and adjacent to the Vλ2-11 gene segment (preferably the 5′ end of the Vλ2-11 gene segment) within the engineered endogenous immunoglobulin κ light chain locus. In some embodiments, each of the one or more human Vλ non-coding sequences is or contains an intron.

[0014] In some embodiments, the engineered endogenous immunoglobulin κ light chain locus comprises one or more human Jλ non-coding sequences, each of which is adjacent to at least one of one or more human Jλ gene segments, wherein the one or more human Jλ non-coding sequences naturally occur adjacent to the human Jλ gene segment within the endogenous human immunoglobulin κ light chain locus. In some embodiments, each of the one or more human Jλ non-coding sequences is or contains an intron. In some embodiments, the engineered endogenous immunoglobulin κ light chain locus comprises one or more human Jκ non-coding sequences, each of which is adjacent to at least one of one or more human Jλ gene segments, wherein the one or more human Jκ non-coding sequences naturally occur adjacent to the human Jκ gene segment within the endogenous human immunoglobulin κ light chain locus. For example, refer to... Figure 21 The first exemplary endogenous human Jκ non-coding sequence naturally occurs at the endogenous human immunoglobulin κ light chain locus. The engineered endogenous immunoglobulin κ light chain locus containing the first exemplary endogenous human Jκ non-coding sequence may have a non-coding sequence at a location adjacent to a Jλ gene segment (e.g., Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7) within the engineered endogenous immunoglobulin κ light chain locus. In some embodiments, each of the one or more human Jκ non-coding sequences is or contains an intron.

[0015] In some embodiments, the engineered endogenous immunoglobulin κ light chain locus comprises one or more human Vλ non-coding sequences, wherein each of the one or more human Vλ non-coding sequences is adjacent to Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, etc., of the engineered endogenous immunoglobulin κ light chain locus. Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3 -27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10 Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1, and each of said one or more human Vλ non-coding sequences is naturally adjacent to Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5- 45. Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1. In some embodiments, the engineered endogenous immunoglobulin κ light chain locus includes one or more human Jλ non-coding sequences, each of which is adjacent to Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 of the engineered endogenous immunoglobulin κ light chain locus, and each of which is naturally adjacent to Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 of the endogenous human immunoglobulin κ light chain locus. In some embodiments, the engineered endogenous immunoglobulin κ light chain locus includes one or more human Jκ non-coding sequences, each of which is adjacent to Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 of the engineered endogenous immunoglobulin κ light chain locus, and each of which naturally occurs adjacent to Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 of the endogenous human immunoglobulin κ light chain locus.

[0016] In some embodiments, the engineered endogenous immunoglobulin κ light chain locus includes a κ light chain non-coding sequence located between the one or more human Vλ gene segments and the one or more human Jλ gene segments. In some embodiments, the κ light chain non-coding sequence is a human κ light chain non-coding sequence. In some embodiments, the human κ light chain non-coding sequence has a sequence that naturally occurs between the human Vκ4-1 gene segment and the human Jκ1 gene segment in the endogenous human immunoglobulin κ light chain locus.

[0017] In some embodiments, the rodents described herein are homozygous for the engineered endogenous immunoglobulin κ light chain locus. In some embodiments, the rodents described herein are heterozygous for the engineered endogenous immunoglobulin κ light chain locus. In some embodiments, the rodent germline genome includes a second engineered endogenous immunoglobulin κ light chain locus, which comprises:

[0018] (a) One or more human Vκ gene segments, and

[0019] (b) One or more human Jκ gene segments,

[0020] The one or more human Vκ gene segments and the one or more human Jκ gene segments are operatively linked to the Cκ gene.

[0021] In some embodiments, the rodent genome further comprises a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element. In some embodiments, the transcriptional control element comprises a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin κ light chain transcriptional control element, an immunoglobulin λ light chain transcriptional control element, or any combination thereof. In some embodiments, the nucleic acid sequence encoding the exogenous TdT is located at an immunoglobulin κ light chain locus, an immunoglobulin λ light chain locus, an immunoglobulin heavy chain locus, a RAG1 locus, or a RAG2 locus. In some embodiments, the TdT is human TdT. In some embodiments, the TdT is a short isoform of TdT (TdTS).

[0022] In some embodiments, the rodents described herein contain nucleic acid sequences encoding exogenous terminal deoxynucleotidyl transferases (TdTs) operably linked to transcriptional control elements in their germline genomes; and exhibit at least a 1.2-fold, at least a 1.5-fold, at least a 1.75-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, or at least a 5-fold increase in light chains (e.g., expressing light chain variable domains therein) in connectivity diversity relative to comparable mice (e.g., littermates) that do not contain exogenous terminal deoxynucleotidyl transferases (TdTs) operably linked to transcriptional control elements in their germline genomes. In some embodiments, connectivity diversity is measured numerically by a unique CDR3 / 10,000 reading.

[0023] In some embodiments, the rodents described herein comprise a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element in the phylogenetic genome of the rodent, and at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, and at least 65% of the light chains (e.g., λ and / or κ light chains) produced by the rodent exhibit non-template addition.

[0024] In some implementations, the phylogenetic genome of the rodents described herein includes:

[0025] An engineered endogenous immunoglobulin heavy chain locus, wherein the engineered endogenous immunoglobulin heavy chain locus comprises:

[0026] (a) One or more people V H Gene segments,

[0027] (b) One or more people D H Gene segments, and

[0028] (c) One or more people J H Gene segments,

[0029] The one or more persons V H Gene segments, one or more individuals D H Gene segments and one or more individuals J H The gene segment is operatively linked to the rodent immunoglobulin heavy chain constant region at the engineered endogenous immunoglobulin heavy chain locus.

[0030] In some implementation schemes, one or more persons V H Gene segment, one or more individuals D H Gene segments and one or more individuals J H Gene segment replacing one or more rodent VH Gene segment, one or more rodent D H Gene segment, one or more rodent J H Gene segments or any combination thereof. In some implementations, one or more individuals V H Gene segment, one or more individuals D H Gene segments and one or more individuals J H Gene segment replacement of one or more rodent V H Gene segment, one or more rodent D H Gene segment, one or more rodent J H Gene segments or any combination thereof.

[0031] In some implementation schemes, one or more persons V H Gene segment includes V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, VH 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1 or any combination thereof. In some implementations, one or more persons V H Gene segment includes V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2 and V H 6-1.

[0032] In some implementation schemes, one or more persons D H Gene segments include D H 1-1, D H2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H 7-27 or any combination thereof. In some implementations, one or more persons D H Gene segments include D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26 and D H 7-27.

[0033] In some implementations, one or more persons J H Gene segments include J H 1. J H 2. J H 3. J H 4. J H 5. J H6 or any combination thereof. In some implementations, one or more persons J H Gene segments include J H 1. J H 2. J H 3. J H 4. J H 5 and J H 6.

[0034] In some implementations, the engineered endogenous immunoglobulin heavy chain locus contains one or more human V... H Non-coding sequence, the one or more persons V H Each of the non-coding sequences is adjacent to the one or more persons V H At least one of the gene segments, wherein the one or more V H Each of the non-coding sequences is naturally adjacent to human V at the endogenous human immunoglobulin heavy chain locus. H Gene segments appear. In some implementations, the one or more individuals V H Each of the non-coding sequences is or contains an intron. In some embodiments, the engineered endogenous immunoglobulin heavy chain locus contains one or more human D... H Non-coding sequences, the one or more people D H Each of the non-coding sequences is adjacent to one or more persons D H At least one of the gene segments, wherein the one or more D H Each of the non-coding sequences is naturally adjacent to the human D locus in the endogenous human immunoglobulin heavy chain gene. H Gene segments appear. In some implementations, the one or more individuals D H Each of the non-coding sequences is or contains an intron. In some embodiments, the engineered endogenous immunoglobulin heavy chain locus contains one or more human J... H Non-coding sequences, the one or more people J H Each of the non-coding sequences is adjacent to one or more people J. H At least one of the gene segments, wherein the one or more J H Each of the non-coding sequences is naturally adjacent to the human J locus in the endogenous human immunoglobulin heavy chain gene. H Gene segments appear. In some implementations, the one or more individuals J H Each of the non-coding sequences is or contains an intron.

[0035] In some implementations, the rodents described herein are homozygous for the engineered endogenous immunoglobulin heavy chain locus.

[0036] In some implementations, the rodent immunoglobulin heavy chain constant region is the endogenous rodent immunoglobulin heavy chain constant region.

[0037] In some embodiments, the endogenous Vλ gene segment, the endogenous Jλ gene fragment, and the endogenous Cλ gene are all or partially deleted. In some embodiments, the rodents described herein do not detectably express the endogenous immunoglobulin λ light chain variable domain. In some embodiments, the rodents described herein do not detectably express the endogenous immunoglobulin κ light chain variable domain.

[0038] In some embodiments, the engineered endogenous immunoglobulin heavy chain locus lacks a functional endogenous rodent Adam6 gene. In some embodiments, the rodent germline genome contains one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments. In some embodiments, one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are expressed (e.g., in cells of the male reproductive system, such as testicular cells).

[0039] In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are contained on the same chromosome as the engineered endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are contained within the engineered endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are located in a first-person V... H Gene segments and the second person V H Between gene segments. In some implementations, the first person V H The gene segment is V H 1-2, and the second person V H The gene segment is V H6-1. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments replace the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments replace the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are located in human V H Gene segments and human D H Between gene segments.

[0040] In some embodiments, the rodents described herein comprise a population of B cells expressing antibodies comprising immunoglobulin λ light chains, each containing a variable domain of the human immunoglobulin λ light chain. In some embodiments, the variable domain of the human immunoglobulin λ light chain is encoded by a rearranged variable region sequence of the human immunoglobulin λ light chain, the rearranged variable region sequence comprising (i) one or more human Vλ gene segments or a somatic hypermutated variant thereof, and (ii) one or more human Jλ gene segments or a somatic hypermutated variant thereof.

[0041] In some embodiments, the rodents described herein comprise a population of B cells expressing antibodies comprising immunoglobulin heavy chains, each of which contains a human immunoglobulin heavy chain variable domain. In some embodiments, the human immunoglobulin heavy chain variable domain is encoded by a rearranged human immunoglobulin heavy chain variable region sequence, the rearranged human immunoglobulin heavy chain variable region sequence comprising (i) one or more human V H (ii) one or more human D H One of the gene segments or its somatic hypermutated variant, and (ii) one or more human J H One of the gene segments or its somatic hypermutation variant.

[0042] In some embodiments, the rodents described herein generate a population of B cells in response to immunization with an antigen comprising one or more epitopes. In some embodiments, the rodents generate a population of B cells expressing antibodies that bind (e.g., specifically bind) to one or more epitopes of a target antigen. In some embodiments, the antibodies expressed by the B cell population generated in response to the antigen comprise a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence, and / or a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein. In some embodiments, the antibodies expressed by the B cell population generated in response to the antigen comprise a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence; and / or a κ light chain having a human κ light chain variable domain encoded by a human κ light chain variable region sequence as described herein.

[0043] In some embodiments, rodents generate a population of B cells expressing antibodies that bind to one or more epitopes of a target antigen, wherein the antibodies expressed by the B cell population generated in response to the antigen comprise: (i) a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence; (ii) a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein; (iii) a κ light chain having a human κ light chain variable domain encoded by a human κ light chain variable region sequence as described herein; or (iv) any combination thereof.

[0044] In some embodiments, the human heavy chain variable region sequence, the human λ light chain variable region sequence, and / or the human κ light chain variable region sequence, as described herein, are somatic hypermutated. In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90% of the B cells in response to an antigen-generated population contain the human heavy chain variable region sequence, the λ light chain variable region sequence, and / or the somatic hypermutated κ light chain variable region sequence.

[0045] In some implementations, the rodents described herein are mice or rats.

[0046] In some embodiments, this document describes cells and / or tissues (e.g., isolated cells and / or tissues) provided from rodents. In some embodiments, the provided cells and tissues include, for example, lymphoid tissue, spleen cells, B cells, stem cells, and / or germ cells. In some embodiments, the provided cells are isolated. In some embodiments, the isolated cells are or include progenitor B cells, pre-B cells, immature B cells, mature naïve B cells, activated B cells, memory B cells, B lineage lymphocytes, and / or plasma cells. In some embodiments, the isolated cells include stem cells (e.g., embryonic stem cells) and / or germ cells (e.g., sperm, oocytes).

[0047] In some embodiments, this disclosure provides an isolated rodent cell, the rodent's germline genome comprising:

[0048] An engineered endogenous immunoglobulin κ light chain locus, wherein the engineered endogenous immunoglobulin κ light chain locus comprises:

[0049] (a) One or more human Vλ gene segments

[0050] (b) One or more human Jλ gene segments, and

[0051] (c) Cλ gene,

[0052] The one or more human Vλ gene segments and the one or more human Jλ gene segments are operatively linked to the Cλ gene.

[0053] In some embodiments, the isolated rodent cells described herein lack the rodent Cκ gene at an engineered endogenous immunoglobulin κ gene locus.

[0054] In some embodiments, the isolated rodent cells described herein are rodent embryonic stem (ES) cells.

[0055] In some embodiments, this disclosure provides a rodent embryo produced from rodent ES cells described herein.

[0056] In some embodiments, this disclosure provides an immortalized cell derived from rodent cells isolated herein.

[0057] In some embodiments, the present invention provides a method for preparing rodents whose germline genome contains an engineered endogenous immunoglobulin κ light chain locus, the method comprising the following steps:

[0058] (a) Introducing one or more DNA fragments into the germline genome of rodent ES cells, wherein the one or more DNA fragments comprise:

[0059] (i) One or more human Vλ gene segments

[0060] (ii) One or more human Jλ gene segments, and

[0061] (iii) One or more Cλ genes

[0062] The one or more human Vλ gene segments, the one or more human Jλ gene segments, and the one or more Cλ genes are introduced into the germline genome of the rodent ES cells at the endogenous immunoglobulin κ light chain locus, and the one or more human Vλ gene segments, the one or more human Jλ gene segments, and the one or more Cλ genes are operatively linked; and

[0063] (b) Using the rodent ES cells produced in (a) to produce rodents.

[0064] In some embodiments, a method for preparing a rodent germline genome containing an engineered endogenous immunoglobulin κ light chain locus includes the step of introducing a κ light chain non-coding sequence into the germline genome of the rodent ES cells such that the κ light chain non-coding sequence is located between one or more human Vλ gene segments and one or more human Jλ gene segments in the germline genome of the rodent ES cells.

[0065] In some embodiments, this disclosure provides a method for preparing rodents whose germline genome contains an engineered endogenous immunoglobulin κ light chain locus, the method comprising the following steps:

[0066] The endogenous immunoglobulin κ light chain locus in the germline genome was engineered to include:

[0067] (a) One or more human Vλ gene segments

[0068] (b) One or more human Jλ gene segments, and

[0069] (c) One or more Cλ genes.

[0070] The one or more human Vλ gene segments and the one or more human Jλ gene segments are operatively linked to the one or more Cλ genes, and

[0071] One or more Cλ genes are inserted to replace the rodent Cκ gene at the endogenous immunoglobulin κ gene locus.

[0072] In some implementations, the Cλ gene replaces the rodent Cκ gene at the endogenous immunoglobulin κ gene locus.

[0073] In some embodiments, one or more human Vλ gene segments include Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof. In some implementations, one or more human Vλ gene segments include Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some implementations, one or more human Vλ gene segments include Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1.

[0074] In some embodiments, one or more human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any combination thereof. In some embodiments, one or more human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0075] In some embodiments, the engineered endogenous immunoglobulin κ light chain locus comprises one or more human Vλ non-coding sequences, each of which is adjacent to at least one of the one or more human Vλ gene segments, wherein the one or more human Vλ non-coding sequences naturally occur adjacent to the human Vλ gene segment within the endogenous human immunoglobulin κ light chain locus. In some embodiments, each of the one or more human Vλ non-coding sequences is or contains an intron. In some embodiments, the engineered endogenous immunoglobulin κ light chain locus comprises one or more human Jλ non-coding sequences, each of which is adjacent to at least one of the one or more human Jλ gene segments, wherein the one or more human Jλ non-coding sequences naturally occur adjacent to the human Jλ gene segment within the endogenous human immunoglobulin κ light chain locus. In some embodiments, each of the one or more human Jλ non-coding sequences is or contains an intron. In some embodiments, the engineered endogenous immunoglobulin κ light chain locus comprises one or more human Jκ non-coding sequences, each of which is adjacent to at least one of one or more human Jλ gene segments, wherein each of the one or more human Jκ non-coding sequences naturally occurs adjacent to the human Jκ gene segment within the endogenous human immunoglobulin κ light chain locus. In some embodiments, each of the one or more human Jκ non-coding sequences is or contains an intron.

[0076] In some embodiments, the Cλ gene is or includes the rodent Cλ gene. In some embodiments, the rodent Cλ gene has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the mouse Cλ1, mouse Cλ2, or mouse Cλ3 genes. In some embodiments, the rodent Cλ gene is or includes the mouse Cλ1 gene. In some embodiments, the rodent Cλ gene is or includes the rat Cλ gene. In some embodiments, the rat Cλ gene has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the rat Cλ1, rat Cλ2, rat Cλ3, or rat Cλ4 genes.

[0077] In some embodiments, one or more DNA fragments contain at least one selection marker. In some embodiments, one or more DNA fragments contain at least one site-specific recombination site.

[0078] In some implementations, the rodent germline genome includes:

[0079] An engineered endogenous immunoglobulin heavy chain locus, wherein the engineered endogenous immunoglobulin heavy chain locus comprises:

[0080] (a) One or more people V H Gene segments,

[0081] (b) One or more people D H Gene segments, and

[0082] (c) One or more people J H Gene segments,

[0083] The one or more persons V H Gene segments, one or more individuals D H Gene segments and one or more individuals J H The gene segment is operatively linked to the rodent immunoglobulin heavy chain constant region.

[0084] In some embodiments, the step of engineering an endogenous immunoglobulin κ light chain locus in a germline genome is performed in rodent ES cells, the germline genome of which contains an engineered endogenous immunoglobulin heavy chain locus, the engineered endogenous immunoglobulin heavy chain locus comprising one or more human V loci operatively linked to a rodent immunoglobulin heavy chain constant region. H Gene segment, one or more individuals D H Gene segments and one or more individuals J H Gene segments.

[0085] In some implementations, the engineered endogenous immunoglobulin heavy chain locus contains one or more human V... H Non-coding sequence, the one or more persons V H Each of the non-coding sequences is adjacent to one or more people V H At least one of the gene segments, wherein the one or more individuals V H Each of the non-coding sequences is naturally adjacent to human V at the endogenous human immunoglobulin heavy chain locus. H Gene segments appear. In some implementations, the one or more individuals V H Each of the non-coding sequences is or contains an intron. In some embodiments, the engineered endogenous immunoglobulin heavy chain locus contains one or more human D... H Non-coding sequences, the one or more people D H Each of the non-coding sequences is adjacent to one or more people D. H At least one of the gene segments, wherein the one or more D HEach of the non-coding sequences is naturally adjacent to the human D locus in the endogenous human immunoglobulin heavy chain gene. H Gene segments appear. In some implementations, the one or more individuals D H Each of the non-coding sequences is or contains an intron. In some embodiments, the engineered endogenous immunoglobulin heavy chain locus contains one or more human J... H Non-coding sequences, the one or more people J H Each of the non-coding sequences is adjacent to one or more persons J H At least one of the gene segments, wherein the one or more J H Each of the non-coding sequences is naturally adjacent to the human J locus in the endogenous human immunoglobulin heavy chain gene. H Gene segments appear. In some implementations, the one or more individuals J H Each of the non-coding sequences is or contains an intron.

[0086] In some embodiments, this disclosure provides a method for generating antibodies in rodents, the method comprising the following steps:

[0087] (i) Immunize rodents with the target antigen.

[0088] The rodents described therein possess a phylogenetic genome, which comprises:

[0089] The engineered endogenous immunoglobulin κ light chain locus includes:

[0090] (a) One or more human Vλ gene segments

[0091] (b) One or more human Jλ gene segments, and

[0092] (c) One or more Cλ genes.

[0093] The one or more human Vλ gene segments and the one or more human Jλ gene segments are operatively linked to the Cλ gene, and

[0094] The one or more Cλ genes therein replace the rodent Cκ gene at the engineered endogenous immunoglobulin κ light chain locus.

[0095] The rodents are maintained under conditions sufficient to induce an immune response to the target antigen; and

[0096] Antibodies bound to the target antigen are recovered from the rodent, the rodent's cells, or cells derived from the rodent's cells.

[0097] In some embodiments, in response to the immunization step, rodents generate B cells expressing antibodies that bind to the target antigen. In some embodiments, the antibody expressed by the B cells comprises a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence; and / or a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence, as described herein. In some embodiments, the antibody expressed by the B cells comprises (i) a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence; (ii) a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence; (iii) a κ light chain having a human κ light chain variable domain encoded by a human κ light chain variable region sequence; or (iv) any combination thereof.

[0098] In some embodiments, in response to the immunization step, the rodent generates B cells that express antibodies that bind to the target antigen. In some embodiments, the antibody expressed by the B cell population generated in response to the antigen comprises a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence and / or a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein. In some embodiments, the antibody expressed by the B cell population generated in response to the antigen comprises (i) a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence; (ii) a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein; (iii) a κ light chain having a human κ light chain variable domain encoded by a human κ light chain variable region sequence as described herein; or (iv) any combination thereof.

[0099] In some embodiments, in response to the immunization step, a rodent generates a population of B cells expressing antibodies that bind to one or more epitopes of a target antigen, wherein the antibodies expressed by the B cell population generated in response to the antigen comprise: (i) a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence, (ii) a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein, (iii) a κ light chain having a human κ light chain variable domain encoded by a human κ light chain variable region sequence as described herein, or (iv) any combination thereof.

[0100] In some embodiments, the human heavy chain variable region sequence, the human λ light chain variable region sequence, and / or the human κ light chain variable region sequence, as described herein, are somatic hypermutated. In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90% of the B cells in response to an antigen-generated population contain the human heavy chain variable region sequence, the λ light chain variable region sequence, and / or the somatic hypermutated κ light chain variable region sequence.

[0101] In some embodiments, antibodies binding to the target antigen are isolated, recovered, or identified from rodent B cells. In some embodiments, antibodies binding to the target antigen are isolated, recovered, or identified from hybridomas prepared using rodent B cells.

[0102] In some implementations, the antigen comprises one or more epitopes, and the antibody that binds to the target antigen binds to one or more epitopes.

[0103] In some embodiments, the Cλ gene is or includes the rodent Cλ gene. In some embodiments, the rodent Cλ gene has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the mouse Cλ1, mouse Cλ2, or mouse Cλ3 genes. In some embodiments, the rodent Cλ gene is or includes the mouse Cλ1 gene. In some embodiments, the rodent Cλ gene is or includes the rat Cλ gene. In some embodiments, the rat Cλ gene has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the rat Cλ1, rat Cλ2, rat Cλ3, or rat Cλ4 genes.

[0104] In some embodiments, one or more human Vλ gene segments include Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof. In some implementations, one or more human Vλ gene segments include Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1. In some implementations, one or more human Vλ gene segments include Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, and Vλ3-1.

[0105] In some embodiments, one or more human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any combination thereof. In some embodiments, one or more human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7.

[0106] In some embodiments, the engineered endogenous immunoglobulin κ light chain locus comprises one or more human Vλ non-coding sequences, wherein each of the one or more human Vλ non-coding sequences is adjacent to Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ1-52, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ1-52, Vλ1-51, Vλ2-18 ...9, Vλ1-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ1-52, Vλ1-51, Vλ1-52, Vλ1-51, Vλ1-52, Vλ1-51, Vλ1-52, Vλ1-53, Vλ1-54, Vλ1-51, λ3-9, Vλ2-8, Vλ4-3, or Vλ3-1, and each of the one or more human Vλ non-coding sequences is naturally adjacent to Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, or Vλ1-40 of the endogenous human immunoglobulin λ light chain locus. , Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ 2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 appear. In some embodiments, the engineered endogenous immunoglobulin κ light chain locus includes one or more human Jλ non-coding sequences, each of which is adjacent to Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 of the engineered endogenous immunoglobulin κ light chain locus, and each of which is naturally adjacent to Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 of the endogenous human immunoglobulin κ light chain locus. In some embodiments, the engineered endogenous immunoglobulin κ light chain locus includes one or more human Jκ non-coding sequences, each of which is adjacent to Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 of the engineered endogenous immunoglobulin κ light chain locus, and each of which is naturally adjacent to Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 of the endogenous human immunoglobulin κ light chain locus.

[0107] In some embodiments, the rodent possesses a germline genome comprising an engineered endogenous immunoglobulin heavy chain locus, the engineered endogenous immunoglobulin heavy chain locus comprising:

[0108] (a) One or more people V H Gene segments,

[0109] (b) One or more people D H Gene segments, and

[0110] (c) One or more people J H Gene segments,

[0111] The one or more persons V H Gene segments, one or more individuals D H Gene segments and one or more individuals J H The gene segment is operatively linked to the rodent immunoglobulin heavy chain constant region.

[0112] In some implementation schemes, one or more persons V H Gene segment includes V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1 or any combination thereof. In some implementations, one or more persons V H Gene segment includes V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2 and V H 6-1.

[0113] In some implementation schemes, one or more persons D H Gene segments include DH 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H 7-27 or any combination thereof. In some implementations, one or more persons D H Gene segments include D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26 and D H 7-27.

[0114] In some implementations, one or more persons J H Gene segments include J H 1. J H 2. J H 3. J H 4. J H 5. JH 6 or any combination thereof. In some implementations, one or more persons J H Gene segments include J H 1. J H 2. J H 3. J H 4. J H 5 and J H 6.

[0115] In some implementations, the engineered endogenous immunoglobulin heavy chain locus contains one or more human V... H Non-coding sequences, wherein one or more persons V H Each of the non-coding sequences is adjacent to V in the engineered endogenous immunoglobulin heavy chain locus. H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, VH 4-4, V H 1-3, V H 1-2 or V H 6-1, and wherein one or more persons V H Each of the non-coding sequences is naturally adjacent to the V locus of the endogenous human immunoglobulin heavy chain gene. H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2 or V H 6-1. In some implementations, the engineered endogenous immunoglobulin heavy chain locus contains one or more human D... H Non-coding sequences, wherein one or more persons D HEach of the non-coding sequences is adjacent to the D in the engineered endogenous immunoglobulin heavy chain locus. H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26 or D H 7-27, and wherein one or more of the persons D H Each of the non-coding sequences is naturally adjacent to the D locus of the endogenous human immunoglobulin heavy chain gene. H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26 or D H It appeared on 7-27. In some implementations, the engineered endogenous immunoglobulin heavy chain locus contains one or more human J... H Non-coding sequences, wherein one or more people J HEach of the non-coding sequences is adjacent to J in the engineered endogenous immunoglobulin heavy chain locus. H 1. J H 2. J H 3. J H 4. J H 5 or J H 6, and wherein one or more of the persons J H Each of the non-coding sequences is naturally adjacent to the J locus of the endogenous human immunoglobulin heavy chain gene. H 1. J H 2. J H 3. J H 4. J H 5 or J H 6 appears. In some embodiments, the recovered rodent cells are B cells. In some embodiments, the cells derived from rodent cells are hybridomas.

[0116] In some implementations, the nucleotide sequences encoding the human heavy chain variable region sequence, the human λ light chain variable region sequence, and / or the human κ light chain variable region sequence are obtained from B cells.

[0117] In some embodiments, the engineered endogenous immunoglobulin heavy chain locus lacks a functional endogenous rodent Adam6 gene. In some embodiments, the rodent germline genome contains one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments. In some embodiments, one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are expressed (e.g., in cells of the male reproductive system, such as testicular cells).

[0118] In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are contained on the same chromosome as the engineered endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are contained within the engineered endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are located in a first-person V... H Gene segments and the second person V H Between gene segments. In some implementations, the first person V H The gene segment is V H1-2, and the second person V H The gene segment is V H 6-1. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments replace the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments replace the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are located in human V H Gene segments and human D H Between gene segments.

[0119] In some implementations, the rodent is a mouse or a rat.

[0120] In some embodiments, this disclosure provides a rodent whose germline genome includes a homozygous engineered endogenous immunoglobulin κ light chain locus, the homozygous engineered endogenous immunoglobulin κ light chain locus comprising:

[0121] (i) One or more human Vλ gene segments, wherein the one or more human Vλ gene segments include Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof.

[0122] (ii) One or more human Jλ gene segments, wherein the one or more human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, Jλ7 or any combination thereof, and

[0123] (iii) Cλ gene in rodents;

[0124] The one or more human Vλ gene segments, the one or more human Jλ gene segments, and the rodent Cλ gene are operatively linked to each other.

[0125] The rodent Cλ gene therein replaces the rodent Cκ gene at the endogenous immunoglobulin κ light chain locus.

[0126] The engineered endogenous immunoglobulin κ light chain locus described herein comprises:

[0127] (a) One or more human Vλ non-coding sequences, wherein each of the one or more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4 sequences at the engineered endogenous immunoglobulin κ light chain locus. -3 or Vλ3-1, and each of the one or more human Vλ non-coding sequences is naturally adjacent to the occurrence of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 at the endogenous human immunoglobulin λ light chain locus.

[0128] (b) One or more human Jκ non-coding sequences, wherein each of the one or more human Jκ non-coding sequences is adjacent to Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 at the engineered endogenous immunoglobulin κ light chain locus, and wherein each of the one or more human Jκ non-coding sequences naturally occurs adjacent to Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 at the endogenous human immunoglobulin κ light chain locus.

[0129] The immunoglobulin κ light chain locus contains a human κ light chain non-coding sequence located between the one or more human Vλ gene segments and the one or more human Jλ gene segments, the human κ light chain non-coding sequence having a sequence that naturally occurs between the human Vκ4-1 gene segment and the human Jκ1 gene segment of the endogenous human immunoglobulin κ light chain locus.

[0130] In some implementations, the rodent Cλ gene is the mouse Cλ1 gene.

[0131] In some implementations, the engineered endogenous immunoglobulin κ light chain locus contains the rodent immunoglobulin κ light chain enhancer Eκ. i And Eκ3'.

[0132] In some embodiments, the engineered endogenous immunoglobulin κ light chain locus includes the deletion of one or more rodent Vκ gene segments and / or one or more Jκ gene segments. In some embodiments, the engineered endogenous immunoglobulin κ light chain locus includes the deletion of all functional rodent Vκ and / or Jκ gene segments.

[0133] In some embodiments, this disclosure provides a rodent whose phylogenetic genome comprises:

[0134] (a) A homozygous endogenous immunoglobulin heavy chain locus, said homozygous endogenous immunoglobulin heavy chain locus comprising one or more human V genes operatively linked to one or more endogenous immunoglobulin heavy chain constant region genes. H Gene segment, one or more individuals D H Gene segments and one or more individuals J H Gene segments are provided to enable the rodents to express immunoglobulin heavy chains, each of which comprises a human heavy chain variable domain sequence and a rodent heavy chain constant domain sequence.

[0135] (b) A first engineered endogenous immunoglobulin κ light chain locus, comprising one or more human Vκ gene segments and one or more Jκ gene segments operatively linked to an endogenous rodent Cκ region gene, such that the rodent expresses immunoglobulin light chains, each of the immunoglobulin light chains comprising a human κ light chain variable domain sequence and a rodent κ light chain constant domain sequence, and

[0136] (c) A second engineered endogenous immunoglobulin κ light chain locus, wherein the second engineered endogenous immunoglobulin κ light chain locus comprises:

[0137] (i) One or more human Vλ gene segments, wherein the one or more human Vλ gene segments include Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof.

[0138] (ii) One or more human Jλ gene segments, wherein the one or more human Jλ gene segments include Jλ1, Jλ2, Jλ3, Jλ6, Jλ7 or any combination thereof, and

[0139] (iii) Cλ gene in rodents;

[0140] The one or more human Vλ gene segments, the one or more human Jλ gene segments, and the rodent Cλ gene are operatively linked to each other.

[0141] The rodent Cλ gene therein replaces the rodent Cκ gene at the endogenous immunoglobulin κ light chain locus.

[0142] The engineered endogenous immunoglobulin κ light chain locus described herein comprises:

[0143] (a) One or more human Vλ non-coding sequences, wherein each of the one or more human Vλ non-coding sequences is adjacent to the Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4 sequences at the engineered endogenous immunoglobulin κ light chain locus. -3 or Vλ3-1, and each of the one or more human Vλ non-coding sequences is naturally adjacent to the occurrence of Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 at the endogenous human immunoglobulin λ light chain locus.

[0144] (b) One or more human Jκ non-coding sequences, wherein each of the one or more human Jκ non-coding sequences is adjacent to Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 at the engineered endogenous immunoglobulin κ light chain locus, and wherein each of the one or more human Jκ non-coding sequences naturally occurs adjacent to Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 at the endogenous human immunoglobulin κ light chain locus.

[0145] The immunoglobulin κ light chain locus contains a human κ light chain non-coding sequence located between the one or more human Vλ gene segments and the one or more human Jλ gene segments, the human κ light chain non-coding sequence having a sequence that naturally occurs between the human Vκ4-1 gene segment and the human Jκ1 gene segment in the endogenous human immunoglobulin κ light chain locus;

[0146] This enables the rodents to express immunoglobulin light chains, each of which comprises a human λ light chain variable domain sequence and a rodent λ light chain constant domain sequence.

[0147] In some embodiments, the rodents described herein contain an inactivated endogenous immunoglobulin λ light chain locus. In some embodiments, the rodents described herein are heterozygous for the inactivated endogenous immunoglobulin λ light chain locus. In some embodiments, the rodents described herein are homozygous for the inactivated endogenous immunoglobulin λ light chain locus.

[0148] In some embodiments, the rodent genome further comprises a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element. In some embodiments, the transcriptional control element comprises a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin κ light chain transcriptional control element, an immunoglobulin λ light chain transcriptional control element, or any combination thereof. In some embodiments, the nucleic acid sequence encoding the exogenous TdT is located at an immunoglobulin κ light chain locus, an immunoglobulin λ light chain locus, an immunoglobulin heavy chain locus, a RAG1 locus, or a RAG2 locus. In some embodiments, the TdT is human TdT. In some embodiments, the TdT is a short isoform of TdT (TdTS).

[0149] In some embodiments, the rodents described herein comprise a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element in the rodent's germline genome; and exhibit at least a 1.2-fold, at least a 1.5-fold, at least a 1.75-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, or at least a 5-fold increase in light chains (e.g., expression of light chain variable domains therein) in connectivity diversity relative to comparable mice (e.g., littermates) that do not contain an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element in their germline genome. In some embodiments, connectivity diversity is measured using unique CDR3 / 10,000 readings.

[0150] In some embodiments, the rodents described herein comprise a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element in the rodent's germline genome, and at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, and at least 65% of the light chains (e.g., λ and / or κ light chains) produced by the rodent exhibit non-template addition.

[0151] In some implementations, the rodents described herein are rats or mice.

[0152] In some embodiments, this disclosure provides an antibody prepared by a method comprising the following steps:

[0153] (a) Provide the rodents described herein;

[0154] (b) Immunize the rodents with the target antigen;

[0155] (c) Maintaining the rodent under conditions sufficient to induce an immune response in the rodent to the target antigen; and

[0156] (d) Recovering antibodies bound to the target antigen from the rodent or rodent cells or cells derived from the rodent's cells.

[0157] The antibody in (d) comprises a human heavy chain variable domain and a human λ light chain variable domain.

[0158] In some embodiments, this disclosure provides an antibody prepared by a method comprising the following steps:

[0159] (a) Immunization of the rodents described herein with the target antigen;

[0160] (b) Maintaining the rodent under conditions sufficient to induce an immune response in the rodent to the target antigen; and

[0161] (c) Recovering antibodies bound to the target antigen from the rodent or rodent cells or cells derived from the rodent's cells.

[0162] The antibody in (c) comprises a human heavy chain variable domain and a human λ light chain variable domain.

[0163] In some embodiments, rodents do not detectably express the endogenous immunoglobulin κ light chain variable domain. In some embodiments, rodents do not detectably express the endogenous immunoglobulin λ light chain variable domain.

[0164] In some embodiments, the rodents described herein generate a population of B cells in response to immunization with an antigen comprising one or more epitopes. In some embodiments, the rodents generate a population of B cells expressing antibodies that bind (e.g., specifically bind) to one or more epitopes of a target antigen. In some embodiments, the antibodies expressed by the B cell population generated in response to the antigen comprise a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence and / or a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein. In some embodiments, the antibodies expressed by the B cell population generated in response to the antigen comprise (i) a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence; (ii) a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein; (iii) a κ light chain having a human κ light chain variable domain encoded by a human κ light chain variable region sequence as described herein; or (iv) any combination thereof.

[0165] In some embodiments, rodents generate a population of B cells expressing antibodies that bind to one or more epitopes of a target antigen, wherein the antibodies expressed by the B cell population generated in response to the antigen comprise: (i) a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence; (ii) a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein; and / or (iii) a κ light chain having a human κ light chain variable domain encoded by a human κ light chain variable region sequence as described herein.

[0166] In some embodiments, the human heavy chain variable region sequence, the human λ light chain variable region sequence, and / or the human κ light chain variable region sequence, as described herein, are somatic hypermutated. In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90% of the B cells in response to an antigen-generated population contain the human heavy chain variable region sequence, the λ light chain variable region sequence, and / or the somatic hypermutated κ light chain variable region sequence.

[0167] In some embodiments, this disclosure provides a method for preparing antibodies, the method comprising:

[0168] (i) Expressing a first nucleotide sequence encoding an immunoglobulin heavy chain in a host cell, wherein the first nucleotide sequence comprises a human heavy chain variable region sequence;

[0169] (ii) Expression in host cells of a second nucleotide sequence encoding the immunoglobulin λ light chain, wherein the second nucleotide sequence comprises a human λ light chain variable region sequence, which is identified from the germline genome as being expressed (e.g., expressed and / or isolated) by rodents:

[0170] An engineered endogenous immunoglobulin κ light chain locus, wherein the engineered endogenous immunoglobulin κ light chain locus comprises:

[0171] (a) One or more human Vλ gene segments

[0172] (b) One or more human Jλ gene segments, and

[0173] (c) One or more Cλ genes.

[0174] The one or more human Vλ gene segments and the one or more human Jλ gene segments are operatively linked to the one or more Cλ genes, and

[0175] The rodents in question lack the rodent Cκ gene at the engineered endogenous immunoglobulin κ gene locus.

[0176] (iii) Culturing the host cells to express immunoglobulin light chains and immunoglobulin heavy chains and to form antibodies; and

[0177] (iv) Obtain the antibody from the host cell and / or host cell culture.

[0178] In some embodiments, the first nucleotide sequence contains the human heavy chain constant region. In some embodiments, the antibody is a fully human antibody.

[0179] In some implementations, the second nucleotide comprises a human λ light chain constant region sequence.

[0180] In some embodiments, the antibody is a trans-chimeric antibody. In some embodiments, the first nucleotide sequence comprises the rodent heavy chain constant region. In some embodiments, the second nucleotide sequence comprises the rodent λ light chain constant region sequence.

[0181] In some embodiments, this disclosure provides a rodent whose phylogenetic genome comprises:

[0182] (a) A first engineered endogenous immunoglobulin κ light chain locus, wherein the first engineered endogenous immunoglobulin κ light chain locus comprises:

[0183] (i) One or more human Vλ gene segments

[0184] (ii) One or more human Jλ gene segments, and

[0185] (iii) Cλ gene,

[0186] The one or more human Vλ gene segments and the one or more human Jλ gene segments are operatively linked to the Cλ gene, and

[0187] The rodents described therein lack the rodent Cκ gene at the first engineered endogenous immunoglobulin κ gene locus; and

[0188] (b) A second engineered endogenous immunoglobulin κ light chain locus, wherein the second engineered endogenous immunoglobulin κ light chain locus further comprises:

[0189] (i) One or more human Vκ gene segments, and

[0190] (ii) One or more human Jκ gene segments

[0191] The one or more human Vκ gene segments and the one or more human Jκ gene segments are operatively linked to the Cκ gene.

[0192] In some implementations, the Cκ gene is an endogenous rodent Cκ gene.

[0193] In some embodiments, the rodent genome further comprises a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element. In some embodiments, the transcriptional control element comprises a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin κ light chain transcriptional control element, an immunoglobulin λ light chain transcriptional control element, or any combination thereof. In some embodiments, the nucleic acid sequence encoding the exogenous TdT is located at the immunoglobulin κ light chain locus, the immunoglobulin λ light chain locus, the immunoglobulin heavy chain locus, the RAG1 locus, or the RAG2 locus. In some embodiments, the TdT is human TdT. In some embodiments, the TdT is a short isoform of TdT (TdTS).

[0194] In some embodiments, the rodent genome further comprises a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element. In some embodiments, the transcriptional control element comprises a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin κ light chain transcriptional control element, an immunoglobulin λ light chain transcriptional control element, or any combination thereof. In some embodiments, the nucleic acid sequence encoding the exogenous TdT is located at the immunoglobulin κ light chain locus, the immunoglobulin λ light chain locus, the immunoglobulin heavy chain locus, the RAG1 locus, or the RAG2 locus. In some embodiments, the TdT is human TdT. In some embodiments, the TdT is a short isoform of TdT (TdTS).

[0195] In some embodiments, the rodents described herein comprise a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element in the rodent's germline genome; and exhibit at least a 1.2-fold, at least a 1.5-fold, at least a 1.75-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, or at least a 5-fold increase in light chains (e.g., expression of light chain variable domains thereof) in connectivity diversity relative to comparable mice (e.g., littermates) that do not contain an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element in their germline genome. In some embodiments, connectivity diversity is measured by unique CDR3 / 10,000 readings.

[0196] In some embodiments, the rodents described herein comprise a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element in the rodent's germline genome, and at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, and at least 65% of the light chains (e.g., λ and / or κ light chains) produced by the rodent exhibit non-template addition.

[0197] In various embodiments, the non-human animal, non-human cell, or non-human tissue described herein is a rodent, rodent cell, or rodent tissue; in some embodiments, it is a mouse, mouse cell, or mouse tissue; in some embodiments, it is a rat, rat cell, or rat tissue. In some embodiments, the mouse, mouse cell, or mouse tissue described herein contains a genetic background, said genetic background including the 129 strain, the BALB / c strain, the C57BL / 6 strain, a mixture of 129xC57BL / 6 strains, or combinations thereof. Attached Figure Description

[0198] The accompanying drawings, which constitute the main body of this document, are for illustrative purposes only and are not intended to be limiting.

[0199] Figure 1A and 1B A non-scale illustration shows an exemplary embodiment of a strategy for constructing a targeting vector used in an embodiment of generating rodents according to the present disclosure (described in Example 1.1).

[0200] Figure 2A A non-scale illustration shows an exemplary embodiment of inserting a targeting vector into an engineered Igκ light chain locus of a rodent embryonic stem cell (ES) clone (described in Example 1.1), wherein the ES cell clone is used to generate an embodiment according to this disclosure.

[0201] Figure 2B A non-scale illustration of an exemplary embodiment of the removal of one or more select cassettes mediated by recombinases in an engineered Igκ light chain locus caused by the insertion of a targeting vector (described in Example 1.1) used in an embodiment of producing rodents according to the present disclosure.

[0202] Figure 3 A non-scale illustration shows an exemplary embodiment of a strategy for constructing a targeting vector used in an embodiment of generating rodents according to the present disclosure (described in Example 1.2).

[0203] Figure 4A A non-scale illustration is shown of an engineered Igκ light chain locus of a rodent embryonic stem cell (ES) clone into which a targeting vector is inserted (described in Example 1.2) for use in generating rodents according to the present disclosure.

[0204] Figure 4B A non-scale illustration of an exemplary embodiment of the removal of one or more select cassettes mediated by a recombinase in an engineered Igκ light chain locus caused by the insertion of a targeting vector (described in Example 1.2) used in an embodiment producing rodents according to this disclosure.

[0205] Figure 5 The results derived from representative embodiments according to this disclosure show single-cell-gated spleen cells harvested from wild-type (WT) and 6558HO (LiK, homozygous) mice. The top row shows the expression of CD19 (y-axis) and CD3 (x-axis), and the bottom row shows CD19 expressing immunoglobulin D (IgD, y-axis) and immunoglobulin M (IgM, x-axis). +-Gated spleen cells.

[0206] Figure 6 The results derived from representative embodiments according to this disclosure include representative single-cell-gated bone marrow samples harvested from wild-type (WT) and 6558HO (LiK, homozygous) mice. The top row shows the expression of CD19 (y-axis) and CD3 (x-axis), and the bottom row shows the expression of immunoglobulin M (IgD, y-axis) and B220 (x-axis).

[0207] Figure 7 The results shown are derived from representative embodiments according to this disclosure, including representative CD19 samples harvested from wild-type (WT) and 6558HO (LiK, homozygous) mice. + Gated spleen cells indicate the expression of immunoglobulin light chains containing the mouse Igλ (y-axis) or mouse Igκ (x-axis) constant regions.

[0208] Figure 8 Results derived from representative embodiments according to this disclosure, including representative single-cell-gated spleen cells harvested from various specified humanized mice, illustrate the expression of CD19 (y-axis) and CD3 (x-axis). HOH / LiK / λ - / - Mice – mice homozygous for humanized immunoglobulin heavy chains (see, for example, U.S. Patent Nos. 8,642,835 and 8,697,940), homozygous for the LiK locus, and homozygous for the inactivated endogenous immunoglobulin λ light chain locus; HOH / KoK / LiK / λ - / - Mice – mice that are homozygous for humanized immunoglobulin heavy chain (see, for example, U.S. Patent Nos. 8,642,835 and 8,697,940), hemizygous for one κ locus containing the LiK locus and a second κ locus containing the humanized immunoglobulin κ light chain locus, and homozygous for the inactivated endogenous immunoglobulin λ light chain locus; HOH / KoK mice – control mice that are homozygous for humanized immunoglobulin heavy chain and homozygous for humanized immunoglobulin κ light chain.

[0209] Figure 9 The results obtained from representative embodiments according to this disclosure are shown, including representative CD19 samples harvested from various specified humanized mice. + Gated spleen cells indicate the expression of immunoglobulin light chains containing the mouse Igλ (y-axis) or mouse Igκ (x-axis) constant regions.

[0210] Figure 10Results obtained from representative embodiments according to this disclosure are shown, including representative single-cell-gated bone marrow harvested from various specified humanized mice, illustrating the expression of immunoglobulin M (IgD, y-axis) and B220 (x-axis).

[0211] Figure 11 The results obtained from representative embodiments according to this disclosure include the expression of immunoglobulin light chains containing the mouse Igλ (y-axis) or mouse Igκ (x-axis) constant regions in immature (apical) and mature (basal) B cells from representative single-cell-gated bone marrow harvested from various specified humanized mice.

[0212] Figure 12 The illustration is a non-scale schematic diagram of an exemplary embodiment of the present disclosure, showing an engineered endogenous immunoglobulin κ light chain locus and rearranged locus as described herein to form an mRNA molecule.

[0213] Figure 13 Results obtained from representative embodiments according to this disclosure are shown, including representative SDS-PAGE protein immunoblotting (protein blotting) using serum isolated from wild-type (WT) and 6558 homozygous (LiK HO) mice, as described in Example 3.3.

[0214] Figure 14 The results of tests conducted according to embodiments of this disclosure are shown, illustrating the expression of CD19 (y-axis) and CD3 (x-axis) in representative single-cell-gated spleen cells harvested from humanized mice. HOH / LiK / λ - / - / TdT mice – mice homozygous for humanized immunoglobulin heavy chains (see, for example, U.S. Patent Nos. 8,642,835 and 8,697,940), homozygous for the LiK locus and homozygous for the inactivated endogenous immunoglobulin λ light chain locus, said mice comprising a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT); and HOH / KoK / LiK / λ - / - / TdT mice – mice that are homozygous for the humanized immunoglobulin heavy chain (see, for example, U.S. Patent Nos. 8,642,835 and 8,697,940), hemizygous for a κ locus containing the LiK locus and a second κ locus containing the humanized immunoglobulin κ light chain locus, and homozygous for the inactivated endogenous immunoglobulin λ light chain locus, said mice containing a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT).

[0215] Figure 15 The results of tests conducted according to embodiments of this disclosure are shown, demonstrating representative CD19 samples harvested from various specified humanized mice.+ Gated spleen cells indicate the expression of immunoglobulin light chains containing the constant regions of mouse Igλ (y-axis) and mouse Igκ (x-axis).

[0216] Figure 16 The results of tests conducted according to embodiments of the present disclosure are shown, illustrating the expression of immunoglobulin M (IgM, y-axis) and B220 (x-axis) in representative single-cell-gated bone marrow harvested from various specified humanized mice.

[0217] Figure 17 The results of tests conducted according to embodiments of this disclosure show representative single-cell-gated bone marrow samples harvested from various specified humanized mice, illustrating the expression of mouse Igλ (y-axis) or mouse Igκ (x-axis) immunoglobulin light chains in immature (apical) and mature (basal) B cells.

[0218] Figure 18 The results of the test according to the embodiments of the present disclosure are shown in a graph comparing the immune responses of LiK / VI-3, LiK / VI-3 / TdT and VI-3 / TdT mouse strains after immunization with a protein immunogen.

[0219] Figure 19 The test results of the embodiments of the present invention are shown in the figure, which compares the immune response to the His tag in LiK / VI-3, LiK / VI-3 / TdT and VI-3 / TdT mouse strains after immunization with an unrelated protein antigen fused with the HIS tag.

[0220] Figure 20 A non-scale illustration showing a portion of the endogenous human immunoglobulin λ light chain locus. Figure 20 The diagram includes a first arrow pointing to a representation of a first exemplary endogenous human Vλ non-coding sequence at the endogenous human immunoglobulin λ light chain locus. As shown, the first exemplary endogenous human Vλ non-coding sequence (represented by lines) at the endogenous human immunoglobulin λ light chain locus occurs naturally adjacent to the human Vλ3-12 gene region (represented by dark gray squares) and the human Vλ2-11 gene region (represented by dark gray squares) at the endogenous human immunoglobulin Igλ light chain locus. Figure 20 It also includes a second arrow pointing to a representation of a second exemplary endogenous human Vλ coding sequence at the endogenous human immunoglobulin λ light chain locus. As shown, the second exemplary endogenous human Vλ non-coding sequence (represented by lines) at the endogenous human immunoglobulin λ light chain locus occurs naturally adjacent to the human Vλ2-11 gene region (represented by dark gray squares) and the human Vλ3-10 gene region (represented by dark gray squares) at the endogenous human immunoglobulin λ light chain locus.

[0221] Figure 21 An off-scale illustration of a portion of the endogenous human immunoglobulin κ light chain locus. Figure 21 Includes a first arrow pointing to a representation of a first exemplary endogenous human Jκ non-coding sequence at the endogenous human immunoglobulin κ light chain locus. As shown, the first exemplary endogenous human Jκ non-coding sequence (represented by lines) at the endogenous human immunoglobulin κ light chain locus occurs naturally adjacent to the human Jκ1 gene segment (represented by dark gray squares) and the human Jκ2 gene segment (represented by dark gray squares) at the endogenous human immunoglobulin κ light chain locus. Figure 21 It also includes a second arrow pointing to a representation of a second exemplary endogenous human Jκ non-coding sequence at the endogenous human immunoglobulin κ light chain locus. As shown, the second exemplary endogenous human Jκ non-coding sequence (represented by lines) at the endogenous human immunoglobulin κ light chain locus occurs naturally adjacent to the human Jκ2 gene segment (represented by dark gray squares) and the human Jκ3 gene segment (represented by dark gray squares) at the endogenous human immunoglobulin κ light chain locus.

[0222] Brief description of the sequence selection in the sequence list

[0223] The following are representative nucleic acid and amino acid sequences of various immunoglobulin constant regions from the mouse, rat, or human λ gene. Nucleic acid and amino acid sequences of immunoglobulin genes and peptides are available from the International Immunogenetic Information System website www.imgt.org.

[0224] Mouse Cλ1 DNA (SEQ ID NO:1):

[0225] GCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCTCTGAAGAGCTCGAGACTAACAAGGCCACACTGGTGTGTACGATCACTGATTTCTACCCAGGTGTGGTGACAGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGTATG GAGACAACCCAGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGACCCTGACAGCAAGAGCATGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCATGAAGGTCACACTGTGGAGAAGAGTTTGTCCCGTGCTGACTGTTCC

[0226] Mouse Cλ1 amino acids (SEQ ID NO:2):

[0227] GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVT HEGHTVEKSL SRADCS

[0228] Mouse Cλ2 DNA (SEQ ID NO:3):

[0229] GTCAGCCCAAGTCCACTCCCACTCTCACCGTGTTTCCACCTTCCTCTGAGGAGCTCAAGGAAAACAAAGCCACACTGGTGTGTCTGATTTCCAACTTTTCCCCGAGTGGTGTGACAGTGGCCTGGAAGGCAAATGGTACACCTATCACCCAGGGTGTGGACACTTCAAATCCCACCAAAGAGGGCAACAAGTTCATGGCCAGCAGCTTCCTACATTTGACATCGGACCAGTGGAGATCTCACAACAGTTTTACCTGTCAAGTTACACATGAAGGGGACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTCTC

[0230] Mouse Cλ2 amino acids (SEQ ID NO:4):

[0231] GQPKSTPTLTVFPPSSEELKENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTKEGNKFMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL

[0232] Mouse Cλ3 DNA (SEQ ID NO:5):

[0233] GTCAGCCCAAGTCCACTCCCACACTCACCATGTTTCCACCTTCCCCTGAGGAGCTCCAGGAAAACAAAGCCACACTCGTGTGTCTGATTTCCAATTTTTCCCCAAGTGGTGTGACAGTGGCCTGGAAGGCAAATGGTACACCTATCACCCAGGGTGTGGACACTTCAAATCCCACCAAAGAGGACAACAAGTACATGGCCAGCAGCTTCTTACATTTGACATCGGACCAGTGGAGATCTCACAACAGTTTTACCTGCCAAGTTACACATGAAGGGGACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATG TCTC

[0234] Mouse Cλ3 amino acids (SEQ ID NO:6):

[0235] GQPKSTPTLTMFPPSPEELQENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTKEDNKYMASSFLHLTSDQWRSHNSFTCQVTH EGDTVEKSLSPAECL

[0236] Rat Cλ1 DNA (SEQ ID NO:7):

[0237] GTCAGCCCAAGTCCACTCCCACACTCACAGTATTTCCACCTTCAACTGAGGAGCTCCAGGGAAACAAAGCCACACTGGTGTGTCTGATTTCTGATTTCTACCCGAGTGATGTGGAAGTGGCCTGGAAGGCAAATGGTGCACCTATCTCCCAGGGTGTGGACACTGCAAATCCCACCAAACAGGGCAACAAATACATCGCCAGCAGCTTCTTACGTTTGACAGCAGAACAGTGGAGATCTCGCAACAGTTTTACCTGCCAAGTTACACATGAAGGGAACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTGTC

[0238] Rat Cλ1 amino acids (SEQ ID NO:8):

[0239] GQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKANGAPISQGVDTANPTKQGNKYIASSFLRLTAEQWRSRNSFTCQVTHEGNTVEKSLSPAECV

[0240] Rat Cλ2 DNA (SEQ ID NO:9):

[0241] ACCAACCCAAGGCTACGCCCTCAGTCACCCTGTTCCCACCTTCCTCTGAAGAGCTCAAGACTGACAAGGCTACACTGGTGTGTATGGTGACAGATTTCTACCCTGGTGTTATGACAGTGGTCTGGAAGGCAGATGGTACCCCTATCACTCAGGGTGTGGAGACTACCCAGCCTTTCAAACAGAACAACAAGTACATGGCTACCAGCTACCTGCTTTTGACAGCAAAAGCATGGGAGACTCATAGCAATTACAGCTGCCAGGTCACTCACGAAGAGAACACTGTGGAGAAGAGTTTGTCCCGTGCTGAGTGTTCC

[0242] Rat Cλ2 amino acids (SEQ ID NO:10):

[0243] DQPKATPSVTLFPPSSEELKTDKATLVCMVTDFYPGVMTVVWKADGTPITQGVETTQPFKQNNKYMATSYLLLTAKAWETHSNYSCQVTHEENTVEKSLSRAECS

[0244] Rat Cλ3 DNA (SEQ ID NO:11):

[0245] GTCAGCCCAAGTCCACTCCCACACTCACAGTATTTCCACCTTCAACTGAGGAGCTCCAGGGAAACAAAGCCACACTGGTGTGTCTGATTTCTGATTTCTACCCGAGTGATGTGGAAGTGGCCTGGAAGGCAAATGGTGCACCTATCTCCCAGGGTGTGGACACTGCAAATCCCACCAAACAGGGCAACAAATACATCGCCAGCAGCTTCTTACGTTTGACAGCAGAACAGTGGAGATCTCGCAACAGTTTTACCTGCCAAGTTACACATGAAGGGAACACTGTGGAAAAGAGTCTGTCTCCTGCAGAGTGTGTC

[0246] Rat Cλ3 amino acids (SEQ ID NO:12):

[0247] GQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKANGAPISQGVDTANPTKQGNKYIASSFLRLTAEQWRSRNSFTCQVTHEGNTVEKSLSPAECV

[0248] Rat Cλ4 DNA (SEQ ID NO:13):

[0249] ACCAACCCAAGGCTACGCCCTCAGTCACCCTGTTCCCACCTTCCTCTGAAGAGCTCAAGACTGACAAGGCTACACTGGTGTGTATGGTGACAGATTTCTACCCTGGTGTTATGACAGTGGTCTGGAAGGCAGATGGTACCCCTATCACTCAGGGTGTGGAGACTACCCAGCCTTTCAAACAGAACAACAAGTACATGGCTACCAGCTACCTGCTTTTGACAGCAAAAGCATGGGAGACTCATAGCAATTACAGCTGCCAGGTCACTCACGAAGAGAACACTGTGGAGAAGAGTTTGTCCCGTGCTGAGTGTTCC

[0250] Rat Cλ4 amino acids (SEQ ID NO:14):

[0251] DQPKATPSVTLFPPSSEELKTDKATLVCMVTDFYPGVMTVVWKADGTPITQGVETTQPFKQNNKYMATSYLLLTAKAWETHSNYSCQVTHEENTVEKSLSRAECS

[0252] Human Cλ1 DNA (SEQ ID NO:15):

[0253] CCCAAGGCCAACCCCACGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTCCAAGCCAACAAGGCCACACTAGTGTGTCTGATCAGTGACTTCTACCCGGGAGCTGTGACAGTGGCTTGGAAGGCAGATGGCAGCCCCGTCAAGGCGGGAGTGGAGACGACCAAACCCTCCAAACAGAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATAG

[0254] Human Cλ1 amino acid (SEQ ID NO:16):

[0255] PKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0256] Human Cλ2 DNA (SEQ ID NO:17):

[0257] GTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCTTGGAAAGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0258] Human Cλ2 amino acids (SEQ ID NO:18):

[0259] QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0260] Human Cλ3 DNA (SEQ ID NO:19):

[0261] CCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTTGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGGGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTTGCCCCTACGGAATGTTCATAG

[0262] Human Cλ3 amino acids (SEQ ID NO:20):

[0263] PKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0264] Human Cλ6 DNA (SEQ ID NO:21):

[0265] GGTCAGCCCAAGGCTGCCCCATCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGCCTGATCAGTGACTTCTACCCGGGAGCTGTGAAAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAACACGGGAGTGGAGACCACCACACCCTCCAAACAGAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGTTCATAG

[0266] Human Cλ6 amino acid (SEQ ID NO:22):

[0267] QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPVNTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS

[0268] Human Cλ7 DNA (SEQ ID NO:23):

[0269] GTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCGTAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGTGGGAGTG GAGACCACCAAACCCTCCAAACAAAGCAACAACAAGTATGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCGGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGCTCT

[0270] Human Cλ7 amino acid (SEQ ID NO:24):

[0271] QPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS

[0272] definition

[0273] The scope of this invention is defined by the appended claims and is not limited to certain embodiments described herein. Those skilled in the art will recognize that various modifications are equivalent to such described embodiments or otherwise fall within the scope of the claims. Generally, unless explicitly stated otherwise, the terminology used herein has the same meaning as understood in the art. Explicit definitions of certain terms are provided below; the meaning of these and other terms in particular instances of this specification will be clear to those skilled in the art from the context. Further definitions of the following and other terms are set forth throughout this specification. All patent and non-patent references or relevant portions thereof cited in this specification are incorporated herein by reference in their entirety.

[0274] The use of sequential terms, such as “first,” “second,” “third,” etc., to modify claim elements does not imply any priority, precedence, or order of action of one claim element over another, but is merely a marker to distinguish one claim element with a certain name from another element with the same name (if sequential terms were not used).

[0275] As used in this application, the terms “about” and “approximately” are used as equivalents. Any numbers used in this application, whether or not they are used with “about” or “approximately”, are intended to cover any normal fluctuations as understood by one of ordinary skill in the art.

[0276] Unless explicitly stated to the contrary, the article "a / an" in the specification and claims shall be understood to include plural indicators. Unless otherwise stated or otherwise apparent from the context, a claim or description including "or" among one or more members of the group is considered satisfied if one, more than one, or all members of the group are present, used, or otherwise associated with a given product or method. The invention includes embodiments in which exactly one member of the group appears, is used, or is otherwise associated with a given product or process. The invention also includes embodiments in which more than one or all members of the group are present, used, or otherwise associated with a given product or method. Furthermore, it should be understood that the invention covers all variations, combinations, and permutations, wherein one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims are introduced into all variations, combinations, and permutations in another claim dependent on the same basic claim (or any other related claim), unless otherwise stated or unless contradiction or inconsistency would be obvious to those skilled in the art. Where elements are presented as a list (e.g., Markush group or similar format), it should be understood that various subgroups of said elements are also disclosed, and any element may be removed from said group. It should be understood that, generally, when an aspect of the invention is referred to as containing a particular element, feature, etc., certain embodiments of the invention or an aspect thereof consist of, or are substantially consist of, such elements, features, etc. For simplicity, those embodiments are not described in such detail in any instance herein. It should also be understood that any embodiment or aspect of the invention may be expressly excluded from the claims, whether or not a specific exclusion is set forth in the specification.

[0277] Administration: As used herein, includes administering the composition to a subject or system (e.g., cells, organs, tissues, organisms or related components or groups thereof). Those skilled in the art will understand that the route of administration may vary depending on, for example, the subject or system to which the composition is administered, the nature of the composition, the purpose of administration, etc. For example, in some embodiments, administration to an animal subject (e.g., to a human or rodent) may be via bronchial (including bronchial infusion), buccal, enteric, intradermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intravenous, intracardiac, mucosal, nasal, oral, rectal, subcutaneous, sublingual, local, tracheal (including intratracheal infusion), percutaneous, vaginal, and / or vitreous administration. In some embodiments, administration may include intermittent dosing. In some embodiments, administration may include continuous dosing (e.g., infusion) for at least a selected period of time.

[0278] Improvement: As used herein, includes the prevention, reduction, or mitigation of a condition, or an improvement in the condition of the subject. Improvement includes, but does not require, a complete recovery from or complete prevention of disease, symptom, or illness.

[0279] Approximately: When applied to one or more target values, this includes values ​​similar to the stated reference value. In some implementations, the terms "approximately" or "about" refer to a range of values ​​falling within ±10% (greater than or less than) of the stated reference value, unless otherwise stated or otherwise apparent from the context (except where such a value would exceed 100% of the possible value).

[0280] Biological activity: As used herein, refers to the property of any agent that is active in a biological system, in vitro, or in vivo (e.g., within an organism). For example, an agent that has a biological effect when present in an organism is considered biologically active. In a particular embodiment, where a protein or polypeptide is biologically active, a portion of a protein or polypeptide sharing at least one biological activity of the protein or polypeptide is generally referred to as a “biologically active” portion.

[0281] Comparable, as used herein, refers to two or more agents, entities, situations, condition groups, etc., that may not be identical but are similar enough to allow for comparison between them so that conclusions can be reasonably drawn based on observed differences or similarities. In this context, those skilled in the art will understand that, in any given situation, a degree of equivalence is required for two or more such agents, entities, situations, condition groups, etc., to be considered comparable.

[0282] Conservatism: As used herein, this refers to instances describing conserved amino acid substitutions, including replacing an amino acid residue with another amino acid residue having a side-chain R group that has similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the target functional properties of a protein, such as its ability to bind to ligands. Examples of amino acid groups with side chains having similar chemical properties include: aliphatic side chains such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (Ile, I); aliphatic hydroxyl side chains such as serine (Ser, S) and threonine (Thr, T); amide-containing side chains such as asparagine (Asn, N) and glutamine (Gln, Q); aromatic side chains such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains such as lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains such as aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfur-containing side chains such as cysteine ​​(Cys, C) and methionine (Met, M). Conserved amino acid substituents include, for example, valine / leucine / isoleucine (Val / Leu / Ile, V / L / I), phenylalanine / tyrosine (Phe / Tyr, F / Y), lysine / arginine (Lys / Arg, K / R), alanine / valine (Ala / Val, A / V), glutamic acid / aspartic acid (Glu / Asp, E / D), and asparagine / glutamine (Asn / Gln, N / Q). In some embodiments, the conserved amino acid substitution may be the substitution of any native residue in the protein with alanine, for example, for alanine scanning mutagenesis. In some embodiments, a conserved substitution is performed that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445, which is incorporated herein by reference in its entirety. In some implementations, the substitution is a moderately conservative substitution, wherein the substitution has a non-negative value in the PAM250 log-likelihood matrix.

[0283] Control: As used herein, "control" means in the art as a standard for comparison with the results. Controls are typically used to enhance the integrity of experiments by separating variables, thereby drawing conclusions about those variables. In some embodiments, a control is a reaction or assay performed concurrently with the test reaction or assay to provide a comparison. "Control" also includes "control animal." A "control animal" may have modifications as described herein, modifications other than those described herein, or no modifications (i.e., wild-type animals). In one experiment, the "test" (i.e., the variable being tested) is applied. In a second experiment, the "control" is used without the variable being tested. In some embodiments, a control is a historical control (i.e., a previously performed test or assay, or a previously known quantity or result). In some embodiments, a control is or includes a printed or otherwise preserved record. A control may be a positive control or a negative control.

[0284] Disruption: As used herein, disruption refers to the result of a homologous recombination event with a DNA molecule (e.g., having an endogenous homologous sequence, such as a gene or locus). In some embodiments, disruption can achieve or represent an insertion, deletion, substitution, replacement, missense mutation, or frameshift of a DNA sequence, or any combination thereof. Insertion may include the insertion of a complete gene or gene fragment, such as an exon, which may have a source other than an endogenous sequence (e.g., a heterologous sequence). In some embodiments, disruption can increase the expression and / or activity of a gene or gene product (e.g., a polypeptide encoded by a gene). In some embodiments, disruption can decrease the expression and / or activity of a gene or gene product. In some embodiments, disruption can alter the sequence of a gene or encoded gene product (e.g., an encoded polypeptide). In some embodiments, disruption can truncate or fragment a gene or encoded gene product (e.g., an encoded polypeptide). In some embodiments, disruption can extend a gene or encoded gene product. In some such embodiments, disruption can achieve the assembly of a fusion polypeptide. In some embodiments, disruption can affect the level of a gene or gene product but not its activity. In some embodiments, disruption may affect the activity of a gene or gene product, but not its level. In some embodiments, disruption may have no significant effect on the level of a gene or gene product. In some embodiments, disruption may have no significant effect on the activity of a gene or gene product. In some embodiments, disruption may have no significant effect on the level or activity of a gene or gene product.

[0285] Determine, measure, evaluate, assess, measure, and analyze: These terms are used interchangeably throughout this document to refer to any form of measurement and include determining the presence or absence of an element. These terms include quantitative and / or qualitative determinations. Determination can be relative or absolute. “Determining its presence” can determine the quantity of something present and / or determine its existence.

[0286] Endogenous promoters: As used in this article, these are promoters that are naturally associated with endogenous genes, such as in wild-type organisms.

[0287] Engineered: As used herein, this generally refers to aspects of artificial manipulation. For example, in some embodiments, a polynucleotide may be considered "engineered" when two or more sequences not naturally linked together are artificially manipulated to link together in an engineered polynucleotide. In some embodiments, an engineered polynucleotide may contain a regulatory sequence found in nature that is operatively associated with a first coding sequence but not with a second coding sequence, and is artificially linked to make it operatively associated with the second coding sequence. Alternatively, in some embodiments, first and second nucleic acid sequences, each encoding a polypeptide element or domain that is not naturally linked to each other, may be linked together in a single engineered polynucleotide. In contrast, in some embodiments, a cell or organism may be considered "engineered" if it has been manipulated to alter its genetic information (e.g., a new genetic material not previously present has been introduced, or a previously present genetic material has been altered or removed). As will be understood by those skilled in the art, the progeny of engineered polynucleotides or cells are generally still referred to as "engineered," even if the actual manipulation was performed on the original entity. Furthermore, as those skilled in the art will understand, various methods can be used to achieve the “engineering” described herein. For example, in some embodiments, “engineering” may involve screening or designing (e.g., nucleic acid sequences, polypeptide sequences, cells, tissues, and / or organisms), or otherwise analyzing, suggesting, and / or selecting sequences, altering, etc., by using a computer system programmed to perform analysis or comparison. Alternatively, in some embodiments, “engineering” may involve using in vitro chemical synthesis methods and / or recombinant nucleic acid technologies, such as nucleic acid amplification (e.g., via polymerase chain reaction), hybridization, mutation, transformation, transfection, etc., and / or any of various controlled pairing methods. As those skilled in the art will understand, various established techniques of this kind (e.g., for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipid transfection, etc.)) are well known in the art and are described in the various general and more specific references cited and / or discussed in this specification.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Principles of Gene Manipulation: An Introduction to Genetic Manipulation, 5th ed., Old, RW and SBPrimrose, Blackwell Science, Inc., 1994, which are incorporated herein by reference in their entirety.

[0288] Functionality, as used herein, refers to the form or segment of an entity (e.g., a gene or gene segment) that exhibits specific properties (e.g., forming part of a coding sequence) and / or activities. For example, in the case of immunoglobulins, variable regions are encoded by unique gene segments (i.e., V, D, and / or J) that are assembled (or recombined) to form a functional coding sequence. When present in the genome, gene segments are organized in clusters, although variations do occur. A “functional” gene segment is a gene segment represented in an expressed sequence (i.e., variable region) where the corresponding genomic DNA has been isolated (i.e., copied) and identified by sequence. Some immunoglobulin gene segment sequences have open reading frames and are considered functional, even though they are not present in an expression library, while other immunoglobulin gene segment sequences contain mutations (e.g., point mutations, insertions, deletions, etc.) and / or truncated sequences that result in stop codons, which subsequently prevent such gene segment sequences from exhibiting one or more properties and / or one or more activities associated with one or more non-mutated sequences. These types of sequences are not represented in the expressed sequences and are therefore classified as pseudogenes.

[0289] Gene: As used herein, a gene refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes a coding sequence (i.e., a sequence that encodes a specific product). In some embodiments, a gene includes a non-coding sequence. In some specific embodiments, a gene may include coding (e.g., exons) and non-coding (e.g., introns) sequences. In some embodiments, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences, which, for example, control or influence one or more aspects of gene expression (e.g., cell type-specific expression, inducible expression, etc.). For clarity, it is noted that, as used herein, the term "gene" generally refers to a portion of nucleic acid encoding a polypeptide or a fragment thereof; the term may optionally include regulatory sequences, as will be clear from the context to those skilled in the art. This definition is not intended to exclude the application of the term "gene" to non-protein-coding expression units, but rather to clarify that, in most cases, the term used herein refers to nucleic acid encoding a polypeptide.

[0290] Heterogeneity: As used herein, refers to an agent or entity derived from a different source. For example, when used to refer 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) is artificially engineered; 2) is artificially (e.g., via engineering) introduced into a cell or organism (or its precursor); 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). Heterogeneity also includes substances that are normally present in a particular natural cell or organism but have been altered or modified, for example, through mutation or insertion controlled by a non-naturally associated and, in some embodiments, non-endogenous regulatory element (e.g., a promoter).

[0291] Host cell: As used herein, this refers to a cell into which nucleic acids or proteins have been introduced. Those skilled in the art will understand upon reading this disclosure that such terms refer not only to a specific subject cell but also to the offspring of such cells. Because certain modifications can occur in offspring due to mutations or environmental influences, such offspring may actually differ from the parent cells but are still included within the scope of the term "host cell." In some embodiments, the host cell is or includes prokaryotic or eukaryotic cells. Generally, a host cell is any cell suitable for receiving and / or producing heterologous nucleic acids or proteins, regardless of which kingdom of life the cell belongs to. Exemplary cells include prokaryotes and eukaryotes (single-celled or multi-celled), bacterial cells (e.g., *Escherichia coli*, *Bacillus* spp., *Streptomyces* spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., *Saccharomyces cerevisiae*, *Schizosaccharomyces pombe*, *Pichia pastoris*, *Pichia methanolica*, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, *Ophiopogon japonicus*, etc.), non-human animal cells, human cells, or cell fusions, such as hybridomas or tetramorphic hybridomas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells are eukaryotic cells selected from the following: CHO (e.g., CHO K1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Cetori cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cells contain one or more viral genes, such as retinal cells expressing viral genes (e.g., (Cells). In some embodiments, the host cell is or comprises isolated cells. In some embodiments, the host cell is part of a tissue. In some embodiments, the host cell is part of an organism.

[0292] Identity: As used herein in conjunction with sequence comparison, identity refers to that determined by a variety of different algorithms known in the art for measuring the identity of nucleotide and / or amino acid sequences. In some implementations, identity as described herein is determined using ClustalWv.1.83 (slow) alignment, with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a Gonnet similarity matrix (MACVECTOR). TM 10.0.2, MacVector Inc., 2008).

[0293] Replacement: As used herein, this refers to a positional substitution where the first nucleic acid sequence is located at the position of the second nucleic acid sequence in the chromosome (e.g., where the second nucleic acid sequence was previously (e.g., initially) located in the chromosome, for example, at an endogenous locus of the second nucleic acid sequence). The phrase “replacement” does not require the removal of the second nucleic acid sequence from, for example, a locus or chromosome. In some embodiments, the second nucleic acid sequence and the first nucleic acid sequence are comparable to each other in that, for example, the first and second sequences are homologous to each other, contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.), and / or have similar or identical sequences. In some embodiments, the first and / or second nucleic acid sequences comprise one or more promoters, enhancers, splice donor sites, splice acceptor sites, introns, exons, and untranslated regions (UTRs); in some embodiments, the first and / or second nucleic acid sequences comprise one or more coding sequences. In some embodiments, the first nucleic acid sequence is a homolog or variant (e.g., a mutant) of the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence is a direct homolog or homolog of the second sequence. In some embodiments, the first nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, the first nucleic acid sequence is or includes a human nucleic acid sequence, and the second nucleic acid sequence is or includes a rodent sequence (e.g., a mouse or rat sequence). In some embodiments, the first nucleic acid sequence is or includes a human nucleic acid sequence, and the second nucleic acid sequence is or includes a human sequence. In some embodiments, the first nucleic acid sequence is a variant or mutant of the second sequence (i.e., a sequence containing one or more sequence differences (e.g., substitutions) compared to the second sequence). The nucleic acid sequence thus positioned may include one or more regulatory sequences that are a portion of the source nucleic acid sequence used to obtain the sequence thus positioned (e.g., a promoter, enhancer, 5'- or 3'-untranslated region, etc.). For example, in various embodiments, the first nucleic acid sequence is a heterologous sequence replacing the endogenous sequence, which causes the nucleic acid sequence thus placed (containing the heterologous sequence) to produce a gene product but does not express the endogenous sequence; the first nucleic acid sequence has an endogenous genomic sequence having a nucleic acid sequence encoding a polypeptide having a similar function to the polypeptide encoded by the endogenous sequence (e.g., the endogenous genomic sequence encodes a non-human variable region polypeptide (all or part), and the DNA fragment encodes one or more human variable region polypeptides (all or part)). In various embodiments, a human immunoglobulin gene segment or fragment thereof replaces the endogenous non-human immunoglobulin gene segment or fragment.

[0294] In vitro: As used herein, this refers to events that occur in an artificial environment, such as in test tubes or reaction vessels, in cell cultures, etc., and not in multicellular organisms.

[0295] In vivo: As used herein, this refers to events occurring within a multicellular organism, such as humans and / or non-human animals. In the context of cell-based systems, the term may be used to refer to events occurring within living cells (e.g., as opposed to in vitro systems).

[0296] Separated: As used herein, means a substance and / or entity (1) separated from at least some of the components with which it was originally associated at the time of its formation (whether in nature or in an experimental apparatus), and / or (2) designed, produced, prepared and / or manufactured artificially. A separated substance and / or entity 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 it was originally associated. In some embodiments, the separated agent is separated from other components initially associated with it in amounts of 10%-100%, 15%-100%, 20%-100%, 25%-100%, 30%-100%, 35%-100%, 40%-100%, 45%-100%, 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 96%-100%, 97%-100%, 98%-100%, or 99%-100%. In some embodiments, the separated agent is separated from other components initially associated with it in amounts of 10%-100%, 10%-99%, 10%-98%, 10%-97%, 10%-96%, 10%-95%, 10%-90%, 10%-85%, 10%-80%, 10%-75%, 10%-70%, 10%-65%, 10%-60%, 10%-55%, 10%-50%, 10%-45%, 10%-40%, 10%-35%, 10%-30%, 10%-25%, 10%-20%, or 10%-15%. In some embodiments, the separated agent is separated from other components initially associated with it in amounts of 11%-99%, 12%-98%, 13%-97%, 14%-96%, 15%-95%, 20%-90%, 25%-85%, 30%-80%, 35%-75%, 40%-70%, 45%-65%, 50%-60%, or 55%-60%. In some embodiments, the separated agent is 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 greater than about 99% purity. In some embodiments, the separating agent is 80%-99%, 85%-99%, 90%-99%, 95%-99%, 96%-99%, 97%-99%, or 98%-99% pure. In some embodiments, the separating agent is 80%-99%, 80%-98%, 80%-97%, 80%-96%, 80%-95%, 80%-90%, or 80%-85% pure.In some embodiments, the isolated agent is 85%-98%, 90%-97%, or 95%-96% pure. In some embodiments, the substance is considered "pure" if it is substantially free of other components. In some embodiments, as those skilled in the art will understand, the substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percentage or purity of the substance is calculated without such carriers or excipients. By way of example only, in some embodiments, a biological polymer, such as a naturally occurring polypeptide or polynucleotide, is considered "isolated" when: a) its source or derived source is not related to some or all of the components that accompany it in its natural state; b) it is substantially free of other polypeptides or nucleic acids of the same species that produce it in nature; or c) it is expressed by cells or other expression systems (which are not the species that naturally produce it), or associated with components from said cells or other expression systems. Therefore, for example, in some embodiments, a polypeptide synthesized chemically or in a system different from the natural cellular system from which it is produced is considered an "isolated" polypeptide. Or, additionally, in some embodiments, a polypeptide subjected to one or more purification techniques may be considered an "isolated" polypeptide, which is separated to some extent from other components: a) those naturally associated with it; and / or b) those originally associated with it at the time of its production.

[0297] A locus, as used herein, refers to the location or position of a gene (or important sequence), DNA sequence, or polypeptide-coding sequence on the chromosome of an organism's genome. For example, an "immunoglobulin locus" may refer to an immunoglobulin gene segment (e.g., V, D, J, or C), an immunoglobulin gene segment DNA sequence, an immunoglobulin gene segment-coding sequence, or the location of an immunoglobulin gene segment on the chromosome of an organism's genome, the location of which has been identified. An "immunoglobulin locus" may contain regulatory elements of an immunoglobulin gene segment, including but not limited to enhancers, promoters, 5' and / or 3' regulatory sequences or regions, or combinations thereof. An "immunoglobulin locus" may contain intergenetic DNA, such as DNA between gene segments that are typically located or present in wild-type loci. Those skilled in the art will understand that in some embodiments, chromosomes may contain hundreds or even thousands of genes, and physical co-location of similar genetic loci may be demonstrated when compared between different species. Such genetic loci may be described as having shared homolinearity.

[0298] Naturally occurring: As used herein, the term "biological element" (e.g., nucleic acid sequence) means that the biological element is found in a specific context and / or location and has not been engineered (e.g., genetically engineered) in a cell or organism (e.g., an animal). In other words, a sequence that naturally occurs in a specified context and / or location is not in a specified context and / or location as a result of engineering (e.g., genetic engineering). For example, a sequence that naturally occurs adjacent to the human Jκ1 gene segment at the endogenous human immunoglobulin κ light chain locus is a sequence that can be found adjacent to the human Jκ1 gene segment at the endogenous human immunoglobulin κ light chain locus (without genetic engineering). In some embodiments, the sequence can be obtained, acquired, and / or isolated from the cell or organism in which it naturally occurs. In some embodiments, the cell or organism is not the direct source of the sequence that naturally occurs in the cell or organism. For example, the corresponding sequence in the cell or organism can be identified and then generated or replicated by mechanisms known in the art.

[0299] Non-human animals: As used herein, refers to any non-human vertebrate organism. In some embodiments, non-human animals are cyclostomes, bony fish, cartilaginous fish (e.g., sharks or rays), amphibians, reptiles, mammals, and birds. In some embodiments, non-human animals are mammals. In some embodiments, non-human mammals are primates, goats, sheep, pigs, dogs, cattle, or rodents. In some embodiments, non-human animals are rodents, such as rats or mice.

[0300] Nucleic acid: As used herein, refers to any compound and / or substance incorporated into or potentially incorporated into an oligonucleotide chain. In some embodiments, “nucleic acid” is a compound and / or substance incorporated into or potentially incorporated into an oligonucleotide chain via phosphodiester bonds. As will become clear from the context, in some embodiments, “nucleic acid” refers to a single nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain containing a single nucleic acid residue. In some embodiments, “nucleic acid” is or comprises RNA; in some embodiments, “nucleic acid” is or comprises DNA. In some embodiments, “nucleic acid” is, comprises, or consists of one or more native nucleic acid residues. In some embodiments, “nucleic acid” is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from “nucleic acid” in that it does not utilize a phosphodiester backbone. For example, in some embodiments, “nucleic acid” is, comprises, or consists of one or more “peptide nucleic acids” that are known in the art and have peptide bonds, rather than phosphodiester bonds in the backbone. Alternatively, in some embodiments, the "nucleic acid" has one or more thiophosphate and / or 5'-N-phosphamide bonds, rather than phosphodiester bonds. In some embodiments, the "nucleic acid" is, comprises, or is composed of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) or is composed of said one or more natural nucleosides. In some embodiments, "nucleic acid" is, comprises, or is composed of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 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-deazoadenosine, 7-deazoguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof) or is composed of one or more of these nucleoside analogs. In some embodiments, compared to those in natural nucleic acids, "nucleic acid" comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). In some embodiments, the "nucleic acid" has a nucleotide sequence that encodes a functional gene product, such as RNA or a polypeptide. In some embodiments, the "nucleic acid" comprises one or more introns. In some embodiments, the "nucleic acid" comprises one or more exons.In some implementations, "nucleic acid" is prepared by one or more of the following methods: isolation from natural sources, enzymatic synthesis (in vivo or in vitro) via polymerization based on complementary templates, propagation in recombinant cells or systems, and chemical synthesis. In some implementations, the length of the "nucleic acid" is at least, for example, but not limited to, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues. In some embodiments, the "nucleic acid" is single-stranded; in some embodiments, the "nucleic acid" is double-stranded. In some embodiments, the "nucleic acid" has a nucleotide sequence comprising at least one element that encodes a polypeptide or is a complementary sequence to a sequence encoding a polypeptide. In some embodiments, the "nucleic acid" has enzymatic activity.

[0301] Operablely linked: As used herein, this refers to the juxtaposition of the components described herein in a relationship that allows them to function in a predetermined manner. A control sequence “operably linked” to a coding sequence is linked in such a manner that the expression of the coding sequence is achieved under conditions compatible with said control sequence. “Operably linked” sequences include both expression control sequences adjacent to the target gene and expression control sequences that function trans- or at a distance to control the expression of the target gene (or target sequence). The term “expression control sequence” includes polynucleotide sequences necessary to influence the expression and processing of the coding sequences to which they are linked. “Expression control sequences” include: appropriate transcription initiation sequences, termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals, such as splicing signals and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance polypeptide stability; and sequences that enhance protein secretion when needed. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence, while in eukaryotes, they typically include a promoter and a transcription termination sequence. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0302] Physiological conditions: As used herein, refer to conditions as understood in the art relating to the survival and / or replication of cells or organisms. In some embodiments, the term includes external or internal conditions that occur naturally for an organism or cellular system. In some embodiments, physiological conditions are conditions present in humans or non-human animals, particularly those present at and / or within surgical sites. Physiological conditions typically include, for example, a temperature range of 20°C–40°C, an atmospheric pressure of 1 atmosphere, a pH of 6–8, a glucose concentration of 1–20 mM, an atmospheric level of oxygen concentration, and gravity on Earth. In some embodiments, laboratory conditions are manipulated and / or maintained at physiological conditions. In some embodiments, physiological conditions are encountered in an organism.

[0303] Polypeptide: As used herein, refers to any polymeric chain of amino acids. In some embodiments, the polypeptide has an amino acid sequence that is naturally occurring. In some embodiments, the polypeptide has an amino acid sequence that is not naturally occurring. In some embodiments, the polypeptide has an amino acid sequence containing portions that are separate from each other in nature (i.e., from two or more different organisms, such as human and non-human portions). In some embodiments, the polypeptide has an amino acid sequence that has been engineered to be artificially designed and / or generated. In some embodiments, the polypeptide has an amino acid sequence encoded by a sequence that is not naturally occurring (e.g., an engineered sequence that is artificially designed and / or generated to encode the polypeptide).

[0304] Recombinant: As used herein, refers to polypeptides designed, engineered, prepared, expressed, created, or isolated via recombinant means, such as polypeptides expressed by transfection into host cells using a recombinant expression vector; polypeptides isolated from recombinant, combinatorial human polypeptide libraries (Hoogenboom, HR, 1997, TIB Tech. 15:62-70; Azzazy, H. and W. Highsmith, 2002, Clin. Biochem. 35:425-45; Gavilondo, JV and JW. Larrick, 2002, BioTechniques 29:128-45; Hoogenboom H. and P. Chames, 2000, Immunol. Today 21:371-8, all of which are incorporated herein by reference in their entirety); and antibodies isolated from animals (e.g., mice) that have been genetically engineered to include human immunoglobulin genes (see, for example, Taylor, LD et al., 1992, Nucl. Acids). Res. 20:6287-95; Kellermann, SA. and LL Green, 2002, Curr. Opin. Biotechnol. 13:593-7; Little, M. et al., 2000, Immunol. Today 21:364-70; Osborn, MJ et al., 2013, J. Immunol. 190:1481-90; Lee, EC. et al., 2014, Nat. Biotech. 32(4):356-63; Macdonald, LE et al., 2014, Proc. Natl. Acad. Sci. USA 111(14):5147-52; Murphy, AJ et al., 2 014, Proc. Natl. Acad. Sci. USA 111(14): 5153-8, which is incorporated herein by reference in its entirety, or a polypeptide prepared, expressed, generated, or isolated by any other means involving splicing selected sequence elements together. In some embodiments, one or more such selected sequence elements are found in nature. In some embodiments, one or more such selected sequence elements are designed on a computer. In some embodiments, one or more such selected sequence elements are derived from the mutagenesis (e.g., in vivo or in vitro) of known sequence elements, such as from natural or synthetic (e.g., artificial) sources. For example, in some embodiments, the recombinant polypeptide consists of sequences found in the genome of a target source organism (e.g., human, mouse, etc.).In some embodiments, the recombinant polypeptide has an amino acid sequence generated by mutagenesis (e.g., in vitro or in vivo, for example, in a non-human animal) such that the amino acid sequence of the recombinant polypeptide (when derived from and related to the polypeptide sequence) may not be naturally present in the genome of a non-human animal.

[0305] Reference material: As used herein, refers to a standard or control agent, animal, group, individual, population, sample, sequence, or value compared to the agent, animal, group, individual, population, sample, sequence, or value. In some embodiments, the reference agent, animal, group, individual, population, sample, sequence, or value is tested and / or measured substantially simultaneously with the target agent, animal, group, individual, population, sample, sequence, or value to be tested or determined. In some embodiments, the reference agent, animal, group, individual, population, sample, sequence, or value is an empirical reference, optionally embodied in a tangible medium. In some embodiments, the reference material refers to a control group. "Reference material" also includes "reference animal." A "reference animal" may have modifications as described herein, modifications other than those described herein, or no modifications (i.e., wild-type animal). Generally, as understood by those skilled in the art, the reference agent, animal, group, individual, population, sample, sequence, or value is to be determined or identified under comparable conditions for determining or identifying the target agent, animal (e.g., mammal), group, individual, population, sample, sequence, or value.

[0306] Replacement: As used herein, refers to the process of removing a “replaced” nucleic acid sequence (e.g., a gene) found at a host locus (e.g., in a genome) from said locus and placing a different “replacement” nucleic acid in its place. In some embodiments, the replaced nucleic acid sequence and the replacement nucleic acid sequence are equivalent to each other in that, for example, they are homologous to each other, contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.), and / or have similar or identical sequences. In some embodiments, the replaced nucleic acid sequence comprises one or more of a promoter, enhancer, splice donor site, splice acceptor site, intron, exon, and untranslated region (UTR); in some embodiments, the replacement nucleic acid sequence comprises one or more coding sequences. In some embodiments, the replacement nucleic acid sequence is a homolog or variant (e.g., a mutant) of the replaced nucleic acid sequence. In some embodiments, the replacement nucleic acid sequence is a direct homolog or homolog of the replaced sequence. In some embodiments, the replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, the substitution nucleic acid sequence is or contains a human nucleic acid sequence, and the substituted nucleic acid sequence is or contains a rodent sequence (e.g., a mouse or rat sequence). In some embodiments, the substitution nucleic acid sequence is or contains a human nucleic acid sequence, and the substituted nucleic acid sequence is or contains a human sequence. In some embodiments, the substitution nucleic acid sequence is a variant or mutant of the substituted sequence (i.e., a sequence containing one or more sequence differences (e.g., substitutions) compared to the substituted sequence). The nucleic acid sequence thus placed may contain one or more regulatory sequences, said one or more regulatory sequences being a portion of the source nucleic acid sequence used to obtain the sequence thus placed (e.g., a promoter, enhancer, 5'- or 3'-untranslated region, etc.). For example, in various embodiments, the substitution is to replace the endogenous sequence with a heterologous sequence, the substitution resulting in the production of a gene product from the nucleic acid sequence (containing the heterologous sequence) but without expressing the endogenous sequence; the substitution is an endogenous genomic sequence having a nucleic acid sequence encoding a polypeptide with a similar function to a polypeptide encoded by the endogenous sequence (e.g., the endogenous genomic sequence encodes a non-human variable region polypeptide (whole or part), and the DNA fragment encodes one or more human variable region polypeptides (whole or part)). In various embodiments, an endogenous non-human immunoglobulin gene segment or fragment thereof is substituted with a human immunoglobulin gene segment or fragment thereof.

[0307] Essentially: As used herein, this refers to qualitative conditions that exhibit all or nearly all or some degree of the target characteristics or properties. Those generally skilled in the art of biology will understand that biological and chemical phenomena rarely (if at all) complete, and / or fully proceed or achieve or avoid absolute results. Therefore, the term "essentially" is used to encompass the potential lack of completeness inherent in many biological and chemical phenomena.

[0308] Fundamental similarity: As used herein, this refers to a comparison between amino acid or nucleic acid sequences. As understood by those skilled in the art, two sequences are generally considered “fundamentally similar” if they have similar residues (e.g., amino acids or nucleotides) at corresponding positions. As understood by those skilled in the art, while similar residues may be identical residues (see also fundamental identity below), similar residues may also be non-identical residues with appropriately comparable structural and / or functional characteristics. For example, as is well known to those skilled in the art, certain amino acids are typically classified as “hydrophobic” or “hydrophilic” amino acids, and / or have “polar” or “nonpolar” side chains. Substituting one amino acid with another of the same type is generally considered a “conserved” substitution. Typical amino acid classifications are summarized in the table below.

[0309]

[0310]

[0311]

[0312] As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, vacancy BLAST, and PSI-BLAST for amino acid sequences. Exemplary procedures of this kind are described in Altschul, SF et al., 1990, J. Mol. Biol., 215(3):403-10; Altschul, SF et al., 1996, Meth. Enzymol. 266:460-80; Altschul, SF et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, AD and BFFOuellette (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, all of which are incorporated herein by reference in their entirety. In addition to identifying similar sequences, the procedures described above typically provide an indication of the degree of identity. In some embodiments, two sequences are considered substantially similar if corresponding residues of two sequences are similar at least, for example, but not limited to, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more on the relevant segment residues (e.g., identical or containing conserved substitutions). In some embodiments, the relevant segment is a complete sequence (e.g., a gene sequence, a gene segment, a sequence encoding a domain, a polypeptide, or a domain). In some embodiments, the relevant segment comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues. In some embodiments, the relevant segment comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues. In some embodiments, the relevant segment comprises continuous residues along the complete sequence. In some embodiments, the relevant segment comprises discontinuous residues along the complete sequence, for example, discontinuous residues aggregated by the folding conformation of a polypeptide or a portion thereof.

[0313] Fundamental identity: As used herein, this refers to a comparison between amino acid or nucleic acid sequences. As understood by those skilled in the art, two sequences are generally considered "fundamentally identical" if they contain the same residues (e.g., amino acids or nucleotides) at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, vacancy BLAST, and PSI-BLAST for amino acid sequences. Exemplary procedures of this kind are described in Altschul, SF et al., 1990, J. Mol. Biol., 215(3):403-10; Altschul, SF et al., 1996, Meth. Enzymol. 266:460-80; Altschul, SF et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, AD and BFFOuellette (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, all of which are incorporated herein by reference in their entirety. In addition to identifying identical sequences, the procedures described above typically provide an indication of the degree of identity. In some embodiments, two sequences are considered substantially identical if at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of corresponding residues are identical at the relevant segment residues. In some embodiments, the relevant segment residues are complete sequences. In some embodiments, the relevant segment residues are, for example, but not limited to, at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[0314] Targeted construct or targeted vector: As used herein, refers to a polynucleotide molecule containing a target region. The target region contains a sequence identical or substantially identical to a sequence in a target cell, tissue, or animal, and the targeted construct is integrated into the genome of the cell, tissue, or animal via homologous recombination. This document also includes and describes targeted regions targeted using site-specific recombinase recognition sites (e.g., loxP or Frt sites). In some embodiments, the targeted construct as described herein further comprises a specific target nucleic acid sequence or gene, a selection marker, a control sequence and / or regulatory sequence, and other nucleic acid sequences that allow recombination mediated via exogenous addition of proteins that facilitate or promote recombination involving such sequences. In some embodiments, the targeted construct as described herein further comprises all or part of a target gene, wherein the target gene is a heterologous gene encoding a polypeptide that has all or part of a function similar to a protein encoded by an endogenous sequence. In some embodiments, the targeted construct as described herein further comprises all or part of a target humanized gene, wherein the humanized target gene encodes all or part of a polypeptide that has a function similar to a polypeptide encoded by an endogenous sequence. In some embodiments, the targeting construct (or targeting vector) may comprise artificially manipulated nucleic acid sequences. For example, in some embodiments, the targeting construct (or targeting vector) may be constructed to contain engineered or recombinant polynucleotides comprising two or more sequences that are not linked together in the natural order but are artificially manipulated to directly link the engineered or recombinant polynucleotides together.

[0315] Transgenic or transgenic construct: As used herein, refers to a nucleic acid sequence (encoding, for example, a target polypeptide (whole or part)) introduced into a cell through human manipulation, such as by the methods described herein. A transgenic may be partially or completely heterologous, i.e., foreign, relative to the genetically engineered animal or cell into which it is introduced. A transgenic may contain one or more transcriptional regulatory sequences and any other nucleic acid, such as introns or promoters, which may be necessary for the expression of the selected nucleic acid sequence.

[0316] Genetically modified nonhuman animals or genetically engineered nonhuman animals: These terms are used interchangeably herein and refer to any nonhuman animal that is not naturally occurring, wherein one or more cells of the nonhuman animal contain a heterologous nucleic acid and / or a gene encoding a target polypeptide (in whole or in part). For example, in some embodiments, “genetically modified nonhuman animal” or “genetically engineered nonhuman animal” refers to a nonhuman animal containing a transgene or transgenic construct, as described herein. In some embodiments, the heterologous nucleic acid and / or gene are introduced into cells directly or indirectly through intentional genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term “genetic manipulation” does not include typical breeding techniques but refers to the introduction of recombinant DNA molecules. The molecule may be integrated into a chromosome or may be extrachromosomal replicated DNA. The phrase “genetically modified nonhuman animal” or “genetically engineered nonhuman animal” refers to an animal that is heterozygous or homozygous for the heterologous nucleic acid and / or gene, and / or an animal having a single copy or multiple copies of the heterologous nucleic acid and / or gene.

[0317] Vector: As used herein, a vector is a nucleic acid molecule capable of transporting another nucleic acid to its associated nucleic acid. In some embodiments, vectors are capable of extrachromosomal replication and / or expression of nucleic acids they are linked in host cells (such as eukaryotic and / or prokaryotic cells). Vectors capable of guiding the operatively linked expression of genes are referred to herein as “expression vectors”.

[0318] Wild-type: As used herein, refers to an entity that has the structure and / or activity found in nature in a “normal” state or context (as opposed to mutants, diseased, altered, etc.). Those skilled in the art will understand that wild-type genes and peptides often exist in many different forms (e.g., alleles). Detailed Implementation

[0319] This disclosure provides, in particular, engineered nonhuman animals having heterologous genetic material encoding a human Vλ domain, the heterologous genetic material comprising human Vλ and Jλ gene sequences (i.e., gene segments), and other sequences (e.g., recombinant signal sequences (RSS)) that are appropriately rearranged and expressed, providing antibodies having Igλ light chains containing human and nonhuman portions or antibodies having fully human Igλ light chains. For example, in various embodiments, when human gene segments are present in the genome of the engineered nonhuman animal, corresponding recombinant signal sequences (e.g., VλRSS with Vλ gene segments, JλRSS with Jλ gene segments, VκRSS with Vκ gene segments, JκRSS with Jκ gene segments, etc.) may also be present. In various embodiments, the provided engineered nonhuman animal contains heterologous genetic material inserted in a manner that causes antibodies containing light chains having human Vλ domains and nonhuman or human Cλ domains to be expressed in a nonhuman animal antibody library. Furthermore, the provided engineered nonhuman animals contain heterologous genetic material inserted in a manner that causes antibodies containing light chains with human Vλ domains and nonhuman or human Cλ domains to be expressed by an engineered Igκ light chain locus, the locus containing human and nonhuman Igλ gene sequences (e.g., gene segments), and in some embodiments, human Igκ light chain sequences are expressed in the germline genome of the nonhuman animal.

[0320] Without being bound by any particular theory, it is anticipated that non-human animals, as described herein, can provide improved in vivo systems that utilize antibodies expressing human Vλ domains for the production of therapeutic antibodies. In some embodiments, it is also anticipated that non-human animals, as described herein, can provide alternative engineered forms of light chain loci (e.g., Igκ light chain loci) containing heterologous genetic material for the development of human antibody-based therapeutics (e.g., human monoclonal antibodies, multispecific binders, scFvs, fusion peptides, etc.) to disease targets associated with biased antibody responses (e.g., antibody responses characterized by the majority of the κ or λ light chain). Therefore, the provided non-human animals are particularly suitable for developing human antibodies and human antibody-based molecules (e.g., multispecific binders, scFvs, fusion peptides, etc.) against targets (e.g., viruses) associated with poor immunogenicity partly due to antibody library and / or response bias.

[0321] This disclosure particularly describes an immunoglobulin κ light chain locus comprising one or more human Vλ gene segments, one or more human Jλ gene segments, and a Cλ gene. This locus is referred to as the "λ in κ" locus or "LiK".

[0322] Specifically, this disclosure describes the generation of non-human animals (e.g., rodents) with germline genomes containing engineered Igκ light chain loci. In some embodiments, the engineered Igκ light chain loci are characterized by the introduction of multiple human Vλ and Jλ gene segments and the introduction of a non-human or human Cλ gene in place of the non-human Cκ gene, such that the multiple human Vλ and Jλ gene segments are operatively linked to the non-human or human Cλ gene. As described herein, the generation of such engineered Igκ light chain loci results in the expression of antibodies containing light chains comprising human Vλ domains and non-human or human Cλ domains from the engineered Igκ light chain locus in the germline genome of the non-human animal. In some embodiments, the provided non-human animal germline genome contains an Igκ light chain locus containing a human Igλ light chain sequence. In some embodiments, the provided non-human animal germline genome includes (i) an Igκ light chain locus containing a human Igλ light chain sequence, and (ii)(a) an Igκ light chain locus containing a human Igλ light chain sequence, or (ii)(b) an Igκ light chain locus containing a human Igκ light chain sequence. In some embodiments, the provided non-human animal germline genome includes an Igκ light chain locus as described herein, and also includes (i) a humanized IgH locus or (ii) a humanized IgH locus, and a functionally silenced or otherwise non-functional endogenous Igλ light chain locus. As described herein, the provided non-human animal expresses an antibody library containing an Igλ light chain comprising a human Vλ domain.

[0323] In some embodiments, the non-human animal described herein contains a human Igλ light chain sequence within the Igκ light chain locus. In some embodiments, the non-human animal described herein contains both human and non-human Igλ light chain sequences within the Igκ light chain locus. In some embodiments, the non-human animal described herein contains both human Igλ and human Igκ light chain sequences within the Igκ light chain locus. In some embodiments, the non-human animal described herein contains human Igλ, human Igκ, and rodent Igκ, and / or rodent Igλ light chain sequences within the Igκ light chain locus. In some embodiments, the non-human animal described herein contains a human Igλ light chain sequence, a non-human Igλ light chain sequence, a human Igκ light chain sequence, a non-human Igκ light chain sequence, or a combination thereof within the Igκ light chain locus. In many embodiments of the non-human animal described herein, the non-human sequence is or includes a rodent sequence (e.g., mouse or rat).

[0324] In some embodiments, the Igκ and / or Igλ light chain sequences comprise intergenic DNA of human or rodent origin. In some embodiments, the Igκ and / or Igλ light chain sequences comprise intergenic DNA that is engineered and based on sequences of human or rodent origin. In some embodiments, the intergenic DNA has the same immunoglobulin locus, wherein the intergenic DNA is placed, inserted, positioned, or engineered (e.g., Igκ intergenic DNA in the Igκ light chain locus). In some embodiments, the intergenic DNA has different immunoglobulin loci, wherein the intergenic DNA is placed, inserted, positioned, or engineered (e.g., Igλ intergenic DNA in the Igκ light chain locus). In some embodiments, the non-human animal as described herein contains an engineered Igκ light chain locus containing intergenic DNA comprising Igκ light chain sequences, Igλ light chain sequences, and / or combinations thereof.

[0325] In various embodiments, the humanized immunoglobulin heavy chain locus contains at least one human V gene operatively linked to a non-human immunoglobulin heavy chain constant region (e.g., an endogenous non-human immunoglobulin heavy chain constant region containing one or more immunoglobulin heavy chain constant region genes, such as IgM, IgD, IgG, IgE, IgA, etc.). H At least one person D H and at least one person J H Gene segments, such as multiple human V genes operatively linked to the constant region of the non-human immunoglobulin heavy chain, H D H and J H Genetic regions. In some embodiments, the provided non-human animal has a germline genome containing one or more immunoglobulin loci depicted in the figures. Such engineered non-human animals provide a source of human antibodies and human antibody fragments, and provide an improved in vivo system suitable for generating human therapeutic antibodies using human Vλ sequences.

[0326] As described in the embodiments section below, a non-human animal is provided, the non-human animal having a genome containing a human heavy chain (i.e., V) replacing the non-human variable region gene segment at the endogenous immunoglobulin gene locus. H D H and J H At least one of the following gene segments: ) and light chain (e.g., Vλ and Jλ at the endogenous κ locus) variable regions, such as multiple human heavy chains (i.e., Vλ and Jλ). H D H and J HHuman immunoglobulin loci contain human non-coding intergenic DNA, including variable region gene segments (e.g., Vλ and Jλ at endogenous κ loci) and light chain segments (e.g., Vλ and Jλ at endogenous κ loci). Such intergenic DNA includes, for example, promoters, leader sequences, and recombination signal sequences that allow appropriate recombination and expression of human gene segments in the context of antibody variable domains. Those skilled in the art will understand that non-human immunoglobulin loci also contain such non-coding intergenic DNA. Upon reading this invention, those skilled in the art will understand that such loci can be constructed using other or non-human intergenic DNA to produce the same expression of human variable domains in the context of antibodies in non-human animals. Such similar loci only require human coding sequences (i.e., exons) containing the desired human gene segments to achieve antibody expression containing human variable domains.

[0327] Various aspects of certain embodiments are described in detail in the following sections, each of which can be applied to any aspect or embodiment as described herein. The purpose of these sections is not to limit.

[0328] Antibody library in non-human animals

[0329] Immunoglobulins (also called antibodies) are large (approximately 150 kDa), Y-shaped glycoproteins produced by B cells of the host's immune system to neutralize pathogens (e.g., viruses, bacteria, etc.). Each immunoglobulin (Ig) consists of two identical heavy chains and two identical light chains, each possessing two structural components: a variable domain and a constant domain. The variable regions of the heavy and light chains differ in antibodies produced by different B cells, but are identical for all antibodies produced by a single B cell or a B cell clone. The variable regions of the heavy and light chains of each antibody collectively contain the antigen-binding region (or antigen-binding site). Immunoglobulins can exist in different variant forms, referred to as isotypes or classes, based on the constant regions (or domains) of their heavy chains. The constant regions of the heavy chains are identical in all antibodies of the same isotype, but differ in antibodies of different isotypes. The table below summarizes nine antibody isotypes in mice and humans.

[0330]

[0331] Other isotypes have been found in other species. Due to the different structural features between different isotypes, they impart specific biological properties to antibodies and are found in different locations within the animal body (cells, tissues, etc.). Initially, B cells produce IgM and IgD with the same antigen-binding region. Upon activation, B cells convert to different isotypes through a process called class switching, which involves changes in the constant region of the antibody produced by the B cell, while the variable region remains the same, thus preserving the antigen specificity of the original antibody (B cell).

[0332] Two separate loci (Igκ and Igλ) contain gene segments that, after rearrangement, encode the light chain of the antibody and exhibit allele and allotype exclusion. + With λ + B cell expression ratios vary across species. For example, humans exhibit a ratio of approximately 60:40 (κ:λ). In mice and rats, a ratio of 95:5 (κ:λ) has been observed. Interestingly, the κ:λ ratio (5:95) observed in cats is the opposite of that in mice and rats. Several studies have been conducted to elucidate the possible reasons behind these observed ratios, and the complexity of the locus (i.e., the number of gene segments, particularly the V gene segments) and the efficiency of gene segment rearrangements have been proposed as fundamental principles. The human Igλ light chain locus extends over 1,000 kb and contains approximately 70 Vλ gene segments (29 to 33 functional) and 7 Jλ-Cλ gene segment pairs (4 to 5 functional) grouped into three clusters (see, for example, Figure 1 of U.S. Patent No. 9,006,511, which is incorporated herein by reference in its entirety). The Vλ regions observed in expressed antibody libraries are mostly encoded by gene segments contained within the nearest-end cluster (referred to as cluster A). The mouse Igλ light chain locus is significantly different from the human locus and, depending on the strain, contains only a few Vλ and Jλ gene segments that form two distinct gene clusters (see, for example, Figure 2 in U.S. Patent No. 9,006,511, which is incorporated herein by reference in its entirety).

[0333] The development of therapeutic antibodies for treating various human diseases has focused primarily on the production of engineered non-human animals, particularly engineered rodent strains whose genomes contain varying amounts of genetic material corresponding to human immunoglobulin genes (reviewed in Brüggemann, M. et al., 2015, Arch. Immunol. Ther. Exp. 63:101-8, which is incorporated herein by reference in its entirety). Initial efforts to generate such genetically engineered rodent strains focused on integrating portions of human immunoglobulin loci that themselves could support recombination of gene segments and the generation of fully human heavy and / or light chains, while simultaneously inactivating endogenous immunoglobulin loci (see, for example, Brüggemann, M. et al., 1989, Proc. Nat. Acad. Sci. USA 86: 67-09-13; Brüggemann, M. et al., 1991, Eur. J. Immunol. 21: 1323-6; Taylor, LD et al., 1992, Nucl. Acids). Res. 20:6287-6295; Davies, NP et al., 1993, Biotechnol. 11:911-4; Green, LL et al., 1994, Nat. Genet. 7:13-21; Lonberg, N. et al., 1994, Nature 368:856-9; Taylor, LD et al., 1994, Int. Immunol. 6:579-91; Wagner, SD et al., 1994, Eur. J. Immunol. 24:2672-81; Fishwild DM et al., 1996, Nat. Biotechnol. 14: 845-51; Wagner, SD et al., 1996, Genomics 35: 405-14; Mendez, MJ et al., 1997, Nat. Genet. 15: 146-56; Green, LL 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, SA and LL Green, 2002, Cur. Opin. Biotechnol. 13: 593-7 (the entire references are incorporated herein by reference).In particular, some efforts have involved the integration of human Igλ light chain sequences (see, for example, U.S. Patent Application Publications 2002 / 0088016A1, 2003 / 0217373A1 and 2011 / 0236378A1; U.S. Patents 6,998,514 and 7,435,871; Nicholson, IC et al., 1999, J. Immunol. 163:6898-906; Popov, AV et al., 1999, J. Exp. Med. 189(10):1611-19, all of which are incorporated herein by reference in their entirety). Such efforts have focused on the random integration of yeast artificial chromosomes containing human Vλ, Jλ, and Cλ sequences to produce mouse strains expressing the complete human Igλ light chain (i.e., human Vλ and Cλ domains). Recent efforts have employed a similar strategy, using constructs that also contain human Vλ, Jλ, and Cλ sequences (Osborn, MJ et al., 2013, J. Immunol. 190: 1481-90; Lee, EC et al., 2014, Nat. Biotech. 32(4): 356-63, each of which is incorporated herein by reference in its entirety).

[0334] However, other efforts include the specific insertion of human Vλ and Jλ gene segments into endogenous rodent Ig light chain loci (κ and λ) such that the human Vλ and Jλ gene segments are operatively linked to endogenous Ig light chain constant regions (see, for example, U.S. Patent Nos. 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662, and 9,163,092, all of which are incorporated herein by reference in their entirety). In such animals, human Vλ gene segments from clusters A and B, along with one or four human Jλ gene segments, are inserted into endogenous Igκ and Igλ light chain loci. As a result, several different human Vλ and Jλ gene segments exhibited appropriate rearrangements at two engineered rodent Ig light chain loci to form functional light chains expressed in rodent antibody libraries, which include human Vλ domains in the context of endogenous Cκ and Cλ regions (see, for example, Table 7 of U.S. Patent No. 9,006,511). Figure 11-13(The patents mentioned herein are incorporated herein by reference in their entirety.) In particular, mice with engineered Igκ light chain loci containing human Vλ and Jλ gene segments exhibit a human λ to endogenous λ ratio (by IgCκ to IgCλ ratio) of approximately 1:1 in the spleen compartment (see, for example, Table 4 of U.S. Patent No. 9,006,511, which is incorporated herein by reference in its entirety). Indeed, both engineered mouse strains (i.e., engineered Igκ or engineered Igλ light chain loci) have demonstrated that the human Vλ domain can be expressed by endogenous Ig light chain loci in rodents, which typically exhibits a large bias in light chain expression (see above). This disclosure provides the understanding that an alternative engineered Ig light chain locus structure can be generated to maximize the use of human Vλ and Jλ gene segments in an antibody library for therapeutic targets in non-human animals, particularly where the light chain locus lacks the complexity and robustness typically associated with the human Igλ light chain locus (i.e., such loci found in human cells) compared to non-human animals containing the Igλ light chain locus. Such alternative engineered Ig light chain locus structures offer the ability to generate unique antibody libraries through their design.

[0335] This disclosure exemplifies the successful generation of non-human animals whose germline genome contains an engineered endogenous Igκ light chain locus comprising multiple human Vλ and Jλ gene segments operatively linked to a non-human or human Igλ light chain constant region gene, wherein the non-human or human Igλ light chain constant region gene is a non-human Igκ light chain constant region gene location inserted into the endogenous Igκ light chain locus. Specifically, this invention demonstrates the successful generation of (1) engineered non-human animals expressing antibodies having human variable regions and non-human constant regions, wherein the antibodies comprise light chains containing human Vλ and non-human Cλ domains, and (2) engineered non-human animals expressing antibodies having human variable regions and human constant regions, wherein the antibodies comprise light chains containing human Vλ and Cλ domains. As specifically illustrated herein, the expression of such light chains is achieved by inserting the aforementioned human Vλ and Jλ gene segments into endogenous Igκ light chain loci (or alleles). In some embodiments, the provided non-human animals are engineered to inactivate the expression of the variable region of the endogenous Igλ light chain (e.g., via gene deletion).

[0336] In some embodiments, the provided non-human animal is engineered to inactivate the expression of the endogenous Igκ light chain variable region (e.g., by substitution or replacement). In some embodiments, the provided non-human animal is engineered to express the human Igλ light chain variable region from an engineered endogenous Igκ light chain locus, as well as the Igκ light chain variable region from an engineered endogenous human Igκ light chain locus. Therefore, in at least some embodiments, the present invention includes developing an improved in vivo system for generating human antibodies by providing an engineered non-human animal containing another engineered Igκ light chain locus, which can express an antibody library comprising a human Vλ domain and a non-human or human Cλ domain.

[0337] Nucleic acid constructs

[0338] Typically, a polynucleotide molecule or a portion thereof containing a human Igλ light chain sequence (e.g., human Vλ and Jλ gene segments) is linked (e.g., inserted) to a vector (preferably a DNA vector) to replicate the polynucleotide molecule in a host cell.

[0339] Human Igλ light chain sequences can be cloned directly from known sequences or sources (e.g., libraries) or synthesized from computer-designed germline sequences based on publicly available sequences obtainable from GenBank or other publicly available databases (e.g., IMGT). Furthermore, bacterial artificial chromosome (BAC) libraries can provide target immunoglobulin DNA sequences (e.g., human Vλ and Jλ sequences, and combinations thereof). BAC libraries can contain inserts of 100–150 kb in size and are capable of having inserts up to 300 kb (Shizuya, et al., 1992, Proc. Natl. Acad. Sci., USA 89:8794–8797; Switek, et al., 1993, Genes and Development 7:2071–2084; Kim, et al., 1996, Genomics 34 213–218; all of which are incorporated herein by reference in their entirety). For example, human BAC libraries with average insert sizes of 164–196 kb have been described (Osoegawa, K. et al., 2001, Genome Res. 11(3):483–96; Osoegawa, K. et al., 1998, Genomics 52:1–8, article number GE985423, which are incorporated herein by reference in their entirety). Human and mouse genomic BAC libraries have been constructed and are commercially available (e.g., ThermoFisher). Genomic BAC libraries can also serve as sources of immunoglobulin DNA sequences and transcriptional control regions.

[0340] Alternatively, immunoglobulin DNA sequences can be isolated, cloned, and / or transferred from yeast artificial chromosomes (YACs). For example, the nucleotide sequence of the human Igλ light chain locus has been determined (see, for example, Dunham, I. et al., 1999, Nature 402:489-95, which is incorporated herein by reference in its entirety). Furthermore, YACs have previously been used to assemble transgenes of the human Igλ light chain locus (see, for example, Popov, AV et al., 1996, Gene 177:195-201; Popov, AV et al., 1999, J. Exp. Med. 189(10):1611-19, which is incorporated herein by reference in its entirety). The complete Igλ light chain locus (human or rodent) can be cloned and contained in several YACs. If multiple YACs are used and contain regions of similar overlap, they can be recombined within a yeast host strain to produce a single construct representing the entire locus or a necessary portion of the locus (e.g., the region targeted by a targeting vector). YAC arms can be engineered to be further modified with mammalian selection boxes to facilitate the introduction of the construct into embryonic stem cells or embryos using methods known in the art and / or described herein.

[0341] The DNA and amino acid sequences used to construct the human Igλ light chain gene segment of the engineered Igκ light chain locus as described herein can be obtained from publicly available databases (e.g., GenBank, IMGT, etc.) and / or publicly available antibody sequences. In some embodiments, the nucleic acid construct containing the human Igλ light chain gene segment includes a J region (i.e., a genomic sequence containing multiple light chain J gene segments), wherein the J region contains the coding sequence of the human Jλ gene segment and its corresponding 12RSS, wherein the 12RSS has been located in non-coding intergenic DNA and is typically associated with the coding sequence of the human Jκ gene segment and its corresponding 23RSS.

[0342] In some embodiments, this sequence may be referred to as the engineered light chain J region. In some embodiments, the nucleic acid construct containing the human Igλ light chain gene segment comprises human Vλ and Jλ sequences operatively linked to a human or non-human Igλ light chain constant region (Cλ) gene. In some embodiments, the nucleic acid construct containing the human Igλ light chain gene segment comprises human Vλ and Jλ sequences operatively linked to one or more non-human Igκ light chain enhancer regions (or enhancer sequences). In some embodiments, the nucleic acid construct containing the human Igλ light chain gene segment comprises human Vλ and Jλ sequences operatively linked to a non-human or human Cλ region gene and a non-human Igκ light chain enhancer region (or enhancer sequence).

[0343] In some embodiments, the nucleic acid construct containing human Vλ and Jλ sequences further comprises intergenic DNA of human and / or mouse origin. In some embodiments, the intergenic DNA is or comprises a non-coding mouse Igκ light chain sequence, a non-coding human Igκ light chain sequence, a non-coding mouse Igλ light chain sequence, a non-coding human Igλ light chain sequence, or a combination thereof.

[0344] Nucleic acid constructs can be prepared using methods known in the art. For example, nucleic acid constructs can be prepared as part of a larger plasmid. This preparation allows for the cloning and selection of the correct constructs in a manner known in the art. As described herein, nucleic acid constructs (in whole or in part) containing the human Igλ light chain sequence can be located between restriction sites on the plasmid so that they can be isolated from the remaining plasmid sequence for incorporation into the desired non-human animal.

[0345] Various methods for preparing nucleic acid constructs (e.g., plasmids) and transforming host organisms are known in the art. For other suitable expression systems for prokaryotic and eukaryotic cells, as well as general recombination procedures, see Principles of Gene Manipulation: An Introduction to Genetic Manipulation, 5th ed., Old, RW and SBPrimrose, Blackwell Science, Inc., 1994 and Molecular Cloning: A Laboratory Manual, 2nd ed., Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989, each of which is incorporated herein by reference in its entirety.

[0346] Targeted vector

[0347] Targeting vectors can be used to introduce nucleic acid constructs into genomic target loci and contain the nucleic acid constructs, as well as homologous arms located flanking the nucleic acid constructs; those skilled in the art will recognize the various choices and features generally applicable to the design, structure, and / or use of targeting vectors. For example, targeting vectors may be linear or circular, and they may be single-stranded or double-stranded. Targeting vectors may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). For ease of reference, homologous arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homologous arms. This terminology refers to the relative position of the homologous arm to the nucleic acid construct within the targeting vector. The 5' and 3' homologous arms correspond to regions within the target locus or regions within another targeting vector, and are referred to herein as the "5' target sequence" and the "3' target sequence," respectively. In some embodiments, the homologous arms may also serve as either the 5' or 3' target sequence.

[0348] In some embodiments, the methods described herein use two, three, or more targeting vectors capable of recombination with each other. In various embodiments, the targeting vector is a large targeting vector (LTVEC) as described elsewhere herein. In such embodiments, the first, second, and third targeting vectors each contain 5' and 3' homologous arms. The 3' homologous arm of the first targeting vector contains a sequence that overlaps with the 5' homologous arm of the second targeting vector (i.e., an overlapping sequence), which allows homologous recombination between the first and second LTVECs.

[0349] In the dual-targeting approach, the 5' homologous arm of the first targeting vector and the 3' homologous arm of the second targeting vector can be similar to the corresponding fragment (i.e., the target sequence) within the target genome locus, which can promote homologous recombination of the first and second targeting vectors with the corresponding genomic fragments and modify the target genome locus.

[0350] In the triple-targeting approach, the 3' homologous arm of the second targeting vector may contain a sequence that overlaps with the 5' homologous arm of the third targeting vector (i.e., an overlapping sequence), which allows homologous recombination between the second and third LTVECs. The 5' homologous arm of the first targeting vector and the 3' homologous arm of the third targeting vector are similar to the corresponding fragments (i.e., target sequences) within the target genome loci, which can promote homologous recombination between the first and third targeting vectors and the corresponding genomic fragments, and modify the target genome loci.

[0351] Homologous arms and target sequences, or two homologous arms, "correspond" or "corresponding" to each other when the two regions share a sufficient level of sequence identity to act as substrates for homologous recombination reactions. The sequence identity between a given target sequence found on the target vector (i.e., the overlapping sequence) and the corresponding homologous arm can be any level of sequence identity that allows homologous recombination to occur. For illustrative purposes only, the sequence identity shared by a homologous arm of a targeting vector (or a fragment thereof) and the target sequence of another targeting vector, or the target sequence of a target genomic locus (or a fragment thereof), may be, for example, but not limited to, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, such that the sequence can be homologously recombinated.

[0352] Furthermore, the corresponding similarity (e.g., identity) region between the homologous arm and the corresponding target sequence can have any length sufficient to promote homologous recombination at the target genome locus. For example, a given homologous arm and / or corresponding target sequence may contain a corresponding similarity region of length, for example, but not limited to, about 5-10kb, 5-15kb, 5-20kb, 5-25kb, 5-30kb, 5-35kb, 5-40kb, 5-45kb, 5-50kb, 5-55kb, 5-60kb, 5-65kb, 5-70kb, 5-75kb, 5-80kb, 5-85kb, 5-90kb, 5-95kb, 5-100kb, 100-200kb, or 200-300kb (as described elsewhere herein), such that the homologous arm has sufficient similarity to homologous recombination with the corresponding target sequence within a target genomic locus of the cell or within another targeting vector. In some implementations, a given homologous arm and / or corresponding target sequence contains a corresponding similarity region of length, such as, but not limited to, about 10-100 kb, 15-100 kb, 20-100 kb, 25-100 kb, 30-100 kb, 35-100 kb, 40-100 kb, 45-100 kb, 50-100 kb, 55-100 kb, 60-100 kb, 65-100 kb, 70-100 kb, 75-100 kb, 80-100 kb, 85-100 kb, 90-100 kb, or 95-100 kb (as described elsewhere herein), to ensure that the homologous arm has sufficient similarity to undergo homologous recombination with the corresponding target sequence within a target genomic locus of the cell or within another targeting vector.

[0353] The overlapping sequence of the 3' homologous arm of the first targeting vector and the 5' homologous arm of the second targeting vector, or the overlapping sequence of the 3' homologous arm of the second targeting vector and the 5' homologous arm of the third targeting vector, may have any length sufficient to promote homologous recombination between the targeting vectors. For example, a given overlapping sequence of a homologous arm may contain a corresponding overlapping region of approximately 1-5 kb, 5-10 kb, 5-15 kb, 5-20 kb, 5-25 kb, 5-30 kb, 5-35 kb, 5-40 kb, 5-45 kb, 5-50 kb, 5-55 kb, 5-60 kb, 5-65 kb, 5-70 kb, 5-75 kb, 5-80 kb, 5-85 kb, 5-90 kb, 5-95 kb, 5-100 kb, 100-200 kb, or 200-300 kb, so that the homologous arm has sufficient similarity to homologously recombine with the corresponding target sequence within the cell's target genome locus or within another targeting vector. In some implementations, the given overlapping sequence of the homologous arm includes an overlapping region of approximately 1-100 kb, 5-100 kb, 10-100 kb, 15-100 kb, 20-100 kb, 25-100 kb, 30-100 kb, 35-100 kb, 40-100 kb, 45-100 kb, 50-100 kb, 55-100 kb, 60-100 kb, 65-100 kb, 70-100 kb, 75-100 kb, 80-100 kb, 85-100 kb, 90-100 kb, or 95-100 kb, such that the overlapping sequence of the homologous arm has sufficient similarity to homologous recombination with a corresponding target sequence within a target genome locus of the cell or within another targeting vector. In some embodiments, the overlapping sequence is 1-5 kb (including end values). In some embodiments, the overlapping sequence is about 1 kb to about 70 kb (including end values). In some embodiments, the overlapping sequence is about 10 kb to about 70 kb (including end values). In some embodiments, the overlapping sequence is about 10 kb to about 50 kb (including end values). In some embodiments, the overlapping sequence is at least 10 kb. In some embodiments, the overlapping sequence is at least 20 kb.For example, overlapping sequences can be approximately 1kb to approximately 5kb (including end values), approximately 5kb to approximately 10kb (including end values), approximately 10kb to approximately 15kb (including end values), approximately 15kb to approximately 20kb (including end values), approximately 20kb to approximately 25kb (including end values), approximately 25kb to approximately 30kb (including end values), approximately 30kb to approximately 35kb (including end values), approximately 35kb to approximately 40kb (including end values), approximately 40kb to approximately 45kb (including end values), approximately 45kb to approximately 50kb (including end values), approximately 50kb to approximately 60kb (including end values), approximately 60kb to approximately 70kb (including end values), and approximately 70kb to approximately 80kb (including end values). Approximately 80kb to approximately 90kb (including end values), approximately 90kb to approximately 100kb (including end values), approximately 100kb to approximately 120kb (including end values), approximately 120kb to approximately 140kb (including end values), approximately 140kb to approximately 160kb (including end values), approximately 160kb to approximately 180kb (including end values), approximately 180kb to approximately 200kb (including end values), approximately 200kb to approximately 220kb (including end values), approximately 220kb to approximately 240kb (including end values), approximately 240kb to approximately 260kb (including end values), approximately 260kb to approximately 280kb (including end values), or approximately 280kb to approximately 300kb (including end values). For illustrative purposes only, overlapping sequences may be approximately 20kb to approximately 60kb (including end values). In addition, the overlapping sequence may be at least 1kb, at least 5kb, at least 10kb, at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 35kb, at least 40kb, at least 45kb, at least 50kb, at least 60kb, at least 70kb, at least 80kb, at least 90kb, at least 100kb, at least 120kb, at least 140kb, at least 160kb, at least 180kb, at least 200kb, at least 220kb, at least 240kb, at least 260kb, at least 280kb, or at least 300kb. In some implementations, the overlapping sequence may be up to 400 kb, up to 350 kb, up to 300 kb, up to 280 kb, up to 260 kb, up to 240 kb, up to 220 kb, up to 200 kb, up to 180 kb, up to 160 kb, up to 140 kb, up to 120 kb, up to 100 kb, up to 90 kb, up to 80 kb, up to 70 kb, up to 60 kb, or up to 50 kb.

[0354] In some embodiments, homologous arms correspond to loci natural to the cell (e.g., target loci), or alternatively, they correspond to heterologous or exogenous DNA fragments integrated into the cell's genome, including, for example, transgenes, expression cassettes, or heterologous or exogenous regions of DNA. In some embodiments, homologous arms may correspond to a region on a target vector in the cell. In some embodiments, homologous arms of the target vector may correspond to yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), artificial chromosomes, or any other engineered region contained in a suitable host cell. Furthermore, homologous arms of the target vector may correspond to or be derived from a BAC library, granule library, or P1 phage library. In some embodiments, homologous arms of the target vector correspond to original, heterologous, or exogenous prokaryotes, yeast, birds (e.g., chickens), non-human mammals, rodents, humans, rats, mice, hamsters, rabbits, pigs, cattle, deer, sheep, goats, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys), domesticated mammals, agricultural mammals, or any other target organism. In some embodiments, the homologous arm corresponds to a cellular locus that exhibits limited sensitivity to targeting using conventional methods or shows relatively low levels of successful integration at the target site, and / or significant levels of off-target integration, in the absence of nuclease-induced nicks or double-strand breaks (e.g., Cas proteins). In some embodiments, the homologous arm is designed to contain engineered DNA.

[0355] In some embodiments, the 5' and 3' homologous arms of the targeting vector correspond to the target genome. Alternatively, the homologous arms correspond to a related genome. For example, the target genome is the genome of a first strain of mice, and the targeting arms correspond to the genome of a second strain of mice, wherein the first and second strains are different. In some embodiments, the homologous arms correspond to the genome of the same animal, or a genome from the same strain; for example, the target genome is the genome of a first strain of mice, and the targeting arms correspond to the genome of a mouse from the same mouse or the same strain.

[0356] The homologous arm of the targeting vector may have any length sufficient to promote homologous recombination events with the corresponding target sequence, including, for example, lengths of 1-5 kb (including the end value), 5-10 kb (including the end value), 5-15 kb (including the end value), 5-20 kb (including the end value), 5-25 kb (including the end value), 5-30 kb (including the end value), 5-35 kb (including the end value), 5-40 kb (including the end value), 5-45 kb (including the end value), and 5-50 kb (including the end value). (Value), 5-55kb (inclusive of end value), 5-60kb (inclusive of end value), 5-65kb (inclusive of end value), 5-70kb (inclusive of end value), 5-75kb (inclusive of end value), 5-80kb (inclusive of end value), 5-85kb (inclusive of end value), 5-90kb (inclusive of end value), 5-95kb (inclusive of end value), 5-100kb (inclusive of end value), 100-200kb (inclusive of end value), or 200-300kb (inclusive of end value). In some embodiments, the homologous arm of the targeting vector has a length sufficient to facilitate homologous recombination with the corresponding target sequence, said length being 1-100 kb (including the end value), 5-100 kb (including the end value), 10-100 kb (including the end value), 15-100 kb (including the end value), 20-100 kb (including the end value), 25-100 kb (including the end value), 30-100 kb (including the end value), 35-100 kb (including the end value), 40-100 kb (including the end value). b (including end value), 45-100kb (including end value), 50-100kb (including end value), 55-100kb (including end value), 60-100kb (including end value), 65-100kb (including end value), 70-100kb (including end value), 75-100kb (including end value), 80-100kb (including end value), 85-100kb (including end value), 90-100kb (including end value), or 95-100kb (including end value). As described in this article, large target carriers can employ target arms with greater lengths.

[0357] Nucleases (e.g., CRISPR / Cas systems) can be used in combination with targeting vectors to facilitate the modification of target loci (e.g., modification of Igκ light chain loci, or modification of previously modified or engineered Igκ light chain loci). Such nucleases can promote homologous recombination between the targeting vector and the target locus. When a nuclease is used in combination with a targeting vector, the targeting vector may contain 5' and 3' homologous arms corresponding to 5' and 3' target sequences located sufficiently close to the nuclease cleavage site to facilitate homologous recombination events between the target sequence and the homologous arms through a nick or double-strand break at the nuclease cleavage site. The term "nuclease cleavage site" includes a DNA sequence at which a nick or double-strand break is created by a nuclease (e.g., the Cas9 cleavage site). The target sequences within the target locus corresponding to the 5' and 3' homologous arms of the target vector are "sufficiently close" to the nuclease cleavage site, and if said distance is at a nick or double-strand break at the recognition site, it promotes homologous recombination events between the 5' and 3' target sequences and the homologous arms. Therefore, in some embodiments, the target sequences corresponding to the 5' and / or 3' homologous arms of the target vector are within at least one nucleotide of a given recognition site, or within at least 10 nucleotides to about 14 kb of a given recognition site. In some embodiments, the nuclease cleavage site is adjacent to at least one or both of the target sequences.

[0358] The spatial relationship of the target sequence corresponding to the homologous arm of the target vector and the nuclease cleavage site can vary. For example, the target sequence can be located at the 5' of the nuclease cleavage site, at the 3' of the recognition site, or flanked by the nuclease cleavage site.

[0359] Compared to using a targeting vector alone, combining a targeting vector (including, for example, a large targeting vector) with a nuclease can increase targeting efficiency. For example, compared to using a targeting vector alone, when a targeting vector is combined with a nuclease, the targeting efficiency of the targeting vector can be increased by at least two, three, four, five, six, seven, eight, nine, or ten times, or within the range of these integers, such as 2-10 times.

[0360] Some targeting vectors are “large targeting vectors” or “LTVECs”, which include a targeting vector containing homologous arms corresponding to and derived from a nucleic acid sequence (larger than those typically used by other methods intended for homologous recombination in cells). The length of an LTVEC can be, for example, at least 10 kb, or the sum of the 5' and 3' homologous arms can be, for example, at least 10 kb. LTVECs also include targeting vectors containing nucleic acid constructs larger than those typically used by other methods for homologous recombination in cells. For example, due to the size limitations of LTVECs, LTVECs can enable large locus modifications that cannot be accommodated by conventional plasmid-based targeting vectors. For example, the targeting locus can be (i.e., the 5' and 3' homologous arms can correspond to) a cellular locus that cannot be targeted using conventional methods in the absence of nuclease-induced nicks or double-strand breaks (e.g., Cas proteins), or can only be mistargeted or targeted with significantly low efficiency.

[0361] In some implementations, the methods described herein use two or three LTVECs capable of recombinating with each other and with target genomic loci in three- or four-way recombination events. Such methods enable large locus modifications that cannot be achieved using a single LTVEC.

[0362] Examples of LTVECs include vectors derived from bacterial artificial chromosomes (BACs), artificial chromosomes, or yeast artificial chromosomes (YACs). LTVECs can be linear or circular. Examples of LTVECs and methods for preparing them are described, for example, in U.S. Patent Nos. 6,586,251, 6,596,541, and 7,105,348; and International Patent Application Publication No. WO 2002 / 036789, each of which is incorporated herein by reference in its entirety.

[0363] Provide non-human animals, cells and tissues

[0364] A nonhuman animal expressing (e.g., its B cell expression) an antibody is provided, the antibody containing a light chain comprising a human Vλ domain resulting from the integration of genetic material corresponding to at least a portion of the human Igλ light chain locus (i.e., at least a portion of the human Vλ and Jλ gene segments), and the light chain encoding the human Vλ domain (i.e., a rearranged human Vλ-Jλ sequence) located at the corresponding nonhuman Igκ light chain variable region sequence in the germline genome of the nonhuman animal. Suitable examples described herein include, but are not limited to, rodents, particularly mice.

[0365] This disclosure provides improved in vivo systems for identifying and developing, for example, novel antibodies, antibody components (e.g., antigen-binding moieties and / or compositions or forms comprising them), and / or antibody-based therapeutics that can be used to treat various diseases affecting humans. Furthermore, this disclosure covers the recognition that non-human animals (e.g., rodents) having engineered immunoglobulin loci, such as engineered immunoglobulin (Ig)κ(κ) ​​light chain loci, and / or otherwise expressing, producing, or containing antibody libraries, are useful, said non-human animals being characterized by light chains having the human Vλ(λ) region. For example, in some embodiments, such non-human animals can be used to utilize the diversity of human Vλ sequences in the identification and development of novel antibody-based therapeutics. In some embodiments, the non-human animals described herein provide improved in vivo systems for developing antibodies and / or antibody-based therapeutics for administration to humans. In some embodiments, the non-human animals described herein provide improved in vivo systems for developing antibodies and / or antibody-based therapeutics containing a human Vλ domain, characterized by improved performance (e.g., expression and / or demonstration in an antigen-specific antibody library) compared to antibodies and / or antibody-based therapeutics obtained from existing in vivo systems containing human Vλ region sequences.

[0366] This disclosure particularly provides non-human animals possessing an Igκ light chain locus containing an engineered immunoglobulin light chain variable region and an engineered immunoglobulin light chain constant region gene. As described herein, the provided non-human animals contain an immunoglobulin κ light chain locus in their germline genome, the immunoglobulin κ light chain locus containing an engineered immunoglobulin κ light chain variable region characterized by the presence of one or more human Vλ gene segments and one or more human Jλ gene segments, the one or more human Vλ gene segments and the one or more human Jλ gene segments being operatively linked to an immunoglobulin λ light chain constant region (Cλ) gene located at the non-human immunoglobulin κ light chain constant region (Cκ) gene at the endogenous immunoglobulin κ locus of the non-human animal. In some embodiments, the provided non-human animal contains an Igκ light chain locus containing intergenic DNA originating from immunoglobulin λ light chains and / or immunoglobulin κ light chains and combinations thereof.

[0367] In many embodiments, the engineered immunoglobulin κ light chain variable region further comprises an immunoglobulin κ light chain sequence located or inserted between one or more human Vλ gene segments and one or more human Jλ gene segments. In some embodiments, the immunoglobulin κ light chain sequence located or inserted between one or more human Vλ gene segments and one or more human Jλ gene segments is or comprises a rodent (e.g., rat or mouse) sequence. In some embodiments, the immunoglobulin κ light chain sequence located or inserted between one or more human Vλ gene segments and one or more human Jλ gene segments is or comprises a human sequence. For example, in some embodiments, the human immunoglobulin κ light chain sequence is or comprises a genomic sequence that naturally occurs between the human Vκ4-1 gene segment and the human Jκ1 gene segment at the human immunoglobulin κ light chain locus.

[0368] In some embodiments, the provided non-human animal contains at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 functional human Vλ gene segments. In some embodiments, the provided non-human animal contains 5 to 25, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, or 5 to 6 functional human Vλ gene segments. In some embodiments, the provided non-human animal contains 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 11 to 25, 12 to 25, 13 to 25, 14 to 25, 15 to 25, 16 to 25, 17 to 25, 18 to 25, 19 to 25, 20 to 25, 21 to 25, 22 to 25, 23 to 25, or 24 to 25 functional human Vλ gene segments. In some embodiments, the provided non-human animal contains 6 to 24, 7 to 23, 8 to 22, 9 to 21, 10 to 20, 11 to 19, 12 to 18, 13 to 17, 14 to 16, or 15 to 16 functional human Vλ gene segments. In some implementations, the provided non-human animals contain 6 to 24, 7 to 23, 8 to 22, 9 to 21, 10 to 20, 11 to 19, 12 to 18, 13 to 17, or 14 to 16 functional human Vλ gene segments.

[0369] In some embodiments, the provided non-human animal contains a total of 10 to 70, 10 to 65, 10 to 60, 10 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, or 10 to 15 human Vλ gene segments. In some embodiments, the provided non-human animal contains a total of 15 to 70, 20 to 70, 25 to 70, 30 to 70, 35 to 70, 40 to 70, 45 to 70, 50 to 70, 55 to 70, 60 to 70, or 65 to 70 human Vλ gene segments. In some embodiments, the provided non-human animal contains a total of 15 to 65, 20 to 60, 25 to 55, 20 to 50, 25 to 45, 30 to 40, 30 to 35, or 35 to 40 human Vλ gene segments.

[0370] In some embodiments, the provided non-human animal contains human Vλ and / or Jλ gene segments in a natural or germline configuration (e.g., a DNA sequence containing multiple human Vλ and / or Jλ gene segment coding sequences interspersed with non-coding human immunoglobulin λ light chain sequences). In some embodiments, the provided non-human animal contains human Vλ and / or Jλ gene segments in a configuration distant from or deviating from the natural or germline configuration (e.g., a DNA sequence containing multiple human Vλ and / or Jκ gene segment coding sequences interspersed with non-coding immunoglobulin κ light chain sequences (e.g., human or mouse)). In some embodiments, the provided non-human animal contains human Vλ and / or Jλ gene segments in a configuration not naturally present in the human immunoglobulin λ light chain locus of the germline genome of human cells.

[0371] In some embodiments, the provided non-human animal contains a DNA sequence at an endogenous non-human Igκ light chain locus, said DNA sequence comprising multiple human Vλ and Jλ coding sequences interspersed (or juxtaposed, associated, etc.) with non-coding human immunoglobulin light chain sequences (e.g., κ, λ, and combinations thereof). In some embodiments, the provided non-human animal contains a DNA sequence at an endogenous non-human Igλ light chain locus, said DNA sequence comprising multiple human Vλ and Jλ coding sequences interspersed with non-coding non-human (e.g., rodent) immunoglobulin λ light chain sequences.

[0372] In some embodiments, the provided nonhuman animal is characterized by expressing an antibody from an endogenous immunoglobulin κ light chain locus in the germline genome of the nonhuman animal, said antibody containing (1) a human Vλ domain and (2) a nonhuman or human Cλ domain. In some embodiments, the provided nonhuman animal is characterized by improved utilization of the human Vλ region from the engineered immunoglobulin κ light chain locus (e.g., but not limited to about 2-fold) compared to one or more reference engineered nonhuman animals.

[0373] In some embodiments, a non-human animal, non-human cell, or non-human tissue is provided, the germline genome of which includes an endogenous immunoglobulin κ light chain locus, the endogenous immunoglobulin κ light chain locus comprising: (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and (c) a Cλ gene, wherein (a) and (b) are operatively linked to (c), and wherein the rodent lacks the rodent Cκ gene at the endogenous immunoglobulin κ light chain locus.

[0374] In some embodiments, a non-human animal, non-human cell, or non-human tissue is provided, the germline genome of which includes an endogenous immunoglobulin κ light chain locus. This endogenous immunoglobulin κ light chain locus includes an insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and a Cλ gene. The human Vλ and Jλ gene segments are operatively linked to the Cλ gene, and the Cλ gene is inserted at a non-human Cκ gene at the endogenous immunoglobulin κ light chain locus. In many embodiments of the non-human animal, non-human cell, or non-human tissue, the Cλ gene inserted at the non-human Cκ gene at the endogenous immunoglobulin κ light chain locus is a non-human or human Cλ gene. In some embodiments, the non-human Cλ gene is or includes mammalian Cλ genes selected from the group consisting of primates, goats, sheep, pigs, dogs, cattle, or rodents.

[0375] In some implementations, the non-human Cλ gene is or includes the rodent Cλ gene.

[0376] In some embodiments, the rodent Cλ gene is or includes the mouse Cλ gene. In some embodiments, the mouse Cλ gene contains at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the sequence identical to the mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3. In some embodiments, the mouse Cλ gene contains a sequence substantially identical or identical to the sequence of the mouse Cλ gene selected from the group consisting of mouse Cλ1, mouse Cλ2, and mouse Cλ3. In some embodiments, the mouse Cλ1 gene is or includes SEQ ID NO:1. In some embodiments, the mouse Cλ2 gene is or includes SEQ ID NO:3. In some embodiments, the mouse Cλ3 gene is or includes SEQ ID NO:5. In some embodiments, the mouse Cλ gene contains the same sequence as the mouse Cλ1 gene.

[0377] In some embodiments, the mouse Cλ gene contains 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% of the mouse Cλ gene selected from the group consisting of: mouse Cλ1, mouse Cλ2, and mouse Cλ3. In some embodiments, the mouse Cλ gene contains 80% to 98%, 80% to 95%, 80% to 90%, or 80% to 85% of the mouse Cλ gene selected from the group consisting of: mouse Cλ1, mouse Cλ2, and mouse Cλ3. In some embodiments, the mouse Cλ gene contains 85% to 98%, 90% to 95%, or 88% to 93% of the mouse Cλ gene selected from the group consisting of: mouse Cλ1, mouse Cλ2, and mouse Cλ3.

[0378] In some embodiments, the rodent Cλ gene is or comprises the rat Cλ gene. In some embodiments, the rat Cλ gene comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the rat Cλ gene selected from the group consisting of: rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes. In some embodiments, the rat Cλ gene comprises a sequence that is substantially identical or identical to the rat Cλ gene selected from the group consisting of rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes. In some embodiments, the rat Cλ1 gene is or comprises SEQ ID NO:7. In some embodiments, the rat Cλ2 gene is or comprises SEQ ID NO:9. In some embodiments, the rat Cλ3 gene is or comprises SEQ ID NO:11. In some embodiments, the rat Cλ4 gene is or comprises SEQ ID NO:13.

[0379] In some embodiments, the rat Cλ gene contains 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% of the rat Cλ gene selected from the following groups: rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes. In some embodiments, the rat Cλ gene contains 80% to 98%, 80% to 95%, 80% to 90%, or 80% to 85% of the rat Cλ gene selected from the following groups: rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes. In some embodiments, the rat Cλ gene contains 85% to 98%, 90% to 95%, or 88% to 93% of the rat Cλ gene selected from the following groups: rat Cλ1, rat Cλ2, rat Cλ3, and rat Cλ4 genes.

[0380] In some embodiments, the human Cλ gene comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the human Cλ genes selected from the following groups: human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes. In some embodiments, the human Cλ gene comprises a sequence that is substantially identical or identical to the human Cλ genes selected from the following groups: human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes. In some embodiments, the human Cλ gene comprises a sequence identical to the human Cλ gene selected from the human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 gene groups. In some embodiments, the human Cλ1 gene is or comprises SEQ ID NO:15. In some embodiments, the human Cλ2 gene is or comprises SEQ ID NO:17. In some embodiments, the human Cλ3 gene is or comprises SEQ ID NO:19. In some embodiments, the human Cλ6 gene is or includes SEQ ID NO:21. In some embodiments, the human Cλ7 gene is or includes SEQ ID NO:23. In some embodiments, the human Cλ gene is or includes the human Cλ2 gene.

[0381] In some embodiments, the human Cλ gene contains 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% of the human Cλ gene selected from the following groups: human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes. In some embodiments, the human Cλ gene contains 80% to 98%, 80% to 95%, 80% to 90%, or 80% to 85% of the human Cλ gene selected from the following groups: human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes. In some embodiments, the human Cλ gene contains 85% to 98%, 90% to 95%, or 88% to 93% of the human Cλ gene selected from the following groups: human Cλ1, human Cλ2, human Cλ3, human Cλ6, and human Cλ7 genes.

[0382] In some embodiments of the provided non-human animal, non-human cell, or non-human tissue, the insertion of one or more human Vλ gene segments and one or more human Jλ gene segments replaces the non-human Vκ and Jκ gene segments at the endogenous immunoglobulin κ light chain locus. In some embodiments, the insertion comprises human non-coding DNA naturally occurring between the human Vλ gene segment and the Jλ gene segment, and combinations thereof. In some embodiments of the provided non-human animal, non-human cell, or non-human tissue, the insertion of one or more human Vλ gene segments and one or more human Jλ gene segments replaces or replaces the non-human Vκ and Jκ gene segments at the endogenous immunoglobulin κ light chain locus. In some embodiments of the provided non-human animal, non-human cell, or non-human tissue, the immunoglobulin κ light chain locus comprises the insertion of at least 24, at least 34, at least 52, at least 61, or at least 70 human Vλ gene segments, and at least 1, at least 2, at least 3, at least 4, or at least 5 human Jλ gene segments. In some embodiments of the provided non-human animals, non-human cells, or non-human tissues, the immunoglobulin κ light chain locus includes insertions of 39 human Vλ gene segments and at least 5 human Jλ gene segments. In some embodiments of the provided non-human animals, non-human cells, or non-human tissues, the immunoglobulin κ light chain locus includes insertions of human Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, and Vλ... 1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1 or any combination thereof, and J Jλ1, Jλ2, Jλ3, Jλ6, Jλ7 or any combination thereof.In some embodiments, the insertion includes human non-coding DNA naturally adjacent to human Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36 at endogenous human λ light chain loci. The presence of Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, or Vλ3-1; and human non-coding DNA (in whole or in part) naturally occurring adjacent to human Jλ1, Jλ2, Jλ3, Jλ6, or Jλ7 at the endogenous human λ light chain locus. In some embodiments, the insertion comprises human non-coding DNA naturally adjacent to human Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1- 36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 appear; and human non-coding DNA, said human non-coding DNA naturally adjacent to human Jκ1, Jκ2, Jκ3, Jκ4 or Jκ5 at the endogenous human κ light chain locus.In some implementation schemes, human Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23 The insertion of Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof comprises human non-coding DNA naturally adjacent to human Vλ4-69, Vλ8-61, Vλ4-60, Vλ3-21, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof at the endogenous human λ light chain locus. λ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-4 0. Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2 -14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 appear, and the insertion of human Jλ1, Jλ2, Jλ3, Jλ6, Jλ7 or any combination thereof comprises human non-coding DNA (whole or part) that is naturally adjacent to human Jλ1, Jλ2, Jλ3, Jλ6, Jλ7 appearing in the endogenous human λ light chain locus.In some implementation schemes, human Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23 The insertion of Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof comprises human non-coding DNA naturally adjacent to human Vλ4-69, Vλ8-61, Vλ4-60, Vλ3-21, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof at the endogenous human λ light chain locus. λ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-4 0. Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2 -14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 or Vλ3-1 appear, and the insertion of human Jλ1, Jλ2, Jλ3, Jλ6, Jλ7 or any combination thereof comprises human non-coding DNA (whole or part) that is naturally adjacent to human Jκ1, Jκ2, Jκ3, Jκ4 or Jκ5 at the endogenous human κ light chain locus.

[0383] In some embodiments of the provided non-human animals, non-human cells, or non-human tissues, the immunoglobulin κ light chain locus, as described herein, further comprises a human immunoglobulin κ light chain sequence located between one or more human Vλ gene segments, one or more human Jλ gene segments, one or more human Vλ gene segments, and one or more human Jλ gene segments, or combinations thereof. In some embodiments, the human immunoglobulin κ light chain sequence, as described herein, is or comprises a genomic sequence naturally occurring between the human Vκ4-1 gene segment and the human Jκ1 gene segment at the immunoglobulin κ light chain locus.

[0384] In some embodiments of the provided non-human animal, non-human cell, or non-human tissue, the germline genome of the non-human animal, non-human cell, or non-human tissue further includes an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus containing insertions of one or more human V... H Gene segment, one or more individuals D HGene segments and one or more individuals J H Gene segment, the human V H D H and J H The gene segment is operatively linked to the non-human immunoglobulin heavy chain constant region at the endogenous immunoglobulin heavy chain locus (see, for example, U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940 and 8,791,323, each of which is incorporated herein by reference in its entirety).

[0385] In some implementations, one or more people V are inserted. H Gene segment, one or more individuals D H Gene segments and one or more individuals J H Gene segment substitution or replacement ( wholly or partially) of non-human V H D H and J H Gene segments (e.g., using human V) H D H J H The coding sequence of the gene segment is specifically replaced or substituted for non-human V. H D H J H (The coding sequence of the gene segment). In some embodiments, the insertion includes human non-coding DNA, which is naturally present in the human V region. H D H and J H Among gene segments and combinations thereof. In some embodiments, the non-human immunoglobulin heavy chain constant region is or includes an endogenous non-human immunoglobulin heavy chain constant region. In many embodiments, the non-human immunoglobulin heavy chain constant region (e.g., endogenous) includes one or more non-human immunoglobulin heavy chain constant region genes or gene segments (e.g., IgM, IgD, IgG, IgE, IgA, etc.). In some embodiments, the immunoglobulin heavy chain locus as described herein includes an insertion of human V. H Gene segment V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, VH 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1 or any combination thereof, person D H Gene segment D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H 7-27 or any combination thereof, and human J H Gene segment J H1. J H 2. J H 3. J H 4. J H 5. J H 6 or any combination thereof. In some embodiments, the insertion comprises human non-coding DNA naturally adjacent to human V at an endogenous heavy chain locus. H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, or V H 6-1 appears; human non-coding DNA, which is naturally adjacent to human DNA. H 1-1, D H 2-2, D H 3-3, DH 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26 or D H 7-27 appeared; and human non-coding DNA, said human non-coding DNA naturally adjacent to human J at the endogenous heavy chain locus. H 1. J H 2. J H 3. J H 4. J H 5 or J H Appear.

[0386] In some embodiments, the non-human animals described herein contain the Adam6 gene in their genome (e.g., their germline genome), which encodes the ADAM6 polypeptide, its functional ortholog, functional homolog, or functional fragment (see, for example, U.S. Patents 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety). In some embodiments, the ADAM6 polypeptide, its functional ortholog, functional homolog, or functional fragment is expressed by the Adam6 gene. In some embodiments, the Adam6 gene is not derived from a non-human animal containing the Adam6 gene (e.g., a mouse containing the rat Adam6 gene or a mouse Adam6 gene obtained from another mouse strain). In some embodiments, the non-human animals described herein include ectopic Adam6 genes. As used herein, an ectopic Adam6 gene means an Adam6 gene in a different context than the Adam6 gene found in wild-type non-human animals. For example, the Adam6 gene may be located on a different chromosome, at a different locus, or near a different sequence. An exemplary ectopic Adam6 gene is the mouse Adam6 gene located within a human immunoglobulin sequence (e.g., a segment of the human heavy chain variable region gene). In some embodiments, the non-human animals described herein include inserted or integrated Adam6 genes.

[0387] In some embodiments, the non-human animals described herein include those whose genomes (e.g., their germline genomes) contain one or more nucleotide sequences encoding one or more non-human Adam6 peptides, their functional orthologs, functional homologs, or functional fragments.

[0388] In some embodiments, the non-human animals described herein contain one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments in their genome (e.g., their germline genome). In some embodiments, the non-human animals described herein contain the mouse Adam6a gene and / or the mouse Adam6b gene in their genome (e.g., their germline genome). In some embodiments, the non-human animals described herein contain one or more nucleotide sequences encoding mouse ADAM6a, its functional orthologs, functional homologs, or functional fragments, and / or mouse ADAM6b, its functional orthologs, functional homologs, or functional fragments.

[0389] In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are inserted and / or located on the same chromosome as the endogenous immunoglobulin heavy chain gene locus. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are inserted and / or located in a position such that the one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are adjacent to the human immunoglobulin heavy chain variable region gene segment. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are inserted and / or located in a position such that the one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are adjacent to the human immunoglobulin heavy chain variable region gene segment. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 peptides, their functional orthologs, functional homologs, or functional fragments are inserted and / or located such that the one or more nucleotide sequences encoding one or more non-human ADAM6 peptides, their functional orthologs, functional homologs, or functional fragments are located between segments of the human immunoglobulin heavy chain variable region gene. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 peptides, their functional orthologs, functional homologs, or functional fragments are inserted and / or located between the first and second human V HBetween gene segments. In some implementations, the first person V H The gene segment is human V H 1-2, and the second person V H The gene segment is human V H 6-1. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 peptides, their functional orthologs, functional homologs, or functional fragments are inserted and / or located at the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 peptides, their functional orthologs, functional homologs, or functional fragments are inserted into the human V... H Gene segments and human D H Between gene segments.

[0390] In some embodiments, the non-human animals described herein include the Adam6 gene, which restores or enhances ADAM6 activity. In some embodiments, the Adam6 gene restores ADAM6 activity to the level of comparable non-human animals containing a functional, endogenous Adam6 gene. In some embodiments, the Adam6 gene enhances ADAM6 activity to a level at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times the ADAM6 activity of comparable non-human animals not containing a functional Adam6 gene.

[0391] In some embodiments, the non-human animals described herein include the Adam6 gene, which restores or enhances the fertility of male non-human animals. In some embodiments, the Adam6 gene restores the fertility of male non-human animals to the level of comparable non-human animals containing a functional, endogenous Adam6 gene. In some embodiments, the Adam6 gene restores the fertility of male non-human animals such that the number of offspring produced by the male non-human animal through mating is at least 70%, at least 80%, at least 90%, or at least 95% of the number of offspring produced by comparable mating of comparable male non-human animals not containing the functional Adam6 gene. In some embodiments, the Adam6 gene enhances the fertility of male non-human animals such that the number of offspring produced by the male non-human animal through mating is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times the number of offspring produced by comparable mating of comparable male non-human animals not containing the functional Adam6 gene.

[0392] In some embodiments, the non-human immunoglobulin heavy chain locus described herein lacks at least one endogenous non-human Adam6 gene. In some embodiments, the lack of at least one endogenous non-human Adam6 gene reduces ADAM6 activity and / or fertility in male mice lacking the endogenous non-human Adam6 gene. In some embodiments, the non-human immunoglobulin heavy chain locus described herein includes disruption of at least one endogenous non-human Adam6 gene. In some embodiments, disruption of at least one endogenous non-human Adam6 gene reduces ADAM6 activity and / or fertility in male mice lacking the endogenous non-human Adam6 gene.

[0393] In some embodiments of non-human animals, non-human cells, or non-human tissues, the non-human animals, non-human cells, or non-human tissues are homozygous or heterozygous for the endogenous immunoglobulin heavy chain locus, as described herein.

[0394] In some embodiments of non-human animals, non-human cells, or non-human tissues, the non-human animals, non-human cells, or non-human tissues are homozygous or heterozygous for the endogenous immunoglobulin κ light chain locus, as described herein.

[0395] In some embodiments of the provided nonhuman animals, nonhuman cells, or nonhuman tissues, the endogenous immunoglobulin λ light chain locus is completely or partially absent. In some embodiments of the provided nonhuman animals, nonhuman cells, or nonhuman tissues, the endogenous immunoglobulin λ light chain locus is functionally silenced or otherwise nonfunctional (e.g., through gene targeting). In some embodiments of the provided nonhuman animals, nonhuman cells, or nonhuman tissues, the nonhuman animal, nonhuman cell, or nonhuman tissue is homozygous or heterozygous for the functionally silenced or otherwise nonfunctional endogenous immunoglobulin λ light chain locus, as described herein.

[0396] In some implementations, the non-human animals, non-human cells, or non-human tissues described herein do not detectably express endogenous immunoglobulin λ light chain, endogenous immunoglobulin κ light chain, or endogenous immunoglobulin λ light chain and endogenous immunoglobulin κ light chain.

[0397] In some implementations, the non-human animal, non-human cell, or non-human tissue described herein has a genome that further contains a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element.

[0398] In some implementations, the transcriptional control element includes the RAG1 transcriptional control element, the RAG2 transcriptional control element, the immunoglobulin heavy chain transcriptional control element, the immunoglobulin κ light chain transcriptional control element, the immunoglobulin λ light chain transcriptional control element, or any combination thereof.

[0399] In some implementations, the nucleic acid sequence encoding exogenous TdT is located at the immunoglobulin κ light chain locus, the immunoglobulin λ light chain locus, the immunoglobulin heavy chain locus, the RAG1 locus, or the RAG2 locus.

[0400] In some embodiments, the TdT is a human TdT. In some embodiments, the TdT is a short isoform (TdTS) of TdT.

[0401] In some embodiments, the human Igλ light chain sequence comprises genetic material from (e.g., isolated or obtained from) the human Igλ light chain locus or the same locus as the human Igλ light chain locus, wherein the human Igλ light chain sequence encodes an Ig light chain, and the Ig light chain comprises a coding portion of the genetic material from the human Igλ light chain locus. In some embodiments, the human Igλ light chain sequence as described herein comprises at least one human Vλ gene segment and at least one human Jλ gene segment, and one or more sequences (e.g., recombination signal sequences) necessary to facilitate the rearrangement of the at least one human Vλ gene segment with the at least one human Jλ gene segment to form a functional rearrangement encoding a human Vλ domain, the human Vλ-Jλ sequence. In many embodiments, the human Igλ light chain sequence comprises multiple human Vλ and Jλ gene segments, and one or more sequences necessary to facilitate the rearrangement of the human Vλ gene segments with the Jλ gene segments. In many embodiments, the human Igλ light chain sequence comprises at least the coding sequence (e.g., exons) of one or more human Vλ gene segments and at least the coding sequence (e.g., exons) of one or more human Jλ gene segments. In some embodiments, the human Igλ light chain sequence as described herein is a genomic sequence of a human Igλ light chain locus (e.g., isolated and / or cloned from a bacterial artificial chromosome) and contains multiple germline human Vλ gene segments. In some embodiments, the human Igλ light chain sequence comprises germline human Vλ and Jλ sequences (i.e., gene segments) (i.e., multiple human Vλ gene segments isolated by DNA intervention containing sequences necessary for and promoting recombination; and multiple Jλ gene segments isolated by DNA intervention containing sequences necessary for and promoting recombination).

[0402] In some embodiments, the human Igλ light chain sequence described herein is an engineered sequence and includes multiple human Jλ gene segments with a conformation different from that found at the human Igλ light chain locus in human cells. In some embodiments, the human Igλ light chain sequence described herein is an engineered sequence and includes multiple human Vλ and Jλ gene segments with a conformation similar to or analogous to that found at the Igκ light chain locus in wild-type mouse or human cells. In some embodiments, the human Igλ light chain sequence comprises an engineered human Jλ sequence (i.e., a coding sequence of a human Jλ gene segment prepared by de novo DNA synthesis, the coding sequence comprising a sequence necessary for and promoting recombination with one or more human Vλ gene segments). In some embodiments, the human Igλ light chain sequence comprises Igκ and Igλ sequences that occur naturally in the Igκ and Igλ genome sequences, respectively. In some embodiments, the human Igλ light chain sequence comprises an Igκ sequence, particularly a Jκ region (i.e., a sequence containing both coding and non-coding sequences that occur in a region containing multiple Jκ gene segments), the Igκ sequence being naturally present in the Igκ light chain. In the locus, besides the Igκ sequence containing the coding sequences for the Jλ gene segment and Jλ12RSS at the corresponding coding sequences of the Jκ gene segment and Jκ23RSS, respectively, in some embodiments, the human Igλ light chain sequence contains multiple Jλ gene segments and Jλ12RSS at the Jκ gene segment and Jκ23RSS positions of the Jκ region sequence. In various embodiments, the intervention (or intergenetic) DNA containing sequences essential for and promoting recombination comprises human Igκ and / or human Igλ genome sequences. Alternatively, and in some embodiments, the intervention (or intergenetic) DNA containing sequences essential for and promoting recombination comprises murine Igκ and / or murine Igλ genome sequences.

[0403] In some embodiments, the human Igλ light chain sequence is or includes the sequence shown in the figures. In some embodiments, the human Igλ light chain sequence encodes or is capable of encoding (e.g., after rearrangement of human gene segments) a Vλ domain polypeptide present in immunoglobulins, particularly immunoglobulins expressed by human B cells. Also provided are non-human animals, embryos, cells, and targeted constructs for preparing non-human animals, non-human embryos, and cells containing the human Igλ light chain sequence at the corresponding non-human Igκ light chain sequence (e.g., the endogenous rodent Igκ light chain locus).

[0404] In some embodiments, the human Igλ light chain sequence is inserted into the corresponding non-human Igκ light chain sequence within the germline genome of a non-human animal. In some embodiments, the human Igλ light chain sequence is inserted upstream of a non-human Igλ light chain sequence (e.g., a non-human Igλ light chain constant region gene sequence), the non-human Igλ light chain sequence being located within the non-human Igκ light chain sequence (e.g., a non-human Igκ light chain constant region gene sequence). In some embodiments, the human Igκ light chain sequence is inserted into the middle segment of the human Igλ light chain sequence (i.e., between the human Vλ and Jλ gene segments) such that the human Igκ light chain sequence is juxtaposed with the human Igλ light chain sequence.

[0405] In some embodiments, all or substantially all of the variable regions at the non-human Igκ light chain locus are replaced or replaced by one or more human Igλ light chain sequences (as described herein), and said one or more human Igλ light chain sequences are operatively linked to a non-human or human Igλ light chain constant region gene. In some embodiments, the non-human Igκ light chain constant region gene is missing or replaced in non-human animals including human Igλ light chain sequences as described herein. In a non-limiting example, in the case of inserting a human Igλ light chain sequence at a non-human Igκ light chain locus, the insertion is performed in a manner that maintains the integrity of the non-human Igκ light chain enhancer region (or enhancer sequence) near the insertion point (e.g., a non-human Igκ intron enhancer, and / or a non-human Igκ 3' enhancer). Therefore, such non-human animals possess wild-type Igκ light chain enhancer regions (or enhancer sequences) operablely linked to human and non-human Igλ light chain sequences (e.g., human Vλ and Jλ gene segments and non-human Cλ region genes) or operablely linked to human Igλ light chain sequences (e.g., human Vλ and Jλ gene segments and human Cλ region genes). In some embodiments, non-human Igκ light chain loci are modified, substituted, disrupted, deleted, replaced, or engineered with one or more human Igλ light chain sequences as described herein as mouse Igκ light chain loci. In some embodiments, one or more human Igλ light chain sequences as described herein are inserted into one copy (i.e., an allele) of a non-human Igκ light chain locus from two copies of the non-human Igκ light chain locus, thereby producing non-human animals that are heterozygous for human Igκ light chain sequences. In some embodiments of non-human animals heterozygous for the human Igκ light chain sequence, the non-human animal comprises one or more human Igκ light chain sequences inserted into another copy (i.e., an allele) of the non-human Igκ light chain locus. In some embodiments, a non-human animal is provided that is homozygous for the Igκ light chain locus, which comprises one or more human Igλ light chain sequences as described herein.

[0406] In some embodiments, the engineered nonhuman Igκ light chain locus described herein includes a human Vλ gene segment and a Jλ gene segment operatively linked to a nonhuman or human Igλ light chain constant region gene, wherein the nonhuman or human Igλ light chain constant region gene is located at a nonhuman Igκ light chain constant region gene that is present in a wild-type Igκ light chain locus of a nonhuman animal of the same species.

[0407] In some embodiments, one or more endogenous non-human Igλ light chain sequences (or portions thereof) of the endogenous non-human Igλ light chain locus are not deleted. In some embodiments, one or more endogenous non-human Igλ light chain sequences (or portions thereof) of the endogenous non-human Igλ light chain locus are deleted. In some embodiments, one or more endogenous non-human Igλ light chain sequences (e.g., V, J, and / or C, or any combination thereof) of the endogenous non-human Igλ light chain locus are altered, substituted, disrupted, deleted, or replaced to render the non-human Igλ light chain locus functionally silent. In some embodiments, one or more endogenous non-human Igλ light chain sequences (e.g., V, J, and / or C, or any combination thereof) of the endogenous non-human Igλ light chain locus are altered, substituted, disrupted, deleted, or replaced with a targeting vector to render the non-human Igλ light chain locus functionally inactivated (i.e., unable to produce functional light chains that can be expressed and / or detected as antibodies in a non-human animal antibody library). For example, U.S. Patent No. 9,006,511 (see, for example, Figure 2) provides guidance on the inactivation of endogenous nonhuman Igλ light chain loci, which is incorporated herein by reference in its entirety.

[0408] In some embodiments, the non-human animal contains an engineered Igκ light chain locus as described herein, which is randomly integrated into its genome (e.g., as part of a randomly integrated human Igλ light chain sequence). Thus, such non-human animals can be described as having a human Igλ light chain transgene containing multiple operatively linked to non-human or human Igλ light chain constant regions, and personal Vλ and Jλ gene segments of a non-human Igκ light chain enhancer region (or enhancer sequence) such that the human Vλ and Jλ gene segments are rearranged to encode an Ig light chain of an antibody in a non-human animal expression library, wherein the Ig light chain comprises a human Vλ domain and a non-human Cλ domain, or the Ig light chain comprises both human Vλ and Cλ domains. Various methods can be used to detect the engineered Igκ light chain locus or transgene as described herein, including methods such as PCR, Western blotting, RNA blotting, restriction fragment length polymorphism (RFLP), or allele gain or deletion assays. In some embodiments, the non-human animal described herein is heterozygous for the engineered Igκ light chain locus, as described herein. In some embodiments, the engineered Igκ light chain locus in the non-human animal described herein is hemizygous, as described herein. In some embodiments, the non-human animal described herein contains one or more copies of the engineered Igκ light chain locus or the transgene, as described herein. In some embodiments, the non-human animal described herein contains the Igκ light chain locus as shown in the figure.

[0409] This disclosure recognizes that non-human animals, as described herein, utilize variable regions of the human heavy chain, λ light chain, and κ light chain contained in their genomes in their antibody selection and production mechanisms (e.g., recombination and somatic hypermutation). Therefore, in various embodiments, the variable domains of the human heavy chain, λ light chain, and κ light chain in human immunoglobulins produced by the non-human animals described herein are encoded by variable regions of the human heavy chain, λ light chain, and κ light chain, which are respectively contained in their genomes or in somatic hypermutated variants thereof.

[0410] In some embodiments, a non-human animal is provided whose genome contains an engineered endogenous immunoglobulin κ light chain locus, wherein the non-human animal contains B cells containing human heavy chain variable region sequences, human λ light chain variable region sequences, and / or somatic hypermutated human κ light chain variable region sequences. In some embodiments, the human heavy chain variable region sequences, human λ light chain and / or human κ light chain variable region sequences present in the B cells of the mice of this disclosure have 1, 2, 3, 4, 5 or more somatic hypermutations. Methods for identifying the source gene segments in mature antibody sequences are known to those skilled in the art. For example, various tools can be used to assist in such analysis, such as DNAPLOT, IMGT / V-QUEST, JOINSOLVER, SoDA, and Ab-origin.

[0411] This disclosure particularly provides cells and tissues from non-human animals as described herein. In some embodiments, spleen cells (and / or other lymphoid tissues) from non-human animals as described herein are provided. In some embodiments, B cells from non-human animals as described herein are provided. In some embodiments, progenitor B cells from non-human animals as described herein are provided. In some embodiments, pre-B cells from non-human animals as described herein are provided. In some embodiments, immature B cells from non-human animals as described herein are provided. In some embodiments, mature naïve B cells from non-human animals as described herein are provided. In some embodiments, activated B cells from non-human animals as described herein are provided. In some embodiments, memory B cells from non-human animals as described herein are provided. In some embodiments, B lineage lymphocytes from non-human animals as described herein are provided. In some embodiments, plasma or plasma cells from non-human animals as described herein are provided. In some embodiments, stem cells from non-human animals as described herein are provided. In some embodiments, the stem cells are embryonic stem cells. In some embodiments, germ cells from non-human animals as described herein are provided. In some embodiments, the germ cells are oocytes. In some embodiments, the germ cells are sperm cells. In some embodiments, the sperm cells of the non-human animal described herein express one or more ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments. In some embodiments, any cells or tissues from the non-human animal described herein may be isolated. In some embodiments, isolated cells and / or isolated tissues from the non-human animal described herein are provided. In some embodiments, a hybridoma is provided, wherein the hybridoma is prepared from non-human animal B cells described herein. In some embodiments, the hybridoma is prepared from non-human animal B cells immunized with a target antigen. In some embodiments, the hybridoma is prepared from non-human animal B cells expressing antibodies that bind (e.g., specifically bind) to epitopes on the target antigen.

[0412] Any non-human animal described herein may be immunized with one or more target antigens under conditions and for a duration sufficient to induce an immune response in the non-human animal to one or more target antigens. Methods for immunizing non-human animals are known to those skilled in the art. Exemplary, non-limiting methods for immunizing non-human animals can be found in US 2007 / 0280945A1, which is incorporated herein by reference in its entirety.

[0413] This disclosure particularly provides immunized nonhuman animals as described herein, and cells and tissues isolated from said nonhuman animals. In some embodiments, the nonhuman animals described herein generate a population of B cells in response to immunization with an antigen comprising one or more epitopes. In some embodiments, the nonhuman animals generate a population of B cells expressing antibodies that bind (e.g., specifically bind) to one or more epitopes of a target antigen. In some embodiments, the antibodies expressed by the B cell population generated in response to the antigen comprise a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence, and / or a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein. In some embodiments, the antibody expressed by a population of B cells in response to an antigen comprises (i) a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence, (ii) a λ light chain having a human λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein, (iii) a κ light chain having a human κ light chain variable domain encoded by a human κ light chain variable region sequence as described herein, or (iv) any combination thereof.

[0414] In some embodiments, a non-human animal generates a population of B cells expressing antibodies that bind to one or more epitopes of a target antigen, wherein the antibodies expressed by the B cell population generated in response to the antigen comprise: (i) a heavy chain having a human heavy chain variable domain encoded by a human heavy chain variable region sequence, (ii) a λ light chain having a λ light chain variable domain encoded by a human λ light chain variable region sequence as described herein, (iii) a κ light chain having a human κ light chain variable domain encoded by a human κ light chain variable region sequence as described herein, or (iv) any combination thereof. In some embodiments, the human heavy chain variable region sequence, the human λ light chain variable region sequence, and / or the human κ light chain variable region sequence as described herein are somatic hypermutated. In some implementations, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90% of the B cells in the antigen-response B cell population contain human heavy chain variable region sequences, human λ light chain variable region sequences, and / or human κ light chain variable region sequences that are somatic hypermutated.

[0415] In some embodiments, the non-human animals provided herein contain in their germline genome (1) an engineered endogenous immunoglobulin κ light chain locus comprising (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments and (c) a Cλ gene, wherein the one or more human Vλ gene segments and the one or more human Jλ gene segments are operatively linked to the Cλ gene, (2) lacking the rodent Cκ gene at the engineered endogenous immunoglobulin κ locus, and (3) an engineered endogenous immunoglobulin κ light chain locus comprising (a) one or more human Vκ gene segments, (b) one or more human Jκ gene segments and (c) a Cκ gene, wherein the one or more human Vκ gene segments and the one or more human Jκ gene segments are operatively linked to the Cκ gene. In some embodiments, the percentage of light chains (which are λ light chains) in the spleen cells of such non-human animals (e.g., as detected or observed by flow cytometry (see example 3)) is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%. In some embodiments, the percentage of light chains (which are λ light chains) in the spleen cells of such non-human animals (e.g., as detected or observed by flow cytometry (see example 3)) is between 35% and 80%, between 35% and 75%, between 40% and 80%, between 40% and 75%, between 50% and 80%, between 50% and 75%, between 55% and 80%, between 55% and 75%, between 60% and 80%, or between 60% and 75%. In some implementations, the percentage of light chains (which are κ light chains) in the spleen cells of such non-human animals (e.g., as detected or observed by flow cytometry (see example 3)) is up to 65%, up to 60%, up to 55%, up to 50%, up to 45%, up to 40%, or up to 35%. In some embodiments, the percentage of light chains (which are κ light chains) in the spleen cells of such non-human animals (e.g., as detected or observed by flow cytometry (see example 3)) is between 20% and 65%, between 25% and 65%, between 20% and 60%, between 25% and 60%, between 20% and 55%, between 25% and 55%, between 20% and 50%, between 25% and 50%, between 20% and 45%, between 25% and 45%, between 20% and 40%, or between 25% and 40%.In some implementations, the ratio of κ:λ light chains in the spleen cells of such non-human animals (e.g., as detected or observed by flow cytometry (see example 3)) is between 0.5:1 and 3:1, between 0.65:1 and 3:1, between 0.8:1 and 3:1, between 1:1 and 3:1, between 1.2:1 and 3:1, between 1:1 and 2.3:1, between 1.1:1 and 1.8:1, between 1.2:1 and 2.3:1, or between 1.2:1 and 1.8:1.

[0416] The method for preparing the provided non-human animals

[0417] Compositions and methods for preparing non-human animals are provided, the germline genome of which comprises an engineered Igκ light chain locus, the engineered Igκ light chain locus comprising one or more human Igλ light chain sequences (e.g., human Vλ and Jλ gene segments) located at a non-human Igκ light chain sequence position, including a human Igλ light chain coding sequence comprising specific polymorphic forms of the human Vλ and Jλ segments (e.g., specific V and / or J alleles or variants). Compositions and methods for preparing non-human animals expressing antibodies comprising an Igλ light chain, said light chain containing a human variable region and a non-human or human constant region assembled from an Igκ light chain locus, said Igκ light chain locus containing human Vλ and Jλ gene segments operatively linked to a non-human or human Igλ light chain constant region gene, said non-human or human Igλ light chain constant region gene being located at the position of a non-human Igκ light chain constant region gene, said non-human Igκ light chain constant region gene typically found in wild-type non-human Igκ light chain loci. In some embodiments, compositions and methods for preparing non-human animals expressing such antibodies under the control of an endogenous Igκ enhancer and / or an endogenous Igκ regulatory sequence are also provided. In some embodiments, compositions and methods for preparing non-human animals expressing such antibodies under the control of a heterologous Igκ enhancer and / or a heterologous Igκ regulatory sequence are also provided.

[0418] The method described herein involves inserting human Vλ and Jλ sequences that encode a human Vλ domain upstream of a non-human or human Igλ light chain constant region gene (e.g., a mouse or human Cλ region gene), the non-human or human Igλ light chain constant region gene being located at the position of a non-human Igκ light chain constant region gene, the non-human Igκ light chain constant region gene typically present in wild-type non-human Igκ light chain loci, to express an antibody characterized by the presence of a light chain containing a human Vλ domain and a non-human Cλ domain (e.g., a rodent Cλ domain), or the presence of a light chain containing a human Vλ and a non-human Cλ domain (e.g., one or more rodent Cλ domains), and expression on the surface of B cells and in the serum of non-human animals.

[0419] In some embodiments, the method includes inserting genetic material containing human Vλ and Jλ gene segments into an Igκ light chain locus (e.g., a wild-type, modified, or engineered Igκ light chain locus). In some embodiments, the method includes inserting genetic material containing human Jλ gene segments into an Igκ light chain locus of a modified or engineered strain. In some embodiments, the genetic material containing the human Igλ light chain sequence may be engineered or genomicized (e.g., cloned from a bacterial artificial chromosome). In some embodiments, the genetic material containing the human Igλ light chain sequence may be designed from publicly available sources and / or bacterial artificial chromosomes such that the genetic material contains human Vλ and Jλ segments with orientations different from those found in the human Igλ light chain locus, but the genetic material still contains sequences supporting rearrangements of the human Vλ and Jλ segments to encode a functional human Vλ domain of the Ig light chain. For example, the guidelines provided herein can be used to design genetic material corresponding to multiple human Vλ and Jλ gene segments to construct a human Igλ light chain sequence containing human Vλ and Jλ segments with a sequence and / or arrangement different from that found at human Igλ light chain loci in human cells (e.g., an arrangement similar to or analogous to human or rodent Igκ light chain loci, such as a series of V gene segments inserted at 3' followed by a series of J gene segments). In this example, the genetic material of the human Vλ and Jλ gene segments will be equivalent to the corresponding segments in human cells; however, the sequence and arrangement may differ. When constructing engineered Igκ light chain loci to produce non-human animals as described herein, the necessary recombination signal sequences can be configured so that the human V and J gene segments can be correctly rearranged to form a functional human Vλ domain. Guidelines for the germline conformations of human Vλ and Jλ gene segments and the sequences necessary for appropriate recombination can be found, for example, in Molecular Biology of B Cells, London: Elsevier Academic Press, 2004, Honjo, T., Alt, FW, Neuberger, M. (eds.), Chapter 4 (pp. 37–59) and Chapter 5 (pp. 61–82); these references are incorporated herein by reference in their entirety.

[0420] In some embodiments, the method includes multiple insertions within a single ES cell clone. In some embodiments, the method includes sequential insertions within successive ES cell clones. In some embodiments, the method includes a single insertion within an engineered ES cell clone.

[0421] In some embodiments, the method includes a DNA insertion upstream of the mouse Cλ1 gene (or the human Cλ2 gene) such that the DNA insertion is operatively linked to the mouse Cλ1 gene (or the human Cλ2 gene), the DNA insertion comprising the human Vλ gene segments Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5- 37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1 or any combination thereof, and human Jλ gene segments Jλ1, Jλ2, Jλ3, Jλ6, Jλ7 or any combination thereof, and the mouse Cλ1 gene (or human Cλ2 gene) is located at the mouse Cκ gene position of the endogenous Igκ light chain locus.

[0422] In some embodiments, the method includes a DNA insertion downstream of the human Vλ3-1 gene segment and upstream of a non-human Igκ intron enhancer region (or enhancer sequence) at an engineered Igκ light chain locus, such that the DNA insertion is operatively linked to a mouse Cλ1 gene (or a human Cλ2 gene), the DNA insertion comprising a human Igκ genome sequence naturally occurring between the human Vκ4-1 gene segment and the human Jκ1 gene segment at the human Igκ light chain locus and one or more human Jλ gene segments (e.g., I, II, III, IV, V, VI, or VII), the mouse Cλ1 gene (or human Cλ2 gene) located at the mouse Cκ gene position at the endogenous non-human Igκ light chain locus. In some embodiments, the method includes a DNA insertion between a human Vλ3-1 gene segment and a non-human Igκ intron enhancer, the DNA insertion comprising a human Vκ-Jκ sequence naturally occurring between human Vκ4-1 and Jκ1 gene segments at the human Igκ light chain locus and five human Jλ gene segments (e.g., Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7). In various embodiments, the DNA insertion including the human Jλ gene segment comprises human Jκ genomic DNA having coding sequences for the human Jλ gene segment and the human Jλ12RSS.

[0423] The methods described herein enable the insertion of additional human Vλ and Jλ fragments to further supplement the diversity of engineered Igλ light chain loci. For example, in some embodiments, the method may include inserting approximately 270 kb of DNA upstream of a murine Cλ1 gene (or human Cλ2 gene) at an engineered Igκ light chain locus, such that the DNA is operatively linked to the murine Cλ1 gene (or human Cλ2 gene), wherein the DNA comprises human Vλ gene segments Vλ10-54, Vλ6-57, Vλ4-60, Vλ8-61, and Vλ4-69. In such embodiments, the DNA is inserted upstream of a human Vλ5-52 gene segment operatively linked to a murine Cλ1 gene (or human Cλ2 gene) at an engineered Igκ light chain locus, the DNA comprising human Vλ gene segments Vλ10-54, Vλ6-57, Vλ4-60, Vλ8-61, and Vλ4-69. In some embodiments, the DNA comprises the human VpreB gene. The aforementioned additional human Vλ gene segments can be cloned directly from a commercially available BAC clone and arranged in a smaller DNA fragment using recombination techniques described herein or known in the art. Alternatively, the aforementioned additional human Vλ gene segments can be synthesized as engineered DNA fragments and added to the engineered Igκ light chain locus as described above using molecular biology techniques known in the art. Similarly, additional human Jλ gene segments can be obtained from commercially available BAC clones or synthesized directly from published sequences. An exemplary illustration of the engineered Igκ light chain locus in non-human animals as described herein is presented in Figure 2B Or in 4B.

[0424] Where appropriate, the human Igλ light chain sequence encoding the human Vλ domain (i.e., the sequence comprising segments of the human Vλ and Jλ genes) may be modified to include codons optimized for expression in non-human animals (e.g., see U.S. Patent Nos. 5,670,356 and 5,874,304). The codon-optimized sequence is an engineered sequence and preferably encodes the same polypeptide (or a bioactive fragment of the full-length polypeptide having substantially the same activity as the full-length polypeptide) encoded by a non-codon-optimized parental polynucleotide. In some embodiments, the human Igλ light chain sequence encoding the human Vλ domain may include modified sequences to optimize codon usage for a specific cell type (e.g., rodent cells). For example, the codons for each nucleotide sequence to be inserted into the genome of a non-human animal (e.g., rodent) may be optimized for expression in non-human animal cells. Such a sequence may be described as a codon-optimized sequence.

[0425] The insertion of a nucleotide sequence encoding a human Vλ domain uses minimal modification of a nonhuman phylogenetic genome as described herein and results in the expression of an antibody containing a light chain having a human Vλ domain expressed by an endogenous engineered Igκ light chain locus. Methods for generating engineered nonhuman animals (including knockout and knock-in) are known in the art (see, for example, Gene Targeting: A Practical Approach, ed. Joyner, Oxford University Press, Inc., 2000; which is incorporated herein by reference in its entirety). For example, the generation of genetically engineered rodents may optionally involve disrupting a locus of one or more endogenous rodent genes (or gene segments) and introducing one or more heterologous genes (or gene segments or nucleotide sequences) into the rodent genome, at the same location as the endogenous rodent gene (or gene segment) in some embodiments. In some embodiments, the nucleotide sequence encoding the human Vλ domain is introduced upstream of a randomly inserted, engineered light chain transgenic mouse or human Igλ light chain constant region gene in the rodent germline genome. In some embodiments, the nucleotide sequence encoding the human Vλ domain is introduced upstream of a mouse or human Igλ light chain constant region gene at an endogenous Igκ light chain locus in the rodent germline genome; in some embodiments, the endogenous Igκ light chain locus is altered, modified, or engineered to contain a human Igλ gene segment (e.g., human V and J) operatively linked to the mouse Cλ1 gene or operatively linked to the human Cλ2 gene.

[0426] A schematic diagram (not to scale) of an exemplary method for constructing an engineered Igκ light chain locus as described herein is provided. Figure 1A , 1B In 2A, 2B, 3, 4A, and 4B. Specifically, Figure 1A and 1B Exemplary strategies for constructing engineered Igκ light chain loci are listed, characterized by the insertion of nucleotide sequences containing multiple human Vλ and Jλ gene segments. For example... Figure 1A and 1BAs shown, an engineered fragment containing a DNA fragment of the human Vκ-Jκ intergenic region (see U.S. Patent Nos. 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662, and 9,163,092) and a set of human Jλ gene segments (e.g., human Jλ1, Jλ2, Jλ3, Jλ6, and Jλ7) is operatively linked to a rodent Igκ intron enhancer region (or enhancer sequence) via a series of steps using various molecular biology techniques described in Example 1. This engineered fragment is also engineered to contain a rodent Igλ light chain constant region operatively linked to a human Jλ gene segment. A selection cassette (e.g., neomycin and hygromycin) is contained in a targeting vector to allow selection of positive clones in bacterial and mammalian cells (e.g., embryonic stem cells). As shown, the neomycin resistance gene is flanked by a lox2372 specific recombination site (lox) and located between the human Vκ-Jκ region and the human Jλ gene segment, while the hygromycin selector cassette is flanked by a loxP specific recombination site and located at the 3' of the rodent Igλ light chain constant region (mCλ1) gene. This DNA fragment is then combined with a DNA fragment containing a rodent Igκ light chain 3' enhancer to produce the final targeting vector ( Figure 1B The resulting targeting vector (constructor G) was linearized and electroporated into rodent embryonic stem (ES) cells to generate rodents whose germline genome contained an engineered Igκ light chain locus. As described in the Examples section below, the electroporated rodent ES cells used for the targeting vector contained an engineered Igκ light chain locus, as previously described in U.S. Patent Nos. 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662, and 9,163,092, which are incorporated herein by reference in their entirety. Figure 3As described herein, homologous recombination with a targeting vector can generate an engineered Igκ light chain locus characterized by operatively linking to multiple human Vλ and Jλ gene segments of the murine Cλ1 gene located at the position of the murine Cκ gene, which is naturally present in the wild-type Igκ light chain locus. The human Jλ gene segment is uniquely engineered into a sequence not naturally present in the human Jκ region of the genome, but possessing the human Jλ coding sequence and associated 12 RSS, located at the human Jκ coding sequence and associated 23 RSS. Positive rodent ES cell clones are confirmed using screening methods described herein and / or known in the art. Any remaining selection cassettes can be deleted as needed via recombinase-mediated deletion (see Example 2).

[0427] Alternatively, the human Cλ gene can be used in the targeting vector instead of the mouse Cλ gene. This is just one example. Figure 3 A targeting vector constructed in a similar manner to that described above is presented, except that the sequence encoding the human Cλ2 gene is engineered into the targeting vector and operatively linked to five human Jλ gene segments. This approach offers additional benefits in the development of human antibody therapeutics because the DNA encoding the variable and constant regions of the light chain can be isolated together, eliminating any subsequent cloning steps, thereby allowing for the linking to the human light chain constant region for the preparation of fully human antibodies.

[0428] Targeting vectors for constructing engineered Igκ light chain loci as described herein can be incorporated into the germline genome of non-human cells (e.g., rodent embryonic stem cells). In some embodiments, the targeting vectors as described herein are incorporated into the wild-type Igκ light chain locus of a non-human cell germline genome, said cells also containing human V proteins operatively linked to one or more immunoglobulin heavy chain constant region genes. H D H and J H Genomic DNA (e.g., containing multiple human V) H D H and J H (See, for example, U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, and 8,791,323, each of which is incorporated herein by reference in its entirety.) In some embodiments, the targeting vector as described herein is incorporated into a modified or engineered endogenous immunoglobulin κ light chain locus in the germline genome of a nonhuman cell, the nonhuman cell further comprising human V that is operatively linked to one or more immunoglobulin heavy chain constant region genes. H D H and J H Genomic DNA (e.g., containing multiple human V)H D H and J H (See, for example, U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, 8,791,323, 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662 and 9,163,092, each of which is incorporated herein by reference in its entirety.)

[0429] A targeting vector is introduced into rodent (e.g., mouse) embryonic stem cells via electroporation to enable non-human cells or non-human animals (e.g., mice) to express antibodies having a light chain containing a human Vλ domain and a non-human or human Cλ domain, and the light chain being expressed by an endogenous engineered immunoglobulin κ light chain locus. As described herein, genetically engineered rodents are produced in which an engineered endogenous immunoglobulin κ light chain locus (e.g., an endogenous immunoglobulin κ light chain locus containing a human Igλ light chain sequence (i.e., multiple human Vλ and Jλ gene segments) is generated in the rodent germline genome, operably linked to a rodent or human Cλ gene located at the endogenous rodent Cκ gene). Antibodies are expressed on the surface of rodent B cells and in the serum of the rodents, the antibodies being characterized by a light chain having a human Vλ domain and a non-human or human Cλ domain. When the endogenous immunoglobulin κ light chain locus in the rodent germline genome is not targeted by the targeting vector, it is preferable to insert an engineered immunoglobulin κ light chain transgene at a location different from the endogenous rodent immunoglobulin κ light chain locus (e.g., a randomly inserted transgene).

[0430] As described above, the generation of an engineered immunoglobulin κ light chain locus in non-human animals provides engineered rodent strains that produce antibodies comprising immunoglobulin λ light chains expressed from such an engineered immunoglobulin κ light chain locus (which has a human Vλ domain and a non-human or human Cλ domain). This utilizes multiple human Vλ light chains containing genes operatively linked to the immunoglobulin heavy chain constant region. H D H and J H The presence of an engineered immunoglobulin heavy chain locus in the gene segment resulted in an engineered rodent strain that produces antibodies and antibody components for the development of human antibody-based therapeutics. Thus, a single engineered rodent strain was realized that provides another in vivo system for exploring the human Vλ domain to develop novel antibody-based drugs for treating human diseases.

[0431] In some embodiments, a method for preparing a nonhuman animal whose germline genome contains an engineered endogenous immunoglobulin κ light chain locus is provided. The method includes (a) introducing a DNA fragment into a nonhuman embryonic stem cell, the DNA fragment containing a nucleotide sequence comprising (i) one or more human Vλ gene segments, (ii) one or more human Jλ gene segments, and (iii) a Cλ gene (e.g., nonhuman or human), wherein (i)-(iii) are operatively linked, and wherein the nucleotide sequence further comprises an immunoglobulin κ light chain sequence between (i) and (ii); (b) obtaining the nonhuman embryonic stem cells generated in (a); and (c) generating a rodent using the rodent embryonic stem cells from (b).

[0432] In some embodiments, a method for preparing a nonhuman animal is provided, the germline genome of which comprises an engineered endogenous immunoglobulin κ light chain gene, the method comprising (a) introducing a DNA fragment into nonhuman embryonic stem cells, the DNA fragment comprising a nucleotide sequence comprising one or more human Jλ gene segments, one or more nonhuman immunoglobulin κ light chain enhancers, and a nonhuman or human Cλ gene, the human Jλ gene segments being operatively linked to the one or more nonhuman immunoglobulin κ light chain enhancers and the nonhuman or human Cλ gene; (b) obtaining the nonhuman embryonic stem cells generated in (a); and (c) using the rodent embryonic stem cells from (b) to generate rodents.

[0433] In some embodiments, a method for preparing a non-human animal is provided, the germline genome of which includes an engineered endogenous immunoglobulin κ light chain locus, wherein the engineered endogenous immunoglobulin κ light chain locus includes insertions of one or more human Vλ gene segments, one or more human Jλ gene segments, and a non-human or human Cλ gene, wherein the human Vλ and Jλ gene segments are operatively linked to the non-human or human Cλ gene, and the non-human or human Cλ gene is inserted at the endogenous immunoglobulin κ locus into a non-human Cλ gene. At the gene site, the method includes modifying the germline genome of a non-human animal such that the germline genome contains an engineered endogenous immunoglobulin κ light chain locus, the engineered endogenous immunoglobulin κ light chain locus comprising the insertion of one or more human Vλ gene segments, one or more human Jλ gene segments, and a non-human Cλ or human Cλ gene, the human Vλ and Jλ gene segments being operatively linked to the non-human or human Cλ gene, and the non-human or human Cλ gene being inserted at the non-human Cκ gene located at the endogenous immunoglobulin κ gene locus.

[0434] In some embodiments of the method for preparing a non-human animal, one or more human Vλ gene segments include at least 24, at least 34, at least 52, at least 61, or at least 70 human Vλ gene segments. In some embodiments of the method for preparing a non-human animal, one or more human Vλ gene segments include 39 human Vλ gene segments. In some embodiments of the method for preparing non-human animals, one or more human Vλ gene segments include human Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43, Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof. In some embodiments, one or more human Vλ gene segments comprise human non-coding DNA that occurs naturally adjacent to the relevant human Vλ gene segment in the endogenous human λ light chain locus.

[0435] In some embodiments of the method for preparing a non-human animal, one or more human Jλ gene segments include at least one, at least two, at least three, at least four, or at least five human Jλ gene segments. In some embodiments of the method for preparing a non-human animal, one or more human Jλ gene segments include five human Jλ gene segments. In some embodiments of the method for preparing a non-human animal, one or more human Jλ gene segments include human Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any combination thereof. In some embodiments, one or more human Jλ gene segments include human non-coding DNA (all or part) that naturally occurs adjacent to the relevant human Jλ gene segment at the endogenous human λ light chain locus. In some embodiments, one or more human Jλ gene segments include human non-coding DNA that naturally occurs adjacent to human Jκ1-Jκ5 at the endogenous human κ light chain locus.

[0436] In some embodiments of the method for preparing non-human animals, the DNA fragment includes intergenic DNA containing non-coding immunoglobulin DNA (e.g., DNA naturally occurring coding sequences between two V gene segments, between V and J gene segments, or between two J gene segments). In many embodiments, the non-coding immunoglobulin DNA is non-coding immunoglobulin light chain DNA (e.g., human or mouse). In some embodiments, the non-coding immunoglobulin light chain DNA is immunoglobulin κ light chain DNA, immunoglobulin λ light chain DNA, or a combination thereof.

[0437] In some embodiments of the method for preparing non-human animals, the DNA fragment further comprises one or more selection markers. In some embodiments of the method for preparing non-human animals, the DNA fragment further comprises one or more site-specific recombination sites. In some embodiments of the method for preparing non-human animals, the DNA fragment further comprises one or more sets of site-specific recombination sites recombined with the same recombinase. In some embodiments of the method for preparing non-human animals, the DNA fragment further comprises one or more sets of site-specific recombination sites recombined with different recombinases.

[0438] In some embodiments of the method for preparing a non-human animal, the DNA fragment comprises an engineered sequence comprising an immunoglobulin κ light chain sequence and an immunoglobulin λ light chain sequence together in a continuous sequence. In some embodiments of the method for preparing a non-human animal, the DNA fragment comprises an engineered sequence comprising an immunoglobulin κ light chain sequence and an immunoglobulin λ light chain sequence together in a single sequence but interrupted by a non-immunoglobulin sequence (e.g., a recombination signal sequence, an resistance gene, or a combination thereof). In some embodiments of the method for preparing a non-human animal, the engineered sequence comprises portions of the Jκ region and portions of the Jλ region. In some embodiments, the engineered sequence comprises a portion of the human Jκ region and a portion of the human Jλ region. In some embodiments, the portion of the human Jκ region comprises a non-coding sequence of the human Jκ region naturally occurring at the human immunoglobulin κ light chain locus in human cells. In some embodiments, the portion of the human Jλ region comprises a coding sequence of one or more human Jλ gene segments and a recombination signal sequence (RSS). In some embodiments of the method for preparing non-human animals, the DNA fragment comprises an engineered sequence characterized in some embodiments by the presence of a coding sequence and a recombination signal sequence (RSS) of one or more human Jλ gene segments, wherein the one or more human Jλ gene segments are positionally substituted or replaced (i.e., located at) a corresponding coding sequence and recombination signal sequence (RSS) on a human Jκ gene segment such that the coding sequence and recombination signal sequence (RSS) of the one or more human Jλ gene segments are located within, adjacent to, contiguous with, or juxtaposed with the non-coding sequence of the one or more human Jκ gene segments.

[0439] In some embodiments of the method for preparing non-human animals, a DNA fragment is introduced into non-human embryonic stem cells, the germline genome of which comprises one or more engineered immunoglobulin loci (e.g., immunoglobulin heavy chain, immunoglobulin κ light chain, immunoglobulin λ light chain, and combinations thereof). In some embodiments, the engineered immunoglobulin loci are endogenous engineered immunoglobulin loci.

[0440] In some embodiments of the method for preparing non-human animals, a DNA fragment is introduced into non-human embryonic stem cells, the germline genome of which contains an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus containing one or more human V... H Gene segment, one or more individuals D H Gene segments and one or more individuals J H Gene segment, wherein human V H D H and J HThe gene segment is operatively linked to the non-human immunoglobulin heavy chain constant region.

[0441] In some embodiments of the method for preparing a non-human animal, a DNA fragment is introduced into a non-human embryonic stem cell whose germline genome includes an endogenous immunoglobulin κ light chain locus. The endogenous immunoglobulin κ light chain locus includes insertion of one or more human Vλ and one or more human Jλ gene segments, the human Vλ and Jλ gene segments being operatively linked to a non-human immunoglobulin κ light chain constant region gene. In some embodiments of the method for preparing a non-human animal, a DNA fragment is introduced into a non-human embryonic stem cell whose germline genome includes an endogenous immunoglobulin κ light chain locus. The endogenous immunoglobulin κ light chain locus includes insertion of one or more human Vλ and one or more human Jλ gene segments, and a human immunoglobulin κ light chain sequence is positioned, placed, or located between the one or more human Vλ gene segments and the one or more human Jλ gene segments, wherein the human Vλ and Jλ gene segments are operatively linked to a non-human immunoglobulin κ light chain constant region gene.

[0442] In some embodiments of the method for preparing non-human animals, the germline genome of the non-human animal is modified to include an engineered immunoglobulin κ light chain locus in non-human embryonic stem cells, the germline genome of which includes an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus containing one or more human V... H Gene segment, one or more individuals D H Gene segments and one or more individuals J H Gene segment, the human V H D H and J H The gene segment is operatively linked to the non-human immunoglobulin heavy chain constant region.

[0443] In some embodiments of the method for preparing non-human animals, the germline genome of the non-human animal is modified to include an engineered immunoglobulin κ light chain locus in non-human embryonic stem cells, the germline genome of the non-human embryonic stem cells including an endogenous immunoglobulin κ light chain locus, the endogenous immunoglobulin κ light chain locus including the insertion of one or more human Vλ and one or more human Jλ gene segments, the human Vλ and Jλ gene segments being operatively linked to the non-human immunoglobulin κ light chain constant region gene. In some embodiments of the method for preparing non-human animals, the germline genome of the non-human animal is modified to include an engineered immunoglobulin κ light chain locus in non-human embryonic stem cells. The germline genome of the non-human embryonic stem cells includes an endogenous immunoglobulin κ heavy chain locus comprising one or more human Vλ gene segments, one or more human Jλ gene segments, and a human immunoglobulin κ light chain sequence, positioned, placed, or located between the one or more human Vλ gene segments and the one or more human Jλ gene segments, the human Vλ and Jλ gene segments being operatively linked to a non-human immunoglobulin κ light chain constant region gene.

[0444] In some embodiments of the method for preparing non-human animals, one or more human V... H Gene segment, one or more individuals D H Gene segments and one or more individuals J H Genetic segments including those naturally adjacent to human V H The gene segment appears in human non-coding DNA and is naturally adjacent to human DNA. H The human non-coding DNA segment appearing in the gene region and the human J gene naturally adjacent to the endogenous human immunoglobulin locus. H Human non-coding DNA appearing in gene segments.

[0445] In some implementations, non-human animals prepared, generated, manufactured, obtained, or available by methods as described herein are provided.

[0446] In some embodiments, the genome of the non-human animal described herein further includes one or more human immunoglobulin heavy chain variable regions, as described in U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, and 8,791,323, each of which is incorporated herein by reference in its entirety. Alternatively, the engineered endogenous immunoglobulin κ light chain locus described herein can be engineered into embryonic stem cells of different modified strains, for example... Strains (see, for example, U.S. Patent Nos. 8,502,018 and / or 8,642,835; which are incorporated herein by reference in their entirety). Homozygosity of the engineered Igκ light chain locus as described herein can then be achieved through breeding. Alternatively, in the case of random insertion of an engineered immunoglobulin κ light chain transgene (as described above), rodent strains can be selected based on the expression of the human Vλ domain from said transgene, etc. In some embodiments, Mice can be 1(VI-1) mice, which included 18 human V H Gene segments, all people D H Gene segments and all J H Gene segments. VI-1 mice may also include 16 human Vκ gene segments and all human Jκ gene segments. In some implementations, Mice can be 2(VI-2) mice, which included 39 human V H Gene segments, all people D H Gene segments and all J H Gene segments. VI-2 mice may also include 30 human Vκ gene segments and all human Jκ gene segments. In some implementations, Mice can be 3(VI-3) mice, which included 80 human V H Gene segments, all people D H Gene segments and all J H Gene segments. VI-3 mice may also include 40 human Vκ gene segments and all human Jκ gene segments.

[0447] Alternatively and / or additionally, in some embodiments, the germline genome of the non-human animal as described herein also includes deleted, inactivated, functionally silenced, or other non-functional endogenous immunoglobulin λ light chain loci. Genetically modified genes or loci can be created by using the methods described herein and / or methods known in the art.

[0448] Genetically engineered proto-human animals can be identified based on the presence of an engineered Igκ light chain locus in their germline genome and / or the expression of antibodies with human Vλ domains and non-human or human Cλ domains in non-human animal tissues or cells. Genetically engineered proto-human animals can then be used to breed other non-human animals carrying the engineered endogenous immunoglobulin κ light chain locus, thereby producing a group of non-human animals, each carrying one or more copies of the engineered endogenous immunoglobulin κ light chain locus. Furthermore, genetically engineered non-human animals carrying the engineered immunoglobulin κ light chain locus as described herein can be further bred together with other genetically engineered non-human animals carrying other transgenes (e.g., human immunoglobulin genes) or engineered immunoglobulin loci.

[0449] Genetically engineered nonhuman animals containing selected systems can also be produced, which allow for the regulation, direction, induction, and / or cell type-specific expression of transgenic or integrated sequences. For example, nonhuman animals as described herein can be engineered to contain one or more sequences encoding the human Vλ domain of conditionally expressed antibodies (e.g., reviewed in Rajewski, K. et al., 1996, J. Clin. Invest. 98(3):600-3, which is incorporated herein by reference in its entirety). Exemplary systems include the Cre / loxP recombinase system of phage P1 (see, for example, Lakso, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6, which is incorporated herein by reference in its entirety), and the FLP / Frt recombinase system of Saccharomyces cerevisiae (O'Gorman, S. et al., 1991, Science 251:1351-5, which is incorporated herein by reference in its entirety). Such animals can be provided by constructing “dual” genetically engineered animals, for example by mating two genetically engineered animals, one containing a transgene with selected modifications (e.g., an engineered Igκ light chain locus, as described herein), and the other containing a transgene encoding a recombinase (e.g., Cre recombinase).

[0450] The non-human animals described herein can be prepared as described above, or using methods known in the art, to contain additional human, humanized, or other engineered genes, generally depending on the intended use of the non-human animal. The genetic material of such human, humanized, or other engineered genes can be introduced by further altering the genome of cells (e.g., embryonic stem cells) having the genetically modified or altered cells described above, or as needed by breeding techniques known in the art and other genetically modified or engineered strains. In some embodiments, non-human animals as described herein are prepared to further contain human IgH and / or Igκ light chain genes or gene segments (see, for example, Murphy, AJ et al., (2014) Proc. Natl. Acad. Sci. USA 111(14):5153-5158; U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940 and 8,791,323; U.S. Patent No. 8,791,323; and U.S. Patent Application Publication No. 2013 / 0096287A1; each of which is incorporated herein by reference in its entirety).

[0451] In some implementations, non-human animals, as described herein, can be prepared by introducing the targeting vector described herein into cells from modified or engineered strains. For example, the targeting vector described herein can be introduced... In mice. Mice express antibodies having both fully human variable regions and mouse constant regions. In another example, targeting vectors as described herein can be introduced into engineered mice, as described in U.S. Patent Nos. 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662, and 9,163,092, which are incorporated herein by reference in their entirety. In some embodiments, non-human animals as described herein are prepared to further comprise human immunoglobulin genes (variable and / or constant region genes). In some embodiments, the non-human animals as described herein comprise an engineered Igκ light chain locus as described herein, and genetic material from a heterologous species (e.g., human), wherein said genetic material (in whole or in part) encodes one or more human heavy chain and / or Igκ light chain variable regions.

[0452] For example, as described herein, non-human animals containing the engineered Igκ light chain locus as described herein may also contain (e.g., via crossbreeding or multiple gene targeting strategies) one or more modifications, as described in Murphy, AJ et al., (2014) Proc. Natl. Acad. Sci. USA 111(14):5153-8; Macdonald, LE et al., 2014, Proc. Natl. Acad. Sci. USA 111(14):5147-52; US Patent Nos. 8,502,018, 8,642,835, 8,697,940 and 8,791,323, all of which are incorporated herein by reference in their entirety. In some embodiments, rodents containing the engineered endogenous immunoglobulin κ light chain locus as described herein are hybridized with rodents containing humanized immunoglobulin heavy chain and / or immunoglobulin κ light chain variable region loci (see, for example, U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940 and / or 8,791,323, which are incorporated herein by reference in their entirety). In some embodiments, rodents containing the engineered immunoglobulin κ light chain locus as described herein are hybridized with rodents containing humanized immunoglobulin heavy chain loci (see, for example, U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940 and / or 8,791,323, which are incorporated herein by reference in their entirety) and inactivated endogenous immunoglobulin λ light chain loci (see, for example, U.S. Patent Nos. 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662 and 9,163,092, which are incorporated herein by reference in their entirety).

[0453] Although embodiments for constructing engineered immunoglobulin κ light chain loci in mice (i.e., mice having engineered immunoglobulin κ light chain loci characterized by the presence of multiple human Vλ and Jλ gene segments operatively linked to a mouse or human Cλ gene located at the mouse Cκ gene, such that antibodies containing human Vλ and mouse or human Cλ domains are expressed) have been described extensively herein, other non-human animals comprising engineered endogenous immunoglobulin κ light chain loci are also provided. Such non-human animals include any animal that can be genetically modified to express antibodies as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys), etc. For example, for non-human animals from which suitable genetically modifiable ES cells are not readily available, other methods are used to prepare said genetically modified non-human animals. Such methods include, for example, modifying the genome of a non-ES cell (e.g., fibroblasts or induced pluripotent cells) and using somatic cell nuclear transfer (SCNT) technology to transfer the genetically modified genome into a suitable cell, such as an enucleated oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo.

[0454] Methods for modifying the germline genomes of nonhuman animals (e.g., pig, cattle, rodent, chicken, etc.) include, for example, using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or Cas proteins (i.e., the CRISPR / Cas system) to include the engineered immunoglobulin κ light chain locus as described herein. Guidelines for methods of modifying the germline genomes of nonhuman animals can be found in U.S. Patent Applications Nos. 14 / 747,461 (filed June 23, 2015), 14 / 948,221 (filed November 20, 2015), and 14 / 974,623 (filed December 18, 2015); these patent applications are incorporated herein by reference in their entirety.

[0455] In some embodiments, the non-human animals described herein are mammals. In some embodiments, the non-human animals described herein are small mammals, such as jerboas (Dipodoidea) or the Muroidea superfamily. In some embodiments, the genetically modified animals described herein are rodents. In some embodiments, the rodents described herein are selected from mice, rats, and hamsters. In some embodiments, the rodents described herein are selected from the Muroidea superfamily. In some embodiments, the genetically modified animals described herein are selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock climbing mice, tailed rats, Madagascar rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., moles, bamboo rats, and mole rats). In some embodiments, the genetically modified rodents described herein are selected from true mice or rats (Muridae), gerbils, spiny mice, and crested rats. In some embodiments, the genetically modified mice described herein are members of the Muridae family. In some embodiments, the non-human animals described herein are rodents. In some embodiments, the rodents described herein are selected from mice and rats. In some embodiments, the non-human animals described herein are mice.

[0456] In some embodiments, the non-human animal described herein is a rodent, which is a mouse selected from the following C57BL strains: C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In some implementations, the mice described herein are selected from the following groups of 129 strains: strains 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, for example, Festing et al., 1999, Mammalian Genome 10:836; Auerbach, W. et al., 2000, Biotechniques 29(5):1024-1028,1030,1032, each of which is incorporated herein by reference in its entirety). In some embodiments, the genetically modified mice described herein are a mixture of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In some embodiments, the mice described herein are a mixture of the aforementioned 129 strain or a mixture of the aforementioned BL / 6 strain. In some embodiments, the 129 strain in the mixed strains described herein is the 129S6 (129 / SvEvTac) strain. In some embodiments, the mice described herein are the BALB strain, such as the BALB / c strain. In some embodiments, the mice described herein are a mixture of the BALB strain and another of the aforementioned strains.

[0457] In some embodiments, the non-human animal described herein is a rat. In some embodiments, the rats described herein are selected from Wistar rats, the LEA strain, the Sprague Dawley strain, the Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strains described herein are mixtures of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0458] Rat pluripotent and / or totipotent cells can be derived from any rat strain, including, for example, the ACI rat strain (originally derived from an inbred strain of the August and Copenhagen strains), the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or the Fisher rat strain, such as Fisher F344 or Fisher F6. Rat pluripotent and / or totipotent cells can also be derived from mixed strains of two or more of the above strains. For example, rat pluripotent and / or totipotent cells can be derived from the DA strain or the ACI strain. The ACI rat strain is characterized by black wild rat markings, a white abdomen and feet, and the RT1av1 haplotype. These strains can be obtained from a variety of sources, including Harlan Laboratories. An example of a rat ES cell line derived from ACI rats is the ACI.G1 rat ES cell. The DA rat strain is characterized by guinea pig skin and the RT1av1 haplotype. These rats can be obtained from a variety of sources, including Charles River and Harlan Laboratories. Examples of rat ES cell lines derived from DA rats are the DA.2B and DA.2C rat ES cell lines. In some embodiments, rat pluripotent cells and / or pluripotent cells are derived from inbred rat strains (see, for example, U.S. Patent Application Publication No. 2014-0235933A1, published August 21, 2014, which is incorporated herein by reference in its entirety). Guidelines for modifying the rat genome (e.g., in rat ES cells) using the methods and / or constructs described herein can be found in U.S. Patent Application Publications Nos. 2014-0310828 and 2017-0204430, both of which are incorporated herein by reference in their entirety.

[0459] Specific exemplary implementation scheme - immunoglobulin heavy chain locus

[0460] In some embodiments, the provided non-human animal contains an engineered immunoglobulin κ light chain locus as described herein, and also contains an engineered IgH locus (or allele), characterized by the presence of multiple human V genes arranged in a germline configuration and operatively linked to the non-human immunoglobulin heavy chain constant region genes, enhancers, and regulatory regions. H D H With J H Gene segments. In some embodiments, the engineered immunoglobulin heavy chain locus (or allele) as described herein comprises one or more human V gene segments operatively linked to the non-human immunoglobulin heavy chain constant region. H Gene segment, one or more individuals D HGene segments and one or more individuals J H Gene segment. In some embodiments, the engineered immunoglobulin heavy chain locus (or allele) contains at least one human V gene. H Gene segment V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1 or any combination thereof. In some embodiments, the engineered IgH locus (or allele) contains at least one human D H Gene segment D H 1-1, D H 2-2, D H 3-3, D H 4-4, DH 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H 7-27 or any combination thereof. In some embodiments, the engineered immunoglobulin heavy chain locus (or allele) contains at least one human J H Gene segment J H 1. J H 2. J H 3. J H 4. J H 5. J H 6 or any combination thereof.

[0461] This disclosure recognizes that non-human animals, as described herein, utilize human heavy chain variable region gene segments contained in their genomes in their antibody selection and production mechanisms (e.g., recombination and somatic hypermutation). Therefore, in various embodiments, the human immunoglobulin heavy chain variable domain produced by the non-human animals described herein is encoded by human heavy chain variable region gene segments contained in their genomes or somatic hypermutated variants.

[0462] In some embodiments, a non-human animal is provided whose genome contains an engineered endogenous immunoglobulin κ light chain locus, wherein the non-human animal contains B cells containing human heavy chain variable region sequences, human λ light chain variable region sequences, and / or somatic hypermutated human κ light chain variable region sequences. In some embodiments, the human heavy chain variable region sequences, human λ light chain variable region sequences, and / or human κ light chain variable region sequences present in the B cells of the mice of this disclosure have 1, 2, 3, 4, 5, or more somatic hypermutations. Methods for identifying the source gene segment in a mature antibody sequence are known to those skilled in the art. For example, various tools can be used to assist in this analysis, such as DNAPLOT, IMGT / V-QUEST, JOINSOLVER, SoDA, and Ab-origin.

[0463] In some embodiments, the non-human immunoglobulin heavy chain constant region comprises one or more non-human immunoglobulin heavy chain constant region genes, such as immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin E (IgE), and immunoglobulin A (IgA). In some embodiments, the non-human immunoglobulin heavy chain constant region comprises rodent IgM, rodent IgD, rodent IgG3, rodent IgG1, rodent IgG2b, rodent IgG2a, rodent IgE, and rodent IgA constant region genes. In some embodiments, the human V H D H and J H The gene segment is operatively linked to one or more non-human immunoglobulin heavy chain enhancers (i.e., enhancer sequences or enhancer regions). In some embodiments, the human V H D H and J H The gene segment is operatively linked to one or more non-human immunoglobulin heavy chain regulatory regions (or regulatory sequences). In some embodiments, the human V H D H and J H The gene segment is operatively linked to one or more non-human immunoglobulin heavy chain enhancers (or enhancer sequences) and one or more non-human immunoglobulin heavy chain regulatory regions (or regulatory sequences).

[0464] In some embodiments, the engineered immunoglobulin heavy chain locus described herein does not contain an endogenous Adam6 gene. In some embodiments, the engineered immunoglobulin heavy chain locus described herein does not contain an endogenous Adam6 gene (or Adam6 coding sequence) from the same phylogenetic genome found in the germline genome of a wild-type nonhuman animal of the same species. In some embodiments, the engineered immunoglobulin heavy chain locus described herein does not contain the human Adam6 pseudogene. In some embodiments, the engineered immunoglobulin heavy chain locus described herein includes an insertion of at least one nucleotide sequence encoding one or more nonhuman (e.g., rodent) Adam6 polypeptides, their functional orthologs, functional homologs, or functional fragments. In some embodiments, the insertion may be located outside the engineered immunoglobulin heavy chain locus described herein (e.g., but not limited to, V...). H The 5' most upstream of the gene segment, within an engineered immunoglobulin heavy chain locus, or at other locations in the germline genome of non-human animals (e.g., but not limited to, randomly introduced non-human Adam6 coding sequences), cells, or tissues.

[0465] In various implementation schemes, such as those described herein, non-human animals, non-human cells, or non-human tissues do not detectably express (all or part) endogenous non-human V within the antibody molecule. H Region. In various embodiments, the non-human animal, non-human cell, or non-human tissue provided herein does not contain (or lacks or contains deletions) one or more nucleotide sequences that encode endogenous non-human V in the antibody molecule. H Region (e.g., V) H D H , and / or J H In various embodiments, such as the non-human animal, non-human cell, or non-human tissue provided herein, the germline genome includes endogenous non-human V. H D H and J H Deletion (complete or partial) of a gene segment. In various implementation schemes, the provided non-human animal is fertile.

[0466] Guidelines for generating targeting vectors, nonhuman cells, and animals carrying such engineered immunoglobulin heavy chain loci (or alleles) are found in U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, and 8,791,323, each of which is incorporated herein by reference in its entirety. Various techniques in the art are known to those skilled in the art for achieving such genetic engineering and / or manipulating nonhuman animal (e.g., mammalian) genomes, or for preparing, providing, or manufacturing such sequences for introduction into the germline genomes of nonhuman animals.

[0467] Specific exemplary implementation scheme - Immunoglobulin κ light chain locus

[0468] In some embodiments, the provided non-human animal comprises an engineered endogenous immunoglobulin κ light chain locus, said engineered endogenous immunoglobulin κ light chain locus being characterized by the presence of multiple human Vλ and Jλ gene segments arranged in a germline conformation (i.e., unrearranged and associated with recombination signal sequences) and inserted and operatively linked upstream of a non-human or human Cλ gene, said non-human or human Cλ gene being inserted at the location of the non-human Cκ gene. As described herein, such engineered endogenous immunoglobulin κ light chain locus also comprises a non-human immunoglobulin κ light chain enhancer region (or enhancer sequence). In some embodiments, the engineered endogenous immunoglobulin κ light chain locus comprises one or more human Vλ gene segments and one or more human Jλ gene segments operatively linked to a non-human or human Cλ gene. In some embodiments, the engineered endogenous immunoglobulin κ light chain locus (or allele) comprises at least one human Vλ gene segment appearing in cluster A of the human immunoglobulin λ light chain locus; in some embodiments, clusters A and B of the human immunoglobulin λ light chain locus; in some embodiments, clusters A, B, and C of the human immunoglobulin λ light chain locus. In some embodiments, the engineered immunoglobulin κ light chain locus (or allele) comprises at least one human Vλ gene segment: Vλ4-69, Vλ8-61, Vλ4-60, Vλ6-57, Vλ10-54, Vλ5-52, Vλ1-51, Vλ9-49, Vλ1-47, Vλ7-46, Vλ5-45, Vλ1-44, Vλ7-43. Vλ1-40, Vλ5-39, Vλ5-37, Vλ1-36, Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, or any combination thereof. In some embodiments, the engineered Igκ light chain locus (or allele) contains at least one human Jλ gene segment Jλ1, Jλ2, Jλ3, Jλ6, Jλ7, or any combination thereof.

[0469] The present invention recognizes that non-human animals, as described herein, will utilize human λ light chain variable region gene segments contained in their genomes in their antibody selection and production mechanisms (e.g., recombination and somatic hypermutation). Therefore, in various embodiments, the human immunoglobulin λ light chain variable domain produced by the non-human animals described herein is encoded by human λ light chain variable region gene segments contained in their genomes or in their somatic hypermutations.

[0470] In some embodiments, a non-human animal is provided whose genome contains an eng...

Claims

1. A method for generating antibodies, the method comprising the following steps: (a) Expressing a first nucleotide sequence encoding an immunoglobulin heavy chain in a host cell, wherein the first nucleotide sequence comprises a human heavy chain variable region sequence; (b) Expression in host cells of a second nucleotide sequence encoding the immunoglobulin λ light chain, wherein the second nucleotide sequence comprises a human λ light chain variable region sequence encoding a human λ light chain variable domain, the human λ light chain variable domain being identified in genetically modified mice whose germline genome comprises: The first engineered endogenous immunoglobulin κ light chain locus comprises: (i) One or more human Vλ gene segments, (ii) One or more human Jλ gene segments, and (iii) A mouse Cλ gene or a human Cλ gene, Wherein, the one or more human Vλ gene segments in (i) and the one or more human Jλ gene segments in (ii) replace one or more endogenous mouse Vκ gene segments and one or more endogenous mouse Jκ gene segments. Wherein the one or more human Vλ gene segments of (i) and the one or more human Jλ gene segments of (ii) are operatively linked to the one mouse Cλ gene or the one human Cλ gene of (iii); and The genetically modified mice in this case lack the mouse Cκ gene at the first engineered endogenous immunoglobulin κ light chain locus. (c) Culturing the host cells to express immunoglobulin light chains and immunoglobulin heavy chains and to form antibodies, and (d) Obtaining the antibody from the host cell or host cell culture, wherein the antibody comprises an immunoglobulin λ light chain, the immunoglobulin λ light chain comprising a human immunoglobulin λ light chain variable domain.

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

3. The method of claim 1, wherein the genetically modified mouse is heterozygous for the first engineered endogenous immunoglobulin κ light chain locus.

4. The method of claim 3, wherein the germline genome of the genetically modified mouse comprises a second engineered endogenous immunoglobulin κ light chain locus, the second engineered endogenous immunoglobulin κ light chain locus comprising: (a) One or more human Vκ gene segments, and (b) One or more human Jκ gene segments, The one or more human Vκ gene segments and the one or more human Jκ gene segments are operatively linked to the Cκ gene.

5. The method of any of the preceding claims, wherein the germline genome of the mouse further comprises: An engineered endogenous immunoglobulin heavy chain locus, wherein the engineered endogenous immunoglobulin heavy chain locus comprises: (a) one or more human V H gene segments, (b) one or more human D H gene segments, and (c) one or more human J H gene segments, One or more persons V in (a) H Gene segments, (b) of one or more individuals D H Gene segments and (c) of one or more individuals J H The gene segment is operatively linked to one or more mouse immunoglobulin heavy chain constant region genes at the engineered endogenous immunoglobulin heavy chain gene locus.

6. The method of claim 5, wherein the one or more human V H gene segments of (a), the one or more human D H gene segments of (b), and the one or more human J H gene segments of (c) are replaced with one or more mouse V H gene segments, one or more mouse D H gene segments, or one or more mouse J H gene segments, or a combination thereof.​​​​​​ 7. The method of claim 5, wherein the engineered endogenous immunoglobulin heavy chain locus further comprises: (i) one or more human V H non-coding sequences, each of which is naturally adjacent to a human V H non-coding sequences, each of which is naturally adjacent to a human V H at least one of the gene segments, wherein the one or more V H non-coding sequences, each of which is naturally adjacent to a human V H gene segments occur within an endogenous human immunoglobulin heavy chain locus; (ii) One or more people D H Non-coding sequences, the one or more people D H Each of the non-coding sequences is adjacent to one or more persons D H At least one of the gene segments, wherein the one or more D H Each of the non-coding sequences is naturally adjacent to the human D locus in the endogenous human immunoglobulin heavy chain gene. H Gene segments appear; (iii) One or more people J H Non-coding sequences, the one or more people J H Each of the non-coding sequences is adjacent to one or more persons J H At least one of the gene segments, wherein the one or more J H Each of the non-coding sequences is naturally adjacent to the human J locus in the endogenous human immunoglobulin heavy chain gene. H Gene segment appears; or (iv) Any combination thereof.

8. The method of claim 5, wherein the one or more mouse immunoglobulin heavy chain constant region genes are one or more endogenous mouse immunoglobulin heavy chain constant region genes.

9. The method of claim 5, wherein: (i) the one or more persons V H Gene segment includes V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1 or any combination thereof, (ii) the one or more persons D H Gene segments include D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H 7-27 or any combination thereof, and (iii) The one or more persons J H Gene segments include J H 1. J H 2. J H 3. J H 4. J H 5. J H 6 or any combination thereof.

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

11. The method of any one of claims 1-4, wherein (i) the one or more human Vλ gene segments and (ii) the one or more human Jλ gene segments replace one or more mouse Vκ gene segments, one or more mouse Jκ gene segments, or a combination thereof.

12. The method of any one of claims 1-4, wherein the first engineered endogenous immunoglobulin κ light chain locus further comprises a κ light chain non-coding sequence located between the one or more human Vλ gene segments and the one or more human Jλ gene segments.

13. The method of claim 12, wherein the κ light chain non-coding sequence has a sequence that naturally occurs between the human Vκ4-1 gene segment and the human Jκ1 gene segment at the endogenous human immunoglobulin κ light chain locus.

14. The method according to any one of claims 1-4, wherein the endogenous Vλ gene segment, the endogenous Jλ gene segment, and the endogenous Cλ gene are all or partially deleted.

15. The method of any one of claims 1-4, wherein each of the human immunoglobulin λ light chain variable domains is encoded by a rearranged human immunoglobulin λ light chain variable region sequence, the rearranged human immunoglobulin λ light chain variable region sequence comprising (i) one of the one or more human Vλ gene segments or a somatic hypermutant variant thereof, and (ii) one of the one or more human Jλ gene segments or a somatic hypermutant variant thereof.

16. The method of any one of claims 1-4, wherein the germline genome further comprises a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element.

17. The method of any one of claims 1-4, wherein the mouse Cλ gene or human Cλ gene is a mouse Cλ gene.

18. The method of any one of claims 1-4, wherein the mouse Cλ gene or the human Cλ gene is a human Cλ gene.

19. A method for generating a sequence of variable structural domains in a human λ light chain, the method comprising the following steps: (a) Immunizing genetically modified mice with a target antigen, said genetically modified mice having a germline genome, said germline genome containing a first engineered endogenous immunoglobulin κ light chain locus, said first engineered endogenous immunoglobulin κ light chain locus comprising: (i) One or more human Vλ gene segments, (ii) One or more human Jλ gene segments, and (iii) A mouse Cλ gene or a human Cλ gene, Wherein, (i) the one or more human Vλ gene segments and (ii) the one or more human Jλ gene segments replace one or more endogenous mouse Vκ gene segments and one or more endogenous mouse Jκ gene segments; Wherein, the one or more human Vλ gene segments in (i) and the one or more human Jλ gene segments in (ii) are operatively linked to the one mouse Cλ gene or the one human Cλ gene in (iii), and The genetically modified mice in this case lack the mouse Cκ gene at the first engineered endogenous immunoglobulin κ light chain locus. (b) Recovery from the genetically modified mice: (i) An antibody that binds to the target antigen, or (ii) Cells expressing antibodies that bind to the target antigen; and (c) Confirm the sequence of the variable domain of the human immunoglobulin λ light chain in the antibody, which specifically binds to the target antigen and is produced by the genetically modified mouse.

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

21. The method of claim 19, wherein the genetically modified mouse is heterozygous for the first engineered endogenous immunoglobulin κ light chain locus.

22. The method of claim 21, wherein the germline genome of the genetically modified mouse comprises a second engineered endogenous immunoglobulin κ light chain locus, the second engineered endogenous immunoglobulin κ light chain locus comprising: (a) One or more human Vκ gene segments, and (b) One or more human Jκ gene segments, The one or more human Vκ gene segments and the one or more human Jκ gene segments are operatively linked to the Cκ gene.

23. The method of any of the preceding claims, wherein the germline genome of the mouse further comprises: An engineered endogenous immunoglobulin heavy chain locus, wherein the engineered endogenous immunoglobulin heavy chain locus comprises: (a) One or more people V H Gene segments, (b) One or more people D H Gene segments, and (c) One or more people J H Gene segments, One or more persons V in (a) H Gene segments, (b) of one or more individuals D H Gene segments and (c) of one or more individuals J H The gene segment is operatively linked to one or more mouse immunoglobulin heavy chain constant region genes at the engineered endogenous immunoglobulin heavy chain gene locus.

24. The method of claim 23, wherein the one or more persons in (a) V H Gene segments, (b) of one or more individuals D H Gene segments and (c) of one or more individuals J H Gene segment replacement of one or more mouse V H Gene segment, one or more mouse D H Gene segment, one or more mouse J H Gene segments, or combinations thereof.

25. The method of claim 23, wherein the engineered endogenous immunoglobulin heavy chain locus further comprises: (i) One or more people V H Non-coding sequence, the one or more persons V H Each of the non-coding sequences is adjacent to the one or more persons V H At least one of the gene segments, wherein the one or more V H Each of the non-coding sequences is naturally adjacent to human V at the endogenous human immunoglobulin heavy chain locus. H Gene segments appear; (ii) One or more people D H Non-coding sequences, the one or more people D H Each of the non-coding sequences is adjacent to one or more persons D H At least one of the gene segments, wherein the one or more D H Each of the non-coding sequences is naturally adjacent to the human D locus in the endogenous human immunoglobulin heavy chain gene. H Gene segments appear; (iii) One or more people J H Non-coding sequences, the one or more people J H Each of the non-coding sequences is adjacent to one or more persons J H At least one of the gene segments, wherein the one or more J H Each of the non-coding sequences is naturally adjacent to the human J locus in the endogenous human immunoglobulin heavy chain gene. H Gene segment appears; or (iv) Any combination thereof.

26. The method of claim 23, wherein the one or more mouse immunoglobulin heavy chain constant region genes are one or more endogenous mouse immunoglobulin heavy chain constant region genes.

27. The method of claim 23, wherein: (i) the one or more persons V H Gene segment includes V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1 or combinations thereof, (ii) the one or more persons D H Gene segments include D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H 7-27 or combinations thereof, and (iii) The one or more persons J H Gene segments include J H 1. J H 2. J H 3. J H 4. J H 5. J H 6 or combinations thereof.

28. The method of claim 23, wherein the genetically modified mouse is homozygous for the engineered endogenous immunoglobulin heavy chain locus.

29. The method of any one of claims 19-22, wherein (i) the one or more human Vλ gene segments and (ii) the one or more human Jλ gene segments replace one or more mouse Vκ gene segments, one or more mouse Jκ gene segments, or combinations thereof.

30. The method of any one of claims 19-22, wherein the first engineered endogenous immunoglobulin κ light chain locus further comprises a κ light chain non-coding sequence located between the one or more human Vλ gene segments and the one or more human Jλ gene segments.

31. The method of claim 30, wherein the κ light chain non-coding sequence has a sequence that naturally occurs between the human Vκ4-1 gene segment and the human Jκ1 gene segment at the endogenous human immunoglobulin κ light chain locus.

32. The method according to any one of claims 19-22, wherein the endogenous Vλ gene segment, the endogenous Jλ gene segment, and the endogenous Cλ gene are all or partially deleted.

33. The method of any one of claims 19-22, wherein each of the human immunoglobulin λ light chain variable domains is encoded by a rearranged human immunoglobulin λ light chain variable region sequence comprising (i) one of the one or more human Vλ gene segments or a somatic hypermutant variant thereof, and (ii) one of the one or more human Jλ gene segments or a somatic hypermutant variant thereof.

34. The method of any one of claims 19-22, wherein the germline genome further comprises a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element.

35. The method of any one of claims 19-22, wherein the mouse Cλ gene or human Cλ gene is a mouse Cλ gene.

36. The method of any one of claims 19-22, wherein the mouse Cλ gene or the human Cλ gene is a human Cλ gene.

37. A method for generating a human λ light chain variable region sequence, the method comprising the following steps: (a) Immunizing genetically modified mice with a target antigen, said genetically modified mice having a germline genome, said germline genome containing a first engineered endogenous immunoglobulin κ light chain locus, said first engineered endogenous immunoglobulin κ light chain locus comprising: (i) One or more human Vλ gene segments, (ii) One or more human Jλ gene segments, and (iii) A mouse Cλ gene or a human Cλ gene, Wherein, (i) the one or more human Vλ gene segments and (ii) the one or more human Jλ gene segments replace one or more endogenous mouse Vκ gene segments and one or more endogenous mouse Jκ gene segments; Wherein, the one or more human Vλ gene segments in (i) and the one or more human Jλ gene segments in (ii) are operatively linked to the one mouse Cλ gene or the one human Cλ gene in (iii), and The genetically modified mice in question lack the mouse Cκ gene at the first engineered endogenous immunoglobulin κ light chain locus. (b) Recovery from the genetically modified mice: (i) An antibody that binds to the target antigen, or (ii) Cells expressing antibodies that bind to the target antigen; and (c) Identify the human immunoglobulin λ light chain variable region sequence encoding the variable domain of the human immunoglobulin λ light chain in the antibody, which specifically binds to the target antigen and is produced by the genetically modified mouse.

38. The method of claim 37, wherein the genetically modified mouse is homozygous for the first engineered endogenous immunoglobulin κ light chain locus.

39. The method of claim 37, wherein the genetically modified mouse is heterozygous for the first engineered endogenous immunoglobulin κ light chain locus.

40. The method of claim 39, wherein the germline genome of the genetically modified mouse comprises a second engineered endogenous immunoglobulin κ light chain locus, the second engineered endogenous immunoglobulin κ light chain locus comprising: (a) One or more human Vκ gene segments, and (b) One or more human Jκ gene segments, The one or more human Vκ gene segments and the one or more human Jκ gene segments are operatively linked to the Cκ gene.

41. The method of any of the preceding claims, wherein the germline genome of the mouse further comprises: An engineered endogenous immunoglobulin heavy chain locus, wherein the engineered endogenous immunoglobulin heavy chain locus comprises: (a) One or more people V H Gene segments, (b) One or more people D H Gene segments, and (c) One or more people J H Gene segments, One or more persons V in (a) H Gene segments, (b) of one or more individuals D H Gene segments and (c) of one or more individuals J H The gene segment is operatively linked to one or more mouse immunoglobulin heavy chain constant region genes at the engineered endogenous immunoglobulin heavy chain gene locus.

42. The method of claim 41, wherein the one or more persons in (a) V H Gene segments, (b) of one or more individuals D H Gene segments and (c) of one or more individuals J H Gene segment replacement of one or more mouse V H Gene segment, one or more mouse D H Gene segment, one or more mouse J H Gene segments, or combinations thereof.

43. The method of claim 41, wherein the engineered endogenous immunoglobulin heavy chain locus further comprises: (i) One or more people V H Non-coding sequence, the one or more persons V H Each of the non-coding sequences is adjacent to the one or more persons V H At least one of the gene segments, wherein the one or more V H Each of the non-coding sequences is naturally adjacent to human V at the endogenous human immunoglobulin heavy chain locus. H Gene segments appear; (ii) One or more people D H Non-coding sequences, the one or more people D H Each of the non-coding sequences is adjacent to one or more persons D H At least one of the gene segments, wherein the one or more D H Each of the non-coding sequences is naturally adjacent to the human D locus in the endogenous human immunoglobulin heavy chain gene. H Gene segments appear; (iii) One or more people J H Non-coding sequences, the one or more people J H Each of the non-coding sequences is adjacent to one or more persons J H At least one of the gene segments, wherein the one or more J H Each of the non-coding sequences is naturally adjacent to the human J locus in the endogenous human immunoglobulin heavy chain gene. H Gene segment appears; or (iv) Any combination thereof.

44. The method of claim 41, wherein the one or more mouse immunoglobulin heavy chain constant region genes are one or more endogenous mouse immunoglobulin heavy chain constant region genes.

45. The method of claim 41, wherein: (i) the one or more persons V H Gene segment includes V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1 or combinations thereof, (ii) the one or more persons D H Gene segments include D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H 7-27 or combinations thereof, and (iii) The one or more persons J H Gene segments include J H 1. J H 2. J H 3. J H 4. J H 5. J H 6 or combinations thereof.

46. ​​The method of claim 41, wherein the genetically modified mouse is homozygous for the engineered endogenous immunoglobulin heavy chain locus.

47. The method of any one of claims 37-40, wherein (i) the one or more human Vλ gene segments and (ii) the one or more human Jλ gene segments replace one or more mouse Vκ gene segments, one or more mouse Jκ gene segments, or combinations thereof.

48. The method of any one of claims 37-40, wherein the first engineered endogenous immunoglobulin κ light chain locus further comprises a κ light chain non-coding sequence located between the one or more human Vλ gene segments and the one or more human Jλ gene segments.

49. The method of claim 48, wherein the κ light chain non-coding sequence has a sequence that naturally occurs between the human Vκ4-1 gene segment and the human Jκ1 gene segment at the endogenous human immunoglobulin κ light chain locus.

50. The method of any one of claims 37-40, wherein the endogenous Vλ gene segment, the endogenous Jλ gene segment, and the endogenous Cλ gene are all or partially deleted.

51. The method of any one of claims 37-40, wherein each of the human immunoglobulin λ light chain variable domains is encoded by a rearranged human immunoglobulin λ light chain variable region sequence comprising (i) one of the one or more human Vλ gene segments or a somatic hypermutant variant thereof, and (ii) one of the one or more human Jλ gene segments or a somatic hypermutant variant thereof.

52. The method of any one of claims 37-40, wherein the germline genome further comprises a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element.

53. The method of any one of claims 37-40, wherein the mouse Cλ gene or human Cλ gene is a mouse Cλ gene.

54. The method of any one of claims 37-40, wherein the mouse Cλ gene or the human Cλ gene is a human Cλ gene.

55. A method for preparing genetically modified embryonic stem (ES) cells, the method comprising genetically modifying the ES cells such that their genome includes: The first engineered endogenous immunoglobulin κ light chain locus comprises: (a) One or more human Vλ gene segments, (b) One or more human Jλ gene segments, and (c) A mouse Cλ gene or a human Cλ gene, Wherein, the one or more human Vλ gene segments in (a) and the one or more human Jλ gene segments in (b) replace one or more endogenous mouse Vκ gene segments and one or more endogenous mouse Jκ gene segments, and The one or more human Vλ gene segments in (a) and the one or more human Jλ gene segments in (b) are operatively linked to the one mouse Cλ gene or the one human Cλ gene in (c). and The genetically modified ES cells therein lack the mouse Cκ gene at the first engineered endogenous immunoglobulin κ light chain locus.

56. The method of claim 55, wherein the genetically modified ES cells are homozygous for the first engineered endogenous immunoglobulin κ light chain locus.

57. The method of claim 55, wherein the genetically modified ES cells are heterozygous for the first engineered endogenous immunoglobulin κ light chain locus.

58. The method of claim 57, wherein the genome of the genetically modified ES cell comprises a second engineered endogenous immunoglobulin κ light chain locus, the second engineered endogenous immunoglobulin κ light chain locus comprising: (a) One or more human Vκ gene segments, and (b) One or more human Jκ gene segments, The one or more human Vκ gene segments and the one or more human Jκ gene segments are operatively linked to the Cκ gene.

59. The method of any of the preceding claims, wherein the genome of the genetically modified ES cell further comprises: An engineered endogenous immunoglobulin heavy chain locus, wherein the engineered endogenous immunoglobulin heavy chain locus comprises: (a) One or more people V H Gene segments, (b) One or more people D H Gene segments, and (c) One or more people J H Gene segments, One or more persons V in (a) H Gene segments, (b) of one or more individuals D H Gene segments and (c) of one or more individuals J H The gene segment is operatively linked to one or more mouse immunoglobulin heavy chain constant region genes at the engineered endogenous immunoglobulin heavy chain gene locus.

60. The method of claim 59, wherein the one or more persons in (a) V H Gene segments, (b) of one or more individuals D H Gene segments and (c) of one or more individuals J H Gene segment replacement of one or more mouse V H Gene segment, one or more mouse D H Gene segment, one or more mouse J H Gene segments, or combinations thereof.

61. The method of claim 59, wherein the engineered endogenous immunoglobulin heavy chain locus further comprises: (i) One or more people V H Non-coding sequence, the one or more persons V H Each of the non-coding sequences is adjacent to the one or more persons V H At least one of the gene segments, wherein the one or more V H Each of the non-coding sequences is naturally adjacent to human V at the endogenous human immunoglobulin heavy chain locus. H Gene segments appear; (ii) One or more people D H Non-coding sequences, the one or more people D H Each of the non-coding sequences is adjacent to one or more persons D H At least one of the gene segments, wherein the one or more D H Each of the non-coding sequences is naturally adjacent to the human D locus in the endogenous human immunoglobulin heavy chain gene. H Gene segments appear; (iii) One or more people J H Non-coding sequences, the one or more people J H Each of the non-coding sequences is adjacent to one or more persons J H At least one of the gene segments, wherein the one or more J H Each of the non-coding sequences is naturally adjacent to the human J locus in the endogenous human immunoglobulin heavy chain gene. H Gene segment appears; or (iv) Any combination thereof.

62. The method of claim 59, wherein the one or more mouse immunoglobulin heavy chain constant region genes are one or more endogenous mouse immunoglobulin heavy chain constant region genes.

63. The method of claim 59, wherein: (i) the one or more persons V H Gene segment includes V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1 or combinations thereof, (ii) the one or more persons D H Gene segments include D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, D H 7-27 or combinations thereof, and (iii) The one or more persons J H Gene segments include J H 1. J H 2. J H 3. J H 4. J H 5. J H 6 or combinations thereof.

64. The method of claim 59, wherein the genetically modified ES cells are homozygous for the engineered endogenous immunoglobulin heavy chain locus.

65. The method of any one of claims 55-58, wherein (i) the one or more human Vλ gene segments and (ii) the one or more human Jλ gene segments replace one or more mouse Vκ gene segments, one or more mouse Jκ gene segments, or combinations thereof.

66. The method of any one of claims 55-58, wherein the first engineered endogenous immunoglobulin κ light chain locus further comprises a κ light chain non-coding sequence located between the one or more human Vλ gene segments and the one or more human Jλ gene segments.

67. The method of claim 66, wherein the κ light chain non-coding sequence has a sequence that naturally occurs between the human Vκ4-1 gene segment and the human Jκ1 gene segment at the endogenous human immunoglobulin κ light chain locus.

68. The method according to any one of claims 55-58, wherein the endogenous Vλ gene segment, the endogenous Jλ gene segment, and the endogenous Cλ gene are all or partially deleted.

69. The method of any one of claims 55-58, wherein the human immunoglobulin λ light chain variable domain is encoded by a rearranged human immunoglobulin λ light chain variable region sequence, the rearranged human immunoglobulin λ light chain variable region sequence comprising (i) one of the one or more human Vλ gene segments or a somatic hypermutant variant thereof, and (ii) one of the one or more human Jλ gene segments or a somatic hypermutant variant thereof.

70. The method of any one of claims 55-58, wherein the germline genome further comprises a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element.

71. The method of any one of claims 55-58, wherein the mouse Cλ gene or human Cλ gene is a mouse Cλ gene.

72. The method of any one of claims 55-58, wherein the mouse Cλ gene or the human Cλ gene is a human Cλ gene.

73. Light chain loci, which contain: (a) One or more human Vλ gene segments, (b) One or more human Jλ gene segments, and (c) A mouse Cλ gene or a human Cλ gene, Wherein, the one or more human Vλ gene segments in (a) and the one or more human Jλ gene segments in (b) replace one or more endogenous mouse Vκ gene segments and one or more endogenous mouse Jκ gene segments; and Wherein, the one or more human Vλ gene segments in (a) and the one or more human Jλ gene segments in (b) are operatively linked to the mouse Cλ gene or the human Cλ gene in (c), and The light chain locus mentioned therein lacks the mouse Cκ gene.

74. The light chain locus of claim 73, wherein the one or more human Vλ gene segments of (a) and the one or more human Jλ gene segments of (b) replace one or more mouse Vκ gene segments, one or more mouse Jκ gene segments, or a combination thereof.

75. The light chain locus of claim 73 or 74, wherein the light chain locus further comprises a κ light chain non-coding sequence located between the one or more human Vλ gene segments and the one or more human Jλ gene segments.

76. The light chain locus of claim 75, wherein the κ light chain non-coding sequence has a sequence that naturally occurs between the human Vκ4-1 gene segment and the human Jκ1 gene segment in the endogenous human immunoglobulin κ light chain locus.

77. The light chain locus of any one of claims 73-76, wherein the light chain locus further comprises a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operatively linked to a transcriptional control element.

78. The light chain locus according to any one of claims 73-77, wherein the mouse Cλ gene or the human Cλ gene is a mouse Cλ gene.

79. The light chain locus according to any one of claims 73-77, wherein the mouse Cλ gene or the human Cλ gene is a human Cλ gene.

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