Humanized rodents for testing therapeutic agents

Abstract

Provided herein are methods and compositions related to in vivo testing of therapeutic agents comprising a human Fc in genetically modified rodents (e.g., testing the pharmacokinetic and / or pharmacodynamic properties 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 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). In certain embodiments, the genetically modified rodent comprises one or more Fc receptors with human extracellular domains (e.g., neonatal Fc receptor (FcRn), β-2-microglobulin polypeptide (β2M), Fcε receptor 1α (FcεR1α), Fcγ receptor 1α (FcγR1a), Fcγ receptor 2a (FcγR2a), Fcγ receptor 2b (FcγR2b), Fcγ receptor 3a (FcγR3a), Fcγ receptor 3b (FcγR3b), Fcγ receptor 2c (FcγR2c)). The transmembrane and cytoplasmic domains of the receptor can be human or non-human (e.g., rodent).

Description

[0001] Related applications

[0002] This application is a divisional application of the Chinese invention patent application with application number 201980031749.3, application date March 25, 2019, and invention name “Humanized rodents for testing therapeutic agents”. The original application claims the priority benefit of U.S. provisional patent application serial number 62 / 648,197 filed on March 26, 2018 and U.S. provisional patent application serial number 62 / 689,628 filed on June 25, 2018, each of which is hereby incorporated by reference in its entirety. Background Art

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

[0004] Due to the small size and fully characterized physiology of rodents, they have long been used as animal models for preclinical testing of therapeutic agents. In addition, rodents can be highly genetically modified using well-established technology, and therefore, many disease models that are unavailable in large mammals are available in rodents. However, the performance of complex therapeutic agents such as antibodies and Fc fusion proteins in rodents is often different from that in humans. For example, compared to when the therapeutic agent is administered to humans, they often exhibit extremely different pharmacokinetic properties when administered to rodents, thereby limiting the usefulness of rodent models as predictors of safety, efficacy and optimal administration of complex therapeutic agents in humans. This in turn reduces the usefulness of the animal model in preclinical testing.

[0005] Therefore, there is a great need for new animal models and methods that allow for accurate preclinical testing of complex therapeutics in rodents, producing results that are more predictive of the properties of the therapeutics in human patients. Summary of the Invention

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

[0007] In certain embodiments, provided herein are genetically modified rodents wherein administration of human antibodies and / or human Fc fusion proteins induces a reduced anti-human Fc immune response (e.g., mouse anti-human antibodies or MAHA responses in mice). In some embodiments, the genetically modified rodents (e.g., mice or rats) express antibodies comprising human Fc (e.g., human IgG1 Fc, 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).

[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 the Fc receptors of a human patient will interact with the Fc fusion protein or antibody. Thus, in certain embodiments, the genetically modified rodent comprises one or more Fc receptors with human extracellular domains (e.g., neonatal Fc receptor (FcRn), beta-2-microglobulin polypeptide (β2M), Fcε receptor 1α (FcεR1α), Fcγ receptor 1α (FcγR1a), Fcγ receptor 2a (FcγR2a), Fcγ receptor 2b (FcγR2b), Fcγ receptor 3a (FcγR3a), Fcγ receptor 3b (FcγR3b), Fcγ receptor 2c (FcγR2c)). The transmembrane and cytoplasmic domains of the receptors can be human or non-human (e.g., 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) an immunoglobulin heavy chain variable region comprising one or more V H Gene segment, one or more D H gene segments and one or more J H Gene segments (e.g. rat or mouse V H Gene segment, D H Gene segments and J H gene segments); and (ii) an immunoglobulin heavy chain constant region comprising one or more C sequences encoding an IgG constant domain. H gene segment, the IgG constant domain comprises a human C H 1 domain, human hinge region, human C H 2 domains, human C H 3 domains, human or rodent IgG transmembrane domains and human or rodent IgG cytoplasmic domains. In some embodiments, all C HIn some embodiments, the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region so that the rodent produces a protein comprising a heavy chain variable region derived from V H Gene segment, D H Gene segments and J H The variable domains of gene segments and C H In some embodiments, the locus is located at an endogenous rodent immunoglobulin heavy chain locus. In some embodiments, the locus is located at an endogenous rodent immunoglobulin heavy chain locus. H The human antibodies and Fc fusion proteins of the Fc isotype matching of the encoding induce a reduced immune response when applied to the rodent. For example, in some embodiments, human IgG1 antibodies are applied to rodents expressing antibodies with human variable domains and human IgG1 constant domains as provided herein. In some embodiments, human IgG2 antibodies are applied to rodents expressing antibodies with human variable domains and human IgG2 constant domains as provided herein. In some embodiments, human IgG3 antibodies are applied to rodents expressing antibodies with human variable domains and human IgG3 constant domains as provided herein. In some embodiments, human IgG4 antibodies are applied to rodents expressing antibodies with human variable domains and human IgG4 constant domains as provided herein. In some embodiments, human antibodies with κ light chains are applied to rodents expressing antibodies with human κ constant domains as provided herein, or rodents expressing antibodies with human κ variable domains and constant domains. In some embodiments, human antibodies with lambda light chains are administered to rodents expressing antibodies with human lambda light chains, e.g., antibodies with human lambda constant domains, or antibodies with human lambda variable domains and constant domains, as provided herein. 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 are rodents (e.g., rats or mice) comprising a genetically modified immunoglobulin kappa (Igκ) chain locus in their genome. In some embodiments, the Igκ locus comprises: (1) an immunoglobulin kappa chain variable region comprising one or more human V κ Gene segments and one or more human J κ gene segment; and (2) an immunoglobulin kappa chain constant region comprising a human C κ In some embodiments, the immunoglobulin kappa chain variable region is operably linked to the immunoglobulin kappa chain constant region, such that the rodent produces a protein comprising a V segment derived from a human V κGene segments and human J κ The light chain variable domain of the gene segment and the C κ In some embodiments, the locus is located at the endogenous rodent immunoglobulin kappa chain locus. In some embodiments, human antibodies comprising human kappa chains elicit a reduced immune response when administered to the rodent. In some embodiments, provided herein are animal models and methods for testing human antibodies using the rodents.

[0011] In certain aspects, provided herein are rodents (e.g., rats or mice) comprising a genetically modified immunoglobulin lambda (Igλ) chain locus in their genome. In certain embodiments, the Igλ locus comprises one or more human V λ Gene segment, one or more human J λ gene segment and one or more C λ In some embodiments, the human J λ Gene segments and C λ Gene segments are arranged into one or more J λ -C λ In some embodiments, the human J λ Gene segments and C λ Gene segments are arranged to make human J λ Gene segments are co-located on one or more C λ In some embodiments, the upstream of the human J λ Gene segments and C λ Gene segments are arranged to make human J λ Gene segments and C λ Some of the gene segments are arranged into one or more J λ -C λ cluster, while other J λ Gene segments are co-located on one or more C λ In some embodiments, the human V λ Gene segments and human J λ The gene segment is operably linked to human C λ gene segments, so that rodents produce V λ Gene segments and human J λ The light chain variable domain of the gene segment and the C λIn some embodiments, the locus is located at the endogenous rodent immunoglobulin lambda chain locus. In some embodiments, human antibodies comprising human lambda chains elicit a reduced immune response when administered to the rodent. In some embodiments, provided herein are animal models and methods for testing human antibodies using the rodents.

[0012] In certain aspects, provided herein are rodents (e.g., rats or mice) comprising a genetically modified neonatal Fc receptor (FcRn) locus in their genome. In a certain embodiment, 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, the locus is located at an endogenous rodent FcRn locus. In some embodiments, rodents also comprise a β-2-microglobulin (β2M) locus in their genome, the β-2-microglobulin locus comprising a nucleic acid sequence encoding a human β-2-microglobulin (β2M) polypeptide. In some embodiments, the nucleic acid sequence encoding a human β2M polypeptide is located at an endogenous rodent β2M locus. In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using the rodents.

[0013] In certain aspects, provided herein are rodents (e.g., rats or mice) comprising a genetically modified Fcε receptor 1α (FcεR1α) locus in their genome, the genetically modified Fcε receptor 1α locus comprising a nucleic acid sequence encoding an FcεR1α polypeptide, the 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 at an endogenous rodent FcεR1α locus. In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using the rodents.

[0014] In certain aspects, provided herein are rodents (e.g., rats or mice) comprising a genetically modified Fcγ receptor 1a (FcγR1a) α chain locus in their genome, the genetically modified Fcγ receptor 1a α chain locus comprising a nucleic acid sequence encoding an FcγR1a α chain polypeptide, the 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, provided herein are genetically modified rodents further comprising a functional FcRγ chain. In certain embodiments, the functional FcRγ chain is a rodent FcRγ chain (e.g., a genetically modified rodent endogenous FcRγ chain). In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using the rodents.

[0015] In certain aspects, provided herein are rodents (e.g., rats or mice) comprising a genetically modified Fcγ receptor 2a α chain (FcγR2a) locus in their genome, the genetically modified Fcγ receptor 2a α chain locus comprising a nucleic acid sequence encoding a human FcγR2a α chain polypeptide. In some embodiments, the nucleic acid sequence encoding the FcγR2a α chain polypeptide is located at an endogenous rodent low-affinity FcγR α chain locus. In certain embodiments, provided herein are animal models and methods for using the rodents to test human antibodies and Fc fusion proteins.

[0016] In certain aspects, provided herein are rodents (e.g., rats or mice) comprising a genetically modified Fcγ receptor 2b (FcγR2b) α chain locus in their genome, wherein the genetically modified Fcγ receptor 2b α chain locus comprises a nucleic acid sequence encoding a human FcγR2b α chain polypeptide. In some embodiments, the nucleic acid sequence encoding the FcγR2b α chain polypeptide is located at an endogenous rodent low-affinity FcγR α chain locus. In certain embodiments, provided herein are animal models and methods for using the rodents to test human antibodies and Fc fusion proteins.

[0017] In certain aspects, provided herein are rodents (e.g., rats or mice) comprising a genetically modified Fcγ receptor 3a (FcγR3a) α chain locus in their genome, the genetically modified Fcγ receptor 3a α chain locus comprising a nucleic acid sequence encoding a human FcγR3a α chain polypeptide. In some embodiments, the nucleic acid sequence encoding the FcγR3a α chain polypeptide is located at an endogenous rodent low-affinity FcγR α chain locus. In some embodiments, provided herein are genetically modified rodents further comprising a functional FcRγ chain. In certain embodiments, the functional FcRγ chain is a rodent FcRγ chain (e.g., a genetically modified rodent endogenous FcRγ chain). In certain embodiments, provided herein are animal models and methods for testing human antibodies and Fc fusion proteins using the rodents.

[0018] In certain aspects, provided herein are rodents (e.g., rats or mice) comprising a genetically modified Fcγ receptor 3b (FcγR3b) α chain locus in their genome, wherein the genetically modified Fcγ receptor 3b α chain locus comprises a nucleic acid sequence encoding a human FcγR3b α chain polypeptide. In some embodiments, the nucleic acid sequence encoding the FcγR3b α chain polypeptide is located at an endogenous rodent low-affinity FcγR α chain locus. In certain embodiments, provided herein are animal models and methods for using the rodents to test human antibodies and Fc fusion proteins.

[0019] In certain aspects, provided herein are rodents (e.g., rats or mice) comprising a genetically modified Fcγ receptor 2c (FcγR2c) α chain locus in their genome, the genetically modified Fcγ receptor 2c α chain locus comprising a nucleic acid sequence encoding a human FcγR2c α chain polypeptide. In some embodiments, the nucleic acid sequence encoding the FcγR2c α chain polypeptide is located at an endogenous rodent low-affinity FcγR α chain locus. In certain embodiments, provided herein are animal models and methods for using the rodents to test human antibodies and Fc fusion proteins.

[0020] In certain aspects, provided herein are rodents (such as rats or mice) comprising a combination of genetically modified loci provided herein in their genomes. For example, in some embodiments, provided herein are rodents comprising one or more selected from the following genetically modified loci: provided herein are genetically modified IgH loci, provided herein are genetically modified Igκ loci, provided herein are genetically modified Igλ loci, provided herein are genetically modified FcRn loci, provided herein are genetically modified β2M loci, provided herein are genetically modified FcεR1α loci, provided herein are genetically modified FcγR1a loci, provided herein are genetically modified FcγR2a loci, provided herein are genetically modified FcγR2b loci, provided herein are genetically modified FcγR2c loci, provided herein are genetically modified FcγR3a loci and / or provided herein are genetically modified FcγR3b loci. In certain embodiments, provided herein are rodents comprising genetically modified IgH loci and / or genetically modified Igκ loci provided herein. In some embodiments, provided herein are rodents comprising genetically modified IgH loci and / or genetically modified Igλ loci provided herein. In some embodiments, provided herein are rodents comprising genetically modified IgH loci, genetically modified Igκ and / or Igλ loci provided herein, genetically modified FcRn loci provided herein, and genetically modified β2M loci provided herein. In some embodiments, provided herein are rodents comprising genetically modified IgH loci, genetically modified FcRn loci, genetically modified β2M loci provided herein, genetically modified FcεR1α loci provided herein, genetically modified FcγR1a loci provided herein, genetically modified FcγR2a loci provided herein, genetically modified FcγR2b loci provided herein, genetically modified FcγR2c loci provided herein, genetically modified FcγR3a loci provided herein, and / or genetically modified FcγR3b loci provided herein.

[0021] In certain embodiments, provided herein is a method for testing a therapeutic protein (e.g., a human antibody or Fc fusion protein) comprising a human Fc domain, the method 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, one or more pharmacokinetic parameters include, but are not limited to, area under plasma concentration versus time (AUC), in vivo recovery (IVR), clearance (CL), mean residence time (MRT), half-life (t1 / 2), and volume of distribution (Vss) at steady state. In some embodiments, the method further comprises measuring the therapeutic efficacy of the administered therapeutic protein (e.g., the ability of the administered dose of therapeutic protein to alleviate 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 degree to which the administered dose of therapeutic protein produces one or more adverse effects in an animal model). 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)). In some embodiments, the method further comprises measuring the extent to which administration of the therapeutic protein induces an anti-human Fc immune response in the rodent. In some embodiments, the method further comprises evaluating the safety and / or efficacy of the dosing regimen of the therapeutic protein.

[0022] In certain embodiments, provided herein is an animal model for testing therapeutic proteins (such as human antibodies or Fc fusion proteins) comprising human Fc domains. In some embodiments, the animal model includes administering therapeutic proteins to rodents (such as mice or rats) provided herein. In some embodiments, the animal model also includes measuring one or more pharmacokinetic properties of the administered therapeutic protein. In some embodiments, one or more pharmacokinetic parameters include but are not limited to area under plasma concentration versus time (AUC), in vivo recovery (IVR), clearance (CL), mean residence time (MRT), drug half-life (t1 / 2) and volume of distribution (Vss) at steady state. In some embodiments, the animal model also includes measuring the therapeutic efficacy of the administered therapeutic protein (such as the ability of the administered dose of therapeutic protein to alleviate or eliminate one or more disease symptoms in an animal model). In some embodiments, the animal model also includes measuring the safety of the administered therapeutic protein (such as the degree to which the administered dose of therapeutic protein produces one or more adverse effects in an animal model). In certain embodiments, the animal model 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)). In some embodiments, the animal model further comprises measuring the extent to which administration of the therapeutic protein induces an anti-human Fc immune response in the rodent. In some embodiments, the animal model further comprises evaluating the safety and / or efficacy of the dosing regimen of the therapeutic protein.

[0023] In some embodiments, when administered to a rodent provided herein, the administered therapeutic agent elicits a reduced immune response. In some embodiments, the administered human antibody or Fc fusion protein has a human C-terminal domain that is identical to a human C-terminal domain in a genetically modified IgH locus of a rodent provided herein. H In some embodiments, the isotype and / or allotype of the Fc domain encoded by the rodent is matched to the isotype and / or allotype of the human C HThe human antibodies and Fc fusion proteins of the Fc isotype matching of the encoding induce a reduced immune response when applied to the rodent. For example, in some embodiments, human IgG1 antibodies are applied to rodents expressing antibodies with human variable domains and human IgG1 constant domains as provided herein. In some embodiments, human IgG2 antibodies are applied to rodents expressing antibodies with human variable domains and human IgG2 constant domains as provided herein. In some embodiments, human IgG3 antibodies are applied to rodents expressing antibodies with human variable domains and human IgG3 constant domains as provided herein. In some embodiments, human IgG4 antibodies are applied to rodents expressing antibodies with human variable domains and human IgG4 constant domains as provided herein. In some embodiments, human antibodies with κ light chains are applied to rodents expressing antibodies with human κ constant domains as provided herein, or rodents expressing antibodies with human κ variable domains and constant domains. In some embodiments, a human antibody having a lambda light chain is administered to a rodent that expresses an antibody having a human lambda light chain, e.g., an antibody having a human lambda constant domain, or an antibody having a human lambda variable domain and a constant domain. In some embodiments, the agent is a human IgG1 antibody, and the rodent comprises a genetically modified IgH locus comprising C1, hinge, CH2, and CH3 domains encoding human IgG1. H In some embodiments, the agent is a human IgG4 antibody and the rodent comprises a genetically modified IgH locus comprising C sequences encoding human IgG4 CH1, hinge, CH2, and CH3 domains. H In some embodiments, the therapeutic agent is a human antibody having an Igκ light chain, and the rodent comprises a genetically modified Igκ locus as 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 as 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 (Langerhans cell), eosinophils, mast cells, and basophils) comprising one or more of the genetically modified loci provided herein. For example, in some embodiments, provided herein are rodent cells (e.g., rodent ES cells) comprising genetically modified IgH loci and / or genetically modified Igκ loci provided herein. In some embodiments, provided herein are rodent cells (e.g., rodent ES cells) comprising genetically modified IgH loci and / or genetically modified Igλ loci provided herein. In some embodiments, provided herein are rodent cells (e.g., rodent ES cells) comprising genetically modified IgH loci, genetically modified Igκ and / or Igλ loci provided herein, genetically modified FcRn loci provided herein, and genetically modified β2M loci 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 for preparing 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) so that it comprises one or more of the genetically modified loci provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A A schematic overview not to scale of exemplary modified immunoglobulin heavy chain loci according to certain exemplary embodiments provided herein is shown. As shown, exemplary mouse heavy chain locus 1 comprises human variable regions (comprising human V H 、People D H Kazuto J H Gene segments, for simplicity, not shown for human V Hgene segments; only a few are schematically represented—for a 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, p. 458 (2001), which are incorporated herein by reference), mouse intronic enhancer, mouse C μ Gene segment, mouse C δ Gene segment, mouse C γ3 Gene segment, mouse C γ1 Gene segment, mouse C γ2b Gene segment encoding a hybrid C of the extracellular domain of human IgG1 and the transmembrane and cytoplasmic domains of mouse IgG2a H Gene segment, mouse C ε Gene segment, mouse C α As shown, the exemplary mouse heavy chain locus 2 comprises the human variable regions (comprising human V H 、People D H Kazuto J H Gene segments, for simplicity, not shown for human V H gene segments; only a few are schematically represented—for a 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, p. 458 (2001), which are incorporated herein by reference), mouse intronic enhancer, mouse C μ Gene segment, mouse C δ Gene segment, mouse C γ3 Gene segment, mouse C γ1 Gene segment, mouse C γ2b Gene segment, human C γ1 Gene segments (both the IgG1 extracellular coding sequence and the transmembrane / cytoplasmic coding sequence are human), mouse C ε Gene segment, mouse C α As shown, the exemplary mouse heavy chain locus 3 comprises the human variable regions (comprising human VH 、People D H Kazuto J H Gene segments, for simplicity, not shown for human V H gene segments; only a few are shown schematically—for a 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, p. 458 (2001), which are incorporated herein by reference), mouse intronic enhancer, mouse C μ Gene segment, mouse C δ Gene segment, mouse C γ3 Gene segment encoding a hybrid C of the extracellular domain of human IgG4 and the transmembrane and cytoplasmic domains of mouse IgG1 H Gene segment, mouse C γ2b Gene segment, mouse C γ2a Gene segment, mouse C ε Gene segment, mouse C α As shown, the exemplary mouse heavy chain locus 4 comprises the human variable regions (comprising human V H 、People D H Kazuto J H Gene segments, for simplicity, not shown for human V H gene segments; only a few are schematically represented—for a 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, p. 458 (2001), which are incorporated herein by reference), mouse intronic enhancer, mouse C μ Gene segment, mouse C δ Gene segment, mouse C γ3 Gene segment, human C γ4 Gene segments (IgG4 extracellular coding sequence and transmembrane / cytoplasmic domain coding sequence are both human), mouse C γ2b Gene segment, mouse C γ2a Gene segment, mouse C εGene segment, mouse C α As shown, the exemplary mouse heavy chain locus 5 comprises the human variable regions (comprising human V H 、People D H Kazuto J H Gene segments, for simplicity, not shown for human V H gene segments; only a few are shown schematically—for a 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, p. 458 (2001), which are incorporated herein by reference), human intronic enhancers, human C μ Gene segment, human C δ Gene segment, human C γ3 Gene segment, human C γ1 As shown, the exemplary mouse heavy chain locus 6 comprises the human variable regions (comprising human V H 、People D H Kazuto J H Gene segments, for simplicity, not shown for human V H gene segments; only a few are shown schematically—for a 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, p. 458 (2001), which are incorporated herein by reference), human intronic enhancers, human C μ Gene segment, human C δ Gene segment, human C γ3 Gene segment, human C γ1 Gene segment, human C γ2 Gene segment, human C γ4 As shown, the exemplary mouse heavy chain locus 7 comprises the mouse variable regions (comprising mouse V H , mouse D H and mouse J HGene segments, for simplicity, not shown for mouse V H Gene segments; only a few are shown schematically – for a complete repertoire of possible V, D, and J gene segments, see imgt.org), mouse intronic enhancers, human C μ Gene segment, human C δ Gene segment, human C γ3 Gene segment, human C γ1 As shown, the exemplary mouse heavy chain locus 8 comprises the mouse variable regions (comprising mouse V H , mouse D H and mouse J H Gene segments, for simplicity, not shown for mouse V H Gene segments; only a few are shown schematically – for a complete repertoire of possible V, D, and J gene segments, see imgt.org), mouse intronic enhancers, human C μ Gene segment, human C δ Gene segment, human C γ3 Gene segment, human C γ1 Gene segment, human C γ2 Gene segment, human C γ4 Gene segments and mouse 3 'regulatory regions. Although not shown, the loci in these embodiments comprise functional mouse Adam6 genes (e.g., Adam6a and / or Adam6b). Unless otherwise indicated (e.g., for lox sites, etc.), hollow shapes and double lines represent human sequences, while solid shapes and single lines represent mouse sequences.

[0027] Figure 1B Show the method for creating Figure 1A Schematic overview not to scale of an exemplary method for modifying an exemplary modified immunoglobulin heavy chain locus 5 depicted in FIG. For simplicity, human V H Gene segments and the mouse Adam6 gene. Unless otherwise indicated (e.g., for lox sites, etc.), hollow shapes and double lines represent human sequences, while solid shapes and single lines represent mouse sequences.

[0028] Figure 1C Show the method for creating Figure 1A Schematic overview not to scale of an exemplary method for modifying an exemplary modified immunoglobulin heavy chain locus 6 depicted in FIG. For simplicity, human V H Gene segments and the mouse Adam6 gene. Unless otherwise indicated (e.g., for lox sites, etc.), hollow shapes and double lines represent human sequences, while solid shapes and single lines represent mouse sequences.

[0029] Figure 1D Show the method for creating Figure 1A Schematic overview not to scale of an exemplary method for modifying an exemplary immunoglobulin heavy chain locus 7 depicted in FIG. For simplicity, mouse V is not shown in each schematic diagram. H Unless otherwise indicated (e.g., for lox sites, etc.), open shapes and double lines represent human sequences, while solid shapes and single lines represent mouse sequences.

[0030] Figure 1E Show the method for creating Figure 1A Schematic overview not to scale of an exemplary method for modifying an exemplary modified immunoglobulin heavy chain locus 8 depicted in FIG. For simplicity, mouse V is not shown in each schematic diagram. H Unless otherwise indicated (e.g., for lox sites, etc.), open shapes and double lines represent human sequences, while solid shapes and single lines represent mouse sequences.

[0031] Figure 2A Shown are schematic overviews not to scale of exemplary modified immunoglobulin light chain loci according to certain exemplary embodiments provided herein. The upper exemplary schematic depicts a locus comprising a human variable region, a mouse intronic enhancer, a human C κ The constant region gene segments and the mouse 3' enhancer of the Igκ locus. λ Gene segment, human J λ -C λ Series pair, person J λ7 and mouse C λ1 Gene segments, human enhancers 1, 2, and 3, and the Igλ locus of the mouse 3' enhancer. Unless otherwise indicated (e.g., for loxp sites, etc.), open shapes and double lines represent human sequences, while solid shapes and single lines represent mouse sequences.

[0032] Figure 2B Show the method for creating Figure 2A A schematic overview not to scale of an exemplary method for modifying an exemplary immunoglobulin kappa chain locus depicted in . Unless otherwise indicated (e.g., for loxp sites, etc.), open shapes and double lines represent human sequences, while solid shapes and single lines represent mouse sequences.

[0033] Figure 3A Shown in hVs-hIgM-hIgD-hIgG3-hIgG1 mice (locus 5), Levels of mouse IgG, mouse IgM, human IgM, human IgG1, and human IgG3 in mouse serum and normal human serum.

[0034] Figure 3B FACS plots showing hIgM versus mIgM and hIgD versus mIgD for hVs-hIgM-hIgD-hIgG3-hIgG1 mice (locus 5) and Allele utilization and the existence of allelic exclusion in mice.

[0035] Figure 3C Shown are the levels of mouse IgG, mouse IgM, human IgM, human IgG1, and human IgG4 present in the sera of the indicated mice.

[0036] Figure 3D Shown are the concentrations of mouse anti-human antibodies in the indicated mice at day 0 and day 34 after injection of human IgG4 antibody.

[0037] Figure 3E Shown are the concentrations of human IgG4 antibodies in the indicated mice during the first 34 days after human IgG4 antibody injection.

[0038] Figure 4 Shown is a not-to-scale schematic overview of an exemplary method for creating an exemplary modified FcRn locus as described in Example 3. Unless otherwise indicated (e.g., for loxp sites, etc.), open shapes and double lines represent human sequence, while solid shapes and single lines represent mouse sequence.

[0039] Figure 5 Shown is a schematic overview, not to scale, of an exemplary method for creating mice comprising exemplary modified FcRn and β2M loci as described in Example 3. Unless otherwise indicated (e.g., for loxp sites, etc.), open shapes and double lines represent human sequences, while solid shapes and single lines represent mouse sequences.

[0040] Figure 6 Shown are antibody clearance of human antibodies by mice comprising exemplary modified FcRn and β2M loci compared to wild-type mice (depicted as antibody concentration).

[0041] Figure 7 Shown is a schematic overview, not to scale, of an exemplary method for creating mice comprising exemplary modified FcγR1α (shown in the figure as FCGR1), FcγRIIIa, FcγRIIa, FcγRIIc, FcγRIIb, FcγRIIIb, and heavy chain loci as described in Example 4. Open shapes and double lines represent human sequences, while solid shapes and single lines represent mouse sequences.

[0042] Figure 8A Shown are FACS plots depicting the utilization of mouse IgD relative to mouse IgM, and human IgD relative to human IgM in mice carrying human high and low affinity FcγRs and human heavy chain constant regions.

[0043] Figure 8B Shown are the numbers or percentages of T cells (mCD3 positive) and B cells (mCD19 positive) present in the spleens of mice bearing human high-affinity FcγRs and human low-affinity FcγRs and human heavy chain constant regions compared to wild-type mice.

[0044] Figure 8C Shown are serum concentrations of human IgM, human IgG1, and human IgG3 in mice bearing human high-affinity FcγRs and human low-affinity FcγRs and human heavy chain constant regions compared to normal human serum.

[0045] Figure 9 Shown is a schematic overview, not to scale, of an exemplary method for creating mice comprising an exemplary modified FcεR1α locus as described in Example 5. Unless otherwise indicated (e.g., for loxp sites, etc.), open shapes and double lines represent human sequence, while solid shapes and single lines represent mouse sequence.

[0046] Figure 10 On the left panel is shown a FACS graph demonstrating expression of humanized FcεR1α on the surface of basophils in exemplary mice comprising the humanized FcεR1α locus disclosed herein. On the right panel, the graph shows the percentage of mouse or human FcεR1α+ basophils present in the spleen of mice comprising the humanized FcεR1α locus disclosed herein compared to wild-type mice.

[0047] Figure 11 The exemplary humanized FcεR1α loci provided herein were shown to be functional. Mice were sensitized by intradermal injection of the ear with human allergen-specific IgE or IgG as a negative control. One day later, the mice were challenged IV with the allergen diluted in Evans blue dye. Evans blue dye extravasation in the ear was measured as a readout of mast cell degranulation. Because human IgE does not bind to mouse FcεR1, responses in mice containing the humanized FcεR1α locus indicate the production of functional FcεR1α.

[0048] Figure 12The exemplary humanized FcεR1α loci provided herein are shown to be functional. Mice were sensitized with IV injections of human allergen-specific IgE. One day later, mice were challenged with IV injections of allergens, and temperature changes were monitored over a 4-hour period following injection. A decrease in temperature was a readout for anaphylaxis.

[0049] Figure 13 A not-to-scale schematic overview of an exemplary method for creating a mouse comprising an exemplary humanized FcRn, β2M, FcεR1α, FcγR1α (shown in the figure as FCGR1), FcγRIIIa, FcγRIIa, FcγRIIc, FcγRIIb, FcγRIIIb, a heavy chain constant region locus, and a heavy chain variable region locus as described in Example 6 is shown. For simplicity, only a few gene segments are schematically represented in loci F and F' - for a 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, p. 458 (2001), which are incorporated herein by reference. Open shapes and double lines represent human sequences, while solid shapes and single lines represent mouse sequences. DETAILED DESCRIPTION

[0050] summary

[0051] Provided herein are methods and compositions related to in vivo testing of therapeutic agents comprising human Fc in genetically modified rodents (e.g., testing the pharmacokinetic and / or pharmacodynamic properties and dosing regimens of the therapeutic agents in genetically modified rodents). In some embodiments, the genetically modified rodents express antibodies comprising human Fc (e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc) or human light chain constant regions. In some embodiments, the rodents express fully human antibodies (i.e., antibodies having human heavy chains and human light (γ or κ) chains). In certain embodiments, the genetically modified rodent comprises one or more Fc receptors having human extracellular domains (e.g., neonatal Fc receptor (FcRn), beta-2-microglobulin polypeptide (β2M), Fcε receptor 1α (FcεR1α), Fcγ receptor 1α (FcγR1a), Fcγ receptor 2a (FcγR2a), Fcγ receptor 2b (FcγR2b), Fcγ receptor 3a (FcγR3a), Fcγ receptor 3b (FcγR3b), Fcγ receptor 2c (FcγR2c)). The transmembrane and cytoplasmic domains of the receptors can be human or non-human (e.g., rodent).

[0052] Human Fc-containing therapeutic agents, such as therapeutic human antibodies and human Fc fusion proteins, are typically tested in non-human species before being administered to humans. Although such agents are often tested in non-human primates or other relatively large mammals, such testing is expensive and places a significant financial burden on drug developers. Furthermore, non-human primates and other relatively large mammals are often not amenable to genetic modification, which limits the disease models available in these organisms.

[0053] 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 ability to undergo genetic modification. Unfortunately, agents comprising human Fc regions often exhibit very different pharmacokinetic and pharmacodynamic properties when administered to prior art rodents compared to when administered to humans. For example, when therapeutic agents with human Fc regions are administered to conventional rodents, the human sequences in the Fc region are often identified as foreign by the rodent (e.g., rat or mouse) immune system. As a result, rodents can mount an immune response (known as a mouse anti-human response or MAHA) against the administered therapeutic agent, which affects the pharmacokinetic and pharmacodynamic properties of the administered agent. In addition, compared to the interaction of the human Fc region of the therapeutic agent with human Fc receptors in patients, they may interact with rodent (e.g., rat or mouse) Fc receptors in a different manner in rodents, which can also affect the pharmacokinetic and pharmacodynamic properties of the administered agent. Therefore, conventional rodent (e.g., rat or mouse) models are often poor predictors of human therapeutic response to therapeutic agents containing human Fc. Therefore, mice with human immunoglobulin locus regions (e.g., see the loci in Figures 1 and 2) can help reduce or eliminate the MAHA response.

[0054] Having molecules that can specifically bind drugs is extremely useful for research and diagnostic purposes. When the drug is a human monoclonal antibody, it is possible to create a vector with mouse variable sequences and human constant sequences (e.g. Figure 1A Mice expressing the mouse locus 8 in the human gene provide an improved method for generating anti-drug antibodies. Any MAHA response generated by injecting the mice with human antibodies will be directed against the variable region of the antibody. This eliminates background responses against the constant region of the antibody and makes the generation of drug-specific antibodies more efficient.

[0055] Provided herein are in vivo systems for developing, screening, and testing human antibodies and Fc fusion proteins for therapeutic use. In certain embodiments, provided herein are genetically modified rodents with reduced rodent anti-human immune responses after administration of therapeutic agents comprising human Fc. As demonstrated herein, this can be achieved by using rodents that have been genetically modified so that they express human Fc that matches the Fc present in the administered antibody or Fc fusion protein. The mice can be, for example, engineered by incorporating fully or partially (e.g., IgG C) nucleic acid sequences encoding the constant regions of human immunoglobulins heavy chains. H 1-HC H 2-C H3) Inserted into the position of a sequence encoding a corresponding portion of an endogenous non-human immunoglobulin heavy chain constant region gene segment. Such animals recognize human Fc as a "self" protein and are therefore less likely to mount an immune response to administered human Fc-containing therapeutics.

[0056] In addition, in some embodiments, provided herein are genetically modified rodents (eg, mice or rats) expressing Fc receptors that can interact with human Fc in a manner similar to Fc receptors expressed by human patients. For example, in certain embodiments, provided herein are genetically modified rodents expressing one or more Fc receptors with at least a human extracellular domain (eg, transmembrane and cytoplasmic domains can be human or rodent). Thus, in certain embodiments, provided herein are mice expressing human or partial human FcRn, human or partial human β2M, human or partial human FcεR1α, human or partial human FcγR1a, human or partial human FcγR2a, human or partial human FcγR2b, human or partial human FcγR3a, human or partial human FcγR3b, and / or human or partial human FcγR2c. Thus, compared to rodents with completely non-human Fc receptors, the mice are able to more accurately mimic the human Fc response of human patients.

[0057] Therefore, provided herein is a rodent (e.g., mouse or rat) that provides a novel in vivo system for developing, selecting and testing therapeutic human antibodies and Fc fusion proteins by evaluating the effector function within the internal environment of the immune system, wherein the development, selection and testing are based not only on the specificity and / or affinity of the selected antibody to the antigen, but also on the relevant overall biological function of the selected antibody. In this way, it is possible to develop and select human therapeutic candidates based on the prediction of the therapeutic potential evaluated with relevant biological responses (e.g., cellular responses) at the entire molecular level rather than based only on the prediction of the individual components evaluated separately. Therefore, provided herein is a rodent that provides a system that is more suitable for predicting clinical human therapeutic antibody function in vivo.

[0058] definition

[0059] The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0060] The term "amino acid" is intended to include all molecules, whether natural or synthetic, that include both amino and acid functionalities and that can be included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and congeners thereof; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing.

[0061] As used herein, the term "antibody" may refer to both intact antibodies and their antigen-binding fragments. A complete 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 chain variable domain and the light chain variable domain can be further subdivided into hypervariable domains referred to as complementary determining regions (CDRs), which are interspersed with more conserved regions referred to as framework regions (FRs). Each heavy chain variable domain and light chain variable domain consists of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable domains of the heavy and light chains contain binding domains that interact with the antigen.

[0062] As used herein, the terms "antigen-binding fragment" and "antigen-binding portion" of an antibody refer to one or more fragments of an antibody that retain the ability to bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab, Fab', F(ab')2, 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.

[0063] 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 specified period of time, such as 12, 18, 24, 36, 48, or 72 hours, or for infinity using extrapolation based on the slope of the curve. Unless otherwise specified herein, AUC is determined for infinity (AUC 无穷 AUC can also be calculated on a per-dose basis. As with many other PK parameters, AUC can be determined in a single animal or in a group of animals over which its mean is calculated.

[0064] As used herein, the term "clearance" or "CL" refers to a measure of the body's ability to eliminate a drug and is expressed as the volume of plasma that clears the drug over time.

[0065] The phrase "derived from" when used in relation to a rearranged variable region gene that is "derived from" an unrearranged variable region and / or an unrearranged variable region gene segment refers to the ability to trace the sequence of the rearranged variable region gene back to a set of unrearranged variable region gene segments that are rearranged to form genes expressing variable domains (taking into account splicing differences and somatic mutations, where applicable). For example, a rearranged variable region gene that has been mutated by a recipient cell is still derived from an unrearranged variable region gene segment. In some embodiments, when the endogenous locus is replaced by a universal light chain or heavy chain locus, the term "derived from" indicates that the source of the sequence can be traced back to the rearranged locus, even though the sequence may have been mutated by the recipient cell.

[0066] 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-bred organism (whether wild-type or engineered).

[0067] The term "in vivo recovery" or "IVR" refers to the incremental recovery (K value), which is the peak activity observed minus the pre-dose level, then divided by the dose. The IVR can also be calculated on a percentage basis. The average IVR can be determined in a population of animals, or a single IVR can be determined in a single animal.

[0068] As used herein, the term "locus" refers to a position on a chromosome containing a group of related genetic elements (e.g., genes, gene segments, regulatory elements). For example, an unrearranged immunoglobulin locus may include immunoglobulin variable region gene segments, one or more immunoglobulin constant region genes, and related regulatory elements (e.g., promoters, enhancers, switch elements, etc.) for directing V (D) J recombination and immunoglobulin expression. The locus may be endogenous or non-endogenous. The term "endogenous locus" refers to the position of a naturally occurring specific genetic element on a chromosome. In some embodiments, the endogenous locus has a sequence found in nature. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the endogenous locus is an engineered locus. For example, the endogenous mouse immunoglobulin heavy chain locus refers to the position on mouse chromosome 12 in wild-type mice that includes the immunoglobulin heavy chain variable region gene segments and constant region genes, the endogenous mouse immunoglobulin λ light chain locus refers to the position on mouse chromosome 16 in wild-type mice that includes the immunoglobulin λ light chain variable region gene segments and constant region genes, and the endogenous mouse immunoglobulin κ light chain locus refers to the position on mouse chromosome 6 in wild-type mice that includes the immunoglobulin κ light chain variable region gene segments and constant region genes.

[0069] 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 in conjunction with the constant region gene as a polypeptide chain of an antigen binding protein.

[0070] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to a polymeric form of deoxyribonucleotides or ribonucleotides of any length or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (loci / locus) determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA with any sequence, isolated RNA with any sequence, nucleic acid probes and primers. Polynucleotides can include modified nucleotides such as methylated nucleotides and nucleotide analogs. If present, the modification of the nucleotide structure can be imparted before or after the assembly of the polymer. Polynucleotides can be further modified, such as by being conjugated to a labeling component. In all nucleic acid sequences provided herein, U nucleotides can be interchanged with T nucleotides.

[0071] The term "unrearranged" includes the following state of an immunoglobulin variable region locus or variable region gene segment: in which the V gene segments and J gene segments (and for heavy variable regions, the D gene segments) are maintained separately but are capable of joining to form a single V, (D), J rearranged V(D)J gene ("variable region gene") comprising the V(D)J lineage.

[0072] As used herein, the term "volume of distribution at steady state" or "Vss" refers to the apparent space (volume) into which a drug distributes. More specifically, Vss represents the amount of drug in an animal's body divided by the plasma concentration at steady state.

[0073] As used herein, the term "C H gene segments” (e.g., Cγ1 gene segments, C γ2a Gene 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 Gene segment, etc.) refers to the DNA sequence segment encoding the constant region of the immunoglobulin heavy chain and can be combined with C 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, Cγ1 gene segment or Cγ1 gene refers to the DNA sequence segment encoding the IgG1 constant region. H A gene segment locus is a naturally occurring locus on a chromosome. H Gene segment or C H The location of the gene.

[0074] Genetically modified loci

[0075] In certain aspects, provided herein are genetically modified rodents (e.g., mice or rats) that can be used to test therapeutic agents comprising human Fc in vivo in genetically modified rodents (e.g., testing the pharmacokinetic and / or pharmacodynamic properties of such therapeutic agents in genetically modified rodents). In some embodiments, the genetically modified rodents comprise 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 rodents comprise a genetically modified locus encoding a full or partial 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 having a human extracellular domain (e.g., neonatal Fc receptor (FcRn) alpha chain, beta-2-microglobulin polypeptide (β2M), Fcε receptor 1 alpha (FcεR1α) alpha chain, Fcγ receptor 1 alpha (FcγR1a) alpha chain, Fcγ receptor 2a (FcγR2a) alpha chain, Fcγ receptor 2b (FcγR2b) alpha chain, Fcγ receptor 3a (FcγR3a) alpha chain, Fcγ receptor 3b (FcγR3b) alpha chain, Fcγ receptor 2c (FcγR2c) alpha chain). In certain embodiments, the transmembrane and cytoplasmic domains encoded by the loci can be human or non-human (e.g., rodent).

[0076] Humanized immunoglobulin heavy chain locus

[0077] In certain aspects, provided herein are rodents (e.g., mice or rats) comprising a genetically modified immunoglobulin (Ig) heavy chain locus. The locus typically comprises 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 The constant region locus includes one or more Ig heavy chain constant region gene segments (C H In certain embodiments, the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region such that the rodent produces an immunoglobulin heavy chain variable region comprising a heavy chain constant region derived from V H Gene segment, D H Gene segments and J H The variable domains of gene segments and C H heavy chain constant domain gene segments of antibodies.

[0078] In some embodiments, the variable region will be an unrearranged variable region and will contain an unrearranged Ig variable region gene segment. In some embodiments, the variable region will be a rearranged variable region and will therefore contain a rearranged variable region gene. In certain embodiments, the Ig variable region gene segment is a human variable region gene segment. In certain embodiments, the Ig variable region gene segment is a rodent (e.g., rat or mouse) variable region gene segment (e.g., rat or mouse variable region gene segment). Therefore, in certain embodiments, the Ig heavy chain variable region locus will contain a human Ig variable region gene segment. 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 hereby incorporated by reference, and in U.S. Patent Publication Nos. 2008 / 0098490, 2012 / 0167237, 2013 / 0145484, 2013 / 0326647, 2014 / 013275, and 2014 / 093908, each of which is hereby incorporated by reference.

[0079] 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 persons D H Segment and one or more persons J H In some embodiments, the unrearranged human Ig variable region gene segment comprises at least 3 V H Gene segment, at least 18 V H Gene segment, at least 20 V H Gene segment, at least 30 V H Gene segment, at least 40 V H Gene segment, at least 50 V H Gene segment, at least 60 V H Gene segment, at least 70 V H Gene segment or at least 80 V H In some embodiments, the engineered IgH locus (or allele) comprises a human V segment found in a human IgH locus occurring in nature. H 3-74 gene segment and human V H All or substantially all functional human V HIn certain 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, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2 and V H 6-1. In some embodiments, the non-human animals provided herein are characterized by having a single polymorphic human V H Gene segments, multiple D H Gene segments and multiple J H In some embodiments, V HThe 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 (a universal heavy chain variable region or a common heavy chain coding sequence, e.g., as described in U.S. Patent Publication No. 20140245468 and U.S. Patent Nos. 9,204,624 and 9,930,871, each of which is hereby incorporated by reference in its entirety). In some embodiments, the non-human animals provided herein comprise a human unrearranged immunoglobulin light chain, e.g., a kappa gene segment, operably linked to a heavy chain constant region gene at an immunoglobulin heavy chain locus (e.g., U.S. Patent No. 9,516,868, which is incorporated by reference in its entirety).

[0080] In other embodiments, the non-human organism may comprise a heavy chain immunoglobulin locus in its germline and / or genome, the heavy chain immunoglobulin locus including insertions and / or replacements of histidine codons designed to introduce pH-dependent binding properties into antibodies produced in the non-human organism. In some of the embodiments, a histidine codon is inserted and / or replaced in the nucleic acid sequence encoding CDR3. Various such heavy immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, U.S. Patent Application Publication Nos. 2013 / 0247236, 20140013456, which are incorporated herein by reference.

[0081] 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 certain embodiments, the engineered IgH locus (or allele) comprises a human D gene segment found in a human IgH locus occurring in nature. H 1-1 gene segment and human D H All or substantially all functional human D between the 7-27 gene segments (including both ends) H In certain embodiments, the engineered IgH locus (or allele) comprises at least 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 H5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26 and D H 7-27. In some embodiments, the unrearranged human Ig gene segments include all human D H Gene segment.

[0082] In some embodiments, the engineered IgH locus (or allele) comprises 1, 2, 3, 4, 5, 6 or more functional human J H In certain embodiments, the engineered IgH locus (or allele) comprises a human J gene segment found in a human IgH locus occurring in nature. H 1 gene segment and human J H All or substantially all functional human J between the 6 gene segments (including both ends) H In certain embodiments, the engineered IgH locus (or allele) comprises at least a human J H Gene segment J H 1. J H 2. J H 3. J H 4. J H 5 and J H 6. In some embodiments, the unrearranged human Ig gene segments include all human J H Gene segment.

[0083] In some embodiments, the engineered IgH locus as described herein does not contain an endogenous Adam6 gene. In some embodiments, the engineered IgH locus as described herein does not contain an endogenous Adam6 gene (or Adam6 coding sequence) in the same germline genomic position as found in the germline genome of a wild-type non-human animal of the same species. In some embodiments, the engineered IgH locus as described herein does not contain a human Adam6 pseudogene. In some embodiments, the engineered IgH locus as described herein comprises at least one insertion of a nucleotide sequence encoding one or more non-human (e.g., rodent) Adam6 polypeptides. The insertion may be outside the engineered immunoglobulin heavy chain locus as described herein (e.g., at the most 5'V Hgene segment), within the engineered IgH locus, or elsewhere in the germline genome of a non-human animal, cell, or tissue (e.g., random introduction of a non-human Adam6 coding sequence).

[0084] In some embodiments, the engineered endogenous immunoglobulin heavy chain locus lacks a functional endogenous rodent Adam6 gene. In some embodiments, the rodent's germline genome comprising an engineered heavy chain locus includes one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments. In some embodiments, one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs, or functional fragments are expressed (e.g., in cells of the male reproductive system, such as testicular cells).

[0085] In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are included on the same chromosome as the engineered endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are included in the engineered endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are included in the engineered endogenous immunoglobulin heavy chain locus. H Gene segment and second human V H In some embodiments, the first person V H The gene segment is V H 1-2, and the second person V H The gene segment is V H 6-1. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments replace the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments replace the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments replace the human Adam6 pseudogene. H Gene segment and human D H between gene segments.

[0086] Exemplary Ig variable regions comprising 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 hereby incorporated by reference. Such mice are described, for example, in U.S. Patent Nos. 8,642,835 and 8,697,940, which are incorporated by reference herein.

[0087] In some embodiments, the Ig heavy chain variable gene locus comprising an unrearranged human Ig heavy chain variable region gene segment also includes a human Ig heavy chain variable region intergenic sequence. In some embodiments, the Ig heavy chain variable gene locus includes a non-human (e.g., rodent, rat, mouse) Ig heavy chain variable region intergenic sequence. In some embodiments, the IgH locus includes 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 includes an IgM enhancer (Eμ). In some embodiments, the IgM enhancer is a non-human Eμ (e.g., rodent Eμ, such as mouse or rat Eμ).

[0088] 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 human rearranged 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 hereby incorporated by reference.

[0089] In certain embodiments, the immunoglobulin constant region comprises a C encoding an IgG constant domain. H gene segment, the IgG constant domain comprises a human C H 1 domain, human hinge region, human C H 2 domains, human C H 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 region). In certain embodiments, the IgG connecting region is a human IgG connecting region.

[0090] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domains and human C H The 3 domain is an IgG1 domain. In some embodiments, the IgG1 domain is encoded by an allele selected from IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04, and IGHG1*05.

[0091] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domains and human C H The IgG2 domain is an IgG2 domain. In some embodiments, the IgG2 domain is encoded by an allele selected from the group consisting of IGHG2*01, IGHG2*02, IGHG2*03, IGHG2*04, IGHG2*05, and IGHG2*06.

[0092] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domains and human C H In some embodiments, the IgG3 domain is an IgG3 domain. In some embodiments, the IgG3 domain is encoded by an allele selected from the group consisting of 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.

[0093] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domains and human C H The IgG4 domain is an IgG4 domain. In some embodiments, the IgG4 domain is encoded by an allele selected from the group consisting of IGHG4*01, IGHG4*02, IGHG4*03, and IGHG4*04.

[0094] In some embodiments, C HThe gene segment encodes a variant human immunoglobulin heavy chain constant region sequence (i.e., a human immunoglobulin heavy chain constant region sequence comprising one or more additions, deletions and / or substitutions relative to an appropriate reference human immunoglobulin heavy chain constant region sequence), wherein the variant human immunoglobulin heavy chain constant region sequence is characterized in that the effector function and / or affinity for FcR is enhanced or diminished relative to the reference human immunoglobulin heavy chain constant region.

[0095] In some embodiments, C H The gene segment encodes a human immunoglobulin heavy chain constant region characterized by altered affinity for activating and / or inhibitory receptors. H The gene segment encodes a human immunoglobulin heavy chain constant region characterized by enhanced or diminished binding to the FcRn receptor at acidic pH compared to neutral pH. H The gene segment fully or partially encodes a human immunoglobulin heavy chain constant region, encoding a human immunoglobulin heavy chain constant region with one or more amino acid modifications. Exemplary amino acid modifications include, but are not limited to, substitutions at position 297 (e.g., N297A), substitutions at position 250 (e.g., 250E or 250Q), substitutions at position 252 (e.g., 252L, 252Y, 252F, 252W, or 252T), substitutions at position 254 (e.g., 254S or 254T), substitutions at position 256 (e.g., 256S, 256R, 256Q, 256E, 256D, or 256T), substitutions at position 257 (e.g., 257A, 257B, 257C, 257D, or 257E), substitutions at position 258 (e.g., 258E, 258D, or 258E), substitutions at position 259 (e.g., 261S, 261R, 261Q, 261E, 261D, or 261T), substitutions at position 262 (e.g., 261S, 261R, 261Q, 261E, 261D, or 261T), substitutions at position 263 (e.g., 261S, 261R, 261Q, 261E, 261D, or 261T), substitutions at position 264 (e.g., 261S, 261R, 261Q, 261E, 261D, or 261T), substitutions at position 26 307 (e.g., 307P or 307A), a substitution at position 308 (e.g., 308F or 308V), a substitution at position 428 (e.g., 428L or 428F), a substitution at position 433 (e.g., 433H, 433Lm, 433R, 433S, 433P, 433Q, or 433K), a substitution at position 434 (e.g., 434A, 434W, 434H, 434F, or 434Y), and combinations thereof. In some embodiments, C HThe gene segment encodes a human immunoglobulin heavy chain constant region having one or more pairs or one or more 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).

[0096] In some embodiments, C H The gene segments encode chimeric immunoglobulin heavy chain constant domains that include segments or portions derived from (or present in) more than one human immunoglobulin isotype. For example, such chimeric C H The region may comprise a C region derived from a human IgG1, human IgG2 or human IgG4 molecule. H 2 domains and C from human IgG1, human IgG2 or human IgG4 molecules H In certain 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 derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues from positions 216 to 227 according to EU numbering) and a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues from positions 228 to 236 according to EU numbering). In certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2.

[0097] In certain embodiments, the modified C H The gene segment is located in the endogenous C H In certain embodiments, the modified C H The gene segment is located in the 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 Gene segment locus.

[0098] The endogenous immunoglobulin heavy chain constant region gene structure can vary between rodents. For example, the Norwegian rat does not have C at the endogenous immunoglobulin heavy chain locus. γ3 gene segments, however, mouse strains typically have. Even for the same species, the immunoglobulin heavy chain constant region gene structure can vary between strains. For example, although some mouse strains have C at the endogenous immunoglobulin heavy chain locus, γ2a gene segment, but other mouse strains (e.g., those with the Igh1-b allele) instead have the C γ2c gene segment, and some mouse strains may have C γ2a Gene segment and C γ2c Therefore, the rodent C disclosed in the Figures, Examples and / or description herein γ1 、C γ2a 、C γ2b and C γ3 The gene segment is an exemplary rodent C H gene segments, and those skilled in the art will appreciate that specific constant region gene structures will vary between rodent strains. Thus, for example, those skilled in the art will appreciate that the present disclosure encompasses proteins comprising proteins that replace or are in addition to any disclosed rodent C γ2a Rodent C gene segment γ2c gene segments of rodents.

[0099] In some embodiments, the modified C H The gene segment is human C γ1 gene segment (or at least the human C γ1 The C gene segment encoding the IgG1 constant domain H 1 domain, hinge region, C H 2 domains and C H 3 domain), and it is located in the endogenous C γ2a In some embodiments, human C γ1 The C gene segment encoding the IgG1 constant domain H 1 domain, hinge region, C H 2 domains and C H The 3 domain portion is operably linked to endogenous rodent C γ2a The portion of the gene segment encoding the IgG2a transmembrane and / or cytoplasmic domains.

[0100] In some embodiments, the modified C H The gene segment is human C γ1 gene segment (or at least the human C γ1The C gene segment encoding the IgG1 constant domain H 1 domain, hinge region, C H 2 domains and C H 3 domain), and it is located in the endogenous C γ2c In some embodiments, human C γ1 The C gene segment encoding the IgG1 constant domain H 1 domain, hinge region, C H 2 domains and C H The 3 domain portion is operably linked to endogenous rodent C γ2c The portion of the gene segment encoding the IgG2c transmembrane and / or cytoplasmic domains.

[0101] In some embodiments, the modified C H The gene segment is human C γ4 gene segment (or at least the human C γ4 The C gene segment encoding the IgG4 constant domain H 1 domain, hinge region, C H 2 domains and C H 3 domain), and it is located in the endogenous C γ1 In some embodiments, human C γ4 The C gene segment encoding the IgG4 constant domain H 1 domain, hinge region, C H 2 domains and C H The 3 domain portion is operably linked to endogenous rodent C γ1 The portion of the gene segment encoding the IgG1 transmembrane and / or cytoplasmic domains.

[0102] In certain embodiments, the modified C H Gene segment replaces endogenous C H In certain embodiments, the modified C H Gene segment replaces endogenous C γ1 Gene segment, endogenous C γ2a Gene segment, endogenous C γ2b Gene segment, endogenous C γ2c gene segment or endogenous C γ3 All or part of a gene segment.

[0103] In some embodiments, the modified C H The gene segment is human C γ1 gene segment (or at least the human C γ1 The C gene segment encoding the IgG1 constant domainH 1 domain, hinge region, C H 2 domains and C H 3 domain), and it replaces the endogenous C γ2a In some embodiments, human C γ1 The gene segment encoding the IgG1 constant domain C H 1 domain, hinge region, C H 2 domains and C H Partial replacement of endogenous rodent C γ2a The C gene segment encoding the IgG2a constant domain H 1 domain, hinge region, C H 2 domains and C H 3 domains, so that human C γ1 The C gene segment encoding the IgG1 constant domain H 1 domain, hinge region, C H 2 domains and C H The 3 domain portion is operably linked to endogenous rodent C γ2a The portion of the gene segment encoding the IgG2a transmembrane and / or cytoplasmic domains.

[0104] In some embodiments, the modified C H The gene segment is human C γ4 gene segment (or at least the human C γ4 The C gene segment encoding the IgG4 constant domain H 1 domain, hinge region, C H 2 domains and C H 3 domain), and it replaces the endogenous C γ1 In some embodiments, human C γ4 The C gene segment encoding the IgG4 constant domain H 1 domain, hinge region, C H 2 domains and C H Partial replacement of endogenous rodent C γ1 The C gene segment encoding the IgG1 constant domain H 1 domain, hinge region, C H 2 domains and C H 3 domains, so that human C γ4 The C gene segment encoding the IgG4 constant domain H 1 domain, hinge region, C H 2 domains and C H The 3 domain portion is operably linked to endogenous rodent Cγ1 The portion of the gene segment encoding the IgG1 transmembrane and / or cytoplasmic domains.

[0105] In certain embodiments, the Ig heavy chain constant region comprises one or more rodent (eg, rat or mouse) C H In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C μ In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C δ In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C γ1 In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C γ2a In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C γ2b In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C γ2c In some embodiments, the Ig constant region comprises a rodent (eg, mouse) C γ3 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 ε In some embodiments, the one or more rodent constant region gene segments are endogenous constant region gene segments. In some embodiments, the modified C H The gene segment is the only modified C in the Ig heavy chain constant region H Gene segment.

[0106] In some embodiments, the modified C H The gene segment is a multiple modified C in the constant region of the Ig heavy chain H In some embodiments, the Ig heavy chain constant region comprises a human or partially human C μ In some embodiments, the Ig heavy chain constant region comprises a human or partially human C δ In some embodiments, the Ig heavy chain constant region comprises a human or partially human C γ1 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C γ2 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C γ3In some embodiments, the Ig heavy chain constant region comprises a human or partially human C γ4 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C ε In some embodiments, the Ig heavy chain constant region comprises a human or partially human C α In 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 further 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 Gene segment.

[0107] 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 (eg, rat or mouse) or human 3' regulatory region (3'RR).

[0108] In some embodiments, the modified immunoglobulin heavy chain locus is located at an endogenous immunoglobulin heavy chain locus. In some embodiments, the immunoglobulin heavy chain locus replaces all or a portion of the endogenous immunoglobulin heavy chain locus. In certain 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 deleting, repositioning, and / or inverting all or a portion of the endogenous Ig heavy chain locus).

[0109] 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). In some embodiments, one or more C H The gene segments are altered, disrupted, deleted or replaced, in particular with immunoglobulin heavy chain constant region sequences as described herein (e.g. encoding human IgG C H 1-HC H 2-C H3 polypeptide sequence) is operably linked to one or more transmembrane and cytoplasmic coding sequences (e.g., M1 and / or M2 coding sequences) of a non-human immunoglobulin heavy chain IgG constant region gene, and in some embodiments, to an immunoglobulin heavy chain constant region sequence (e.g., encoding human IgE C H 1-C H 2-C H 3-C H 4 polypeptide sequence) is operably linked to one or more transmembrane and cytoplasmic coding sequences of the IgE constant region gene. In some embodiments, all of the immunoglobulin heavy chain constant region is substantially replaced by a heterologous immunoglobulin heavy chain constant region. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operably linked to the 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 the transmembrane and cytoplasmic coding sequences (e.g., M1 and M2 exons) of the IgE constant region gene. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operably linked to the transmembrane and cytoplasmic coding sequences (e.g., one or more M exons) of the IgA constant region gene. In some embodiments, in the immunoglobulin heavy chain constant region comprising a heterologous immunoglobulin heavy chain constant region sequence operably linked to the transmembrane and cytoplasmic coding sequences of one or more constant region genes as described herein, one or more C H Gene segments (e.g. C μ 、C δ In 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 inserted into an IgG constant region gene (e.g., C γ1 、C γ2a 、C γ2b, 、C γ2c or C γ3 ) in one copy (i.e., allele) of a gene encoding a heterologous immunoglobulin heavy chain constant region gene, and the IgG constant region gene has two copies, thereby producing a non-human animal that is heterozygous for a heterologous immunoglobulin heavy chain constant region sequence. In some embodiments, a non-human animal that is homozygous for an immunoglobulin heavy chain constant region that includes a heterologous immunoglobulin heavy chain constant region sequence as described herein is provided.

[0110] In some embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises one or more IgG encoding CH Gene segments, each of which comprises a human extracellular domain encoding sequence (e.g., human IgG C1-C2) operably linked to a non-human transmembrane and cytoplasmic domain encoding sequence (e.g., non-human IgG M1-M2) of the same or different IgG subclass. H 1-HC H 2-C H 3).

[0111] In some embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises one or more engineered IgG encoding C H gene segments, and further comprising a wild-type (eg, unmodified non-human such as rat or mouse) C μ Constant region genes.

[0112] In some embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises one or more engineered IgG encoding C H gene segments, and further comprising a wild-type (eg, unmodified non-human such as rat or mouse) C μ and C δ Constant region genes.

[0113] In various embodiments, an engineered IgG encoding a C-terminal fragment comprising a human immunoglobulin heavy chain constant region sequence as described herein is H The gene segment is selected from C γ1 、C γ2a 、C γ2b 、C γ2c or C γ3 The engineered IgG encoding C H Gene segment.

[0114] In certain embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises an engineered C γ2a A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG1 C H 1-HC H 2-C H 3, and is operably linked to a non-human (eg, rat or mouse) C γ2a Sequences of the M1 and M2 exons of the gene segment.

[0115] In certain embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises an engineered C γ2a A gene segment comprising a C H1-HC H 2-C H The location of exons 3-M1-M2 encodes human IgG1C H 1-HC H 2-C H 3-M1-M2, and is operably linked to a non-human (eg, rat or mouse) C γ2a The sequence of the switch region of the gene segment.

[0116] In certain embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises an engineered C γ2c A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG1 C H 1-HC H 2-C H 3, and is operably linked to a non-human (eg, rat or mouse) C γ2c Sequences of the M1 and M2 exons of the gene segment.

[0117] In certain embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises an engineered C γ2c A gene segment comprising a C H 1-HC H 2-C H The location of exons 3-M1-M2 encodes human IgG1C H 1-HC H 2-C H 3-M1-M2, and is operably linked to a non-human (eg, rat or mouse) C γ2c The sequence of the switch region of the gene segment.

[0118] In certain embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises an engineered C γ1 A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG4 C H 1-HC H 2-C H 3, and is operably linked to a non-human (eg, rat or mouse) C γ1 Sequences of the M1 and M2 exons of the gene segment.

[0119] In certain embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises an engineered Cγ1 A gene segment comprising a C H 1-HC H 2-C H The location of exon 3-M1-M2 encodes human IgG4C H 1-HC H 2-C H 3-M1-M2, and is operably linked to a non-human (eg, rat or mouse) C γ1 The sequence of the switch region of the gene segment.

[0120] In certain embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises an engineered C γ2a A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG1 C H 1-HC H 2-C H 3, and is operably linked to a non-human (eg, rat or mouse) C γ2a The sequences of M1 and M2 exons of the gene segment; and the engineered C γ1 A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG4 C H 1-HC H 2-C H 3, and is operably linked to a non-human (eg, rat or mouse) C γ1 Sequences of the M1 and M2 exons of the gene segment.

[0121] In certain embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises an engineered C γ2a A gene segment comprising a C H 1-HC H 2-C H The location of exons 3-M1-M2 encodes human IgG1C H 1-HC H 2-C H 3-M1-M2, and is operably linked to a non-human (eg, rat or mouse) C γ2a The sequence of the switch region of the gene segment; and the engineered C γ1 A gene segment comprising a C H 1-HC H 2-C HThe position of exon 3-M1-M2 encodes human IgG4 C H 1-HC H 2-C H 3-M1-M2, and is operably linked to a non-human (eg, rat or mouse) C γ1 The sequence of the switch region of the gene segment.

[0122] In certain embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises an engineered C γ2c A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG1 C H 1-HC H 2-C H 3, and is operably linked to a non-human (eg, rat or mouse) C γ2c The sequences of M1 and M2 exons of the gene segment; and the engineered C γ1 A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG4 C H 1-HC H 2-C H 3, and is operably linked to a non-human (eg, rat or mouse) C γ1 Sequences of the M1 and M2 exons of the gene segment.

[0123] In certain embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises an engineered C γ2c A gene segment comprising a C H 1-HC H 2-C H The location of exons 3-M1-M2 encodes human IgG1C H 1-HC H 2-C H 3-M1-M2, and is operably linked to a non-human (eg, rat or mouse) C γ2c The sequence of the switch region of the gene segment; and the engineered C γ1 A gene segment comprising a C H 1-HC H 2-C H The position of exon 3-M1-M2 encodes human IgG4 C H 1-HC H 2-C H3-M1-M2, and is operably linked to a non-human (eg, rat or mouse) C γ1 The sequence of the switch region of the gene segment.

[0124] In various embodiments, the engineered immunoglobulin heavy chain constant region as described herein comprises one or more additional modifications, including modifications such that, in addition to one or more modifications comprising a constant region encoding human IgG C H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H The constant region genes (i.e., isotypes) other than the IgG constant region (e.g., IgG1 and / or IgG2a) of the sequence of 3-M1-M2 are non-functional, for example, by completely or partially deleting, completely or partially changing, completely or partially destroying, completely or partially replacing one or more of the genes encoding IgD, IgE, IgA, and the isotypes that do not themselves contain genes encoding human IgGC as described herein. H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H The invention also provides an engineered non-human embryo, a cell, and a targeting vector for preparing the non-human animal, embryo, and cell.

[0125] In certain embodiments, the engineered immunoglobulin heavy chain constant regions provided herein comprise wild-type (e.g., unmodified non-human such as rat or mouse) C μ Gene segment; C γ1 A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG4 C H 1-HC H 2-C H 3, and is operably linked to the C γ1 The sequence of the M1-M2 exons of the gene segment; and δ 、C γ2a、 C γ2c 、C γ2b 、C γ3、 C ε and C α Deletion of gene segments.

[0126] In certain embodiments, the engineered immunoglobulin heavy chain constant regions provided herein comprise wild-type (e.g., unmodified non-human such as rat or mouse) C μ Gene segment; C γ1 A gene segment comprising a C H 1-HC H 2-C H The position of exon 3-M1-M2 encodes human IgG4 C H 1-HC H 2-C H 3-M1-M2, and is operably connected to the C γ1 The sequence of the switch region of the gene segment; and C δ 、C γ2a、 C γ2c 、C γ2b 、C γ3 、C ε and C α Deletion of gene segments.

[0127] In certain embodiments, the engineered immunoglobulin heavy chain constant regions provided herein comprise wild-type (e.g., unmodified non-human such as rat or mouse) C μ Gene segment; C γ2a A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG1 C H 1-HC H 2-C H 3, and is operably linked to the C γ2a The sequence of the M1-M2 exons of the gene segment; and δ 、C γ1 、C γ2b 、C γ2c 、C γ3 、C ε and C α Deletion of gene segments.

[0128] In certain embodiments, the engineered immunoglobulin heavy chain constant regions provided herein comprise wild-type (e.g., unmodified non-human such as rat or mouse) C μ Gene segment; C γ2a A gene segment comprising a C H 1-HC H 2-C H The position of exon 3-M1-M2 encodes human IgG1 C H 1-HC H 2-CH 3-M1-M2, and is operably connected to the C γ2a The sequence of the switch region of the gene segment; and C δ 、C γ1 、C γ2b 、C γ2c 、C γ3 、C ε and C α Deletion of gene segments.

[0129] In certain embodiments, the engineered immunoglobulin heavy chain constant regions provided herein comprise wild-type (e.g., unmodified non-human such as rat or mouse) C μ Gene segment; C γ2c A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG1 C H 1-HC H 2-C H 3, and is operably linked to the C γ2c The sequence of the M1-M2 exons of the gene segment; and δ 、C γ1 、C γ2a 、C γ2b 、C γ3 、C ε and C α Deletion of gene segments.

[0130] In certain embodiments, the engineered immunoglobulin heavy chain constant regions provided herein comprise wild-type (e.g., unmodified non-human such as rat or mouse) C μ Gene segment; C γ2c A gene segment comprising a C H 1-HC H 2-C H The position of exon 3-M1-M2 encodes human IgG1 C H 1-HC H 2-C H 3-M1-M2, and is operably connected to the C γ2c The sequence of the switch region of the gene segment; and C δ 、C γ1 、C γ2a 、C γ2b 、C γ3 、C ε and C α Deletion of gene segments.

[0131] In various embodiments, the engineered immunoglobulin heavy chain constant region as described herein comprises one or more additional modifications, including modifications to one or more heavy chain constant regions encoding human IgG C H 1-HC H2 -C H 3 or human IgG C H 1-HC H 2-C H The constant region gene (i.e., isotype) other than the IgG constant region (e.g., IgG1 and / or IgG2a) of the sequence of 3-M1-M2 is engineered to be changed, modified, replaced, engineered, etc.: the human immunoglobulin heavy chain constant region sequence as described herein is inserted into IgD, IgE, IgA, and IgA sequences that do not themselves contain the constant region sequence encoding the human IgG C sequence as described herein. H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H In one or more immunoglobulin constant region genes of IgG (eg, IgG2b and / or IgG3) having the sequence of 3-M1-M2.

[0132] Also provided are engineered non-human embryos, cells, and targeting vectors for making non-human animals, embryos, and cells comprising immunoglobulin loci having engineered constant regions as described herein.

[0133] In certain embodiments, the engineered immunoglobulin heavy chain constant regions provided herein comprise wild-type C μ Gene segment; wild type C δ Gene segment; C γ3 A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG3C H 1-HC H 2-C H 3, and is operably linked to the C γ3 The sequence of the M1-M2 exon of the gene segment; C γ1 A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG4 C H 1-HC H 2-C H 3, and is operably linked to the C γ1The sequence of the M1-M2 exon of the gene segment; C γ2b A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG2 C H 1-HC H 2-C H 3, and is operably linked to the C γ2b The sequence of the M1-M2 exon of the gene segment; C γ2a A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG1 C H 1-HC H 2-C H 3, and is operably linked to the C γ2a The sequence of the M1-M2 exons of the gene segment (and / or C γ2c A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG1 C H 1-HC H 2-C H 3, and is operably linked to the C γ2c The sequence of the M1-M2 exon of the gene segment); C ε A gene segment comprising a C H 1-C H 2-C H 3-C H The location of exon 4 encoding human IgEC H 1-C H 2-C H 3-C H 4, and is operably linked to the C ε The sequence of the M1-M2 exons of the gene segment; and α A gene segment comprising a C H 1-HC H 2-C H The position of exon 3 encodes human IgA1 or IgA2 C H 1-HC H 2-C H 3, and is operably linked to the C α The sequence of one or more M exons of a gene segment.

[0134] In certain embodiments, the engineered immunoglobulin heavy chain constant regions provided herein comprise wild-type C μ Gene segment; wild type C δ Gene segment; C γ3 A gene segment comprising a C H 1-HC H 2-C H The position of exon 3-M1-M2 encodes human IgG3 C H 1-HC H 2-C H 3-M1-M2, and is operably connected to the C γ3 The sequence of the switch region of the gene segment; C γ1 A gene segment comprising a C H 1-HC H 2-C H The location of exon 3-M1-M2 encodes human IgG4C H 1-HC H 2-C H 3-M1-M2, and is operably connected to the C γ1 The sequence of the switch region of the gene segment; C γ2b A gene segment comprising a C H 1-HC H 2-C H The location of exons 3-M1-M2 encodes human IgG2C H 1-HC H 2-C H 3-M1-M2, and is operably connected to the C γ2b The sequence of the switch region of the gene segment; C γ2a A gene segment comprising a C H 1-HC H 2-C H The location of exons 3-M1-M2 encodes human IgG1C H 1-HC H 2-C H 3-M1-M2, and is operably connected to the C γ2a The sequence of the switch region of the gene segment (and / or C γ2c A gene segment comprising a C H 1-HC H 2-C H The location of exons 3-M1-M2 encodes human IgG1C H 1-HC H 2-C H3-M1-M2, and is operably connected to the C γ2c sequence of the switch region of the gene segment); C ε A gene segment comprising a C H 1-C H 2-C H 3-C H The location of exon 4 encoding human IgE C H 1-C H 2-C H 3-C H 4, and is operably linked to the C ε The sequence of the M1-M2 exons of the gene segment; and α A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgA1 or IgA2C H 1-HC H 2-C H 3, and is operably linked to the C α The sequence of one or more M exons of a gene segment.

[0135] In various embodiments, provided herein are genetically engineered heavy chain immunoglobulin loci depicted in Figure 1 and genetically modified rodents (e.g., rats or mice), ES and other cells and tissues comprising the loci. Thus, in one embodiment, provided herein is a genetically engineered heavy chain immunoglobulin locus comprising human heavy chain variable gene segments (human heavy chain V, D and J gene segments); rodent (e.g., rat or mouse) Ei enhancer; rodent (e.g., rat or mouse) C μ 、C δ 、C γ3 、C γ1 、C γ2b Gene segment; Chimeric C γ2a A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG1C H 1-HC H 2-C H 3, and is operably linked to a rodent (e.g., rat or mouse) C γ2aThe sequence of the M1-M2 exons of the gene segment (e.g., such that the exons encoding the extracellular domain of human IgG1 are operably linked to the exons encoding the transmembrane and cytoplasmic domains of rodent, e.g., rat or mouse IgG2a) (and / or chimeric C γ2c A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG1C H 1-HC H 2-C H 3, and is operably linked to a rodent (e.g., rat or mouse) C γ2c The sequence of the M1-M2 exons of the gene segment (e.g., such that the exons encoding the extracellular domain of human IgG1 are operably linked to the exons encoding the transmembrane and cytoplasmic domains of rodent, such as rat or mouse IgG2c); rodent (e.g., rat or mouse) C ε and C α Gene segment; 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 FIG1 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 the genetically engineered locus are also provided.

[0136] In one embodiment, a genetically engineered heavy chain immunoglobulin locus is provided herein, wherein the genetically engineered heavy chain immunoglobulin locus comprises human heavy chain variable gene segments (human heavy chain V, D and J gene segments); a rodent (e.g., rat or mouse) Ei enhancer; a rodent (e.g., rat or mouse) C μ 、C δ 、C γ3 、C γ1 、C γ2b Gene segment; human C γ1 A gene segment encoding human IgG1C H 1-HC H 2-C H 3-M1-M2 sequence, such that both the IgG1 extracellular coding sequence and the transmembrane / cytoplasmic coding sequence are human; rodent (e.g., rat or mouse) C ε and C αGene segment; 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 FIG1 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 the genetically engineered locus are also provided.

[0137] In one embodiment, a genetically engineered heavy chain immunoglobulin locus is provided herein, wherein the genetically engineered heavy chain immunoglobulin locus comprises human heavy chain variable gene segments (human heavy chain V, D and J gene segments); a rodent (e.g., rat or mouse) Ei enhancer; a rodent (e.g., rat or mouse) C μ 、C δ 、C γ3 Gene segment; Chimeric C γ1 A gene segment comprising a C H 1-HC H 2-C H The location of exon 3 encodes human IgG4 C H 1-HC H 2-C H 3, and is operably linked to a rodent (e.g., rat or mouse) C γ1 The sequence of the M1-M2 exons of the gene segment (e.g., such that the exons encoding the extracellular domain of human IgG4 are operably linked to the exons encoding the transmembrane and cytoplasmic domains of rodent, such as rat or mouse IgG1); rodent (e.g., rat or mouse) C γ2b 、C γ2a (and / or C γ2c ), C ε 、C α Gene segment; 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 FIG1 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 the genetically engineered locus are also provided.

[0138] In one embodiment, a genetically engineered heavy chain immunoglobulin locus is provided herein, wherein the genetically engineered heavy chain immunoglobulin locus comprises human heavy chain variable gene segments (human heavy chain V, D and J gene segments); a rodent (e.g., rat or mouse) Ei enhancer; a rodent (e.g., rat or mouse) C μ 、C δ 、C γ3 Gene segment; human C γ4 A gene segment encoding human IgG4 C H 1-HC H 2-C H 3-M1-M2 sequence, so that both the IgG4 extracellular coding sequence and the transmembrane / cytoplasmic coding sequence are human; rodent (e.g., rat or mouse) C γ2b 、C γ2a (and / or C γ2c ) gene segment; rodent (eg, rat or mouse) C ε and C α Gene segment; 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 FIG1 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 the genetically engineered locus are also provided.

[0139] In one embodiment, a genetically engineered heavy chain immunoglobulin locus is provided herein, wherein the genetically engineered heavy chain immunoglobulin locus comprises human heavy chain variable gene segments (human heavy chain V, D and J gene segments); human Ei enhancer; human C μ 、C δ 、C γ3 and C γ1 Gene segment; 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 FIG1 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 the genetically engineered locus are also provided.

[0140] In one embodiment, a genetically engineered heavy chain immunoglobulin locus is provided herein, wherein the genetically engineered heavy chain immunoglobulin locus comprises human heavy chain variable gene segments (human heavy chain V, D and J gene segments); human Ei enhancer; human C μ 、C δ 、C γ3 、C γ1 、C γ2 and C γ4 Gene segment; 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 FIG1 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 the genetically engineered locus are also provided.

[0141] In one embodiment, a genetically engineered heavy chain immunoglobulin locus is provided herein, comprising rodent (e.g., rat or mouse) heavy chain variable gene segments (rodent (e.g., rat or mouse) heavy chain V, D, and J gene segments); rodent (e.g., rat or mouse) Ei enhancer; human C μ 、C δ 、C γ3 and C γ1 gene segment; 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 FIG1 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 the genetically engineered locus are also provided.

[0142] In one embodiment, a genetically engineered heavy chain immunoglobulin locus is provided herein, comprising rodent (e.g., rat or mouse) heavy chain variable gene segments (rodent (e.g., rat or mouse) heavy chain V, D, and J gene segments); rodent (e.g., rat or mouse) Ei enhancer; human C μ 、C δ 、C γ3 、C γ1 、C γ2 and C γ4gene segment; 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 FIG1 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 the genetically engineered locus are also provided.

[0143] Also provided herein are methods for producing engineered immunoglobulin heavy chain loci. In some embodiments, the methods for producing the engineered immunoglobulin heavy chain loci described herein comprise introducing a human IgG1 C locus comprising a polypeptide encoding an immunoglobulin heavy chain constant domain. H 1-HC H 2-C H 3 nucleotide sequence of about 1.6kb DNA inserted into the C γ2a C gene segment H 1-HC H 2-C H The location of exon 3 in order to make the human IgG1C H 1-HC H 2-C H 3 nucleotide sequence is operably linked to the C γ2a M1 and M2 exons of the gene segment.

[0144] In some embodiments, a method for generating an engineered immunoglobulin heavy chain locus described herein comprises inserting a human IgG1 C locus comprising a polypeptide encoding an immunoglobulin heavy chain constant domain. H 1-HC H 2-C H The approximately 6.8 kb DNA of the 3-M1-M2 nucleotide sequence was inserted into the C γ2a C gene segment H 1-HC H 2-C H 3-M1-M2 exon position, so that the human IgG1 C H 1-HC H 2-C H The 3-M1-M2 nucleotide sequence is operably linked to the C γ2a conversion region of a gene segment.

[0145] Also provided herein are methods for producing engineered immunoglobulin heavy chain loci. In some embodiments, the methods for producing the engineered immunoglobulin heavy chain loci described herein comprise introducing a human IgG1 C locus comprising a polypeptide encoding an immunoglobulin heavy chain constant domain. H1-HC H 2-C H 3 nucleotide sequence of about 1.6kb DNA inserted into the C γ2c C gene segment H 1-HC H 2-C H The location of exon 3 in order to make the human IgG1C H 1-HC H 2-C H 3 nucleotide sequence is operably linked to the C γ2c M1 and M2 exons of the gene segment.

[0146] In some embodiments, a method for generating an engineered immunoglobulin heavy chain locus described herein comprises inserting a human IgG1 C locus comprising a polypeptide encoding an immunoglobulin heavy chain constant domain. H 1-HC H 2-C H The approximately 6.8 kb DNA of the 3-M1-M2 nucleotide sequence was inserted into the C γ2c C gene segment H 1-HC H 2-C H 3-M1-M2 exon position, so that the human IgG1 C H 1-HC H 2-C H The 3-M1-M2 nucleotide sequence is operably linked to the C γ2c conversion region of a gene segment.

[0147] In some embodiments, a method for generating an engineered immunoglobulin heavy chain locus described herein comprises inserting a human IgG4 C locus comprising a polypeptide encoding an immunoglobulin heavy chain constant domain. H 1-HC H 2-C H 3 nucleotide sequence of about 1.5kb DNA inserted into the C γ1 C gene segment H 1-HC H 2-C H The location of exon 3 in order to make the human IgG4C H 1-HC H 2-C H 3 nucleotide sequence is operably linked to the C γ1 M1 and M2 exons of the gene segment.

[0148] In some embodiments, a method for generating an engineered immunoglobulin heavy chain locus described herein comprises inserting a human IgG4 C locus comprising a polypeptide encoding an immunoglobulin heavy chain constant domain. H 1-HC H 2-C H The approximately 5.8 kb DNA of the 3-M1-M2 nucleotide sequence was inserted into the C γ1 C gene segment H 1-HC H 2-C H 3-M1-M2 exon position, so that the human IgG4 C H 1-HC H 2-C H The 3-M1-M2 nucleotide sequence is operably linked to the C γ1 conversion region of a gene segment.

[0149] In some embodiments, the method for generating the engineered immunoglobulin heavy chain loci described herein comprises inserting a locus comprising a human C μ 、C δ 、C γ3 、C γ1 In other embodiments, the method further comprises inserting a DNA comprising a human C γ2 and C γ4 An additional approximately 43 kb of DNA was sequenced.

[0150] In the various methods described above for generating engineered immunoglobulin heavy chain loci, DNA can be introduced into an endogenous rodent (e.g., rat or mouse) immunoglobulin heavy chain locus comprising endogenous rodent (e.g., rat or mouse) heavy chain variable gene segments, or an immunoglobulin heavy chain locus comprising human heavy chain variable gene segments. The DNA can be introduced by homologous recombination or by other methods known in the art or described herein.

[0151] In some embodiments, rodents (e.g., rats or mice) are heterozygous for the modified immunoglobulin heavy chain loci described herein. In certain embodiments, rodents (e.g., rats or mice) are homozygous for the modified immunoglobulin heavy chain loci described herein.

[0152] Humanized immunoglobulin kappa locus

[0153] In certain aspects, provided herein are rodents (e.g., mice or rats) comprising a genetically modified Igκ chain locus. The locus comprises a κ variable region and a κ constant region. The κ variable region comprises an Igκ chain variable region gene segment (i.e., at least V κ Gene segments and Jκ Gene segment). The constant region includes the Igκ chain constant region (C κ In certain embodiments, an immunoglobulin kappa chain variable region, such as a human Ig kappa variable region, is operably linked to an immunoglobulin kappa chain constant region, such that a rodent (e.g., a rat or mouse) produces an immunoglobulin kappa chain constant region comprising a human V κ Gene segments and human J κ The light chain variable domain of the gene segment and the C κ In some embodiments, the antibody comprises a light chain constant domain of a gene segment. In some embodiments, the Igκ variable region will be an unrearranged Igκ variable region, and therefore will contain an unrearranged Igκ variable region gene segment. In some embodiments, the Igκ variable region will be a rearranged Igκ variable region, and therefore will contain 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 (eg, a rat or mouse variable region gene segment). In some embodiments, the Igκ constant region locus comprises partially or completely human Igκ constant region gene segments. In some embodiments, the Igκ chain locus described herein is located at an endogenous Igκ chain locus.

[0154] In certain embodiments, the Igκ variable region comprises an unrearranged human Igκ variable region gene segment. In certain embodiments, the engineered Igκ light chain locus (or allele) comprises at least a human V κ A gene segment that occurs in the distal variable cluster (or distal arm or distal repeat) of a naturally occurring human Igκ light chain locus. In certain embodiments, the engineered Igκ light chain locus (or allele) comprises at least a human V κ A gene segment that occurs in the proximal variable cluster (or proximal arm or proximal repeat) of a naturally occurring human Igκ light chain locus. In certain embodiments, the engineered Igκ light chain locus (or allele) comprises a human V κ Gene segments that occur in the distal and proximal variable clusters of a naturally occurring human Igκ light chain locus. In certain embodiments, the engineered Igκ light chain locus (or allele) comprises human V segments found in naturally occurring human Igκ light chain loci. κ 2-40 (or V κ 3D-7) gene segment and human V κ All or substantially all functional human V κ Gene segment.

[0155] In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises a plurality of human V κ Segment and one or more persons J κ In some embodiments, the immunoglobulin variable region gene segment comprises four functional V κ Section and Owner J κ In some embodiments, the immunoglobulin variable region gene segment comprises 16 functional V κ Section and Owner J κ In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises a human V κ All and Everyone in SegmentJ κ section.

[0156] In certain 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 certain 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 κ 2D-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 κ1-17, 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 4-1. In certain embodiments, the engineered Igκ light chain locus (or allele) comprises at least 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 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 2D-40. In certain embodiments, the engineered Igκ light chain locus (or allele) comprises at least a human V κ Gene 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 κ5-2 and V κ 4-1.

[0157] In some embodiments, the non-human animals provided herein are characterized by having at most two human V L Gene segments and multiple J L In some embodiments, V L The gene segment is V κ In some embodiments, V L The gene segment is V λ In some embodiments, V κ The gene segment is V κ 3-20 and V κ 1-39.

[0158] In some embodiments, the engineered Igκ light chain locus (or allele) comprises 1, 2, 3, 4, 5 or more functional human J κ In certain embodiments, the engineered Igκ light chain locus (or allele) comprises a human J gene segment found in a naturally occurring human Igκ light chain locus. κ 1 gene segment and human J κ All or substantially all functional human J between the 5 gene segments (including both ends) κ In certain 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 5.

[0159] In other embodiments, the non-human organism may comprise a light chain immunoglobulin locus in its germline and / or genome, the light chain immunoglobulin locus including insertion and / or replacement of histidine codons designed to introduce pH-dependent binding properties into antibodies produced in the non-human organism. In some of the embodiments, a histidine codon is inserted and / or replaced in the nucleic acid sequence encoding CDR3. Various light chain immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, U.S. Patent Application Publication Nos. 2013 / 0247236, 20140013456, which are incorporated herein by reference.

[0160] 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 hereby incorporated by reference. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises all of a human Jκ segment.

[0161] In some embodiments, the Igκ variable gene locus containing unrearranged human Igκ variable region gene segments further 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).

[0162] In some embodiments, the Igκ variable region locus is a rearranged variable region locus (universal light chain variable region) comprising an Igκ variable region gene. In some embodiments, the rearranged Igκ variable region gene is a human rearranged Igκ variable region gene. The use of a universal light chain variable region facilitates the production of bispecific antibodies. Exemplary rearranged Ig light chain variable regions are provided in U.S. Patent Publication No. 2013 / 0185821, which is hereby incorporated by reference.

[0163] In some embodiments, the Ig kappa 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 kappa chain locus comprises a rodent (e.g., rat or mouse) or human intronic kappa enhancer (E κi In some embodiments, the IgH locus comprises a rodent (eg, rat or mouse) or human 3′ kappa enhancer (E κ3′ ).

[0164] In some embodiments, the modified immunoglobulin kappa chain locus is located at an endogenous immunoglobulin kappa chain locus. In some embodiments, the immunoglobulin kappa chain locus replaces all or a portion of the endogenous immunoglobulin kappa chain locus. In certain embodiments, the modified Ig kappa chain locus is located on a transgene that is positioned outside the endogenous locus. In some embodiments, the endogenous Ig kappa chain locus is inactivated (e.g., by deleting, relocating, and / or inverting all or a portion of the endogenous Ig kappa chain locus).

[0165] In some embodiments, the method for generating the engineered immunoglobulin κ light chain locus described herein comprises inserting approximately 0.5 kb of DNA comprising a human Igκ constant nucleotide sequence encoding an immunoglobulin κ light chain constant domain polypeptide into a position within 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.

[0166] In some embodiments, rodents (e.g., rats or mice) are heterozygous for the modified immunoglobulin kappa chain loci described herein. In certain embodiments, rodents (e.g., rats or mice) are homozygous for the modified immunoglobulin kappa chain loci described herein.

[0167] Humanized immunoglobulin λ locus

[0168] In certain aspects, provided herein are rodents (eg, mice or rats) comprising a genetically modified Ig lambda chain locus. The locus comprises an Ig lambda chain variable region gene segment (i.e., at least V λ Gene segments and J λ The modified λ locus also includes at least one Igλ chain constant region (C λ ) gene segment. In certain embodiments, V λ Gene segments and J λ Gene segments such as human V λ Gene segments and human J λ The gene segment is operably linked to C λ , so that rodents (such as rats or mice) produce V λ Gene segments and human J λ The light chain variable domain of the gene segment and the C λ In some embodiments, the V λ Gene segments and J λ The gene segment will be unrearranged V λ and J λ In some embodiments, V λ Gene segments and J λ The gene segment will be the rearranged V λ and J λ gene segments, and thus will be in the form of rearranged variable region genes. In certain embodiments, the Ig λ The variable region gene segments are human variable region gene segments. In certain embodiments, the Ig λ The variable region gene segments are rodent variable region gene segments (eg, rat or mouse variable region gene segments). In some embodiments, the Ig λThe constant region locus comprises a lambda constant region gene segment that is partially or completely human. In some embodiments, the Ig lambda chain locus described herein is located at an endogenous Ig lambda chain locus. Exemplary variable regions comprising Ig lambda gene segments are provided, for example, U.S. Patent Publication Nos. 2012 / 0073004 and 2002 / 0088016 and U.S. Patent Application No. 15 / 803,513 (filed on November 3, 2017; published as US2018 / 0125043), each of which is hereby incorporated by reference.

[0169] In some embodiments, the Igλ variable gene locus containing unrearranged human Igκ variable region gene segments further comprises human Igλ variable region intergenic sequences. In some embodiments, the Igλ variable gene locus comprises non-human (e.g., rodent, rat, mouse) Igλ variable region intergenic sequences. In some embodiments, the Igλ 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).

[0170] In some embodiments, the human Igλ light chain locus comprises genetic material from a human Igλ light chain locus. In some embodiments, the human Igλ light chain locus as described herein comprises at least one human V λ Gene segment, at least one person J λ Gene segment, at least one person C λ gene segments, and one or more genes for promoting the at least one human V λ The gene segment is associated with at least one human J λ The gene segments rearrange to form the coding human V λ Functional rearrangement of domains in human V λ -J λ In many embodiments, the human Igλ light chain sequence comprises multiple human V λ gene segments and one or more to promote the human V λ Gene segment associated with at least one person λ In some embodiments, the human Igλ light chain sequence as described herein is a genomic sequence of a human Igλ light chain locus (e.g., isolated and / or cloned from a bacterial artificial chromosome) and contains multiple human V λ In some embodiments, the human Igλ light chain sequence comprises a human V λ 、J λ and C λ Sequence (ie, as described for human V λ 、Jλ and C λ The sequence is present in the Igλ light chain locus in human cells, in other words, J λ and C λ Sequence J λ C λ In some embodiments, the human Igλ light chain sequence fully or partially encodes an Igλ light chain polypeptide that is present in immunoglobulins, particularly immunoglobulins expressed by human B cells. Also provided are non-human animals, embryos, cells, and targeting constructs for preparing non-human animals, non-human embryos, and cells that contain the human Igλ light chain sequence in the location of a corresponding non-human Igλ light chain sequence (e.g., an endogenous rodent Igλ light chain locus).

[0171] In some embodiments, a human Igλ light chain sequence is inserted into the position of a corresponding non-human Igλ light chain sequence within the germline genome of a non-human animal. In some embodiments, a human Igλ light chain sequence is inserted upstream of a non-human Igλ light chain sequence (e.g., a non-human Igλ light chain constant region sequence). In some embodiments, a 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.

[0172] In certain embodiments, the Ig lambda 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, V λ 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 comprises V λ 3-27, V λ 1-36, V λ 5-37, V λ 5-39, V λ 1-40, Vλ 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 comprises V λ 10-54, V λ 6-57, V λ 4-60, V λ 8-61 and V λ 4-69. In some embodiments, the Ig lambda chain locus comprises one or more pairs of human J λ -C λ For example, in certain embodiments, the Ig lambda chain locus is in the human V λ The downstream of the gene segment contains 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 Chain locus in human V λ The downstream of the gene segment contains 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. People λ 7 and mouse C λ 1, such as Figure 2A Described in.

[0173] In some embodiments, the Igλ locus is a rearranged locus comprising an Igλ variable region gene (universal light chain variable region). In some embodiments, the rearranged Igλ variable region gene is a human rearranged Igλ 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 binding. Exemplary rearranged Ig light chain variable regions are provided in U.S. Patent Publication No. 2013 / 0185821, which is hereby incorporated by reference.

[0174] In some embodiments, the Ig lambda 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 lambda chain locus comprises a rodent (e.g., rat or mouse) lambda enhancer 2.4. In some embodiments, the Ig lambda chain locus comprises a human or rodent (e.g., rat or mouse) 3′ lambda enhancer. In some embodiments, the Ig lambda chain locus comprises a rodent (e.g., rat or mouse) lambda enhancer 3.1.

[0175] In some embodiments, the modified immunoglobulin lambda chain locus is located at an endogenous immunoglobulin lambda chain locus. In some embodiments, the immunoglobulin lambda chain locus replaces all or a portion of the endogenous immunoglobulin lambda chain locus. In certain embodiments, the modified Ig lambda chain locus is located on a transgene that is positioned outside the endogenous locus. In some embodiments, the endogenous Ig lambda chain locus is inactivated (e.g., by deleting, relocating, and / or inverting all or a portion of the endogenous Ig lambda chain locus).

[0176] In some embodiments, the rodent (e.g., rat or mouse) is heterozygous for the modified immunoglobulin lambda chain locus. In certain embodiments, the rodent (e.g., rat or mouse) is homozygous for the modified immunoglobulin lambda chain locus.

[0177] In some embodiments, the non-human organism comprises a light chain immunoglobulin locus in its germline and / or genome, and the light chain immunoglobulin locus comprises a limited light chain variable gene segment pedigree (e.g., a double light chain variable region comprising two light chain variable gene segments). In some embodiments, the light chain variable gene segments in the limited light chain gene segment pedigree are human light chain gene segments. Exemplary double light chain variable regions are provided in U.S. Patent Publication No. 2013 / 0198880, which is hereby incorporated by reference. In some embodiments, a non-human organism comprising double light chain variable regions is used to generate bispecific antibodies.

[0178] Humanized CD79a and CD79b loci

[0179] In some embodiments, rodents as described herein (such as mice or rats) include people or humanized B cell antigen receptor complex-associated protein α chain (CD79a or Igα) and / or B cell antigen receptor complex-associated protein β chain (CD79b or Igβ) locus. Rodents containing people or humanized CD79a and CD79b genes express people or humanized CD79a and CD79b polypeptides in a heterodimer form on the surface of B cells, and the polypeptides associate with membrane-expressed immunoglobulins in a non-covalent manner to form B cell receptors (BCR). BCR associates with antigens and plays a role in signal transduction and internalization after engaging with antigens. In some embodiments, rodents as described herein include people or humanized CD79a and CD79b genes. In certain embodiments, the rodent as 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 the 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 the 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; in certain embodiments, the transmembrane and intracellular domains of endogenous CD79a and CD79b polypeptides, respectively. In some embodiments, the human and endogenous portions are operably linked to endogenous CD79a or CD79b promoters, respectively.

[0180] In some embodiments, the rodent as 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, it encodes a sequence comprising amino acids 33-119 of a human CD79a polypeptide, in one embodiment, it encodes a sequence comprising amino acids 33-143 of a human CD79a polypeptide, in one embodiment, it encodes 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 further comprises a human stalk region; in a certain embodiment, the chimeric CD79a polypeptide further comprises a human transmembrane domain; and in some embodiments, the chimeric CD79a polypeptide further comprises a rodent (e.g., mouse) cytoplasmic domain. In some embodiments, the rodent comprises a chimeric CD79a gene comprising a portion of the human region described herein and a sequence encoding a human or rodent (e.g., mouse) CD79a signal peptide; in one embodiment, the sequence encoding the signal peptide is a mouse CD79a sequence encoding amino acids 1-28 of mouse CD79a.

[0181] In some embodiments, the rodent as 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, it encodes a sequence comprising amino acids 29-159 of a human CD79b polypeptide, in one embodiment, it encodes 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 further comprises a human stalk region; in a certain embodiment, the chimeric CD79b polypeptide further comprises a human transmembrane domain; and in some embodiments, the chimeric CD79b polypeptide further comprises a rodent (e.g., mouse) cytoplasmic domain. In some embodiments, the rodent comprises a chimeric CD79b gene comprising a portion of the human region described herein and a sequence encoding a human or rodent (e.g., mouse) CD79b signal peptide; in one embodiment, the sequence encoding the signal peptide is a mouse CD79b sequence encoding amino acids 1-25 of mouse CD79b.

[0182] Representative source sequences of human CD79A genes and human CD79A polypeptides from which desired human portions can be obtained are provided by GenBank Accession Nos. NP_001774.1, NM_001783.3, NP_067612.1, and NM_021601.3, and UniProt ID P 11912. Representative source sequences of human CD79B genes and human CD79B polypeptides from which desired human portions can be obtained are provided by 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.

[0183] In some embodiments, the rodents (e.g., mice or rats) provided herein further comprise one or more human CD79A and CD79B genes as described in U.S. Patent Application Publication Nos. 2011-0093963A1 and 2009-0053210A1; International Patent Application Publication No. WO 2008 / 027986; and European Patent No. 2 064 325B1, each of which is hereby incorporated 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 a 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 a 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 of a human CD79a polypeptide, a sequence comprising amino acids 33-143 of a human CD79a polypeptide, or a sequence comprising amino acids 33-165 of a human CD79a polypeptide), and wherein the human CD79b portion encodes a sequence comprising amino acids 29-135 (e.g., a sequence comprising amino acids 29-159 of a human CD79a polypeptide, or a sequence comprising amino acids 29-184 of a human CD79a polypeptide). In some embodiments, the endogenous CD79a and CD79b portions each encode at least the intracellular domain of the endogenous CD79a and CD79b polypeptides, respectively; in certain embodiments, both the transmembrane and intracellular domains of the endogenous CD79a and CD79b polypeptides, respectively.

[0184] Humanized neonatal Fc receptor locus

[0185] In some embodiments, the rodents provided herein (e.g., mice or rats) express and / or comprise a humanized or human neonatal Fc receptor (FcRn) locus in their genome. FcRn, also known as the Brambell receptor, is a protein expressed by endothelial cells and associated with beta-2-microglobulin (β2M) and binding to the Fc domain of IgG antibodies and serum albumin. FcRn extends the half-life of IgG and serum albumin. In particular, by binding to IgG and serum albumin in a pH-dependent manner, FcRn is able to rescue these serum proteins from lysosomal degradation achieved by endothelial cells, thereby increasing the serum half-life of the protein.

[0186] 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 rodent (e.g., mouse or rat) cytoplasmic domain.

[0187] In some embodiments, the nucleic acid sequence encoding the FcRn polypeptide is located at the 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 in the endogenous FcRn locus is replaced by a nucleic acid sequence encoding the extracellular domain of human FcRn, so that the rodent comprising such a locus expresses an FcRn having a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domain. 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., 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).

[0188] In certain embodiments, the mouse exons encoding the α1, α2, and α3 domains of the mouse FcRn gene (exons 3, 4, and 5 as the first three coding exons) are replaced with human exons encoding the α1, α2, and α3 domains of the human FcRn gene (exons 3, 4, and 5) (see Figure 4 In some embodiments, the FcRn gene comprises mouse exon 1 (non-coding exon), mouse exon 2 (comprising a nucleic acid sequence encoding a signal peptide), and human exons 3-6, 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.

[0189] GenBank accession numbers NC_000019.10 (49512279-49526428), NM_001136019.1, and NP_001129491.1 provide representative source sequences of human FcRn genes, cDNAs, and polypeptides from which the desired human portions can be obtained. GenBank accession numbers NC_000073.6 (45092992-45103846), NM_010189.1, and NP_034319.1 provide representative source sequences of mouse FcRn genes, cDNAs, and polypeptides from which the desired mouse portions can be obtained and / or which can be used to design targeting vector homology arms.

[0190] In some embodiments, the rodent is heterozygous for a genetically modified FcRn locus. In some embodiments, the rodent is homozygous for a genetically modified FcRn locus.

[0191] Humanized β-2-microglobulin

[0192] In some embodiments, the genetically modified rodents (e.g., rats or mice) and ES cells described herein express and / or comprise in their genome a locus encoding a humanized beta-2-microglobulin (β2M) polypeptide, which lacks a transmembrane region and associates with FcRn and class I MHC molecules.

[0193] 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 the 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, rodents do not express rodent β2M, or do not express functional rodent β2M polypeptides. In some embodiments, the β2M gene 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).

[0194] 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, a non-human animal (e.g., a mouse) comprises a humanized β2M gene, wherein the gene comprises exons 2, 3, and 4 of a human β2M gene, and in some embodiments, the humanized β2M gene comprises exon 1 of a non-human (e.g., mouse) β2M gene. The humanized β2M locus is schematically depicted in Figure 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.

[0195] Humanized Fcε receptor 1α

[0196] In some embodiments, the rodents provided herein (e.g., mice or rats) express and / or contain a humanized or human Fcε receptor 1α (FcεR1α) locus in their genome. FcεR1α associates with FcεR1β and FcεR1γ to form FcεR1, a high-affinity IgE receptor expressed on epidermal Langerhans cells, eosinophils, mast cells, and basophils. The IgE binding site of FcεR1 is found in the FcεR1α subunit.

[0197] In some embodiments, the FcεR1α locus comprises a nucleic acid sequence encoding an FcεR1α 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 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 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. Figure 9 middle.

[0198] In some embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide is located at the endogenous rodent FcεR1α locus. In certain 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 in the endogenous FcεR1α locus is replaced by a nucleic acid sequence encoding the extracellular domain of human FcεR1α, so that the rodent comprising such a locus expresses FcεR1α with a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domain. In some embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide is located at the endogenous rodent FcεR1α locus, and the FcεR1α polypeptide comprises a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain. In some embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide replaces all or part of the endogenous rodent FcεR1α gene, and the FcεR1α polypeptide comprises a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain. In some embodiments, the rodent does not express rodent FcεR1α, or does not express functional rodent FcεR1α. In some embodiments, the FcεR1α 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).

[0199] In certain embodiments, a portion of mouse coding exon 1, coding exon 2, coding exon 3, coding exon 4, and coding exon 5 of mouse FcεRIα are replaced with a portion of human coding exon 1, coding exon 2, coding exon 3, coding exon 4, and coding exon 5 of a human FcεRIα gene. In some embodiments, the FcεRIα gene comprises chimeric mouse / human exon 1 (comprising a mouse promoter and 5'UTR), human coding exons 2-5 until a stop codon, a human 3'UTR and polyadenylation (polyA), followed by a mouse 3'UTR and polyadenylation. In some embodiments, chimeric gene exons 1 (partially) and 2 encode a signal peptide, exons 3 and 4 encode two Ig-like domains of FcεRIα that are believed to interact with IgE, and exon 5 encodes the cytoplasmic and transmembrane domains of the protein (see Figure 9 ).

[0200] Representative source sequences of human FcεR1α genes, cDNAs, and polypeptides from which desired human portions can be obtained are provided by GenBank Accession Nos. NC_000001.11 (159283888-159308224), NM_002001.3, and NP_001992.1. Representative source sequences of mouse FcεR1α genes, cDNAs, and polypeptides from which desired mouse portions can be obtained and / or can be used to design targeting vector homology arms are provided by GenBank Accession Nos. NC_000067.6 (173221269-173227232), NM_010184.1, and NP_034314.1.

[0201] In some embodiments, the rodent is heterozygous for a genetically modified FcεR1α locus. In some embodiments, the rodent is homozygous for a genetically modified FcεR1α locus.

[0202] Humanized Fcγ receptor 1a

[0203] In some embodiments, the rodents provided herein (e.g., mice or rats) express and / or comprise in their genome a humanized or human Fcγ receptor 1a (FcγR1a, often labeled FcγR1 in the figures). FcγR1a is a high-affinity FcγR protein expressed on monocytes that binds the Fc portion of IgG and leads to activation of the host cell.

[0204] 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 rodent (e.g., mouse or rat) cytoplasmic domain.

[0205] In some embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide is located at the endogenous rodent FcγR1a locus. In certain 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 in the endogenous FcγR1a locus is replaced by a nucleic acid sequence encoding the extracellular domain of human FcεR1α, so that the rodent comprising such a locus expresses FcγR1a with a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domain. 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., 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).

[0206] Humanized FcγR1a polypeptides, 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.

[0207] In some embodiments, the rodent is heterozygous for the genetically modified FcyR1a locus. In some embodiments, the rodent is homozygous for the genetically modified FcyR1a locus.

[0208] Humanized low-affinity Fcγ receptor

[0209] In some embodiments, the genetically modified rodents (e.g., rats or mice) and ES cells described herein express and / or comprise in their genome a locus encoding a human low-affinity Fcγ receptor (FcγR) polypeptide (e.g., a human FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, or FcγRIIIb polypeptide).

[0210] 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 located at an endogenous rodent low-affinity FcγR locus. In certain embodiments, the nucleic acid sequence encoding the FcγRIIa polypeptide replaces all or a portion of the endogenous rodent low-affinity FcγR locus. In a specific embodiment, the human FcγRIIa gene comprises a polymorphism, wherein the polymorphism is selected from the 131His low-response polymorphism and the 131Arg high-response polymorphism. In a specific embodiment, the FcγRIIa polymorphism is the 131His low-response polymorphism. In some embodiments, the rodent does not express a rodent low-affinity FcγR polypeptide (e.g., does not express a rodent FcγRIIb, FcγRIV, and / or FcγRIII polypeptide, or does not express a functional rodent FcγRIIb, FcγRIV, and / or FcγRIII polypeptide). In some embodiments, the FcγRIIa gene 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).

[0211] 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 located at an endogenous rodent low-affinity FcγR locus. In certain embodiments, 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 187Ile or 187Thr substitution. 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 FcγRIIb gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory element is a rodent regulatory element (eg, a rat or mouse promoter or enhancer).

[0212] 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 the human FcγRIIc polypeptide is located at an endogenous rodent low-affinity FcγR locus. In certain embodiments, 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 57 stop (57Stop) variant and 57Q variant. In some embodiments, rodents do not express rodent low-affinity FcγR polypeptides (e.g., rodent FcγRIIB, FcγRIV and / or FcγRIII polypeptides are not expressed). In some embodiments, the FcγRIIc 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).

[0213] 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 located at an endogenous rodent low-affinity FcγR locus. In certain embodiments, 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 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 one embodiment, the FcγRIIIa gene is a specific allelic variant, wherein the allelic variant is selected from a 176Val variant and a 176Phe variant. In a specific embodiment, the FcγRIIIa allelic variant is a 176Val variant. In some embodiments, the FcγRIIIa gene locus comprises non-human regulatory elements (eg, non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (eg, rat or mouse promoter or enhancer).

[0214] 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 located at an endogenous rodent low-affinity FcγR locus. In certain embodiments, the nucleic acid sequence encoding the FcγRIIIb polypeptide replaces all or part of the endogenous rodent low-affinity FcγR locus. In a specific embodiment, the FcγRIIIb gene is a specific allelic variant, wherein the allelic variant is selected from the group consisting of NA1 variants and NA2 variants. In another specific embodiment, the FcγRIIIb allelic variant is an NA2 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 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 (eg, non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (eg, rat or mouse promoter or enhancer).

[0215] In some embodiments, provided herein are rodents (e.g., rats or mice) comprising one or more low-affinity FcγR genes as 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, provided herein are rodents (e.g., rats or mice) comprising at least two low-affinity human FcγR genes and endogenous rodent (e.g., rat or mouse) Fcγ chain genes, wherein the low-affinity human FcγR genes are selected from the group consisting of human FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In certain embodiments, provided herein are rodents (e.g., rats or mice) comprising human FcγRIIa and FcγRIIIb, and endogenous rodent (e.g., rat or mouse) Fcγ chain genes. In certain embodiments, provided herein are rodents (e.g., rats or mice) comprising FcγRIIa, FcγRIIIa, FcγRIIb, FcγRIIc, and FcγRIIId genes, as well as endogenous rodent (e.g., rat or mouse) Fcγ chain genes. In various embodiments, rodents (e.g., rats or mice) comprising one or more human FcγRs are further comprised of homozygous disruptions in endogenous rodent (e.g., rat or mouse) FcγRIIB, FcγRIV, and FcγRIII genes (i.e., endogenous rodent FcγRIIb, FcγRIV, and FcγRIIIα chain coding sequences). In various embodiments, rodents (e.g., rats or mice) comprising one or more human low-affinity FcγRs as described herein do not express endogenous rodent low-affinity FcγR polypeptides (e.g., endogenous low-affinity FcγRα chain polypeptides) in a detectable manner.

[0216] 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.

[0217] Genetically modified non-human animals and ES cells

[0218] In certain aspects, provided herein are genetically modified non-human animals (e.g., rodents such as rats or mice) comprising one or more of the humanized loci disclosed herein, and genetically modified non-human animal ES cells that can be used to prepare the non-human animals.

[0219] 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, a non-human animal or ES cell comprises an IgH locus provided herein in its germline and / or genome. In certain embodiments, a non-human animal or ES cell further comprises an Igκ and / or Igλ locus provided herein. In some embodiments, a non-human animal or ES cell comprises a CD79a and / or CD79b locus provided herein in its germline and / or genome. In certain embodiments, a non-human animal or ES cell comprises an FcRn locus provided herein in its germline and / or genome. In certain embodiments, a non-human animal or ES cell comprises a β2M locus provided herein in its germline and / or genome. In certain embodiments, a non-human animal or ES cell comprises an FcεR1α locus provided herein in its germline and / or genome. In certain embodiments, a non-human animal or ES cell comprises an FcγR1a locus provided herein in its germline and / or genome. In certain embodiments, non-human animals or ES cells comprise the FcγR2a locus provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells comprise the FcγR2b locus provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells comprise the FcγR3a locus provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells comprise the FcγR3b locus provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells comprise the FcγR2c locus provided herein in their germline and / or genome. In some embodiments, non-human animals or ES cells are heterozygous for one or more of the loci provided herein, such as genetically engineered loci. In some embodiments, non-human animals or ES cells are homozygous for one or more of the loci provided herein, such as genetically engineered loci.

[0220] In some embodiments, non-human animal can be any non-human animal. In some embodiments, non-human animal is a vertebrate. In some embodiments, non-human animal is a mammal. In some embodiments, genetically modified non-human animal as described herein can be selected from the group consisting of mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, llamas, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals to which genetically modified ES cells are suitable, alternative methods can be used to prepare non-human animals comprising genetically modified as described herein. The method includes, for example, modifying non-ES cell genomes (e.g., fibroblasts or induced pluripotent cells) and adopting nuclear transfer to transfer modified genomes to suitable cells such as oocytes, and incubating modified cells (e.g., modified oocytes) under conditions suitable for forming embryos in non-human animals.

[0221] In various embodiments, provided herein are non-human animals such as rodents (such as rats or mice) comprising genetically engineered immunoglobulin heavy chain gene seat. The exemplary embodiments of the genetically engineered gene seat are schematically depicted in Figure 1, and are described in detail herein. In some embodiments, non-human animals as described herein include Adam6 genes in its genome (such as its germline genome), and the Adam6 genes encode ADAM6 polypeptides, its functional orthologs, functional homologs or functional fragments (see, for example, U.S. Patent number 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety). In some embodiments, ADAM6 polypeptides, its functional orthologs, functional homologs or functional fragments are expressed from Adam6 genes. In some embodiments, Adam6 genes do not originate from the non-human animals (such as mice including rat Adam6 genes or mice Adam6 genes obtained from another mouse strain) comprising Adam6 genes. In some embodiments, non-human animals as described herein include dystopy Adam6 genes. As used herein, an "ectopic" Adam6 gene refers to an Adam6 gene that is in a different situation than the Adam6 gene appears in a wild-type non-human animal. For example, the Adam6 gene can 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 include an inserted or integrated Adam6 gene.

[0222] In some embodiments, the non-human animal described herein comprises an insertion into its genome (e.g., its germline genome) of one or more nucleotide sequences encoding one or more non-human Adam6 polypeptides, their functional orthologs, functional homologs, or functional fragments.

[0223] In some embodiments, non-human animals as described herein include one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments in its genome (e.g., its germline genome). In some embodiments, non-human animals as described herein include mouse Adam6a genes and / or mouse Adam6b genes in its genome (e.g., its germline genome). In some embodiments, non-human animals as described herein include one or more nucleotide sequences encoding mouse ADAM6a, its functional orthologs, functional homologs or functional fragments and / or mouse ADAM6b, its functional orthologs, functional homologs or functional fragments.

[0224] In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are inserted into and / or located on the chromosome identical to the endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are inserted into and / or located in a certain position, so that the nucleotide sequences of the one or more encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are adjacent to human immunoglobulin heavy chain variable region gene segments. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are inserted into and / or located in a certain position, so that the nucleotide sequences of the one or more encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are adjacent to human immunoglobulin heavy chain variable region gene segments. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are inserted into and / or located at a certain position so that the one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are located between the human immunoglobulin heavy chain variable region gene segments. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are inserted into and / or located between the first and second human VH In some embodiments, the first person V H The gene segment is human V H 1-2, and the second person V H The gene segment is human V H 6-1. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are inserted into and / or located in the position of a human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more non-human ADAM6 polypeptides, their functional orthologs, functional homologs or functional fragments are inserted into a human V H Gene segment and human D H between gene segments.

[0225] In some embodiments, non-human animals as described herein include an Adam6 gene that restores or enhances ADAM6 activity. In some embodiments, the Adam6 gene restores ADAM6 activity to the level of a similar non-human animal including a functional endogenous Adam6 gene. In some embodiments, the Adam6 gene enhances ADAM6 activity to a level that is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times the ADAM6 activity of a similar non-human animal that does not include a functional Adam6 gene.

[0226] In some embodiments, the non-human animals described herein include an Adam6 gene that restores or enhances the fertility of male non-human animals. In some embodiments, the Adam6 gene restores the fertility of male non-human animals to the level of similar non-human animals that include a functional endogenous Adam6 gene. In some embodiments, the Adam6 gene restores the fertility of male non-human animals so that the number of offspring produced by mating the male non-human animals is at least 70%, at least 80%, at least 90%, or at least 95% of the number of offspring produced by similar mating of similar male non-human animals that do not include a functional Adam6 gene. In some embodiments, the Adam6 gene enhances the fertility of male non-human animals so that the number of offspring produced by mating the male non-human animals includes at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times the number of offspring produced by similar mating of similar male non-human animals that do not include a functional Adam6 gene.

[0227] In some embodiments, the non-human immunoglobulin heavy chain locus as described herein lacks at least one endogenous non-human Adam6 gene. In some embodiments, the lack of at least one endogenous non-human Adam6 gene reduces ADAM6 activity and / or fertility in male mice lacking endogenous non-human Adam6 genes. In some embodiments, the non-human immunoglobulin heavy chain locus as described herein includes the destruction of at least one endogenous non-human Adam6 gene. In some embodiments, the destruction of at least one endogenous non-human Adam6 gene reduces ADAM6 activity and / or fertility in male mice lacking endogenous non-human Adam6 genes.

[0228] In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human animal is a small mammal such as Dipodoidea or Muroidea. In some embodiments, the non-human animal is a rodent. In certain 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 group consisting of Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (e.g., true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (e.g., climbing mice, rock mice, white-tailed rats, Malagasy rats, and mice), Platacanthomyidae (e.g., spiny dormouse), and Spalacidae (e.g., mole rats, bamboo rats, and zokors). In some embodiments, the rodent is selected from the group consisting of true mice or rats (Muridae), gerbils, spiny mice, and crested rats. In some embodiments, the mouse is from a member of the family Muridae. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent is selected from the group consisting of mice and rats. In some embodiments, the non-human animal is a mouse.

[0229] 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 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 hybrid of the 129 strain and the C57BL strain. In some embodiments, the mouse is a hybrid of the 129 strain and / or a hybrid of the C57BL / 6 strain. In some embodiments, the hybrid 129 strain is the 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse is a BALB strain (e.g., BALB / c). In some embodiments, the mouse is a mixture of the BALB strain and another strain, such as the C57BL strain and / or the 129 strain. In some embodiments, the non-human animal provided herein may be a mouse obtained from any combination of the above-mentioned strains.

[0230] 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.

[0231] In certain embodiments, genetically modified non-human animals or ES cells include multiple loci provided herein in their genomes and / or germlines, such as multiple genetically engineered loci provided herein. For example, in some embodiments, non-human animals or ES cells include IgH loci provided herein and Igκ and / or Igλ loci provided herein in their germlines and / or genomes. In some embodiments, non-human animals or ES cells include IgH loci provided herein, Igκ and / or Igλ loci provided herein in their germlines and / or genomes, and optionally include CD79a and / or CD79b loci provided herein. In some embodiments, non-human animals or ES cells include IgH loci provided herein, Igκ and / or Igλ loci provided herein, FcRn loci provided herein, and β2M loci provided herein in their germlines and / or genomes. 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.

[0232] In certain aspects, the genetically modified non-human animal expresses one or more of the 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.

[0233] Genetically modified non-human animals and ES cells can be produced using any suitable method known in the art. For example, the genetically modified non-human animal ES cells can be produced using methods described in U.S. Patent Nos. 6,586,251, 6,596,541, 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. The invention relates to a method for modifying an ES cell line using a CRISPR / Cas system, a CRISPR / Cas system, a transcription activator-like effector nuclease (TALEN) system, or a zinc finger nuclease (ZFN) system. In some embodiments, the invention relates to a method for modifying an ES cell line using a CRISPR / Cas system, a CRISPR / Cas system as described in, for example, 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 preparing genetically modified non-human animals and ES cells are also provided herein in Examples 1 and 3-5.

[0234] The ES cells described herein can then be used to generate non-human animals using methods known in the art. For example, the mouse non-human ES cells described herein can be used to generate non-human animals using methods described in U.S. Patent No. 7,294,754 and Poueymirou et al., Nature Biotech 25:91-99 (2007). The method produces genetically modified mice, each of which is hereby incorporated by reference. The resulting mice can be bred to obtain homozygosity.

[0235] Methods for testing human Fc-containing therapeutics

[0236] In certain aspects, provided herein are methods for testing therapeutic proteins (e.g., human antibodies or Fc fusion proteins) comprising a human Fc domain, the method comprising administering the therapeutic protein to a rodent (e.g., a mouse or rat) as provided herein. In certain embodiments, provided herein are animal models for performing the methods.

[0237] In some embodiments, the administered human antibody or Fc fusion protein has a human C-terminal domain that is identical to a human C-terminal domain in a genetically modified IgH locus of a rodent provided herein. HFor example, in some embodiments, the agent is a human IgG1 antibody and the rodent comprises a genetically modified IgH locus comprising C1, hinge, CH2, and CH3 domains encoding human IgG1. H In some embodiments, the agent is a human IgG4 antibody and the rodent comprises a genetically modified IgH locus comprising C sequences 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 C1 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 C1 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 C1, hinge, CH2, CH3, M1, and M2 domains encoding human IgG3. H In some embodiments, the agent is a human IgG4 antibody and the rodent comprises a genetically modified IgH locus comprising C1, hinge, CH2, CH3, M1, and M2 domains encoding human IgG4. H In some embodiments, the agent is a human IgM antibody and the rodent comprises a genetically modified IgH locus comprising C1, C2, C3, and C4 domains encoding human IgM. H In some embodiments, the agent is a human IgD antibody and the rodent comprises a genetically modified IgH locus comprising C1, H1, H2, CH2, CH3, M1, and M2 domains encoding human IgD H In some embodiments, the therapeutic agent is a human antibody having an Igκ light chain, and the rodent comprises a genetically modified Igκ locus as 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 as provided herein.

[0238] In some embodiments, the method includes measuring one or more pharmacokinetic properties of the administered therapeutic protein. In certain embodiments, the animal model for determining the pharmacokinetic properties of the administered human antibody or fusion protein is a genetically modified rodent comprising a modified IgH locus provided herein. In certain embodiments, the animal model for determining the pharmacokinetic properties of the administered human antibody or fusion protein is a genetically modified rodent comprising a modified IgH locus provided herein and a modified Igκ locus and / or modified Igλ locus provided herein. In some embodiments, the administered human antibody or Fc fusion protein has a humanized C locus that is different from that of the humanized C locus in the genetically modified IgH locus. H In some embodiments, the rodent further comprises a modified FcRn locus as provided herein. In some embodiments, the rodent further comprises a modified β2M as provided herein.

[0239] 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 (CL), mean residence time (MRT), half-life of the agent (t1 / 2), 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., 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).

[0240] In some embodiments, the method further comprises measuring the therapeutic efficacy of the administered therapeutic protein (e.g., the ability of the administered 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, the level of cytokine expression, the proliferation of immune cells, tissue damage, and / or animal survival. In some embodiments, the animal model is an infectious disease model, and disease symptoms may include, for example, the level of infectious agent, tissue damage, and / or animal survival.

[0241] In some embodiments, the method further comprises measuring the safety and dosing of the administered therapeutic protein (e.g., the extent to which the administered 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, hair loss, allergies, anemia, lack of appetite, loss of balance, bleeding, blood clots, difficulty breathing, bronchitis, bruising, low white blood cell counts, low red blood cell counts, low platelet counts, cardiotoxicity, conjunctivitis, constipation, coughing, dehydration, diarrhea, electrolyte imbalance, loss of fertility, fever, hair loss, heart failure, infection, injection site reactions, iron deficiency, renal failure, leukopenia, abnormal liver function, pneumonia, rapid heartbeat, rectal bleeding, seizures, weight loss, and weight gain. For example, in certain embodiments, provided herein is a method for measuring allergic reactions induced by a therapeutic agent using a passive cutaneous anaphylaxis (PCA) and / or passive systemic anaphylaxis (PSA) model.

[0242] 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 is a method for screening a therapeutic agent comprising a human Fc region of a human antibody, the method comprising: (a) administering an agent comprising an Fc region of a human antibody to a rodent as provided herein, wherein the agent binds to a target cell in the rodent; (b) measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against the target cell; and (c) comparing the amount of ADCC in step (b) to a control, wherein an increase in target cell killing indicates that the agent has an increased ability to mediate ADCC.

[0243] In some embodiments, the method further comprises measuring the extent to which administration of the therapeutic protein induces an anti-human Fc immune response in the rodent.

[0244] In some embodiments, when administered to a rodent provided herein, the administered therapeutic agent elicits a reduced immune response. In some embodiments, the administered human antibody or Fc fusion protein has a human C-terminal domain that is identical to a human C-terminal domain in a 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 comprises a genetically modified IgH locus comprising C1, hinge, CH2, and CH3 domains encoding human IgG1. H In some embodiments, the agent is a human IgG4 antibody and the rodent comprises a genetically modified IgH locus comprising C sequences 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 C1 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 C1, hinge, CH2, CH3, M1, and M2 domains encoding human IgG2. H In some embodiments, the agent is a human IgG3 antibody and the rodent comprises a genetically modified IgH locus comprising C1, hinge, CH2, CH3, M1, and M2 domains encoding human IgG3. H In some embodiments, the agent is a human IgG4 antibody and the rodent comprises a genetically modified IgH locus comprising C1, hinge, CH2, CH3, M1, and M2 domains encoding human IgG4. H In some embodiments, the agent is a human IgM antibody and the rodent comprises a genetically modified IgH locus comprising C1, C2, C3, and C4 domains encoding human IgM. H In some embodiments, the agent is a human IgD antibody and the rodent comprises a genetically modified IgH locus comprising C1, H1, H2, CH2, CH3, M1, and M2 domains encoding human IgD HIn some embodiments, the therapeutic agent is a human antibody having an Igκ light chain, and the rodent comprises a genetically modified Igκ locus as 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 as provided herein.

[0245] In certain embodiments, provided herein are methods comprising testing therapeutic antibodies comprising human Fc. In some embodiments, the antibody tested comprises human heavy chain variable domains. In some embodiments, the antibody comprises human heavy chain constant domains. In some embodiments, provided herein are antibodies comprising IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant domains. The sequence of human heavy chain constant domains is known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH publication number 91-3242 and the IMGT database available at www.imgt.org).

[0246] In some embodiments, the antibodies tested comprise a modified Fc domain (e.g., a mutation that alters the interaction between the Fc and an Fc receptor). For example, in some embodiments, the antibodies provided herein comprise modifications to their 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 modification is 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 antibodies comprise multiple modifications to their Fc domains. In some embodiments, the plurality of modifications is selected from the group consisting of: 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 / A 330I, S239D / D265S / S298A / I332E, S239E / D265S / H268D / I332E, S239E / D265S / I332E, S239E / S267E / H268D, S239E / A 327L / A330I, D265E / S267D / A330S, S267G / H268E / D270E, H268D / E269L / S298A / K326A / A327H, H268D / / K326A / A327H.Additional Fc modifications and combinations of Fc modifications are provided 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, 8,218,805, 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 hereby incorporated by reference.

[0247] In some embodiments, the antibody tested is a bispecific antibody. In some embodiments, the two antigen-binding domains of the bispecific antibody have different heavy chain variable domains, but have the same light chain variable domains. In some embodiments, the Fc domain of the heavy chain comprises modifications to facilitate the formation of heavy chain heterodimers and / or to inhibit the formation of heavy chain homodimers. Such modifications are, for example, provided in U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, and 8,679,785 and in U.S. Patent Publication No. 2013 / 0195849, each of which is hereby incorporated by reference.

[0248] In some embodiments, the antibody tested in the methods provided herein has a human light chain variable domain. In some embodiments, the light chain variable domain is a lambda light chain variable domain. In some embodiments, the light chain variable domain is a kappa 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 lambda light chain constant domain. In some embodiments, the light chain constant domain is a kappa light chain constant domain. The sequence of the human light chain constant domain is known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH publication number 91-3242 and the IMGT database available at www.imgt.org).

[0249] In certain embodiments, a therapeutic agent is administered to a rodent provided herein as part of a pharmaceutical composition, eg, a pharmaceutical composition containing a human antibody or Fc fusion protein formulated together with a pharmaceutically acceptable carrier.

[0250] The pharmaceutical compositions provided herein can be specially formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, such as those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, for example, in the form of a sterile solution or suspension or sustained-release formulation.

[0251] Pharmaceutical compositions suitable for parenteral administration comprise a human antibody or Fc fusion protein in combination with one or more of the following: a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution, dispersion, suspension or emulsion, or a sterile powder for reconstitution into a sterile injectable solution or dispersion immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostatics, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0252] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (such as 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 maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0253] In certain embodiments, the composition comprises a human antibody or Fc fusion protein at a w / v concentration that produces a suitable dosage. The antibody can 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. g / 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.

[0254] In some embodiments, the composition is prepared by mixing the human antibody or Fc fusion protein with optional physiologically acceptable carriers, excipients, or stabilizers, including but not limited to buffers, sugars, salts, surfactants, solubilizers, polyols, diluents, binders, stabilizers, salts, lipophilic solvents, amino acids, chelating agents, preservatives, etc. (Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th edition, L. Brunton et al.; and Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1999)) in the form of a lyophilized composition or aqueous solution at 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, citrate, glycine, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexahydroxyquaternium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; 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 nonionic surfactants such as Tween, polysorbate 80, or polyethylene glycol (PEG).

[0255] In some embodiments, the buffer 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.

[0256] In some embodiments, the composition comprises a buffer or pH adjuster to provide improved pH control. Such a composition can have a pH between about 3.0 and about 9.0, between about 4.0 and about 8.0, between about 5.0 and about 8.0, between about 5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.5 and about 8.0, between about 5.5 and about 7.0, or between about 5.5 and about 6.5. In another embodiment, the composition has 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. It will be appreciated by those skilled in the art that the pH of the composition generally should not be equal to the isoelectric point of the human antibody or Fc fusion protein to be used in the composition. Typically, the buffer is a salt prepared from an organic or inorganic acid or base. Representative buffers 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, tromethamine hydrochloride, or phosphate buffers. Additionally, amino acid components may also contribute to buffering capacity. Representative amino acid components that can be used in the composition as buffers include, but are not limited to, glycine and histidine. In certain embodiments, the buffer is selected from histidine, citrate, phosphate, glycine, and acetate. In a specific embodiment, the buffer is histidine. In another specific embodiment, the buffer is citrate. In another specific embodiment, the buffer is glycine. The purity of the buffer 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 the histidine or glycine as understood in the art, for example, as described in The Merck Index, 13th edition, ed. O'Neil et al. (Merck & Co., 2001).

[0257] In certain embodiments, the composition comprises histidine as a buffer. In 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 between about 1 mM and about 200 mM, between about 1 mM and about 150 mM, between about 1 mM and about 100 mM, between about 1 mM and about 75 mM, between about 10 mM and about 200 mM, between about 10 mM and about 150 mM, between about 10 mM and about 100 mM, between about 10 mM and about 75 mM, between about 10 mM and about 50 mM, between about 10 mM and about 40 mM, between about 10 mM and about 30 mM, between about 20 mM and about 75 mM, between about 20 mM and about 50 mM, between about 20 mM and about 40 mM, or between about 20 mM and about 30 mM histidine. In another embodiment, 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 histidine, or no histidine.

[0258] In some embodiments, compositions comprises carbohydrate excipient.Carbohydrate excipient can for example serve as viscosity enhancer, stabilizer, bulking agent, solubilizing agent and / or similar agent.By weight or volume, carbohydrate excipient is usually present between about 1% to about 99%, for example, between about 0.1% to about 20%, between about 0.1% to about 15%, between about 0.1% to about 5%, between about 1% to about 20%, between about 5% to about 15%, between about 8% to about 10%, between about 10% and about 15%, between about 15% and about 20%, between 0.1% to 20%, between 5% to 15%, between 8% to 10%, between 10% and 15%, between 15% and 20%, between about 0.1% to about 5%, between about 5% to about 10%, or between about 15% to about 20%. In other specific embodiments, the carbohydrate excipient is present at 1%, or at 1.5%, or at 2%, or at 2.5%, or at 3%, or at 4%, or at 5%, or at 10%, or at 15%, or at 20%.

[0259] In some embodiments, the composition comprises a carbohydrate excipient. Suitable carbohydrate excipients for use in the composition include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), etc. In certain embodiments, the carbohydrate excipient for the composition 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 another specific embodiment, the carbohydrate excipient is sucrose. In 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%.

[0260] In some embodiments, the composition comprises trehalose. In certain 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 another embodiment, the composition is included 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 another embodiment, 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.

[0261] In certain embodiments, the composition comprises an excipient. In a specific embodiment, the composition comprises at least one excipient selected from the group consisting of a sugar, a salt, a surfactant, an amino acid, a polyol, a chelating agent, an emulsifier, and a preservative. In certain embodiments, the composition comprises a salt, such as a salt selected from the group consisting of NaCl, KCl, CaCl2, and MgCl2. In a specific embodiment, the composition comprises NaCl.

[0262] 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 amino acid. In another embodiment, the composition may comprise between about 1 mM and about 100 mM, between about 10 mM and about 150 mM, between about 25 mM and about 250 mM, between about 25 mM and about 300 mM, between about 25 mM and about 350 mM, between about 25 mM and about 400 mM, between about 50 mM and about 250 mM, between about 50 mM and about 300 mM, or between about 50 mM and about 300 mM. In another embodiment, 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 an amino acid.

[0263] In some embodiments, the composition comprises a surfactant. As used herein, the term "surfactant" refers to an organic substance with an amphiphilic structure; that is, they are composed of groups with opposite solubility tendencies, which are typically oil-soluble hydrocarbon chains and water-soluble ionic groups. Depending on the charge of the surface active portion, surfactants can be classified as anionic surfactants, cationic surfactants, and nonionic surfactants. Surfactants are often used as wetting agents, emulsifiers, solubilizers, and dispersants for various pharmaceutical compositions and formulations of biological substances. A pharmaceutically acceptable surfactant such as polysorbate (e.g., polysorbate 20 or 80); polyoxamer (e.g., poloxamer 188); Triton; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or Stearyl sarcosine; linoleyl betaine, myristyl betaine, or cetyl betaine; lauramidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, or isostearamidopropyl betaine (e.g., lauramidopropyl betaine); myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g. PF68, etc.). In certain embodiments, the composition comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. If a pump or plastic container is used to administer the composition, a surfactant is particularly useful. The presence of a pharmaceutically acceptable surfactant mitigates the tendency of the protein 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 a 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%.

[0264] In some embodiments, compositions comprises other excipients and / or additives, including but not limited to diluents, adhesives, stabilizers, lipophilic solvents, preservatives, adjuvants etc. Pharmaceutically acceptable excipients and / or additives can be used in compositions provided herein. Can optionally be added in compositions with commonly used excipients / additives so that aggregation is reduced, such as pharmaceutically acceptable chelating agents (for example but not limited to EDTA, DTPA or EGTA). If pump or plastic container are used for applying compositions, these additives are particularly useful.

[0265] In some embodiments, composition comprises antiseptic.Optionally antiseptic can be added in composition such as between about 0.001% to about 5% or any suitable concentration of any scope or numerical value, described antiseptic is such as phenol, meta-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (such as but not limited to magnesium chloride hexahydrate), alkyl parahydroxybenzoate (methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or their mixture.The concentration of the antiseptic used in composition is the concentration that is enough to produce microbial influence.Described concentration depends on selected antiseptic, and is easy to determine by those skilled in the art.

[0266] In some embodiments, the composition is isotonic with human blood, wherein the composition has an osmotic pressure substantially the same as human blood. The isotonic composition will typically have an osmotic pressure of from about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, using a vapor pressure type or ice freezing type osmometer. The tension of the composition is adjusted by using a tension modifier. A "tonicity modifier" is a pharmaceutically acceptable inert substance that can be added to a composition to provide the isotonicity of the composition. Suitable tension modifiers for the compositions provided herein include, but are not limited to, sugars, salts, and amino acids.

[0267] In certain embodiments, the composition is a pyrogen-free composition that is substantially free of endotoxins and / or related pyrogenic substances. Endotoxins include toxins that are confined to the interior of a microorganism and are released only when the microorganism is decomposed or dies. Pyrogenic substances also include heat-stable substances that induce fever from the outer membranes of bacteria and other microorganisms. If administered to a human, both of these substances can cause fever, hypotension, and shock. Due to potential harmful effects, even low amounts of endotoxins must be removed from pharmaceutical drug solutions administered intravenously. For intravenous drug administration, the Food and Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per kilogram of body weight per dose in a single hour period (The United States Pharmacopeial Convention, Pharmacopeial Forum 26(1):223(2000)). When therapeutic proteins are administered in amounts of hundreds or thousands of milligrams per kilogram of body weight, as can be the case with target proteins (e.g., antibodies), even trace amounts of harmful and dangerous endotoxins must be removed. In some embodiments, the endotoxin and pyrogen levels in the composition are 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.

[0268] When used for in vivo administration, the compositions described herein should be sterile. The compositions can be sterilized by various sterilization methods, including aseptic filtration, radiation, etc. In certain embodiments, the compositions are sterilized by filtration using a pre-sterilized 0.22 micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical specifications as described in "Remington: The Science & Practice of Pharmacy ", 21st edition, Lippincott Williams & Wilkins, (2005). Compositions comprising target proteins (e.g., antibodies) such as those disclosed herein will typically be stored in lyophilized form or in solution form. It is expected that the sterile composition comprising the target protein (e.g., antibody) will be placed in a container with a sterile access port, such as an intravenous solution bag or a bottle with an adapter allowing the compositions to be recovered, such as a stopper pierced by a hypodermic needle. In certain embodiments, the compositions are provided in a pre-filled syringe form.

[0269] In certain embodiments, the composition is a lyophilized formulation.The term "lyophilized" or "freeze-dried" includes the state of a substance that has been subjected to a drying procedure, such as lyophilization, wherein at least 50% of the water has been removed.

[0270] Regardless of the route of administration selected, the agents provided herein that can 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.

[0271] In the methods provided herein, human antibodies, Fc fusion proteins and / or pharmaceutical compositions can be delivered by any suitable route of administration, including oral, nasal, rectal, vaginal, parenteral, intracisternal, and topical, including buccal and sublingual delivery, such as by powders, ointments or drops. In certain embodiments, the pharmaceutical composition is delivered systemically (e.g., orally or parenterally).

[0272] In certain embodiments, actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied to determine an amount of the active ingredient that is effective to achieve the desired therapeutic response in the animal models, composition, and mode of administration, without being toxic in the animal models.

[0273] For example, in certain embodiments, non-human animals as described herein are used to determine the pharmacokinetic profiles of one or more human antibody candidates. In various embodiments, one or more non-human animals as described herein and one or more controls 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 / 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 larger doses). Candidate therapeutic antibodies can be administered by any desired route of administration, including parenteral and non-parenteral administration. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intraventricular, intracranial, intrapleural or other injection routes. The non-parenteral route includes, for example, oral, nasal, transdermal, lung, rectal, buccal, vaginal, ocular. Administration can also be carried out by continuous infusion, topical application, sustained release from implants (gels, films, etc.) and / or intravenous injection. Blood is separated from non-human animals (humanization and control) at various time points (e.g., 0 hour, 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 days). Various assays can be performed to measure the pharmacokinetic profile of the candidate therapeutic antibody administered using samples obtained from non-human animals as described herein, including but not limited to total IgG, anti-therapeutic antibody responses, agglutination, etc.

[0274] In various embodiments, non-human animals as described herein are used to measure the effects of treatments that block or modulate the activity of a polypeptide of interest, as well as the effects on gene expression due to cellular changes, or in the case of receptor polypeptides, the effects on the density of receptor polypeptides on the surface of cells in the non-human animal. In various embodiments, non-human animals as described herein or cells isolated therefrom are exposed to a candidate therapeutic agent that binds to a polypeptide of interest, and after a subsequent period of time, the effects on specific cellular processes associated with the polypeptide of interest, such as ligand-receptor interactions or signal transduction, are analyzed.

[0275] Non-human animals as described herein provide a kind of for developing and selecting the improvement in vivo system of the people's antibody used in oncology and / or infectious disease.In various embodiments, non-human animals as described herein and control non-human animals (for example, there is genetic modification different from genetic modification as described herein or do not have genetic modification, i.e. wild type) can be implanted with tumor (or tumor cell) or infected with virus (for example influenza virus, HIV, HCV, HPV etc.).After implantation or infection, candidate therapeutic agent can be used to non-human animals.Before using candidate therapeutic agent, tumor or virus can be allowed to have enough time to set up in one or more positions in non-human animals.Alternatively and / or in addition, the immune response in the non-human animals can be monitored to characterize and select the potential human antibodies that can be used as therapeutic agent development.

[0276] Methods for preparing human antibodies

[0277] In certain aspects, provided herein are methods for preparing human antibodies using rodents (e.g., mice or rats) comprising humanized heavy chain loci provided herein and human light chain loci (e.g., humanized κ and / or λ light chain loci provided herein). In some embodiments, rodents also include humanized CD79a loci provided herein and / or humanized CD79b loci provided herein. In some embodiments, rodents also include humanized FcRn loci provided herein and / or humanized β2M loci provided herein. In certain embodiments, rodents also include humanized FcγR1a loci provided herein. In some embodiments, rodents also include humanized FcεR1α loci provided herein. In some embodiments, rodents also include humanized FcγR2b loci provided herein, humanized FcγR2c loci provided herein, humanized FcγR3a loci provided herein, and / or humanized FcγR3b loci provided herein.

[0278] Rodents provided herein can be used to prepare antibodies (e.g., human antibodies) using standard methods known in the art. For example, in some embodiments, the rodents provided herein are immunized with a target antigen, the immunization being sufficient to cause the rodent to produce an immune response to the target antigen and continuing to be sufficient to cause the rodent to produce an immune response to the target antigen. In some embodiments, the target antigen is a human antibody (e.g., a human therapeutic antibody) or an Fc fusion protein (e.g., a therapeutic Fc fusion protein). Antibodies are separated from rodents (or one or more cells, such as one or more B cells) and characterized using various assays measuring, for example, affinity, specificity, epitope mapping, the ability to block ligand-receptor interactions, inhibition of receptor activation, etc.

[0279] In some embodiments, a method for producing antibodies in a rodent (e.g., a mouse or rat) is provided, comprising the steps of: (a) immunizing a rodent producing human antibodies as described herein with a target antigen, (b) maintaining the rodent under conditions sufficient for the rodent to produce an immune response to the target antigen, and (c) recovering antibodies that bind to the target antigen from the rodent or rodent cells. In some embodiments, the method further comprises destroying immune tolerance to the antigen in the rodent, or otherwise depleting the target antigen, such as by employing a CRISPR / Cas9 system using multiple guide RNAs to reduce or eliminate expression of an autoantigen that is homologous or shares a target epitope with the target antigen used to immunize the rodent (e.g., as described in U.S. Patent Publication No. 2017 / 0332610, which is hereby incorporated by reference).

[0280] In some embodiments, a method for producing nucleic acids encoding human heavy and / or light chains in a rodent is provided, the method comprising the steps of: (a) immunizing a rodent expressing human antibodies as described herein with an antigen of interest, (b) maintaining the rodent under conditions sufficient for the rodent to generate an immune response to the antigen of interest, and (c) recovering nucleic acids encoding human heavy and / or light chains from the rodent or rodent cells.

[0281] In some embodiments, provided herein are rodents that can be used to prepare anti-drug antibodies (e.g., anti-idiotypic antibodies). For example, in some embodiments, human therapeutic antibodies are used to immunize rodents provided herein, and the immunity is sufficient to cause the rodent to produce an immune response to the human therapeutic antibodies under conditions sufficient to cause the rodent to produce an immune response to the human therapeutic antibodies and to continue for a time sufficient to cause the rodent to produce an immune response to the human therapeutic antibodies. In some embodiments, human therapeutic antibodies have the same heavy chain constant domains as the heavy chain constant domains encoded by the humanized immunoglobulin heavy chain loci of the rodents provided herein. In some embodiments, human therapeutic antibodies have the same light chain constant domains as the light chain constant domains encoded by the humanized immunoglobulin light chain loci (e.g., humanized κ and / or λ light chain loci) of the rodents provided herein. In some embodiments, rodents do not include one or more of the human variable region gene segments from which therapeutic antibodies are obtained in their genome (e.g., one or more immunoglobulin loci in rodents include rodent variable regions or portions thereof). Anti-drug antibodies (e.g., anti-idiotypic antibodies) are isolated from rodents (or one or more cells, such as one or more B cells) and characterized using various assays that measure, for example, affinity, specificity, epitope location, ability to block antigen-therapeutic antibody interactions, etc. The anti-drug antibodies (e.g., anti-idiotypic antibodies) generated can be used for pharmacokinetic (PK) analysis of human therapeutic antibodies, or for analyzing the immunogenicity of human therapeutic antibodies during preclinical analysis, or for localizing human therapeutic antibodies.

[0282] In some embodiments, a method of producing anti-drug antibodies (e.g., anti-idiotypic antibodies) in a rodent (e.g., a mouse or rat) is provided, the method comprising the steps of: (a) immunizing a rodent as described herein that produces antibodies comprising one or more human constant regions with a human therapeutic antibody that is isotype matched to a human constant region present in the rodent, (b) maintaining the rodent under conditions sufficient for the rodent to generate an immune response to the human therapeutic antibody, and (c) recovering antibodies that bind to the human therapeutic antibody from the rodent or rodent cells.

[0283] Methods for preparing genetically modified non-human animals and ES cells

[0284] In certain aspects, provided herein are methods for preparing non-human animals (such as 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 for preparing non-human animals (such as mice or rats) and ES cells comprising humanized heavy chain loci provided herein and / or human light chain loci provided herein (such as humanized κ and / or λ light chain loci provided herein). In some embodiments, provided herein are methods for preparing non-human animals (such as mice or rats) and ES cells further comprising humanized CD79a loci provided herein and / or humanized CD79b loci provided herein. In some embodiments, provided herein are methods for preparing non-human animals (such as mice or rats) and ES cells further comprising humanized FcRn loci provided herein and / or humanized β2M loci provided herein. In some embodiments, provided herein are methods for preparing non-human animals (such as mice or rats) and ES cells further comprising humanized FcεR1α loci provided herein. In certain embodiments, provided herein is a method for preparing a non-human animal (eg, mouse or rat) and an ES cell further comprising a humanized FcγR1a locus provided herein. In some embodiments, provided herein is a method for preparing a non-human animal (eg, mouse or rat) and an ES cell further comprising a humanized FcεR1α locus provided herein. In some embodiments, provided herein is a method for preparing a non-human animal (eg, mouse or rat) and an ES cell further comprising 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 and ES cell. In certain embodiments, provided herein is a method for preparing a non-human animal (eg, mouse or rat) and an ES cell comprising a humanized FcRn locus provided herein and / or a humanized β2M locus provided herein. In certain embodiments, provided herein are methods for preparing non-human animals (eg, mice or rats) and ES cells comprising the humanized FcεR1α loci provided herein. Exemplary methods for preparing the genetically modified non-human animals and ES cells provided herein are described in the description, examples, and / or figures herein.

[0285] Reagent test kit

[0286] Provided herein is a package or test kit comprising one or more containers, the containers being equipped with at least one non-human animal, non-human cell, DNA fragment and / or targeting vector as described herein, in the examples and / or in the accompanying drawings. Test kits can be used in any applicable method (e.g., research method). Optionally accompanied by the one or more containers may be a notification in the form specified by a government agency regulating the manufacture, use or sale of a drug or biological product, the notification reflecting (a) approval for manufacture, use or sale for human administration by the agency, (b) instructions for use, (c) protocols for the transfer of management materials and / or biological products (e.g., non-human animals or non-human cells as described herein) between two or more entities, and combinations thereof.

[0287] Additional exemplary embodiments

[0288] In exemplary embodiment 1, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a V H Gene segment, D H Gene segments and J H gene segments; and (ii) an immunoglobulin heavy chain constant region comprising a C encoding an IgG constant domain. H gene segment, the IgG constant domain comprises a human C H 1 domain, human hinge region, human C H 2 domains, human C H 3 domains, an IgG transmembrane domain, and an IgG cytoplasmic domain, wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces a protein comprising proteins derived from the V H Gene segment, the D H Gene segments and the J H The variable domains of the gene segments and the C H Gene segments of the heavy chain constant domain of IgG antibodies.

[0289] In exemplary embodiment 2, provided herein is the rodent of embodiment 1, wherein the IgG transmembrane domain is a rodent IgG transmembrane domain.

[0290] In exemplary embodiment 3, provided herein is the rodent of embodiment 1, wherein the IgG transmembrane domain is a human IgG transmembrane domain.

[0291] In exemplary embodiment 4, provided herein is the rodent of any one of embodiments 1 to 3, wherein the IgG cytoplasmic domain is a rodent IgG cytoplasmic domain.

[0292] In exemplary embodiment 5, provided herein is the rodent of any one of embodiments 1 to 3, wherein the IgG cytoplasmic domain is a human IgG cytoplasmic domain.

[0293] In exemplary embodiment 6, provided herein is the rodent of any one of embodiments 1 to 5, wherein the human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H The 3 domain is an IgG1 domain.

[0294] In exemplary embodiment 7, provided herein is the rodent of embodiment 6, wherein the IgG1 domain is encoded by an allele selected from the group consisting of IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04, and IGHG1*05.

[0295] In exemplary embodiment 8, provided herein is the rodent of any one of embodiments 1 to 5, wherein the human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H The 3 domain is an IgG2 domain.

[0296] In exemplary embodiment 9, provided herein is the rodent of embodiment 8, wherein the IgG2 domain is encoded by an allele selected from the group consisting of IGHG2*01, IGHG2*02, IGHG2*03, IGHG2*04, IGHG2*05, and IGHG2*06.

[0297] In exemplary embodiment 10, provided herein is the rodent of any one of embodiments 1 to 5, wherein the human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H The 3 domain is an IgG3 domain.

[0298] In exemplary embodiment 11, provided herein is the rodent of embodiment 10, wherein the IgG3 domain is encoded by an allele selected from the group consisting of 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.

[0299] In exemplary embodiment 12, provided herein is the rodent of any one of embodiments 1 to 5, wherein the human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H The 3 domain is an IgG4 domain.

[0300] In exemplary embodiment 13, provided herein is the rodent of embodiment 12, wherein the IgG4 domain is encoded by an allele selected from the group consisting of IGHG4*01, IGHG4*02, IGHG4*03, and IGHG4*04.

[0301] In exemplary embodiment 14, provided herein is the rodent of any one of embodiments 6 to 13, wherein the human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H 3 domains of the C H The gene segment is located in the endogenous C γ2a or C γ2c Gene segment locus.

[0302] In exemplary embodiment 15, provided herein is the rodent of embodiment 14, wherein the encoding human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H 3 domains of the C H Gene segment replaces endogenous C γ2a or C γ2c Gene segment.

[0303] In exemplary embodiment 16, provided herein is the rodent of any one of embodiments 6 to 13, wherein the human C H 1 domain, the human hinge region, the human C H2 domains and the human C H 3 domains of the C H The gene segment is located in the endogenous C γ1 Gene segment locus.

[0304] In exemplary embodiment 17, provided herein is the rodent of embodiment 16, wherein the encoding human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H 3 domains of the C H Gene segment replaces endogenous C γ1 Gene segment.

[0305] In exemplary embodiment 18, provided herein is the rodent of any one of embodiments 6 to 13, wherein the human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H 3 domains of the C H The gene segment is located in the endogenous C γ2b Gene segment locus.

[0306] In exemplary embodiment 19, provided herein is the rodent of embodiment 18, wherein the encoding human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H 3 domains of the C H Gene segment replaces endogenous C γ2b Gene segment.

[0307] In exemplary embodiment 20, provided herein is the rodent of any one of embodiments 6 to 13, wherein the human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H 3 domains of the C H The gene segment is located in the endogenous C γ3 Gene segment locus.

[0308] In exemplary embodiment 21, provided herein is the rodent of embodiment 20, wherein the encoding human C H 1 domain, the human hinge region, the human C H 2 domains and the human C H 3 domains of the C H Gene segment replaces endogenous C γ3 Gene segment.

[0309] In exemplary embodiment 22, provided herein is the rodent of any one of embodiments 1 to 21, wherein the immunoglobulin heavy chain constant region further comprises a rodent C μ Gene segment.

[0310] In exemplary embodiment 23, provided herein is the rodent of any one of embodiments 1 to 21, wherein the immunoglobulin heavy chain constant region further comprises a human C μ Gene segment.

[0311] In exemplary embodiment 24, provided herein is the rodent of any one of embodiments 1 to 23, wherein the immunoglobulin heavy chain constant region further comprises a rodent C δ Gene segment.

[0312] In exemplary embodiment 25, provided herein is the rodent of any one of embodiments 1 to 23, wherein the immunoglobulin heavy chain constant region further comprises a human C δ Gene segment.

[0313] In exemplary embodiment 26, provided herein is the rodent of any one of embodiments 1 to 25, wherein the immunoglobulin heavy chain constant region further comprises a rodent C γ1 Gene segment.

[0314] In exemplary embodiment 27, provided herein is the rodent of any one of embodiments 1 to 26, wherein the immunoglobulin heavy chain constant region further comprises a rodent C γ2a and / or C γ2c Gene segment.

[0315] In exemplary embodiment 28, provided herein is the rodent of any one of embodiments 1 to 27, wherein the immunoglobulin heavy chain constant region further comprises a rodent C γ2b Gene segment.

[0316] In exemplary embodiment 29, provided herein is the rodent of any one of embodiments 1 to 28, wherein the immunoglobulin heavy chain constant region further comprises a rodent C γ3 Gene segment.

[0317] In exemplary embodiment 30, provided herein is the rodent of any one of embodiments 1 to 29, wherein the immunoglobulin heavy chain constant region further comprises a rodent C ε Gene segment.

[0318] In exemplary embodiment 31, provided herein is the rodent of any one of embodiments 1 to 29, wherein the immunoglobulin heavy chain constant region further comprises a human C ε Gene segment.

[0319] In exemplary embodiment 32, provided herein is the rodent of any one of embodiments 1 to 31, wherein the immunoglobulin heavy chain constant region further comprises a rodent C α Gene segment.

[0320] In exemplary embodiment 33, provided herein is the rodent of any one of embodiments 1 to 31, wherein the immunoglobulin heavy chain constant region further comprises a human C α Gene segment.

[0321] In exemplary embodiment 34, provided herein is the rodent of any one of embodiments 1 to 21, wherein the immunoglobulin heavy chain constant region comprises a human C μ Gene segment, human C δ Gene segment, human C γ3 gene segments and human C γ1 Gene segment.

[0322] In exemplary embodiment 35, provided herein is the rodent of embodiment 34, wherein the immunoglobulin heavy chain constant region further comprises a human C γ2 gene segments and human C γ4 Gene segment.

[0323] In exemplary embodiment 36, provided herein is the rodent of embodiment 34 or embodiment 35, wherein the immunoglobulin heavy chain constant region further comprises a human C α Gene segment.

[0324] In exemplary embodiment 37, provided herein is the rodent of any one of embodiments 34 to 36, wherein the immunoglobulin heavy chain constant region further comprises a human C ε Gene segment.

[0325] In exemplary embodiment 38, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a V H Gene segment, D H Gene segments and J H gene segment; and (ii) an immunoglobulin heavy chain constant region comprising a C encoding an IgM constant domain Hgene segment, the IgM constant domain comprises a human C H 1 domain, human C H 2 domains, human C H 3 domains, human C H 4 domains, an IgM transmembrane domain, and an IgM cytoplasmic domain, wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces an immunoglobulin heavy chain variable region comprising a heavy chain constant region derived from the V H Gene segment, the D H Gene segments and the J H The variable domains of the gene segments and the C H IgM antibody heavy chain constant domain gene segments.

[0326] In exemplary embodiment 39, provided herein is the rodent of embodiment 38, wherein the immunoglobulin heavy chain constant region further comprises a human C δ Gene segment.

[0327] In exemplary embodiment 40, provided herein is the rodent of embodiment 38 or 39, wherein the immunoglobulin heavy chain constant region further comprises a human C γ3 Gene segment.

[0328] In exemplary embodiment 41, provided herein is the rodent of any one of embodiments 38 to 40, wherein the immunoglobulin heavy chain constant region further comprises a human C γ1 Gene segment.

[0329] In exemplary embodiment 42, provided herein is the rodent of any one of embodiments 38 to 41, wherein the immunoglobulin heavy chain constant region further comprises a human C γ2 Gene segment.

[0330] In exemplary embodiment 43, provided herein is the rodent of any one of embodiments 38 to 42, wherein the immunoglobulin heavy chain constant region further comprises a human C γ4 Gene segment.

[0331] In exemplary embodiment 44, provided herein is the rodent of any one of embodiments 38 to 43, wherein the immunoglobulin heavy chain constant region comprises a human C μ Gene segment, human C δ Gene segment, human C γ3 gene segments and human C γ1 Gene segment.

[0332] In exemplary embodiment 45, provided herein is the rodent of embodiment 44, wherein the immunoglobulin heavy chain constant region further comprises a human C γ2 gene segments and human C γ4 Gene segment.

[0333] In exemplary embodiment 46, provided herein is the rodent of embodiment 44 or embodiment 45, wherein the immunoglobulin heavy chain constant region further comprises a human C α Gene segment.

[0334] In exemplary embodiment 47, provided herein is the rodent of any one of embodiments 44 to 46, wherein the immunoglobulin heavy chain constant region further comprises a human C ε Gene segment.

[0335] In exemplary embodiment 48, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a V H Gene segment, D H Gene segments and J H gene segment; and (ii) an immunoglobulin heavy chain constant region comprising a C encoding an IgD constant domain H gene segment, the IgD constant domain comprises a human C H 1 domain, human hinge H1 domain, human hinge H2 domain, human C H 2 domains, human C H 3 domains, a human IgD transmembrane domain, and a human IgD cytoplasmic domain, wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces an immunoglobulin heavy chain variable region comprising a heavy chain constant region derived from the V H Gene segment, the D H Gene segments and the J H The variable domains of the gene segments and the C H Gene segments of the heavy chain constant domain of IgD antibodies.

[0336] In exemplary embodiment 49, provided herein is the rodent of embodiment 48, wherein the immunoglobulin heavy chain constant region further comprises a human C μ Gene segment.

[0337] In exemplary embodiment 50, provided herein is the rodent of embodiment 48 or 49, wherein the immunoglobulin heavy chain constant region further comprises a human C γ3 Gene segment.

[0338] In exemplary embodiment 51, provided herein is the rodent of any one of embodiments 48 to 50, wherein the immunoglobulin heavy chain constant region further comprises a human C γ1 Gene segment.

[0339] In exemplary embodiment 52, provided herein is the rodent of any one of embodiments 48 to 51, wherein the immunoglobulin heavy chain constant region further comprises a human C γ2 Gene segment.

[0340] In exemplary embodiment 53, provided herein is the rodent of any one of embodiments 48 to 52, wherein the immunoglobulin heavy chain constant region further comprises a human C γ4 Gene segment.

[0341] In exemplary embodiment 54, provided herein is the rodent of any one of embodiments 48 to 53, wherein the immunoglobulin heavy chain constant region comprises a human C μ Gene segment, human C δ Gene segment, human C γ3 gene segments and human C γ1 Gene segment.

[0342] In exemplary embodiment 55, provided herein is the rodent of embodiment 54, wherein the immunoglobulin heavy chain constant region further comprises a human C γ2 gene segments and human C γ4 Gene segment.

[0343] In exemplary embodiment 56, provided herein is the rodent of embodiment 54 or embodiment 55, wherein the immunoglobulin heavy chain constant region further comprises a human C α Gene segment.

[0344] In exemplary embodiment 57, provided herein is the rodent of any one of embodiments 54 to 56, wherein the immunoglobulin heavy chain constant region further comprises a human C ε Gene segment.

[0345] In exemplary embodiment 58, provided herein is the rodent of any one of embodiments 1 to 57, wherein the engineered immunoglobulin heavy chain locus further comprises a rodent intronic enhancer (E i ).

[0346] In exemplary embodiment 59, provided herein is the rodent of any one of embodiments 1 to 57, wherein the engineered immunoglobulin heavy chain locus further comprises a human intronic enhancer (Ei ).

[0347] In exemplary embodiment 60, provided herein is the rodent of any one of embodiments 1 to 59, wherein the engineered immunoglobulin heavy chain locus further comprises a rodent 3' regulatory region (3'RR).

[0348] In exemplary embodiment 61, provided herein is the rodent of any one of embodiments 1 to 5, wherein the engineered immunoglobulin heavy chain locus further comprises a human 3' regulatory region (3'RR).

[0349] In exemplary embodiment 62, provided herein is the rodent of any one of embodiments 1 to 61, wherein the engineered immunoglobulin heavy chain locus further comprises a rodent S μ Conversion site.

[0350] In exemplary embodiment 63, provided herein is the rodent of any one of embodiments 1 to 62, wherein the engineered immunoglobulin heavy chain locus further comprises a rodent S γ3 Conversion site.

[0351] In exemplary embodiment 64, provided herein is the rodent of any one of embodiments 1 to 63, wherein the engineered immunoglobulin heavy chain locus further comprises a rodent S γ1 Conversion site.

[0352] In exemplary embodiment 65, provided herein is the rodent of any one of embodiments 1 to 64, wherein the engineered immunoglobulin heavy chain locus further comprises a rodent S γ2b Conversion site.

[0353] In exemplary embodiment 66, provided herein is the rodent of any one of embodiments 1 to 65, wherein the engineered immunoglobulin heavy chain locus further comprises a rodent S γ2a and / or S γ2c Conversion site.

[0354] In exemplary embodiment 67, provided herein is the rodent of any one of embodiments 1 to 66, wherein the engineered immunoglobulin heavy chain locus further comprises a rodent S ε Conversion site.

[0355] In exemplary embodiment 68, provided herein is the rodent of any one of embodiments 1 to 67, wherein the engineered immunoglobulin heavy chain locus further comprises a rodent S αConversion site.

[0356] In exemplary embodiment 69, provided herein is the rodent of any one of embodiments 1 to 61, wherein the engineered immunoglobulin heavy chain locus further comprises a human S μ Conversion site.

[0357] In exemplary embodiment 70, provided herein is the rodent of any one of embodiments 1 to 61 and 69, wherein the engineered immunoglobulin heavy chain locus further comprises a human S γ3 Conversion site.

[0358] In exemplary embodiment 71, provided herein is the rodent of any one of embodiments 1 to 61 and 69-70, wherein the engineered immunoglobulin heavy chain locus further comprises a human S γ1 Conversion site.

[0359] In exemplary embodiment 72, provided herein is the rodent of any one of embodiments 1 to 61 and 69-71, wherein the engineered immunoglobulin heavy chain locus further comprises a human S γ2 Conversion site.

[0360] In exemplary embodiment 73, provided herein is the rodent of any one of embodiments 1 to 61 and 69-72, wherein the engineered immunoglobulin heavy chain locus further comprises a human S γ4 Conversion site.

[0361] In exemplary embodiment 74, provided herein is the rodent of any one of embodiments 1 to 61 and 69-73, wherein the engineered immunoglobulin heavy chain locus further comprises a human S ε Conversion site.

[0362] In exemplary embodiment 75, provided herein is the rodent of any one of embodiments 1 to 61 and 69-74, wherein the engineered immunoglobulin heavy chain locus further comprises a human S α Conversion site.

[0363] In exemplary embodiment 76, provided herein is the rodent of any one of embodiments 1 to 75, wherein the V H The gene segment is rodent V H gene segment, the D H The gene segment is rodent D H gene segment, and the J H The gene segment is rodent J H Gene segment.

[0364] In exemplary embodiment 77, provided herein is the rodent of embodiment 76, wherein the rodent V H Gene segment, the rodent D H Gene segments and the rodent J H The gene segment is an endogenous rodent gene segment.

[0365] In exemplary embodiment 78, provided herein is the rodent of any one of embodiments 1 to 75, wherein the V H The gene segment is human V H gene segment, the D H The gene segment is human D H gene segment, and the J H The gene segment is human J H Gene segment.

[0366] In exemplary embodiment 79, provided herein is the rodent of embodiment 78, wherein the immunoglobulin heavy chain variable region comprises at least 3 human V H Gene segment.

[0367] In exemplary embodiment 80, provided herein is the rodent of embodiment 78 or embodiment 79, wherein the immunoglobulin heavy chain variable region comprises a human D H All in the gene segment.

[0368] In exemplary embodiment 81, provided herein is the rodent of any one of embodiments 78 to 80, wherein the immunoglobulin heavy chain variable region comprises a human J H All in the gene segment.

[0369] In exemplary embodiment 82, provided herein is the rodent of any one of embodiments 78 to 81, wherein the immunoglobulin heavy chain variable region lacks a functional endogenous rodent Adam6 gene.

[0370] In exemplary embodiment 83, provided herein is a rodent as 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.

[0371] In exemplary embodiment 84, provided herein is the rodent of embodiment 83, wherein the functional rodent Adam6 polypeptide, the functional ortholog thereof, the functional homolog or the functional fragment thereof is expressed.

[0372] In exemplary embodiment 85, provided herein is a rodent as described in embodiment 83 or embodiment 84, wherein the nucleotide sequence encoding the rodent ADAM6 polypeptide, the functional homolog thereof, the functional homolog or the functional fragment is included on the same chromosome as the immunoglobulin heavy chain variable region.

[0373] In exemplary embodiment 86, provided herein is a rodent as described in any one of embodiments 83 to 85, wherein the nucleotide sequence encoding the rodent ADAM6 polypeptide, the functional homolog thereof, the functional homolog or the functional fragment is included in the engineered immunoglobulin heavy chain locus.

[0374] In exemplary embodiment 87, provided herein is a rodent as described in any one of embodiments 83 to 86, wherein the nucleotide sequence encoding the rodent ADAM6 polypeptide, the functional ortholog thereof, the functional homolog or the functional fragment replaces the human Adam6 pseudogene.

[0375] In exemplary embodiment 88, provided herein is a rodent as described in any one of embodiments 83 to 87, wherein the nucleotide sequence encoding the rodent ADAM6 polypeptide, the functional ortholog thereof, the functional homolog or the functional fragment replaces the human Adam6 pseudogene.

[0376] In exemplary embodiment 89, provided herein is the rodent of any one of embodiments 83 to 88, wherein the nucleotide sequence encoding the rodent ADAM6 polypeptide, the functional ortholog thereof, the functional homolog, or the functional fragment thereof is in the first human V H Gene segment and second human V H between gene segments.

[0377] In exemplary embodiment 90, provided herein is the rodent of embodiment 89, wherein the first human V H The gene segment is V H 1-2, and the second person V H The gene segment is V H 6-1.

[0378] In exemplary embodiment 91, provided herein is the rodent of any one of embodiments 83 to 86, wherein the nucleotide sequence encoding the rodent ADAM6 polypeptide, the functional ortholog thereof, the functional homolog or the functional fragment thereof is in the human V H Gene segment and human D H between gene segments.

[0379] In exemplary embodiment 92, provided herein is the rodent of any one of embodiments 1 to 91, wherein the engineered immunoglobulin heavy chain locus is located at an endogenous immunoglobulin heavy chain locus.

[0380] In exemplary embodiment 93, provided herein is the rodent of embodiment 92, wherein the engineered immunoglobulin heavy chain locus replaces all or a portion of the endogenous immunoglobulin heavy chain locus.

[0381] In exemplary embodiment 94, provided herein is the rodent of any one of embodiments 1 to 93, wherein the rodent is heterozygous for the engineered immunoglobulin heavy chain locus.

[0382] In exemplary embodiment 95, provided herein is the rodent of any one of embodiments 1 to 93, wherein the rodent is homozygous for the engineered immunoglobulin heavy chain locus.

[0383] In exemplary embodiment 96, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a human V H Gene segment, human D H Gene segments and human J H gene segment; (ii) rodent intronic enhancer (E i ); (iii) an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region comprising: (a) a rodent C μ Gene segment; (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) encoding human IgG1 C H 1 domain, human IgG1 hinge region, human IgG1 C H 2 domains, human IgG1 C H 3 domains, a rodent IgG2a transmembrane domain, and a modified C of the rodent IgG2a cytoplasmic domain H Gene segment; (g) rodent C ε gene segment; and (h) rodent C αgene segment; and (iv) a rodent 3′ regulatory region (3′RR), wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces a protein comprising a heavy chain variable region derived from the human V H gene segment, the human D H Gene segments and the human J H The variable domain of the gene segment and the variable domain derived from the modified C H Gene segments of the heavy chain constant domain of IgG antibodies.

[0384] In exemplary embodiment 97, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a human V H Gene segment, human D H Gene segments and human J H gene segment; (ii) rodent intronic enhancer (E i ); (iii) an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region comprising: (a) a rodent C μ Gene segment; (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) encoding human IgG1 C H 1 domain, human IgG1 hinge region, human IgG1 C H 2 domains, human IgG1 C H 3 domains, human IgG1 transmembrane domain and human IgG1 cytoplasmic domain. H Gene segment; (g) rodent C ε gene segment; and (h) rodent C α gene segment; and (iv) a rodent 3′ regulatory region (3′RR), wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces a protein comprising a heavy chain variable region derived from the human V H gene segment, the human D H Gene segments and the human J H The variable domain of the gene segment and the variable domain derived from the modified C H Gene segments of the heavy chain constant domain of IgG antibodies.

[0385] In exemplary embodiment 98, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a human V H Gene segment, human D H Gene segments and human J H gene segment; (ii) rodent intronic enhancer (E i ); (iii) an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region comprising: (a) a rodent C μ Gene segment; (b) rodent C δ Gene segment; (c) rodent C γ3 Gene segment; (d) encoding human IgG4 C H 1 domain, human IgG4 hinge region, human IgG4 C H 2 domains, human IgG4 C H 3 domains, a rodent IgG1 transmembrane domain, and a modified C of the 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 segment; and (h) rodent C α gene segment; and (iv) a rodent 3′ regulatory region (3′RR), wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces a protein comprising a heavy chain variable region derived from the human V H gene segment, the human D H Gene segments and the human J H The variable domain of the gene segment and the variable domain derived from the modified C H Gene segments of the heavy chain constant domain of IgG antibodies.

[0386] In exemplary embodiment 99, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a human V H Gene segment, human D H Gene segments and human J H gene segment; (ii) rodent intronic enhancer (E i ); (iii) an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region comprising: (a) a rodent Cμ Gene segment; (b) rodent C δ Gene segment; (c) rodent C γ3 Gene segment; (d) encoding human IgG4 C H 1 domain, human IgG4 hinge region, human IgG4 C H 2 domains, human IgG4 C H 3 domains, human IgG4 transmembrane domain and 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 segment; and (h) rodent C α gene segment; and (iv) a rodent 3′ regulatory region (3′RR), wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces a protein comprising a heavy chain variable region derived from the human V H gene segment, the human D H Gene segments and the human J H The variable domain of the gene segment and the variable domain derived from the modified C H Gene segments of the heavy chain constant domain of IgG antibodies.

[0387] In exemplary embodiment 100, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a human V H Gene segment, human D H Gene segments and human J H gene segment; (ii) human intronic enhancer (E i ); (iii) an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region comprising: (a) a human C μ Gene segment; (b) human C δ Gene segment; (c) human C γ3 Gene segment; (d) human C γ1 gene segment; and (iv) a rodent 3' regulatory region (3'RR), wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces IgG antibodies comprising fully human heavy chains.

[0388] In exemplary embodiment 101, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a human V H Gene segment, human D H Gene segments and human J H gene segment; (ii) human intronic enhancer (E i ); (iii) an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region comprising: (a) a human C μ Gene segment; (b) human C δ Gene segment; (c) human C γ3 Gene segment; (d) human C γ1 Gene segment; (c) human C γ2 Gene segment; (d) human C γ4 gene segment; and (iv) a rodent 3' regulatory region (3'RR), wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces IgG antibodies comprising fully human heavy chains.

[0389] In exemplary embodiment 102, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a rodent V H Gene segment, rodent D H Gene segments and rodent J H gene segment; (ii) rodent intronic enhancer (E i ); (iii) an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region comprising: (a) a human C μ Gene segment; (b) human C δ Gene segment; (c) human C γ3 Gene segment; (d) human C γ1 gene segment; and (iv) a rodent 3' regulatory region (3'RR), wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces IgG antibodies comprising heavy chains comprising rodent variable domains and human constant domains.

[0390] In exemplary embodiment 103, provided herein is a rodent comprising in its genome: an engineered immunoglobulin heavy chain locus comprising: (i) an immunoglobulin heavy chain variable region comprising a rodent V H Gene segment, rodent D H Gene segments and rodent J H gene segment; (ii) rodent intronic enhancer (E i ); (iii) an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region comprising: (a) a human C μ Gene segment; (b) human C δ Gene segment; (c) human C γ3 Gene segment; (d) human C γ1 Gene segment; (c) human C γ2 Gene segment; (d) human C γ4 gene segment; and (iv) a rodent 3' regulatory region (3'RR), wherein the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region, such that the rodent produces IgG antibodies comprising heavy chains comprising rodent variable domains and human constant domains.

[0391] In exemplary embodiment 104, provided herein is the rodent of any one of embodiments 96 to 103, wherein the engineered immunoglobulin heavy chain locus is located at an endogenous immunoglobulin heavy chain locus.

[0392] In exemplary embodiment 105, provided herein is the rodent of embodiment 104, wherein the engineered immunoglobulin heavy chain locus replaces all or a portion of the endogenous immunoglobulin heavy chain locus.

[0393] In exemplary embodiment 106, provided herein is the rodent of any one of embodiments 96 to 105, wherein the rodent is heterozygous for the engineered immunoglobulin heavy chain locus.

[0394] In exemplary embodiment 107, provided herein is the rodent of any one of embodiments 96 to 105, wherein the rodent is homozygous for the engineered immunoglobulin heavy chain locus.

[0395] In exemplary embodiment 108, provided herein is a rodent as described in any one of embodiments 1 to 107, further comprising in its genome: an engineered immunoglobulin kappa (kappa) chain locus, the engineered immunoglobulin kappa chain locus comprising: (1) an immunoglobulin kappa chain variable region comprising a human V κ Gene segments and human J κ gene segment; and (2) an immunoglobulin kappa chain constant region comprising a human C κ gene segment, wherein the immunoglobulin kappa chain variable region is operably linked to the immunoglobulin kappa chain constant region, such that the rodent produces a protein comprising a variable region derived from the human V κ Gene segments and the human J κ The light chain variable domain of the gene segment and the light chain variable domain of the human C κ light chain constant domain gene segments of antibodies.

[0396] In exemplary embodiment 109, provided herein is the rodent of embodiment 108, wherein the engineered immunoglobulin kappa chain locus further comprises a rodent intronic kappa enhancer (E κi ).

[0397] In exemplary embodiment 110, provided herein is the rodent of embodiment 108, wherein the engineered immunoglobulin kappa chain locus further comprises a human intronic kappa enhancer (E κi ).

[0398] In exemplary embodiment 111, provided herein is the rodent of any one of embodiments 108 to 110, wherein the engineered immunoglobulin kappa chain locus further comprises a rodent 3′ kappa enhancer (E κ3′ ).

[0399] In exemplary embodiment 112, provided herein is the rodent of any one of embodiments 108 to 110, wherein the engineered immunoglobulin kappa chain locus further comprises a human 3′ kappa enhancer (E κ3′ ).

[0400] In exemplary embodiment 113, provided herein is the rodent of any one of embodiments 108 to 112, wherein the immunoglobulin kappa chain variable region comprises at least 6 human V κ Gene segment.

[0401] In exemplary embodiment 114, provided herein is the rodent of any one of embodiments 108 to 113, wherein the immunoglobulin kappa chain variable region comprises a human Jκ All in the gene segment.

[0402] In exemplary embodiment 115, provided herein is the rodent of any one of embodiments 108 to 114, wherein the engineered immunoglobulin kappa chain locus is located at an endogenous immunoglobulin kappa chain locus.

[0403] In exemplary embodiment 116, provided herein is the rodent of embodiment 115, wherein the engineered immunoglobulin kappa chain locus replaces all or a portion of the endogenous immunoglobulin kappa chain locus.

[0404] In exemplary embodiment 117, provided herein is the rodent of any one of embodiments 108 to 116, wherein the rodent is heterozygous for the engineered immunoglobulin kappa chain locus.

[0405] In exemplary embodiment 118, provided herein is the rodent of any one of embodiments 108 to 116, wherein the rodent is homozygous for the engineered immunoglobulin kappa chain locus.

[0406] In exemplary embodiment 119, provided herein is the rodent of any one of embodiments 1 to 118, further comprising in its genome: an engineered immunoglobulin lambda chain locus comprising: a human V λ Gene segment, human J λ gene segments and human C λ gene segment, wherein the human V λ Gene segments and the human J λ The gene segment is operably linked to the human C λ gene segments, so that the rodent produces a gene comprising a gene derived from the human V λ Gene segments and the human J λ The light chain variable domain of the gene segment and the light chain variable domain of the human C λ light chain constant domain gene segments of antibodies.

[0407] In exemplary embodiment 120, provided herein is the rodent of embodiment 119, wherein the human C λ The gene segment is human C λ1 Gene segment.

[0408] In exemplary embodiment 121, provided herein is the rodent of embodiment 120, wherein the human J λ The gene segment is human J λ1Gene segment.

[0409] In exemplary embodiment 122, provided herein is the rodent of embodiment 119, wherein the human C λ The gene segment is human C λ2 Gene segment.

[0410] In exemplary embodiment 123, provided herein is the rodent of embodiment 122, wherein the human J λ The gene segment is human J λ2 Gene segment.

[0411] In exemplary embodiment 124, provided herein is the rodent of embodiment 119, wherein the human C λ The gene segment is human C λ3 Gene segment.

[0412] In exemplary embodiment 125, provided herein is the rodent of embodiment 124, wherein the human J λ The gene segment is human J λ3 Gene segment.

[0413] In exemplary embodiment 126, provided herein is the rodent of embodiment 119, wherein the human C λ The gene segment is human C λ6 Gene segment.

[0414] In exemplary embodiment 127, provided herein is the rodent of embodiment 126, wherein the human J λ The gene segment is human J λ6 Gene segment.

[0415] In exemplary embodiment 128, provided herein is the rodent of embodiment 119, wherein the human J λ The gene segment is human J λ7 Gene segment.

[0416] In exemplary embodiment 129, provided herein is the rodent of any one of embodiments 119 to 128, wherein the engineered immunoglobulin lambda chain locus comprises a human C λ1 Gene segment, human C λ2 Gene segment, human C λ3 Gene segment, human C λ6 gene segments and rodent C λ1 Gene segment.

[0417] In exemplary embodiment 130, provided herein is the rodent of embodiment 129, wherein the engineered immunoglobulin lambda chain locus comprises 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 Gene segment.

[0418] In exemplary embodiment 131, provided herein is the rodent ES cell of embodiment 130, wherein the engineered immunoglobulin lambda chain locus comprises a human J 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 Gene segment clusters.

[0419] In exemplary embodiment 132, provided herein is the rodent of any one of embodiments 119 to 131, wherein the engineered immunoglobulin lambda chain locus comprises at least 7 human V λ Gene segment.

[0420] In exemplary embodiment 133, provided herein is the rodent of any one of embodiments 119 to 132, wherein the engineered immunoglobulin lambda chain locus further comprises a rodent lambda enhancer 2.4.

[0421] In exemplary embodiment 134, provided herein is the rodent of any one of embodiments 119 to 133, wherein the engineered immunoglobulin lambda chain locus further comprises a rodent 3' lambda enhancer.

[0422] In exemplary embodiment 135, provided herein is the rodent of any one of embodiments 119 to 134, wherein the engineered immunoglobulin lambda chain locus further comprises rodent lambda enhancer 3.1.

[0423] In exemplary embodiment 136, provided herein is the rodent of any one of embodiments 119 to 135, wherein the engineered immunoglobulin lambda chain locus further comprises a human 3′ lambda enhancer.

[0424] In exemplary embodiment 137, provided herein is the rodent of any one of embodiments 119 to 136, wherein the engineered immunoglobulin lambda chain locus is located at an endogenous immunoglobulin lambda chain locus.

[0425] In exemplary embodiment 138, provided herein is the rodent of embodiment 137, wherein the engineered immunoglobulin lambda chain locus replaces all or a portion of the endogenous immunoglobulin lambda chain locus.

[0426] In exemplary embodiment 139, provided herein is the rodent of any one of embodiments 119 to 138, wherein the rodent is heterozygous for the engineered immunoglobulin lambda chain locus.

[0427] In exemplary embodiment 140, provided herein is the rodent of any one of embodiments 119 to 138, wherein the rodent is homozygous for the engineered immunoglobulin lambda chain locus.

[0428] In exemplary embodiment 141, provided herein is the rodent of any one of embodiments 1 to 140, further comprising in its genome an engineered neonatal Fc receptor (FcRn) locus comprising a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain.

[0429] In exemplary embodiment 142, provided herein is the rodent of embodiment 141, wherein the FcRn polypeptide further comprises a rodent transmembrane domain.

[0430] In exemplary embodiment 143, provided herein is the rodent of embodiment 141, wherein the FcRn polypeptide further comprises a human transmembrane domain.

[0431] In exemplary embodiment 144, provided herein is the rodent of any one of embodiments 141 to 143, wherein the FcRn polypeptide further comprises a rodent cytoplasmic domain.

[0432] In exemplary embodiment 145, provided herein is the rodent of any one of embodiments 141 to 143, wherein the FcRn polypeptide further comprises a human cytoplasmic domain.

[0433] In exemplary embodiment 146, provided herein is the rodent of any one of embodiments 141 to 145, wherein the nucleic acid sequence encoding the FcRn polypeptide is located at an endogenous rodent FcRn locus.

[0434] In exemplary embodiment 147, provided herein is the rodent of embodiment 146, wherein the nucleic acid sequence encoding the FcRn polypeptide replaces all or a portion of an endogenous rodent FcRn gene.

[0435] In exemplary embodiment 148, provided herein is the rodent of embodiment 141, wherein the nucleic acid sequence encoding the FcRn extracellular domain replaces an endogenous nucleic acid sequence encoding the rodent FcRn extracellular domain.

[0436] In exemplary embodiment 149, provided herein is the rodent of any one of embodiments 141 to 148, wherein the rodent does not express rodent FcRn.

[0437] In exemplary embodiment 150, provided herein is the rodent of any one of embodiments 141 to 149, wherein the rodent is heterozygous for the engineered FcRn locus.

[0438] In exemplary embodiment 151, provided herein is the rodent of any one of embodiments 141 to 149, wherein the rodent is homozygous for the engineered FcRn locus.

[0439] In exemplary embodiment 152, provided herein is the rodent of any one of embodiments 141 to 151, further comprising in its genome an engineered beta-2-microglobulin (β2M) locus comprising a nucleic acid sequence encoding a human or humanized beta-2-microglobulin (β2M) polypeptide.

[0440] In exemplary embodiment 153, provided herein is the rodent of embodiment 152, wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide is located at an endogenous rodent β2M locus.

[0441] In exemplary embodiment 154, provided herein is the rodent of embodiment 153, wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide replaces all or a portion of the endogenous rodent β2M gene.

[0442] In exemplary embodiment 155, provided herein is the rodent of any one of embodiments 152 to 154, wherein the nucleic acid sequence comprises exons 2-4 of the human β2M gene.

[0443] In exemplary embodiment 156, provided herein is the rodent of any one of embodiments 152 to 155, wherein the rodent does not express a rodent β2M polypeptide.

[0444] In exemplary embodiment 157, provided herein is the rodent of any one of embodiments 152 to 156, wherein the rodent is heterozygous for the engineered β2M locus.

[0445] In exemplary embodiment 158, provided herein is the rodent of any one of embodiments 152 to 156, wherein the rodent is homozygous for the engineered β2M locus.

[0446] In exemplary embodiment 159, provided herein is a rodent as described in any one of embodiments 1 to 158, further comprising in its genome an engineered Fcε receptor 1α (FcεR1α) locus comprising a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain.

[0447] In exemplary embodiment 160, provided herein is the rodent of embodiment 159, wherein the FcεR1α polypeptide further comprises a rodent transmembrane domain.

[0448] In exemplary embodiment 161, provided herein is the rodent of embodiment 159, wherein the FcεR1α polypeptide further comprises a human transmembrane domain.

[0449] In exemplary embodiment 162, provided herein is the rodent of any one of embodiments 159 to 161, wherein the FcεR1α polypeptide further comprises a rodent cytoplasmic domain.

[0450] In exemplary embodiment 163, provided herein is the rodent of any one of embodiments 159 to 161, wherein the FcεR1α polypeptide further comprises a human cytoplasmic domain.

[0451] In exemplary embodiment 164, provided herein is the rodent of any one of embodiments 159 to 163, wherein the nucleic acid sequence encoding the FcεR1α polypeptide is located at an endogenous rodent FcεR1α locus.

[0452] In exemplary embodiment 165, provided herein is the rodent of embodiment 164, wherein the nucleic acid sequence encoding the FcεR1α polypeptide replaces all or a portion of an endogenous rodent FcεR1α gene.

[0453] 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.

[0454] In exemplary embodiment 167, provided herein is the rodent of embodiment 159, wherein the nucleic acid sequence encoding the human FcεR1α extracellular domain replaces the endogenous nucleic acid sequence encoding the rodent FcεR1α extracellular domain.

[0455] In exemplary embodiment 168, provided herein is the rodent of any one of embodiments 159 to 167, wherein the rodent does not express rodent FcεR1α.

[0456] In exemplary embodiment 169, provided herein is the rodent of any one of embodiments 159 to 168, wherein the rodent is heterozygous for the engineered FcεR1α locus.

[0457] In exemplary embodiment 170, provided herein is the rodent of any one of embodiments 159 to 168, wherein the rodent is homozygous for the engineered FcεR1α locus.

[0458] In exemplary embodiment 171, provided herein is the rodent of any one of embodiments 1 to 170, further comprising in its genome an engineered Fcγ receptor 1a (FcγR1a) locus comprising a nucleic acid sequence encoding an FcγR1a polypeptide comprising a human extracellular domain.

[0459] In exemplary embodiment 172, provided herein is the rodent of embodiment 171, wherein the FcγR1a polypeptide further comprises a rodent transmembrane domain.

[0460] In exemplary embodiment 173, provided herein is the rodent of embodiment 171, wherein the FcγR1a polypeptide further comprises a human transmembrane domain.

[0461] In exemplary embodiment 174, provided herein is the rodent of any one of embodiments 171 to 173, wherein the FcγR1a polypeptide further comprises a rodent cytoplasmic domain.

[0462] In exemplary embodiment 175, provided herein is the rodent of any one of embodiments 171 to 173, wherein the FcγR1a polypeptide further comprises a human cytoplasmic domain.

[0463] In exemplary embodiment 176, provided herein is the rodent of any one of embodiments 171 to 175, wherein the nucleic acid sequence encoding the FcγR1a polypeptide is located at an endogenous rodent FcγR1a locus.

[0464] In exemplary embodiment 177, provided herein is the rodent of embodiment 176, wherein the nucleic acid sequence encoding the FcγR1a polypeptide replaces all or a portion of an endogenous rodent FcγR1a gene.

[0465] In exemplary embodiment 178, provided herein is the rodent of embodiment 171, wherein the nucleic acid sequence encoding the extracellular domain of human FcγR1a replaces the endogenous nucleic acid sequence encoding the extracellular domain of rodent FcγR1a.

[0466] In exemplary embodiment 179, provided herein is the rodent of any one of embodiments 171 to 178, wherein the rodent does not express rodent FcγR1a.

[0467] In exemplary embodiment 180, provided herein is the rodent of any one of embodiments 171 to 179, wherein the rodent is heterozygous for the engineered FcγR1a locus.

[0468] In exemplary embodiment 181, provided herein is the rodent of any one of embodiments 171 to 179, wherein the rodent is homozygous for the engineered FcγR1a locus.

[0469] In exemplary embodiment 182, provided herein is the rodent of any one of embodiments 1 to 181, further comprising in its genome an engineered Fcγ receptor 2a (FcγR2a) locus comprising a nucleic acid sequence encoding a human FcγR2a polypeptide.

[0470] In exemplary embodiment 183, provided herein is the rodent of embodiment 182, wherein the nucleic acid sequence encoding the FcγR2a polypeptide is located at an endogenous rodent low-affinity FcγR locus.

[0471] In exemplary embodiment 184, provided herein is the rodent of embodiment 183, wherein the nucleic acid sequence encoding the human FcγR2a polypeptide replaces all or a portion of an endogenous rodent low affinity FcγR gene.

[0472] In exemplary embodiment 185, provided herein is the rodent of any one of embodiments 182 to 184, wherein the rodent is heterozygous for the engineered FcγR2a locus.

[0473] In exemplary embodiment 186, provided herein is the rodent of any one of embodiments 182 to 184, wherein the rodent is homozygous for the engineered FcγR2a locus.

[0474] In exemplary embodiment 187, provided herein is the rodent of any one of embodiments 1 to 186, further comprising in its genome an engineered Fcγ receptor 2b (FcγR2b) locus comprising a nucleic acid sequence encoding a human FcγR2b polypeptide.

[0475] In exemplary embodiment 188, provided herein is the rodent of embodiment 187, wherein the nucleic acid sequence encoding the FcγR2b polypeptide is located at an endogenous rodent low-affinity FcγR locus.

[0476] In exemplary embodiment 189, provided herein is the rodent of embodiment 188, wherein the nucleic acid sequence encoding the human FcγR2b polypeptide replaces all or a portion of an endogenous rodent low affinity FcγR gene.

[0477] In exemplary embodiment 190, provided herein is the rodent of any one of embodiments 187 to 189, wherein the rodent is heterozygous for the engineered FcγR2b locus.

[0478] In exemplary embodiment 191, provided herein is the rodent of any one of embodiments 187 to 189, wherein the rodent is homozygous for the engineered FcγR2b locus.

[0479] In exemplary embodiment 192, provided herein is the rodent of any one of embodiments 1 to 191, further comprising in its genome an engineered Fcγ receptor 3a (FcγR3a) locus comprising a nucleic acid sequence encoding a human FcγR3a polypeptide.

[0480] In exemplary embodiment 193, provided herein is the rodent of embodiment 192, wherein the nucleic acid sequence encoding the FcγR3a polypeptide is located at an endogenous rodent low-affinity FcγR locus.

[0481] In exemplary embodiment 194, provided herein is the rodent of embodiment 193, wherein the nucleic acid sequence encoding the human FcγR3a polypeptide replaces all or a portion of an endogenous rodent low affinity FcγR gene.

[0482] In exemplary embodiment 195, provided herein is the rodent of any one of embodiments 192 to 194, wherein the rodent is heterozygous for the engineered FcγR3a locus.

[0483] In exemplary embodiment 196, provided herein is the rodent of any one of embodiments 192 to 194, wherein the rodent is homozygous for the engineered FcγR3a locus.

[0484] In exemplary embodiment 197, provided herein is the rodent of any one of embodiments 1 to 196, further comprising in its genome an engineered Fcγ receptor 3b (FcγR3b) locus comprising a nucleic acid sequence encoding a human FcγR3b polypeptide.

[0485] In exemplary embodiment 198, provided herein is the rodent of embodiment 197, wherein the nucleic acid sequence encoding the FcγR3b polypeptide is located at an endogenous rodent low-affinity FcγR locus.

[0486] In exemplary embodiment 199, provided herein is the rodent of embodiment 197, wherein the nucleic acid sequence encoding the human FcγR3b polypeptide replaces all or a portion of an endogenous rodent low affinity FcγR gene.

[0487] In exemplary embodiment 200, provided herein is the rodent of any one of embodiments 197 to 199, wherein the rodent is heterozygous for the engineered FcγR3b locus.

[0488] In exemplary embodiment 201, provided herein is the rodent of any one of embodiments 197 to 199, wherein the rodent is homozygous for the engineered FcγR3b locus.

[0489] In exemplary embodiment 202, provided herein is the rodent of any one of embodiments 1 to 201, further comprising in its genome an engineered Fcγ receptor 2c (FcγR2c) locus comprising a nucleic acid sequence encoding a human FcγR2c polypeptide.

[0490] In exemplary embodiment 203, provided herein is the rodent of embodiment 202, wherein the nucleic acid sequence encoding the FcγR2c polypeptide is located at an endogenous rodent low-affinity FcγR locus.

[0491] In exemplary embodiment 204, provided herein is the rodent of embodiment 203, wherein the nucleic acid sequence encoding the human FcγR2c polypeptide replaces all or a portion of an endogenous rodent low-affinity FcγR gene.

[0492] In exemplary embodiment 205, provided herein is the rodent of any one of embodiments 202 to 204, wherein the rodent does not express rodent FcγR2c.

[0493] In exemplary embodiment 206, provided herein is the rodent of any one of embodiments 202 to 205, wherein the rodent is heterozygous for the engineered FcγR2c locus.

[0494] In exemplary embodiment 207, provided herein is the rodent of any one of embodiments 202 to 205, wherein the rodent is homozygous for the engineered FcγR2c locus.

[0495] In exemplary embodiment 208, provided herein is a rodent comprising in its genome an engineered neonatal Fc receptor (FcRn) locus comprising a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain.

[0496] In exemplary embodiment 209, provided herein is the rodent of embodiment 208, wherein the FcRn polypeptide further comprises a rodent transmembrane domain.

[0497] In exemplary embodiment 210, provided herein is the rodent of embodiment 208, wherein the FcRn polypeptide further comprises a human transmembrane domain.

[0498] In exemplary embodiment 211, provided herein is the rodent of any one of embodiments 208 to 210, wherein the FcRn polypeptide further comprises a rodent cytoplasmic domain.

[0499] In exemplary embodiment 212, provided herein is the rodent of any one of embodiments 208 to 210, wherein the FcRn polypeptide further comprises a human cytoplasmic domain.

[0500] In exemplary embodiment 213, provided herein is the rodent of any one of embodiments 208 to 212, wherein the nucleic acid sequence encoding the FcRn polypeptide is located at an endogenous rodent FcRn locus.

[0501] In exemplary embodiment 214, provided herein is the rodent of embodiment 213, wherein the nucleic acid sequence encoding the FcRn polypeptide replaces all or a portion of an endogenous rodent FcRn gene.

[0502] In exemplary embodiment 215, provided herein is the rodent of embodiment 208, wherein the nucleic acid sequence encoding the extracellular domain of human FcRn replaces the endogenous nucleic acid sequence encoding the extracellular domain of rodent FcRn.

[0503] In exemplary embodiment 216, provided herein is the rodent of any one of embodiments 208 to 215, wherein the rodent does not express rodent FcRn.

[0504] In exemplary embodiment 217, provided herein is the rodent of any one of embodiments 208 to 216, wherein the rodent is heterozygous for the engineered FcRn locus.

[0505] In exemplary embodiment 218, provided herein is the rodent of any one of embodiments 208 to 216, wherein the rodent is homozygous for the engineered FcRn locus.

[0506] In exemplary embodiment 219, provided herein is the rodent of any one of embodiments 208 to 218, further comprising in its genome a beta-2-microglobulin (β2M) locus comprising a nucleic acid sequence encoding a human or humanized beta-2-microglobulin (β2M) polypeptide.

[0507] In exemplary embodiment 220, provided herein is the rodent of embodiment 219, wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide is located at an endogenous rodent β2M locus.

[0508] In exemplary embodiment 221, provided herein is the rodent of embodiment 220, wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide replaces all or a portion of the endogenous rodent β2M gene.

[0509] In exemplary embodiment 222, provided herein is the rodent of any one of embodiments 219 to 221, wherein the nucleic acid sequence comprises exons 2-4 of the human β2M gene.

[0510] In exemplary embodiment 223, provided herein is the rodent of any one of embodiments 219 to 222, wherein the rodent does not express a rodent β2M polypeptide.

[0511] In exemplary embodiment 224, provided herein is the rodent of any one of embodiments 219 to 223, wherein the rodent is heterozygous for the engineered β2M locus.

[0512] In exemplary embodiment 225, provided herein is the rodent of any one of embodiments 219 to 223, wherein the rodent is homozygous for the engineered β2M locus.

[0513] In exemplary embodiment 226, provided herein is a rodent comprising in its genome an engineered Fcε receptor 1α (FcεR1α) locus comprising a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain.

[0514] In exemplary embodiment 227, provided herein is the rodent of embodiment 226, wherein the FcεR1α polypeptide further comprises a rodent transmembrane domain.

[0515] In exemplary embodiment 228, provided herein is the rodent of embodiment 226, wherein the FcεR1α polypeptide further comprises a human transmembrane domain.

[0516] In exemplary embodiment 229, provided herein is the rodent of any one of embodiments 226 to 228, wherein the FcεR1α polypeptide further comprises a rodent cytoplasmic domain.

[0517] In exemplary embodiment 230, provided herein is the rodent of any one of embodiments 226 to 228, wherein the FcεR1α polypeptide further comprises a human cytoplasmic domain.

[0518] In exemplary embodiment 231, provided herein is the rodent of any one of embodiments 226 to 230, wherein the nucleic acid sequence encoding the FcεR1α polypeptide is located at an endogenous rodent FcεR1α locus.

[0519] In exemplary embodiment 232, provided herein is the rodent of embodiment 231, wherein the nucleic acid sequence encoding the FcεR1α polypeptide replaces all or a portion of an endogenous rodent FcεR1α gene.

[0520] In exemplary embodiment 233, provided herein is the rodent of embodiment 232, wherein the FcεR1α polypeptide comprises a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain.

[0521] In exemplary embodiment 234, provided herein is the rodent of embodiment 226, wherein the nucleic acid sequence encoding the human FcεR1α extracellular domain replaces the endogenous nucleic acid sequence encoding the rodent FcεR1α extracellular domain.

[0522] In exemplary embodiment 235, provided herein is the rodent of any one of embodiments 226 to 234, wherein the rodent does not express rodent FcεR1α.

[0523] In exemplary embodiment 236, provided herein is the rodent of any one of embodiments 226 to 235, wherein the rodent is heterozygous for the engineered FcεR1α locus.

[0524] In exemplary embodiment 237, provided herein is the rodent of any one of embodiments 226 to 235, wherein the rodent is homozygous for the engineered FcεR1α locus.

[0525] In exemplary embodiment 238, provided herein is the rodent of any one of embodiments 1 to 237, wherein the rodent is a mouse.

[0526] In exemplary embodiment 239, provided herein is the rodent of any one of embodiments 1 to 237, wherein the rodent is a rat.

[0527] In exemplary embodiment 240, provided herein is a method of testing a therapeutic protein comprising a human Fc domain, the method comprising administering the therapeutic protein to a rodent as described in any one of embodiments 1 to 207, and measuring one or more pharmacokinetic properties of the administered therapeutic protein.

[0528] In exemplary embodiment 241, provided herein is a method as in embodiment 240, wherein the one or more pharmacokinetic properties are selected from one or more of the following: area under plasma concentration versus time (AUC), in vivo recovery (IVR), clearance (CL), mean residence time (MRT), half-life of the agent (t1 / 2), and volume of distribution at steady state (Vss).

[0529] In exemplary embodiment 242, provided herein is a method of testing the therapeutic efficacy of a therapeutic protein comprising a human Fc domain, the method comprising administering the therapeutic protein to a rodent as described in any one of embodiments 1 to 207, and measuring the therapeutic efficacy of the administered therapeutic protein.

[0530] In exemplary embodiment 243, provided herein is a method for determining a therapeutically effective dose of a therapeutic protein comprising a human Fc domain, the method comprising administering multiple doses of the therapeutic protein to a rodent as described in any one of embodiments 1 to 207, and determining the therapeutic efficacy of each dose of the therapeutic protein.

[0531] In exemplary embodiment 244, provided herein is a method for determining a safe dose of a therapeutic protein comprising a human Fc domain, the method comprising administering multiple doses of the therapeutic protein to a rodent as described in any one of embodiments 1 to 207, and determining the safety of the therapeutic protein at each dose.

[0532] In exemplary embodiment 245, provided herein is a method for determining a tolerable dose of a therapeutic protein comprising a human Fc domain, the method comprising administering multiple doses of the therapeutic protein to a rodent as described in any one of embodiments 1 to 207, and determining the tolerability of each dose of the therapeutic protein.

[0533] In exemplary embodiment 246, provided herein is a method of testing a therapeutic protein comprising a human Fc domain, the method comprising administering the therapeutic protein to a rodent as described in any one of embodiments 148 to 239, and measuring one or more Fc receptor-mediated responses in the rodent.

[0534] In exemplary embodiment 247, provided herein is a method as described in embodiment 246, wherein the one or more Fc receptor-mediated responses include an ADCC response.

[0535] In exemplary embodiment 248, provided herein is a method for screening a therapeutic agent comprising a human Fc region of a human antibody, the method comprising: (a) administering an agent comprising an Fc region of a human antibody to a rodent as described in any one of embodiments 148 to 239, wherein the agent binds to a target cell in the mouse; (b) measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against the target cell; and (c) comparing the amount of ADCC in step (b) to a control, wherein an increase in target cell killing indicates that the agent has an increased ability to mediate ADCC.

[0536] In exemplary embodiment 249, provided herein is a method for measuring an immune response generated by a rodent against a therapeutic protein comprising a human Fc domain, the method comprising administering the therapeutic protein to the rodent of any one of embodiments 1 to 207, and measuring the immune response generated by the rodent against the therapeutic protein.

[0537] In exemplary embodiment 250, provided herein is a method as described in any one of embodiments 240 to 249, wherein the therapeutic protein is a human antibody.

[0538] In exemplary embodiment 251, provided herein is a method as described in any one of embodiments 240 to 249, wherein the therapeutic protein is an Fc fusion protein.

[0539] In exemplary embodiment 252, provided herein is a method as described in any one of embodiments 240 to 251, wherein the human Fc domain is a human IgG1 Fc domain.

[0540] In exemplary embodiment 253, provided herein is a method as in embodiment 252, wherein the therapeutic protein is a human IgG1 antibody.

[0541] In exemplary embodiment 254, provided herein is a method as described in embodiment 252, wherein the therapeutic protein is an Fc fusion protein comprising a human IgG1 Fc domain.

[0542] In exemplary embodiment 255, provided herein is a method as described in any one of embodiments 252 to 254, wherein the encoding comprises human C H 1 domain, human hinge region, human C H 2 domains, human C H The C of the IgG constant domain comprising the IgG 3 domain, the IgG transmembrane domain and the IgG cytoplasmic domain H The gene segment is C γ1 Gene segment.

[0543] In exemplary embodiment 256, provided herein is a method as described in any one of embodiments 240 to 251, wherein the human Fc domain is a human IgG2 Fc domain.

[0544] In exemplary embodiment 257, provided herein is a method as in embodiment 256, wherein the therapeutic protein is a human IgG2 antibody.

[0545] In exemplary embodiment 258, provided herein is a method as described in embodiment 256, wherein the therapeutic protein is an Fc fusion protein comprising a human IgG2 Fc domain.

[0546] In exemplary embodiment 259, provided herein is a method as described in any one of embodiments 256 to 258, wherein the encoding comprises human C H 1 domain, human hinge region, human C H 2 domains, human C H The C of the IgG constant domain comprising the IgG 3 domain, the IgG transmembrane domain and the IgG cytoplasmic domain H The gene segment is C γ2 Gene segment.

[0547] In exemplary embodiment 260, provided herein is a method as described in any one of embodiments 240 to 251, wherein the human Fc domain is a human IgG3 Fc domain.

[0548] In exemplary embodiment 261, provided herein is a method as in embodiment 260, wherein the therapeutic protein is a human IgG3 antibody.

[0549] In exemplary embodiment 262, provided herein is a method as described in embodiment 260, wherein the therapeutic protein is an Fc fusion protein comprising a human IgG3 Fc domain.

[0550] In exemplary embodiment 263, provided herein is a method as described in any one of embodiments 260 to 262, wherein the encoding comprises human C H 1 domain, human hinge region, human C H 2 domains, human C H The C of the IgG constant domain comprising the IgG 3 domain, the IgG transmembrane domain and the IgG cytoplasmic domain H The gene segment is C γ3 Gene segment.

[0551] In exemplary embodiment 264, provided herein is a method as described in any one of embodiments 240 to 251, wherein the human Fc domain is a human IgG4 Fc domain.

[0552] In exemplary embodiment 265, provided herein is a method as in embodiment 264, wherein the therapeutic protein is a human IgG4 antibody.

[0553] In exemplary embodiment 266, provided herein is a method as in embodiment 264, wherein the therapeutic protein is an Fc fusion protein comprising a human IgG4 Fc domain.

[0554] In exemplary embodiment 267, provided herein is a method as described in any one of embodiments 264 to 266, wherein the encoding comprises human C H 1 domain, human hinge region, human C H 2 domains, human C H The C of the IgG constant domain comprising the IgG 3 domain, the IgG transmembrane domain and the IgG cytoplasmic domain H The gene segment is C γ4 Gene segment.

[0555] In exemplary embodiment 268, provided herein is a method as described in any one of embodiments 250, 252, 253, 255-257, 259-261, 263-265, and 267, wherein the therapeutic protein is a human antibody with a kappa light chain and the rodent expresses antibodies comprising human kappa light chains.

[0556] In exemplary embodiment 269, provided herein is a method as described in any one of embodiments 250, 252, 253, 255-257, 259-261, 263-265, and 267, wherein the therapeutic protein is a human antibody having a lambda light chain and the rodent expresses antibodies comprising human lambda light chains.

[0557] In exemplary embodiment 270, provided herein is a method as described in any one of embodiments 240 to 269, wherein the rodent is a mouse.

[0558] In exemplary embodiment 271, provided herein is a method as described in any one of embodiments 240 to 269, wherein the rodent is a rat.

[0559] In exemplary embodiment 272, provided herein is an animal model for testing a therapeutic protein comprising a human Fc domain, wherein the model comprises administering the therapeutic protein to a rodent as described in any one of embodiments 1 to 207, and measuring one or more pharmacokinetic properties of the administered therapeutic protein.

[0560] In exemplary embodiment 273, provided herein is an animal model as described in embodiment 272, wherein the one or more pharmacokinetic properties are selected from one or more of the following: area under plasma concentration versus time (AUC), in vivo recovery (IVR), clearance (CL), mean residence time (MRT), half-life of the agent (t1 / 2), and volume of distribution at steady state (Vss).

[0561] In exemplary embodiment 274, provided herein is an animal model for testing the therapeutic efficacy of a therapeutic protein comprising a human Fc domain, the animal model comprising administering th...

Claims

1. A method for producing a rodent comprising an engineered neonatal Fc receptor (FcRn) locus in its genome, wherein the engineered neonatal FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain and a rodent cytoplasmic domain, the method comprising modifying the genome of the rodent to comprise an FcRn locus comprising a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain and a rodent cytoplasmic domain, wherein the nucleic acid sequence encoding the FcRn polypeptide is located at an endogenous rodent FcRn locus.

2. The method of claim 1, wherein the FcRn polypeptide further comprises a rodent transmembrane domain or a human transmembrane domain.

3. The method of claim 1, wherein the nucleic acid sequence encoding the FcRn polypeptide replaces all or part of an endogenous rodent FcRn gene. 4 . The method of claim 3 , wherein the nucleic acid sequence encoding the extracellular domain of human FcRn replaces the endogenous nucleic acid sequence encoding the extracellular domain of rodent FcRn.

5. The method of claim 1, wherein the rodent does not express rodent FcRn.

6. The method of claim 1, wherein the rodent is heterozygous for the engineered FcRn locus.

7. The method of claim 1, wherein the rodent is homozygous for the engineered FcRn locus.

8. The method of claim 1, wherein the rodent further comprises a beta-2-microglobulin (β2M) locus in its genome, the beta-2-microglobulin locus comprising a nucleic acid sequence encoding a human or humanized beta-2-microglobulin (β2M) polypeptide.

9. The method of claim 8, wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide is located at an endogenous rodent β2M locus.

10. The method of claim 9, wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide replaces all or a portion of the endogenous rodent β2M gene.

11. The method of claim 8, wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide comprises exons 2-4 of the human β2M gene.

12. The method of claim 8, wherein the rodent does not express rodent β2M polypeptide.

13. The method of claim 8, wherein the rodent is homozygous for the β2M locus.

14. The method of claim 1, wherein the rodent further comprises an engineered Fcε receptor 1α (FcεR1α) locus in its genome, wherein the engineered Fcε receptor 1α locus comprises a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain.

15. The method of claim 14, wherein the FcεR1α polypeptide further comprises (a) a rodent transmembrane domain or a human transmembrane domain, and (b) Rodent cytoplasmic domain or human cytoplasmic domain. 16 . The method of claim 14 , wherein the nucleic acid sequence encoding the FcεR1α polypeptide is located at an endogenous rodent FcεR1α locus. 17 . The method of claim 16 , wherein the nucleic acid sequence encoding the FcεR1α polypeptide replaces all or a portion of an endogenous rodent FcεR1α gene.

18. The method of claim 16, wherein the rodent is homozygous for the FcεR1α locus.

19. The method of claim 14, wherein the rodent does not express rodent FcεR1α.

20. The method of any one of claims 1-19, wherein the rodent is a mouse.

21. The method of claim 20, wherein the engineered FcRn locus comprises exons 1-2 of a mouse FcRn gene, exons 3-5 of a human FcRn gene, and exons 6-7 of a mouse FcRn gene.

22. A method for preparing a genetically modified rodent embryonic stem (ES) cell, the method comprising modifying the ES cell genome to comprise an engineered neonatal Fc receptor (FcRn) locus, the FcRn locus comprising a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain and a rodent cytoplasmic domain, wherein the nucleic acid sequence encoding the FcRn polypeptide is located at an endogenous rodent FcRn locus.

23. The method of claim 22, wherein the FcRn polypeptide further comprises a rodent transmembrane domain or a human transmembrane domain.

24. The method of claim 22, wherein the nucleic acid sequence encoding the FcRn polypeptide replaces all or a portion of an endogenous rodent FcRn gene.

25. The method of claim 22, wherein the nucleic acid sequence encoding the extracellular domain of human FcRn replaces an endogenous nucleic acid sequence encoding the extracellular domain of rodent FcRn.

26. The method of claim 22, wherein the rodent ES cell genome is homozygous for the FcRn locus.

27. The method of claim 22, wherein the rodent ES cell genome further comprises a β-2-microglobulin (β2M) locus comprising a nucleic acid sequence encoding a human or humanized β-2-microglobulin (β2M) polypeptide.

28. The method of claim 27, wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide is located at an endogenous rodent β2M locus.

29. The method of claim 27, wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide replaces all or a portion of an endogenous rodent β2M gene.

30. The method of claim 27, wherein the nucleic acid sequence encoding the human or humanized β2M polypeptide comprises exons 2-4 of the human β2M gene.

31. The method of claim 27, wherein the rodent ES cell genome is homozygous for the β2M locus.

32. The method of claim 27, wherein the rodent ES cell genome further comprises an engineered Fcε receptor 1α (FcεR1α) locus, wherein the engineered Fcε receptor 1α locus comprises a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain, a rodent transmembrane domain or a human transmembrane domain, and a rodent cytoplasmic domain or a human cytoplasmic domain.

33. The method of claim 32, wherein the nucleic acid sequence encoding the FcεR1α polypeptide is located at an endogenous rodent FcεR1α locus.

34. The method of claim 32, wherein the nucleic acid sequence encoding the FcεR1α polypeptide replaces all or a portion of an endogenous rodent FcεR1α gene.

35. The method of claim 32, wherein the rodent ES cell genome is homozygous for the FcεR1α locus.

36. The method of any one of claims 22-35, wherein the rodent ES cells are mouse ES cells.

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