Humanized rodents for testing therapeutics

Abstract

To provide humanized rodents for testing therapeutic agents.SOLUTION: Provided herein are methods and compositions relating to the in vivo testing of therapeutic agents comprising a human Fc in genetically modified rodents (e.g., the testing of pharmacokinetic and / or pharmacodynamic properties of such a therapeutic agent in genetically modified rodents). In some embodiments, the genetically modified rodents express antibodies comprising a human Fc (e.g., a human IgG1 Fc, a human IgG4 Fc). In some embodiments, the rodents express fully human antibodies (i.e., antibodies having human heavy chains and human light (γ or κ) chains).SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 648,197, filed March 26, 2018, and U.S. Provisional Patent Application No. 62 / 689,628, filed June 25, 2018, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on March 20, 2019 is titled RPB-01925_(28744-01925)_SL.txt and is 12,008 bytes in size. [Background technology]

[0003] The safety, efficacy, and pharmacokinetic properties of therapeutic drugs are typically tested in animal models before administering such drugs to humans. The use of large animals, such as non-human primates, in such preclinical studies is expensive, and suitable disease models are often unavailable, limiting the usefulness of such animals in drug efficacy testing.

[0004] Rodents have long been used as animal models for preclinical testing of therapeutics due to their small size and well-characterized physiology. Furthermore, rodents are highly amenable to genetic modification using well-established techniques, allowing for the availability of many disease models unavailable in larger mammals. However, complex therapeutics, such as antibodies and Fc fusion proteins, often behave differently in rodents than in humans. For example, such therapeutics often exhibit significantly different pharmacokinetic properties when administered to rodents than when administered to humans, limiting the utility of rodent models as predictors of safety, efficacy, and optimal dosing of complex therapeutics in humans. This reduces the usefulness of these animal models in preclinical testing.

[0005] Thus, there is a critical need for new animal models and methods that allow accurate preclinical testing of complex therapeutics in rodents that will yield results more predictive of the therapeutic's properties in human patients. Summary of the Invention

[0006] Provided herein are methods and compositions for in vivo testing of therapeutic agents comprising a human Fc in genetically modified rodents (e.g., mice or rats) (e.g., testing the pharmacokinetic and / or pharmacodynamic properties and dosing regimens of such therapeutic agents).

[0007] In certain embodiments, provided herein are genetically modified rodents in which administration of human antibodies and / or human Fc fusion proteins induces a reduced anti-human Fc immune response (e.g., a mouse anti-human antibody response or MAHA response in mice). In some embodiments, the genetically modified rodent (e.g., a mouse or rat) expresses antibodies comprising a human Fc (e.g., human IgG1 Fc, human IgG4 Fc). In some embodiments, the rodent expresses fully human antibodies (i.e., antibodies having a human heavy chain and a human light chain (gamma or kappa chain)).

[0008] In some embodiments, provided herein are rodents that express one or more humanized or partially humanized Fc receptors that interact with an Fc fusion protein or antibody in a manner that reflects how Fc receptors in a human patient interact with such Fc fusion proteins or antibodies. Thus, in certain embodiments, the genetically modified rodent comprises one or more Fc receptors that comprise a human extracellular domain (e.g., fetal Fc receptor (FcRn), beta-2-microglobulin polypeptide (β2M), Fcε receptor 1α (FcεR1α), Fcγ receptor 1 alpha (FcγR1a), Fc gamma receptor 2a (FcγR2a), Fc gamma receptor 2b (FcγR2b), Fc gamma receptor 3a (FcγR3a), Fc gamma receptor 3b (FcγR3b), Fc gamma receptor 2c (FcγR2c)). The transmembrane and cytoplasmic domains of such receptors can be human or non-human (eg, rodent, such as rat or mouse).

[0009] In certain aspects, provided herein are rodents (e.g., rats or mice) and rodent ES cells that comprise a genetically modified immunoglobulin heavy chain (IgH) locus in their genome. In certain embodiments, the IgH locus comprises (i) one or more V H Gene segment, one or more D H A gene segment and one or more J H gene segments (e.g., rat or mouse V H Gene segment, D H Gene segments and J H (ii) an immunoglobulin heavy chain variable region comprising a human C H 1 domain, human hinge region, human C H 2 domains, human C H one or more C3 domains encoding IgG constant domains including a human or rodent IgG transmembrane domain and a human or rodent IgG cytoplasmic domain H and an immunoglobulin heavy chain constant region comprising gene segments. In some embodiments, all C in the immunoglobulin heavy chain constant region. HIn some embodiments, the gene segments are human. In some embodiments, the immunoglobulin heavy chain variable region is a rodent, such as V H Gene segment, D H Gene segments and J H variable domains derived from gene segments and C H and a heavy chain constant domain derived from a gene segment operably linked to an immunoglobulin heavy chain constant region to produce an IgG antibody. In certain embodiments, the locus is located at an endogenous rodent immunoglobulin heavy chain locus. In some embodiments, the human C gene segment is located within the rodent immunoglobulin heavy chain locus. H Human antibodies and Fc fusion proteins isotype-matched to the Fc encoded by elicit a reduced immune response when administered to such rodents. For example, in some embodiments, a human IgG1 antibody is administered to a rodent provided herein that expresses an antibody having a human variable domain and a human IgG1 constant domain. In some embodiments, a human IgG2 antibody is administered to a rodent provided herein that expresses an antibody having a human variable domain and a human IgG2 constant domain. In some embodiments, a human IgG3 antibody is administered to a rodent provided herein that expresses an antibody having a human variable domain and a human IgG3 constant domain. In some embodiments, a human IgG4 antibody is administered to a rodent provided herein that expresses an antibody having a human variable domain and a human IgG4 constant domain. In some embodiments, a human antibody having a κ light chain is administered to a rodent provided herein that expresses an antibody having a human κ light chain, e.g., expressing an antibody having a human κ constant domain, or expressing an antibody having human κ variable and constant domains. In some embodiments, a human antibody having a λ light chain is administered to a rodent provided herein that expresses an antibody having a human λ light chain, e.g., expresses an antibody having a human λ constant domain, or expresses an antibody having human λ variable and constant domains. In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using such rodents.

[0010] In certain aspects, provided herein is a rodent (e.g., a rat or mouse) that comprises a genetically modified immunoglobulin kappa (Igκ) chain locus in its genome. In some embodiments, the Igκ locus is a genetically modified Igκ locus that is derived from (1) one or more human V κ A gene segment and one or more human J κ (2) a human C κ and an immunoglobulin kappa chain constant region comprising gene segments. In some embodiments, the immunoglobulin kappa chain variable region is a region of interest for the rodent, such as a human V κ Gene segments and human J κ a light chain variable domain derived from a gene segment and a C κ and a light chain constant domain derived from a gene segment operably linked to an immunoglobulin κ chain constant region to produce an antibody comprising the gene segment. In certain embodiments, this locus is located at an endogenous rodent immunoglobulin κ chain locus. In some embodiments, a human antibody comprising a human κ chain elicits a reduced immune response when administered to such a rodent. In certain embodiments, provided herein are animal models and methods for testing human antibodies using such rodents.

[0011] In certain aspects, provided herein is a rodent (e.g., a rat or a mouse) that comprises a genetically modified immunoglobulin lambda (Igλ) chain locus in its genome. In certain embodiments, the Igλ locus comprises one or more human V λ gene segment, one or more human J λ A gene segment and one or more C λ In some embodiments, the human J λ Gene segments and C λ A gene segment consists of one or more J λ -C λ In some embodiments, the human J λ Gene segments and C λThe gene segment is human J λ One or more gene segments C λ In some embodiments, the human J λ Gene segments and C λ The gene segment is human J λ Gene segments and C λ Some gene segments have one or more J λ -C λ Arranged as a cluster, other J λ One or more gene segments C λ In some embodiments, the human V λ Gene segments and human J λ The gene segment is similar to that of the rodent V λ Gene segments and human J λ a light chain variable domain derived from a gene segment and a C λ and a light chain constant domain derived from a human C gene segment. λ The human antibody is operably linked to a gene segment. In certain embodiments, the locus is located at an endogenous rodent immunoglobulin λ chain locus. In some embodiments, a human antibody comprising a human λ chain elicits a reduced immune response when administered to such a rodent. In certain embodiments, provided herein are animal models and methods for testing human antibodies using such rodents.

[0012] In certain aspects, provided herein is a rodent (e.g., a rat or mouse) comprising a genetically modified fetal Fc receptor (FcRn) locus in its genome. In certain embodiments, the FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain, a rodent or human transmembrane domain, and a rodent or human cytoplasmic domain. In certain embodiments, this locus is located at an endogenous rodent FcRn locus. In some embodiments, the rodent further comprises a β-2-microglobulin (β2M) locus in its genome comprising a nucleic acid sequence encoding a human β2M polypeptide. In some embodiments, the nucleic acid sequence encoding the human β2M polypeptide is located at the endogenous rodent β2M locus. In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using such rodents.

[0013] In certain aspects, provided herein is a rodent (e.g., a rat or mouse) comprising in its genome a genetically modified FcεR1α locus comprising a nucleic acid sequence encoding an Fc epsilon receptor 1 alpha (FcεR1α) polypeptide comprising a human extracellular domain, a rodent or human transmembrane domain, and a rodent or human cytoplasmic domain. In some embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide is located in the endogenous rodent FcεR1α locus. In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using such rodents.

[0014] In certain aspects, provided herein is a rodent (e.g., a rat or mouse) comprising, in its genome, a genetically modified FcγR1a α-chain locus comprising a nucleic acid sequence encoding an Fc gamma receptor 1a (FcγR1a) α-chain polypeptide comprising a human extracellular domain, a rodent or human transmembrane domain, and a rodent or human cytoplasmic domain. In some embodiments, the nucleic acid sequence encoding the FcγR1a α-chain polypeptide is located at an endogenous rodent FcγR1a α-chain locus. In some embodiments, the genetically modified rodent provided herein further comprises a functional FcR γ-chain. In certain embodiments, the functional FcR γ-chain is a rodent FcR γ-chain (e.g., an FcR γ-chain endogenous to the genetically modified rodent). In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc-fusion proteins using such rodents.

[0015] In certain aspects, provided herein is a rodent (e.g., a rat or mouse) comprising in its genome a genetically engineered FcγR2a α-chain locus comprising a nucleic acid sequence encoding a human Fc gamma receptor 2a (FcγR2a) α-chain polypeptide. In some embodiments, the nucleic acid sequence encoding the FcγR2a α-chain polypeptide is positioned at the endogenous rodent low-affinity FcγR α-chain locus. In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using such rodents.

[0016] In certain aspects, provided herein is a rodent (e.g., a rat or mouse) comprising in its genome a genetically engineered FcγR2b α-chain locus comprising a nucleic acid sequence encoding a human Fc gamma receptor 2b (FcγR2b) α-chain polypeptide. In some embodiments, the nucleic acid sequence encoding the FcγR2b α-chain polypeptide is positioned at the endogenous rodent low-affinity FcγR α-chain locus. In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using such rodents.

[0017] In certain aspects, provided herein is a rodent (e.g., a rat or mouse) comprising in its genome a genetically modified FcγR3a α-chain locus comprising a nucleic acid sequence encoding a human Fc gamma receptor 3a (FcγR3a) α-chain polypeptide. In some embodiments, the nucleic acid sequence encoding the FcγR3a α-chain polypeptide is positioned at an endogenous rodent low-affinity FcγR α-chain locus. In some embodiments, the genetically modified rodent provided herein further comprises a functional FcR γ-chain. In certain embodiments, the functional FcR γ-chain is a rodent FcR γ-chain (e.g., an FcR γ-chain endogenous to the genetically modified rodent). In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using such rodents.

[0018] In certain aspects, provided herein is a rodent (e.g., a rat or mouse) comprising in its genome a genetically engineered FcγR3b α-chain locus comprising a nucleic acid sequence encoding a human Fc gamma receptor 3b (FcγR3b) α-chain polypeptide. In some embodiments, the nucleic acid sequence encoding the FcγR3b α-chain polypeptide is positioned at the endogenous rodent low-affinity FcγR α-chain locus. In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using such rodents.

[0019] In certain aspects, provided herein is a rodent (e.g., a rat or mouse) comprising in its genome a genetically engineered FcγR2c α-chain locus comprising a nucleic acid sequence encoding a human Fc gamma receptor 2c (FcγR2c) α-chain polypeptide. In some embodiments, the nucleic acid sequence encoding the FcγR2c α-chain polypeptide is positioned at the endogenous rodent low-affinity FcγR α-chain locus. In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using such rodents.

[0020] In certain aspects, provided herein are rodents (e.g., rats or mice) that comprise in their genome a combination of genetically modified loci provided herein. For example, in some embodiments, the rodents provided herein comprise one or more genetically modified loci selected from a genetically modified IgH locus provided herein, a genetically modified Igκ locus provided herein, a genetically modified Igλ locus provided herein, a genetically modified FcRn locus provided herein, a genetically modified β2M locus provided herein, a genetically modified FcεR1α locus provided herein, a genetically modified FcγR1a locus provided herein, a genetically modified FcγR2a locus provided herein, a genetically modified FcγR2b locus provided herein, a genetically modified FcγR2c locus provided herein, a genetically modified FcγR3a locus provided herein, and / or a genetically modified FcγR3b locus provided herein. In certain embodiments, the rodents provided herein comprise a genetically modified IgH locus provided herein and / or a genetically modified Igκ locus provided herein. In some embodiments, the rodents provided herein comprise a genetically modified IgH locus provided herein and / or a genetically modified Igλ locus provided herein. In some embodiments, the rodents provided herein comprise a genetically modified IgH locus provided herein, a genetically modified Igκ and / or Igλ locus provided herein, a genetically modified FcRn locus provided herein, and a genetically modified β2M locus provided herein.In some embodiments, the rodent provided herein comprises a genetically modified IgH locus provided herein, a genetically modified FcRn locus provided herein, a genetically modified β2M locus provided herein, a genetically modified FcεR1α locus provided herein, a genetically modified FcγR1a locus provided herein, a genetically modified FcγR2a locus provided herein, a genetically modified FcγR2b locus provided herein, a genetically modified FcγR2c locus provided herein, a genetically modified FcγR3a locus provided herein, and / or a genetically modified FcγR3b locus provided herein.

[0021] In certain embodiments, provided herein are methods for testing a therapeutic protein comprising a human Fc domain (e.g., a human antibody or an Fc fusion protein), comprising administering the therapeutic protein to a rodent (e.g., a mouse or rat) provided herein. In some embodiments, the method further comprises measuring one or more pharmacokinetic properties of the administered therapeutic protein. In some embodiments, the one or more pharmacokinetic parameters include, but are not limited to, area under plasma concentration versus time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), drug half-life (t), and volume of distribution at steady state (Vss). In some embodiments, the method further comprises measuring the therapeutic efficacy of the administered therapeutic protein (e.g., the ability of a dose of the therapeutic protein to reduce or eliminate one or more disease symptoms in an animal model). In some embodiments, the method further comprises measuring the safety of the administered therapeutic protein (e.g., the extent to which a dose of the therapeutic protein produces one or more adverse effects in an animal model). In certain embodiments, the methods further comprise measuring the extent to which the therapeutic protein induces one or more Fc receptor-mediated responses in the rodent (e.g., the extent to which the therapeutic protein induces antibody-dependent cell-mediated cytotoxicity (ADCC)). In some embodiments, the methods further comprise measuring the extent to which administration of the therapeutic protein induces an anti-human Fc immune response in the rodent. In some embodiments, the methods further comprise evaluating the safety and / or efficacy of the therapeutic protein administration regimen.

[0022] In certain embodiments, provided herein are animal models for testing therapeutic proteins (e.g., human antibodies or Fc fusion proteins) comprising a human Fc domain. In some embodiments, the animal model comprises administering a therapeutic protein to a rodent (e.g., a mouse or rat) provided herein. In some embodiments, the animal model further comprises measuring one or more pharmacokinetic properties of the administered therapeutic protein. In some embodiments, the one or more pharmacokinetic parameters include, but are not limited to, area under plasma concentration versus time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), drug half-life (t), and volume of distribution at steady state (Vss). In some embodiments, the animal model further comprises measuring the therapeutic efficacy of the administered therapeutic protein (e.g., the ability of a dose of the therapeutic protein to reduce or eliminate one or more disease symptoms in the animal model). In some embodiments, the animal model further comprises measuring the safety of the administered therapeutic protein (e.g., the extent to which a dose of the therapeutic protein produces one or more adverse effects in the animal model). In certain embodiments, the animal model further comprises measuring the extent to which a therapeutic protein induces one or more Fc receptor-mediated responses in a rodent (e.g., the extent to which a therapeutic protein induces antibody-dependent cell-mediated cytotoxicity (ADCC)). In some embodiments, the animal model further comprises measuring the extent to which administration of a therapeutic protein induces an anti-human Fc immune response in a rodent. In some embodiments, the animal model further comprises assessing the safety and / or efficacy of an administration regimen of a therapeutic protein.

[0023] In some embodiments, the administered therapeutic agent elicits a reduced immune response when administered to a rodent provided herein. In some embodiments, the administered human antibody or Fc fusion protein encodes a human C1 gene within the genetically modified IgH locus of a rodent provided herein. HIn some embodiments, the Fc domain of a rodent has an isotype and / or allotype that matches the isotype and / or allotype of the Fc domain encoded by a human C within the rodent immunoglobulin heavy chain locus. H Human antibodies and Fc fusion proteins isotype-matched to the Fc encoded by elicit a reduced immune response when administered to such rodents. For example, in some embodiments, a human IgG1 antibody is administered to a rodent provided herein that expresses an antibody having a human variable domain and a human IgG1 constant domain. In some embodiments, a human IgG2 antibody is administered to a rodent provided herein that expresses an antibody having a human variable domain and a human IgG2 constant domain. In some embodiments, a human IgG3 antibody is administered to a rodent provided herein that expresses an antibody having a human variable domain and a human IgG3 constant domain. In some embodiments, a human IgG4 antibody is administered to a rodent provided herein that expresses an antibody having a human variable domain and a human IgG4 constant domain. In some embodiments, a human antibody having a κ light chain is administered to a rodent provided herein that expresses an antibody having a human κ light chain, e.g., expressing an antibody having a human κ constant domain, or expressing an antibody having human κ variable and constant domains. In some embodiments, a human antibody having a λ light chain is administered to a rodent provided herein that expresses an antibody having a human λ light chain, e.g., expresses an antibody having a human λ constant domain, or expresses an antibody having human λ variable and constant domains. In some embodiments, the agent is a human IgG1 antibody, and the rodent is administered a C nucleotide sequence encoding the human IgG1 CH1, hinge, CH2, and CH3 domains. H In some embodiments, the agent is a human IgG4 antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgG4 CH1, hinge, CH2, and CH3 domains. HThe rodent comprises a genetically modified IgH locus comprising a gene segment. In some embodiments, the therapeutic agent is a human antibody having an Igκ light chain, and the rodent comprises a genetically modified Igκ locus provided herein. In some embodiments, the therapeutic agent is a human antibody having an Igλ light chain, and the rodent comprises a genetically modified Igλ locus provided herein.

[0024] In certain aspects, provided herein are rodent cells (e.g., ES cells, immune cells, endothelial cells, B cells, NK cells, macrophages, dendritic cells, Langerhans cells, eosinophils, mast cells, and basophils) comprising one or more of the genetically modified loci provided herein. For example, in some embodiments, rodent cells (e.g., rodent ES cells) provided herein comprise a genetically modified IgH locus provided herein and / or a genetically modified Igκ locus provided herein. In some embodiments, rodent cells (e.g., rodent ES cells) provided herein comprise a genetically modified IgH locus provided herein and / or a genetically modified Igλ locus provided herein. In some embodiments, a rodent cell (e.g., a rodent ES cell) provided herein comprises a genetically modified IgH locus provided herein, a genetically modified Igκ and / or Igλ locus provided herein, a genetically modified FcRn locus provided herein, and a genetically modified β2M locus provided herein. In some embodiments, the rodent cells provided herein (e.g., rodent ES cells) comprise a genetically modified IgH locus provided herein, a genetically modified FcRn locus provided herein, a genetically modified β2M locus provided herein, a genetically modified FcεR1α locus provided herein, a genetically modified FcγR1a locus provided herein, a genetically modified FcγR2a locus provided herein, a genetically modified FcγR2b locus provided herein, a genetically modified FcγR2c locus provided herein, a genetically modified FcγR3a locus provided herein, and / or a genetically modified FcγR3b locus provided herein.

[0025] In some embodiments, provided herein are methods of producing the genetically modified rodents (e.g., rats or mice) and rodent ES cells (e.g., rat or mouse ES cells) provided herein. In certain embodiments, the methods include genetically modifying the genome of a rodent (e.g., rat or mouse) or rodent ES cell (e.g., rat or mouse ES cell) to include one or more of the genetically modified loci provided herein. The present invention provides, for example, the following items. (Item 1) 1. A method of testing a human antibody, comprising administering the antibody to a rodent, the rodent having in its genome: an immunoglobulin heavy chain locus, the immunoglobulin heavy chain locus comprising: (i)V H Gene segment, D H Gene segments and J H an immunoglobulin heavy chain variable region comprising a gene segment; (ii) an immunoglobulin heavy chain constant region, (a) Human IgG1 C H 1 domain, human IgG1 hinge region, human IgG1 C H 2 domain, human IgG1 C H C, which encodes the IgG constant domain containing three domains: the IgG transmembrane domain and the IgG cytoplasmic domain. H gene segments, and / or (b) Human IgG4 C H 1 domain, human IgG4 hinge region, human IgG4 C H 2 domain, human IgG2 C H C, which encodes the IgG constant domain containing three domains: the IgG transmembrane domain and the IgG cytoplasmic domain. H and optionally, the immunoglobulin heavy chain constant region comprising a gene segment, The immunoglobulin heavy chain constant region is optionally human C γ2 Gene segment, human C γ3 Gene segment, human or rodent C μ Gene segment, human or rodent C δ Gene segment, human or rodent C ε gene segments, and / or human or rodent C α further comprising a gene segment, The immunoglobulin heavy chain variable region is H gene segment, H Gene segments and the J H a variable domain derived from said C gene segment;H a heavy chain constant domain derived from the gene segment, operably linked to the immunoglobulin heavy chain constant region to produce an IgG antibody, The method, wherein the human antibody being tested comprises human CH1, human CH2, and human CH3 domains of a human isotype present in the rodent immunoglobulin heavy chain constant region. (Item 2) Said C H 2. The method of claim 1, wherein the IgG transmembrane domain encoded by one or more of the gene segments is a rodent or human IgG transmembrane domain. (Item 3) Said C H 3. The method of claim 1 or 2, wherein the IgG cytoplasmic domain encoded by one or more of the gene segments is a rodent or human IgG cytoplasmic domain. (Item 4) (a) the IgG1 domain is encoded by an allele selected from IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04 and IGHG1*05; (b) the IgG4 domain is encoded by an allele selected from IGHG4*01, IGHG4*02, IGHG4*03 and IGHG4*04; (c)C above γ2 the gene segment is an allele selected from IGHG2*01, IGHG2*02, IGHG2*03, IGHG2*04, IGHG2*05 and IGHG2*06; (c)C above γ3 the gene segment is an allele selected from IGHG3*01, IGHG3*02, IGHG3*03, IGHG3*04, IGHG3*05, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*13, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18 and IGHG3*19; 2. The method of claim 1, wherein the heavy chain constant domains of the human antibodies are encoded by the same allele. (Item 5) (a) The above C H one of the gene segments is located at an endogenous Cγ2a or Cγ2c gene segment locus and optionally replaces an endogenous Cγ2a or Cγ2c gene segment; (b) The above C H One of the gene segments is endogenous C γ1 Located at a gene segment locus, optionally an endogenous C γ1 Replace gene segments, (c)C above H One of the gene segments is endogenous C γ3 Located at a gene segment locus, optionally an endogenous C γ3 Replace gene segments, and / or (d) The above C H One of the gene segments is endogenous C γ2b Located at a gene segment locus, optionally an endogenous C γ2b The method of item 1, wherein a gene segment is replaced. (Item 6) The immunoglobulin heavy chain constant region is human C μ Gene segment, human C δ Gene segment, human C γ3 Gene segments and human C γ1 2. The method of claim 1, comprising a gene segment. (Item 7) The immunoglobulin heavy chain constant region is human C γ2 Gene segments and human C γ4 7. The method of item 6, further comprising a gene segment. (Item 8) The immunoglobulin heavy chain locus contains a rodent or human intronic enhancer (E i ), and / or a rodent or human 3' regulatory region (3'RR). (Item 9) The immunoglobulin heavy chain locus is a rodent or human S μ Switch site, rodent or human S γ3 Switch site, rodent or human S γ1 Switch site, rodent S γ2b Switch site, rodent S γ2a , rodent S γ2c Switch site, human S γ2 Switch site, human S γ4 Switch site, rodent or human S ε switch site, and / or rodent or human S α Item 10. The method of any one of the preceding items, further comprising a switch site. (Item 10) The above V H The gene segment is a rodent V H a gene segment, H The gene segment is a rodent D H a gene segment, H The gene segment is rodent J H gene segment, and optionally the rodent V H gene segments, said rodent D H gene segments and the rodent J H 10. The method of any one of the preceding items, wherein the gene segment is an endogenous rodent gene segment. (Item 11) The above V H The gene segment is human V H a gene segment, H The gene segment is human D H a gene segment, H The gene segment is human J H 10. The method according to any one of items 1 to 9, wherein the target gene is a gene segment. (Item 12) 10. The method of any one of the preceding items, wherein the immunoglobulin heavy chain locus is positioned at an endogenous immunoglobulin heavy chain locus, and optionally the immunoglobulin heavy chain locus replaces all or a portion of the endogenous immunoglobulin heavy chain locus. (Item 13) The rodent has in its genome: an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus comprising: (i) Human V H Gene segment, human D H Gene segments and human J H an immunoglobulin heavy chain variable region comprising a gene segment; (ii) rodent intronic enhancer (E i )and, (iii) an immunoglobulin heavy chain constant region, (a) Rodent C μ gene segments, (b) Rodent C δ gene segments, (c) Rodent C γ3 gene segments, (d) Rodent C γ1 gene segments, (e) Rodent C γ2b gene segments, (f) Human IgG1 C H 1 domain, human IgG1 hinge region, human IgG1 C H 2 domain, human IgG1 C H A modified C3 domain encoding a rodent IgG2a transmembrane domain and a rodent IgG2a cytoplasmic domain. H gene segments, (g) Rodent C ε gene segments, and (h) Rodents C α the immunoglobulin heavy chain constant region comprising a gene segment; (iv) a rodent 3' regulatory region (3'RR), The immunoglobulin heavy chain variable region is a human V H gene segment, the human D H gene segments and the human J H a variable domain derived from the gene segment and the modified C H and a heavy chain constant domain derived from the gene segment, operably linked to the immunoglobulin heavy chain constant region so as to produce an IgG antibody. (Item 14) The rodent has in its genome: an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus comprising: (i) Human V H Gene segment, human D H Gene segments and human J H an immunoglobulin heavy chain variable region comprising a gene segment; (ii) rodent intronic enhancer (E i )and, (iii) an immunoglobulin heavy chain constant region, (a) Rodent C μ gene segments, (b) Rodent C δ gene segments, (c) Rodent C γ3 gene segments, (d) Rodent C γ1 gene segments, (e) Rodent C γ2b gene segments, (f) Human IgG1 C H 1 domain, human IgG1 hinge region, human IgG1 C H 2 domain, human IgG1 C H A modified C3 domain encoding the human IgG1 transmembrane domain and the human IgG1 cytoplasmic domain. H gene segments, (g) Rodent C ε gene segments, and (h) Rodent C α the immunoglobulin heavy chain constant region comprising a gene segment; (iv) a rodent 3' regulatory region (3'RR), The immunoglobulin heavy chain variable region is a human V H gene segment, the human D H gene segments and the human J H a variable domain derived from the gene segment and the modified C H and a heavy chain constant domain derived from the gene segment, operably linked to the immunoglobulin heavy chain constant region so as to produce an IgG antibody. (Item 15) The rodent has in its genome: an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus comprising: (i) Human V H Gene segment, human D H Gene segments and human J H an immunoglobulin heavy chain variable region comprising a gene segment; (ii) rodent intronic enhancer (E i )and, (iii) an immunoglobulin heavy chain constant region, (a) Rodent C μ gene segments, (b) Rodent C δ gene segments, (c) Rodent C γ3 gene segments, (d) Human IgG4 C H 1 domain, human IgG4 hinge region, human IgG4 C H 2 domain, human IgG4 C H a modified C encoding three domains, a rodent IgG1 transmembrane domain, and a rodent IgG1 cytoplasmic domain H gene segments, (e) Rodent C γ2b gene segments, (f) Rodent C γ2a and / or C. γ2c gene segments, (g) Rodent C ε gene segments, and (h) Rodent C α the immunoglobulin heavy chain constant region comprising a gene segment; (iv) a rodent 3' regulatory region (3'RR), The immunoglobulin heavy chain variable region is a human V H gene segment, the human D H gene segments and the human J H a variable domain derived from the gene segment and the modified C H and a heavy chain constant domain derived from the gene segment, operably linked to the immunoglobulin heavy chain constant region so as to produce an IgG antibody. (Item 16) The rodent has in its genome: an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus comprising: (i) Human V H Gene segment, human D H Gene segments and human J H an immunoglobulin heavy chain variable region comprising a gene segment; (ii) rodent intronic enhancer (E i )and, (iii) an immunoglobulin heavy chain constant region, (a) Rodent C μ gene segments, (b) Rodent C δ gene segments, (c) Rodent C γ3 gene segments, (d) Human IgG4 C H 1 domain, human IgG4 hinge region, human IgG4 C H 2 domain, human IgG4 C H A modified C3 domain encoding the human IgG4 transmembrane domain and the human IgG4 cytoplasmic domain. H gene segments, (e) Rodent C γ2b gene segments, (f) Rodent C γ2a and / or C. γ2c gene segments, (g) Rodent C ε gene segments, and (h) Rodent C α the immunoglobulin heavy chain constant region comprising a gene segment; (iv) a rodent 3' regulatory region (3'RR), The immunoglobulin heavy chain variable region is a human V H gene segment, the human D H gene segments and the human J H a variable domain derived from the gene segment and the modified C H and a heavy chain constant domain derived from the gene segment, operably linked to the immunoglobulin heavy chain constant region so as to produce an IgG antibody. (Item 17) The rodent has in its genome: an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus comprising: (i) Human V H Gene segment, human D H Gene segments and human J H an immunoglobulin heavy chain variable region comprising a gene segment; (ii) Human intronic enhancer (E i )and, (iii) an immunoglobulin heavy chain constant region, (a) Human C μ gene segments, (b) Human C δ gene segments, (c) Human C γ3 gene segments, (d) Human C γ1 the immunoglobulin heavy chain constant region comprising a gene segment; (iv) a rodent 3' regulatory region (3'RR), 2. The method of claim 1, wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region such that the rodent produces an IgG antibody comprising a fully human heavy chain. (Item 18) The rodent has in its genome: an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus comprising: (i) Human V H Gene segment, human D H Gene segments and human J H an immunoglobulin heavy chain variable region comprising a gene segment; (ii) Human intronic enhancer (E i )and, (iii) an immunoglobulin heavy chain constant region, (a) Human C μ gene segments, (b) Human C δ gene segments, (c) Human C γ3 gene segments, (d) Human C γ1 gene segments, (c) Human C γ2 gene segments, (d) Human C γ4 the immunoglobulin heavy chain constant region comprising a gene segment; (iv) a rodent 3' regulatory region (3'RR), 2. The method of claim 1, wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region such that the rodent produces an IgG antibody comprising a fully human heavy chain. (Item 19) The rodent has in its genome: an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus comprising: (i) Rodent V H Gene segment, rodent D H Gene segments and rodent J H an immunoglobulin heavy chain variable region comprising a gene segment; (ii) rodent intronic enhancer (E i )and, (iii) an immunoglobulin heavy chain constant region, (a) Human C μ gene segments, (b) Human C δ gene segments, (c) Human C γ3 gene segments, (d) Human C γ1 the immunoglobulin heavy chain constant region comprising a gene segment; (iv) a rodent 3' regulatory region (3'RR), 2. The method of claim 1, wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region such that the rodent produces an IgG antibody comprising a heavy chain comprising a rodent variable domain and a human constant domain. (Item 20) The rodent has in its genome: an endogenous immunoglobulin heavy chain locus, the endogenous immunoglobulin heavy chain locus comprising: (i) Rodent V H Gene segment, rodent D H Gene segments and rodent J H an immunoglobulin heavy chain variable region comprising a gene segment; (ii) rodent intronic enhancer (E i )and, (iii) an immunoglobulin heavy chain constant region, (a) Human C μ gene segments, (b) Human C δ gene segments, (c) Human C γ3 gene segments, (d) Human C γ1 gene segments, (c) Human C γ2 gene segments, (d) Human C γ4 the immunoglobulin heavy chain constant region comprising a gene segment; (iv) a rodent 3' regulatory region (3'RR), 2. The method of claim 1, wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region such that the rodent produces an IgG antibody comprising a heavy chain comprising a rodent variable domain and a human constant domain. (Item 21) The rodent has in its genome: The immunoglobulin kappa (κ) chain locus may further comprise: (1) Human V κ Gene segments and human J κ an immunoglobulin kappa chain variable region comprising a gene segment; (2) Human C κ an immunoglobulin κ chain constant region comprising a gene segment; The immunoglobulin κ chain variable region is a human V κ gene segments and the human J κ a light chain variable domain derived from a gene segment and the human C κ a light chain constant domain derived from a gene segment operably linked to the immunoglobulin κ chain constant region to produce an antibody, 21. The method of any one of items 1 to 20, wherein the human antibody to be tested comprises a κ light chain. (Item 22) 22. The method of claim 21, wherein the immunoglobulin κ chain locus is positioned at an endogenous immunoglobulin κ chain locus, and optionally the immunoglobulin κ chain locus replaces all or a portion of the endogenous immunoglobulin κ chain locus. (Item 23) The rodent has in its genome: and further comprising an immunoglobulin lambda (λ) chain locus, wherein the immunoglobulin λ chain locus comprises: a human Vλ gene segment, a human Jλ gene segment, and a human Cλ gene segment; Human V λ gene segments and the human J λ The gene segment is selected from the group consisting of: λ gene segments and the human J λ a light chain variable domain derived from a gene segment and the human C λ and a light chain constant domain derived from the human C gene segment. λ operably linked to a gene segment, 21. The method of any one of items 1 to 20, wherein the human antibody to be tested comprises a λ light chain. (Item 24) 24. The method of claim 23, wherein the immunoglobulin λ chain locus is positioned at an endogenous immunoglobulin λ chain locus, and optionally the immunoglobulin λ chain locus replaces all or a portion of the endogenous immunoglobulin λ chain locus. (Item 25) The rodent has in its genome: (a) an FcRn locus comprising a nucleic acid sequence encoding a fetal Fc receptor (FcRn) polypeptide comprising a human extracellular domain, a human or rodent transmembrane domain, and a human or rodent cytoplasmic domain, optionally wherein the nucleic acid sequence encoding the FcRn polypeptide is located in an endogenous rodent FcRn locus; (b) a β-2-microglobulin (β2M) locus comprising a nucleic acid sequence encoding a human or humanized β2M polypeptide, optionally wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide is located in an endogenous rodent β2M locus; (c) an FcεR1α locus comprising a nucleic acid sequence encoding an Fc epsilon receptor 1 alpha (FcεR1α) polypeptide comprising a human extracellular domain, a human or rodent transmembrane domain, and a human or rodent cytoplasmic domain, optionally wherein the nucleic acid sequence encoding the FcεR1α polypeptide is located in an endogenous rodent FcεR1α locus; (d) an Fc gamma receptor 1a (FcγR1a) locus comprising a nucleic acid sequence encoding an FcγR1a polypeptide comprising a human extracellular domain, a human or rodent transmembrane domain, and a human or rodent cytoplasmic domain, optionally wherein the nucleic acid sequence encoding the FcγR1a polypeptide is located in an endogenous rodent FcγR1a locus; (e) an FcγR2a locus comprising a nucleic acid sequence encoding a human Fc gamma receptor 2a (FcγR2a) polypeptide, optionally wherein the nucleic acid sequence encoding the FcγR2a polypeptide is located in an endogenous rodent FcγR2a locus; (f) a human Fc gamma receptor 3a (FcγR3a) locus comprising a nucleic acid sequence encoding an FcγR3a polypeptide, optionally wherein the nucleic acid sequence encoding the FcγR3a polypeptide is located in an endogenous rodent low affinity FcγR locus; (g) a human Fc gamma receptor 3b (FcγR3b) locus comprising a nucleic acid sequence encoding an FcγR3b polypeptide, optionally wherein the nucleic acid sequence encoding the FcγR3b polypeptide is located in an endogenous rodent low affinity FcγR locus; and / or (h) The method of any one of items 1 to 25, further comprising an FcγR2c locus comprising a nucleic acid sequence encoding a human Fc gamma receptor 2c (FcγR2c) polypeptide, optionally wherein the nucleic acid sequence encoding the FcγR2c polypeptide is located in an endogenous rodent low affinity FcγR locus. (Item 26) Item 10. The method of any one of the preceding items, wherein the rodent is a mouse. (Item 27) A rodent comprising in its genome a fetal Fc receptor (FcRn) locus comprising a nucleic acid sequence encoding a fetal FcRn polypeptide comprising a human extracellular domain and a rodent cytoplasmic domain. (Item 28) A rodent comprising in its genome an Fc epsilon receptor 1 alpha (FcεR1α) locus comprising a nucleic acid sequence encoding an Fc epsilon receptor 1 alpha (FcεR1α) polypeptide comprising a human extracellular domain and a rodent cytoplasmic domain. (Item 29) 27. The method of any one of items 1 to 26, further comprising measuring the therapeutic efficacy of the administered human antibody. (Item 30) 27. The method of any one of items 1 to 26, further comprising administering multiple doses of the human antibody and determining the therapeutic efficacy, safety, and / or tolerability of each dose of the human antibody. (Item 31) 27. The method of any one of items 1 to 26, further comprising measuring an immune response raised by the rodent against the human antibody. [Brief explanation of the drawings]

[0026] [Figure 1A]1 shows, not to scale, a schematic diagram of an exemplary modified immunoglobulin heavy chain locus according to certain exemplary embodiments provided herein. As shown, the exemplary mouse heavy chain locus 1 comprises, in 5' to 3' order, human variable region (human VH, human D, and human JH) gene segments; for simplicity, the human VH gene segment is not shown, and only a few gene segments are represented diagrammatically. For the complete repertoire of possible V, D, and J gene segments, see imgt.org, Lefranc, M.-P., Exp. Clin. Immunogenet., 18, 100-116 (2001), and Lefranc, M.-P. and Lefranc, G., The Immunoglobulin Facts Book, Academic Press, London, 458, which are incorporated herein by reference. (See, e.g., pages (2001)), a mouse intronic enhancer, a mouse Cμ gene segment, a mouse Cδ gene segment, a mouse Cγ3 gene segment, a mouse Cγ1 gene segment, a mouse Cγ2b gene segment, a hybrid C H gene segment encoding the human IgG1 extracellular domain and the mouse IgG2a transmembrane and cytoplasmic domains, a mouse Cε gene segment, a mouse Cα gene segment, and a mouse 3′ regulatory region. As shown, the exemplary mouse heavy chain locus 2 includes, in 5′ to 3′ order, a human variable region (human V H , human D , and human J H gene segments; for simplicity, the human V H gene segment is not shown, and only a few gene segments are represented schematically).For the complete repertoire of possible V, D, and J gene segments, see imgt.org, Lefranc, M.-P., Exp. Clin. Immunogenet., 18, 100-116 (2001), and Lefranc, M.-P. and Lefranc, G., The Immunoglobulin Facts Book, Academic Press, London, 458 pages (2001), which are incorporated herein by reference), mouse intronic enhancers, mouse Cμ gene segments, mouse Cδ gene segments, mouse Cγ3 gene segments, mouse Cγ1 gene segments, mouse Cγ2b gene segments, human Cγ1 gene segments (both the IgG1 extracellular and transmembrane / cytoplasmic encoding sequences are human), mouse Cε gene segments, mouse Cα gene segments, and mouse 3' regulatory regions. As shown, an exemplary mouse heavy chain locus 3 includes, in 5' to 3' order, human variable region (human VH, human D, and human JH gene segments; for simplicity, the human VH gene segment is not shown, and only a few gene segments are represented diagrammatically. For the complete repertoire of possible V, D, and J gene segments, see imgt.org, Lefranc, M.-P., Exp. Clin. Immunogenet., 18, 100-116 (2001), and Lefranc, M.-P. and Lefranc, G., The Immunoglobulin Facts Book, Academic Press, London, 458 (2002), which are incorporated herein by reference. pages (2001)), a mouse intronic enhancer, a mouse Cμ gene segment, a mouse Cδ gene segment, a mouse Cγ3 gene segment, a hybrid C H gene segment encoding the human IgG4 extracellular domain and the mouse IgG1 transmembrane and cytoplasmic domains, a mouse Cγ2b gene segment, a mouse Cγ2a gene segment, a mouse Cε gene segment, a mouse Cα gene segment, and a mouse 3' regulatory region.As shown, the exemplary mouse heavy chain locus 4 comprises, in 5' to 3' order, human variable region (human VH, human D, and human JH gene segments; for simplicity, the human VH gene segment is not shown, and only a few gene segments are represented diagrammatically. For the complete repertoire of possible V, D, and J gene segments, see imgt.org, Lefranc, M.-P., Exp. Clin. Immunogenet., 18, 100-116 (2001), and Lefranc, M.-P. and Lefranc, G., The Immunoglobulin Facts Book, Academic Press, London, 458 (2002), which are incorporated herein by reference. pages (2001)), a mouse intronic enhancer, a mouse Cμ gene segment, a mouse Cδ gene segment, a mouse Cγ3 gene segment, a human Cγ4 gene segment (the sequences encoding the IgG4 extracellular and transmembrane / cytoplasmic domains are both human), a mouse Cγ2b gene segment, a mouse Cγ2a gene segment, a mouse Cε gene segment, a mouse Cα gene segment, and a mouse 3' regulatory region. As shown, the exemplary mouse heavy chain locus 5 includes, from 5' to 3', a human variable region (comprising a human VH, human D, and human JH gene segment; for simplicity, the human VH gene segment is not shown and only a few gene segments are represented diagrammatically. For the complete repertoire of possible V, D, and J gene segments, see imgt.org, Lefranc, M.-P., Exp. Clin. Immunogenet., 18, 100-116 (2001), and Lefranc, M.-P. and Lefranc, G., The Immunoglobulin Facts Book, Academic Press, London, 458 pages (2001), which are incorporated herein by reference), a human intronic enhancer, a human Cμ gene segment, a human Cδ gene segment, a human Cγ3 gene segment, a human Cγ1 gene segment, and a mouse 3' regulatory region.As shown, the exemplary mouse heavy chain locus 6 comprises, in 5' to 3' order, human variable region (human VH, human D, and human JH gene segments; for simplicity, the human VH gene segment is not shown, and only a few gene segments are represented diagrammatically. For the complete repertoire of possible V, D, and J gene segments, see imgt.org, Lefranc, M.-P., Exp. Clin. Immunogenet., 18, 100-116 (2001), and Lefranc, M.-P. and Lefranc, G., The Immunoglobulin Facts Book, Academic Press, London, 458 (2002), which are incorporated herein by reference. pages (2001)), a human intronic enhancer, a human Cμ gene segment, a human Cδ gene segment, a human Cγ3 gene segment, a human Cγ1 gene segment, a human Cγ2 gene segment, a human Cγ4 gene segment, and a mouse 3′ regulatory region. As shown, exemplary mouse heavy chain locus 7 includes, from 5′ to 3′, a mouse variable region (including a mouse VH, a mouse DH, and a mouse JH gene segment; for simplicity, the mouse VH gene segment is not shown, and only a few gene segments are represented diagrammatically. For the complete repertoire of possible V, D, and J gene segments, see imgt.org), a mouse intronic enhancer, a human Cμ gene segment, a human Cδ gene segment, a human Cγ3 gene segment, a human Cγ1 gene segment, and a mouse 3′ regulatory region. As shown, the exemplary mouse heavy chain locus 8 includes, in 5' to 3' order, mouse variable region (mouse VH, mouse D, and mouse JH gene segments; for simplicity, the mouse VH gene segment is not shown, and only a few gene segments are represented schematically.For a complete repertoire of possible V, D, and J gene segments, see imgt.org), mouse intronic enhancers, human Cμ gene segments, human Cδ gene segments, human Cγ3 gene segments, human Cγ1 gene segments, human Cγ2 gene segments, human Cγ4 gene segments, and mouse 3' regulatory regions. Although not shown, the locus in these embodiments includes a functional mouse Adam6 gene (e.g., Adam6a and / or Adam6b). Unless otherwise specified (e.g., for lox sites, etc.), open shapes and double underlines represent human sequences, and filled shapes and single underlines represent mouse sequences. [Figure 1B]

[0023] Figure 1 shows, not to scale, a schematic diagram of an exemplary method for generating the exemplary modified immunoglobulin heavy chain locus 5 shown in Figure 1A, as described in Example 1.1. For simplicity, the human VH gene segments and the mouse Adam6 gene are not shown in either schematic diagram. Unless otherwise specified (e.g., for lox sites, etc.), open shapes and double underlines represent human sequences, and filled shapes and single underlines represent mouse sequences. [Figure 1C]

[0023] Figure 1 shows, not to scale, a schematic diagram of an exemplary method for generating the exemplary modified immunoglobulin heavy chain locus 6 shown in Figure 1A, as described in Example 1.1. For simplicity, the human VH gene segments and the mouse Adam6 gene are not shown in either schematic diagram. Unless otherwise specified (e.g., for lox sites, etc.), open shapes and double underlines represent human sequences, and filled shapes and single underlines represent mouse sequences. [Figure 1D]

[0023] Figure 1 shows, not to scale, a schematic diagram of an exemplary method for generating the exemplary modified immunoglobulin heavy chain locus 7 shown in Figure 1A, as described in Example 1.2. For simplicity, mouse VH gene segments are not shown in either schematic diagram. Unless otherwise specified (e.g., for lox sites, etc.), open shapes and double underlines represent human sequences, and filled shapes and single underlines represent mouse sequences. [Figure 1E]

[0023] Figure 1 shows, not to scale, a schematic diagram of an exemplary method for generating the exemplary modified immunoglobulin heavy chain locus 8 shown in Figure 1A, as described in Example 1.2. For simplicity, mouse VH gene segments are not shown in either schematic diagram. Unless otherwise specified (e.g., for lox sites, etc.), open shapes and double underlines represent human sequences, and filled shapes and single underlines represent mouse sequences. [Figure 2A] Schematics, not to scale, of exemplary modified immunoglobulin light chain loci according to certain exemplary embodiments provided herein are shown. The top exemplary schematic shows an Igκ locus, including a human variable region, a mouse intronic enhancer, a human Cκ constant region gene segment, and a mouse 3′ enhancer. The bottom exemplary schematic shows an Igλ locus, including a Vλ gene segment, a human Jλ-Cλ tandem pair, a human Jλ7 and a mouse Cλ1 gene segment, human enhancers 1, 2, and 3, and a mouse 3′ enhancer. Unless otherwise specified (e.g., for loxp sites, etc.), open shapes and double underlines represent human sequences, and filled shapes and single underlines represent mouse sequences. [Figure 2B] 2A, as described in Example 1.3. Unless otherwise specified (e.g., for loxp sites, etc.), open shapes and double lines represent human sequences, and filled shapes and single lines represent mouse sequences. [Figure 3A]1 shows the levels of mouse IgG, mouse IgM, human IgM, human IgG1, and human IgG3 present in the serum of hVs-hIgM-hIgD-hIgG3-hIgG1 mice (locus 5), VELOCIMMUNE® mice, and in normal human serum. [Figure 3B] FACS plots for hIgM vs. mIgM and hIgD vs. mIgD are shown to demonstrate allelic usage and the presence of allelic exclusion in hVs-hIgM-hIgD-hIgG3-hIgG1 mice (locus 5) and VELOCIMMUNE® mice. [Figure 3C] The levels of mouse IgG, mouse IgM, human IgM, human IgG1 and human IgG4 present in the serum of the indicated mice are shown. [Figure 3D] Shown are concentrations of mouse anti-human antibodies in the indicated mice at days 0 and 34 after injection with human IgG4 antibody. [Figure 3E] Shown are concentrations of human IgG4 antibodies in the indicated mice over the first 34 days after injection of the human IgG4 antibodies. [Figure 4] 1 shows, not to scale, a schematic diagram of an exemplary method for generating an exemplary modified FcRn locus, as described in Example 3. Unless otherwise specified (e.g., for loxp sites, etc.), open shapes and double lines represent human sequences, and filled shapes and single lines represent mouse sequences. [Figure 5] 1 shows, not to scale, a schematic diagram of an exemplary method for generating mice containing exemplary modified FcRn and β2M loci, as described in Example 3. Unless otherwise specified (e.g., for loxp sites, etc.), open shapes and double underlines represent human sequences, and filled shapes and single underlines represent mouse sequences. [Figure 6] Antibody clearance of a human antibody by mice containing exemplary modified FcRn and β2M loci is shown compared to wild-type mice (shown as antibody concentration). [Figure 7]1 shows, not to scale, a schematic diagram of an exemplary method for generating a mouse containing exemplary modified FcγR1α (designated FCGR1 in the figure), FcγRIIIa, FcγRIIa, FcγRIIc, FcγRIIb, FcγRIIIb and heavy chain loci, as described in Example 4. Open shapes and double underlines represent human sequences, and filled shapes and single underlines represent mouse sequences. [Figure 8A] 1 shows FACS plots illustrating the utilization of mouse IgD versus mouse IgM, and human IgD versus human IgM in mice carrying human high-affinity FcγR and human low-affinity FcγR and human heavy chain constant regions. [Figure 8B] The numbers or percentages of T cells (mCD3 positive) and B cells (mCD19 positive) present in the spleens of mice carrying human high-affinity FcγR and human low-affinity FcγR and human heavy chain constant regions are shown compared to wild-type mice. [Figure 8C] Serum concentrations of human IgM, human IgG1, and human IgG3 in mice carrying human high-affinity FcγR and human low-affinity FcγR and human heavy chain constant regions are shown compared to normal human serum. [Figure 9] 1 shows a schematic diagram, not to scale, of an exemplary method for generating a mouse containing an exemplary modified FcεR1α locus, as described in Example 5. Unless otherwise specified (e.g., for loxp sites, etc.), open shapes and double lines represent human sequences, and filled shapes and single lines represent mouse sequences. [Figure 10] The left panel shows a FACS plot demonstrating that humanized FcεR1α is expressed on the surface of basophils from an exemplary mouse comprising a humanized FcεR1α locus as disclosed herein. The right panel shows the percentage of mouse or human FcεR1α + basophils present in the spleen of a mouse comprising a humanized FcεR1α locus as disclosed herein compared to a wild-type mouse. [Figure 11]This demonstrates that the exemplary humanized FcεR1α locus provided herein is functional. Mice were sensitized by intradermal injection of either human allergen-specific IgE or IgG as a negative control into the ear. One day later, the mice were challenged by intravenous injection of allergen diluted in Evans blue dye. Extravasation of Evans blue dye into the ear was measured as an indicator of mast cell degranulation. Because human IgE does not bind to mouse FcεR1, responses in mice containing the humanized FcεR1α locus indicate that functional FcεR1α was produced. [Figure 12] This paper demonstrates that the exemplary humanized FcεR1α locus provided herein is functional. Mice are sensitized by IV injection of human allergen-specific IgE. One day later, mice are challenged by IV injection of allergen, and temperature changes are monitored for 4 hours after injection. A drop in body temperature is an indication of systemic anaphylaxis. [Figure 13] 1 shows, not to scale, a schematic diagram of an exemplary method for generating a mouse containing exemplary humanized FcRn, β2M, FcεR1α, FcγR1α (designated FCGR1 in the figure), FcγRIIIa, FcγRIIa, FcγRIIc, FcγRIIb, FcγRIIIb, heavy chain constant region loci, and heavy chain variable region loci, as described in Example 6. For simplicity, only a few gene segments are represented schematically in loci F and F′. For the complete repertoire of possible V, D, and J gene segments, see imgt.org, Lefranc, M.-P., Exp. Clin. Immunogenet., 18, 100-116 (2001), and Lefranc, M.-P. and Lefranc, G., The Immunoglobulin Facts Book, Academic Press, London, 458 pages (2001), which are incorporated herein by reference. Open shapes and double lines represent human sequences, and filled shapes and single lines represent mouse sequences. DETAILED DESCRIPTION OF THE INVENTION

[0027] overview Provided herein are methods and compositions for in vivo testing of therapeutic agents comprising a human Fc in genetically modified rodents (e.g., testing the pharmacokinetic and / or pharmacodynamic properties and dosing regimens of such therapeutic agents in genetically modified rodents). In some embodiments, the genetically modified rodents express antibodies comprising a human Fc (e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc) or a human light chain constant region. In some embodiments, the rodents express fully human antibodies (i.e., antibodies having a human heavy chain and a human light chain (gamma or kappa chain)). In certain embodiments, the genetically modified rodent comprises one or more Fc receptors comprising a human extracellular domain (e.g., fetal Fc receptor (FcRn), beta-2-microglobulin polypeptide (β2M), Fcε receptor 1α (FcεR1α), Fcγ receptor 1 alpha (FcγR1a), Fc gamma receptor 2a (FcγR2a), Fc gamma receptor 2b (FcγR2b), Fc gamma receptor 3a (FcγR3a), Fc gamma receptor 3b (FcγR3b), Fc gamma receptor 2c (FcγR2c)). The transmembrane and cytoplasmic domains of such receptors can be human or non-human (e.g., rodent).

[0028] Therapeutic drugs containing human Fc, such as therapeutic human antibodies and human Fc fusion proteins, are typically tested in non-human species before being administered to humans. Such drugs are often tested in non-human primates or other relatively large mammals, but such testing is expensive and imposes a significant economic burden on drug developers. Furthermore, non-human primates and other relatively large mammals are often not amenable to genetic modification, and there are limited disease models available in such organisms.

[0029] In contrast, rodent species (e.g., rats and mice) are convenient animal models for testing therapeutic antibodies and Fc fusion proteins due to their small size, well-characterized physiology, and amenability to genetic modification. Unfortunately, drugs containing human Fc regions often exhibit significantly different pharmacokinetic and pharmacodynamic properties when administered to prior art rodents compared to humans. For example, when therapeutic drugs containing human Fc regions are administered to conventional rodents, the human sequences within the Fc region are often recognized as foreign by the rodent's (e.g., rat or mouse) immune system. As a result, rodents can mount an immune response to the administered therapeutic drug (known as a mouse anti-human response, or MAHA), which affects the pharmacokinetic and pharmacodynamic properties of the administered drug. Furthermore, the human Fc region of a therapeutic drug may interact differently with rodent (e.g., rat or mouse) Fc receptors than with human Fc receptors in patients, which may also affect the pharmacokinetic and pharmacodynamic properties of the administered drug. Thus, traditional rodent (e.g., rat or mouse) models are often poor predictors of human therapeutic response to human Fc-containing therapeutics. Therefore, mice bearing human immunoglobulin locus regions (see, e.g., loci in Figures 1 and 2) would be beneficial for reducing or eliminating MAHA responses.

[0030] Having a molecule that can specifically bind to a drug is extremely useful for research and diagnostic purposes. The generation of mice containing mouse variable and human constant loci (e.g., mouse locus 8 in Figure 1A) offers an improved method for generating anti-drug antibodies when the drug is a human monoclonal antibody. Any MAHA response elicited by injecting a mouse with a human antibody is directed against the variable region of the antibody. This eliminates background responses against the constant region of the antibody, making the generation of drug-specific antibodies more efficient.

[0031] Provided herein are in vivo systems for the development, screening, and testing of human antibodies and Fc fusion proteins for therapeutic use. In certain embodiments, provided herein are genetically modified rodents that exhibit a reduced rodent anti-human immune response following administration of a therapeutic agent comprising a human Fc. As demonstrated herein, this can be achieved through the use of rodents that have been genetically modified to express a human Fc that matches the Fc present in the administered antibody or Fc fusion protein. Such mice can be engineered, for example, by harboring a nucleic acid sequence encoding, in whole or in part, a human immunoglobulin heavy chain constant region (e.g., IgG C H 1-HC H 2-C H 3) can be generated by inserting the human Fc into an endogenous non-human immunoglobulin heavy chain constant region gene segment in place of the sequence encoding the corresponding portion of the gene. Such animals will recognize the human Fc as a "self" protein and will therefore be less likely to mount an immune response to administered human Fc-containing therapeutics.

[0032] Additionally, in some embodiments, provided herein are genetically modified rodents (e.g., mice or rats) that express Fc receptors capable of interacting with human Fc in a manner similar to Fc receptors expressed by human patients. For example, in certain embodiments, the genetically modified rodents provided herein express one or more Fc receptors having at least a human extracellular domain (e.g., the transmembrane and cytoplasmic domains may be either human or rodent). Thus, in certain embodiments, the mice provided herein express human or partially human FcRn, human or partially human β2M, human or partially human FcεR1α, human or partially human FcγR1a, human or partially human FcγR2a, human or partially human FcγR2b, human or partially human FcγR3a, human or partially human FcγR3b, and / or human or partially human FcγR2c. Such mice are therefore capable of more accurately recapitulating the human Fc responses of human patients compared to rodents that contain completely non-human Fc receptors.

[0033] Thus, the rodents (e.g., mice or rats) provided herein provide a novel in vivo system for developing, selecting, and testing therapeutic human antibodies and Fc fusion proteins based not only on specificity and / or affinity for antigen, but also on the entire relevant biological function of the selected antibody through evaluation of effector functions within the internal milieu of the immune system. In this way, human therapeutic candidates can be developed and selected based on their therapeutic potential assessed at the whole-molecule level for relevant biological responses (e.g., cellular responses), rather than predictors based solely on individual components evaluated in isolation. Thus, the rodents provided herein provide a system that is particularly well suited to predicting in vivo clinical human therapeutic antibody function.

[0034] definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0035] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both an amino functional group and an acid functional group and that are capable of being included in a natural amino acid polymer. Exemplary amino acids include the natural amino acids; their analogs, derivatives, and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing.

[0036] As used herein, the term "antibody" refers to both intact antibodies and antigen-binding fragments thereof. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable domain and a heavy chain constant domain. Each light chain comprises a light chain variable domain and a light chain constant domain. The heavy and light chain variable domains can be further divided into hypervariable domains called complementarity-determining regions (CDRs), which are flanked by highly conserved regions called framework regions (FRs). Each heavy and light chain variable domain is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The heavy and light chain variable domains contain the binding domains that interact with antigens.

[0037] The terms "antigen-binding fragment" and "antigen-binding portion" of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to bind to an antigen. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include Fab, Fab', F(ab'), Fv, scFv, disulfide-linked Fv, Fd, single-chain antibodies, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable domain of an intact antibody. These antibody fragments can be obtained using conventional recombinant and / or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.

[0038] As used herein, the term "area under the plasma concentration versus time curve" or "AUC" refers to the rate and extent of elimination of a therapeutic agent following administration. In some embodiments, AUC is determined over a specific period of time, such as 12, 18, 24, 36, 48, or 72 hours, or over an infinite time period using extrapolation based on the slope of the curve. Unless otherwise specified herein, AUC is used to measure the area under the plasma concentration versus time curve (AUC INF ) AUC can also be calculated based on the dose per administration. As with many other PK parameters, AUC determinations can be performed on individual animals or on populations of animals (in which case the average is calculated).

[0039] As used herein, the term "clearance rate" or "CL" refers to a measure of the body's ability to eliminate a drug and is expressed as the volume of plasma from which the drug is removed over time.

[0040] The term "derived from," when used with reference to an unrearranged variable region and / or a rearranged variable region gene "derived from" an unrearranged variable region gene segment, refers to the fact that the sequence of the rearranged variable region gene can be traced back to the set of unrearranged variable region gene segments that were rearranged to form the gene expressing that variable domain (taking into account alternative splicing and somatic mutations, if applicable). For example, a rearranged variable region gene that has undergone somatic mutation is also derived from an unrearranged variable region gene segment. In some embodiments in which the endogenous locus has been replaced with a universal light chain or heavy chain locus, the term "derived from" indicates that the origin of the sequence can be traced back to the rearranged locus, even if the sequence has undergone somatic mutation.

[0041] As used herein, the phrase "endogenous gene" or "endogenous gene segment" refers to a gene or gene segment found in a parent or reference organism prior to the introduction of a disruption, deletion, substitution, alteration, or modification as described herein. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is an engineered organism. In some embodiments, the reference organism is a laboratory-raised organism (whether wild-type or engineered).

[0042] The term "in vivo recovery" or "IVR" refers to the incremental recovery (K value) obtained by subtracting the pre-dose level from the peak activity observed and then dividing by the dose. IVR can also be calculated on a percentage basis. An average IVR may be determined for a population of animals, or an individual IVR may be determined for each individual animal.

[0043] As used herein, the term "locus" refers to a location on a chromosome containing a set of related genetic elements (e.g., genes, gene segments, regulatory elements). For example, an unrearranged immunoglobulin locus can include an immunoglobulin variable region gene segment, one or more immunoglobulin constant region genes, and associated regulatory elements (e.g., promoters, enhancers, switch elements, etc.) that direct V(D)J recombination and immunoglobulin expression. A locus can be endogenous or non-endogenous. The term "endogenous locus" refers to the location on a chromosome where a particular genetic element is naturally found. In some embodiments, an endogenous locus has a sequence found in nature. In some embodiments, an endogenous locus is a wild-type locus. In some embodiments, an endogenous locus is an engineered locus. For example, an endogenous mouse immunoglobulin heavy chain locus refers to a location on mouse chromosome 12 that contains a wild-type mouse immunoglobulin heavy chain variable region gene segment and constant region gene, an endogenous mouse immunoglobulin λ light chain locus refers to a location on mouse chromosome 16 that contains a wild-type mouse immunoglobulin λ light chain variable region gene segment and constant region gene, while an endogenous mouse immunoglobulin κ light chain locus refers to a location on mouse chromosome 6 that contains a wild-type mouse immunoglobulin κ light chain variable region gene segment and constant region gene.

[0044] An unrearranged variable region gene segment can be said to be "operably linked" to an adjacent constant region gene if the unrearranged variable region gene segment is capable of rearranging to form a rearranged variable region gene that is expressed together with the constant region gene as a polypeptide chain of an antigen binding protein.

[0045] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and perform any function. Non-limiting examples of polynucleotides include: coding or non-coding regions of a gene or gene fragment, locus(s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Polynucleotides can be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides can be interchangeable with T nucleotides.

[0046] The term "unrearranged" refers to the state of an immunoglobulin variable region locus or variable region gene segment in which the V gene segment and the J gene segment (and, in the case of a heavy chain variable region, also the D gene segment) are maintained separately but can connect to form a rearranged V(D)J gene ("variable region gene") comprising one V, (D), and J of a V(D)J repertoire.

[0047] As used herein, the term "steady-state volume of distribution" or "Vss" refers to the apparent space (volume) in which a drug is distributed. More specifically, Vss represents the amount of drug in an animal divided by the steady-state plasma concentration.

[0048] As used herein, "C H gene segment" (e.g., Cγ1 gene segment, C γ2aGene segment, C γ2c gene segment, Cμ gene segment, C γ2b Gene segment, C γ3 Gene segment, C δ gene segment, Cε gene segment, C α The term gene segment (e.g., a segment of DNA sequence encoding an immunoglobulin heavy chain constant region) refers to a segment of DNA sequence encoding an immunoglobulin heavy chain constant region, H Genes (e.g., Cγ gene, C γ2a Gene, C γ2c Gene, C γ3 Gene, C γ2b gene, Cμ gene, C δ gene, Cε gene, C α For example, the term Cγ1 gene segment or Cγ1 gene refers to a segment of DNA sequence that encodes the IgG1 constant region. H The term "gene segment locus" refers to H Gene segment or C H Refers to the location on a chromosome where a gene is naturally found.

[0049] Genetically modified loci In certain aspects, provided herein are genetically modified rodents (e.g., mice or rats) useful for in vivo testing of therapeutic agents comprising a human Fc in the genetically modified rodent (e.g., testing the pharmacokinetic and / or pharmacodynamic properties of such therapeutic agents in the genetically modified rodent). In some embodiments, the genetically modified rodent comprises a genetically modified locus encoding an antibody heavy chain comprising a human Fc (e.g., human IgG1 Fc, human IgG4 Fc). In some embodiments, the rodent comprises a genetically modified locus encoding a fully or partially human light chain (e.g., encoding a gamma light chain or a kappa light chain). In certain embodiments, the genetically modified rodent comprises one or more loci encoding an Fc receptor comprising a human extracellular domain (e.g., fetal Fc receptor (FcRn) α-chain, β-2-microglobulin polypeptide (β2M), Fcε receptor 1 α (FcεR1α) α-chain, Fcγ receptor 1 alpha (FcγR1a) α-chain, Fc gamma receptor 2a (FcγR2a) α-chain, Fc gamma receptor 2b (FcγR2b) α-chain, Fc gamma receptor 3a (FcγR3a) α-chain, Fc gamma receptor 3b (FcγR3b) α-chain, Fc gamma receptor 2c (FcγR2c) α-chain). In certain embodiments, the transmembrane and cytoplasmic domains encoded by such loci can be human or non-human (e.g., rodent).

[0050] Humanized immunoglobulin heavy chain locus In certain aspects, provided herein are rodents (e.g., mice or rats) that comprise a genetically modified immunoglobulin (Ig) heavy chain locus. Such loci generally comprise a variable region and a constant region. The variable region comprises an Ig heavy chain variable region gene segment (e.g., at least V H Gene segment, D H Gene segments and J H A constant region locus contains one or more Ig heavy chain constant region gene segments (C H In certain embodiments, the immunoglobulin heavy chain variable region is selected from the group consisting of: V HGene segment, D H Gene segments and J H variable domains derived from gene segments and C H and a heavy chain constant domain derived from the gene segment, operably linked to an immunoglobulin heavy chain constant region to produce an antibody.

[0051] In some embodiments, the variable region is an unrearranged variable region and contains unrearranged Ig variable region gene segments. In some embodiments, the variable region is a rearranged variable region and thus contains rearranged variable region genes. In certain embodiments, the Ig variable region gene segments are human variable region gene segments. In certain embodiments, the Ig variable region gene segments are rodent (e.g., rat or mouse) variable region gene segments (e.g., rat or mouse variable region gene segments). Thus, in certain embodiments, the Ig heavy chain variable region locus contains human Ig variable region gene segments. Exemplary variable region loci comprising human variable region gene segments have been described in the art. For example, such loci are described in U.S. Pat. Nos. 5,770,429, 5,814,318, 6,114,598, 6,998,514, 8,232,449, 8,502,018, and 8,697,940, each of which is incorporated herein by reference, and U.S. Patent Publication Nos. 2008 / 0098490, 2012 / 0167237, 2013 / 0145484, 2013 / 0326647, 2014 / 013275, and 2014 / 093908, each of which is incorporated herein by reference.

[0052] In certain embodiments, the Ig heavy chain variable region locus contains unrearranged human Ig heavy chain variable region gene segments. In some embodiments, the unrearranged human Ig variable region gene segments comprise one or more human V H Segment, one or more human D Hsegment and one or more human J H In some embodiments, the unrearranged human Ig variable region gene segment comprises at least three V segments. H Gene segments, at least 18 V H Gene segments, at least 20 V H Gene segments, at least 30 V H Gene segments, at least 40 V H Gene segment, at least 50 V H Gene segments, at least 60 V H Gene segments, at least 70 V H gene segments, or at least 80 V H In some specific embodiments, the engineered IgH locus (or allele) comprises a human V gene segment of a naturally occurring human IgH locus. H 3-74 and Human V H All or substantially all functional human V sequences found between (and including) the 6-1 gene segment H In some specific embodiments, the engineered IgH locus (or allele) comprises at least a 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, 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, VH 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 In some embodiments, the non-human animals provided herein comprise a single polymorphic human V H Gene segments, multiple D H Gene segments and multiple J H In some embodiments, the V has a restricted immunoglobulin heavy chain locus characterized by gene segments (e.g., as described in U.S. Patent Publication No. 2013 / 0096287, which is incorporated herein by reference). H The gene segment is V H 1-2 or V H 1-69. In some embodiments, the non-human animals provided herein have a rearranged heavy chain variable region (universal heavy chain variable region or consensus heavy chain coding sequence; e.g., those described in U.S. Patent Publication No. 20140245468 and U.S. Patent Nos. 9,204,624 and 9,930,871, each of which is incorporated by reference in its entirety). In some embodiments, the non-human animals provided herein comprise an unrearranged human immunoglobulin light chain (e.g., κ) gene segment (e.g., U.S. Patent No. 9,516,868, incorporated by reference in its entirety) operably linked to a heavy chain constant region gene of an immunoglobulin heavy chain locus.

[0053] In still other embodiments, a non-human organism may comprise in its germline and / or genome a heavy chain immunoglobulin locus that comprises an insertion and / or substitution of a histidine codon designed to introduce pH-dependent binding properties into antibodies produced in such non-human organism. In some such embodiments, the histidine codon is inserted and / or substituted into the nucleic acid sequence encoding CDR3. Examples of such various heavy chain immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, and U.S. Patent Application Publication Nos. 2013 / 0247236 and 20140013456, which are incorporated herein by reference.

[0054] In some embodiments, the engineered IgH locus (or allele) comprises 5, 10, 15, 20, 25 or more (e.g., 26, 27, etc.) human D H In some specific embodiments, the engineered IgH locus (or allele) comprises a human D gene segment of a naturally occurring human IgH locus. H 1-1 and Human D H All or substantially all functional human D sequences found between (and including) the 7-27 gene segment H In some specific embodiments, the engineered IgH locus (or allele) comprises at least one human 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, DH 3-22, D H 6-25, D H 1-26 and D H 7-27. In some embodiments, the unrearranged human Ig gene segment comprises a human D H Includes all gene segments.

[0055] In some embodiments, the engineered IgH locus (or allele) comprises 1, 2, 3, 4, 5, 6 or more functional human J H In some specific embodiments, the engineered IgH locus (or allele) comprises a human J gene segment of a naturally occurring human IgH locus. H 1 and Human J H All or substantially all functional human J sequences found between (and including) the six gene segments H In some specific embodiments, the engineered IgH locus (or allele) comprises at least a human J gene segment. H Gene segment J H 1. J H 2. J H 3. J H 4. J H 5 and J H In some embodiments, the unrearranged human Ig gene segment comprises a human J H Includes all gene segments.

[0056] In some embodiments, the engineered IgH loci described herein do not contain an endogenous Adam6 gene. In some embodiments, the engineered IgH loci described herein do not contain an endogenous Adam6 gene (or Adam6 coding sequence) at the same germline genomic location as found in the germline genome of a wild-type non-human animal of the same species. In some embodiments, the engineered IgH loci described herein do not contain a human Adam6 pseudogene. In some embodiments, the engineered IgH loci described herein comprise an insertion of at least one nucleotide sequence encoding one or more non-human (e.g., rodent) Adam6 polypeptides. The insertion is located outside of the engineered immunoglobulin heavy chain loci described herein (e.g., near the 5'-most V H The location may be upstream of the gene segment), within an engineered IgH locus, or elsewhere in the germline genome of the non-human animal, cell, or tissue (e.g., a randomly introduced non-human Adam6 coding sequence).

[0057] In some embodiments, the engineered endogenous immunoglobulin heavy chain locus lacks a functional endogenous rodent Adam6 gene. In some embodiments, the rodent germline genome comprising the engineered heavy chain locus comprises one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are expressed (e.g., in cells of the male reproductive system, e.g., testicular cells).

[0058] In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are contained on the same chromosome as the engineered endogenous immunoglobulin heavy chain locus. ... first human V H Gene segments and the second human V H In some embodiments, the first human V H The gene segment is V H 1-2 and the second human V H The gene segment is V H 6-1. In some embodiments, the one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are in place of the human Adam6 pseudogene. In some embodiments, the one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof replace the human Adam6 pseudogene. In some embodiments, the one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are in place of the human Adam6 pseudogene. H Gene segments and human D H Between gene segments.

[0059] Exemplary Ig variable regions, including Ig heavy chain gene segments, are provided, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and supplementary information, which are incorporated herein by reference. Such mice are described, for example, in U.S. Patent Nos. 8,642,835 and 8,697,940, which are incorporated herein by reference.

[0060] In some embodiments, the Ig heavy chain variable gene locus comprising an unrearranged human Ig heavy chain variable region gene segment also comprises a human Ig heavy chain variable region intergenic sequence. In some embodiments, the Ig heavy chain variable gene locus comprises a non-human (e.g., rodent, rat, mouse) Ig heavy chain variable region intergenic sequence. In some embodiments, the IgH locus comprises a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer). In some embodiments, the IgH locus comprises an IgM enhancer (Eμ). In some embodiments, the IgM enhancer is a non-human Eμ (e.g., a rodent Eμ, such as a mouse or rat Eμ).

[0061] In some embodiments, the Ig heavy chain variable region is a rearranged variable region comprising an Ig heavy chain variable region gene (universal heavy chain variable region). In some embodiments, the rearranged Ig heavy chain variable region gene is a rearranged human Ig heavy chain variable region gene. Exemplary rearranged Ig heavy chain variable regions are provided in U.S. Patent Publication No. 2014 / 0245468, which is incorporated herein by reference.

[0062] In certain embodiments, the immunoglobulin constant region is human C H 1 domain, human hinge region, human C H 2 domains, human C HC, which encodes the IgG constant domain containing three domains: the IgG transmembrane domain and the IgG cytoplasmic domain. H The IgG connecting region comprises a gene segment. In some embodiments, the IgG transmembrane domain is a rodent IgG transmembrane domain (e.g., a mouse or rat transmembrane domain). In certain embodiments, the transmembrane domain is a human IgG transmembrane domain. In some embodiments, the IgG cytoplasmic domain is a rodent IgG cytoplasmic domain (e.g., a mouse or rat cytoplasmic domain). In some embodiments, the IgG cytoplasmic domain is a human IgG cytoplasmic domain. In some embodiments, the IgG connecting region is a rodent IgG connecting region (e.g., a mouse or rat connecting domain). In certain embodiments, the IgG connecting region is a human IgG connecting region.

[0063] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domain and human C H The three domains are IgG1 domains. In some embodiments, the IgG1 domains are encoded by alleles selected from IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04 and IGHG1*05.

[0064] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domain and human C H The 3 domain is an IgG2 domain. In some embodiments, the IgG2 domain is encoded by an allele selected from IGHG2*01, IGHG2*02, IGHG2*03, IGHG2*04, IGHG2*05 and IGHG2*06.

[0065] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domain and human C HThe 3 domain is an IgG3 domain, in some embodiments, such an IgG3 domain is encoded by an allele selected from IGHG3*01, IGHG3*02, IGHG3*03, IGHG3*04, IGHG3*05, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*13, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18 and IGHG3*19.

[0066] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domain and human C H The 3 domain is an IgG4 domain. In some embodiments, the IgG4 domain is encoded by an allele selected from IGHG4*01, IGHG4*02, IGHG4*03 and IGHG4*04.

[0067] In some embodiments, C H The gene segments encode variant human immunoglobulin heavy chain constant region sequences (i.e., human immunoglobulin heavy chain constant region sequences that contain one or more additions, deletions, and / or substitutions relative to an appropriate reference human immunoglobulin heavy chain constant region sequence) that are characterized by increased or decreased effector function and / or affinity for FcRs compared to a reference human immunoglobulin heavy chain constant region.

[0068] In some embodiments, C H The gene segments encode human immunoglobulin heavy chain constant regions characterized by altered affinities for activating and / or inhibitory receptors. In some embodiments, H The gene segment encodes a human immunoglobulin heavy chain constant region characterized by increased or decreased binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. HThe gene segment encodes, in whole or in part, a human immunoglobulin heavy chain constant region, and encodes a human immunoglobulin heavy chain constant region having one or more amino acid modifications. Exemplary amino acid modifications include those at position 297 (e.g., N297A), position 250 (e.g., 250E or 250Q), position 252 (e.g., 252L, 252Y, 252F, 252W, or 252T), position 254 (e.g., 254S or 254T), position 256 (e.g., 256S, 256R, 256Q, 256E, 256D, or 256T), position 307 (e.g., 307P or 307R), and the like. or 307A), position 308 (e.g., 308F or 308V), position 428 (e.g., 428L or 428F), position 433 (e.g., 433H, 433Lm, 433R, 433S, 433P, 433Q, or 433K), position 434 (e.g., 434A, 434W, 434H, 434F, or 434Y), and combinations thereof. H The gene segment encodes a human immunoglobulin heavy chain constant region having one or more pairs or groups of amino acid modifications selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F).

[0069] In some embodiments, C H The gene segment encodes a chimeric immunoglobulin heavy chain constant domain that includes segments or portions derived from (or occurring in) more than one human immunoglobulin isotype. For example, such a chimeric C H The region is C from a human IgG1, human IgG2, or human IgG4 molecule. H2 domains derived from human IgG1, human IgG2 or human IgG4 molecules H In some particular embodiments, the chimeric C H The region further comprises a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues at positions 228 to 236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. In some specific embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge.

[0070] In certain embodiments, modified C H The gene segment is endogenous C H Located at the gene segment locus. In certain embodiments, modified C H The gene segment is endogenous C γ1 Gene segment locus, endogenous C γ2a Gene segment locus, endogenous C γ2b Gene segment locus, endogenous C γ2c Gene segment locus or endogenous C γ3 Located at the gene segment locus.

[0071] The endogenous immunoglobulin heavy chain constant region gene structure can vary among rodents. For example, the Norway rat has a C γ3 Although mouse strains lack the C gene segment, mouse strains generally do. Even within the same species, immunoglobulin heavy chain constant region gene structure can vary among strains. For example, some mouse strains lack the C gene segment at their endogenous immunoglobulin heavy chain loci. γ2a gene segment, but other mouse strains (e.g., mouse strains containing the Igh1-b allele) instead contain C γ2cSome mouse strains have gene segments, γ2a and C γ2c Thus, the rodent C disclosed in the figures, examples, and / or description herein may have both gene segments. γ1 , C γ2a , C γ2b and C γ3 The gene segments are derived from an exemplary rodent C H The constant region gene segments are gene segments, and one skilled in the art will recognize that the specific constant region gene structures may vary from strain to strain of rodent. Thus, for example, one skilled in the art will recognize that the present disclosure is not intended to be limiting for the purposes of any of the disclosed rodent C γ2a Instead of, or in addition to, gene segments, rodent C γ2c It will be appreciated that rodents containing the gene segments are contemplated.

[0072] In some embodiments, a modified C H The gene segment is human C γ1 gene segment (or at least the C of the IgG1 constant domain) H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ1 part of a gene segment) and endogenous C γ2a In some embodiments, the C of the IgG1 constant domain is located at the gene segment locus. H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ1 The portion of the gene segment is an endogenous rodent C encoding the IgG2a transmembrane domain and / or the cytoplasmic domain. γ2a operably linked to a portion of a gene segment.

[0073] In some embodiments, a modified C H The gene segment is human C γ1 gene segment (or at least the C of the IgG1 constant domain) H 1 domain, hinge region, CH 2 domain and C H Human C3 domain encoding γ1 part of a gene segment) and endogenous C γ2c In some embodiments, the C of the IgG1 constant domain is located at the gene segment locus. H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ1 The portion of the gene segment is an endogenous rodent C encoding the IgG2c transmembrane domain and / or the cytoplasmic domain. γ2c operably linked to a portion of a gene segment.

[0074] In some embodiments, a modified C H The gene segment is human C γ4 gene segment (or at least the C of the IgG4 constant domain) H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ4 part of a gene segment) and endogenous C γ1 In some embodiments, the C of the IgG4 constant domain is located at the gene segment locus. H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ4 The portion of the gene segment is an endogenous rodent C encoding the IgG1 transmembrane domain and / or the cytoplasmic domain. γ1 operably linked to a portion of a gene segment.

[0075] In certain embodiments, modified C H The gene segment is endogenous C H In certain embodiments, a modified C H The gene segment is endogenous C γ1 Gene segment, endogenous C γ2a Gene segment, endogenous C γ2bGene segment, endogenous C γ2c gene segment, or endogenous C γ3 Replace all or part of a gene segment.

[0076] In some embodiments, a modified C H The gene segment is human C γ1 gene segment (or at least the C of the IgG1 constant domain) H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ1 part of a gene segment) and endogenous C γ2a In some embodiments, the C gene segment locus of the IgG1 constant domain is replaced in whole or in part. H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ1 The gene segment is part of the IgG1 constant domain C H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ1 The portion of the gene segment encodes the IgG2a transmembrane domain and / or the cytoplasmic domain of an endogenous rodent C γ2a operably linked to a portion of the gene segment C of the IgG2a constant domain H 1 domain, hinge region, C H 2 domain and C H Endogenous rodent C encoding three domains γ2a Replaces part of a gene segment.

[0077] In some embodiments, a modified C H The gene segment is human C γ4 gene segment (or at least the C of the IgG4 constant domain) H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ4part of a gene segment) and endogenous C γ1 In some embodiments, the C gene segment locus of the IgG4 constant domain is replaced in whole or in part. H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ4 The gene segment is part of the IgG4 constant domain C H 1 domain, hinge region, C H 2 domain and C H Human C3 domain encoding γ4 The portion of the gene segment encodes an endogenous rodent C IgG1 transmembrane domain and / or cytoplasmic domain. γ1 operably linked to a portion of the gene segment, H 1 domain, hinge region, C H 2 domain and C H Endogenous rodent C encoding three domains γ1 Replaces part of a gene segment.

[0078] In certain embodiments, the Ig heavy chain constant region is selected from one or more rodent (e.g., rat or mouse) C H In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C gene segment. μ In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C gene segment. δ In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C gene segment. γ1 In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C gene segment. γ2a In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C gene segment. γ2b In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C gene segment. γ2cIn some embodiments, the Ig constant region comprises a rodent (e.g., mouse) C gene segment. γ In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C ε In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C gene segment. ε In some embodiments, the one or more rodent constant region gene segments are endogenous constant region gene segments. In some embodiments, the one or more rodent constant region gene segments are endogenous constant region gene segments. In some embodiments, the one or more rodent constant region gene segments are endogenous constant region gene segments. H The gene segment is the only modified C in the Ig heavy chain constant region. H It is a gene segment.

[0079] In some embodiments, the modified C H The gene segment contains multiple modified C H In some embodiments, the Ig heavy chain constant region is one of the human or partially human C gene segments (e.g., one of 2, 3, 4, 5, 6, 7, or 8 partially or fully humanized gene segments in the Ig heavy chain constant region). μ In some embodiments, the Ig heavy chain constant region comprises a human or partially human C gene segment. δ In some embodiments, the Ig heavy chain constant region comprises a human or partially human C gene segment. γ1 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C gene segment. γ2 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C gene segment. γ3 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C gene segment. γ4 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C gene segment. ε In some embodiments, the Ig heavy chain constant region comprises a human or partially human C gene segment. αIn some embodiments, the Ig heavy chain constant region comprises a human C μ Gene segment, human C δ Gene segment, human C γ1 Gene segments and human C γ3 In some embodiments, the Ig heavy chain constant region comprises a human C γ2 Gene segments and human C γ4 In some embodiments, the Ig heavy chain constant region further comprises a human C α In some embodiments, the Ig heavy chain constant region further comprises a human C ε It further includes gene segments.

[0080] In some embodiments, the IgH locus comprises a human or rodent (e.g., rat or mouse) regulatory element. In some embodiments, the regulatory element is an endogenous regulatory element. In certain embodiments, the IgH locus comprises a rodent (e.g., rat or mouse) or human intronic enhancer (E i In some embodiments, the IgH locus comprises a rodent (e.g., rat or mouse) or human 3' regulatory region (3'RR).

[0081] In some embodiments, the modified immunoglobulin heavy chain locus is positioned at an endogenous immunoglobulin heavy chain locus. In some embodiments, the immunoglobulin heavy chain locus replaces all or part of the endogenous immunoglobulin heavy chain locus. In particular embodiments, the modified IgH locus is located on a transgene that is positioned outside the endogenous locus. In some embodiments, the endogenous IgH locus is inactivated (e.g., by deletion, rearrangement, and / or inversion of all or part of the endogenous Ig heavy chain locus).

[0082] Thus, in some embodiments, one or more immunoglobulin heavy chain constant regions (or portions thereof) of an immunoglobulin heavy chain locus are not deleted (i.e., are intact). H The gene segment may comprise, inter alia, an immunoglobulin heavy chain constant region sequence described herein (e.g., a human IgG C ) operably linked to transmembrane and cytoplasmic coding sequence(s) of a non-human immunoglobulin heavy chain IgG constant region gene (e.g., an M1 and / or M2 coding sequence). H 1-HC H 2-C H a sequence encoding a 3 polypeptide), and in some embodiments, an immunoglobulin heavy chain constant region sequence (e.g., human IgE C 3 polypeptide) operably linked to the transmembrane and cytoplasmic coding sequence(s) of an IgE constant region gene. H 1-C H 2-C H 3-C H In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operably linked to transmembrane and cytoplasmic coding sequences (e.g., M1 and M2 exons) of one or more IgG constant region genes. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operably linked to transmembrane and cytoplasmic coding sequences (e.g., M1 and M2 exons) of an IgE constant region gene. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operably linked to transmembrane and cytoplasmic coding sequences (e.g., M exon(s)) of an IgA constant region gene. In some specific embodiments, one or more C H Gene segments (e.g., C μ , C δIn some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is a human immunoglobulin heavy chain constant region sequence. In some embodiments, the immunoglobulin heavy chain constant region that is altered, disrupted, deleted, replaced, or engineered with one or more heterologous immunoglobulin heavy chain constant region sequences is a murine immunoglobulin heavy chain constant region. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is an IgG constant region gene (e.g., C γ1 , C γ2a , C γ2b , C γ2c or C γ3 ), resulting in a non-human animal that is heterozygous for the xenogenous immunoglobulin heavy chain constant region sequence. In some embodiments, non-human animals are provided that are homozygous for an immunoglobulin heavy chain constant region comprising a xenogenous immunoglobulin heavy chain constant region sequence described herein.

[0083] In some embodiments, the engineered immunoglobulin heavy chain constant regions described herein comprise a human extracellular domain coding sequence (e.g., human IgG C) operably linked to a non-human transmembrane and cytoplasmic domain coding sequence (e.g., non-human IgG M1-M2) of the same or a different IgG subclass. H 1-HC H 2-C H 3) one or more IgG code C H Contains gene segments.

[0084] In some embodiments, the engineered immunoglobulin heavy chain constant regions described herein are selected from the group consisting of one or more engineered IgG coding regions. H a wild-type (e.g., unmodified non-human (e.g., rat or mouse)) C gene segment; μIt further contains constant region genes.

[0085] In some embodiments, the engineered immunoglobulin heavy chain constant regions described herein are selected from the group consisting of one or more engineered IgG coding regions. H a wild-type (e.g., unmodified non-human (e.g., rat or mouse)) C gene segment; μ and C δ It further contains constant region genes.

[0086] In various embodiments, an engineered IgG encoding C IgG1A comprises a human immunoglobulin heavy chain constant region sequence described herein. H The gene segment is C γ1 , C γ2a , C γ2b , C γ2c , or C γ3 an engineered IgG code C of an IgG subclass selected from H It is a gene segment.

[0087] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions described herein are H 1-HC H 2-C H Human IgG1 C instead of exon 3 H 1-HC H 2-C H 3, and a non-human (e.g., rat or mouse) C γ2a an engineered C operably linked to the M1 and M2 exons of the gene segment; γ2a Contains gene segments.

[0088] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions described herein are H 1-HC H 2-C H Human IgG1 instead of the 3-M1-M2 exon C H 1-HC H 2-C H3-M1-M2 encoding sequences, and γ2a an engineered C operably linked to a switch region of a gene segment; γ2a Contains gene segments.

[0089] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions described herein are H 1-HC H 2-C H Human IgG1 C instead of exon 3 H 1-HC H 2-C H 3, and a non-human (e.g., rat or mouse) C γ2c an engineered C operably linked to the M1 and M2 exons of the gene segment; γ2c Contains gene segments.

[0090] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions described herein are H 1-HC H 2-C H Human IgG1 instead of the 3-M1-M2 exon C H 1-HC H 2-C H 3-M1-M2 encoding sequences, and γ2c an engineered C operably linked to a switch region of a gene segment; γ2c Contains gene segments.

[0091] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions described herein are H 1-HC H 2-C H Human IgG4 C instead of exon 3 H 1-HC H 2-C H 3, and a non-human (e.g., rat or mouse) C γ1an engineered C operably linked to the M1 and M2 exons of the gene segment; γ1 Contains gene segments.

[0092] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions described herein are H 1-HC H 2-C H Human IgG4 instead of the 3-M1-M2 exon C H 1-HC H 2-C H 3-M1-M2 encoding sequences, and γ1 an engineered C operably linked to a switch region of a gene segment; γ1 Contains gene segments.

[0093] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions described herein are H 1-HC H 2-C H Human IgG1 C instead of exon 3 H 1-HC H 2-C H 3, and a non-human (e.g., rat or mouse) C γ2a an engineered C operably linked to the M1 and M2 exons of the gene segment; γ2a gene segments and C H 1-HC H 2-C H Human IgG4 instead of exon 3 C H 1-HC H 2-C H 3, and a non-human (e.g., rat or mouse) C γ1 an engineered C operably linked to the M1 and M2 exons of the gene segment; γ1 and a gene segment.

[0094] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions described herein are H 1-HC H 2-C H Human IgG1 instead of the 3-M1-M2 exon C H 1-HC H 2-C H 3-M1-M2 encoding sequences, and γ2a an engineered C operably linked to a switch region of a gene segment; γ2a gene segments and C H 1-HC H 2-C H Human IgG4 C instead of 3-M1-M2 exons H 1-HC H 2-C H 3-M1-M2 encoding sequences, and γ1 an engineered C operably linked to a switch region of a gene segment; γ1 and a gene segment.

[0095] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions described herein are H 1-HC H 2-C H Human IgG1 C instead of exon 3 H 1-HC H 2-C H 3, and a non-human (e.g., rat or mouse) C γ2c an engineered C operably linked to the M1 and M2 exons of the gene segment; γ2c gene segments and C H 1-HC H 2-C H Human IgG4 instead of exon 3 C H 1-HC H 2-C H 3, and a non-human (e.g., rat or mouse) C γ1an engineered C operably linked to the M1 and M2 exons of the gene segment; γ1 and a gene segment.

[0096] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions described herein are H 1-HC H 2-C H Human IgG1 instead of the 3-M1-M2 exon C H 1-HC H 2-C H 3-M1-M2 encoding sequences, and γ2c an engineered C operably linked to a switch region of a gene segment; γ2c gene segments and C H 1-HC H 2-C H Human IgG4 C instead of 3-M1-M2 exons H 1-HC H 2-C H 3-M1-M2 encoding sequences, and γ1 an engineered C operably linked to a switch region of a gene segment; γ1 and a gene segment.

[0097] In various embodiments, the engineered immunoglobulin heavy chain constant regions described herein are human IgG C H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H One or more constant region genes (i.e., isotypes) other than the IgG constant region, including sequences encoding 3-M1-M2 (e.g., IgG1 and / or IgG2a), can be added to, for example, IgD, IgE, IgA, and IgG (which itself is a human IgG C as described herein). H 1-HC H 2-C H 3 or human IgG C H1-HC H 2-C H and one or more further modifications, including rendering non-human animals, embryos, and cells non-functional through total or partial deletion, total or partial alteration, total or partial disruption, or total or partial replacement of one or more immunoglobulin constant region genes encoding IgGs (e.g., IgG2b and / or IgG3) that do not contain sequences encoding 3-M1-M2. Engineered non-human embryos, cells, and targeting vectors for producing such non-human animals, embryos, and cells are also provided.

[0098] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segments and C γ1 A gene segment comprising: C H 1-HC H 2-C H Human IgG4 C instead of exon 3 H 1-HC H 2-C H 3, and the C γ1 C operably linked to the M1-M2 exon of the gene segment γ1 gene segments and C δ , C γ2a , C γ2c , C γ2b , C γ3 , C ε and C α and deletion of a gene segment.

[0099] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segments and C γ1 A gene segment comprising: C H 1-HC H 2-C H Human IgG4 C instead of 3-M1-M2 exons H 1-HC H2-C H 3-M1-M2 coding sequence, γ1 C operably linked to the switch region of the gene segment γ1 gene segments and C δ , C γ2a , C γ2c , C γ2b , C γ3 , C ε and C α and deletion of a gene segment.

[0100] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segments and C γ2a A gene segment comprising: C H 1-HC H 2-C H Human IgG1 C instead of exon 3 H 1-HC H 2-C H 3, and the C γ2a C operably linked to the M1-M2 exon of the gene segment γ2a gene segments and C δ , C γ1 , C γ2b , C γ2c , C γ3 , C ε and C α and deletion of a gene segment.

[0101] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segments and C γ2a A gene segment comprising: C H 1-HC H 2-C H Human IgG1 C instead of 3-M1-M2 exons H 1-HC H 2-C H3-M1-M2 coding sequence, γ2a C operably linked to the switch region of the gene segment γ2a gene segments and C δ , C γ1 , C γ2b , C γ2c , C γ3 , C ε and C α and deletion of a gene segment.

[0102] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segments and C γ2c A gene segment comprising: C H 1-HC H 2-C H Human IgG1 C instead of exon 3 H 1-HC H 2-C H 3, and the C γ2c C operably linked to the M1-M2 exon of the gene segment γ2c gene segments and C δ , C γ1 , C γ2a , C γ2b , C γ3 , C ε and C α and deletion of a gene segment.

[0103] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segments and C γ2c A gene segment comprising: C H 1-HC H 2-C H Human IgG1 C instead of 3-M1-M2 exons H 1-HC H 2-C H 3-M1-M2 coding sequence,γ2c C operably linked to the switch region of the gene segment γ2c gene segments and C δ , C γ1 , C γ2a , C γ2b , C γ3 , C ε and C α and deletion of a gene segment.

[0104] In various embodiments, the engineered immunoglobulin heavy chain constant regions described herein are human IgG C H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H One or more constant region genes (i.e., isotypes) other than the IgG constant region, including sequences encoding 3-M1-M2 (e.g., IgG1 and / or IgG2a), are selected from the human immunoglobulin heavy chain constant region sequences described herein, including IgD, IgE, IgA, and IgG (which themselves are human IgG C sequences described herein). H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H and one or more further modifications, including engineering the gene(s) to be altered, modified, substituted, engineered, etc. via insertion into one or more immunoglobulin constant region genes of an IgG (e.g., IgG2b and / or IgG3) that do not contain sequences encoding 3-M1-M2.

[0105] Also provided are engineered non-human embryos, cells, and targeting vectors for generating non-human animals, embryos, and cells that contain immunoglobulin loci that include the engineered constant regions described herein.

[0106] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are selected from the group consisting of wild-type C μ gene segment and wild-type C δgene segments and C γ3 A gene segment comprising: C H 1-HC H 2-C H Human IgG3 instead of exon 3 C H 1-HC H 2-C H 3, and the C γ3 C operably linked to the M1-M2 exon of the gene segment γ3 gene segments and C γ1 A gene segment comprising: C H 1-HC H 2-C H Human IgG4 C instead of exon 3 H 1-HC H 2-C H 3, and the C γ1 C operably linked to the M1-M2 exon of the gene segment γ1 gene segments and C γ2b A gene segment comprising: C H 1-HC H 2-C H Human IgG2 C instead of exon 3 H 1-HC H 2-C H 3, and the C γ2b C operably linked to the M1-M2 exon of the gene segment γ2b gene segments and C γ2a A gene segment comprising: C H 1-HC H 2-C H Human IgG1 C instead of exon 3 H 1-HC H 2-C H 3, and the C γ2a C operably linked to the M1-M2 exon of the gene segment γ2a Gene segment (and / or C γ2c A gene segment comprising: C H 1-HC H 2-C HHuman IgG1 instead of exon 3 C H 1-HC H 2-C H 3, and the C γ2c C operably linked to the M1-M2 exon of the gene segment γ2c gene segment) and C ε A gene segment comprising: C H 1-C H 2-C H 3-C H Human IgE instead of exon 4 C H 1-C H 2-C H 3-C H 4, and ε C operably linked to the M1-M2 exon of the gene segment ε gene segments and C α A gene segment comprising: C H 1-HC H 2-C H Human IgA1 or IgA2 C instead of exon 3 H 1-HC H 2-C H 3, and the C α a C operably linked to the M exon(s) of the gene segment; α and a gene segment.

[0107] In some particular embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are selected from the group consisting of wild-type C μ gene segment and wild-type C δ gene segments and C γ3 A gene segment comprising: C H 1-HC H 2-C H Human IgG3 C instead of 3-M1-M2 exons H 1-HC H 2-C H 3-M1-M2 coding sequence, γ3 C operably linked to the switch region of the gene segmentγ3 gene segments and C γ1 A gene segment comprising: C H 1-HC H 2-C H Human IgG4 C instead of 3-M1-M2 exons H 1-HC H 2-C H 3-M1-M2 coding sequence, γ1 C operably linked to the switch region of the gene segment γ1 gene segments and C γ2b A gene segment comprising: C H 1-HC H 2-C H Human IgG2 C instead of 3-M1-M2 exons H 1-HC H 2-C H 3-M1-M2 coding sequence, γ2b C operably linked to the switch region of the gene segment γ2b gene segments and C γ2a A gene segment comprising: C H 1-HC H 2-C H Human IgG1 C instead of 3-M1-M2 exons H 1-HC H 2-C H 3-M1-M2 coding sequence, γ2a C operably linked to the switch region of the gene segment γ2a Gene segment (and / or C γ2c A gene segment comprising: C H 1-HC H 2-C H Human IgG1 C instead of 3-M1-M2 exons H 1-HC H 2-C H 3-M1-M2 coding sequence, γ2 C operably linked to the switch region of the c gene segment γ2c gene segment) and C ε A gene segment comprising: C H 1-CH 2-C H 3-C H Human IgE C instead of exon 4 H 1-C H 2-C H 3-C H 4, and ε C operably linked to the M1-M2 exon of the gene segment ε gene segments and C α A gene segment comprising: C H 1-HC H 2-C H Human IgA1 or IgA2 C instead of exon 3 H 1-HC H 2-C H 3, and the C α a C operably linked to an M exon(s) of a gene segment; α and a gene segment.

[0108] In various embodiments, provided herein are genetically engineered heavy chain immunoglobulin loci shown in Figure 1, as well as genetically modified rodents (e.g., rats or mice), ES and other cells, and tissues comprising such loci. Thus, in one embodiment, provided herein are genetically engineered rodents (e.g., rats or mice), ES and other cells, and tissues comprising such loci, comprising human heavy chain variable gene segments (human heavy chain V, D, and J gene segments), a rodent (e.g., rats or mice) Ei enhancer, and a rodent (e.g., rats or mice) C enhancer. μ , C δ , C γ3 , C γ1 , C γ2b gene segments and C H 1-HC H 2-C H Human IgG1 C instead of exon 3 H 1-HC H 2-C H 3, and a rodent (e.g., rat or mouse) C γ2a a chimeric C operably linked to the M1-M2 exon of the gene segment γ2agene segments (e.g., in a format such that an exon encoding the extracellular domain of human IgG1 is operably linked to an exon encoding the transmembrane and cytoplasmic domains of rodent (e.g., rat or mouse) IgG2a) (and / or H 1-HC H 2-C H Human IgG1 C instead of exon 3 H 1-HC H 2-C H 3, and a rodent (e.g., rat or mouse) C γ2c a chimeric C operably linked to the M1-M2 exon of the gene segment γ2c Gene segments (e.g., in a format in which an exon encoding the extracellular domain of human IgG1 is operably linked to an exon encoding the transmembrane and cytoplasmic domains of rodent (e.g., rat or mouse) IgG2c) and rodent (e.g., rat or mouse) C ε and C α and a rodent (e.g., rat or mouse) 3' regulatory region comprising a rodent (e.g., rat or mouse) 3' enhancer. An exemplary genetically modified heavy chain locus is provided in Figure 1 as Mouse Locus 1. In some embodiments, the genetically modified locus comprises a functional Adam6 gene, as described herein and in U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety. Genetically modified non-human animals, ES cells and other cells, and tissues comprising such genetically modified loci are also provided.

[0109] In one embodiment, provided herein is a method for constructing a human heavy chain variable gene segment (human heavy chain V, D, and J gene segments), a rodent (e.g., rat or mouse) Ei enhancer, and a rodent (e.g., rat or mouse) C enhancer. μ , C δ , C γ3 , C γ1 , Cγ2b Human IgG1 C, in which both the gene segments and the IgG1 extracellular and transmembrane / cytoplasmic coding sequences are human. H 1-HC H 2-C H Human C containing the sequence encoding 3-M1-M2 γ1 Gene segments and rodent (e.g., rat or mouse) C ε and C α and a rodent (e.g., rat or mouse) 3' regulatory region comprising a rodent (e.g., rat or mouse) 3' enhancer. An exemplary genetically modified heavy chain locus is provided in Figure 1 as mouse locus 2. In some embodiments, the genetically modified locus comprises a functional Adam6 gene, as described herein and in U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety. Genetically modified non-human animals, ES cells and other cells, and tissues comprising such genetically modified loci are also provided.

[0110] In one embodiment, provided herein is a method for constructing a human heavy chain variable gene segment (human heavy chain V, D, and J gene segments), a rodent (e.g., rat or mouse) Ei enhancer, and a rodent (e.g., rat or mouse) C enhancer. μ , C δ , C γ3 gene segments and C H 1-HC H 2-C H Human IgG4 C instead of exon 3 H 1-HC H 2-C H 3, and a rodent (e.g., rat or mouse) C γ1 a chimeric C operably linked to the M1-M2 exon of the gene segment γ1Gene segments (e.g., in a format in which an exon encoding the extracellular domain of human IgG4 is operably linked to an exon encoding the transmembrane and cytoplasmic domain of rodent (e.g., rat or mouse) IgG1) and rodent (e.g., rat or mouse) C γ2b , C γ2a (and / or C γ2c ) and C ε , C α and a rodent (e.g., rat or mouse) 3' regulatory region comprising a rodent (e.g., rat or mouse) 3' enhancer. An exemplary genetically modified heavy chain locus is provided in Figure 1 as mouse locus 3. In some embodiments, the genetically modified locus comprises a functional Adam6 gene, as described herein and in U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety. Genetically modified non-human animals, ES cells and other cells, and tissues comprising such genetically modified loci are also provided.

[0111] In one embodiment, provided herein is a method for constructing a human heavy chain variable gene segment (human heavy chain V, D, and J gene segments), a rodent (e.g., rat or mouse) Ei enhancer, and a rodent (e.g., rat or mouse) C enhancer. μ , C δ , C γ3 Human IgG4 C, in which both the gene segments and the IgG4 extracellular and transmembrane / cytoplasmic coding sequences are human. H 1-HC H 2-C H Human C containing the sequence encoding 3-M1-M2 γ4 Gene segments and rodent (e.g., rat or mouse) C γ2b , C γ2a (and / or C γ2c ) gene segments and rodent (e.g., rat or mouse) C ε and C αand a rodent (e.g., rat or mouse) 3' regulatory region comprising a rodent (e.g., rat or mouse) 3' enhancer. An exemplary genetically modified heavy chain locus is provided in Figure 1 as mouse locus 4. In some embodiments, the genetically modified locus comprises a functional Adam6 gene, as described herein and in U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety. Genetically modified non-human animals, ES cells and other cells, and tissues comprising such genetically modified loci are also provided.

[0112] In one embodiment, provided herein is a human heavy chain variable gene segment (human heavy chain V, D, and J gene segments), a human Ei enhancer, and a human C μ , C δ , C γ3 and C γ1 and a rodent (e.g., rat or mouse) 3' regulatory region comprising a rodent (e.g., rat or mouse) 3' enhancer. An exemplary genetically modified heavy chain locus is provided in Figure 1 as mouse locus 5. In some embodiments, the genetically modified locus comprises a functional Adam6 gene, as described herein and in U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety. Genetically modified non-human animals, ES cells and other cells, and tissues comprising such genetically modified loci are also provided.

[0113] In one embodiment, provided herein is a human heavy chain variable gene segment (human heavy chain V, D, and J gene segments), a human Ei enhancer, and a human C μ , C δ , C γ3 , C γ1 , C γ2 and C γ4and a rodent (e.g., rat or mouse) 3' regulatory region comprising a rodent (e.g., rat or mouse) 3' enhancer. An exemplary genetically modified heavy chain locus is provided in Figure 1 as mouse locus 6. In some embodiments, the genetically modified locus comprises a functional Adam6 gene, as described herein and in U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety. Genetically modified non-human animals, ES cells and other cells, and tissues comprising such genetically modified loci are also provided.

[0114] In one embodiment, provided herein is a human heavy chain variable gene segment (rodent (e.g., rat or mouse) heavy chain V, D, and J gene segments), a rodent (e.g., rat or mouse) Ei enhancer, and a human C μ , C δ , C γ3 , and C γ1 and a rodent (e.g., rat or mouse) 3' regulatory region comprising a rodent (e.g., rat or mouse) 3' enhancer. An exemplary genetically modified heavy chain locus is provided in Figure 1 as mouse locus 7. In some embodiments, the genetically modified locus comprises a functional Adam6 gene. Genetically modified non-human animals, ES cells and other cells, and tissues comprising such genetically modified loci are also provided.

[0115] In one embodiment, provided herein is a human heavy chain variable gene segment (rodent (e.g., rat or mouse) heavy chain V, D, and J gene segments), a rodent (e.g., rat or mouse) Ei enhancer, and a human C μ , C δ , C γ3 , C γ1 , Cγ2 and C γ4 and a rodent (e.g., rat or mouse) 3' regulatory region comprising a rodent (e.g., rat or mouse) 3' enhancer. An exemplary genetically modified heavy chain locus is provided in Figure 1 as mouse locus 8. In some embodiments, the genetically modified locus comprises a functional Adam6 gene. Genetically modified non-human animals, ES cells and other cells, and tissues comprising such genetically modified loci are also provided.

[0116] Also provided herein are methods for generating an engineered immunoglobulin heavy chain locus. In some embodiments, the methods for generating an engineered immunoglobulin heavy chain locus described herein comprise: γ2a Gene segment C H 1-HC H 2-C H Human IgG1 C encodes an immunoglobulin heavy chain constant domain polypeptide in place of exon 3 H 1-HC H 2-C H Approximately 1.6 kb of DNA containing the 3 nucleotide sequence was inserted into the human IgG1 C H 1-HC H 2-C H 3 nucleotide sequence of the C γ2a This includes operably linking the M1 and M2 exons of the gene segment.

[0117] In some embodiments, the methods for generating an engineered immunoglobulin heavy chain locus described herein comprise: γ2a Gene segment C H 1-HC H 2-C H Human IgG1 C encodes immunoglobulin heavy chain constant domain polypeptides in place of the 3-M1-M2 exon H 1-HC H 2-C HApproximately 6.8 kb of DNA containing the 3-M1-M2 nucleotide sequence was inserted, and the human IgG1 C H 1-HC H 2-C H 3-M1-M2 nucleotide sequence of the C γ2a This includes operably linking the switch region of a gene segment.

[0118] Also provided herein are methods for generating an engineered immunoglobulin heavy chain locus. In some embodiments, the methods for generating an engineered immunoglobulin heavy chain locus described herein comprise: γ2c Gene segment C H 1-HC H 2-C H Human IgG1 C encodes an immunoglobulin heavy chain constant domain polypeptide in place of exon 3 H 1-HC H 2-C H Approximately 1.6 kb of DNA containing the 3 nucleotide sequence was inserted into the human IgG1 C H 1-HC H 2-C H 3 nucleotide sequence of the C γ2c This includes operably linking the M1 and M2 exons of the gene segment.

[0119] In some embodiments, the methods for generating an engineered immunoglobulin heavy chain locus described herein comprise: γ2c Gene segment C H 1-HC H 2-C H Human IgG1 C encodes immunoglobulin heavy chain constant domain polypeptides in place of the 3-M1-M2 exon H 1-HC H 2-C H Approximately 6.8 kb of DNA containing the 3-M1-M2 nucleotide sequence was inserted, and the human IgG1 C H 1-HC H 2-C H 3-M1-M2 nucleotide sequence of the C γ2cThis includes operably linking the switch region of a gene segment.

[0120] In some embodiments, the methods for generating an engineered immunoglobulin heavy chain locus described herein comprise: γ1 Gene segment C H 1-HC H 2-C H Human IgG4 encodes immunoglobulin heavy chain constant domain polypeptides in place of exon 3 C H 1-HC H 2-C H Approximately 1.5 kb of DNA containing the 3 nucleotide sequence was inserted into the human IgG4 C H 1-HC H 2-C H 3 nucleotide sequence of the C γ1 This includes operably linking the M1 and M2 exons of the gene segment.

[0121] In some embodiments, the methods for generating an engineered immunoglobulin heavy chain locus described herein comprise: γ1 Gene segment C H 1-HC H 2-C H Human IgG4 C encodes immunoglobulin heavy chain constant domain polypeptides in place of the 3-M1-M2 exon H 1-HC H 2-C H Approximately 5.8 kb of DNA containing the 3-M1-M2 nucleotide sequence was inserted, and the human IgG4 C H 1-HC H 2-C H 3-M1-M2 nucleotide sequence of the C γ1 This includes operably linking the switch region of a gene segment.

[0122] In some embodiments, the methods for generating an engineered immunoglobulin heavy chain locus described herein comprise the step of generating a human C μ , C δ , Cγ3 , C γ1 In another embodiment, the method comprises inserting approximately 130 kb of DNA comprising the sequence of human C γ2 and C γ4 The method further comprises inserting approximately 43 kb of additional DNA containing the sequence.

[0123] In the various methods for generating engineered immunoglobulin heavy chain loci described above, the DNA can be introduced into either an endogenous rodent (e.g., rat or mouse) immunoglobulin heavy chain locus that contains endogenous rodent (e.g., rat or mouse) heavy chain variable gene segments, or into an immunoglobulin heavy chain locus that contains human heavy chain variable gene segments. The DNA can be introduced via homologous recombination or any of the other methods known in the art or described herein.

[0124] In some embodiments, the rodent (e.g., rat or mouse) is heterozygous for a modified immunoglobulin heavy chain locus described herein. In certain embodiments, the rodent (e.g., rat or mouse) is homozygous for a modified immunoglobulin heavy chain locus described herein.

[0125] Humanized immunoglobulin kappa locus In certain aspects, provided herein is a rodent (e.g., a mouse or rat) comprising a genetically modified Ig κ chain locus. Such a locus comprises a κ variable region and a κ constant region. The κ variable region comprises an Ig κ chain variable region gene segment (i.e., at least V κ Gene segments and J κ The constant region contains the Ig κ chain constant region (C κ In certain embodiments, an immunoglobulin κ chain variable region, such as a human Igκ variable region, is synthesized by a rodent (e.g., a rat or a mouse) that is capable of expressing a human V κ Gene segments and human J κa light chain variable domain derived from a gene segment and a C κ and a light chain constant domain derived from a gene segment. In some embodiments, the Igκ variable region is an unrearranged Igκ variable region and thus contains an unrearranged Igκ variable region gene segment. In some embodiments, the Igκ variable region is a rearranged Igκ variable region and thus contains a rearranged Igκ variable region gene. In certain embodiments, the Igκ variable region gene segment is a human Igκ variable region gene segment. In certain embodiments, the Igκ variable region gene segment is a rodent Igκ variable region gene segment (e.g., a rat or mouse variable region gene segment). In some embodiments, the Igκ constant region locus comprises a partially or fully human Igκ constant region gene segment. In some embodiments, the Igκ chain locus described herein is located at an endogenous Igκ chain locus.

[0126] In certain embodiments, the Igκ variable region contains unrearranged human Igκ variable region gene segments. In some specific embodiments, the engineered Igκ light chain locus (or allele) comprises at least a human V that appears in the distal variable cluster (or distal arm, or distal duplication) of a naturally occurring human Igκ light chain locus. κ In some particular embodiments, the engineered Igκ light chain locus (or allele) comprises at least a human V allele that appears in the proximal variable cluster (or proximal arm, or proximal duplication) of a naturally occurring human Igκ light chain locus. κ In some specific embodiments, the engineered Igκ light chain locus (or allele) comprises a human V gene segment that occurs in the distal and proximal variable clusters of a naturally occurring human Igκ light chain locus. κ In some specific embodiments, the engineered Igκ light chain locus (or allele) comprises a human V gene segment of a naturally occurring human Igκ light chain locus. κ2-40 (or V κ 3D-7) and human V κ All or substantially all functional human V sequences found between (including the endpoints of) the 4-1 gene segment κ Contains gene segments.

[0127] In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise multiple human V κ segment and one or more human J κ In some embodiments, the immunoglobulin variable region gene segment comprises four functional V κ Segment and all human J κ In some embodiments, the immunoglobulin variable region gene segments comprise 16 functional V κ Segment and all human J κ In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises a human V κ All of the segments and all human J κ Includes segments.

[0128] In some specific embodiments, the engineered Igκ light chain locus (or allele) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or more (e.g., 36, 37, 38, 39, 40, etc.) human V κ In some specific embodiments, the engineered Igκ light chain locus (or allele) comprises a human V κ Gene segment V κ 3D-7, V κ 1D-8, V κ 1D-43, V κ 3D-11, V κ 1D-12, V κ 1D-13, V κ 3D-15, V κ 1D-16, V κ 1D-17, V κ 3D-20, V κ 6D-21, V κ2D-26, V κ 2D-28, V κ 2D-29, V κ 2D-30, V κ 1D-33, V κ 1D-39, V κ 2D-40, V κ 2-40, V κ 1-39, V κ 1-33, V κ 2-30, V κ 2-28, V κ 1-27, V κ 2-24, V κ 6-21, V κ 3-20, V κ 1-17, V κ 1-16, V κ 3-15, V κ 1-12, V κ 3-11, V κ 1-9, V κ 1-8, V κ 1-6, V κ 1-5, V κ 5-2 and V κ In some specific embodiments, the engineered Igκ light chain locus (or allele) comprises at least one human V κ Gene segment V κ 3D-7, V κ 1D-8, V κ 1D-43, V κ 3D-11, V κ 1D-12, V κ 1D-13, V κ 3D-15, V κ 1D-16, V κ 1D-17, V κ 3D-20, V κ 6D-21, V κ 2D-26, V κ 2D-28, V κ 2D-29, V κ 2D-30, V κ 1D-33, V κ 1D-39 and V κ In some specific embodiments, the engineered Igκ light chain locus (or allele) comprises at least one human V κGene segment V κ 2-40, V κ 1-39, V κ 1-33, V κ 2-30, V κ 2-28, V κ 1-27, V κ 2-24, V κ 6-21, V κ 3-20, V κ 1-17, V κ 1-16, V κ 3-15, V κ 1-12, V κ 3-11, V κ 1-9, V κ 1-8, V κ 1-6, V κ 1-5, V κ 5-2 and V κ Including 4-1.

[0129] In some embodiments, the non-human animals provided herein contain no more than two human V L Gene segments and multiple J L In some embodiments, the mouse has a restricted immunoglobulin light chain locus characterized by gene segments (e.g., the dual light chain mouse or DLC described in U.S. Patent Publication No. 2013 / 0198880, incorporated herein by reference). L The gene segment is V κ In some embodiments, the V L The gene segment is V λ In some embodiments, the V κ The gene segment is V κ 3-20 and V κ It is 1-39.

[0130] In some embodiments, the engineered Igκ light chain locus (or allele) comprises one, two, three, four, five, or more functional human J κ In some specific embodiments, the engineered Igκ light chain locus (or allele) comprises a human J gene segment of a naturally occurring human Igκ light chain locus. κ1 and Human J κ All or substantially all functional human J sequences found between (and including) the five gene segments κ In some specific embodiments, the engineered Igκ light chain locus (or allele) comprises at least a human J κ Gene segment J κ 1. J κ 2. J κ 3. J κ 4 and J κ Includes 5.

[0131] In yet other embodiments, a non-human organism may comprise in its germline and / or genome a light chain immunoglobulin locus that comprises an insertion and / or substitution of a histidine codon designed to introduce pH-dependent binding properties into antibodies produced in such non-human organism. In some such embodiments, the histidine codon is inserted and / or substituted into the nucleic acid sequence encoding CDR3. Examples of such various light chain immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, and U.S. Patent Application Publication Nos. 2013 / 0247236 and 20140013456, which are incorporated herein by reference.

[0132] Exemplary variable regions comprising Igκ gene segments are provided, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and supplementary information, which are incorporated herein by reference. In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all of the human Jκ segments.

[0133] In some embodiments, the Igκ variable gene locus containing unrearranged human Igκ variable region gene segments also comprises human Igκ variable region intergenic sequences. In some embodiments, the Igκ variable gene locus comprises non-human (e.g., rodent, rat, mouse) Igκ variable region intergenic sequences. In some embodiments, the Igκ gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0134] In some embodiments, the Igκ variable region locus is a rearranged variable region locus comprising an Igκ variable region gene (universal light chain variable region). In some embodiments, the rearranged Igκ variable region gene is a rearranged human Igκ variable region gene. The use of a universal light chain variable region facilitates the generation of bispecific antibodies. Exemplary rearranged Ig light chain variable regions are provided in U.S. Patent Publication No. 2013 / 0185821, which is incorporated herein by reference.

[0135] In some embodiments, the Ig κ chain locus comprises a human or rodent (e.g., rat or mouse) regulatory element. In some embodiments, the regulatory element is an endogenous regulatory element. In certain embodiments, the Ig κ chain locus comprises a rodent (e.g., rat or mouse) or human intronic κ enhancer (E κi In some embodiments, the IgH locus comprises a rodent (e.g., rat or mouse) or human 3' kappa enhancer (E κ3’ ) is included.

[0136] In some embodiments, the modified immunoglobulin κ chain locus is positioned at the endogenous immunoglobulin κ chain locus. In some embodiments, the immunoglobulin κ chain locus replaces all or part of the endogenous immunoglobulin κ chain locus. In particular embodiments, the modified Ig κ chain locus is located on a transgene that is positioned outside the endogenous locus. In some embodiments, the endogenous Ig κ chain locus is inactivated (e.g., by deletion, rearrangement, and / or inversion of all or part of the endogenous Ig κ chain locus).

[0137] In some embodiments, methods for generating an engineered immunoglobulin κ light chain locus described herein comprise inserting approximately 0.5 kb of DNA comprising a human Igκ constant nucleotide sequence encoding an immunoglobulin κ light chain constant domain polypeptide in place of an Igκ constant exon of an Igκ constant region gene, such that the human Igκ constant nucleotide sequence is operably linked to an enhancer and / or regulatory region of the Igκ constant region gene.

[0138] In some embodiments, the rodent (e.g., rat or mouse) is heterozygous for a modified immunoglobulin κ chain locus described herein. In certain embodiments, the rodent (e.g., rat or mouse) is homozygous for a modified immunoglobulin κ chain locus described herein.

[0139] Humanized immunoglobulin lambda locus In certain aspects, provided herein is a rodent (e.g., a mouse or rat) comprising a genetically modified Ig λ chain locus. Such a locus comprises an Ig λ chain variable region gene segment (i.e., at least V λ Gene segments and J λ The modified λ locus contains at least one Ig λ chain constant region (C λ In certain embodiments, the human V λ Gene segments and human Jλ V, such as gene segment λ Gene segments and J λ The gene segments are derived from a rodent (e.g., rat or mouse) that is similar to the human V λ Gene segments and human J λ a light chain variable domain derived from a gene segment and a C λ and a light chain constant domain derived from a C gene segment. λ In some embodiments, V λ Gene segments and J λ The gene segment is unrearranged V λ and J λ In some embodiments, the V λ Gene segments and J λ The gene segments are rearranged V λ and J λ In certain embodiments, the Ig λ The variable region gene segments are human variable region gene segments. In certain embodiments, Ig λ The variable region gene segments are rodent variable region gene segments (e.g., rat or mouse variable region gene segments). In some embodiments, Ig λ The constant region loci comprise partially or fully human λ constant region gene segments. In some embodiments, the Ig λ chain loci described herein are located at endogenous Ig λ chain loci. Exemplary variable regions comprising Ig λ gene segments are provided, for example, in U.S. Patent Publication Nos. 2012 / 0073004 and 2002 / 0088016, and U.S. Patent Application No. 15 / 803,513 (filed November 3, 2017; published as US2018 / 0125043), each of which is incorporated herein by reference.

[0140] In some embodiments, the Ig λ variable gene locus containing unrearranged human Igκ variable region gene segments also comprises human Ig λ variable region intergenic sequences. In some embodiments, the Ig λ variable gene locus comprises non-human (e.g., rodent, rat, mouse) Ig λ variable region intergenic sequences. In some embodiments, the Ig λ gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0141] A human Ig λ light chain locus, in some embodiments, comprises genetic material from a human Ig λ light chain locus. In some embodiments, the human Ig λ light chain locus described herein comprises at least one human V λ gene segment and at least one human J λ gene segment and at least one human C λ gene segment and the at least one human V λ a gene segment and the at least one human J λ Promoting rearrangement of gene segments with human V λ Functionally rearranged human V encoding domain λ -J λ and one or more sequences (e.g., recombination signal sequence(s)) necessary to form the human Ig λ light chain sequence. In many embodiments, the human Ig λ light chain sequence comprises multiple human V λ gene segment and the human V λ gene segment and at least one human J λ In some embodiments, the human Ig λ light chain sequences described herein are genomic sequences of the human Ig λ light chain locus (e.g., isolated and / or cloned from a bacterial artificial chromosome) and comprise multiple human V sequences in germline configuration. λ In some embodiments, the human Ig λ light chain sequence comprises human V gene segments in germline configuration.λ , J λ and C λ sequence (i.e., the human V λ , J λ and C λ The sequence is as it appears in the Igλ light chain locus in human cells, in other words, J λ and C λ The sequence is J λ C λ (These sequences are intended to be representative of a cluster of Ig λ light chains.) In some embodiments, the human Ig λ light chain sequences encode, in whole or in part, Ig λ light chain polypeptides that appear as immunoglobulins, particularly immunoglobulins expressed by human B cells. Also provided are non-human animals, embryos, cells, and targeting constructs for generating non-human animals, non-human embryos, and cells that contain the human Ig λ light chain sequences in place of corresponding non-human Ig λ light chain sequences (e.g., endogenous rodent Ig λ light chain loci).

[0142] In some embodiments, the human Igλ light chain sequence is inserted in place of the corresponding non-human Igλ light chain sequence in the germline genome of the non-human animal. In some embodiments, the human Igλ light chain sequence is inserted upstream of the non-human Igλ light chain sequence (e.g., the non-human Igλ light chain constant region sequence). In some embodiments, the human Igλ light chain sequence is inserted in the middle of one or more non-human Igλ light chain sequences such that the human Igλ light chain sequence is juxtaposed with the non-human Igλ light chain sequence.

[0143] In certain embodiments, the Ig λ chain locus comprises at least 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, or 40 functional V λ In some embodiments, the locus comprises a human V λ Gene segment V λ 3-10, V λ 3-9, V λ 2-8, V λ 4-3 and V λ 3-1. In some embodiments, the locus comprises V λ 2-11, V λ 3-12, Vλ 2-14, V λ 3-16, V λ 3-19, V λ 3-21, V λ 3-22, V λ 2-23, V λ 3-25 and V λ 3-27. In some embodiments, the locus is V λ 3-27, V λ 1-36, V λ 5-37, V λ 5-39, V λ 1-40, V λ 7-43, V λ 1-44, V λ 5-45, V λ 7-46, V λ 1-47, V λ 9-49, V λ 1-51 and V λ 5-52. In certain embodiments, the locus is V λ 10-54, V λ 6-57, V λ 4-60, V λ 8-61 and V λ In some embodiments, the Ig λ chain locus comprises one or more human J λ -C λ For example, in certain embodiments, the Ig λ chain locus comprises a human V λ Downstream of the gene segment, human J λ 1-C λ 1. J λ 2-C λ 2. J λ 3-C λ 3. J λ 6-C λ 6 and / or J λ 7-C λ 7. In some embodiments, Ig λ The human V chain locus is shown in Figure 2A. λ Downstream of the gene segment, human J λ 1-C λ 1. J λ 2-C λ 2. J λ 3-C λ 3. Jλ 6-C λ 6. Human J λ 7 and mouse C λ Includes 1.

[0144] In some embodiments, the Ig λ locus is a rearranged locus comprising an Ig λ variable region gene (universal light chain variable region). In some embodiments, the rearranged Ig λ variable region gene is a rearranged human Ig λ variable region gene. The use of a universal light chain variable region facilitates the generation of bispecific antibodies in which at least one antigen-binding domain has a binding domain. Exemplary rearranged Ig light chain variable regions are provided in U.S. Patent Publication No. 2013 / 0185821, which is incorporated herein by reference.

[0145] In some embodiments, the Ig λ chain locus comprises a human or rodent (e.g., rat or mouse) regulatory element. In some embodiments, the regulatory element is an endogenous regulatory element. In specific embodiments, the Ig λ chain locus comprises a rodent (e.g., rat or mouse) λ enhancer 2.4. In some embodiments, the Ig λ chain locus comprises a human or rodent (e.g., rat or mouse) 3' λ enhancer. In some embodiments, the Ig λ chain locus comprises a rodent (e.g., rat or mouse) λ enhancer 3.1.

[0146] In some embodiments, the modified immunoglobulin λ chain locus is positioned at an endogenous immunoglobulin λ chain locus. In some embodiments, the immunoglobulin λ chain locus replaces all or part of the endogenous immunoglobulin λ chain locus. In particular embodiments, the modified Ig λ chain locus is located on a transgene that is positioned outside the endogenous locus. In some embodiments, the endogenous Ig λ chain locus is inactivated (e.g., by deletion, rearrangement, and / or inversion of all or part of the endogenous Ig λ chain locus).

[0147] In some embodiments, the rodent (e.g., rat or mouse) is heterozygous for the modified immunoglobulin λ chain locus. In certain embodiments, the rodent (e.g., rat or mouse) is homozygous for the modified immunoglobulin λ chain locus.

[0148] In some embodiments, the non-human organism comprises in its germline and / or genome a light chain immunoglobulin locus comprising a limited repertoire of light chain variable gene segments (e.g., a dual light chain variable region comprising two light chain variable gene segments). In some embodiments, the light chain variable gene segments in the limited repertoire of light chain gene segments are human light chain gene segments. Exemplary dual light chain variable regions are provided in U.S. Patent Publication No. 2013 / 0198880, which is incorporated herein by reference. In some embodiments, the non-human organism comprising the dual light chain variable regions is used to produce bispecific antibodies.

[0149] Humanized CD79a and CD79b loci In some embodiments, rodents (e.g., mice or rats) described herein comprise human or humanized B-cell antigen receptor complex-associated protein alpha chain (CD79a or Igα) and / or B-cell antigen receptor complex-associated protein beta chain (CD79b or Igβ) gene loci. Rodents containing human or humanized CD79a and CD79b genes express human or humanized CD79a and CD79b polypeptides as heterodimers on the surface of B cells, and these polypeptides noncovalently associate with membrane-expressed immunoglobulins to form B-cell receptors (BCRs). The BCRs associate with antigens and are involved in signal transduction and internalization following antigen binding. In some embodiments, rodents described herein comprise human or humanized CD79a and CD79b genes. In some specific embodiments, a rodent described herein further comprises a CD79a gene comprising a rodent CD79a portion and a human CD79a portion, and a CD79b gene comprising a rodent CD79b portion and a human CD79b portion, wherein the human CD79a portion encodes substantially all of the extracellular domain of a human CD79a polypeptide (e.g., amino acids corresponding to residues 33-143 of the human CD79a polypeptide), and the human CD79b portion encodes substantially all of the extracellular domain of a human CD79b polypeptide (e.g., amino acids corresponding to residues 29-159 of the human CD79b polypeptide). In some embodiments, the rodent CD79a and CD79b portions each encode at least the intracellular domain of endogenous CD79a and CD79b polypeptides, respectively, and in some specific embodiments, encode the transmembrane and intracellular domains of endogenous CD79a and CD79b polypeptides, respectively. In some embodiments, the human and endogenous portions are operably linked to the endogenous CD79a or CD79b promoter, respectively.

[0150] In some embodiments, a rodent described herein further comprises a chimeric CD79a gene comprising a rodent CD79a portion and a human CD79a portion, wherein the human CD79a portion encodes a sequence comprising amino acids corresponding to residues 33-116 of a human CD79a polypeptide, in one embodiment, a sequence comprising amino acids 33-119 of a human CD79a polypeptide, in one embodiment, a sequence comprising amino acids 33-143 of a human CD79a polypeptide, and in one embodiment, a sequence comprising amino acids 33-165 of a human CD79a polypeptide. In some embodiments, the chimeric CD79a polypeptide comprises a human Ig C2-like domain, in some embodiments, the chimeric CD79a polypeptide also comprises a human stalk region, in some embodiments, the chimeric CD79a polypeptide also comprises a human transmembrane domain, and in some embodiments, the chimeric CD79a polypeptide further comprises a rodent (e.g., murine) cytoplasmic domain. In some embodiments, the rodent comprises a chimeric CD79a gene, wherein the chimeric CD79a gene comprises a human region portion described herein and a sequence encoding a human or rodent (e.g., mouse) CD79a signal peptide, and in one embodiment, the sequence encoding the signal peptide is a mouse CD79a sequence encoding amino acids 1-28 of mouse CD79a.

[0151] In some embodiments, a rodent described herein further comprises a chimeric CD79b gene comprising a rodent CD79b portion and a human CD79b portion, wherein the human CD79b portion encodes a sequence comprising amino acids corresponding to residues 29-135 of a human CD79b polypeptide, in one embodiment, a sequence comprising amino acids 29-159 of a human CD79b polypeptide, and in one embodiment, a sequence comprising amino acids 29-184 of a human CD79b polypeptide. In some embodiments, the chimeric CD79b polypeptide comprises a human Ig V-like domain, in some embodiments, the chimeric CD79b polypeptide also comprises a human stalk region, in some embodiments, the chimeric CD79b polypeptide also comprises a human transmembrane domain, and in some embodiments, the chimeric CD79b polypeptide further comprises a rodent (e.g., murine) cytoplasmic domain. In some embodiments, the rodent comprises a chimeric CD79b gene, wherein the chimeric CD79b gene comprises a human region portion described herein and a sequence encoding a human or rodent (e.g., mouse) CD79b signal peptide, and in one embodiment, the sequence encoding the signal peptide is a mouse CD79b sequence encoding amino acids 1-25 of mouse CD79b.

[0152] GenBank accession numbers NP_001774.1, NM_001783.3, NP_067612.1 and NM_021601.3 and UniProt ID P11912 provides representative source sequences for the human CD79A gene and human CD79A polypeptide, from which desired human portions can be obtained. GenBank Accession Nos. NP_000617.1, NM_000626.2, NP_001035022.1, NM_001039933.1, NP_067613.1, and NM_021602.2, and UniProt ID P40259 provide representative source sequences for the human CD79B gene and human CD79B polypeptide, from which desired human portions can be obtained.

[0153] In some embodiments, a rodent (e.g., a mouse or rat) provided herein further comprises one or more human CD79A and CD79B genes described in U.S. Patent Application Publication Nos. 2011-0093963A1 and 2009-0053210A1, International Patent Application Publication No. WO2008 / 027986, and European Patent No. 2064325B1, each of which is incorporated herein by reference. In certain embodiments, the rodents provided herein comprise a humanized CD79a gene comprising an endogenous CD79a portion and a human CD79a portion, and a humanized CD79b gene comprising an endogenous CD79b portion and a human CD79b portion, wherein the human CD79a portion encodes substantially all of the extracellular domain of a human CD79a polypeptide (e.g., amino acids corresponding to residues 33-143 of the human CD79a polypeptide), and the human CD79b portion encodes substantially all of the extracellular domain of a human CD79b polypeptide (e.g., amino acids corresponding to residues 29-159 of the human CD79b polypeptide). In some embodiments, the rodents provided herein comprise a humanized CD79a gene comprising an endogenous CD79a portion and a human CD79a portion, and a humanized CD79b gene comprising an endogenous CD79b portion and a human CD79b portion, wherein the human CD79a portion encodes a sequence comprising amino acids 33-116 (e.g., a sequence comprising amino acids 33-119, a sequence comprising amino acids 33-143, or a sequence comprising amino acids 33-165 of the human CD79a polypeptide), and the human CD79b portion encodes a sequence comprising amino acids 29-135 (e.g., a sequence comprising amino acids 29-159 or a sequence comprising amino acids 29-184 of the human CD79a polypeptide). In some embodiments, the endogenous CD79a and CD79b portions each encode at least the intracellular domains of the endogenous CD79a and CD79b polypeptides, respectively, and in some specific embodiments, encode the transmembrane and intracellular domains of the endogenous CD79a and CD79b polypeptides, respectively.

[0154] Humanized fetal Fc receptor locus In some embodiments, rodents (e.g., mice or rats) provided herein express and / or contain a humanized or human fetal Fc receptor (FcRn) locus in their genome. FcRn, also known as Brambell receptor, is a protein expressed by endothelial cells that associates with beta-2-microglobulin (β2M) and binds to both the Fc domain of IgG antibodies and serum albumin. FcRn extends the half-life of IgG and serum albumin. Specifically, FcRn binds to IgG and serum albumin in a pH-dependent manner, thereby preventing these serum proteins from being subjected to lysosomal degradation by endothelial cells, thereby increasing their serum half-life.

[0155] In some embodiments, the FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain, a rodent (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain, and a human (e.g., mouse or rat) cytoplasmic domain.

[0156] In some embodiments, a nucleic acid sequence encoding an FcRn polypeptide is placed in an endogenous rodent FcRn locus. In certain embodiments, the nucleic acid sequence encoding the FcRn polypeptide replaces all or part of the endogenous rodent FcRn gene. For example, in some embodiments, the nucleic acid sequence encoding the extracellular domain of the endogenous FcRn locus is replaced with a nucleic acid sequence encoding the extracellular domain of human FcRn, such that a rodent comprising such a locus expresses an FcRn comprising a human extracellular domain and rodent (e.g., rat or mouse) transmembrane and cytoplasmic domains. In some embodiments, the rodent does not express rodent FcRn or does not express functional rodent FcRn. In some embodiments, the FcRn gene locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., a rat or mouse promoter or enhancer).

[0157] In certain embodiments, the mouse exons encoding the alpha 1, alpha 2, and alpha 3 domains of the mouse FcRn gene (the first three coding exons, exons 3, 4, and 5) are replaced with human exons encoding the alpha 1, alpha 2, and alpha 3 domains of the human FcRn gene (exons 3, 4, and 5) (see Figure 4). In some embodiments, the FcRn gene comprises mouse exon 1 (a non-coding exon), mouse exon 2 (containing a nucleic acid sequence encoding a signal peptide), and human exons 3 to 6, mouse exons 6 and 7 (encoding transmembrane and cytoplasmic domains). In some embodiments, the amino acid sequence of the humanized FcRn encoded by the locus is SEQ ID NO: 16.

[0158] GenBank accession numbers NC_000019.10 (49512279-49526428), NM_001136019.1, and NP_001129491.1 provide representative source sequences for human FcRn genes, cDNAs, and polypeptides from which desired human portions can be derived. GenBank accession numbers NC_000073.6 (45092992-45103846), NM_010189.1, and NP_034319.1 provide representative source sequences for mouse FcRn genes, cDNAs, and polypeptides from which desired mouse portions can be derived and / or used to design homology arms for targeting vectors.

[0159] In some embodiments, the rodent is heterozygous for the genetically modified FcRn locus. In some embodiments, the rodent is homozygous for the genetically modified FcRn locus.

[0160] Humanized beta-2-microglobulin In some embodiments, the genetically modified rodents (e.g., rats or mice) and ES cells described herein express and / or contain in their genome a locus encoding a humanized β-2-microglobulin (β2M) polypeptide, which lacks a transmembrane domain and associates with FcRn and MHC class I molecules.

[0161] In some embodiments, the β2M locus comprises a nucleic acid sequence encoding a human β2M polypeptide. In some embodiments, the nucleic acid sequence encoding the human β2M polypeptide is located at an endogenous rodent β2M locus. In certain embodiments, the nucleic acid sequence encoding the β2M polypeptide replaces all or part of the endogenous rodent β2M gene. In some embodiments, the rodent does not express rodent β2M or does not express a functional rodent β2M polypeptide. In some embodiments, the β2M gene locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., a rat or mouse promoter or enhancer).

[0162] Humanized β2M polypeptides, loci encoding humanized β2M polypeptides, and non-human animals expressing humanized β2M polypeptides are described in U.S. Patent Publication Nos. 2013 / 0111617 and 2013 / 0185819, each of which is incorporated herein by reference. Thus, as described in U.S. Patent Publication Nos. 2013 / 0111617 and 2013 / 0185819, in some embodiments, the non-human animal (e.g., a mouse) comprises a humanized β2M gene, wherein the gene comprises exons 2, 3, and 4 of the human β2M gene; in some embodiments, the humanized β2M gene comprises exon 1 of the non-human (e.g., mouse) β2M gene. The humanized β2M locus is schematically depicted in FIG. 5. In some embodiments, the rodent is heterozygous for the genetically modified β2M locus. In some embodiments, the rodent is homozygous for the genetically modified β2M locus.

[0163] Humanized Fc epsilon receptor 1 alpha In some embodiments, rodents (e.g., mice or rats) provided herein express and / or contain a humanized or human Fc epsilon receptor 1 alpha (FcεR1α) locus in their genome. FcεR1α associates with FcεR1β and FcεR1γ to form FcεR1, a high-affinity receptor for IgE expressed on epidermal Langerhans cells, eosinophils, mast cells, and basophils. The IgE-binding site of FcεR1 is found in the FcεR1α subunit.

[0164] In some embodiments, the FcεR1α locus comprises a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain, a rodent (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcεR1α locus comprises a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain, a human transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcεR1α locus comprises a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain. Exemplary embodiments of engineered FcεR1α loci are shown in Figure 9.

[0165] In some embodiments, a nucleic acid sequence encoding an FcεR1α polypeptide is placed at the endogenous rodent FcεR1α locus. In specific embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide replaces all or part of the endogenous rodent FcεR1α gene. For example, in some embodiments, the nucleic acid sequence encoding the extracellular domain of the endogenous FcεR1α locus is replaced with a nucleic acid sequence encoding the extracellular domain of human FcεR1α, such that a rodent comprising such a locus expresses an FcεR1α comprising a human extracellular domain and rodent (e.g., rat or mouse) transmembrane and cytoplasmic domains. In some embodiments, a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain is placed at the endogenous rodent FcεR1α locus. In some embodiments, a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain replaces all or part of an endogenous rodent FcεR1α gene. In some embodiments, the rodent does not express rodent FcεR1α or does not express a functional rodent FcεR1α. In some embodiments, the FcεR1α gene locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., a rat or mouse promoter or enhancer).

[0166] In certain embodiments, portions of mouse coding exon 1, coding exon 2, coding exon 3, coding exon 4, and coding exon 5 of mouse FcεRIα are replaced with portions of human coding exon 1, coding exon 2, coding exon 3, coding exon 4, and coding exon 5 of the human FcεRIα gene. In some embodiments, the FcεRIα gene comprises a chimeric mouse / human exon 1 (including the mouse promoter and 5′ UTR), human coding exons 2-5 through to a stop codon, human 3′ UTR and polyA, followed by mouse 3′ UTR and polyA. In some embodiments, chimeric gene exons 1 (partial) and 2 encode a signal peptide, exons 3 and 4 encode the two Ig-like domains of FcεRIα that are thought to interact with IgE, and exon 5 encodes the cytoplasmic and transmembrane domains of the protein (see FIG. 9 ).

[0167] GenBank Accession Nos. NC_000001.11 (159283888-159308224), NM_002001.3, and NP_001992.1 provide representative source sequences for the human FcεR1α gene, cDNA, and polypeptide, from which the desired human portion can be derived. GenBank Accession Nos. NC_000067.6 (173221269-173227232), NM_010184.1, and NP_034314.1 provide representative source sequences for the mouse FcεR1α gene, cDNA, and polypeptide, from which the desired mouse portion can be derived and / or used to design homology arms for targeting vectors.

[0168] In some embodiments, the rodent is heterozygous for the genetically modified FcεR1α locus. In some embodiments, the rodent is homozygous for the genetically modified FcεR1α locus.

[0169] Humanized Fc gamma receptor 1a In some embodiments, a rodent (e.g., a mouse or rat) provided herein expresses and / or contains in its genome a humanized or human Fc gamma receptor 1a (FcγR1a; often designated FcγR1 in figures) locus. FcγR1a is a high-affinity FcγR protein expressed on monocytes that binds to the Fc portion of IgG, leading to host cell activation.

[0170] In some embodiments, the FcγR1a locus comprises a nucleic acid sequence encoding an FcγR1a polypeptide comprising a human extracellular domain, a rodent (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcγR1a locus comprises a nucleic acid sequence encoding an FcγR1a polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcγR1a locus comprises a nucleic acid sequence encoding an FcγR1a polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain, and a human (e.g., mouse or rat) cytoplasmic domain.

[0171] In some embodiments, a nucleic acid sequence encoding an FcγR1α polypeptide is placed in an endogenous rodent FcγR1a locus. In specific embodiments, the nucleic acid sequence encoding the FcγR1a polypeptide replaces all or part of the endogenous rodent FcγR1a gene. For example, in some embodiments, the nucleic acid sequence encoding the extracellular domain of the endogenous FcγR1a locus is replaced with a nucleic acid sequence encoding the extracellular domain of human FcεR1α, such that a rodent comprising such a locus expresses an FcγR1a comprising a human extracellular domain and rodent (e.g., rat or mouse) transmembrane and cytoplasmic domains. In some embodiments, the rodent does not express rodent FcγR1a or does not express functional rodent FcγR1a. In some embodiments, the FcγR1a gene locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0172] Humanized FcγR1a polypeptides, genetic loci encoding humanized FcγR1a polypeptides, and non-human animals expressing humanized FcγR1a polypeptides are described in U.S. Patent No. 9,474,255 and U.S. Patent Publication No. 2017 / 0086432, each of which is incorporated herein by reference.

[0173] In some embodiments, the rodent is heterozygous for the genetically modified FcγR1a locus. In some embodiments, the rodent is homozygous for the genetically modified FcγR1a locus.

[0174] Humanized low affinity Fc gamma receptor In some embodiments, the genetically modified rodents (e.g., rats or mice) and ES cells described herein express and / or contain in their genome a genetic locus encoding a human low affinity Fc gamma receptor (FcγR) polypeptide (e.g., a human FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, or FcγRIIIb polypeptide).

[0175] In some embodiments, the low affinity FcγR locus comprises a nucleic acid sequence encoding a human FcγRIIa polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIa polypeptide is positioned at an endogenous rodent low affinity FcγR locus. In a specific embodiment, the nucleic acid sequence encoding the FcγRIIa polypeptide replaces all or part of the endogenous rodent low affinity FcγR locus. In one specific embodiment, the human FcγRIIa gene comprises a polymorphism, wherein the polymorphism is selected from a 131His low-response polymorphism and a 131Arg high-response polymorphism. In one specific embodiment, the FcγRIIa polymorphism is a 131His low-response polymorphism. In some embodiments, the rodent does not express rodent low affinity FcγR polypeptides (e.g., does not express rodent FcγRIIb, FcγRIV, and / or FcγRIII polypeptides, or does not express functional rodent FcγRIIb, FcγRIV, and / or FcγRIII polypeptides). In some embodiments, the FcγRIIa gene locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., a rat or mouse promoter or enhancer).

[0176] In some embodiments, the low affinity FcγR locus comprises a nucleic acid sequence encoding a human FcγRIIb polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIb polypeptide is positioned at an endogenous rodent low affinity FcγR locus. In a specific embodiment, the nucleic acid sequence encoding the FcγRIIb polypeptide replaces all or part of the endogenous rodent low affinity FcγR locus. In a specific embodiment, the human FcγRIIb gene comprises an amino acid substitution, wherein the substitution is selected from a 187Ile or a 187Thr substitution. In some embodiments, the rodent does not express a rodent low affinity FcγR polypeptide (e.g., does not express rodent FcγRIIb, FcγRIV, and / or FcγRIII polypeptide, or does not express functional rodent FcγRIIb, FcγRIV, and / or FcγRIII polypeptide). In some embodiments, the FcγRIIb gene locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., a rat or mouse promoter or enhancer).

[0177] In some embodiments, the low affinity FcγR locus comprises a nucleic acid sequence encoding a human FcγRIIc polypeptide. In some embodiments, the nucleic acid sequence encoding a human FcγRIIc polypeptide is positioned at an endogenous rodent low affinity FcγR locus. In a specific embodiment, the nucleic acid sequence encoding the FcγRIIc polypeptide replaces all or part of the endogenous rodent low affinity FcγR locus. In one embodiment, the FcγRIIc gene is a specific allelic variant, wherein the allelic variant is selected from a 57Stop variant and a 57Q variant. In some embodiments, the rodent does not express a rodent low affinity FcγR polypeptide (e.g., does not express rodent FcγRIIB, FcγRIV, and / or FcγRIII polypeptide). In some embodiments, the FcγRIIc gene locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0178] In some embodiments, the low affinity FcγR locus comprises a nucleic acid sequence encoding a human FcγRIIIa polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIIa polypeptide is positioned at an endogenous rodent low affinity FcγR locus. In a specific embodiment, the nucleic acid sequence encoding the FcγRIIIa polypeptide replaces all or part of the endogenous rodent low affinity FcγR locus. In some embodiments, the rodent does not express a rodent low affinity FcγR polypeptide (e.g., does not express rodent FcγRIIb, FcγRIV and / or FcγRIII polypeptides, or does not express functional rodent FcγRIIb, FcγRIV and / or FcγRIII polypeptides). In one embodiment, the FcγRIIIa gene is a particular allelic variant, wherein the allelic variant is selected from a 176Val variant and a 176Phe variant. In one specific embodiment, the FcγRIIIa allelic variant is the 176Val variant. In some embodiments, the FcγRIIIa gene locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., a rat or mouse promoter or enhancer).

[0179] In some embodiments, the low affinity FcγR locus comprises a nucleic acid sequence encoding a human FcγRIIIb polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIIb polypeptide is positioned at an endogenous rodent low affinity FcγR locus. In a specific embodiment, the nucleic acid sequence encoding the FcγRIIIb polypeptide replaces all or part of the endogenous rodent low affinity FcγR locus. In one specific embodiment, the FcγRIIIb gene is a particular allelic variant, wherein the allelic variant is selected from an NA1 variant and an NA2 variant. In another specific embodiment, the FcγRIIIb allelic variant is an NA2 variant. In some embodiments, the rodent does not express rodent low affinity FcγR polypeptides (e.g., does not express rodent FcγRIIb, FcγRIV, and / or FcγRIII polypeptides, or does not express functional rodent FcγRIIb, FcγRIV, and / or FcγRIII polypeptides). In some embodiments, the FcγRIIIb gene locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., a rat or mouse promoter or enhancer).

[0180] In some embodiments, a rodent (e.g., a rat or a mouse) provided herein comprises one or more human low affinity FcγR genes described in U.S. Patent Nos. 9,221,894, 9,056,130, 9,089,599, 8,658,154, 8,883,496, or 8,658,853. In some embodiments, a rodent (e.g., a rat or a mouse) provided herein comprises at least two low affinity human FcγR genes and one endogenous rodent (e.g., rat or mouse) Fc γ chain gene, wherein the low affinity human FcγR genes are selected from the group consisting of human FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some specific embodiments, a rodent (e.g., a rat or mouse) provided herein comprises human FcγRIIa and FcγRIIIb and endogenous rodent (e.g., rat or mouse) Fc γ chain genes. In some specific embodiments, a rodent (e.g., a rat or mouse) provided herein comprises FcγRIIa, FcγRIIIa, FcγRIIb, FcγRIIc, and FcγRIIId genes and endogenous rodent (e.g., rat or mouse) Fc γ chain genes. In various embodiments, a rodent (e.g., a rat or mouse) comprising one or more human FcγRs further comprises homozygous disruptions in endogenous rodent (e.g., rat or mouse) FcγRIIB, FcγRIV, and FcγRIII genes (i.e., endogenous rodent FcγRIIb, FcγRIV, and FcγRIII α chain coding sequences). In various embodiments, a rodent (e.g., a rat or mouse) comprising one or more human low affinity FcγRs described herein does not detectably express endogenous rodent low affinity FcγR polypeptides (e.g., endogenous low affinity FcγR α chain polypeptides).

[0181] In some embodiments, the rodent is heterozygous for the genetically modified low affinity FcγR locus. In some embodiments, the rodent is homozygous for the genetically modified low affinity FcγR locus.

[0182] Genetically modified non-human animals and ES cells In certain aspects, provided herein are genetically modified non-human animals (e.g., rodents such as rats or mice) that comprise one or more of the humanized loci disclosed herein, and genetically modified non-human animal ES cells useful for producing such non-human animals.

[0183] In certain aspects, provided herein are genetically modified non-human animals and non-human animal ES cells that comprise one or more of the engineered loci described herein in their germline and / or genome. For example, in some embodiments, the non-human animal or ES cell comprises an IgH locus provided herein in its germline and / or genome. In certain embodiments, the non-human animal or ES cell further comprises an Igκ and / or an Igλ locus provided herein. In some embodiments, the non-human animal or ES cell comprises a CD79a and / or CD79b locus provided herein in its germline and / or genome. In certain embodiments, the non-human animal or ES cell comprises an FcRn locus provided herein in its germline and / or genome. In certain embodiments, the non-human animal or ES cell comprises a β2M locus provided herein in its germline and / or genome. In certain embodiments, the non-human animal or ES cell comprises in its germline and / or genome the FcεR1α locus provided herein. In certain embodiments, the non-human animal or ES cell comprises in its germline and / or genome the FcγR1a locus provided herein. In certain embodiments, the non-human animal or ES cell comprises in its germline and / or genome the FcγR2a locus provided herein. In certain embodiments, the non-human animal or ES cell comprises in its germline and / or genome the FcγR2b locus provided herein. In certain embodiments, the non-human animal or ES cell comprises in its germline and / or genome the FcγR3a locus provided herein. In certain embodiments, the non-human animal or ES cell comprises in its germline and / or genome the FcγR3b locus provided herein. In certain embodiments, the non-human animal or ES cell comprises in its germline and / or genome the FcγR2c locus provided herein. In some embodiments, the non-human animal or ES cells are heterozygous for one or more of the genetic loci, eg, the engineered genetic loci provided herein.In some embodiments, the non-human animal or ES cells are homozygous for one or more of the genetic loci, eg, the engineered genetic loci provided herein.

[0184] In some embodiments, the non-human animal can be any non-human animal. In some embodiments, the non-human animal is a vertebrate. In some embodiments, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal described herein can be selected from the group consisting of mice, rats, rabbits, pigs, bovines (e.g., cows, bulls, buffalo), deer, sheep, goats, llamas, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods can be employed to generate non-human animals containing the genetic modifications described herein. Such methods include, for example, modifying the genome of a non-ES cell (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to implant the modified genome into a suitable cell, such as an oocyte, and growing the modified cell (e.g., the modified oocyte) within the non-human animal under conditions suitable for the formation of an embryo.

[0185] In various embodiments, provided herein is a non-human animal, e.g., a rodent (e.g., a rat or mouse), comprising a genetically engineered immunoglobulin heavy chain locus. An exemplary embodiment of such a genetically engineered locus is shown schematically in FIG. 1 and described in detail herein. In some embodiments, a non-human animal described herein comprises an Adam6 gene in its genome (e.g., its germline genome), which gene encodes an ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof (see, e.g., U.S. Pat. Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety). In some embodiments, the ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof is expressed from the Adam6 gene. In some embodiments, the Adam6 gene is not derived from a non-human animal comprising an Adam6 gene (e.g., a mouse comprising a rat Adam6 gene or a mouse Adam6 gene obtained from another mouse strain). In some embodiments, the non-human animals described herein comprise an ectopic Adam6 gene. As used herein, an "ectopic" Adam6 gene refers to an Adam6 gene that is in a different context than the Adam6 gene appears in a wild-type non-human animal. For example, the Adam6 gene may be located on a different chromosome, at a different locus, or adjacent to a different sequence. An exemplary ectopic Adam6 gene is a mouse Adam6 gene located within a human immunoglobulin sequence (e.g., a human heavy chain variable region gene segment). In some embodiments, the non-human animals described herein comprise an inserted or integrated Adam6 gene.

[0186] In some embodiments, a non-human animal described herein comprises in its genome (e.g., its germline genome) an insertion of one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof.

[0187] In some embodiments, a non-human animal described herein comprises in its genome (e.g., its germline genome) one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, a non-human animal described herein comprises in its genome (e.g., its germline genome) a mouse Adam6a gene and / or a mouse Adam6b gene. In some embodiments, a non-human animal described herein comprises one or more nucleotide sequences encoding mouse ADAM6a, a functional ortholog, functional homolog, or functional fragment thereof, and / or mouse ADAM6b, a functional ortholog, functional homolog, or functional fragment thereof.

[0188] In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted and / or located on the same chromosome as an endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted and / or located at a position such that the one or more nucleotide sequences encoding the one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are contiguous with a human immunoglobulin heavy chain variable region gene segment. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted and / or located at a position such that the one or more nucleotide sequences encoding the one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are adjacent to a human immunoglobulin heavy chain variable region gene segment. In some embodiments, the one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted and / or positioned at a location such that the one or more nucleotide sequences encoding the one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are located between human immunoglobulin heavy chain variable region gene segments. In some embodiments, the one or more nucleotide sequences encoding the one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are located between human immunoglobulin heavy chain variable region gene segments. H Gene segments and the second human V H In some embodiments, the first human V H The gene segment is human V H 1-2 and the second human V H The gene segment is human VH In some embodiments, the one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted in place of and / or located in the place of a human Adam6 pseudogene. In some embodiments, the one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are inserted in place of a human Adam6 pseudogene. H Gene segments and human D H It is inserted between gene segments.

[0189] In some embodiments, the non-human animals described herein contain an Adam6 gene that restores or improves ADAM6 activity. In some embodiments, the Adam6 gene restores ADAM6 activity to the level of a comparable non-human animal that contains a functional endogenous Adam6 gene. In some embodiments, the Adam6 gene improves ADAM6 activity to a level that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher than the ADAM6 activity of a comparable non-human animal that does not contain a functional Adam6 gene.

[0190] In some embodiments, the non-human animals described herein comprise an Adam6 gene that restores or improves fertility in male non-human animals. In some embodiments, the Adam6 gene restores the fertility of the male non-human animal to the level of a comparable non-human animal that contains a functional sex endogenous Adam6 gene. In some embodiments, the Adam6 gene restores the fertility of the male non-human animal such that the number of pups produced by mating with the male non-human animal is at least 70%, at least 80%, at least 90%, or at least 95% of the number of pups produced by a similar mating with a comparable male non-human animal that does not contain a functional Adam6 gene. In some embodiments, the Adam6 gene improves the fertility of the male non-human animal such that the number of pups produced by mating with the male non-human animal is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold the number of pups produced by a similar mating with a comparable male non-human animal that does not contain a functional Adam6 gene.

[0191] In some embodiments, the non-human immunoglobulin heavy chain loci described herein lack at least one endogenous non-human Adam6 gene. In some embodiments, the deletion 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 loci described herein comprise a disruption of at least one endogenous non-human Adam6 gene. In some embodiments, the 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.

[0192] In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human animal is a small mammal, such as an animal of the superfamily Dipodoidea or the superfamily Muroidea. In some embodiments, the non-human animal is a rodent. In particular embodiments, the rodent is a mouse, rat, or hamster. In some embodiments, the rodent is selected from the superfamily Muroidea. In some embodiments, the non-human animal is from a family selected from the family Calomyscidae (e.g., mouse-like hamsters), family Cricetidae (e.g., hamsters, New World rats and mice, and voles), family Muridae (e.g., purebred mice and rats, gerbils, spiny mice, and maned rats), family Nesomyidae (e.g., tree mice, rock mice, white-tailed rats, Madagascar rats and mice), family Platacanthomyidae (e.g., spiny dormice), and family Spalacidae (e.g., mole rats, bamboo rats, and mole rats). In some embodiments, the rodent is selected from a pure breed mouse or rat (family Muridae), a gerbil, a spiny mouse, and a maned mouse. In some embodiments, the mouse is from a species of the family Muridae. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent is selected from a mouse and a rat. In some embodiments, the non-human animal is a mouse.

[0193] In some embodiments, the non-human animal is a mouse of the C57BL strain. In some embodiments, the C57BL strain is selected from 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 embodiments, the non-human animal is a mouse of the 129 strain. In some embodiments, the 129 strain is selected from the group consisting of strains that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2. In some embodiments, the genetically modified mouse is a mix of a 129 strain and a C57BL strain. In some embodiments, the mouse is a mix of a 129 strain and / or a C57BL / 6 strain. In some embodiments, the mix of a 129 strain is a 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse is a BALB strain (e.g., BALB / c). In some embodiments, the mice are a mixture of the BALB strain and another strain (e.g., the C57BL strain and / or the 129 strain). In some embodiments, the non-human animals provided herein can be mice derived from any combination of the aforementioned strains.

[0194] In some embodiments, the non-human animal provided herein is a rat. In some embodiments, the rat is selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

[0195] In certain embodiments, a genetically modified non-human animal or ES cell comprises in its genome and / or germline multiple loci provided herein, e.g., multiple engineered loci provided herein. For example, in some embodiments, a non-human animal or ES cell comprises in its germline and / or genome an IgH locus provided herein and an Igκ and / or Igλ locus provided herein. In some embodiments, a non-human animal or ES cell comprises in its germline and / or genome an IgH locus provided herein, an Igκ and / or Igλ locus provided herein, and optionally a CD79a and / or CD79b locus provided herein. In some embodiments, a non-human animal or ES cell comprises in its germline and / or genome an IgH locus provided herein, an Igκ and / or Igλ locus provided herein, an FcRn locus provided herein, and a β2M locus provided herein. In some embodiments, the non-human animal or ES cell comprises in its germline and / or genome an IgH locus provided herein, an Igκ and / or Igλ locus provided herein, an FcRn locus provided herein, a β2M locus provided herein, an FcεR1α locus provided herein, an FcγR1a locus provided herein, an FcγR2a locus provided herein, an FcγR2b locus provided herein, an FcγR3a locus provided herein, an FcγR3b locus provided herein, and / or an FcγR2c locus provided herein, and any combination thereof.

[0196] In certain aspects, the genetically modified non-human animal expresses one or more humanized polypeptides encoded by the humanized loci provided herein. For example, in some embodiments, the non-human animal expresses a humanized Ig heavy chain polypeptide. In certain embodiments, the non-human animal expresses a humanized Igκ polypeptide and / or a humanized Igλ polypeptide. In some embodiments, the non-human animal expresses a humanized CD79a polypeptide and / or a humanized CD79b polypeptide. In certain embodiments, the non-human animal expresses a humanized FcRn polypeptide. In certain embodiments, the non-human animal expresses a humanized β2M polypeptide. In certain embodiments, the non-human animal expresses a humanized FcεR1α polypeptide. In certain embodiments, the non-human animal expresses a humanized FcγR1a polypeptide. In certain embodiments, the non-human animal or ES cell non-human animal expresses a humanized FcγR2a polypeptide. In certain embodiments, the non-human animal expresses a humanized FcγR2b locus polypeptide. In certain embodiments, the non-human animal expresses a humanized FcγR3a polypeptide. In certain embodiments, the non-human animal expresses a humanized FcγR3b polypeptide. In certain embodiments, the non-human animal expresses a humanized FcγR2c polypeptide.

[0197] Genetically modified non-human animals and ES cells can be produced using any suitable method known in the art. For example, such genetically modified non-human animal ES cells can be produced using VELOCIGENE® technology, which is described in U.S. Patent Nos. 6,586,251, 6,596,541, and 7,105,348, and Valenzuela et al. (2003) "High-throughput engineering of the mouse genome coupled with high-resolution expression analysis" Nat. Biotech. 21(6):652-659, each of which is incorporated herein by reference. Modifications can also be made using genome-targeting nuclease systems, such as CRISPR / Cas systems, transcription activator-like effector nuclease (TALEN) systems, or zinc finger nuclease (ZFN) systems. In some embodiments, the modifications are performed using a CRISPR / Cas system, such as those described in U.S. Patent Application Nos. 14 / 314,866, 14 / 515,503, 14 / 747,461, and 14 / 731,914, each of which is incorporated herein by reference. Exemplary methods for generating such genetically modified non-human animals and ES cells are also provided in Examples 1 and 3-5 herein.

[0198] Furthermore, the ES cells described herein can be used to generate non-human animals using methods known in the art. For example, the mouse non-human animal ES cells described herein can be used to generate genetically modified mice using the VELOCIMOUSE® method, as described in U.S. Patent No. 7,294,754 and Poueymirou et al., Nature Biotech 25:91-99 (2007), each of which is incorporated herein by reference. The resulting mice can be bred to homozygotes.

[0199] Testing methods for human Fc-containing therapeutics In certain aspects, provided herein are methods for testing a therapeutic protein comprising a human Fc domain (e.g., a human antibody or an Fc fusion protein), comprising administering the therapeutic protein to a rodent provided herein (e.g., a mouse or rat provided herein). In certain embodiments, provided herein are animal models for carrying out such methods.

[0200] In some embodiments, the administered human antibody or Fc fusion protein comprises a human C in the genetically modified IgH locus of the rodent provided herein. H For example, in some embodiments, the agent is a human IgG1 antibody and the rodent is harboring a C nucleotide sequence encoding the human IgG1 CH1, hinge, CH2, and CH3 domains. H In some embodiments, the agent is a human IgG4 antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgG4 CH1, hinge, CH2, and CH3 domains. H In some embodiments, the agent is a human IgG1 antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgG1 CH1, hinge, CH2, CH3, M1, and M2 domains. H In some embodiments, the agent is a human IgG2 antibody and the rodent is a human IgG2 C encoding the CH1, hinge, CH2, CH3, M1 and M2 domains H In some embodiments, the agent is a human IgG3 antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgG3 CH1, hinge, CH2, CH3, M1, and M2 domains. HIn some embodiments, the agent is a human IgG4 antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgG4 CH1, hinge, CH2, CH3, M1, and M2 domains. H In some embodiments, the agent is a human IgM antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgM CH1, CH2, CH3, and CH4 domains. H In some embodiments, the agent is a human IgD antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding the human IgD CH1, H1, H2, CH2, CH3, M1, and M2 domains. H The rodent comprises a genetically modified IgH locus comprising a gene segment. In some embodiments, the therapeutic agent is a human antibody having an Igκ light chain, and the rodent comprises a genetically modified Igκ locus provided herein. In some embodiments, the therapeutic agent is a human antibody having an Igλ light chain, and the rodent comprises a genetically modified Igλ locus provided herein.

[0201] In some embodiments, the method comprises measuring one or more pharmacokinetic properties of the administered therapeutic protein. In certain embodiments, the animal model used to determine the pharmacokinetic properties of the administered human antibody or fusion protein is a genetically modified rodent provided herein comprising a modified IgH locus provided herein. In certain embodiments, the animal model used to determine the pharmacokinetic properties of the administered human antibody or fusion protein is a genetically modified rodent provided herein comprising a modified IgH locus provided herein and a modified Igκ locus and / or a modified Igλ locus provided herein. In some embodiments, the administered human antibody or Fc fusion protein comprises a humanized C locus within the genetically modified IgH locus. HIn some embodiments, the rodent further comprises a modified FcRn locus provided herein. In some embodiments, the rodent further comprises a modified β2M provided herein.

[0202] In some embodiments, the one or more pharmacokinetic parameters include, but are not limited to, area under plasma concentration versus time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), drug half-life (t), and volume of distribution at steady state (Vss). Generally, the pharmacokinetic properties of an administered therapeutic agent are determined by administering a selected dose of the therapeutic agent (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / mg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg or more) and then determining how the plasma concentration of the therapeutic agent changes over time (e.g., at 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or more).

[0203] In some embodiments, the method further comprises measuring the therapeutic efficacy of the administered therapeutic protein (e.g., the ability of a dose of the therapeutic protein to reduce or eliminate one or more disease symptoms in an animal model). In some embodiments, the animal model is a cancer model, and disease symptoms may include, for example, tumor size, tumor metastasis, and / or animal survival. In certain embodiments, the animal model is an autoimmune or inflammatory model, and disease symptoms may include, for example, cytokine expression levels, immune cell proliferation, tissue damage, and / or animal survival. In some embodiments, the animal model is an infectious disease model, and disease symptoms may include, for example, pathogen levels, tissue damage, and / or animal survival.

[0204] In some embodiments, the methods further comprise determining the safety and dosage of the administered therapeutic protein (e.g., the extent to which a dose of the therapeutic protein produces one or more adverse effects in an animal model). Adverse effects include, but are not limited to, allergic reactions, alopecia, anaphylaxis, anemia, loss of appetite, loss of balance, bleeding, blood clots, difficulty breathing, bronchitis, bruising, decreased white blood cell count, decreased red blood cell count, decreased platelet count, cardiotoxicity, conjunctivitis, constipation, cough, dehydration, diarrhea, electrolyte imbalance, infertility, fever, hair loss, heart failure, infection, injection site reactions, iron deficiency, kidney failure, leukopenia, liver dysfunction, pneumonia, rapid heartbeat, rectal bleeding, convulsions, weight loss, and weight gain. For example, in certain embodiments, provided herein are methods for determining allergic reactions induced by a therapeutic agent using passive cutaneous anaphylaxis (PCA) and / or passive systemic anaphylaxis (PSA) models.

[0205] In certain embodiments, the method further comprises measuring the extent to which the therapeutic protein induces one or more Fc receptor-mediated responses in the rodent (e.g., the extent to which the therapeutic protein induces antibody-dependent cell-mediated cytotoxicity (ADCC)). For example, in certain embodiments, provided herein are methods of screening for therapeutic agents comprising a human Fc region of a human antibody, the methods comprising: (a) administering to a rodent provided herein an agent comprising the Fc region of a human antibody, wherein the agent binds to target cells in the rodent; (b) measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against the target cells; and (c) comparing the amount of ADCC in step (b) with a control, wherein increased killing of target cells indicates increased ability of the agent to mediate ADCC.

[0206] In some embodiments, the methods further comprise determining the extent to which administration of the therapeutic protein induces an anti-human Fc immune response in the rodent.

[0207] In some embodiments, the administered therapeutic agent elicits a reduced immune response when administered to a rodent provided herein. In some embodiments, the administered human antibody or Fc fusion protein encodes a human C1 gene within the genetically modified IgH locus of a rodent provided herein. H For example, in some embodiments, the agent is a human IgG1 antibody and the rodent is harboring a C nucleotide sequence encoding the human IgG1 CH1, hinge, CH2, and CH3 domains. H In some embodiments, the agent is a human IgG4 antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgG4 CH1, hinge, CH2, and CH3 domains. H In some embodiments, the agent is a human IgG1 antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgG1 CH1, hinge, CH2, CH3, M1, and M2 domains. H In some embodiments, the agent is a human IgG2 antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgG2 CH1, hinge, CH2, CH3, M1, and M2 domains. H In some embodiments, the agent is a human IgG3 antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgG3 CH1, hinge, CH2, CH3, M1, and M2 domains. H In some embodiments, the agent is a human IgG4 antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgG4 CH1, hinge, CH2, CH3, M1, and M2 domains. H In some embodiments, the agent is a human IgM antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding human IgM CH1, CH2, CH3, and CH4 domains. HIn some embodiments, the agent is a human IgD antibody and the rodent comprises a genetically modified IgH locus comprising gene segments encoding the human IgD CH1, H1, H2, CH2, CH3, M1, and M2 domains. H The rodent comprises a genetically modified IgH locus comprising a gene segment. In some embodiments, the therapeutic agent is a human antibody having an Igκ light chain, and the rodent comprises a genetically modified Igκ locus provided herein. In some embodiments, the therapeutic agent is a human antibody having an Igλ light chain, and the rodent comprises a genetically modified Igλ locus provided herein.

[0208] In certain embodiments, the methods provided herein include testing a therapeutic antibody that comprises a human Fc. In some embodiments, the antibody being tested comprises a human heavy chain variable domain. In some embodiments, the antibody comprises a human heavy chain constant domain. In some embodiments, the antibody provided herein comprises an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant domain. Sequences of human heavy chain constant domains are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 and the IMGT database (available at www.imgt.org)).

[0209] In some embodiments, the antibody being tested comprises an altered Fc domain (e.g., a mutation that alters the interaction between Fc and an Fc receptor). For example, in some embodiments, an antibody provided herein comprises a modification in its Fc domain at positions 235, 236, 237, 239, 265, 267, 268, 269, 270, 298, 326, 327, 330, 332, 350, 351, 366, 392, 394, 405, and / or 407 (using the EU numbering system). In some embodiments, the modifications are selected from the group consisting of L235A, G236E, G237F, S239E, S239D, D265E, D265S, S267E, S267D, S267G, H268E, H268D, E269L, D270N, D270E, S298A, K326A, K326D, A327H, A327V, A327L, A330I, A330S, I332E, T350V, L351Y, T366L, K392M, K392L, T394W, F405A and / or Y407V (using the EU numbering system). In some embodiments, the antibody comprises multiple modifications in its Fc domain. In some embodiments, the multiple modifications are D270N / K326D, S239E / S298A / K326A / A327H, L235A / S239E / D265E / A327H, G236E / G237F / S239E, G237F / S239E / D265E, G327F / S239E / H268D, G236E / D270N / A327V / I332E, G237F / S239E / A327H, G237F / A327L / A330I, S23 9D / D265S / S298A / I332E, S239E / D265S / H268D / I332E, S239E / D265S / I332E, S239E / S267E / H268D, S239E / A327L / A330I, D265E / S267D / A330S, S267G / H268E / D270E, H268D / E269L / S298A / K326A / A327H, H268D / / K326A / A327H.Further Fc modifications and combinations of Fc modifications are described in U.S. Patent Nos. 5,624,821, 5,648,260, 6,528,624, 6,737,056, 7,122,637, 7,183,387, 7,297,775, 7,317,091, 7,332,581, 7,632,497, 7,662,925, 7,695,936, 8,093,359, 8,216,805, 8,218,806, and 8,220,807. Nos. 5, 8,388,955 and 8,937,158, and U.S. Patent Publication Nos. 2005 / 0054832, 2006 / 0222653, 2006 / 0275282, 2006 / 0275283, 2007 / 0190063, 2008 / 0154025, 2009 / 0042291, 2013 / 0108623 and 2013 / 0089541, each of which is incorporated herein by reference.

[0210] In some embodiments, the antibody being tested is a bispecific antibody. In some embodiments, the two antigen-binding domains of a bispecific antibody have different heavy chain variable domains but identical light chain variable domains. In some embodiments, the Fc domain of the heavy chain contains a modification that promotes heavy chain heterodimerization and / or inhibits heavy chain homodimerization. Such modifications are provided, for example, in U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, and 8,679,785, and U.S. Patent Publication No. 2013 / 0195849, each of which is incorporated herein by reference.

[0211] In some embodiments, the antibodies tested in the methods provided herein have a human light chain variable domain. In some embodiments, the light chain variable domain is a λ light chain variable domain. In some embodiments, the light chain variable domain is a κ light chain variable domain. In some embodiments, the antibody has a human light chain constant domain. In some embodiments, the light chain constant domain is a λ light chain constant domain. In some embodiments, the light chain constant domain is a κ light chain constant domain. Sequences of human light chain constant domains are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 and the IMGT database (available at www.imgt.org)).

[0212] In certain embodiments, a therapeutic agent is administered to a rodent provided herein as part of a pharmaceutical composition, e.g., a pharmaceutical composition containing a human antibody or Fc fusion protein formulated together with a pharmaceutically acceptable carrier.

[0213] The pharmaceutical compositions provided herein may be specially formulated for administration in solid or liquid form, including those adapted for (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., intended for oral mucosal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue, or (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained-release formulation.

[0214] Pharmaceutical compositions suitable for parenteral administration can include human antibodies or Fc fusion proteins in combination with one or more pharmaceutically acceptable sterile, isotonic, aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, and can contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0215] Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions provided herein include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0216] In certain embodiments, the composition comprises a human antibody or Fc-fusion protein at a concentration that provides a w / v suitable for a desired dosage. The antibody may be at least 1 mg / mL, at least 5 mg / mL, at least 10 mg / mL, at least 15 mg / mL, at least 20 mg / mL, at least 25 mg / mL, at least 30 mg / mL, at least 35 mg / mL, at least 40 mg / mL, at least 45 mg / mL, at least 50 mg / mL, at least 55 mg / mL, at least 60 mg / mL, at least 65 mg / mL, at least 70 mg / mL, at least 75 mg / mL, at least 80 mg / mL, at least 85 mg / mL, at least 90 mg / mL, at least 100 mg / mL, at least 120 mg / mL, at least 140 mg / mL, at least 160 mg / mL, at least 180 mg / mL, at least 190 mg / mL, at least 210 mg / mL, at least 220 mg / mL, at least 230 mg / mL, at least 240 mg / mL, at least 250 mg / mL, at least 260 mg / mL, at least 270 mg / mL, at least 280 mg / mL, at least 290 mg / mL, at least 300 mg / mL, at least 310 mg / mL, at least 320 mg / mL, at least 330 mg / mL, at least 340 mg / mL, at least 350 mg / mL, at least 350 mg / mL, at least 360 mg / mL, at least 370 mg / mL, at least 380 mg / mL, at least 390 mg / mL, at least 400 mg / mL, at least 410 mg / mL, at least 420 mg / mL, at least 430 mg / mL, at least 440 mg mg / mL, at least 90 mg / mL, at least 95 mg / mL, at least 100 mg / mL, at least 105 mg / mL, at least 110 mg / mL, at least 115 mg / mL, at least 120 mg / mL, at least 125 mg / mL, at least 130 mg / mL, at least 135 mg / mL, at least 140 mg / mL, at least 150 mg / mL, at least 200 mg / mL, at least 250 mg / mL, or at least 300 mg / mL.

[0217] In some embodiments, compositions are prepared by combining a human antibody or Fc fusion protein with an optional physiologically acceptable carrier, excipient, or stabilizer, such as, but not limited to, a buffer, a sugar, a salt, a surfactant, a solubilizer, a polyol, a diluent, a binder, a stabilizer, a salt, a lipophilic solvent, an amino acid, a chelating agent, a preservative, or the like (see Goodman and Gilman's The Pharmacological Basis of of Therapeutics, 12th edition, L. Brunton, et al. and Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1999)), and prepared in the form of a lyophilized composition or aqueous solution of the desired final concentration. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as histidine, phosphate, citric acid, glycine, acetic acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than 10 drug residues); serum albumin, gelatin, etc. hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including trehalose, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, polysorbate 80, PLURONICS®, or polyethylene glycol (PEG).

[0218] In some embodiments, the buffering agent is histidine, citrate, phosphate, glycine, or acetate. The sugar excipient can be trehalose, sucrose, mannitol, maltose, or raffinose. The surfactant can be polysorbate 20, polysorbate 40, polysorbate 80, or Pluronic® F68. The salt can be NaCl, KCl, MgCl2, or CaCl2.

[0219] In some embodiments, the compositions include a buffer or pH adjuster to provide improved pH control. Such compositions may have a pH of about 3.0 to about 9.0, about 4.0 to about 8.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.5 to about 8.0, about 5.5 to about 7.0, or about 5.5 to about 6.5. In further embodiments, such compositions have a pH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In a specific embodiment, the composition has a pH of about 6.0. Those skilled in the art will understand that the pH of a composition generally should not be the same as the isoelectric point of the human antibody or Fc fusion protein used in the composition. Typically, the buffer is a salt prepared from an organic or inorganic acid or base. Representative buffering agents include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris buffer, tromethamine hydrochloride buffer, or phosphate buffer. Additionally, amino acid components can function as buffers. Representative amino acid components that can be utilized in the composition as buffers include, but are not limited to, glycine and histidine. In certain embodiments, the buffering agent is selected from histidine, citrate, phosphate, glycine, and acetate. In one specific embodiment, the buffering agent is histidine. In another specific embodiment, the buffering agent is citrate. In yet another specific embodiment, the buffering agent is glycine. The purity of the buffering agent should be at least 98%, or at least 99%, or at least 99.5%. As used herein, the term "purity" in the context of histidine and glycine refers to the chemical purity of histidine or glycine as understood in the art, e.g., as described in The Merck Index, 13th ed., O'Neil et al. ed. (Merck & Co., 2001).

[0220] In certain embodiments, the composition comprises histidine as a buffering agent, hi certain embodiments, the histidine is present in the composition at a concentration of at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 150 mM, or at least about 200 mM histidine. In another embodiment, the composition comprises about 1 mM to about 200 mM, about 1 mM to about 150 mM, about 1 mM to about 100 mM, about 1 mM to about 75 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 10 mM to about 100 mM, about 10 mM to about 75 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 20 mM to about 75 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, or about 20 mM to about 30 mM histidine. In further embodiments, the composition comprises about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM, or about 200 mM histidine. In a specific embodiment, the composition may comprise about 10 mM, about 25 mM, or no histidine.

[0221] In some embodiments, the composition includes a carbohydrate excipient. The carbohydrate excipient may act, for example, as a thickener, stabilizer, bulking agent, solubilizer, and / or the like. The carbohydrate excipient is generally present in an amount of about 1% to about 99% by weight or volume, e.g., about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 5%, about 1% to about 20%, about 5% to about 15%, about 8% to about 10%, about 10% and about 15%, about 15% and about 20%, 0.1% to 20%, 5% to 15%, 8% to 10%, 10% and 15%, 15% and 20%, about 0.1% to about 5%, about 5% to about 10%, or about 15% to about 20%. In yet other specific embodiments, the carbohydrate excipient is present at 1%, or 1.5%, or 2%, or 2.5%, or 3%, or 4%, or 5%, or 10%, or 15%, or 20%.

[0222] In some embodiments, the composition comprises a carbohydrate excipient. Carbohydrate excipients suitable for use in the composition include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, and xylitol sorbitol (glucitol). In certain embodiments, the carbohydrate excipient used in the compositions provided herein is selected from sucrose, trehalose, lactose, mannitol, and raffinose. In a specific embodiment, the carbohydrate excipient is trehalose. In another specific embodiment, the carbohydrate excipient is mannitol. In yet another specific embodiment, the carbohydrate excipient is sucrose. In yet another specific embodiment, the carbohydrate excipient is raffinose. The purity of the carbohydrate excipient should be at least 98%, or at least 99%, or at least 99.5%.

[0223] In some embodiments, the composition comprises trehalose. In specific embodiments, the composition comprises at least about 1%, at least about 2%, at least about 4%, at least about 8%, at least about 20%, at least about 30%, or at least about 40% trehalose. In other embodiments, the composition comprises between about 1% and about 40%, between about 1% and about 30%, between about 1% and about 20%, between about 2% and about 40%, between about 2% and about 30%, between about 2% and about 20%, between about 4% and about 40%, between about 4% and about 30%, or between about 4% and about 20% trehalose. In further embodiments, the composition comprises about 1%, about 2%, about 4%, about 6%, about 8%, about 15%, about 20%, about 30%, or about 40% trehalose. In a specific embodiment, the composition comprises about 4%, about 6%, or about 15% trehalose.

[0224] In certain embodiments, the composition comprises an excipient. In one specific embodiment, the composition comprises at least one excipient selected from sugars, salts, surfactants, amino acids, polyols, chelating agents, emulsifiers, and preservatives. In certain embodiments, the composition comprises a salt selected from, for example, NaCl, KCl, CaCl, and MgCl. In one specific embodiment, the composition comprises NaCl.

[0225] In some embodiments, the composition comprises an amino acid, such as lysine, arginine, glycine, histidine, or an amino acid salt. The composition may comprise at least about 1 mM, at least about 10 mM, at least about 25 mM, at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, or at least about 400 mM of an amino acid. In another embodiment, the composition may contain about 1 mM to about 100 mM, about 10 mM to about 150 mM, about 25 mM to about 250 mM, about 25 mM to about 300 mM, about 25 mM to about 350 mM, about 25 mM to about 400 mM, about 50 mM to about 250 mM, about 50 mM to about 300 mM, about 50 mM to about 350 mM, about 50 mM to about 400 mM, about 100 mM to about 250 mM, about 100 mM to about 300 mM, about 100 mM to about 400 mM, about 150 mM to about 250 mM, about 150 mM to about 300 mM, or about 150 mM to about 400 mM of amino acids. In further embodiments, the composition comprises about 1 mM, 1.6 mM, 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about 400 mM of amino acid.

[0226] In some embodiments, the composition includes a surfactant. As used herein, the term "surfactant" refers to an organic substance with an amphiphilic structure, i.e., consisting of groups with opposite solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Depending on the charge of the surface-active moiety, surfactants can be classified into anionic, cationic, and nonionic surfactants. Surfactants are often used as wetting agents, emulsifiers, solubilizers, and dispersants in various pharmaceutical compositions and preparations of biological materials. Polysorbates (e.g., Polysorbate 20 or 80); poloxamers (e.g., Poloxamer 188); Triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, pyrimidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, pyrimidopropyl-, or isostearamidopropyl-dimethylamine; sodium cocoyl methyl taurate or disodium oleyl methyl taurate; and the MONAQUA® series (Mona Pharmaceutically acceptable surfactants, such as polysorbate 20, polysorbate 40, polysorbate 60, and copolymers of ethylene and propylene glycol (e.g., Polysorbate Industries, Inc., Paterson, NJ), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONICS® PF68, etc.), can optionally be added to the composition to reduce aggregation. In certain embodiments, the composition comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. Surfactants are particularly useful when a pump or plastic container is used to administer the composition. The presence of a pharmaceutically acceptable surfactant mitigates the tendency of proteins to aggregate. The composition may comprise a polysorbate at a concentration ranging from about 0.001% to about 1%, or from about 0.001% to about 0.1%, or from about 0.01% to about 0.1%.In other specific embodiments, the composition comprises polysorbate at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or 0.015%, or 0.02%.

[0227] In some embodiments, the compositions contain other excipients and / or additives, including but not limited to, diluents, binders, stabilizers, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutically acceptable excipients and / or additives can be used in the compositions provided herein. Commonly used excipients / additives, such as pharmaceutically acceptable chelating agents (e.g., but not limited to, EDTA, DTPA, or EGTA), can optionally be added to the compositions to reduce aggregation. These additives are particularly useful when a pump or plastic container is used to administer the composition.

[0228] In some embodiments, the composition includes a preservative. Preservatives such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., but not limited to, hexahydrate), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, can be optionally added to the composition at any suitable concentration, such as about 0.001% to about 5%, or any range or value therein. The concentration of the preservative used in the composition is sufficient to cause microbial action. Such a concentration will depend on the preservative selected and can be easily determined by one of ordinary skill in the art.

[0229] In some embodiments, the composition is isotonic with human blood, meaning that the composition has essentially the same osmotic pressure as human blood. Such isotonic compositions generally have an osmotic pressure of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, by using a vapor pressure or freezing point osmometer. The tonicity of the composition is adjusted by the use of a tonicity adjuster. A "tonicity adjuster" is a pharmaceutically acceptable inert substance that can be added to a composition to render the composition isotonic. Suitable tonicity adjusters for the compositions provided herein include, but are not limited to, sugars, salts, and amino acids.

[0230] In certain embodiments, the compositions are pyrogen-free compositions that are substantially free of endotoxins and / or related pyrogens. Endotoxins include toxins trapped inside microorganisms that are released only when the microorganisms decompose or die. Pyrogens also include heat-inducible, thermostable substances derived from the outer membrane of bacteria and other microorganisms. All of these substances can cause fever, hypotension, and shock when administered to humans. Due to potential harmful effects, endotoxins, even in small amounts, must be removed from pharmaceutical solutions administered intravenously. The U.S. Food and Drug Administration ("FDA") has set a limit of 5 endotoxin units (EU) per kilogram of body weight per hour per dose for intravenous drug administration (The United States Pharmacopeial Convention, Pharmacopeial Forum 26(1):223(2000)). When therapeutic proteins, such as proteins of interest (e.g., antibodies), are administered in amounts of hundreds or thousands of milligrams per kilogram of body weight, harmful and dangerous endotoxins must be removed, even in trace amounts. In some embodiments, the concentration of endotoxins and pyrogens in the composition is less than 10 EU / mg, or less than 5 EU / mg, or less than 1 EU / mg, or less than 0.1 EU / mg, or less than 0.01 EU / mg, or less than 0.001 EU / mg.

[0231] When used for in vivo administration, the compositions described herein must be sterile. The compositions can be sterilized by various sterilization methods, including sterile filtration, radiation, etc. In certain embodiments, the compositions are sterile filtered through a sterile 0.22 micron filter. Sterile compositions for injection can be prepared using the methods described in "Remington: The Science & Practice of Pharmacy", 21st ed., Lippincott It can be formulated according to conventional pharmaceutical practice, as described in Williams & Wilkins, (2005).The composition comprising target protein (for example, antibody) such as that disclosed herein is usually stored in lyophilized form or solution.It is intended that the sterile composition comprising target protein (for example, antibody) is placed in a container with sterile access port, for example, an intravenous solution bag or vial with an adapter that can extract composition, such as the stopper that can be penetrated by a hypodermic needle.In certain embodiments, the composition is provided as a pre-filled syringe.

[0232] In certain embodiments, the composition is a lyophilized formulation. The terms "lyophilization" or "freeze-dried" include a state of matter that has been subjected to a drying procedure, such as lyophilization, in which at least 50% of the water has been removed.

[0233] Regardless of the route of administration selected, the agents provided herein, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions provided herein, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0234] In the methods provided herein, the human antibodies, Fc-fusion proteins, and / or pharmaceutical compositions can be delivered by any suitable route of administration, including, for example, oral, nasal, e.g., by spray, rectal, vaginal, parenteral, intracisternal, and topical, by powder, ointment, or drops, buccal, and sublingual administration. In certain embodiments, the pharmaceutical compositions are administered systemically (e.g., via oral or parenteral administration).

[0235] In certain embodiments, the actual dosage of the active ingredient in the pharmaceutical compositions described herein may be varied to determine the amount of active ingredient that is nontoxic in animal models and effective in achieving the desired therapeutic response in that animal model, composition, and method of administration.

[0236] For example, in certain embodiments, the non-human animals described herein are used to determine the pharmacokinetic profile of one or more human antibody candidates. In various embodiments, one or more non-human animals described herein and one or more control or reference non-human animals are each exposed to one or more human antibody candidates at various dosages (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg or more). The candidate therapeutic antibodies can be administered via any desired route of administration, including parenteral and non-parenteral routes of administration. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intrathecal, intrathecal, intraventricular, intracranial, intrathoracic, or other injection routes. Non-parenteral routes include, for example, oral, nasal, transdermal, pulmonary, rectal, oral mucosal, vaginal, and ocular. Administration may also be by continuous infusion, topical administration, sustained release from an implant (gel, membrane, etc.), and / or intravenous injection. Blood is isolated from non-human animals (humanized and control) at various time points (e.g., 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or more). To determine the pharmacokinetic profile of the administered candidate therapeutic antibody, various assays, including, but not limited to, total IgG, anti-therapeutic antibody response, agglutination, etc., can be performed using samples obtained from the non-human animals described herein.

[0237] In various embodiments, the non-human animals described herein are used to measure the therapeutic effect of blocking or modulating the activity of a polypeptide of interest, and the effect on gene expression as a result of cellular changes, or, in the context of a receptor polypeptide, on the density of the receptor polypeptide on the cell surface of the non-human animal. In various embodiments, the non-human animals described herein, or cells isolated from the animals, are exposed to a candidate therapeutic agent that binds to the polypeptide of interest, and after a period of time, are analyzed for its effect on a particular cellular process associated with the polypeptide of interest, such as ligand-receptor interaction or signal transduction.

[0238] The non-human animals described herein provide improved in vivo systems for the development and selection of human antibodies for use in oncology and / or infectious diseases. In various embodiments, the non-human animals described herein and control non-human animals (e.g., animals with genetic modifications different from those described herein, or animals without genetic modifications, i.e., wild-type animals) can be implanted with a tumor (or tumor cells) or infected with a virus (e.g., influenza, HIV, HCV, HPV, etc.). After implantation or infection, the non-human animals can be administered a candidate therapeutic agent. The tumor or virus may be allowed sufficient time to establish itself in one or more locations within the non-human animal before administration of the candidate therapeutic agent. Alternatively and / or additionally, immune responses in such non-human animals can be monitored to characterize and select potential human antibodies that can be developed as therapeutic agents.

[0239] Methods for producing human antibodies In certain aspects, provided herein are methods of producing human antibodies using a rodent (e.g., a mouse or rat) comprising a humanized heavy chain locus provided herein and a human light chain locus provided herein (e.g., a humanized κ and / or λ light chain locus provided herein). In some embodiments, the rodent further comprises a humanized CD79a locus provided herein and / or a humanized CD79b locus provided herein. In some embodiments, the rodent further comprises a humanized FcRn locus provided herein and / or a humanized β2M locus provided herein. In certain embodiments, the rodent further comprises a humanized FcγR1a locus provided herein. In some embodiments, the rodent further comprises a humanized FcεR1α locus provided herein. In some embodiments, the rodent further comprises a humanized FcγR2b locus provided herein, a humanized FcγR2c locus provided herein, a humanized FcγR3a locus provided herein, and / or a humanized FcγR3b locus provided herein.

[0240] The rodents provided herein can be employed to generate antibodies (e.g., human antibodies) using standard methods known in the art. For example, in some embodiments, the rodents provided herein are immunized with an antigen of interest under conditions and for a time sufficient for the rodent to develop an immune response to the antigen of interest. In some embodiments, the antigen of interest is a human antibody (e.g., a human therapeutic antibody) or an Fc fusion protein (e.g., a therapeutic Fc fusion protein). The antibody is isolated from the rodent (or one or more cells, e.g., one or more B cells) and characterized using various assays that measure, for example, affinity, specificity, epitope mapping, ability to block ligand-receptor interactions, activation of inhibitory receptors, etc.

[0241] In some embodiments, a method of producing antibodies in a rodent (e.g., a mouse or rat) is provided, the method comprising: (a) immunizing a rodent that produces a human antibody described herein with an antigen of interest; (b) maintaining the rodent under conditions sufficient for the rodent to mount an immune response to the antigen of interest; and (c) recovering antibodies that bind to the antigen of interest from the rodent or from rodent cells. In some embodiments, the method further comprises breaking immune tolerance to the antigen in the rodent or otherwise deleting the antigen of interest, for example, employing a CRISPR / Cas9 system using multiple guide RNAs to reduce or eliminate expression of an autoantigen that is homologous to or shares an epitope of interest with the antigen of interest used to immunize the rodent (e.g., as described in U.S. Patent Publication No. 2017 / 0332610, which is incorporated herein by reference).

[0242] In some embodiments, methods are provided for producing nucleic acids encoding human heavy and / or light chains in a rodent, the methods comprising: (a) immunizing a rodent that expresses a human antibody described herein with an antigen of interest; (b) maintaining the rodent under conditions sufficient for the rodent to generate an immune response against the antigen of interest; and (c) recovering nucleic acids encoding the human heavy and / or light chains from the rodent or rodent cells.

[0243] In some embodiments, rodents provided herein can be employed to generate anti-drug antibodies (e.g., anti-idiotypic antibodies). For example, in some embodiments, rodents provided herein are immunized with a human therapeutic antibody under conditions and for a time sufficient for the rodent to develop an immune response to the human therapeutic antibody. In some embodiments, the human therapeutic antibody has a heavy chain constant domain that is the same as a heavy chain constant domain encoded by a humanized immunoglobulin heavy chain locus of a rodent provided herein. In some embodiments, the human therapeutic antibody has a light chain constant domain that is the same as a light chain constant domain encoded by a humanized immunoglobulin light chain locus of a rodent provided herein (e.g., a humanized κ and / or λ light chain locus). In some embodiments, the rodent does not comprise in its genome one or more of the human variable region gene segments from which the therapeutic antibody is derived (e.g., one or more immunoglobulin loci of the rodent comprise a rodent variable region or portion thereof). Anti-drug antibodies (e.g., anti-idiotypic antibodies) are isolated from the rodent (or one or more cells, e.g., one or more B cells) and characterized using various assays that measure, for example, affinity, specificity, epitope mapping, ability to block antigen-therapeutic antibody interactions, etc. The generated anti-drug antibodies (e.g., anti-idiotypic antibodies) may be used in pharmacokinetic (PK) analysis of the human therapeutic antibody, may be used to analyze the immunogenicity of the human therapeutic antibody in preclinical analyses, or may be used to localize the human therapeutic antibody.

[0244] In some embodiments, methods are provided for producing anti-drug antibodies (e.g., anti-idiotypic antibodies) in rodents (e.g., mice or rats), the methods comprising: (a) immunizing a rodent that produces antibodies comprising a human constant region(s) described herein with a human therapeutic antibody that is isotype-matched to the human constant region present in the rodent; (b) maintaining the rodent under conditions sufficient for the rodent to mount an immune response against the human therapeutic antibody; and (c) recovering antibodies from the rodent, or from rodent cells, that bind to the human therapeutic antibody.

[0245] Methods for producing genetically modified non-human animals and ES cells In certain aspects, provided herein are methods of producing non-human animals (e.g., mice or rats) and ES cells comprising one or more of the genetically modified loci provided herein. For example, in some embodiments, provided herein are methods of producing non-human animals (e.g., mice or rats) and ES cells comprising a humanized heavy chain locus provided herein and / or a human light chain locus provided herein (e.g., a humanized κ and / or λ light chain locus provided herein). In some embodiments, provided herein are methods of producing non-human animals (e.g., mice or rats) and ES cells further comprising a humanized CD79a locus provided herein and / or a humanized CD79b locus provided herein. In some embodiments, provided herein are methods of producing non-human animals (e.g., mice or rats) and ES cells further comprising a humanized FcRn locus provided herein and / or a humanized β2M locus provided herein. In some embodiments, provided herein are methods of producing non-human animals (e.g., mice or rats) and ES cells that further comprise a humanized FcεR1α locus provided herein. In specific embodiments, provided herein are methods of producing non-human animals (e.g., mice or rats) and ES cells that further comprise a humanized FcγR1a locus provided herein. In some embodiments, provided herein are methods of producing non-human animals (e.g., mice or rats) and ES cells that further comprise a humanized FcγR1α locus provided herein. In some embodiments, provided herein are methods of producing non-human animals (e.g., mice or rats) and ES cells that further comprise a humanized FcγR2a locus provided herein, a humanized FcγR2b locus provided herein, a humanized FcγR2c locus provided herein, a humanized FcγR3a locus provided herein, and / or a humanized FcγR3b locus provided herein.In certain embodiments, provided herein are methods of producing non-human animals (e.g., mice or rats) and ES cells comprising a humanized FcRn locus provided herein and / or a humanized β2M locus provided herein. In certain embodiments, provided herein are methods of producing non-human animals (e.g., mice or rats) and ES cells comprising a humanized FcεR1α locus provided herein. Exemplary methods of producing the genetically modified non-human animals and ES cells provided herein are described in the description, examples, and / or figures herein.

[0246] kit Provided herein are packs or kits comprising one or more containers filled with at least one non-human animal, non-human cell, DNA fragment, and / or targeting vector described in the descriptions, examples, and / or figures herein. The kits can be used in any applicable method (e.g., research method). Such container(s) may optionally be accompanied by documentation in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, indicating (a) regulatory approval for manufacture, use, or sale for human administration, (b) instructions for use, and (c) agreements governing the transfer of materials and / or biological products (e.g., non-human animals or non-human cells described herein) between two or more entities and combinations thereof.

[0247] Additional Exemplary Embodiments In exemplary embodiment 1, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a V H Gene segment, D H Gene segments and J H (ii) an immunoglobulin heavy chain variable region comprising a human C H 1 domain, human hinge region, human C H 2 domains, human C HC, which encodes the IgG constant domain containing three domains: the IgG transmembrane domain and the IgG cytoplasmic domain. H and an immunoglobulin heavy chain variable region, the immunoglobulin heavy chain constant region comprising gene segments, wherein the rodent has a V H Gene segment, D H Gene segments and J H variable domains derived from gene segments and C H and a heavy chain constant domain derived from a gene segment operably linked to an immunoglobulin heavy chain constant region so as to produce an IgG antibody.

[0248] In exemplary embodiment 2, provided herein is a rodent according to embodiment 1, wherein the IgG transmembrane domain is a rodent IgG transmembrane domain.

[0249] In exemplary embodiment 3, provided herein is a rodent of embodiment 1, wherein the IgG transmembrane domain is a human IgG transmembrane domain.

[0250] In exemplary embodiment 4, provided herein is a rodent according to any one of embodiments 1-3, wherein the IgG cytoplasmic domain is a rodent IgG cytoplasmic domain.

[0251] In exemplary embodiment 5, provided herein is a rodent of any one of embodiments 1-3, wherein the IgG cytoplasmic domain is a human IgG cytoplasmic domain.

[0252] In exemplary embodiment 6, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human C H The rodent according to any one of embodiments 1 to 5, wherein the three domains are IgG1 domains.

[0253] In exemplary embodiment 7, provided herein is a rodent of embodiment 6, wherein the IgG1 domain is encoded by an allele selected from IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04 and IGHG1*05.

[0254] In exemplary embodiment 8, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human C H The rodent according to any one of embodiments 1 to 5, wherein the three domains are IgG2 domains.

[0255] In exemplary embodiment 9, provided herein is a rodent of embodiment 8, wherein the IgG2 domain is encoded by an allele selected from IGHG2*01, IGHG2*02, IGHG2*03, IGHG2*04, IGHG2*05, and IGHG2*06.

[0256] In exemplary embodiment 10, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human C H 6. The rodent of any one of embodiments 1 to 5, wherein the 3 domain is an IgG3 domain.

[0257] In exemplary embodiment 11, provided herein is a rodent of embodiment 10, wherein the IgG3 domain is encoded by an allele selected from IGHG3*01, IGHG3*02, IGHG3*03, IGHG3*04, IGHG3*05, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*13, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18, and IGHG3*19.

[0258] In exemplary embodiment 12, provided herein is a human CH 1 domain, human hinge region, human C H 2 domain and human C H The rodent according to any one of embodiments 1 to 5, wherein the three domains are IgG4 domains.

[0259] In exemplary embodiment 13, provided herein is a rodent of embodiment 12, wherein the IgG4 domain is encoded by an allele selected from IGHG4*01, IGHG4*02, IGHG4*03, and IGHG4*04.

[0260] In exemplary embodiment 14, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human C H C encoding 3 domains H The gene segment is endogenous C γ2a or C γ2c The rodent of any one of embodiments 6 to 13, wherein the gene segment locus is located.

[0261] In exemplary embodiment 15, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human C H C encoding 3 domains H The gene segment is endogenous C γ2a or C γ2c 15. The rodent of embodiment 14, wherein the gene segment is replaced.

[0262] In exemplary embodiment 16, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human C H C encoding 3 domains H The gene segment is endogenous C γ1 The rodent of any one of embodiments 6 to 13, wherein the gene segment locus is located.

[0263] In exemplary embodiment 17, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human C H C encoding 3 domains H The gene segment is endogenous C γ1 17. The rodent of embodiment 16, wherein the gene segment is replaced.

[0264] In exemplary embodiment 18, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human C H C encoding 3 domains H The gene segment is endogenous C γ2b The rodent of any one of embodiments 6 to 13, wherein the gene segment locus is located.

[0265] In exemplary embodiment 19, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human C H C encoding 3 domains H The gene segment is endogenous C γ2b 19. The rodent of embodiment 18, wherein the gene segment is replaced.

[0266] In exemplary embodiment 20, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human C H C encoding 3 domains H The gene segment is endogenous C γ3 The rodent of any one of embodiments 6 to 13, wherein the gene segment locus is located.

[0267] In exemplary embodiment 21, provided herein is a human C H 1 domain, human hinge region, human C H 2 domain and human CH C encoding 3 domains H The gene segment is endogenous C γ3 21. The rodent of embodiment 20, wherein the gene segment is replaced.

[0268] In exemplary embodiment 22, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of a rodent C μ 22. The rodent of any one of embodiments 1 to 21, further comprising a gene segment.

[0269] In exemplary embodiment 23, provided herein is an immunoglobulin wherein the immunoglobulin heavy chain constant region is selected from the group consisting of human C μ 22. The rodent of any one of embodiments 1 to 21, further comprising a gene segment.

[0270] In exemplary embodiment 24, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of a rodent C δ 24. The rodent of any one of embodiments 1 to 23, further comprising a gene segment.

[0271] In exemplary embodiment 25, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C δ 24. The rodent of any one of embodiments 1 to 23, further comprising a gene segment.

[0272] In exemplary embodiment 26, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of a rodent C γ1 26. The rodent of any one of embodiments 1 to 25, further comprising a gene segment.

[0273] In exemplary embodiment 27, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of a rodent C γ2a and / or C. γ2c 27. The rodent of any one of embodiments 1 to 26, further comprising a gene segment.

[0274] In exemplary embodiment 28, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of a rodent C γ2b 28. The rodent of any one of embodiments 1 to 27, further comprising a gene segment.

[0275] In exemplary embodiment 29, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of a rodent C γ3 The rodent of any one of embodiments 1 to 28, further comprising a gene segment.

[0276] In exemplary embodiment 30, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of a rodent C ε The rodent of any one of embodiments 1 to 29, further comprising a gene segment.

[0277] In exemplary embodiment 31, provided herein is an immunoglobulin wherein the immunoglobulin heavy chain constant region is selected from the group consisting of human C ε The rodent of any one of embodiments 1 to 29, further comprising a gene segment.

[0278] In exemplary embodiment 32, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of a rodent C α 32. The rodent of any one of embodiments 1 to 31, further comprising a gene segment.

[0279] In exemplary embodiment 33, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C α 32. The rodent of any one of embodiments 1 to 31, further comprising a gene segment.

[0280] In exemplary embodiment 34, provided herein is an immunoglobulin wherein the immunoglobulin heavy chain constant region is selected from the group consisting of human C μ Gene segment, human C δ Gene segment, human C γ3 Gene segments and human C γ122. The rodent of any one of embodiments 1 to 21, comprising the gene segment.

[0281] In exemplary embodiment 35, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C γ2 Gene segments and human C γ4 35. The rodent of embodiment 34, further comprising a gene segment.

[0282] In exemplary embodiment 36, provided herein is an immunoglobulin wherein the immunoglobulin heavy chain constant region is selected from the group consisting of human C α The rodent of embodiment 34 or embodiment 35, further comprising a gene segment.

[0283] In exemplary embodiment 37, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C ε 37. The rodent of any one of embodiments 34 to 36, further comprising a gene segment.

[0284] In exemplary embodiment 38, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a V H Gene segment, D H Gene segments and J H (ii) an immunoglobulin heavy chain variable region comprising a human C H 1 domain, human C H 2 domains, human C H 3 domains, human C H C, encoding the IgM constant domain containing the human IgM transmembrane domain and the human IgM cytoplasmic domain. H and an immunoglobulin heavy chain variable region, the immunoglobulin heavy chain constant region comprising gene segments, wherein the rodent has a V H Gene segment, D H Gene segments and J H variable domains derived from gene segments and C Hand a heavy chain constant domain derived from a gene segment operably linked to an immunoglobulin heavy chain constant region so as to produce an IgM antibody.

[0285] In exemplary embodiment 39, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C δ 39. The rodent of embodiment 38, further comprising a gene segment.

[0286] In exemplary embodiment 40, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C γ3 40. The rodent of embodiment 38 or 39, further comprising a gene segment.

[0287] In exemplary embodiment 41, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C γ1 41. The rodent of any one of embodiments 38 to 40, further comprising a gene segment.

[0288] In exemplary embodiment 42, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C γ2 42. The rodent of any one of embodiments 38 to 41, further comprising a gene segment.

[0289] In exemplary embodiment 43, provided herein is an immunoglobulin wherein the immunoglobulin heavy chain constant region is selected from the group consisting of human C γ4 43. The rodent of any one of embodiments 38 to 42, further comprising a gene segment.

[0290] In exemplary embodiment 44, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C μ Gene segment, human C δ Gene segment, human C γ3 Gene segments and human C γ1 44. The rodent of any one of embodiments 38 to 43, comprising the gene segment.

[0291] In exemplary embodiment 45, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C γ2 Gene segments and human C γ4 45. The rodent of embodiment 44, further comprising a gene segment.

[0292] In exemplary embodiment 46, provided herein is an immunoglobulin wherein the immunoglobulin heavy chain constant region is selected from the group consisting of human C α The rodent of embodiment 44 or embodiment 45, further comprising a gene segment.

[0293] In exemplary embodiment 47, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C ε 47. The rodent of any one of embodiments 44 to 46, further comprising a gene segment.

[0294] In exemplary embodiment 48, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a V H Gene segment, D H Gene segments and J H (ii) an immunoglobulin heavy chain variable region comprising a human C H 1 domain, human hinge H1 domain, human hinge H2 domain, human C H 2 domains, human C H C, encoding the IgD constant domain containing the human IgD transmembrane domain and the human IgD cytoplasmic domain. H and an immunoglobulin heavy chain variable region, the immunoglobulin heavy chain constant region comprising gene segments, wherein the rodent has a V H Gene segment, D H Gene segments and J H variable domains derived from gene segments and C H and a heavy chain constant domain derived from a gene segment operably linked to an immunoglobulin heavy chain constant region so as to produce an IgD antibody.

[0295] In exemplary embodiment 49, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C μ The rodent of embodiment 48, further comprising a gene segment.

[0296] In exemplary embodiment 50, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C γ3 50. The rodent of embodiment 48 or 49, further comprising a gene segment.

[0297] In exemplary embodiment 51, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C γ1 51. The rodent of any one of embodiments 48 to 50, further comprising a gene segment.

[0298] In exemplary embodiment 52, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C γ2 52. The rodent of any one of embodiments 48 to 51, further comprising a gene segment.

[0299] In exemplary embodiment 53, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C γ4 53. The rodent of any one of embodiments 48 to 52, further comprising a gene segment.

[0300] In exemplary embodiment 54, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C μ Gene segment, human C δ Gene segment, human C γ3 Gene segments and human C γ1 54. The rodent of any one of embodiments 48 to 53, comprising the gene segment.

[0301] In exemplary embodiment 55, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C γ2 Gene segments and human C γ455. The rodent of embodiment 54, further comprising a gene segment.

[0302] In exemplary embodiment 56, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C α 56. The rodent of embodiment 54 or embodiment 55, further comprising a gene segment.

[0303] In exemplary embodiment 57, provided herein is an immunoglobulin heavy chain constant region selected from the group consisting of human C ε 57. The rodent of any one of embodiments 54 to 56, further comprising a gene segment.

[0304] In exemplary embodiment 58, provided herein is an engineered immunoglobulin heavy chain locus comprising a rodent intronic enhancer (E i 58. The rodent of any one of embodiments 1-57, further comprising:

[0305] In exemplary embodiment 59, provided herein is an engineered immunoglobulin heavy chain locus comprising a human intronic enhancer (E i 58. The rodent of any one of embodiments 1-57, further comprising:

[0306] In exemplary embodiment 60, provided herein is a rodent according to any one of embodiments 1 to 59, wherein the engineered immunoglobulin heavy chain locus further comprises a rodent 3' regulatory region (3'RR).

[0307] In exemplary embodiment 61, provided herein is a rodent of any one of embodiments 1-5, wherein the engineered immunoglobulin heavy chain locus further comprises a human 3' regulatory region (3'RR).

[0308] In exemplary embodiment 62, provided herein is a method for producing a recombinant human immunoglobulin heavy chain (IgG) gene comprising: μ62. The rodent of any one of embodiments 1-61, further comprising a switch site.

[0309] In exemplary embodiment 63, provided herein is a method for producing an engineered immunoglobulin heavy chain locus in a rodent S. γ3 63. The rodent of any one of embodiments 1-62, further comprising a switch site.

[0310] In exemplary embodiment 64, provided herein is a method for producing a recombinant human immunoglobulin heavy chain (IgG) gene comprising: γ1 64. The rodent of any one of embodiments 1 to 63, further comprising a switch site.

[0311] In exemplary embodiment 65, provided herein is a method for producing an engineered immunoglobulin heavy chain locus in a rodent S. γ2b 65. The rodent of any one of embodiments 1-64, further comprising a switch site.

[0312] In exemplary embodiment 66, provided herein is a method for producing a recombinant human immunoglobulin heavy chain (IgG) gene comprising: γ2a and / or S. γ2c 66. The rodent of any one of embodiments 1-65, further comprising a switch site.

[0313] In exemplary embodiment 67, provided herein is a method for producing a recombinant human immunoglobulin heavy chain (IgG) gene comprising: ε 67. The rodent of any one of embodiments 1 to 66, further comprising a switch site.

[0314] In exemplary embodiment 68, provided herein is a method for producing an engineered immunoglobulin heavy chain locus in a rodent S. α 68. The rodent of any one of embodiments 1 to 67, further comprising a switch site.

[0315] In exemplary embodiment 69, provided herein is a method for producing an engineered immunoglobulin heavy chain locus comprising the steps of:μ 62. The rodent of any one of embodiments 1-61, further comprising a switch site.

[0316] In exemplary embodiment 70, provided herein is a method for producing an engineered immunoglobulin heavy chain locus comprising the steps of: γ3 70. The rodent of any one of embodiments 1 to 61 and 69, further comprising a switch site.

[0317] In exemplary embodiment 71, provided herein is a method for producing an engineered immunoglobulin heavy chain locus comprising the steps of: γ1 The rodent of any one of embodiments 1-61 and 69-70, further comprising a switch site.

[0318] In exemplary embodiment 72, provided herein is a method for producing an engineered immunoglobulin heavy chain locus comprising the steps of: γ2 The rodent of any one of embodiments 1-61 and 69-71, further comprising a switch site.

[0319] In exemplary embodiment 73, provided herein is a method for producing an engineered immunoglobulin heavy chain locus comprising the steps of: γ4 The rodent of any one of embodiments 1-61 and 69-72, further comprising a switch site.

[0320] In exemplary embodiment 74, provided herein is a method for producing an engineered immunoglobulin heavy chain locus comprising the steps of: ε The rodent of any one of embodiments 1-61 and 69-73, further comprising a switch site.

[0321] In exemplary embodiment 75, provided herein is a method for producing an engineered immunoglobulin heavy chain locus comprising the steps of: α The rodent of any one of embodiments 1-61 and 69-74, further comprising a switch site.

[0322] In exemplary embodiment 76, provided herein is a method for producing a medicament comprising:H The gene segment is a rodent V H gene segment, D H The gene segment is a rodent D H gene segment, J H The gene segment is rodent J H The rodent of any one of embodiments 1 to 75, wherein the gene segment.

[0323] In exemplary embodiment 77, provided herein is a rodent V H Gene segment, rodent D H Gene segments and rodent J H The rodent of embodiment 76, wherein the gene segment is an endogenous rodent gene segment.

[0324] In exemplary embodiment 78, provided herein is a method for producing a medicament comprising: H The gene segment is human V H gene segment, D H The gene segment is human D H gene segment, J H The gene segment is human J H The rodent of any one of embodiments 1 to 75, wherein the gene segment.

[0325] In exemplary embodiment 79, provided herein is an immunoglobulin heavy chain variable region comprising at least three human V H 79. The rodent of embodiment 78, comprising the gene segment.

[0326] In exemplary embodiment 80, provided herein is an immunoglobulin heavy chain variable region comprising a human D H The rodent of embodiment 78 or embodiment 79, comprising all of the gene segments.

[0327] In exemplary embodiment 81, provided herein is an immunoglobulin comprising an immunoglobulin heavy chain variable region selected from the group consisting of human J H81. The rodent of any one of embodiments 78-80, comprising all of the gene segments.

[0328] In exemplary embodiment 82, provided herein is a rodent according to any one of embodiments 78 to 81, wherein the immunoglobulin heavy chain variable region lacks a functional endogenous rodent Adam6 gene.

[0329] In exemplary embodiment 83, provided herein is a rodent described in any one of embodiments 78 to 82, wherein the germline genome further comprises a nucleotide sequence encoding a functional rodent Adam6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof.

[0330] In exemplary embodiment 84, provided herein is a rodent according to embodiment 83, which expresses a functional rodent Adam6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof.

[0331] In exemplary embodiment 85, provided herein is a rodent according to embodiment 83 or embodiment 84, wherein a nucleotide sequence encoding the rodent ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof is contained on the same chromosome as the immunoglobulin heavy chain variable region.

[0332] In exemplary embodiment 86, provided herein is a rodent described in any one of embodiments 83 to 85, wherein a nucleotide sequence encoding a rodent ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof is contained within an engineered immunoglobulin heavy chain locus.

[0333] In exemplary embodiment 87, provided herein is a rodent described in any one of embodiments 83 to 86, wherein the nucleotide sequence encoding the rodent ADAM6 polypeptide, its functional ortholog, functional homolog, or functional fragment is in place of the human Adam6 pseudogene.

[0334] In exemplary embodiment 88, provided herein is a rodent described in any one of embodiments 83 to 87, wherein a nucleotide sequence encoding a rodent ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof replaces the human Adam6 pseudogene.

[0335] In exemplary embodiment 89, provided herein is a method for detecting a rodent ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof, comprising: H Gene segments and the second human V H 89. The rodent of any one of embodiments 83-88, wherein the gene segment is between the

[0336] In exemplary embodiment 90, provided herein is a first human V H Gene segment V H 1-2 and the second human V H Gene segment V H 6-1.

[0337] In exemplary embodiment 91, provided herein is a method for treating a rodent ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof, comprising administering to a mammalian subject the method of claim 1, wherein the nucleotide sequence encoding the rodent ADAM6 polypeptide, a functional ortholog, a functional homolog, or a functional fragment thereof is selected from the group consisting of human V H Gene segments and human D H 87. The rodent of any one of embodiments 83-86, wherein the gene segment is between the

[0338] In exemplary embodiment 92, provided herein is a rodent of any one of embodiments 1 to 91, wherein the engineered immunoglobulin heavy chain locus is placed at the endogenous immunoglobulin heavy chain locus.

[0339] In exemplary embodiment 93, provided herein is the rodent of embodiment 92, wherein the engineered immunoglobulin heavy chain locus replaces all or part of the endogenous immunoglobulin heavy chain locus.

[0340] In exemplary embodiment 94, provided herein is a rodent of any one of embodiments 1-93, wherein the rodent is heterozygous for the engineered immunoglobulin heavy chain locus.

[0341] In exemplary embodiment 95, provided herein is the rodent of any one of embodiments 1-93, wherein the rodent is homozygous for the engineered immunoglobulin heavy chain locus.

[0342] In exemplary embodiment 96, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a human V H Gene segment, human D H Gene segments and human J H (ii) an immunoglobulin heavy chain variable region containing a gene segment; and (iii) a rodent intronic enhancer (E i ) and (iii) an immunoglobulin heavy chain constant region, μ gene segments, (b) rodent C δ gene segment, (c) rodent C γ3 gene segment, (d) rodent C γ1 gene segment, (e) rodent C γ2b gene segment, (f) human IgG1 C H 1 domain, human IgG1 hinge region, human IgG1 C H 2 domain, human IgG1 CH A modified C3 domain encoding a rodent IgG2a transmembrane domain and a rodent IgG2a cytoplasmic domain. H gene segment, (g) rodent C ε gene segments, and (h) rodent C α and (iv) a rodent 3' regulatory region (3'RR), wherein the rodent immunoglobulin heavy chain variable region comprises a human V H Gene segment, human D H Gene segments and human J H variable domains derived from gene segments and modified C H and a heavy chain constant domain derived from a gene segment operably linked to an immunoglobulin heavy chain constant region so as to produce an IgG antibody.

[0343] In exemplary embodiment 97, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a human V H Gene segment, human D H Gene segments and human J H (ii) an immunoglobulin heavy chain variable region containing a gene segment; and (iii) a rodent intronic enhancer (E i ) and (iii) an immunoglobulin heavy chain constant region, μ gene segments, (b) rodent C δ gene segment, (c) rodent C γ3 gene segment, (d) rodent C γ1 gene segment, (e) rodent C γ2b gene segment, (f) human IgG1 C H 1 domain, human IgG1 hinge region, human IgG1 C H 2 domain, human IgG1 C H A modified C3 domain encoding the human IgG1 transmembrane domain and the human IgG1 cytoplasmic domain. H gene segment, (g) rodent Cε gene segments, and (h) rodent C α and (iv) a rodent 3' regulatory region (3'RR), wherein the rodent immunoglobulin heavy chain variable region is a human V H Gene segment, human D H Gene segments and human J H variable domains derived from gene segments and modified C H and a heavy chain constant domain derived from a gene segment operably linked to an immunoglobulin heavy chain constant region so as to produce an IgG antibody.

[0344] In exemplary embodiment 98, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a human V H Gene segment, human D H Gene segments and human J H (ii) an immunoglobulin heavy chain variable region containing a gene segment; and (iii) a rodent intronic enhancer (E i ) and (iii) an immunoglobulin heavy chain constant region, μ gene segments, (b) rodent C δ gene segment, (c) rodent C γ3 gene segment, (d) human IgG4 C H 1 domain, human IgG4 hinge region, human IgG4 C H 2 domain, human IgG4 C H a modified C encoding three domains, a rodent IgG1 transmembrane domain, and a rodent IgG1 cytoplasmic domain H gene segment, (e) rodent C γ2b gene segment, (f) rodent C γ2a and / or C. γ2c gene segment, (g) rodent C ε gene segments, and (h) rodent C αand (iv) a rodent 3' regulatory region (3'RR), wherein the rodent immunoglobulin heavy chain variable region is a human V H Gene segment, human D H Gene segments and human J H variable domains derived from gene segments and modified C H and a heavy chain constant domain derived from a gene segment operably linked to an immunoglobulin heavy chain constant region so as to produce an IgG antibody.

[0345] In exemplary embodiment 99, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a human V H Gene segment, human D H Gene segments and human J H (ii) an immunoglobulin heavy chain variable region containing a gene segment; and (iii) a rodent intronic enhancer (E i ) and (iii) an immunoglobulin heavy chain constant region, μ gene segments, (b) rodent C δ gene segment, (c) rodent C γ3 gene segment, (d) human IgG4 C H 1 domain, human IgG4 hinge region, human IgG4 C H 2 domain, human IgG4 C H A modified C3 domain encoding the human IgG4 transmembrane domain and the human IgG4 cytoplasmic domain. H gene segment, (e) rodent C γ2b gene segment, (f) rodent C γ2a and / or C. γ2c gene segment, (g) rodent C ε gene segments, and (h) rodent C α and (iv) a rodent 3' regulatory region (3'RR), wherein the rodent immunoglobulin heavy chain variable region is a human VH Gene segment, human D H Gene segments and human J H variable domains derived from gene segments and modified C H and a heavy chain constant domain derived from a gene segment operably linked to an immunoglobulin heavy chain constant region so as to produce an IgG antibody.

[0346] In exemplary embodiment 100, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a human V H Gene segment, human D H Gene segments and human J H (ii) an immunoglobulin heavy chain variable region comprising a gene segment; and (iii) a human intronic enhancer (E i ) and (iii) an immunoglobulin heavy chain constant region, comprising: (a) a human C μ gene segments, (b) human C δ gene segment, (c) human C γ3 gene segments, (d) human C γ1 and (iv) a rodent 3' regulatory region (3'RR), wherein the immunoglobulin heavy chain variable region is rodent operably linked to the immunoglobulin heavy chain constant region such that the rodent produces IgG antibodies comprising a fully human heavy chain.

[0347] In exemplary embodiment 101, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a human V H Gene segment, human D H Gene segments and human J H (ii) an immunoglobulin heavy chain variable region comprising a gene segment; and (iii) a human intronic enhancer (E i ) and (iii) an immunoglobulin heavy chain constant region, comprising: (a) a human C μgene segments, (b) human C δ gene segment, (c) human C γ3 gene segments, (d) human C γ1 gene segment, (c) human C γ2 gene segments, (d) human C γ4 and (iv) a rodent 3' regulatory region (3'RR), wherein the immunoglobulin heavy chain variable region is rodent operably linked to the immunoglobulin heavy chain constant region such that the rodent produces IgG antibodies comprising a fully human heavy chain.

[0348] In exemplary embodiment 102, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a rodent V H Gene segment, rodent D H Gene segments and rodent J H (ii) an immunoglobulin heavy chain variable region containing a gene segment; and (iii) a rodent intronic enhancer (E i ) and (iii) an immunoglobulin heavy chain constant region, comprising: (a) a human C μ gene segments, (b) human C δ gene segment, (c) human C γ3 gene segments, (d) human C γ1 and (iv) a rodent 3' regulatory region (3'RR), wherein the immunoglobulin heavy chain variable region is rodent operably linked to the immunoglobulin heavy chain constant region such that the rodent produces an IgG antibody comprising a heavy chain comprising a rodent variable domain and a human constant domain.

[0349] In exemplary embodiment 103, provided herein is a rodent comprising an engineered immunoglobulin heavy chain locus in its genome, the engineered immunoglobulin heavy chain locus comprising: (i) a rodent V H Gene segment, rodent D H Gene segments and rodent JH (ii) an immunoglobulin heavy chain variable region containing a gene segment; and (iii) a rodent intronic enhancer (E i ) and (iii) an immunoglobulin heavy chain constant region, comprising: (a) a human C μ gene segments, (b) human C δ gene segment, (c) human C γ3 gene segments, (d) human C γ1 gene segment, (c) human C γ2 gene segments, (d) human C γ4 and (iv) a rodent 3' regulatory region (3'RR), wherein the immunoglobulin heavy chain variable region is rodent operably linked to the immunoglobulin heavy chain constant region such that the rodent produces an IgG antibody comprising a heavy chain comprising a rodent variable domain and a human constant domain.

[0350] In exemplary embodiment 104, provided herein is a rodent according to any one of embodiments 96 to 103, wherein the engineered immunoglobulin heavy chain locus is placed at the endogenous immunoglobulin heavy chain locus.

[0351] In exemplary embodiment 105, provided herein is the rodent of embodiment 104, wherein the engineered immunoglobulin heavy chain locus replaces all or part of the endogenous immunoglobulin heavy chain locus.

[0352] In exemplary embodiment 106, provided herein is a rodent according to any one of embodiments 96 to 105, wherein the rodent is heterozygous for the engineered immunoglobulin heavy chain locus.

[0353] In exemplary embodiment 107, provided herein is a rodent according to any one of embodiments 96 to 105, wherein the rodent is homozygous for the engineered immunoglobulin heavy chain locus.

[0354] In exemplary embodiment 108, provided herein is a human IgG1-positive ... κ Gene segments and human J κ (2) a human C κ and an immunoglobulin κ chain constant region comprising gene segments, wherein the immunoglobulin κ chain variable region is a human V κ Gene segments and human J κ A light chain variable domain derived from a gene segment and a human C κ and a light chain constant domain derived from the gene segment, operably linked to an immunoglobulin κ chain constant region so as to produce an antibody.

[0355] In exemplary embodiment 109, provided herein is an engineered immunoglobulin κ chain locus comprising a rodent intronic κ enhancer (E κi 109. The rodent of embodiment 108, further comprising:

[0356] In exemplary embodiment 110, provided herein is an engineered immunoglobulin κ chain locus comprising a human intronic κ enhancer (E κi 109. The rodent of embodiment 108, further comprising:

[0357] In exemplary embodiment 111, provided herein is an engineered immunoglobulin κ chain locus comprising a rodent 3′ κ enhancer (E κ3’ 111. The rodent of any one of embodiments 108-110, further comprising:

[0358] In exemplary embodiment 112, provided herein is an engineered immunoglobulin κ chain locus comprising a human 3′ κ enhancer (E κ3’ 111. The rodent of any one of embodiments 108-110, further comprising:

[0359] In exemplary embodiment 113, provided herein is an immunoglobulin comprising an immunoglobulin κ chain variable region comprising at least six human V κ The rodent of any one of embodiments 108 to 112, comprising the gene segment.

[0360] In exemplary embodiment 114, provided herein is an immunoglobulin κ chain variable region comprising a human J κ The rodent of any one of embodiments 108 to 113, comprising all of the gene segments.

[0361] In exemplary embodiment 115, provided herein is a rodent described in any one of embodiments 108 to 114, wherein an engineered immunoglobulin κ chain locus is placed at an endogenous immunoglobulin κ chain locus.

[0362] In exemplary embodiment 116, provided herein is a rodent according to embodiment 115, wherein the engineered immunoglobulin κ chain locus replaces all or part of the endogenous immunoglobulin κ chain locus.

[0363] In exemplary embodiment 117, provided herein is a rodent according to any one of embodiments 108 to 116, wherein the rodent is heterozygous for the engineered immunoglobulin κ chain locus.

[0364] In exemplary embodiment 118, provided herein is a rodent according to any one of embodiments 108 to 116, wherein the rodent is homozygous for the engineered immunoglobulin κ chain locus.

[0365] In exemplary embodiment 119, provided herein is a human IgG1-positive ... λ Gene segment, human J λ Gene segments and human Cλ Contains gene segments, human V λ Gene segments and human J λ The gene segment is similar to that of the rodent V λ Gene segments and human J λ A light chain variable domain derived from a gene segment and a human C λ and a light chain constant domain derived from a human C gene segment. λ The rodent of any one of embodiments 1-118, wherein the gene segment is operably linked to the rodent.

[0366] In exemplary embodiment 120, provided herein is a human C λ The gene segment is human C λ1 120. The rodent of embodiment 119, wherein the gene segment is

[0367] In exemplary embodiment 121, provided herein is a human J λ The gene segment is human J λ1 121. The rodent of embodiment 120, wherein the gene segment.

[0368] In exemplary embodiment 122, provided herein is a human C λ The gene segment is human C λ2 120. The rodent of embodiment 119, wherein the gene segment is

[0369] In exemplary embodiment 123, provided herein is a human J λ The gene segment is human J λ2 123. The rodent of embodiment 122, wherein the gene segment is

[0370] In exemplary embodiment 124, provided herein is a human C λ The gene segment is human C λ3 120. The rodent of embodiment 119, wherein the gene segment is

[0371] In exemplary embodiment 125, provided herein is a human J λ The gene segment is human J λ3 125. The rodent of embodiment 124, wherein the gene segment is

[0372] In exemplary embodiment 126, provided herein is a human C λ The gene segment is human C λ6 120. The rodent of embodiment 119, wherein the gene segment is

[0373] In exemplary embodiment 127, provided herein is a human J λ The gene segment is human J λ6 127. The rodent of embodiment 126, wherein the gene segment is

[0374] In exemplary embodiment 128, provided herein is a human J λ The gene segment is human J λ7 120. The rodent of embodiment 119, wherein the gene segment is

[0375] In exemplary embodiment 129, provided herein is a method for producing an engineered immunoglobulin λ chain locus comprising: λ1 Gene segment, human C λ2 Gene segment, human C λ3 Gene segment, human C λ6 Gene segments and rodent C λ1 The rodent of any one of embodiments 119 to 128, comprising the gene segment.

[0376] In exemplary embodiment 130, provided herein is an engineered immunoglobulin λ chain locus comprising a human J λ1 Gene segment, human J λ2 Gene segment, human J λ3 Gene segment, human J λ6 Gene segments and human J λ7 130. The rodent of embodiment 129, comprising the gene segment.

[0377] In exemplary embodiment 131, provided herein is a method for producing an engineered immunoglobulin λ chain locus comprising: λ1 -C λ1 Gene segment cluster, human J λ2 -C λ2 Gene segment cluster, human J λ3 -C λ3 Gene segment cluster, human J λ6 -C λ6 Gene segment clusters and human J λ7 -Rodent C λ1 131. A rodent ES cell according to embodiment 130, comprising a gene segment cluster.

[0378] In exemplary embodiment 132, provided herein is an engineered immunoglobulin λ chain locus comprising at least seven human V λ 132. The rodent of any one of embodiments 119 to 131, comprising the gene segment.

[0379] In exemplary embodiment 133, provided herein is a rodent according to any one of embodiments 119 to 132, wherein the engineered immunoglobulin λ chain locus further comprises rodent λ enhancer 2.4.

[0380] In exemplary embodiment 134, provided herein is a rodent according to any one of embodiments 119 to 133, wherein the engineered immunoglobulin λ chain locus further comprises a rodent 3′ λ enhancer.

[0381] In exemplary embodiment 135, provided herein is a rodent according to any one of embodiments 119 to 134, wherein the engineered immunoglobulin λ chain locus further comprises rodent λ enhancer 3.1.

[0382] In exemplary embodiment 136, provided herein is a rodent according to any one of embodiments 119 to 135, wherein the engineered immunoglobulin λ chain locus further comprises a human 3′ λ enhancer.

[0383] In exemplary embodiment 137, provided herein is a rodent described in any one of embodiments 119 to 136, wherein the engineered immunoglobulin λ chain locus is positioned at the endogenous immunoglobulin λ chain locus.

[0384] In exemplary embodiment 138, provided herein is the rodent of embodiment 137, wherein the engineered immunoglobulin λ chain locus replaces all or part of the endogenous immunoglobulin λ chain locus.

[0385] In exemplary embodiment 139, provided herein is a rodent according to any one of embodiments 119 to 138, wherein the rodent is heterozygous for the engineered immunoglobulin λ chain locus.

[0386] In exemplary embodiment 140, provided herein is a rodent according to any one of embodiments 119 to 138, wherein the rodent is homozygous for the engineered immunoglobulin λ chain locus.

[0387] In exemplary embodiment 141, provided herein is a rodent of any one of embodiments 1 to 140, further comprising in its genome an engineered Fc receptor (FcRn) locus comprising a nucleic acid sequence encoding a fetal FcRn polypeptide comprising a human extracellular domain.

[0388] In exemplary embodiment 142, provided herein is the rodent of embodiment 141, wherein the FcRn polypeptide further comprises a rodent transmembrane domain.

[0389] In exemplary embodiment 143, provided herein is the rodent of embodiment 141, wherein the FcRn polypeptide further comprises a human transmembrane domain.

[0390] In exemplary embodiment 144, provided herein is a rodent according to any one of embodiments 141-143, wherein the FcRn polypeptide further comprises a rodent cytoplasmic domain.

[0391] In exemplary embodiment 145, provided herein is a rodent according to any one of embodiments 141-143, wherein the FcRn polypeptide further comprises a human cytoplasmic domain.

[0392] In exemplary embodiment 146, provided herein is a rodent according to any one of embodiments 141 to 145, wherein the nucleic acid sequence encoding the FcRn polypeptide is located at the endogenous rodent FcRn locus.

[0393] In exemplary embodiment 147, provided herein is the rodent of embodiment 146, wherein the nucleic acid sequence encoding the FcRn polypeptide replaces all or part of the endogenous rodent FcRn gene.

[0394] In exemplary embodiment 148, provided herein is the rodent of embodiment 141, wherein the nucleic acid sequence encoding the FcRn extracellular domain replaces the endogenous nucleic acid sequence encoding the rodent FcRn extracellular domain.

[0395] In exemplary embodiment 149, provided herein is the rodent of any one of embodiments 141-148, wherein the rodent does not express rodent FcRn.

[0396] In exemplary embodiment 150, provided herein is a rodent according to any one of embodiments 141-149, wherein the rodent is heterozygous for the engineered FcRn locus.

[0397] In exemplary embodiment 151, provided herein is a rodent according to any one of embodiments 141-149, wherein the rodent is homozygous for the engineered FcRn locus.

[0398] In exemplary embodiment 152, provided herein is a rodent described in any one of embodiments 141 to 151, further comprising in its genome an engineered β-2-microglobulin (β2M) locus comprising a nucleic acid sequence encoding a human or humanized β-2-microglobulin (β2M) polypeptide.

[0399] In exemplary embodiment 153, provided herein is a rodent according to embodiment 152, wherein a nucleic acid sequence encoding a human or humanized β2M polypeptide is positioned at the endogenous rodent β2M locus.

[0400] In exemplary embodiment 154, provided herein is a rodent described in embodiment 153, in which a nucleic acid sequence encoding a human or humanized β2M polypeptide replaces all or part of the endogenous rodent β2M gene.

[0401] In exemplary embodiment 155, provided herein is a rodent according to any one of embodiments 152-154, wherein the nucleic acid sequence comprises exons 2-4 of the human β2M gene.

[0402] In exemplary embodiment 156, provided herein is a rodent according to any one of embodiments 152-155, wherein the rodent does not express a rodent β2M polypeptide.

[0403] In exemplary embodiment 157, provided herein is a rodent of any one of embodiments 152-156, wherein the rodent is heterozygous for the engineered β2M locus.

[0404] In exemplary embodiment 158, provided herein is a rodent according to any one of embodiments 152-156, wherein the rodent is homozygous for the engineered β2M locus.

[0405] In exemplary embodiment 159, provided herein is a rodent described in any one of embodiments 1 to 158, further comprising in its genome an engineered Fc epsilon receptor 1 alpha (FcεR1α) locus comprising a nucleic acid sequence encoding an Fc epsilon receptor 1 alpha (FcεR1α) polypeptide comprising a human extracellular domain.

[0406] In exemplary embodiment 160, provided herein is the rodent of embodiment 159, wherein the FcεR1α polypeptide further comprises a rodent transmembrane domain.

[0407] In exemplary embodiment 161, provided herein is the rodent of embodiment 159, wherein the FcεR1α polypeptide further comprises a human transmembrane domain.

[0408] In exemplary embodiment 162, provided herein is a rodent according to any one of embodiments 159-161, wherein the FcεR1α polypeptide further comprises a rodent cytoplasmic domain.

[0409] In exemplary embodiment 163, provided herein is a rodent according to any one of embodiments 159-161, wherein the FcεR1α polypeptide further comprises a human cytoplasmic domain.

[0410] In exemplary embodiment 164, provided herein is a rodent described in any one of embodiments 159 to 163, wherein a nucleic acid sequence encoding an FcεR1α polypeptide is located in the endogenous rodent FcεR1α locus.

[0411] In exemplary embodiment 165, provided herein is a rodent according to embodiment 164, wherein the nucleic acid sequence encoding the FcεR1α polypeptide replaces all or part of the endogenous rodent FcεR1α gene.

[0412] In exemplary embodiment 166, provided herein is the rodent of embodiment 165, wherein the FcεR1α polypeptide comprises a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain.

[0413] In exemplary embodiment 167, provided herein is a rodent described in embodiment 159, in which a nucleic acid sequence encoding a human FcεR1α extracellular domain replaces an endogenous nucleic acid sequence encoding a rodent FcεR1α extracellular domain.

[0414] In exemplary embodiment 168, provided herein is the rodent of any one of embodiments 159-167, wherein the rodent does not express rodent FcεR1α.

[0415] In exemplary embodiment 169, provided herein is a rodent according to any one of embodiments 159 to 168, wherein the rodent is heterozygous for the engineered FcεR1α locus.

[0416] In exemplary embodiment 170, provided herein is a rodent according to any one of embodiments 159-168, wherein the rodent is homozygous for the engineered FcεR1α locus.

[0417] In exemplary embodiment 171, provided herein is a rodent described in any one of embodiments 1 to 170, further comprising in its genome an engineered Fc gamma receptor 1a (FcγR1a) locus comprising a nucleic acid sequence encoding an FcγR1a polypeptide comprising a human extracellular domain.

[0418] In exemplary embodiment 172, provided herein is the rodent of embodiment 171, wherein the FcγR1a polypeptide further comprises a rodent transmembrane domain.

[0419] In exemplary embodiment 173, provided herein is the rodent of embodiment 171, wherein the FcγR1a polypeptide further comprises a human transmembrane domain.

[0420] In exemplary embodiment 174, provided herein is a rodent according to any one of embodiments 171 to 173, wherein the FcγR1a polypeptide further comprises a rodent cytoplasmic domain.

[0421] In exemplary embodiment 175, provided herein is a rodent according to any one of embodiments 171-173, wherein the FcγR1a polypeptide further comprises a human cytoplasmic domain.

[0422] In exemplary embodiment 176, provided herein is a rodent described in any one of embodiments 171 to 175, wherein a nucleic acid sequence encoding an FcγR1a polypeptide is located in the endogenous rodent FcγR1a locus.

[0423] In exemplary embodiment 177, provided herein is a rodent according to embodiment 176, wherein the nucleic acid sequence encoding the FcγR1a polypeptide replaces all or part of the endogenous rodent FcγR1a gene.

[0424] In exemplary embodiment 178, provided herein is a rodent described in embodiment 171, in which a nucleic acid sequence encoding a human FcγR1a extracellular domain replaces an endogenous nucleic acid sequence encoding a rodent FcγR1a extracellular domain.

[0425] In exemplary embodiment 179, provided herein is the rodent of any one of embodiments 171-178, wherein the rodent does not express rodent FcγR1a.

[0426] In exemplary embodiment 180, provided herein is a rodent according to any one of embodiments 171 to 179, wherein the rodent is heterozygous for an engineered FcγR1a locus.

[0427] In exemplary embodiment 181, provided herein is a rodent according to any one of embodiments 171 to 179, wherein the ro...

Claims

1. A genetically modified mouse comprising in its genome a humanized fetal Fc receptor (FcRn) locus located at an endogenous mouse fetal Fc receptor (FcRn) locus, said humanized FcRn locus comprising a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain and a rodent cytoplasmic domain, said humanized FcRn locus comprising exons 1-2 of the mouse FcRn gene, exons 3-5 of the human FcRn gene, and exons 6-7 of the mouse FcRn gene.

2. The mouse described in claim 1, wherein the nucleic acid sequence encoding the FcRn polypeptide replaces all or part of the endogenous mouse FcRn gene.

3. The mouse of claim 1, wherein exons 3-5 of the human FcRn gene replace the endogenous nucleic acid sequence encoding the mouse FcRn extracellular domain.

4. The mouse of any one of claims 1 to 3, wherein the mouse does not express mouse FcRn.

5. The mouse of any one of claims 1 to 4, wherein the mouse is homozygous for the humanized FcRn locus.

6. The mouse of any one of claims 1 to 5, wherein the mouse further comprises in its genome a β-2-microglobulin (β2M) locus comprising a nucleic acid sequence encoding a human or humanized β-2-microglobulin (β2M) polypeptide.

7. The mouse of claim 6 , wherein the nucleic acid sequence encoding a human or humanized β2M polypeptide is located at the endogenous mouse β2M locus.

8. The mouse of claim 7 , wherein the nucleic acid sequence encoding a human or humanized β2M polypeptide replaces all or part of the endogenous mouse β2M gene.

9. The mouse of any one of claims 6 to 8, wherein the nucleic acid sequence encoding a human or humanized β2M polypeptide comprises exons 2 to 4 of the human β2M gene.

10. The mouse of any one of claims 6 to 9, wherein the mouse does not express mouse β2M polypeptide.

11. The mouse of any one of claims 6 to 10, wherein the mouse is homozygous for the β2M locus.

12. 12. A method of producing the mouse of any one of claims 1-11, comprising modifying the genome of a mouse such that the genome of the mouse comprises the humanized fetal Fc receptor (FcRn) locus comprising the nucleic acid sequence encoding the FcRn polypeptide.

13. A mouse embryonic stem (ES) cell comprising in its genome a humanized fetal Fc receptor (FcRn) locus located at an endogenous mouse fetal Fc receptor (FcRn) locus, said humanized FcRn locus comprising a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain and a rodent cytoplasmic domain, said humanized FcRn locus comprising exons 1-2 of the mouse FcRn gene, exons 3-5 of the human FcRn gene, and exons 6-7 of the mouse FcRn gene.

14. The mouse ES cell of claim 13 , wherein the nucleic acid sequence encoding the FcRn polypeptide replaces all or part of an endogenous mouse FcRn gene.

15. The mouse ES cell of claim 13, wherein exons 3 to 5 of the human FcRn gene replace the endogenous nucleic acid sequence encoding the mouse FcRn extracellular domain.

16. The mouse ES cell according to any one of claims 13 to 15, wherein the genome of the mouse ES cell is homozygous for the humanized FcRn locus.

17. The mouse ES cell of any one of claims 13 to 16, wherein the genome of the mouse ES cell further comprises a β-2-microglobulin (β2M) locus comprising a nucleic acid sequence encoding a human or humanized β-2-microglobulin (β2M) polypeptide.

18. The mouse ES cell of claim 17, wherein the nucleic acid sequence encoding a human or humanized β2M polypeptide is located at the endogenous mouse β2M locus.

19. The mouse ES cell of claim 18, wherein the nucleic acid sequence encoding a human or humanized β2M polypeptide replaces all or part of an endogenous mouse β2M gene.

20. The mouse ES cell of any one of claims 17 to 19, wherein the nucleic acid sequence encoding a human or humanized β2M polypeptide comprises exons 2 to 4 of the human β2M gene.

21. The mouse ES cell according to any one of claims 17 to 20, wherein the genome of the mouse ES cell is homozygous for the β2M locus.

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