mice expressing humanized Fc alpha receptors

Abstract

This specification provides methods and compositions relating to mice expressing human or humanized Foot receptor (FcaR) from the FcaR gene locus located in the mouse leukocyte receptor complex (LRC). [Solution] In certain embodiments, such mice are useful for in vivo testing of therapeutic agents containing human IgA Fc (e.g., testing of the pharmacokinetic and / or pharmacodynamic properties of such therapeutic agents, as well as administration planning). The Specified also provides methods for using such mice, cells obtained from such mice, methods for producing such mice, and ES cells including genetically modified cells similar to those of such mice.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 126,326, filed on 16 December 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Mice are essential in vivo models for preclinical trials of therapeutic drugs due to their small size, well-characterized physiology, and relatively low maintenance costs compared to larger mammalian models (such as primates). Mouse models are frequently used to evaluate the toxicity and pharmacokinetics of therapeutic drugs before proceeding to human clinical studies. Despite these advantages, in vivo testing of antibodies and Fc fusion proteins has a significant drawback: these therapeutics may behave differently in mice than in humans. This is particularly true when testing IgA-based therapeutics in mice, as mice do not express Fc receptors that bind to IgA antibodies. Consequently, mouse models have not been extensively characterized and are not used to predict the safety, efficacy, and optimal dosage of IgA-based therapeutics. There is a need for new mouse models and methods to enable accurate preclinical trials of IgA-based therapeutics that predict the characteristics of such therapeutics in human patients. [Overview of the project]

[0003] This specification provides methods and compositions relating to mice expressing human or humanized Fcα receptors (FcαR), as well as methods and compositions relating to in vivo testing of therapeutic agents containing human IgA Fc in such mice (e.g., testing of pharmacokinetic and / or pharmacodynamic properties, and administration plans for such therapeutic agents). As described herein, mice are convenient animal models for testing therapeutic antibodies and Fc fusion proteins due to their small size, well-characterized physiology, and adaptability to genetic modification. Unfortunately, drugs containing the human IgA Fc region (e.g., IgA antibodies and Fcα fusion proteins) often exhibit very different pharmacokinetic and pharmacodynamic properties when administered to wild-type mice compared to when administered to humans, because such mice lack FcαR. Therefore, the mice provided herein can serve as in vivo systems for the development, screening, and testing of human IgA antibodies and Fcα fusion proteins for therapeutic use.

[0004] In certain embodiments, the Specified Provisions provide a mouse having an Fc alpha receptor (FcαR) locus located in the mouse leukocyte receptor complex (LRC) in its genome, wherein the FcαR locus (e.g., human or humanized FcαR locus) contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain. In some embodiments, the mouse expresses the FcαR polypeptide on mouse neutrophils, monocytes, macrophages, eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells). In some embodiments, neutrophils, monocytes, macrophages, eosinophils, and / or dendritic cells (e.g., plasmacytoid dendritic cells) are obtained from mouse blood. In some embodiments, neutrophils, monocytes, macrophages, eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells) are obtained from mouse spleen.

[0005] In some embodiments, the Specified Provision provides mouse embryonic stem cells (ES cells) having an Fc alpha receptor (FcαR) locus located in the leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain.

[0006] In some embodiments, the FcαR locus is located in the intergenetic region between the Tthy1 protein locus and the Rdh13 protein locus. In some embodiments, the FcαR locus is located in the intergenetic region between the Lilra5 protein locus and the Gp6 protein locus. In some embodiments, the FcαR locus is located in the intergenetic region between the Pira6 protein locus and the Gp6 protein locus. In some embodiments, the FcαR locus is located in the intergenetic region between the Pira6 protein locus and the Ncr1 protein locus. In some embodiments, the FcαR locus is located in the intergenetic region between the nucleic acid sequences encoding the Pira6 protein and the Ncr1 protein. In some embodiments, the intergenetic region is a 54kb region between the Pira6 locus and the Ncr1 locus. In some embodiments, the FcαR locus is located on mouse chromosome 7 between coordinates 4,303,905 and 4,312,280 (+ strand, GRCm38 assembly).

[0007] In some embodiments, the FcαR locus includes a nucleic acid sequence encoding the human FcαR polypeptide. In some embodiments, the FcαR locus includes human exons 1-5 of the human Fcα receptor gene. In some embodiments, the FcαR locus includes the non-coding portion of rodent (non-mouse) FcαR exon 1, human FcαR exons 1 and 2, human FcαR exons 3 and 4, and the coding portion of rodent (non-mouse) FcαR exon 5. In some embodiments, the human or humanized FcαR receptor locus includes a genomic sequence (+ strand, GRCh38 assembly) found between coordinates 54,862,297-54,906,185 on human chromosome 19. In some embodiments, the FcαR locus further includes a nucleic acid sequence present in the human KIR3DL2 gene. In some embodiments, the locus further includes a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

[0008] In some embodiments, mouse or mouse ES cells are heterozygous for the FcαR locus. In some embodiments, mouse or mouse ES cells are homozygous for the FcαR locus.

[0009] In some embodiments, the mouse or mouse ES cells of any of the above embodiments or models further include in their genome the human or humanized Fc gamma receptor (FcγR) locus, the human or humanized IgH locus, the human or humanized Igκ locus, the human or humanized Igλ locus, the human or humanized FcRn locus, the human or humanized β2M locus, and / or the human or humanized FcεR1α locus. In some embodiments, the mouse or mouse ES cells are heterozygous for the human or humanized FcγR locus, the human or humanized IgH locus, the human or humanized Igκ locus, the human or humanized Igλ locus, the human or humanized FcRn locus, the human or humanized β2M locus, and / or the human or humanized FcεR1α locus. In some embodiments, mouse or mouse ES cells are homozygous for the human or humanized FcγR locus, human or humanized IgH locus, human or humanized Igκ locus, human or humanized Igλ locus, human or humanized FcRn locus, human or humanized β2M locus, and / or human or humanized FcεR1α locus.

[0010] In some embodiments, mouse or mouse ES cells comprising the FcαR locus of various embodiments described herein contain a human or humanized FcγR locus in the genome comprising a nucleic acid sequence encoding human or humanized FcγR. In some embodiments, the human or humanized FcγR locus comprises one or more nucleic acid sequences encoding low-affinity FcγR selected from Fc-gamma receptor 2a (FcγR2a), Fc-gamma receptor 2b (FcγR2b), Fc-gamma receptor 3a (FcγR3a), Fc-gamma receptor 3b (FcγR3b), and / or Fc-gamma receptor 2c (FcγR2c). In some embodiments, the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more FcγR selected from human or humanized Fc-gamma receptor 1-alpha (FcγR1a), Fc-gamma receptor 2a (FcγR2a), Fc-gamma receptor 2b (FcγR2b), Fc-gamma receptor 3a (FcγR3a), Fc-gamma receptor 3b (FcγR3b), and / or Fc-gamma receptor 2c (FcγR2c). In some embodiments, the human or humanized FcγR comprises a human extracellular domain. In some embodiments, the human or humanized FcγR comprises a mouse transmembrane domain. In some embodiments, the human or humanized FcγR comprises a human transmembrane domain. In some embodiments, the human or humanized FcγR comprises a mouse cytoplasmic domain. In some embodiments, the human or humanized FcγR comprises a human cytoplasmic domain. In some embodiments, the human or humanized FcγR locus is located at the endogenous mouse FcγR locus. In some embodiments, the nucleic acid sequence encoding human or humanized FcγR replaces all or part of the endogenous mouse FcγR gene. In some embodiments, the nucleic acid sequence encoding human or humanized FcγR includes a nucleic acid sequence encoding the human FcγR extracellular domain, which replaces the endogenous nucleic acid sequence encoding the mouse FcγR extracellular domain. In some embodiments, the mouse of any of the above embodiments does not express mouse FcγR.

[0011] In certain embodiments, this specification provides a method for testing a human IgA antibody or Fcα fusion polypeptide, comprising administering the IgA antibody or Fcα fusion polypeptide to a mouse of any of the above embodiments.

[0012] In some embodiments, the method further comprises measuring one or more pharmacokinetic properties of the administered human IgA antibody or Fcα fusion polypeptide. In some embodiments, the one or more pharmacokinetic properties are selected from one or more of the following: plasma concentration versus area under time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), drug half-life (t1 / 2), and / or steady-state volume of distribution (Vss). In some embodiments, the method further comprises measuring the therapeutic efficacy of the administered human antibody or Fcα fusion polypeptide. In some embodiments, the method further comprises administering multiple doses of the human antibody or Fcα fusion polypeptide and determining the therapeutic efficacy of each dose of the human antibody or Fcα fusion polypeptide. In some embodiments, the method further comprises administering multiple doses of the human antibody or Fcα fusion polypeptide and determining the safety of each dose of the human antibody or Fcα fusion polypeptide. In some embodiments, the method further comprises administering multiple doses of a human antibody or Fcα fusion polypeptide and determining the tolerance of each dose of the human antibody or Fcα fusion polypeptide. In some embodiments, the method further comprises measuring one or more Fc receptor-mediated responses in mice. In some embodiments, one or more Fc receptor-mediated responses are antibody-dependent cell-mediated cytotoxicity (ADCC) responses. In some embodiments, the human antibody binds to target cells in mice, and the method further comprises measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against the target cells and comparing the amount of ADCC to a control, where an increase in target cell death indicates an increased ability of the drug to mediate ADCC. In some embodiments, the method further comprises measuring the immune response produced by mice to the human antibody.

[0013] In certain aspects, the present disclosure provides a method of generating a mouse comprising a Fc alpha receptor (FcαR) locus, the method comprising generating mouse embryonic stem (ES) cells comprising, in the genome, a Fc alpha receptor (FcαR) locus located in the leukocyte receptor complex (LRC) of the mouse genome provided herein, and generating a mouse from the ES cells.

[0014] In certain aspects, the present disclosure provides a method of modifying a mouse genome, the method comprising inserting a Fc alpha receptor (FcαR) locus into the leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR locus comprises a nucleic acid sequence encoding a FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain. In some embodiments, the FcαR locus comprises a nucleic acid sequence encoding a human or humanized FcαR polypeptide. In certain embodiments, the FcαR locus is located in an intergenic region between the locus of the Tthy1 protein and the locus of the Rdh13 protein. In some embodiments, the FcαR locus is located in an intergenic region between the locus of the Lilra5 protein and the locus of the Gp6 protein. In some embodiments, the FcαR locus is located in an intergenic region between the locus of the Pira6 protein and the locus of the Gp6 protein. In some embodiments, the FcαR locus is located in an intergenic region between the locus of the Pira6 protein and the locus of the Ncr1 protein. In some embodiments, the intergenic region is a 54 kb region between the Pira6 locus and the Ncr1 locus. In certain embodiments, the FcαR locus is between coordinates 4,303,905 and 4,312,280 on chromosome 7 of the mouse (plus strand, GRCm38 assembly).

[0015] Unless otherwise indicated, in the figures, white boxes are used for human gene sequences, black boxes are used for mouse gene sequences, single lines are used for mouse intergenic sequences, double lines are used for human intergenic sequences, and white boxes with text (e.g., Lox, CM) are used for selection cassettes. [Brief explanation of the drawing]

[0016] [Figure 1A] This diagram (not to scale) shows the insertion of the MAID20277 cassette into the mouse genome to generate heterozygous mice that can be bred to be homozygous. "LRC" indicates the leukocyte receptor complex, and "EP" indicates electroporation. The names and locations of the primers and probes used in the allele modification (MOA) assay (described in the examples) are shown as short lines at the top of the figure. [Figure 1B] This figure demonstrates the treatment of heterozygous ES cells with CRE recombinase to induce deletion of a selection marker for insertion of the MAID20277 cassette into the mouse genome, thereby producing ES cells heterozygous for the MAID20278 cassette (not to scale). The names and locations of the primers and probes used in the allele modification (MOA) assay (described in the examples) are shown as short lines at the top of the figure. [Figure 2] The image shows a targeted vector containing a sequence homologous to the mouse genome ("5' mouse homology arm"), the I-CeuI site, a 44,887 bp human genome insert including the FCAR gene, the lox2372-Ub-Neo-lox2372 cassette, the PI-SceI site, a second sequence homologous to the mouse genome, and a chloramphenicol ("CM") resistance cassette (not to scale). [Figure 3] This shows the humanization-low affinity FcγR locus on mouse chromosome 1, containing the human FcγR2B, FcγR3B, FcγR2C, FcγR3A, and FcγR2A genes (not to scale). The steps for humanizing this locus are described in U.S. Patent No. 8,658,154, which is incorporated herein by reference. [Figure 4A] This shows a flow cytometry plot detecting FcαR expression in cells from blood samples obtained from genetically modified mice using the MAID20278 cassette. [Figure 4B]This shows a flow cytometry plot detecting FcαR expression in spleen cells obtained from genetically modified mice using the MAID20278 cassette. [Modes for carrying out the invention]

[0017] overview This specification provides methods and compositions relating to mice expressing human, humanized, or partially humanized Fcα receptor (FcαR), as well as methods and compositions relating to in vivo testing of therapeutics containing human IgA Fc in such mice (e.g., testing of the pharmacokinetic and / or pharmacodynamic properties of such therapeutics, and dosing protocols). Specifically, the various embodiments described herein relate to mice, ES cells, and methods comprising a gene encoding and / or expressing a fully human or partially human FcαR1 / CD89 gene / receptor (hereinafter referred to as "FcαR"). The genetically modified mouse contains a nucleic acid sequence in its genome that encodes a human or humanized Fcα receptor (FcαR) protein. In some embodiments, the sequence encoding the human or humanized Fcα receptor (FcαR) protein is located in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, the nucleic acid sequence encoding FcαR is located in an intergeneric region between the Tthy1 protein locus and the Rdh13 protein locus, between the Lilra5 polypeptide locus and the Gp6 polypeptide locus, and / or between the Pira6 protein locus and the Ncr1 protein locus (e.g., between the encoding nucleic acid sequence of the Pira6 protein and the encoding nucleic acid sequence of the Ncr1 protein). In some embodiments, the intergeneric region is a 54kb region between the Pira6 locus and the Ncr1 locus. In some embodiments, the mice provided herein express human FcαR at a locus located between coordinates chr7:4,303,905–4,312,280 in the mouse genome (+ strand, GRCm38 assembly). In some embodiments, FcαR includes a human extracellular domain. The transmembrane domain and / or cytoplasmic domain of such a receptor may be human or non-human (e.g., rat).

[0018] Therapeutic drugs containing human IgA Fc, such as therapeutic human antibodies and human Fcα fusion proteins, are typically tested in non-human species before being administered to humans. While such drugs are often tested in non-human primates and other relatively large mammals, such testing is costly and represents a significant financial burden for drug developers. Furthermore, non-human primates and other relatively large mammals are often unsuitable for genetic modification, limiting the available disease models in such organisms.

[0019] On the other hand, mice are a convenient animal model for testing therapeutic antibodies and Fc fusion proteins due to their small size, well-characterized physiology, and adaptability to genetic modification. Unfortunately, drugs containing the human IgA Fc region often exhibit very different pharmacokinetic and pharmacodynamic properties when administered to conventional mice compared to when administered to humans, because wild-type mice lack FcαR. Therefore, the mice provided herein can serve as an in vivo system for the development, screening, and testing of human IgA antibodies and Fcα fusion proteins for therapeutic use.

[0020] The location of a transgene within the mouse genome can significantly influence its expression. For example, mice containing the human FcαR transgene have been reported to express FcαR on neutrophils but only on a subpopulation of monocytes. For instance, in such mice, macrophages isolated from the peritoneal cavity, as well as non-myeloid cells such as lymphocytes, endothelial cells, and hepatocytes, did not show FcαR expression. van Egmond et al., Blood 93(12):4387-4394 (1999). In contrast, in humans, FcαR is typically expressed on neutrophils, monocytes, macrophages (e.g., Kupffer cells), eosinophils, and dendritic cells. Therefore, in certain embodiments, the mice provided herein express human FcαR at locations in the mouse genome corresponding to the location of the human endogenous FcαR locus, or at locations in the human genome corresponding to loci located near the location of the human endogenous FcαR locus. Specifically, in certain embodiments, the sequence encoding the human or humanized Fc alpha receptor (FcαR) protein is located in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, the mice provided herein express human FcαR at a locus located in the intergeneric region between the Tthy1 protein locus and the Rdh13 protein locus, between the Lilra5 polypeptide locus and the Gp6 polypeptide locus, and / or between the Pira6 protein locus and the Ncr1 protein locus (e.g., between the coding nucleic acid sequence of the Pira6 protein and the coding nucleic acid sequence of the Ncr1 protein). In some embodiments, the intergeneric region is a 54kb region between the Pira6 locus and the Ncr1 locus. In some embodiments, the mice provided herein express human FcαR at a locus located between coordinates chr7:4,303,905–4,312,280 in the mouse genome (+ strand, GRCm38 assembly). In some embodiments provided herein, the mice described herein express FcαR on the cell surface of neutrophils, monocytes / macrophages (e.g., blood monocytes / macrophages), eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells).

[0021] In various embodiments of the mice, ES cells, and methods provided herein, the genome contains and / or the mouse expresses the human or humanized FcαR described herein, which associates with wild-type mouse FcRγ chains. Thus, in some embodiments, the human or humanized FcαR is expressed on the surface of mouse cells in association with endogenous mouse FcRγ chains.

[0022] In some embodiments, the mice provided herein further include a reduced anti-human immune response after administration of a therapeutic agent containing human Fcα. This can be achieved by using genetically modified mice that express human Fc that matches the Fc present in the administered antibody or Fc fusion protein. Such mice can be created, for example, by inserting all or part of a nucleic acid sequence encoding the human immunoglobulin heavy chain constant region in place of the sequence encoding the portion corresponding to the 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 express an immune response to the administered human Fc-containing therapeutic agent.

[0023] Furthermore, in some embodiments, the mice provided herein express an Fcα receptor (FcαR) that can interact with human Fcα, similar to the FcαR expressed in human patients. For example, in certain embodiments, the genetically modified mice provided herein express an FcαR having at least a human extracellular domain (e.g., the transmembrane and cytoplasmic domains may be human or rat). In certain embodiments, in addition to expressing human or partially human FcαR, the mice provided herein express human or partially human β2M, human or partially human FcεR1α, human or partially human FcγR1a, human or partially human FcγR2a, human or partially human FcγR2b, human or partially human FcγR3a, human or partially human FcγR3b, and / or human or partially human FcγR2c. Thus, such mice can more accurately mimic the human Fc response of human patients compared to mice with entirely non-human Fc receptors.

[0024] Accordingly, in certain embodiments, the mice provided herein are a novel in vivo system for developing, selecting, and testing therapeutic human IgA antibodies and Fc fusion proteins based not only on specificity and / or affinity to antigens, but also on the overall biological function associated with the selected antibody through evaluation of effector function of the immune system. In this way, human therapeutic candidates can be developed and selected based on therapeutic potential evaluated at the whole-molecule level, involving related biological responses (e.g., cellular responses), rather than predictions based solely on individually evaluated components. Accordingly, the mice disclosed herein provide a suitable system, in particular, for predicting and characterizing human therapeutic antibody function in vivo.

[0025] Transgenic mice expressing human FcαR disclosed herein can be used to study the functional role of the receptor in immune responses at a lower cost compared to larger animal models or humans. Furthermore, mice are a more suitable model for confirming basic biological functions and then confirming them in larger animals or humans. For example, mice expressing human FcαR have long been used to study the role of the receptor in antibody-dependent cytotoxicity (ADCT) and to identify other components required for FcαR-mediated responses. See van Egmond et al. (1999) Blood, 93(12):4387-94.

[0026] IgA antibodies play a crucial role in the onset and progression of certain diseases. Breedveld and van Egmond discuss several diseases and conditions in which IgA antibodies play a role ((2019) Frontiers in Immunology, 10:553). For example, several autoimmune diseases are characterized by increased levels of IgA antibodies (e.g., rheumatoid arthritis and IgA nephropathy). In inflammatory bowel disease, IgA-opsonized bacteria may cause FcαR crosslinking and neutrophil activation, leading to tissue damage. Low IgA levels are also associated with certain diseases. A potential association has been found between low IgA levels and the severity of allergic asthma, while high levels are also found in subjects with allergic rhinitis. Using the mice described herein, it is possible to identify the role of FcαR in disease severity or the effect of FcαR inhibitors on symptom attenuation.

[0027] IgA deficiency may increase susceptibility to infections, particularly mucosal infections. Administering excessive amounts of IgA can enhance FcαR activation, potentially leading to unintended complications. Therefore, the mice described herein can be used to identify appropriate dosage regimens that effectively overcome IgA deficiency without causing side effects. The mice described herein can also be used to evaluate the efficacy of FcαR inhibitors in treating IgA-mediated inflammation.

[0028] In some embodiments, the mice described herein may be treated with an antitumor (or pathogen) candidate IgA therapeutic agent or an IgA-Fc fusion protein, or a bispecific antibody against FcαR and tumor antigens. In other words, the mice described herein may be used for preclinical evaluation of therapeutic agents involving IgA and / or FcαR.

[0029] definition The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "(an) element" means one or more elements.

[0030] The term "amino acid" is intended to encompass all molecules that may be included in polymers of naturally occurring amino acids, whether natural or synthetic, and that contain both amino and acidic functional groups. Exemplary amino acids include naturally occurring amino acids, their analogues, derivatives and homologues, amino acid analogues with variant side chains, and all of the aforementioned stereoisomers.

[0031] As used herein, the term “antibody” may refer to both an intact antibody and its antigen-binding fragment. An intact antibody is a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain and a heavy chain constant domain. Each light chain contains a light chain variable domain and a light chain constant domain. The heavy chain variable domains and light chain variable domains can be further subdivided into hypervariable domains called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). The heavy chain variable domains and light chain variable domains consist of three CDRs and four FRs, respectively, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable domains of the heavy and light chains contain binding domains that interact with the antigen.

[0032] As used herein, the terms “antigen-binding fragment” and “antibody-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 included in the term “antigen-binding fragment” of an antibody include Fab, Fab', F(ab')2, Fv, scFv, disulfide-bound 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 a similar manner to intact antibodies.

[0033] As used herein, the term “Area under the plasma concentration-time curve” or “AUC” refers to the rate and degree of elimination of a therapeutic agent after administration. In some embodiments, the AUC is determined over a specific period, such as 12, 18, 24, 36, 48, or 72 hours, or over infinity using extrapolation based on the slope of the curve. Unless otherwise specified herein, the AUC is infinite (AUC INF It is determined by ). AUC may be calculated for each dose. As with many other PK parameters, AUC may be determined in a single animal or in a population of animals for which the mean is calculated.

[0034] As used herein, the term “clearance rate” or “CL” refers to a measure of the body’s ability to remove a drug, expressed as the amount of plasma from which the drug has been removed over time. When the phrase "derived from" is used in reference to a reconstructed variable region gene "derived from" an unreconstructed variable region and / or unreconstructed variable region gene segment, it means that the sequence of the reconstructed variable region gene can be traced back to a series of unreconstructed variable region gene segments that were reconstructed to form a gene expressing the variable domain (explaining splice differences and somatic mutations, where applicable). For example, a somatically mutated, reconstructed variable region gene still derives from an unreconstructed variable region gene segment. In some embodiments, when an endogenous locus is replaced by a universal light chain or heavy chain locus, the term "derived from" indicates that the origin of the sequence can be traced back to the aforementioned reconstructed locus, even if the sequence has undergone somatic mutation.

[0035] As used herein, the terms “endogenous gene” or “endogenous gene segment” refer to a gene or gene segment found in the parent or reference organism before the introduction of any disruption, deletion, substitution, alteration, or modification described herein. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is a genetically modified organism. In some embodiments, the reference organism is a laboratory-bred organism (either wild-type or genetically modified).

[0036] The term "in vivo recovery rate" or "IVR" refers to the incremental recovery rate (K value), which is calculated by subtracting the pre-administration level from the observed peak activity and then dividing by the administered dose. IVR may also be calculated on a percentage basis. The mean IVR can be determined for an animal population, or individual IVRs can be determined for individual animals.

[0037] As used herein, the term “locus” refers to a location on a chromosome. In some embodiments, a locus contains a set of related genetic elements (e.g., genes, genetic segments, regulatory elements). For example, the human leukocyte receptor complex (LRC) locus is located on chromosome 19 and contains genes including, among others, KIR, FcαR, NCRI, NLRP, GP6, and Rdh13. A mouse locus homologous to the human LRC locus is located on mouse chromosome 7 and contains several genes homologous to the human LRC genes (e.g., Ncr1, Gp6, and Rdh13 genes). Loci can be endogenous or non-endogenous. The term “endogenous locus” refers to a location on a chromosome where a particular genetic element is found naturally. In some embodiments, an endogenous locus has a naturally occurring sequence. In some embodiments, an endogenous locus is a wild-type locus. In some embodiments, an endogenous locus is an engineered locus.

[0038] The terms “polynucleotide” and “nucleic acid” are used interchangeably. These terms refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides can have any three-dimensional structure and perform any function. Non-limiting examples of polynucleotides include: coding or non-coding regions of genes or gene fragments; loci (multiple loci) defined from linkage analysis; exons; introns; messenger RNA (mRNA); transfer RNA; ribosomal RNA; ribozymes; cDNA; recombinant polynucleotides; branched polynucleotides; plasmids; vectors; isolated DNA of any sequence; isolated RNA of any sequence; nucleic acid probes; and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure may be conferred before or after polymer assembly. Polynucleotides may be further modified, for example, by the attachment of labeling components. In all nucleic acid sequences provided herein, U nucleotides are replaceable with T nucleotides.

[0039] As used herein, the terms “volume of distribution at steady state” or “Vss” refer to the apparent space (volume) in which a drug is distributed. More specifically, Vss represents the amount of drug in the animal body divided by the plasma concentration at steady state.

[0040] As used herein, the term "CH gene segment" (e.g., Cγ1 gene segment, Cγ2a gene segment, Cγ2c gene segment, Cμ gene segment, Cγ2b gene segment, Cγ3 gene segment, Cδ gene segment, Cε gene segment, Cα gene segment, etc.) refers to a segment of DNA sequence encoding the constant region of an immunoglobulin heavy chain. H Genes (for example, the Cγ gene, C γ2a Genes, C γ2c Genes, C γ3 Genes, C γ2b gene, Cμ gene, C δgene, Cε gene, C α genes, etc.) and can be used interchangeably. For example, the Cγ1 gene segment or Cγ1 gene refers to a segment of a DNA sequence encoding the IgG1 constant region. "C H gene segment locus" refers to the position on the chromosome where the C H gene segment or C H gene is naturally found.

[0041] genetically modified locus In some embodiments, genetically modified mice and cells (e.g., ES cells) useful for in vivo testing of therapeutic agents (e.g., testing of the pharmacokinetic and / or pharmacodynamic properties of such therapeutic agents) as described herein contain a human IgA antibody or an Fcα fusion protein. The genetically modified mice disclosed herein contain in their genome a nucleic acid sequence encoding human or humanized FcαR, and include at least one locus corresponding to the location of the human endogenous FcαR locus, or a location in the mouse genome corresponding to a locus located near the location of the human endogenous FcαR locus in the human genome. For example, in certain embodiments, the sequence encoding a human or humanized Fcα receptor (FcαR) protein is located in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, the sequence encoding the human or humanized Fc alpha receptor (FcαR) protein is located in the intergenetic region between the Tthy1 protein locus and the Rdh13 protein locus, between the Lilra5 polypeptide locus and the Gp6 polypeptide locus, between the Pira6 protein locus and the Gp6 protein locus, and / or between the Pira6 protein locus and the Ncr1 protein locus. In some embodiments, the sequence encoding the human or humanized Fc alpha receptor (FcαR) protein is located between coordinates chr7:4,303,905~4,312,280 in the mouse genome (+ strand, GRCm38 assembly). In some embodiments, the intergenetic region is a 54kb region between the Pira6 locus and the Ncr1 locus. In some embodiments, the nucleic acid sequence encoding FcαR further comprises a nucleic acid sequence encoding all or part of the human KIR3DL2 gene, and / or a nucleic acid sequence of all or part of the human NCR1 gene, for example, a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

[0042] In some embodiments, genetically modified mice contain genetically modified loci encoding antibody heavy chains, including human Fc (e.g., human IgA1Fc, human IgA2Fc). Such loci are disclosed in WO2019 / 190990, which is incorporated herein by reference in its entirety. In some embodiments, mice and / or ES cells contain genetically modified loci encoding complete or partial human light chains (e.g., λ light chains or κ light chains). Such loci are disclosed in WO2019 / 190990 and U.S. Patent No. 10,820,582, which is incorporated herein by reference in its entirety. In some embodiments, genetically modified mice and ES cells further include one or more loci encoding Fc receptors having human extracellular domains (e.g., neonatal Fc receptor (FcRn) α chain, β-2-microglobulin polypeptide (β2M), Fcε receptor 1α (FcεR1α) α chain, Fcγ receptor 1α (FcγR1a) α chain, Fc gamma receptor 2a (FcγR2a) α chain, Fc gamma receptor 2b (FcγR2b) α chain, Fc gamma receptor 3a (FcγR3a) α chain, Fc gamma receptor 3b (FcγR3b) α chain, Fc gamma receptor 2c (FcγR2c) α chain). In some embodiments, the transmembrane and cytoplasmic domains encoded by such loci may be human or non-human (e.g., rodents such as mice). Such loci are disclosed in WO2019 / 190990 and U.S. Patents 9,474,255 and 8,658,154, each of which is incorporated herein by reference in its entirety.

[0043] Humanized Fc alpha receptor The mouse or embryonic stem (ES) cells provided herein express and / or contain humanized or human Fc alpha receptor 1 (FcαR1) in their genome. Mice do not have FcαR homologs. Therefore, in some embodiments, the mouse or ES cells contain in their genome a nucleic acid sequence encoding human or humanized FcαR, and include at least one locus corresponding to the location of the human endogenous FcαR locus, or a location in the mouse genome corresponding to a locus located near the location of the human endogenous FcαR locus in the human genome. For example, in certain embodiments, the sequence encoding the human or humanized Fc alpha receptor (FcαR) protein is located in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, the sequence encoding the human or humanized Fc alpha receptor (FcαR) protein is located in the intergenetic region between the Tthy1 protein locus and the Rdh13 protein locus, between the Lilra5 polypeptide locus and the Gp6 polypeptide locus, and / or between the Pira6 protein locus and the Ncr1 protein locus. In some embodiments, the sequence encoding the human or humanized Fc alpha receptor (FcαR) protein is located between coordinates chr7:4,303,905~4,312,280 in the mouse genome (+ strand, GRCm38 assembly). In some embodiments, the intergenetic region is a 54kb region between the Pira6 locus and the Ncr1 locus. In some embodiments, the nucleic acid sequence encoding FcαR further comprises a nucleic acid sequence encoding all or part of the human KIR3DL2 gene, and / or a nucleic acid sequence of all or part of the human NCR1 gene, for example, a nucleic acid sequence present in the 5'UTR of the human NCR1 gene. In certain embodiments, the mice provided herein express FcαR on neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells. In some embodiments, neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells are neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells of blood.In some embodiments, neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells are neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells of the spleen.

[0044] FcαR is an activating receptor that forms a complex with the human FcRγ chain and has moderate affinity (K) for human IgA1, IgA2, and secretory IgA. a Approximately 5x10 6 M -1 ) and high binding activity (K) to the IgA complex. a Approximately 5x10 6 M -1 ) possesses. Binding of FcαR to IgA-coated particles causes the release of inflammatory mediators, phagocytosis, and antibody-dependent cell-mediated cytotoxicity. Tumor cells can be effectively lysed by using IgA antitumor therapy to induce neutrophils to enter tumor cells. In addition to the full-length receptor, neutrophils and eosinophils express it in the form of a selectively spliced ​​receptor capable of binding to secreted IgA. In vivo, two FcαR molecules dimerize and interact with the IgA monomer.

[0045] In some embodiments, the FcαR locus comprises a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain, a rodent (non-mouse, e.g., rat) transmembrane domain, and a rodent (non-mouse, e.g., rat) cytoplasmic domain. In some embodiments, the FcαR locus comprises a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain, a human transmembrane domain, and a rodent (non-mouse, e.g., rat) cytoplasmic domain. In some embodiments, the FcαR locus comprises a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain.

[0046] In some embodiments, the FcαR locus includes 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). In another embodiment, the FcαR locus includes human regulatory elements (e.g., human promoters and / or enhancers).

[0047] As described in the art (see, for example, Monteiro and van de Winkel, (2003) Annu. Rev. Immunol. 21:177-204), the human FcαR gene consists of the following five exons: Exon 1 containing the 5'UTR, the ATG translation start codon, and the sequence encoding the leader peptide; Exon 2 encoding the remainder of the leader peptide; Exons 3 and 4 encoding the two extracellular domains of FcαR, EC1 and EC2 (EC1 is the domain that binds to IgA Fc); and Exon 5 encoding the cytoplasmic and transmembrane domains of the protein.

[0048] In some embodiments, the nucleic acids encoding human or humanized FcαR polypeptides provided herein encode fully human FcαR polypeptides. In certain embodiments, such nucleic acids comprise humanized exon 1 (e.g., including rodent (non-mouse, e.g., rat) 5'UTR and human coding sequence), human coding exons 2-5 up to the stop codon, and human or rodent (non-mouse, e.g., rat) 3'UTR and polyA. In some embodiments, the nucleic acids encoding human or humanized FcαR polypeptides comprise exons 1-5 of the human FcαR gene.

[0049] In some embodiments, the nucleic acid is used to generate a targeted vector for insertion into the mouse ES cell genome. In some embodiments, the nucleic acid encoding a human or humanized FcαR polypeptide contains a human genome sequence of approximately 44 kb including the FCAR gene. In some embodiments, the human genome sequence is a genome sequence located on human chromosome 19 and having coordinates chr19:54,862,297~54,906,185 (+ strand, GRCh38 assembly), and includes the 3' end of the KIR3DL2 gene and the entire human FCAR gene.

[0050] In some embodiments, the nucleic acid encoding the FcαR polypeptide is a rodent (non-mouse, e.g., rat) / human chimeric sequence. In some embodiments, the rodent (non-mouse, e.g., rat) / human chimeric sequence comprises human, rat, or rat / human chimeric exon 1 (the nucleic acid sequence encoding the leader sequence is either human or rat); human or rat exon 2, human exons 3-4; and rat exon 5. In these embodiments, the nucleic acid encodes an FcαR polypeptide comprising a human FcαR extracellular domain and rat transmembrane and cytoplasmic domains. In some embodiments, the regulatory region (e.g., promoter and UTR) is a rodent (e.g., rat) regulatory region.

[0051] NCBI reference sequence number NW_016107304.1 is a representative source sequence for the human FcαR gene. NCBI reference sequences NM_002000.4 and NP_001991.1, NM_133269.4 and NP_579803.1, NM_133271.4 and NP_579805.1, NM_133272.4 and NP_579806.1, NM_133273.4 and NP_579807.1, NM_133274.4 and NP_5798 08.1, NM_133277.4 and NP_579811.1, NM_133278.4 and NP_579812.1, XM_011526625.3 and XP_011524927.1, XM_017026473.1 and XP_016881962.1, XM_017026474.2 and XP_016881963.1 are human FcαR This provides representative source sequences for cDNA and polypeptides from which desired human portions can be obtained.

[0052] NCBI reference sequence number NC_005100.4 provides a representative source sequence for the FcαR gene of Rattus norvegicus, and NCBI reference sequence number NM_201992.1→NP_973721.1 provides a representative source sequence for polypeptides that can be used to design homologous arms of targeting vectors that yield the FcαR cDNA of Rattus norvegicus and the desired Rattus norvegicus moiety.

[0053] In some embodiments, mice are heterozygous for the genetically modified FcαR locus. In some embodiments, mice are homozygous for the genetically modified FcαR locus.

[0054] In various embodiments, the human or humanized FcαR polypeptide described herein is expressed on the cell surface in association with wild-type mouse FcRγ chains.

[0055] Humanized low-affinity Fc gamma receptor In some embodiments, humanized or genetically modified mice and ES cells that contain the human FcαR1 locus in their genome further include loci encoding human low-affinity Fc gamma receptor (FcγR) polypeptides (e.g., human FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, or FcγRIIIb polypeptides; see, for example, U.S. Patent No. 8,658,154, which is incorporated herein by reference in its entirety).

[0056] In some embodiments, the low-affinity FcγR locus contains a nucleic acid sequence encoding the human FcγRIIa polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIa polypeptide is located at the endogenous mouse low-affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding the FcγRIIa polypeptide replaces all or part of the endogenous mouse low-affinity FcγR locus. In some embodiments, the human FcγRIIa gene contains a polymorphism, which is selected from the 131His low-reactivity polymorphism and the 131Arg high-reactivity polymorphism. In some embodiments, the FcγRIIa polymorphism is the 131His low-reactivity polymorphism. In some embodiments, the mouse does not express the mouse low-affinity FcγR polypeptide (e.g., does not express mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides, or does not express functional mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides). In some embodiments, the FcγRIIa 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).

[0057] In some embodiments, the low-affinity FcγR locus contains a nucleic acid sequence encoding the human FcγRIIb polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIb polypeptide is located at the endogenous mouse low-affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding the FcγRIIb polypeptide replaces all or part of the endogenous mouse low-affinity FcγR locus. In some embodiments, the human FcγRIIb gene contains an amino acid substitution, which is selected from 187Ile or 187Thr substitutions. In some embodiments, the mouse does not express the mouse low-affinity FcγR polypeptide (e.g., does not express mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides, or does not express functional mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides). In some embodiments, the FcγRIIb locus contains 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 (e.g., a rat or mouse promoter or enhancer).

[0058] In some embodiments, the low-affinity FcγR locus contains a nucleic acid sequence encoding the human FcγRIIc polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIc polypeptide is located at the endogenous mouse low-affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding the FcγRIIc polypeptide replaces all or part of the endogenous mouse low-affinity FcγR locus. In one embodiment, the FcγRIIc gene is a specific allele variant, selected from the 57Stop and 57Q variants. In some embodiments, the mouse does not express the mouse low-affinity FcγR polypeptide (e.g., does not express mouse FcγRIIB, FcγRIV, and / or FcγRIII polypeptides). In some embodiments, the FcγRIIc locus contains 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).

[0059] In some embodiments, the low-affinity FcγR locus contains a nucleic acid sequence encoding the human FcγRIIIa polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIIa polypeptide is located at the endogenous mouse low-affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding the FcγRIIIa polypeptide replaces all or part of the endogenous mouse low-affinity FcγR locus. In some embodiments, the mouse does not express the mouse low-affinity FcγR polypeptide (e.g., does not express mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptide, or does not express functional mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptide). In one embodiment, the FcγRIIIa gene is a specific allele variant, selected from the 176Val and 176Phe variants. In some embodiments, the FcγRIIIa allele variant is the 176Val variant. In some embodiments, the FcγRIIIa 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).

[0060] In some embodiments, the low-affinity FcγR locus contains a nucleic acid sequence encoding the human FcγRIIIb polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIIb polypeptide is located at the endogenous mouse low-affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding the FcγRIIIb polypeptide replaces all or part of the endogenous mouse low-affinity FcγR locus. In some embodiments, the FcγRIIIb gene is a specific allele variant, selected from NA1 and NA2 variants. In another specific embodiment, the FcγRIIIb allele variant is the NA2 variant. In some embodiments, the mouse does not express the mouse low-affinity FcγR polypeptide (e.g., does not express mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides, or does not express functional mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides). In some embodiments, the FcγRIIIb 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 mouse regulatory element (e.g., a rat or mouse promoter or enhancer).

[0061] In some embodiments, the mice provided herein include one or more human low-affinity FcγR genes as described in U.S. Patents 9,221,894, 9,056,130, 9,089,599, 8,658,154, 8,883,496, or 8,658,853 (these references are incorporated herein by reference). In some embodiments, the mice provided herein include at least two low-affinity human FcγR genes and an endogenous mouse Fcγ chain gene, the low-affinity human FcγR genes being selected from the group consisting of human FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some specific embodiments, the mice provided herein include human FcγRIIa and FcγRIIIb, as well as an endogenous mouse Fcγ chain gene. In some specific embodiments, the mice provided herein include the FcγRIIa, FcγRIIIa, FcγRIIb, FcγRIIc, and FcγRIIId genes, as well as the endogenous mouse Fcγ chain gene. In various embodiments, the mouse containing one or more human FcγR further includes homozygous disruption in the endogenous mouse FcγRIIB, FcγRIV, and FcγRIII genes (i.e., the endogenous mouse FcγRIIb, FcγRIV, and FcγRIIIα chain coding sequences). In various embodiments, the mouse containing one or more human low-affinity FcγR described herein does not express the endogenous mouse low-affinity FcγR polypeptide (e.g., endogenous low-affinity FcγRα chain polypeptide) to a detectable level.

[0062] In some embodiments, mice are heterozygous for the genetically modified low-affinity FcγR locus. In some embodiments, mice are homozygous for the genetically modified low-affinity FcγR locus.

[0063] Humanized neonatal Fc receptor locus In some embodiments, genetically modified mice and ES cells whose genomes contain a humanized or human FcαR1 locus, and in some embodiments, a human or humanized FcγR locus (as described above), further contain a humanized or human neonatal Fc receptor (FcRn) locus. FcRn, also known as the Brambell receptor, is a protein expressed by endothelial cells that associates with beta-2 microglobulin (β2M) and binds to both the Fc domain of IgG antibodies and serum albumin. FcRn extends the half-lives of IgG and serum albumin. Specifically, by binding to IgG and serum albumin in a pH-dependent manner, FcRn can rescue these serum proteins from lysosomal degradation by endothelial cells, thereby extending the serum half-life of such proteins.

[0064] In some embodiments, the FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain, a rodent (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain, and a human (e.g., mouse or rat) cytoplasmic domain.

[0065] In some embodiments, the nucleic acid sequence encoding the FcRn polypeptide is located at the endogenous mouse FcRn locus. In certain embodiments, the nucleic acid sequence encoding the FcRn polypeptide replaces all or part of the endogenous mouse FcRn gene. For example, in some embodiments, the nucleic acid sequence encoding the extracellular domain of endogenous FcRn at the endogenous FcRn locus is replaced with a nucleic acid sequence encoding the extracellular domain of human FcRn so that mice containing such a locus express FcRn having a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domain. In some embodiments, the mouse does not express mouse FcRn or does not express functional mouse FcRn. In some embodiments, the FcRn 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).

[0066] In certain embodiments, the mouse exons encoding the alpha-1, alpha-2, and alpha-3 domains of the mouse FcRn gene (the first three coding exons, exons 3, 4, and 5) are replaced with human exons encoding the alpha-1, alpha-2, and alpha-3 domains of the human FcRn gene (exons 3, 4, and 5). In some embodiments, the FcRn gene includes mouse exon 1 (a non-coding exon), mouse exon 2 (containing a nucleic acid sequence encoding a signal peptide), and human exons 3-6, mouse exons 6 and 7 (encoding the transmembrane and cytoplasmic domains). An exemplary humanized FcRn locus is described in WO2019 / 190990 (e.g., Figure 4), which is incorporated herein by reference.

[0067] GenBank accession numbers NC_000019.10 (49512279~49526428), NM_001136019.1, and NP_001129491.1 provide representative source sequences for human FcRn genes, cDNA, and polypeptides from which 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 for mouse FcRn genes, cDNA, and polypeptides from which desired mouse portions can be obtained and / or used in the design of targeted vector homology arms.

[0068] In some embodiments, mice are heterozygous for the genetically modified FcRn locus. In some embodiments, mice are homozygous for the genetically modified FcRn locus.

[0069] Humanized Fc epsilon receptor 1 alpha In some embodiments, genetically modified mice and ES cells that include a humanized or human FcαR1 locus and, in some embodiments, a human or humanized FcγR locus (as described above) in their genome further include a humanized or human Fc epsilon receptor 1 alpha (FcεR1α) locus. FcεR1α associates with FcεR1β and FcεR1γ to form FcεR1, a high-affinity receptor for IgE expressed on epidermal Langerhans cells, eosinophils, mast cells, and basophils. The IgE binding site of FcεR1 is found in the FcεR1α subunit.

[0070] In some embodiments, the FcεR1α locus comprises a nucleic acid sequence encoding an FcεR1α polypeptide including a human extracellular domain, a rodent (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcεR1α locus comprises a nucleic acid sequence encoding an FcεR1α polypeptide including 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 including a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain. Exemplary embodiments of the manipulated FcεR1α locus are described in WO2019 / 190990, which is incorporated herein by reference.

[0071] In some embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide is located at the endogenous mouse FcεR1α locus. In certain embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide replaces all or part of the endogenous mouse FcεR1α gene. For example, in some embodiments, the nucleic acid sequence encoding the extracellular domain of endogenous FcεR1α at the endogenous FcεR1α locus is replaced with a nucleic acid sequence encoding the extracellular domain of human FcεR1α so that mice containing such a locus express FcεR1α having a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domains. In some embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide, including a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain, is located at the endogenous mouse FcεR1α locus. In some embodiments, a nucleic acid sequence encoding the FcεR1α polypeptide, comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain, replaces all or part of the endogenous mouse FcεR1α gene. In some embodiments, the mouse does not express mouse FcεR1α or does not express functional mouse FcεR1α. In some embodiments, the FcεR1α 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).

[0072] In certain embodiments, portions of mouse coding exons 1, 2, 3, 4, and 5 of mouse FcεRIα are replaced by portions of human coding exons 1, 2, 3, 4, and 5 of the human FcεRIα gene. In some embodiments, the FcεRIα gene includes mouse / human chimeric exon 1 (including the mouse promoter and 5'UTR), stop codons from human coding exons 2-5, human 3'UTR and polyA, followed by mouse 3'UTR and polyA. 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 thought to interact with IgE, and exon 5 encodes the cytoplasmic and transmembrane domains of the protein (see Figure 9 in WO2019 / 190990).

[0073] GenBank accession numbers NC_000001.11 (159283888~159308224), NM_002001.3, and NP_001992.1 provide representative source sequences for the human FcεR1α gene, cDNA, and polypeptide from which desired human portions can be obtained. GenBank accession numbers NC_000067.6 (173221269~173227232), NM_010184.1, and NP_034314.1 provide representative source sequences for the mouse FcεR1α gene, cDNA, and polypeptide from which desired mouse portions can be obtained and / or used in the design of homologous arms for targeted vectors.

[0074] In some embodiments, mice are heterozygous for the genetically modified FcεR1α locus. In some embodiments, mice are homozygous for the genetically modified FcεR1α locus.

[0075] Humanized Fc gamma receptor 1a In some embodiments, genetically modified mice and ES cells that contain a humanized or human FcαR1 locus in their genome further contain a humanized or human Fc gamma receptor 1a (FcγR1a) locus. FcγR1a is a high-affinity FcγR protein expressed on monocytes that binds to the Fc portion of IgG and triggers host cell activation.

[0076] In some embodiments, the FcγR1a locus comprises a nucleic acid sequence encoding an FcγR1a polypeptide comprising a human extracellular domain, a rodent (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcγR1a locus comprises a nucleic acid sequence encoding an FcγR1a polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcγR1a locus comprises a nucleic acid sequence encoding an FcγR1a polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain, and a human (e.g., mouse or rat) cytoplasmic domain.

[0077] In some embodiments, the nucleic acid sequence encoding the FcγR1α polypeptide is located at the endogenous mouse FcγR1a locus. In certain embodiments, the nucleic acid sequence encoding the FcγR1a polypeptide replaces all or part of the endogenous mouse FcγR1a gene. For example, in some embodiments, the nucleic acid sequence encoding the extracellular domain of endogenous FcγR1a at the endogenous FcγR1a locus is replaced with the nucleic acid sequence encoding the extracellular domain of human FcγR1α so that mice containing such a locus express FcγR1a having a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domain. In some embodiments, the mouse does not express mouse FcγR1a or does not express functional mouse FcγR1a. In some embodiments, the FcγR1a locus includes 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 (e.g., a rat or mouse promoter or enhancer).

[0078] Humanized FcγR1a polypeptide, the locus encoding humanized FcγR1a polypeptide, and mice expressing humanized FcγR1a polypeptide are described in U.S. Patent No. 9,474,255 and U.S. Patent Publication No. 2017 / 0086432, respectively, which are incorporated herein by reference.

[0079] In some embodiments, mice are heterozygous for the genetically modified FcγR1a locus. In some embodiments, mice are homozygous for the genetically modified FcγR1a locus.

[0080] Humanized immunoglobulin heavy chain locus As discussed above in this specification, in certain embodiments, mice expressing human FcαR provided herein are used to test human Fc-containing drugs (e.g., human Fcα-containing drugs such as therapeutic human IgA antibodies and human Fcα fusion proteins). However, when therapeutic agents containing a human Fc region are administered to wild-type mice, the human sequence within the Fc region is often recognized as a foreign substance by the mouse immune system. As a result, the mice may initiate an immune response to the administered therapeutic agent (known as the mouse anti-human response or MAHA), which affects the pharmacokinetic and pharmacodynamic properties of the administered drug. As disclosed in WO2019 / 190990 (the entire document is incorporated herein by reference), testing human Fc-containing therapeutic agents in mice having a human immunoglobulin locus region can reduce or eliminate the MAHA response because human Fc is less likely to be recognized as a foreign substance in such mice.

[0081] In some embodiments, genetically modified mice and ES cells whose genomes contain a humanized or human FcαR1 locus, and optionally a human or humanized FcγR locus, a human or humanized Igκ locus, a human or humanized Igλ locus, a human or humanized FcRn locus, a human or humanized β2M locus, and / or a human or humanized FcεR1α locus, further contain genetically modified immunoglobulin (Ig) heavy chain loci. Such loci generally include a variable region and a constant region. The variable region includes an Ig heavy chain variable region gene segment (e.g., at least V H Gene segment, D H gene segment, and J H The constant region gene locus contains one or more Ig heavy chain constant region gene segments (C H ) is included. In certain embodiments, the immunoglobulin heavy chain variable region is such that the mouse is V H Gene segment, D H gene segment, and J HVariable domains and C derived from gene segments H It is functionally ligated to the constant region of an immunoglobulin heavy chain so as to produce an antibody containing a heavy chain constant domain derived from a gene segment.

[0082] The ability to produce antibodies has been utilized in genetically modified animals to generate therapeutic antibodies against human targets. Examples of genetically modified mice containing the human V(D)J gene segment for generating therapeutic antibodies include U.S. Patents 5,633,425, 5,770,429, 5,814,318, 6,075,181, 6,114,598, 6,150,584, 6,998,514, 7,795,494, 7,910,798, 8,232,449, 8,703,485, 8,907,157, and the same. This includes the documents described in U.S. Patent Publication No. 9,145,588, each of which is incorporated herein by reference in its entirety, as well as the documents described in U.S. Patent Publication Nos. 2008 / 0098490, 2010 / 0146647, 2013 / 0145484, 2012 / 0167237, 2013 / 0167256, 2013 / 0219535, 2012 / 0207278, and 2015 / 0113668, and the documents described herein. Each of these references is incorporated herein by reference in its entirety, and furthermore, PCT Publications WO2007117410, WO2008151081, WO2009157771, WO2010039900, WO2011004192, WO2011123708, WO2014093908, WO2014093908, WO2006008548, WO2010109165, and WO20160629 These include publications No. 90, WO2018039180, WO2011158009, WO2013041844, WO2013041846, WO2013079953, WO2013061098, WO2013144567, WO2013144566, WO2013171505, and WO2019008123, each of which is incorporated herein by reference in its entirety.Other examples of genetically modified mice containing the human V(D)J gene segment for generating therapeutic antibodies include U.S. Patents 6,596,541, 6,586,251, 8,642,835, 9,706,759, 10,238,093, 8,754,287, 10,143,186, 9,796,788, and 10,130,08 This includes the documents described in U.S. Patent Publications No. 1, No. 9,226,484, No. 9,012,717, No. 10,246,509, No. 9,204,624, and No. 9,686,970, each of which is incorporated herein by reference in its entirety, in addition to U.S. Patent Publications No. 2013 / 0212719, No. 2015 / 0289489, and No. 2017 / 034763. This includes publications No. 3, No. 2019 / 0223418, No. 2018 / 0125043, No. 2019 / 0261612, and No. 2019 / 0380316, each of which is incorporated herein by reference in its entirety, as well as PCT publications WO2013138680, WO2013138712, WO2013138681, and These include WO2015042250, WO2012148873, WO2013134263, WO2013184761, WO2014160179, WO2017214089, WO2016149678, and WO2017123808, as well as Murphy, A., "VelocImmune:Immunoglobulin Variable Region Humanized Mouse," in Recombinant Antibodies for Immunotherapy, New York, NY, Cambridge University Press, 101-107 (2009), each of which is incorporated herein by reference in its entirety.

[0083] In certain embodiments, the Ig heavy chain variable region locus includes an unreconstructed human Ig heavy chain variable region gene segment. In some embodiments, the unreconstructed human Ig variable region gene segment includes one or more human VH Segment, one or more human D H Segments, and one or more human J H Includes a segment. In some embodiments, the unreconstructed human Ig variable region gene segment contains 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 Includes a gene segment. In some specific embodiments, the manipulated IgH locus (or allele) is the human V of the naturally occurring human IgH locus. H 3-74 gene segments and human V H Functional human V found between the 6-1 gene segment (including both ends) H Includes all or substantially all of the gene segment. In some specific embodiments, the manipulated IgH locus (or allele) is at least 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, VH 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 Includes 6-1. In some embodiments, the mice provided herein are single polymorphic human V H gene segment, multiple D H gene segment, and multiple J H It has a restricted immunoglobulin heavy chain locus characterized by a gene segment (for example, described in U.S. Patent Publication 2013 / 0096287, which is incorporated herein by reference). In some embodiments, V H The gene segment is V H 1-2 or V HIt is 1-69. In some embodiments, the mice provided herein have a rearranged heavy chain variable region (e.g., the universal heavy chain variable region or common heavy chain coding sequence described in U.S. Patent Publication No. 20140245468, as well as U.S. Patents Nos. 9,204,624 and 9,930,871, the entireties of which are incorporated herein by reference). In some embodiments, the mice provided herein include an unrearranged human immunoglobulin light chain, e.g., κ, gene segment functionally linked to a heavy chain constant region gene at the immunoglobulin heavy chain locus (e.g., U.S. Patent No. 9,516,868, which is incorporated herein by reference).

[0084] In still other embodiments, the mouse can include a heavy chain immunoglobulin locus in its germline and / or genome that contains insertions and / or substitutions of histidine codons designed to introduce pH-dependent binding characteristics into antibodies produced in such mice. In some of such embodiments, the histidine codons are inserted and / or substituted in the nucleic acid sequence encoding CDR3. Various such heavy chain immunoglobulin loci are provided in U.S. Patents Nos. 9,301,510, 9,334,334, U.S. Patent Application Publication Nos. 2013 / 0247236, 20140013456, the entireties of which are incorporated herein by reference.

[0085] In some embodiments, the engineered IgH locus (or allele) contains 5, 10, 15, 20, 25, or more (e.g., 26, 27, etc.) human D H gene segments. In some particular embodiments, the engineered IgH locus (or allele) contains functional human D H 1-1 gene segments and human D H 7-27 gene segments found between (including both ends) the natural human IgH locus HIncludes all or substantially all of the gene segments. In some particular embodiments, the engineered IgH locus (or allele) comprises at least the human D H Gene segment D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26, and D H 7-27. In some embodiments, the non-rearranged human Ig gene segments comprise all of the human D H gene segments.

[0086] In some embodiments, the engineered IgH locus (or allele) comprises 1, 2, 3, 4, 5, 6, or more functional human J H gene segments. In some particular embodiments, the engineered IgH locus (or allele) comprises all or substantially all of the functional human J H gene segments found between (including both ends) the human J H 1 gene segment and the human J H 6 gene segments of the native human IgH locus. In some particular embodiments, the engineered IgH locus (or allele) comprises at least the human J H gene segment J H 1, J H 2, J H 3, JH 4, J H 5, and J H Includes 6. In some embodiments, the unreconstructed human Ig gene segment is human J H Includes all gene segments.

[0087] In some embodiments, the engineered IgH locus described herein does not contain the endogenous Adam6 gene. In some embodiments, the engineered IgH locus described herein does not contain the endogenous Adam6 gene (or Adam6 coding sequence) at a germline genomic location similar to that found in the germline genome of the same species of wild-type non-human animal. In some embodiments, the engineered IgH locus described herein does not contain the human Adam6 pseudogene. In some embodiments, the engineered IgH locus described herein includes an insertion of at least one nucleotide sequence encoding one or more non-human (e.g., mouse) Adam6 polypeptides. The insertion is located outside the engineered immunoglobulin heavy chain locus described herein (e.g., at the 5' end of the V H This could be in the upstream of a gene segment, within an engineered IgH locus, or in the germline genome of a non-human animal (e.g., a randomly introduced non-human Adam6 coding sequence), or at other locations in cells or tissues.

[0088] In some embodiments, the engineered endogenous immunoglobulin heavy chain locus lacks the functional endogenous mouse Adam6 gene. In some embodiments, the mouse germline genome containing the engineered heavy chain locus contains one or more nucleotide sequences encoding one or more mouse ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments. In some embodiments, one or more mouse ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments are expressed (for example, in cells of the male germline, e.g., testicular cells).

[0089] In some embodiments, one or more mouse ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments, each are encoded on the same chromosome as the manipulated endogenous immunoglobulin heavy chain locus. In some embodiments, one or more mouse ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments, each are encoded on the same chromosome as the manipulated endogenous immunoglobulin heavy chain locus. In some embodiments, one or more mouse ADAM6 polypeptides, their functional orthologues, functional homologs, or functional fragments, each are encoded on the same chromosome as the first human V H Genetic segments and the second human V H It is located between gene segments. In some embodiments, the first human V H The gene segment is V H 1-2, and the second human V H The gene segment is V H 6-1. In some embodiments, one or more mouse ADAM6 polypeptides, one or more nucleotide sequences encoding their functional orthologues, functional homologs, or functional fragments replace the human Adam6 pseudogene. In some embodiments, one or more mouse ADAM6 polypeptides, one or more nucleotide sequences encoding their functional orthologues, functional homologs, or functional fragments replace the human Adam6 pseudogene. In some embodiments, one or more mouse ADAM6 polypeptides, one or more nucleotide sequences encoding their functional orthologues, functional homologs, or functional fragments replace the human V H Genetic segments and human D H It is located between gene segments.

[0090] Exemplary Ig variable regions, including Ig heavy chain gene segments, are provided, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and Supplemental Information, which are incorporated herein by reference. Such mice are described, for example, in U.S. Patent Nos. 8,642,835 and 8,697,940, which are incorporated herein by reference.

[0091] In some embodiments, the Ig heavy chain variable locus, which includes an unreconstructed human Ig heavy chain variable region gene segment, also includes a human Ig heavy chain variable region intergene sequence. In some embodiments, the Ig heavy chain variable locus includes a non-human (e.g., rodent, rat, mouse) Ig heavy chain variable region intergene 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., a rodent Eμ such as mouse or rat Eμ).

[0092] In some embodiments, the Ig heavy chain variable region is a reconstituted variable region containing an Ig heavy chain variable region gene (universal heavy chain variable region). In some embodiments, the reconstituted Ig heavy chain variable region gene is a reconstituted human Ig heavy chain variable region gene. An example of a reconstituted Ig heavy chain variable region is provided in U.S. Patent Publication 2014 / 0245468, which is incorporated herein by reference.

[0093] In certain embodiments, the mice provided herein express an antibody containing human Fc (e.g., human Fcα) by modifying the constant region of human immunoglobulin (e.g., human C) to express an antibody containing human Fc (e.g., human Fcα). αThis includes the constant region of human immunoglobulin. Mice containing the constant region of human immunoglobulin are disclosed, for example, in WO2019 / 190990, which is incorporated herein by reference in its entirety.

[0094] In certain embodiments, the immunoglobulin constant region is human C H 1 domain, human hinge region, human C H 2 domains, Human C H C encoding the IgG constant domain, which includes 3 domains, an IgG transmembrane domain, and an IgG cytoplasmic domain. H It includes a gene segment. In some embodiments, the IgG transmembrane domain is a rodent IgG transmembrane domain (e.g., a mouse or rat transmembrane domain). In certain embodiments, the transmembrane domain is a human IgG transmembrane domain. In some embodiments, the IgG cytoplasmic domain is a rodent IgG cytoplasmic domain (e.g., a mouse or rat cytoplasmic domain). In some embodiments, the IgG cytoplasmic domain is a human IgG cytoplasmic domain. In some embodiments, the IgG conjugate region is a rodent IgG conjugate region (e.g., a mouse or rat conjugate domain). In certain embodiments, the IgG conjugate region is a human IgG conjugate region.

[0095] In a particular embodiment, human C H 1 domain, human hinge region, human C H 2 domains, and human C H The third domain is the IgG1 domain. In some embodiments, such an IgG1 domain is encoded by an allele selected from IHG1*01, IHG1*02, IHG1*03, IHG1*04, and IHG1*05.

[0096] In a particular embodiment, human C H 1 domain, human hinge region, human C H 2 domains, and human C HThe third domain is the IgG2 domain. In some embodiments, such an IgG2 domain is encoded by an allele selected from IHG2*01, IHG2*02, IHG2*03, IHG2*04, IHG2*05, and IHG2*06.

[0097] In a particular embodiment, human C H 1 domain, human hinge region, human C H 2 domains, and human C H The third domain is the IgG3 domain. In some embodiments, such an IgG3 domain is encoded by an allele selected from IGG3*01, IGG3*02, IGG3*03, IGG3*04, IGG3*05, IGG3*06, IGG3*07, IGG3*08, IGG3*09, IGG3*10, IGG3*11, IGG3*12, IGG3*13, IGG3*14, IGG3*15, IGG3*16, IGG3*17, IGG3*18, and IGG3*19.

[0098] In a particular embodiment, human C H 1 domain, human hinge region, human C H 2 domains, and human C H The third domain is the IgG4 domain. In some embodiments, such an IgG4 domain is encoded by an allele selected from IHG4*01, IHG4*02, IHG4*03, and IHG4*04.

[0099] In some embodiments, C H The 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 to a suitable reference human immunoglobulin heavy chain constant region sequence) characterized by increased or decreased effector function and / or affinity for FcR compared to a reference human immunoglobulin heavy chain constant region.

[0100] In some embodiments, C H The gene segment encodes a constant region of the human immunoglobulin heavy chain characterized by altered affinity for activating and / or inhibitory receptors. In some embodiments, C H The gene segment encodes a constant region of the human immunoglobulin heavy chain, characterized by increased or decreased binding to the FcRn receptor at acidic pH compared to neutral pH, for example. In some embodiments, C H The gene segment encodes the human immunoglobulin heavy chain constant region, either entirely or partially, and encodes the human immunoglobulin heavy chain constant region having one or more amino acid modifications. Examples of amino acid modifications include positions 297 (e.g., N297A), 250 (e.g., 250E or 250Q), 252 (e.g., 252L, 252Y, 252F, 252W, or 252T), 254 (e.g., 254S or 254T), 256 (e.g., 256S, 256R, 256Q, 256E, 256D, or 256T), 307 (e.g., 307P or Substitutions include, but are not limited to, positions 307A, 308 (e.g., 308F or 308V), 428 (e.g., 428L or 428F), 433 (e.g., 433H, 433Lm, 433R, 433S, 433P, 433Q, or 433K), 434 (e.g., 434A, 434W, 434H, 434F, or 434Y), and combinations thereof. In some embodiments, C HThe gene segments are 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 N43 Encodes the constant region of a human immunoglobulin heavy chain having one or more pairs or groups of amino acid modifications selected from the group consisting of 4H); 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).

[0101] In some embodiments, C H The gene segment encodes a chimeric immunoglobulin heavy chain constant domain that contains segments or portions derived from (or appearing in) two or more human immunoglobulin isotypes. For example, such a chimeric C H The region is derived from human IgG1, human IgG2, or human IgG4 molecules. H The two domains are derived from human IgG1, human IgG2, or human IgG4 molecules. H It may include in combination with 3 domains. In some specific embodiments, chimeric C H The region further includes a chimeric hinge region. For example, a chimeric hinge may include an "upper hinge" amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region, combined with a "lower hinge" sequence (amino acid residues at positions 228 to 236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. In some specific embodiments, the chimeric hinge region includes amino acid residues derived from the human IgG1 or human IgG4 upper hinge and amino acid residues derived from the human IgG2 lower hinge.

[0102] In certain embodiments, modified CH The gene segment is endogenous C H It is located at the gene segment locus. In certain embodiments, a modified C H The gene segment is endogenous C γ1 Gene segment locus, endogenous C γ2a Gene segment locus, endogenous C γ2b Gene segment locus, endogenous C γ2c Gene segment locus, or endogenous C γ3 It is located at the gene segment locus.

[0103] In some embodiments, modified C H The gene segment is human C γ1 Gene segment (or at least human C γ1 C of the IgG1 constant domain of the gene segment H 1 domain, hinge region, C H 2 domains, and C H This is the part that codes for the 3 domains, and is endogenous C γ2a It is located at the gene segment locus. In some embodiments, human C γ1 C of the IgG1 constant domain of the gene segment H 1 domain, hinge region, C H 2 domains, and C H The part that codes for the 3 domains is endogenous mouse C γ2a It is functionally ligated to the portion of the gene segment that encodes the IgG2a transmembrane domain and / or cytoplasmic domain.

[0104] In some embodiments, modified C H The gene segment is human C γ1 Gene segment (or at least human C γ1 C of the IgG1 constant domain of the gene segment H 1 domain, hinge region, C H 2 domains, and C H This is the part that codes for the 3 domains, and is endogenous C γ2cIt is located at the gene segment locus. In some embodiments, human C γ1 C of the IgG1 constant domain of the gene segment H 1 domain, hinge region, C H 2 domains, and C H The part that codes for the 3 domains is endogenous mouse C γ2c It is functionally ligated to the portion of the gene segment that encodes the IgG2c transmembrane domain and / or cytoplasmic domain.

[0105] In some embodiments, modified C H The gene segment is human C γ4 Gene segment (or at least human C γ4 C of the IgG4 constant domain in the gene segment H 1 domain, hinge region, C H 2 domains, and C H This is the part that codes for the 3 domains, and is endogenous C γ1 It is located at the gene segment locus. In some embodiments, human C γ4 C of the IgG4 constant domain in the gene segment H 1 domain, hinge region, C H 2 domains, and C H The part that codes for the 3 domains is endogenous mouse C γ1 It is functionally ligated to the portion of the gene segment that encodes the IgG1 transmembrane domain and / or cytoplasmic domain.

[0106] In certain embodiments, modified C H The gene segment is endogenous C H Replace all or part of the gene segment. In certain embodiments, modified C H The gene segment is endogenous C γ1 Gene segment, endogenous C γ2a Gene segment, endogenous C γ2b Gene segment, endogenous C γ2c Gene segment, or endogenous C γ3 Replace all or part of a gene segment.

[0107] In some embodiments, modified C H The gene segment is human C γ1 Gene segment (or at least human C γ1 C of the IgG1 constant domain of the gene segment H 1 domain, hinge region, C H 2 domains, and C H This is the part that codes for the 3 domains, and is endogenous C γ2a Replace all or part of a gene segment locus. In some embodiments, human C γ1 C of the IgG1 constant domain of the gene segment H 1 domain, hinge region, C H 2 domains, and C H The part that codes for the 3 domains is endogenous mouse C γ2a C of the IgG2a constant domain of the gene segment H 1 domain, hinge region, C H 2 domains, and C H Replace the part that codes for the 3 domains, Human C γ1 C of the IgG1 constant domain of the gene segment H 1 domain, hinge region, C H 2 domains, and C H The part that codes for the 3 domains is endogenous mouse C γ2a To ensure that the gene segment is functionally ligated to the portion encoding the IgG2a transmembrane domain and / or cytoplasmic domain.

[0108] In some embodiments, modified C H The gene segment is human C γ4 Gene segment (or at least human C γ4 C of the IgG4 constant domain in the gene segment H 1 domain, hinge region, C H 2 domains, and C H This is the part that codes for the 3 domains, and is endogenous C γ1Replace all or part of a gene segment locus. In some embodiments, human C γ4 C of the IgG4 constant domain in the gene segment H 1 domain, hinge region, C H 2 domains, and C H The part that codes for the 3 domains is endogenous mouse C γ1 C of the IgG1 constant domain of the gene segment H 1 domain, hinge region, C H 2 domains, and C H Replace the part that codes for the 3 domains, Human C γ4 C of the IgG4 constant domain in the gene segment H 1 domain, hinge region, C H 2 domains, and C H The part that codes for the 3 domains is endogenous mouse C γ1 To ensure that the gene segment is functionally linked to the portion encoding the IgG1 transmembrane domain and / or cytoplasmic domain.

[0109] In certain embodiments, the Ig heavy chain constant region is one or more C2 cells in a rodent (e.g., rat or mouse). H It includes a gene segment. In some embodiments, the Ig constant region is C in rodents (e.g., rats or mice). μ It includes a gene segment. In some embodiments, the Ig constant region is C in rodents (e.g., rats or mice). δ It includes a gene segment. In some embodiments, the Ig constant region is C in rodents (e.g., rats or mice). γ1 It includes a gene segment. In some embodiments, the Ig constant region is C in rodents (e.g., rats or mice). γ2a It includes a gene segment. In some embodiments, the Ig constant region is C in rodents (e.g., rats or mice). γ2b It includes a gene segment. In some embodiments, the Ig constant region is C in rodents (e.g., rats or mice). γ2cIt includes a gene segment. In some embodiments, the Ig constant region is C in rodents (e.g., mice). γ3 It includes a gene segment. In some embodiments, the Ig constant region is C in rodents (e.g., rats or mice). ε It includes a gene segment. In some embodiments, the Ig constant region is C in rodents (e.g., rats or mice). ε Includes gene segments. In some embodiments, one or more rodent constant region gene segments are endogenous constant region gene segments. In some embodiments, the above modified C H The gene segment is the only modified C in the Ig heavy chain constant region. H This is a gene segment. In some embodiments, the above modified C H The gene segment contains multiple modified C regions within the Ig heavy chain constant region. H One of the gene segments (for example, one of the 2, 3, 4, 5, 6, 7, or 8 modified gene segments that are partially or fully humanized within the Ig heavy chain constant region). In some embodiments, the Ig heavy chain constant region is human or partially human C μ Includes a gene segment. In some embodiments, the Ig heavy chain constant region is human or partially human C δ Includes a gene segment. In some embodiments, the Ig heavy chain constant region is human or partially human C γ1 Includes a gene segment. In some embodiments, the Ig heavy chain constant region is human or partially human C γ2 Includes a gene segment. In some embodiments, the Ig heavy chain constant region is human or partially human C γ3 Includes a gene segment. In some embodiments, the Ig heavy chain constant region is human or partially human C γ4 Includes a gene segment. In some embodiments, the Ig heavy chain constant region is human or partially human C ε Includes a gene segment. In some embodiments, the Ig heavy chain constant region is human or partially human C αIt includes a gene segment. In some embodiments, the Ig heavy chain constant region is human C μ Gene segment, human C δ Gene segment, human C γ1 Gene segment, and human C γ3 It includes a gene segment. In some embodiments, the Ig heavy chain constant region is human C γ2 Gene segments and human C γ4 It further includes a gene segment. In some embodiments, the Ig heavy chain constant region is human C α It further includes a gene segment. In some embodiments, the Ig heavy chain constant region is human C ε It further includes gene segments.

[0110] In some embodiments, the IgH locus contains a regulatory element from a human or rodent (e.g., rat or mouse). In some embodiments, the regulatory element is an endogenous regulatory element. In certain embodiments, the IgH locus contains an intron enhancer (E) from a rodent (e.g., rat or mouse) or a human. i ) includes. In some embodiments, the IgH locus includes a rodent (e.g., rat or mouse) or human 3' regulatory region (3'RR).

[0111] In some embodiments, the modified immunoglobulin heavy chain locus is located at the endogenous immunoglobulin heavy chain locus. In some embodiments, the immunoglobulin heavy chain locus replaces all or part of the endogenous immunoglobulin heavy chain locus. In 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 deletion, transposition, and / or inversion of all or part of the endogenous Ig heavy chain locus).

[0112] Therefore, in some embodiments, one or more constant regions (or parts thereof) of the immunoglobulin heavy chain locus are not deleted (i.e., intact). In some embodiments, one or more C H The gene segment is functionally linked to the transmembrane and cytoplasmic coding sequences (multiple) (e.g., M1 and / or M2 coding sequences) of the non-human immunoglobulin heavy chain IgG constant region gene described herein (e.g., human IgG C H 1-HC H 2-C H 3. A sequence encoding a polypeptide), and in some embodiments, an immunoglobulin heavy chain constant region sequence functionally linked to the transmembrane and cytoplasmic coding sequences (multiple sequences) of the IgE constant region gene (e.g., human IgE C H 1-C H 2-C H 3-C H The sequence encoding the polypeptide is modified, disrupted, deleted, or substituted. In some embodiments, all or substantially all of the immunoglobulin heavy chain constant region is replaced by a heterologous immunoglobulin heavy chain constant region. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is functionally ligated 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 functionally ligated to the transmembrane and cytoplasmic coding sequences (e.g., M1 and M2 exons) of an IgE constant region gene. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is functionally ligated to the transmembrane and cytoplasmic coding sequences (e.g., M exon(s)) of an IgA constant region gene. In some specific embodiments, one or more C H gene segment (for example, C μ , C δThe immunoglobulin heavy chain constant region (e.g., C) is not deleted or substituted in the immunoglobulin heavy chain constant region, which includes a heterologous immunoglobulin heavy chain constant region sequence functionally linked to the transmembrane and cytoplasmic coding sequences of one or more constant region genes described herein. 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 modified, disrupted, deleted, substituted, or manipulated in one or more heterologous immunoglobulin heavy chain constant region sequences is a mouse immunoglobulin heavy chain constant region. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is the IgG constant region gene (e.g., C) of the immunoglobulin heavy chain constant region. γ1 , C γ2a , C γ2b , C γ2c , or C γ3 One copy of the IgG constant region gene (i.e., the allele) out of two copies is inserted, thereby producing a mouse that is heterozygous with respect to the heterologous immunoglobulin heavy chain constant region sequence. In some embodiments, a mouse that is homozygous with respect to the immunoglobulin heavy chain constant region is provided, comprising the heterologous immunoglobulin heavy chain constant region sequence described herein.

[0113] In some embodiments, the manipulated immunoglobulin heavy chain constant region described herein is a human extracellular domain coding sequence (e.g., human IgG C) functionally linked to a non-human transmembrane domain and cytoplasmic domain coding sequence (e.g., non-human IgG M1-M2) of the same or different IgG subclass. H 1-HC H 2-C H 3) One or more IgG codes C containing each of the above H Includes gene segments.

[0114] In some embodiments, the manipulated immunoglobulin heavy chain constant region described herein is one or more manipulated IgG codes described herein. H Includes gene segment and wild type (e.g., unmodified, non-human such as rat or mouse) Cμ It also includes constant region genes.

[0115] In some embodiments, the manipulated immunoglobulin heavy chain constant region described herein is one or more manipulated IgG codes described herein. H Includes gene segment and wild type (e.g., unmodified, non-human such as rat or mouse) C μ and C δ It also includes constant region genes.

[0116] In various embodiments, an engineered IgG code C comprising the human immunoglobulin heavy chain constant region sequence described herein is used. H The gene segment is C γ1 , C γ2a , C γ2b , C γ2c , or C γ3 The manipulated IgG code C of the IgG subclass selected from H It is a gene segment.

[0117] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region described herein is C H 1-HC H 2-C H Instead of exon 3, human IgG1 C H 1-HC H 2-C H Includes a sequence encoding 3, and is non-human (e.g., rat or mouse) C γ2a Modified C, functionally linked to the M1 and M2 exons of the gene segment. γ2a Includes gene segments.

[0118] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region described herein is C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C HContains sequences encoding 3-M1-M2 and is non-human (e.g., rat or mouse) C γ2a A modified C that is functionally linked to the switch region of a gene segment. γ2a Includes gene segments.

[0119] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region described herein is C H 1-HC H 2-C H Instead of exon 3, human IgG1 C H 1-HC H 2-C H Includes a sequence encoding 3, and is non-human (e.g., rat or mouse) C γ2c Modified C, functionally linked to the M1 and M2 exons of the gene segment. γ2c Includes gene segments.

[0120] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region described herein is C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H Contains sequences encoding 3-M1-M2 and is non-human (e.g., rat or mouse) C γ2c A modified C that is functionally linked to the switch region of a gene segment. γ2c Includes gene segments.

[0121] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region described herein is C H 1-HC H 2-C H Instead of exon 3, human IgG4 C H 1-HC H 2-C H Includes a sequence encoding 3, and is non-human (e.g., rat or mouse) C γ1Modified C, functionally linked to the M1 and M2 exons of the gene segment. γ1 Includes gene segments.

[0122] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region described herein is C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG4 C H 1-HC H 2-C H Contains sequences encoding 3-M1-M2 and is non-human (e.g., rat or mouse) C γ1 A modified C that is functionally linked to the switch region of a gene segment. γ1 Includes gene segments.

[0123] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region described herein is C H 1-HC H 2-C H Instead of exon 3, human IgG1 C H 1-HC H 2-C H Includes a sequence encoding 3, and is non-human (e.g., rat or mouse) C γ2a Modified C, functionally linked to the M1 and M2 exons of the gene segment. γ2a gene segment and C H 1-HC H 2-C H Instead of exon 3, human IgG4 C H 1-HC H 2-C H Includes a sequence encoding 3, and is non-human (e.g., rat or mouse) C γ1 Modified C, functionally linked to the M1 and M2 exons of the gene segment. γ1 Includes gene segments.

[0124] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region described herein is C H1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H Contains sequences encoding 3-M1-M2 and is non-human (e.g., rat or mouse) C γ2a A modified C that is functionally linked to the switch region of a gene segment. γ2a gene segment and C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG4 C H 1-HC H 2-C H Contains sequences encoding 3-M1-M2 and is non-human (e.g., rat or mouse) C γ1 A modified C that is functionally linked to the switch region of a gene segment. γ1 Includes gene segments.

[0125] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region described herein is C H 1-HC H 2-C H Instead of exon 3, human IgG1 C H 1-HC H 2-C H Includes a sequence encoding 3, and is non-human (e.g., rat or mouse) C γ2c Modified C, functionally linked to the M1 and M2 exons of the gene segment. γ2c gene segment and C H 1-HC H 2-C H Instead of exon 3, human IgG4 C H 1-HC H 2-C H Includes a sequence encoding 3, and is non-human (e.g., rat or mouse) C γ1 Modified C, functionally linked to the M1 and M2 exons of the gene segment. γ1 Includes gene segments.

[0126] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region described herein is C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H Contains sequences encoding 3-M1-M2 and is non-human (e.g., rat or mouse) C γ2c A modified C that is functionally linked to the switch region of a gene segment. γ2c gene segment and C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG4 C H 1-HC H 2-C H Contains sequences encoding 3-M1-M2 and is non-human (e.g., rat or mouse) C γ1 A modified C that is functionally linked to the switch region of a gene segment. γ1 Includes gene segments.

[0127] In various embodiments, the manipulated immunoglobulin heavy chain constant region described herein is human IgG C H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H One or more constant region genes (i.e., isotypes) other than the IgG constant region containing sequences encoding 3-M1-M2 (e.g., IgG1 and / or IgG2a), for example, IgD, IgE, IgA, and IgG (human IgG C, which itself is described herein). H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C HThis includes one or more further modifications, including rendering non-functional one or more immunoglobulin constant region genes encoding IgG (e.g., IgG2b and / or IgG3) that do not contain the sequence encoding 3-M1-M2, through whole or partial deletion, whole or partial alteration, whole or partial disruption, or whole or partial substitution. Manipulated non-human embryos, cells, and targeted vectors for producing such mice, embryos, and cells are also provided.

[0128] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region provided herein is wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segment and C γ1 A gene segment, C H 1-HC H 2-C H Instead of exon 3, human IgG4 C H 1-HC H 2-C H Includes an array that codes for 3, and C γ1 Functionally linked C to the M1-M2 exons of the gene segment γ1 gene segment and C δ , C γ2a , C γ2c , C γ2b , C γ3 , C ε and C α This includes deletions of gene segments.

[0129] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region provided herein is wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segment and C γ1 A gene segment, C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG4 C H 1-HC H 2-C HIncludes a sequence that codes for 3-M1-M2, and the C γ1 Functionally linked C to the switch region of the gene segment γ1 gene segment and C δ , C γ2a , C γ2c , C γ2b , C γ3 , C ε and C α This includes deletions of gene segments.

[0130] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region provided herein is wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segment and C γ2a A gene segment, C H 1-HC H 2-C H Instead of exon 3, human IgG1 C H 1-HC H 2-C H Includes an array that codes for 3, and C γ2a Functionally linked C to the M1-M2 exons of the gene segment γ2a gene segment and C δ , C γ1 , C γ2b , C γ2c , C γ3 , C ε and C α This includes deletions of gene segments.

[0131] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region provided herein is wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segment and C γ2a A gene segment, C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C HIncludes a sequence that codes for 3-M1-M2, and the C γ2a Functionally linked C to the switch region of the gene segment γ2a gene segment and C δ , C γ1 , C γ2b , C γ2c , C γ3 , C ε and C α This includes deletions of gene segments.

[0132] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region provided herein is wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segment and C γ2c A gene segment, C H 1-HC H 2-C H Instead of exon 3, human IgG1 C H 1-HC H 2-C H Includes an array that codes for 3, and C γ2c Functionally linked C to the M1-M2 exons of the gene segment γ2c gene segment and C δ , C γ1 , C γ2a , C γ2b , C γ3 , C ε and C α This includes deletions of gene segments.

[0133] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region provided herein is wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ gene segment and C γ2c A gene segment, C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C HIncludes a sequence that codes for 3-M1-M2, and the C γ2c Functionally linked C to the switch region of the gene segment γ2c gene segment and C δ , C γ1 , C γ2a , C γ2b , C γ3 , C ε and C α This includes deletions of gene segments.

[0134] In various embodiments, the manipulated immunoglobulin heavy chain constant region described herein is human IgG C H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H One or more constant region genes (i.e., isotypes) other than the IgG constant region containing sequences encoding 3-M1-M2 (e.g., IgG1 and / or IgG2a) are used in the human immunoglobulin heavy chain constant region sequences described herein, including IgD, IgE, IgA, and IgG (which itself is the human IgG C described herein). H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H This includes one or more further modifications, which involve manipulating an IgG (e.g., IgG2b and / or IgG3) that does not contain the sequence encoding 3-M1-M2 to be altered, modified, substituted, manipulated, etc., by insertion into one or more immunoglobulin constant region genes.

[0135] Also provided are engineered mouse embryos, cells, and targeted vectors for producing mice, embryos, and cells containing immunoglobulin loci having the engineered constant region described herein.

[0136] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region provided herein is wild-type C μGene segment and wild-type C δ gene segment and C γ3 A gene segment, C H 1-HC H 2-C H Instead of exon 3, human IgG3 C H 1-HC H 2-C H Includes an array that codes for 3, and C γ3 Functionally linked C to the M1-M2 exons of the gene segment γ3 gene segment and C γ1 A gene segment, C H 1-HC H 2-C H Human IgG4 C instead of exon 3 H 1-HC H 2-C H Includes an array that codes for 3, and C γ1 Functionally linked C to the M1-M2 exons of the gene segment γ1 gene segment and C γ2b A gene segment, C H 1-HC H 2-C H Instead of exon 3, human IgG2 C H 1-HC H 2-C H Includes an array that codes for 3, and C γ2b Functionally linked C to the M1-M2 exons of the gene segment γ2b gene segment and, Cγ2a A gene segment, C H 1-HC H 2-C H Instead of exon 3, human IgG1 C H 1-HC H 2-C H Includes an array that codes for 3, and C γ2a Functionally linked C to the M1-M2 exons of the gene segment γ2a Gene segment (and / or C γ2c A gene segment, C H 1-HC H 2-C HInstead of exon 3, human IgG1 C H 1-HC H 2-C H Includes an array that codes for 3, and C γ2c Functionally linked C to the M1-M2 exons of the gene segment γ2c (Genetic segment) and C ε A gene segment, C H 1-C H 2-C H 3-C H Instead of exon 4, human IgE C H 1-C H 2-C H 3-C H Includes an array that codes for 4, and the C ε Functionally linked C to the M1-M2 exons of the gene segment ε gene segment and C α A gene segment, C H 1-HC H 2-C H Instead of exon 3, human IgA1 or IgA2 C H 1-HC H 2-C H Includes an array that codes for 3, and C α Functionally linked C to the M exon(s) of a gene segment α Includes gene segments.

[0137] In some specific embodiments, the manipulated immunoglobulin heavy chain constant region provided herein is wild-type C μ Gene segment and wild-type C δ gene segment and C γ3 A gene segment, C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG3 C H 1-HC H 2-C H Includes a sequence that codes for 3-M1-M2, and the C γ3 Functionally linked C to the switch region of the gene segment γ3gene segment and C γ1 A gene segment, C H 1-HC H 2-C H Human IgG4 C instead of 3-M1-M2 exons H 1-HC H 2-C H Includes a sequence that codes for 3-M1-M2, and the C γ1 Functionally linked C to the switch region of the gene segment γ1 gene segment and C γ2b A gene segment, C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG2 C H 1-HC H 2-C H Includes a sequence that codes for 3-M1-M2, and the C γ2b Functionally linked C to the switch region of the gene segment γ2b gene segment and C γ2a A gene segment, C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H Includes a sequence that codes for 3-M1-M2, and the C γ2a Functionally linked C to the switch region of the gene segment γ2a Gene segment (and / or C γ2c A gene segment, C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H Includes a sequence that codes for 3-M1-M2, and the C γ2c Functionally linked C to the switch region of the gene segment γ2c (Genetic segment) and C ε A gene segment, C H 1-C H 2-CH 3-C H Instead of exon 4, human IgE C H 1-C H 2-C H 3-C H Includes an array that codes for 4, and the C ε Functionally linked C to the M1-M2 exons of the gene segment ε gene segment and C α A gene segment, C H 1-HC H 2-C H Instead of exon 3, human IgA1 or IgA2 C H 1-HC H 2-C H Includes an array that codes for 3, and C α Functionally linked C to the M exon(s) of a gene segment α Includes gene segments.

[0138] In some embodiments, mice are heterozygous for the modified immunoglobulin heavy chain locus described herein. In certain embodiments, mice are homozygous for the modified immunoglobulin heavy chain locus described herein.

[0139] Therapeutic agents containing human Fc (e.g., therapeutic agents containing human Fcα, such as therapeutic human IgA antibodies) are typically tested in non-human species (e.g., mice) before administration to humans. Unfortunately, drugs containing human Fc regions often exhibit very different pharmacokinetic and pharmacodynamic properties when administered to conventional mice compared to when administered to humans. For example, when a therapeutic agent with a human Fc region is administered to conventional mice, the human sequence within the Fc region is often recognized as a foreign substance by the mouse immune system. As a result, the mouse may initiate an immune response to the administered therapeutic agent (known as the mouse anti-human response or MAHA), which affects the pharmacokinetic and pharmacodynamic properties of the administered drug. Therefore, conventional mouse models are often inadequate predictors of the human therapeutic response to therapeutic agents containing human Fc. In certain embodiments, the mice provided herein exhibit a reduced MAHA response after administration of therapeutic agents containing human Fc. Thus, the mice disclosed herein, having a human immunoglobulin locus region, are useful in reducing or eliminating the MAHA response.

[0140] Therefore, as disclosed in, for example, WO2019 / 190990 (the entire document is incorporated herein by reference), expressing human or humanized FcαR and a human constant region (e.g., human C) α The MAHA response to the administration of human Fc-containing drugs (e.g., human Fcα-containing drugs such as therapeutic human IgA antibodies) can be tested using various mice provided herein, including those in the constant region.

[0141] Humanized immunoglobulin kappa locus In some embodiments, the mice provided herein further include a humanized Igκ chain locus. As disclosed in WO2019 / 190990 (the entire document is incorporated herein by reference), testing a therapeutic agent containing a human κ chain in mice having a humanized Igκ chain locus can reduce or eliminate the MAHA response because the human κ chain is less likely to be recognized as a foreign substance in such mice.

[0142] In some embodiments, genetically modified mice and ES cells that have been humanized or have the human FcαR1 locus in their genome further include a genetically modified Igκ chain locus. Such a locus includes a κ variable region and a κ constant region. The κ variable region includes the Igκ chain variable region gene segment (i.e., at least V κ Gene segment and J κ The gene segment is included. The constant region is the Igκ chain constant region (C κ ) Includes gene segments. In certain embodiments, immunoglobulin κ chain variable regions, such as the human Igκ variable region, are used in mice to obtain human V κ Gene segments and human J κ Light chain variable domains derived from gene segments, and C κ The immunoglobulin κ chain constant region is functionally linked to produce an antibody containing a light chain constant domain derived from a gene segment. In some embodiments, the Igκ variable region is an unreconstituted Igκ variable region and therefore includes an unreconstituted Igκ variable region gene segment. In some embodiments, the Igκ variable region is a reconstituted Igκ variable region and therefore includes a reconstituted Igκ variable region gene. In certain embodiments, the Igκ variable region gene segment is a human Igκ variable region gene segment. In certain embodiments, the Igκ variable region gene segment is a rodent Igκ variable region gene segment (e.g., a rat or mouse variable region gene segment). In some embodiments, the Igκ constant region locus includes a partially or completely human Igκ constant region gene segment. In some embodiments, the Igκ chain locus described herein is located at an endogenous Igκ chain locus.

[0143] In certain embodiments, the Igκ variable region includes an unreconstructed human Igκ variable region gene segment. In some specific embodiments, the manipulated Igκ light chain locus (or allele) is at least human V that appears in the distal variable cluster (or distal arm or distal duplication) of the naturally occurring human Igκ light chain locus. κ Includes a gene segment. In some specific embodiments, the manipulated Igκ light chain locus (or allele) appears in at least human V in the proximal variable cluster (or proximal arm or proximal duplication) of the naturally occurring human Igκ light chain locus. κ Includes gene segments. In some specific embodiments, the manipulated Igκ light chain locus (or allele) appears in the distal and proximal variable clusters of the naturally occurring human Igκ light chain locus. κ Includes a gene segment. In some specific embodiments, the manipulated Igκ light chain locus (or allele) is the same as the naturally occurring human Igκ light chain locus. κ 2-40 (or V κ 3D-7) Gene segment and human V κ Functional human V found between the 4-1 gene segment (including both ends) κ Includes all or substantially all of the gene segment.

[0144] In some embodiments, the unreconstructed human immunoglobulin variable region gene segment is multiple human V κ Segments and one or more human J κ The segment includes. In some embodiments, the immunoglobulin variable region gene segment contains four functional V κ Segment and all human J κ The segment includes. In some embodiments, the immunoglobulin variable region gene segment contains 16 functional V κ Segment and all human J κ Includes a segment. In some embodiments, the unreconstructed human immunoglobulin variable region gene segment is all human V κSegment and all human J κ Includes segments.

[0145] In some specific embodiments, the manipulated Igκ light chain locus (or allele) is 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.) of human V κ Includes a gene segment. In some specific embodiments, the manipulated Igκ light chain locus (or allele) is 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 κ 1-17, V κ 1-16, V κ 3-15, V κ 1-12, V κ 3-11, V κ 1-9, V κ 1-8, V κ 1-6, V κ 1-5, V κ 5-2, and Vκ Includes 4-1. In some specific embodiments, the manipulated Igκ light chain locus (or allele) is at least 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 Includes 2D-40. In some specific embodiments, the manipulated Igκ light chain locus (or allele) is at least 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 κ 5-2, and V κ Includes 4-1.

[0146] In some embodiments, the mice provided herein are two or fewer human V LGene segments and multiple J L It has a restricted immunoglobulin light chain locus characterized by a gene segment (e.g., double light chain mouse or DLC as described in U.S. Patent Publication 2013 / 0198880 (which is incorporated herein by reference)). In some embodiments, V L The gene segment is V κ It is a gene segment. In some embodiments, V L The gene segment is V λ It is a gene segment. In some embodiments, V κ The gene segment is V κ 3-20 and V κ The number is 1-39.

[0147] In some embodiments, the manipulated Igκ light chain locus (or allele) is one, two, three, four, five, or more functional human J κ Includes a gene segment. In some specific embodiments, the manipulated Igκ light chain locus (or allele) is the human J Igκ light chain locus that naturally occurs. κ 1 gene segment and human J κ Functional human J found between the 5 gene segments (including both ends) κ Includes all or substantially all of the gene segment. In some specific embodiments, the manipulated Igκ light chain locus (or allele) is at least human J κ Gene segment J κ 1, J κ 2, J κ 3, J κ 4, and J κ Includes 5.

[0148] In yet another embodiment, a non-human organism may include a light chain immunoglobulin locus in its germline and / or genome that includes insertions and / or substitutions of histidine codons designed to introduce pH-dependent binding properties to antibodies produced in such a non-human organism. In some of these embodiments, the histidine codon is inserted and / or substituted in a nucleic acid sequence encoding CDR3. Various such light chain immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, U.S. Patent Application Publications 2013 / 0247236 and 2014 / 0013456, which are incorporated herein by reference.

[0149] Exemplary variable regions containing the Igκ gene segment are provided, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and Supplementary Information, which are incorporated herein by reference. In some embodiments, the unreconstructed human immunoglobulin variable region gene segment includes all human Jκ segments.

[0150] In some embodiments, an Igκ variable locus containing an unreconstructed human Igκ variable region gene segment also includes a human Igκ variable region inter-gene sequence. In some embodiments, the Igκ variable locus includes a non-human (e.g., rodent, rat, mouse) Igκ variable region inter-gene sequence. In some embodiments, the Igκ 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).

[0151] In some embodiments, the Igκ variable region locus is a reconstituted variable region locus containing an Igκ variable region gene (universal light chain variable region). In some embodiments, the reconstituted Igκ variable region gene is a reconstituted human Igκ variable region gene (e.g., a reconstituted human Igκ variable region containing a human Vκ1-39Jκ or human Vκ3-20Jκ sequence, such as a human Vκ1-39Jκ5 or human Vκ3-20Jκ1 sequence). The use of a universal light chain variable region facilitates the production of bispecific antibodies. An example of a reconstituted Ig light chain variable region is provided in U.S. Patent Publication 2013 / 0185821, which is incorporated herein by reference.

[0152] In some embodiments, the Igκ chain locus contains a regulatory element from a human or rodent (e.g., rat or mouse). In some embodiments, the regulatory element is an endogenous regulatory element. In certain embodiments, the Igκ chain locus contains an intron κ enhancer (E) from a rodent (e.g., rat or mouse) or a human. κi ) includes. In some embodiments, the IgH locus is a rodent (e.g., rat or mouse) or human 3'κ enhancer (E κ3’ ) includes.

[0153] In some embodiments, the modified immunoglobulin κ-chain locus in the genetically modified rodent includes one or more unreconstructed human Vλ gene segments and one or more unreconstructed human Jλ gene segments upstream of the Cλ gene (e.g., functionally linked). An example of a rodent containing such a genetically modified immunoglobulin κ-chain locus is described in U.S. Patent No. 11,051,498 (which is incorporated herein by reference in its entirety). In some embodiments, the modified immunoglobulin κ-chain locus in the genetically modified rodent includes a limited human λ light chain variable region repertoire, which includes a single reconstructed human immunoglobulin λ light chain variable region (Vλ / Jλ). A single reconstructed human λ light chain variable region is ligated to a human Jλ gene segment and includes a human Vλ gene segment functionally ligated to a Cκ or Cλ gene segment (e.g., a rodent Cκ or Cλ gene segment (e.g., a mouse Cλ1 gene segment)). An example of a rodent containing such a genetically modified immunoglobulin κ chain locus is described in WO2020 / 247623 (which is incorporated herein by reference in its entirety).

[0154] In some embodiments, the modified immunoglobulin κ chain locus is located at the endogenous immunoglobulin κ chain locus. In some embodiments, the immunoglobulin κ chain locus replaces all or part of the endogenous immunoglobulin κ chain locus. In certain embodiments, the modified Igκ chain locus is located on a transgene that is positioned outside the endogenous locus. In some embodiments, the endogenous Igκ chain locus is inactivated (e.g., by deletion, transposition, and / or inversion of all or part of the endogenous Igκ chain locus).

[0155] In some embodiments, mice are heterozygous for the modified immunoglobulin κ chain locus described herein. In certain embodiments, mice are homozygous for the modified immunoglobulin κ chain locus described herein.

[0156] Therefore, as disclosed in, for example, WO2019 / 190990 (the entire document is incorporated herein by reference), expressing human or humanized FcαR and the human light chain constant region (and, in some embodiments, also human C α Using various mice provided herein, including those in the constant region, the MAHA response to the administration of human Fc-containing drugs (e.g., human Fcα-containing drugs such as therapeutic human IgA antibodies) can be tested.

[0157] Humanized immunoglobulin lambda locus In some embodiments, the mice provided herein further include a humanized Igλ chain locus. As disclosed in WO2019 / 190990 (the entire document is incorporated herein by reference), testing a therapeutic agent containing a human λ chain in mice having a humanized Igλ chain locus can reduce or eliminate the MAHA response because the human κ chain is less likely to be recognized as a foreign substance in such mice.

[0158] In some embodiments, mice and ES cells genetically modified to contain a humanized or human FcαR1 locus in their genome further contain a genetically modified Igλ chain locus. Such locus contains an Igλ chain variable region gene segment (i.e., at least V λ Gene segment and J λ The modified λ locus contains at least one Igλ chain constant region (C λ ) Further includes gene segments. In certain embodiments, human V λ Gene segments and human J λ V of gene segments, etc. λGene segment and J λ The gene segment is that mice are human V λ Gene segments and human J λ Light chain variable domains derived from gene segments, and C λ To produce an antibody containing a light chain constant domain derived from a gene segment, C λ Functionally connected to V. In some embodiments, V λ Gene segment and J λ The gene segment is unreconstructed V λ and J λ It may be a gene segment. In some embodiments, V λ Gene segment and J λ The gene segment is a rearranged V λ and J λ It can be a gene segment and therefore can be a form of a reconstituted variable region gene. In certain embodiments, Ig λ The variable region gene segment is a human variable region gene segment. In certain embodiments, Ig λ The variable region gene segment is a rodent variable region gene segment (e.g., a rat or mouse variable region gene segment). In some embodiments, Ig λ The constant region loci contain, partially or completely, human λ constant region gene segments. In some embodiments, the Igλ chain loci described herein are located at endogenous Igλ chain loci. Examples of variable regions containing Igλ gene segments include, for example, those provided in U.S. Patent Publications 2012 / 0073004 and 2002 / 0088016, and U.S. Patent Application 15 / 803,513 (filed November 3, 2017, published as U.S.2018 / 0125043), which are incorporated herein by reference.

[0159] In some embodiments, an Igλ variable locus containing an unreconstructed human Igλ variable region gene segment also includes a human Igλ variable region intergene sequence. In some embodiments, the Igλ variable locus includes a non-human (e.g., rodent, rat, mouse) Igλ variable region intergene sequence. In some embodiments, the Igλ 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).

[0160] In some embodiments, the human Igλ light chain locus comprises genetic material derived from the human Igλ light chain locus. In some embodiments, the human Igλ light chain locus described herein comprises at least one human V λ Gene segment, at least one human J λ Gene segment, at least one human C λ gene segment, and the at least one human V λ Gene segment and the at least one human J λ Promote rearrangement with gene segments, Human V λ Functionally reconstructed human V encoding the domain λ -J λ It includes one or more sequences (e.g., recombinant signal sequences [may include more]) necessary to form the sequence. In many embodiments, the human Igλ light chain sequence includes multiple human V λ Gene segment, and the human V λ Gene segment and at least one human J λ It includes one or more sequences necessary to facilitate rearrangement with gene segments. In some embodiments, the human Igλ light chain sequences described herein are genomic sequences of human Igλ light chain loci (e.g., isolated and / or cloned from bacterial artificial chromosomes) and comprise multiple human V λ It includes a gene segment. In some embodiments, the human Igλ light chain sequence is used to make up the germline of human Vλ , J λ , and C λ The sequence includes (i.e., the human V λ , J λ , and C λ Because the sequence appears at the Igλ light chain locus in human cells, in other words, J λ and C λ The array is J λ C λ (Appears as a cluster). In some embodiments, the human Igλ light chain sequence encodes an Igλ light chain polypeptide, which appears in immunoglobulins, particularly those expressed by human B cells. Mice, embryos, cells, and targeting constructs are also provided for producing mice, non-human embryos, and cells containing the human Igλ light chain sequence instead of the corresponding non-human Igλ light chain sequence (e.g., the endogenous mouse Igλ light chain locus).

[0161] In some embodiments, a human Igλ light chain sequence is inserted in place of a corresponding non-human Igλ light chain sequence in 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 centrally of one or more non-human Igλ light chain sequences so that the human Igλ light chain sequence is juxtaposed with the non-human Igλ light chain sequences.

[0162] In certain embodiments, the Igλ chain locus contains at least 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, or 40 functional V λ Includes a gene segment. In some embodiments, the gene locus is human V λ Gene segment V λ 3-10, V λ 3-9, V λ 2-8, V λ 4-3, and V λ Includes 3-1. In some embodiments, the gene locus is V λ 2-11, V λ 3-12, Vλ 2-14, V λ 3-16, V λ 3-19, V λ 3-21, V λ 3-22, λ 2-23, V λ 3-25, and V λ 3-27. In some embodiments, the locus is V λ 3-27, V λ 1-36, V λ 5-37, V λ 5-39, V λ 1-40, V λ 7-43, V λ 1-44, V λ 5-45, V λ 7-46, V λ 1-47, V λ 9-49, V λ 1-51, and V λ 5-52. In certain embodiments, the locus is V λ 10-54, V λ 6-57, V λ 4-60, V λ 8-61, and V λ 4-69. In some embodiments, the Igλ chain locus comprises one or more human J λ -C λ pairs. For example, in certain embodiments, the Igλ chain locus has a human J λ downstream of the human V gene segment, human J λ 1-C λ 1, J λ 2-C λ 2, J λ 3-C λ 3, J λ 6-C λ 6, and / or J λ 7-C λ 7. In some embodiments, the Ig λ chain locus has a human J λ downstream of the human V gene segment, human J λ 1-C λ 1, J λ 2-C λ 2, J λ 3-C λ ​λ 6-C λ 6. Human J λ 7, and mouse C λ Includes 1.

[0163] In some embodiments, the Igλ locus is a reconstituted locus containing an Igλ variable region gene (universal light chain variable region). In some embodiments, the reconstituted Igλ variable region gene is a reconstituted human Igλ variable region gene. The use of the universal light chain variable region facilitates the production of bispecific antibodies to which at least one antigen-binding domain binds. An example of a reconstituted Ig light chain variable region is provided in U.S. Patent Publication 2013 / 0185821, which is incorporated herein by reference.

[0164] In some embodiments, the Igλ locus contains a regulatory element from a human or rodent (e.g., rat or mouse). In some embodiments, the regulatory element is an endogenous regulatory element. In certain embodiments, the Igλ locus contains a rodent (e.g., rat or mouse) λ enhancer 2.4. In some embodiments, the Igλ locus contains a human or rodent (e.g., rat or mouse) 3'λ enhancer. In some embodiments, the Igλ locus contains a rodent (e.g., rat or mouse) λ enhancer 3.1.

[0165] In some embodiments, the modified immunoglobulin λ-chain locus is located at the endogenous immunoglobulin λ-chain locus. In some embodiments, the immunoglobulin λ-chain locus replaces all or part of the endogenous immunoglobulin λ-chain locus. In certain embodiments, the modified Igλ-chain locus is located on a transgene that is located outside the endogenous locus. In certain embodiments, the modified Igλ-chain locus is located at the endogenous immunoglobulin κ-chain locus. In some embodiments, the endogenous Igλ-chain locus is inactivated (e.g., by deletion, transposition, and / or inversion of all or part of the endogenous Igλ-chain locus).

[0166] In some embodiments, mice are heterozygous for the modified immunoglobulin λ-chain locus. In certain embodiments, mice are homozygous for the modified immunoglobulin λ-chain locus.

[0167] In some embodiments, mice have a light chain immunoglobulin locus in their germline and / or genome that contains a limited repertoire of light chain variable gene segments (e.g., a double light chain variable region containing two light chain variable gene segments). In some embodiments, the light chain variable gene segments in the limited repertoire of light chain gene segments are human light chain gene segments. An example of a double light chain variable region is provided in U.S. Patent Publication 2013 / 0198880, which is incorporated herein by reference. In some embodiments, mice containing a double light chain variable region are used to produce bispecific antibodies.

[0168] Therefore, as disclosed in, for example, WO2019 / 190990 (the entire document is incorporated herein by reference), expressing human or humanized FcαR and the human light chain constant region (and, in some embodiments, also human C αUsing various mice provided herein, including those in the constant region, the MAHA response to the administration of human Fc-containing drugs (e.g., human Fcα-containing drugs such as therapeutic human IgA antibodies) can be tested.

[0169] Humanized CD79a and CD79b loci In some embodiments, mice and ES cells genetically modified to contain the humanized or human FcαR1 locus in their genome further contain the human or humanized B cell antigen receptor complex-associated protein alpha chain (CD79a or Igα) and / or B cell antigen receptor complex-associated protein beta chain (CD79b or Igβ) locus. Mice containing the human or humanized CD79a and CD79b genes express the human or humanized CD79a and CD79b polypeptides as heterodimers on the surface of B cells, which non-covalently associate with membrane-expressed immunoglobulins to form B cell receptors (BCRs). BCRs associate with antigens and function in signal transduction and internal translocation after binding to the antigen. In some embodiments, the mice described herein contain the human or humanized CD79a and CD79b genes. In some specific embodiments, the mouse 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, each of the rodent CD79a and CD79b portions encodes at least the intracellular domain of the endogenous CD79a and CD79b polypeptides, respectively, and in some specific embodiments, encodes the transmembrane domain and the intracellular domain of the endogenous CD79a and CD79b polypeptides, respectively. In some embodiments, the human portion and the endogenous portion are functionally linked to an endogenous CD79a promoter or a CD79b promoter, respectively.

[0170] In some embodiments, the mouse 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 the human CD79a polypeptide; in one embodiment, a sequence comprising amino acids 33-119 of the human CD79a polypeptide; in one embodiment, a sequence comprising amino acids 33-143 of the human CD79a polypeptide; and in one embodiment, a sequence comprising amino acids 33-165 of the human CD79a polypeptide. In some embodiments, the chimeric CD79a polypeptide comprises a human Ig C2-like domain; in some embodiments, the chimeric CD79a polypeptide also comprises a human stalk region; in some embodiments, the chimeric CD79a polypeptide also comprises a human transmembrane domain; and in some embodiments, the chimeric CD79a polypeptide further comprises a rodent (e.g., mouse) cytoplasmic domain. In some embodiments, the mouse comprises a chimeric CD79a gene including the human region portion 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.

[0171] In some embodiments, the mouse 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 the human CD79b polypeptide; in one embodiment, it encodes a sequence comprising amino acids 29-159 of the human CD79b polypeptide; in another embodiment, it encodes a sequence comprising amino acids 29-184 of the human CD79b polypeptide. In some embodiments, the chimeric CD79b polypeptide comprises a human IgV-like domain; in some embodiments, the chimeric CD79b polypeptide also comprises a human stalk region; in some embodiments, the chimeric CD79b polypeptide also comprises a human transmembrane domain; and in some embodiments, the chimeric CD79b polypeptide further comprises a rodent (e.g., mouse) cytoplasmic domain. In some embodiments, the mouse comprises a chimeric CD79b gene including the human region portion 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.

[0172] GenBank accession numbers NP_001774.1, NM_001783.3, NP_067612.1, and NM_021601.3, as well as UniProt ID P11912, provide representative source sequences of the human CD79A gene and human CD79A polypeptide from which the desired human portion can be obtained. GenBank accession numbers NP_000617.1, NM_000626.2, NP_001035022.1, NM_001039933.1, NP_067613.1, and NM_021602.2, as well as UniProt ID P40259 provides representative source sequences for the human CD79B gene and human CD79B polypeptide from which the desired human portion can be obtained.

[0173] In some embodiments, the mice provided herein further comprise one or more human CD79A and CD79B genes described in U.S. Patent Publication Nos. 2011-0093963A1 and 2009-0053210A1, International Patent Publication No. WO2008 / 027986, and European Patent No. 2 064 325 B1 (each of which is incorporated herein by reference). In some specific embodiments, the mice provided herein include 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 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 mice provided herein include 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 containing amino acids 33-116 (e.g., a sequence containing amino acids 33-119, a sequence containing amino acids 33-143, or a sequence containing amino acids 33-165 of the human CD79a polypeptide), and the human CD79b portion encodes a sequence containing amino acids 29-135 (e.g., a sequence containing amino acids 29-159, or a sequence containing amino acids 29-184 of the human CD79a polypeptide). In some embodiments, each of the endogenous CD79a and CD79b portions encodes at least the intracellular domain of the endogenous CD79a and CD79b polypeptides, respectively, and in some specific embodiments, encodes the transmembrane domain and the intracellular domain of the endogenous CD79a and CD79b polypeptides, respectively.

[0174] Humanized β-2-microglobulin In some embodiments, mice and ES cells genetically modified to contain a humanized or human FcαR1 locus in their genome further contain a locus encoding a humanized β-2-microglobulin (β2M) polypeptide. β2M is a polypeptide that lacks a transmembrane domain and binds to FcRn and MHC class I molecules.

[0175] In some embodiments, the β2M locus contains 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 mouse β2M locus. In certain embodiments, the nucleic acid sequence encoding the β2M polypeptide replaces all or part of the endogenous mouse β2M gene. In some embodiments, the mouse does not express mouse β2M or does not express a functional mouse β2M polypeptide. In some embodiments, the β2M locus contains 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).

[0176] Humanized β2M polypeptide, the gene locus encoding the humanized β2M polypeptide, and mice expressing the humanized β2M polypeptide are described in U.S. Patent Publications 2013 / 0111617 and 2013 / 0185819, respectively, which are incorporated herein by reference. Accordingly, as described in U.S. Patent Publications 2013 / 0111617 and 2013 / 0185819, in some embodiments the mouse comprises a humanized β2M gene, which comprises exons 2, 3, and 4 of the human β2M gene, and in some embodiments the humanized β2M gene comprises exon 1 of the non-human (e.g., mouse) β2M gene. In some embodiments the mouse is heterozygous for the genetically modified β2M locus. In some embodiments the mouse is homozygous for the genetically modified β2M locus.

[0177] Genetically modified mice and ES cells In certain embodiments, the Specified herein provides genetically modified mice and ES cells comprising one or more of the humanization loci disclosed herein, as well as genetically modified mouse ES cells useful for producing such mice.

[0178] In certain embodiments, genetically modified mice and mouse ES cells containing the humanized or human FcαR1 locus in their germline and / or genome are provided herein. For example, in some embodiments, the mouse or ES cells contain the FcαR locus provided herein in their germline and / or genome. In some embodiments, the mouse or ES cells further contain the IgH locus provided herein. In some embodiments, the mouse or ES cells further contain the Igκ and / or Igλ locus provided herein. In some embodiments, the mouse or ES cells contain the CD79a and / or CD79b locus provided herein in their germline and / or genome. In some embodiments, the mouse or ES cells contain the FcRn locus provided herein in their germline and / or genome. In some embodiments, the mouse or ES cells contain the β2M locus provided herein in their germline and / or genome. In some embodiments, the mouse or ES cells contain the FcεR1α locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR1a locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR2a locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR2b locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR3a locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR3b locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR2c locus provided herein in their germline and / or genome.In some embodiments, mouse or ES cells are heterozygous for one or more gene loci provided herein, for example, a genetically engineered locus. In some embodiments, mouse or ES cells are homozygous for one or more gene loci provided herein, for example, a genetically engineered locus.

[0179] In some embodiments, the mouse is a 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 mouse is a 129 strain. In some embodiments, the 129 strain is selected from the group consisting of the 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2 strains. In some embodiments, the genetically modified mouse is a mixture of the 129 strain and the C57BL strain. In some embodiments, the mouse is a mixture of the 129 strain and / or the C57BL / 6 strain. In some embodiments, the mixed 129 strain is the 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse is the BALB strain (e.g., BALB / c). In some embodiments, the mice are a mixture of the BALB strain and another strain (e.g., the C57BL strain and / or the 129 strain). In some embodiments, the mice provided herein may be derived from any combination of the aforementioned strains.

[0180] Genetically modified mice and ES cells can be produced using any suitable method known in the art. For example, such genetically modified mouse ES cells can be produced using the VELOCIGENE® technology described in U.S. Patents 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, each of which is incorporated herein by reference. Modifications can also be carried out using genome-targeted nuclease systems such as the CRISPR / Cas system, the activator-like effector nuclease (TALEN) system, or the zinc finger nuclease (ZFN) system. In some embodiments, the modifications are carried out using CRISPR / Cas systems described, for example, in U.S. Patent Applications No. 14 / 314,866, No. 14 / 515,503, No. 14 / 747,461, and No. 14 / 731,914, each of which is incorporated herein by reference. Exemplary methods for producing such genetically modified mice and ES cells are also provided in Example 1 herein.

[0181] Subsequently, the ES cells described herein can be used to produce mice using methods known in the art. For example, using the mouse ES cells described herein, genetically modified mice can be produced using the VELOCIMOUSE® method, as described in U.S. Patent No. 7,294,754 and Poueymirou et al., Nature Biotech 25:91-99 (2007), each of which is incorporated herein by reference. The resulting mice may be homozygous.

[0182] Testing methods for human Fcα-containing therapeutic drugs In certain embodiments, this specification provides a method for testing a therapeutic protein (e.g., a human antibody or Fcα fusion protein) containing a human Fcα domain, comprising administering the therapeutic protein to mice provided herein. In some embodiments, this specification provides animal models for carrying out such methods.

[0183] In some embodiments, the administered human antibody or Fc fusion protein is human C at the genetically modified IgH locus of the mouse provided herein. H It has isotypes and / or allotypes that match the isotypes and / or allotypes of the Fc domain encoded by. For example, in some embodiments, the drug is a human IgA1 antibody and the mouse has C encoding the human IgA1 CH1, hinge, CH2, and CH3 domains. H It contains a genetically modified IgH locus that includes a gene segment. In some embodiments, the drug is a human IgA2 antibody, and the mouse is human IgA2 C encoding the CH1, hinge, CH2, and CH3 domains. H It contains a genetically modified IgH locus that includes a gene segment. In some embodiments, the drug is an antibody containing a human heavy chain variable domain (e.g., an IgA1 or IgA2 antibody).

[0184] In some embodiments, the method includes measuring one or more pharmacokinetic properties of an administered therapeutic protein. In some embodiments, the animal model used to determine the pharmacokinetic properties of an administered human antibody or fusion protein is a genetically modified mouse provided herein, which includes the human FcαR locus.

[0185] In some embodiments, one or more pharmacokinetic parameters include, but are not limited to, plasma concentration-to-area-time (AUC), in vivo recovery rate (IVR), clearance rate (CL), mean residence time (MRT), drug half-life (t1 / 2), and steady-state volume of distribution (Vss). Generally, the pharmacokinetic properties of a administered therapeutic agent are determined by administering a selected dose of the 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 / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg or more) and then determining how the plasma concentration of the 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).

[0186] In some embodiments, the method further includes measuring the therapeutic efficacy of the administered therapeutic protein (e.g., the ability of a given dose of therapeutic protein to alleviate 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 some embodiments, the animal model is an autoimmune or inflammatory model, and disease symptoms may include, for example, cytokine expression levels, immune cell proliferation, tissue damage, and / or animal survival. In some embodiments, the animal model is an infectious disease model, and disease symptoms may include, for example, infectivity levels, tissue damage, and / or animal survival.

[0187] In some embodiments, the method further includes measuring the safety and dosage of the administered therapeutic protein (e.g., the extent to which a given dose of therapeutic protein causes one or more adverse effects in an animal model). Adverse effects include, but are not limited to, allergic reactions, alopecia, anaphylaxis, anemia, loss of appetite, balance disorders, bleeding, thrombosis, dyspnea, bronchitis, bruising, decreased white blood cell count, decreased red blood cell count, decreased platelet count, cardiotoxicity, conjunctivitis, constipation, cough, dehydration, diarrhea, electrolyte imbalance, infertility, fever, hair loss, heart failure, infection, injection site reactions, iron deficiency, renal failure, leukopenia, hepatic dysfunction, pneumonia, rapid heartbeat, rectal bleeding, convulsions, weight loss, and weight gain. In some embodiments, provided herein are methods for measuring therapeutic-induced allergic reactions using passive cutaneous anaphylaxis (PCA) and / or passive systemic anaphylaxis (PSA) models.

[0188] In some embodiments, the method further includes measuring the extent to which a therapeutic protein induces one or more FcαR-mediated reactions in mice (e.g., the extent to which a therapeutic protein induces antibody-dependent cell-mediated cytotoxicity (ADCC)). For example, in certain embodiments, provided herein is a method for screening therapeutic agents comprising the human Fcα region of a human antibody, comprising: (a) administering a drug comprising the Fcα region of a human antibody to mice provided herein, wherein the drug binds to target cells in the mice; (b) measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against the target cells; and (c) comparing the amount of ADCC in step (b) to a control, wherein an increase in the killing of target cells indicates an increase in the drug's ability to mediate ADCC.

[0189] In some embodiments, the method further includes measuring the extent to which administration of a therapeutic protein induces an anti-human Fcα immune response in mice. In some embodiments, the antibodies tested by the methods provided herein have a human light chain variable domain. In some embodiments, the light chain variable domain is a λ light chain variable domain. In some embodiments, the light chain variable domain is a κ light chain variable domain. In some embodiments, the antibody has a human light chain constant domain. In some embodiments, the light chain constant domain is a λ light chain constant domain. In some embodiments, the light chain constant domain is a κ light chain constant domain. 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, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242 and the IMGT database (available at www.imgt.org)).

[0190] In some embodiments, the therapeutic agent is administered to mice provided herein as part of a pharmaceutical composition, for example, a pharmaceutical composition containing a human IgA antibody or Fc fusion protein formulated with a pharmaceutically acceptable carrier.

[0191] The pharmaceutical compositions provided herein may be specifically formulated for administration in solid or liquid form, including (1) oral administration, e.g., drenches (aqueous or nonaqueous solutions or suspensions), tablets, e.g., those intended for oral mucosa, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue, or (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., sterile solutions or suspensions, or those adapted for sustained-release formulations.

[0192] Pharmaceutical compositions suitable for parenteral administration include a human IgA antibody or Fc fusion protein in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into a sterile injectable solution or dispersion immediately before use, and may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes that make the preparation isotonic with the blood of the target recipient, or suspending agents or viscosity modifiers.

[0193] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate 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 dispersants, and by the use of surfactants.

[0194] In some embodiments, the composition contains a human antibody or Fc fusion protein at a concentration that yields a w / v suitable for the desired dose. The antibody is 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 It may be present in the composition at concentrations of mg / mL, at least 90 mg / mL, at least 95 mg / mL, at least 100 mg / mL, at least 105 mg / mL, at least 110 mg / mL, at least 115 mg / mL, at least 120 mg / mL, at least 125 mg / mL, at least 130 mg / mL, at least 135 mg / mL, at least 140 mg / mL, at least 150 mg / mL, at least 200 mg / mL, at least 250 mg / mL, or at least 300 mg / mL.

[0195] In some embodiments, the composition is prepared in the form of a lyophilized composition or aqueous solution of a desired final concentration by mixing a human IgA antibody or Fc fusion protein with an optional physiologically acceptable carrier, excipient, or stabilizer, such as, 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)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used, and include buffers, e.g., histidine, phosphates, citrates, glycine, acetates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., These include serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including trehalose, glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., TWEEN, polysorbate 80, PLURONICS®, or polyethylene glycol (PEG).

[0196] In some embodiments, the buffer is histidine, citrate, phosphate, glycine, or acetate. The sugar excipient may be trehalose, sucrose, mannitol, maltose, or raffinose. The surfactant may be polysorbate 20, polysorbate 40, polysorbate 80, or Pluronic F68. The salt may be NaCl, KCl, MgCl2, or CaCl2.

[0197] In some embodiments, the composition includes a buffer or pH adjuster to provide improved pH control. Such compositions may 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 further embodiments, such compositions have a pH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In some embodiments, the pH of the composition is about 6.0. Those skilled in the art will understand that the pH of the composition should generally not be the same as the isoelectric point of the human antibody or Fc fusion protein used in the composition. Typically, buffers are salts prepared from organic or inorganic acids or bases. Typical buffers include, but are not limited to, organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid salts; Tris buffer, tromethamine hydrochloride buffer, or phosphate buffer. In addition, amino acid components can also function as buffers. Typical amino acid components that can be used in compositions as buffers include, but are not limited to, glycine and histidine. In some embodiments, the buffer is selected from histidine, citrate, phosphate, glycine, and acetate. In some embodiments, the buffer is histidine. In another specific embodiment, the buffer is citrate. In yet another embodiment, the buffer is glycine. The purity of the buffer must 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 means the chemical purity of histidine or glycine as understood in the art, for example, as described in The Merck Index, 13th ed., O'Neil et al. ed. (Merck & Co., 2001).

[0198] In some embodiments, the composition contains histidine as a buffer. In some embodiments, histidine is present in the composition at concentrations 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. In another embodiment, the composition contains histidine in amounts of approximately 1 mM to approximately 200 mM, approximately 1 mM to approximately 150 mM, approximately 1 mM to approximately 100 mM, approximately 1 mM to approximately 75 mM, approximately 10 mM to approximately 200 mM, approximately 10 mM to approximately 150 mM, approximately 10 mM to approximately 100 mM, approximately 10 mM to approximately 75 mM, approximately 10 mM to approximately 50 mM, approximately 10 mM to approximately 40 mM, approximately 10 mM to approximately 30 mM, approximately 20 mM to approximately 75 mM, approximately 20 mM to approximately 50 mM, approximately 20 mM to approximately 40 mM, or approximately 20 mM to approximately 30 mM. In further embodiments, the composition contains 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 of histidine. In some embodiments, the composition may contain about 10 mM, about 25 mM of histidine, or may not contain histidine at all.

[0199] In some embodiments, the composition includes a carbohydrate excipient. The carbohydrate excipient may act, for example, as a thickener, stabilizer, bulking agent, solubilizer, and / or similar. The carbohydrate excipient is generally present in amounts of about 1% to about 99% by weight or volume, for example, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 5%, about 1% to about 20%, about 5% to about 15%, about 8% to about 10%, about 10% and about 15%, about 15% and about 20%, 0.1% to about 20%, 5% to about 15%, 8% to 10%, 10% to 15%, 15% to 20%, about 0.1% to about 5%, about 5% to about 10%, or about 15% to about 20%. In yet another specific embodiment, the carbohydrate excipient is present in an amount of 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 15%, or 20%.

[0200] In some embodiments, the composition includes 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, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melegitose, maltodextrin, dextran, and starch; and algitols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol). In some embodiments, the carbohydrate excipient used in the composition provided herein is selected from sucrose, trehalose, lactose, mannitol, and raffinose. In some embodiments, the carbohydrate excipient is trehalose. In another specific embodiment, the carbohydrate excipient is mannitol. In yet another specific embodiment, the carbohydrate excipient is sucrose. In yet another specific embodiment, the carbohydrate excipient is raffinose. The purity of the carbohydrate excipient must be at least 98%, or at least 99%, or at least 99.5%.

[0201] In some embodiments, the composition contains trehalose. In some embodiments, the composition contains 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% of trehalose. In another embodiment, the composition contains about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 2% to about 40%, about 2% to about 30%, about 2% to about 20%, about 4% to about 40%, about 4% to about 30%, or about 4% to about 20% of trehalose. In further embodiments, the composition contains about 1%, about 2%, about 4%, about 6%, about 8%, about 15%, about 20%, about 30%, or about 40% of trehalose. In some embodiments, the composition contains about 4%, about 6%, or about 15% of trehalose.

[0202] In some embodiments, the composition includes an excipient. In some embodiments, the composition includes at least one excipient selected from sugars, salts, surfactants, amino acids, polyols, chelating agents, emulsifiers, and preservatives. In some embodiments, the composition includes a salt, such as a salt selected from NaCl, KCl, CaCl2, and MgCl2. In some embodiments, the composition includes NaCl.

[0203] In some embodiments, the composition comprises amino acids, such as lysine, arginine, glycine, histidine, or amino acid salts. The composition may contain at least about 1 mM, at least about 10 mM, at least about 25 mM, at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, or at least about 400 mM of amino acids. In another embodiment, the composition may contain amino acids in amounts of about 1 mM to about 100 mM, about 10 mM to about 150 mM, about 25 mM to about 250 mM, about 25 mM to about 300 mM, about 25 mM to about 350 mM, about 25 mM to about 400 mM, about 50 mM to about 250 mM, about 50 mM to about 300 mM, about 50 mM to about 350 mM, about 50 mM to about 400 mM, about 100 mM to about 250 mM, about 100 mM to about 300 mM, about 100 mM to about 400 mM, about 150 mM to about 250 mM, about 150 mM to about 300 mM, or about 150 mM to about 400 mM. In further embodiments, the composition contains about 1 mM, 1.6 mM, 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about 400 mM of amino acids.

[0204] In some embodiments, the composition includes a surfactant. As used herein, the term "surfactant" refers to an organic substance having an amphiphilic structure, i.e., a group having opposite solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified into anionic, cationic, and nonionic surfactants depending on the charge of their surfactant moiety. Surfactants are often used as wetting agents, emulsifiers, solubilizers, and dispersants in a variety of pharmaceutical compositions and preparations of biological materials. Polysorbate (e.g., Polysorbate 20 or 80); Poloxamer (e.g., Poloxamer 188); Triton; Sodium Octyl Glycoside; Lauryl-, Myristyl-, Linoleyl-, or Stearyl-Sulfobetaine; Lauryl-, Myristyl-, Linoleyl-, or Stearyl-Sarcosine; Linoleyl-, Myristyl-, or Cetyl-Betaine; Lauroamidopropyl-, Cocamidopropyl-, Linoleamidopropyl-, Myristamidopropyl-, PaImidopropyl-, or Isostearamidopropyl-Betaine (e.g., Lauroamidopropyl); Myristamidopropyl-, PaImidopropyl-, or Isostearamidopropyl-Dimethylamine; Sodium Cocoyl Methyltaurate, or Disodium Oleyl Methyltaurate; and MONAQUA® series (Mona pharmaceutically acceptable surfactants such as polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONICS® PF68) may be optionally added to the composition to reduce aggregation. In some embodiments, the composition comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. Surfactants are particularly useful when a pump or plastic container is used to dispense the composition. The presence of a pharmaceutically acceptable surfactant mitigates the protein's aggregation properties.The composition may contain polysorbate in 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 contains polysorbate in 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%.

[0205] In some embodiments, the composition includes other excipients and / or additives, including, but not limited to, diluents, binders, stabilizers, lipophilic solvents, preservatives, and adjuvants. pharmaceutically acceptable excipients and / or additives may be used in the compositions provided herein. Commonly used excipients / additives, such as pharmaceutically acceptable chelating agents (e.g., EDTA, DTPA, or EGTA), may be optionally added to the composition to reduce aggregation. These additives are particularly useful when a pump or plastic container is used to dispense the composition.

[0206] In some embodiments, the composition contains a preservative. Preservatives such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercury nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate, but not limited to), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, may be optionally added to the composition in any preferred concentration, such as about 0.001% to about 5%, or any range or value within that range. The concentration of the preservative used in the composition is sufficient to produce a microbial activity. Such a concentration depends on the selected preservative and is readily determined by those skilled in the art.

[0207] In some embodiments, the composition is isotonic with human blood and has essentially the same osmotic pressure as human blood. Such isotonic compositions generally have an osmotic pressure of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, by using a vapor pressure or freezing point osmometer. The tonicity of a composition is adjusted using a tonicity modifier. A “tonicity modifier” is a pharmaceutically acceptable inert substance that can be added to a composition to give it isotonicity. Suitable tonicity modifiers for the compositions provided herein include, but are not limited to, sugars, salts, and amino acids.

[0208] In some embodiments, the composition is a pyrogen-free composition that is substantially free of endotoxins and / or associated pyrogens. Endotoxins include toxins trapped inside microorganisms that are released only when the microorganism decomposes or dies. Pyrogens also include pyrogenic, heat-stable substances from the outer membranes of bacteria and other microorganisms. Any of these substances, when administered to humans, can cause fever, hypotension, and shock. Due to their potential adverse effects, endotoxins must be removed from intravenously administered drug solutions, even in small amounts. The U.S. Food and Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) / dose / kilogram body weight per hour for intravenous drug administration (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, such as the target protein (e.g., an antibody), harmful and dangerous endotoxins must be removed, even in trace amounts. In some embodiments, the levels of endotoxin and pyrogens 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.

[0209] When used for in vivo administration, the compositions described herein must be sterile. The compositions may be sterilized by a variety of sterilization methods, including sterile filtration and radiation. In some embodiments, the compositions are sterile filtered through a sterile 0.22 micron filter. Sterile compositions for injection may be formulated in accordance with conventional pharmaceutical practices, such as those described in "Remington: The Science & Practice of Pharmacy," 21st ed., Lippincott Williams & Wilkins, (2005). Compositions containing the protein of interest (e.g., an antibody), such as those disclosed herein, are typically stored in lyophilized form or in solution. Sterile compositions containing the protein of interest (e.g., an antibody) are intended to be contained in intravenous solution bags or vials having an adapter from which the composition can be removed, such as a container with a sterile access port, for example, a stopper pierced by a subcutaneous needle. In some embodiments, the compositions are provided as pre-filled syringes.

[0210] In some embodiments, the composition is a lyophilized formulation. The terms "lyophilized" or "freeze-dried" include a state of a substance subjected to a drying procedure such as lyophilization, from which at least 50% of the moisture has been removed.

[0211] Regardless of the selected route of administration, the agents and / or pharmaceutical compositions provided herein, which can be used in a preferred hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0212] In the methods provided herein, human antibodies, Fc fusion proteins, and / or pharmaceutical compositions may be delivered by any preferred route of administration, including, for example, oral, nasal, e.g., by spray, rectal, vaginal, parenteral, intracisional, and topical, by powder, ointment or drop, oral mucosa, and sublingual administration. In some embodiments, the pharmaceutical composition is administered systemically (e.g., via oral or parenteral administration).

[0213] In some embodiments, the actual dose of the active ingredient in the pharmaceutical composition described herein may be varied to determine the amount of the active ingredient that is effective in achieving a desired therapeutic response in an animal model without toxicity, in that animal model, composition, and administration method.

[0214] For example, in certain embodiments, the mice described herein are used to determine the pharmacokinetic profiles of one or more human antibody candidates. In various embodiments, one or more mice described herein and one or more control or reference mice are exposed to one or more human antibody candidates at various doses (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg or more). The candidate therapeutic antibodies may be administered via any desired route of administration, including parenteral and non-parenteral routes of administration. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intrathecal, intrasacral, lateral ventricle, intracranial, intrathoracic, or other injection routes. Non-parenteral routes include, for example, oral, nasal, percutaneous, pulmonary, rectal, buccal, vaginal, and ocular administration. Administration may be by continuous infusion, local administration, sustained release from implants (gel, membrane, etc.), and / or intravenous injection. Blood is isolated from mice (humanized and control) at various time points (e.g., 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days, or longer). To determine the pharmacokinetic profile of the administered candidate therapeutic antibody or Fc fusion polypeptide, various assays may be performed using, but are not limited to, total IgA, anti-therapeutic antibody reactions, agglutination, etc., using samples obtained from mice as described herein.

[0215] In various embodiments, the mice described herein are used to measure therapeutic effects that block or modulate the activity of a polypeptide of interest, and effects on gene expression as a result of cellular changes, or, in relation to the receptor polypeptide, effects on the density of receptor polypeptides on the cell surface of mice. In various embodiments, the mice described herein or cells isolated from such mice are exposed to a candidate therapeutic agent that binds to the polypeptide of interest, and after a certain period, their effects on specific cellular processes related to the polypeptide of interest, such as ligand-receptor interaction or signaling, are analyzed.

[0216] The mice described herein provide an improved in vivo system for the development and selection of human IgA antibodies or Fc fusion polypeptides for use in oncology and / or infectious diseases. In various embodiments, the mice described herein and control mice (e.g., mice with different genetic modifications than those described herein, or unmodified, i.e., wild-type mice) may be transplanted with tumors (or tumor cells) or infected with viruses (e.g., influenza, HIV, HCV, HPV, etc.). After transplantation or infection, the mice may be administered a candidate therapeutic agent. The tumors or viruses may be given sufficient time to colonize one or more sites within the mouse before administration of the candidate therapeutic agent. Alternatively and / or additionally, the immune response in such mice may be monitored for the characterization and selection of potential human antibodies that may be developed as therapeutic agents.

[0217] Method for producing genetically modified mice and ES cells In certain embodiments, the methods provided herein are for producing mice and ES cells containing one or more of the genetically modified loci provided herein. Exemplary methods for producing the genetically modified mice and ES cells provided herein are described herein in the description, examples, and / or figures. For example, in some embodiments, the methods provided herein are for producing mice and ES cells containing the FcαR locus provided herein. In some embodiments, the FcαR locus is inserted between the Pira6 gene and the Ncr1 gene on mouse chromosome 7. In some embodiments, the FcαR locus is inserted between nucleotide residues 4,303,905–4,312,280 on mouse chromosome 7 (GRCm38 assembly).

[0218] In some embodiments, methods for producing mice and ES cells further comprising the humanized CD79a locus and / or the humanized CD79b locus provided herein. In some embodiments, methods for producing mice and ES cells further comprising the human or humanized FcRn locus and / or the human or humanized β2M locus provided herein. In some embodiments, methods for producing mice and ES cells further comprising the human or humanized FcεR1α locus provided herein. In certain embodiments, methods for producing mice and ES cells further comprising the human or humanized FcγR1a locus provided herein. In some embodiments, methods for producing mice and ES cells further comprising the human or humanized FcγR2a locus, the human or humanized FcγR2b locus, the human or humanized FcγR2c locus, the human or humanized FcγR3a locus, and / or the human or humanized FcγR3b locus provided herein are provided. In some embodiments, methods for producing mice and ES cells further comprising the human or humanized heavy chain and / or light chain locus provided herein are provided. Exemplary methods for producing genetically modified mice and ES cells provided herein are described in the description, examples, and / or figures herein.

[0219] kit This specification provides packs or kits comprising one or more containers filled with at least one mouse, mouse cell, DNA fragment, and / or targeted vector as described herein, in the descriptions, examples, and / or figures. The kits may be used in any applicable method (e.g., research method). Such containers(s) may optionally be accompanied by documentation in a format prescribed by government agencies regulating the manufacture, use, or sale of pharmaceutical or biological products, the documentation indicating (a) an approval by the authority for manufacture, use, or sale for human administration, (b) a method of use, and (c) a contract governing the transfer of materials and / or biological products (e.g., mice or mouse cells as described herein) between two or more entities and combinations thereof.

[0220] Exemplary Embodiments According to Exemplary Embodiment 1, the Specified herein provides a mouse having an Fc alpha receptor (FcαR) locus located in the mouse leukocyte receptor complex (LRC) in its genome, wherein the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain.

[0221] According to an exemplary embodiment 2, the mouse of embodiment 1 is provided wherein the FcαR locus is located in the intergenetic region between the Tthy1 protein locus and the Rdh13 protein locus.

[0222] According to exemplary embodiment 3, the mouse of embodiment 1 or 2 is provided herein, wherein the FcαR locus is located in the intergenetic region between the Lilra5 protein locus and the Gp6 protein locus.

[0223] According to an exemplary embodiment 4, the Specified herein provides a mouse in any one of embodiments 1 to 3, wherein the FcαR locus is located in an intergenetic region between the Pira6 protein locus and the Ncr1 protein locus, such as between the nucleic acid sequences encoding the Pira6 protein and the Ncr1 protein.

[0224] According to an exemplary embodiment 5, the mouse of embodiment 4 is provided herein, wherein the intergenetic region is a 54kb region between the Pira6 locus and the Ncr1 locus.

[0225] According to exemplary embodiment 6, the Specified herein provides a mouse of any one of embodiments 1 to 5, wherein the FcαR locus is located between coordinates 4,303,905 and 4,312,280 (+ strand, GRCm38 assembly) on mouse chromosome 7.

[0226] According to an exemplary embodiment 7, the Specified herein provides a mouse in any one of embodiments 1 to 6, wherein the FcαR locus comprises a nucleic acid sequence encoding a human FcαR polypeptide.

[0227] According to an exemplary embodiment 8, the Specified herein provides a mouse in any one of embodiments 1 to 7, wherein the FcαR locus comprises human exons 1 to 5 of the human Fc alpha receptor gene.

[0228] According to exemplary embodiment 9, this specification provides a mouse of any one of embodiments 1 to 6, wherein the FcαR locus comprises the non-coding portion of non-mouse rodent FcαR exon 1, human FcαR exons 1 and 2, human FcαR exons 3 and 4, and the coding portion of non-mouse rodent FcαR exon 5.

[0229] According to exemplary embodiment 10, the Specified herein provides a mouse of embodiment 7 or 8, wherein the human or humanized FcαR receptor locus comprises a genomic sequence (+ strand, GRCh38 assembly) located between coordinates 54,862,297–54,906,185 on human chromosome 19.

[0230] According to exemplary embodiment 11a, the Specified provides a mouse of embodiment 7 or 8 in which the FcαR locus further comprises a nucleic acid sequence present in the human KIR3DL2 gene. According to exemplary embodiment 11b, the Specified provides a mouse of embodiment 7, 8, or 11a in which the FcαR locus further comprises a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

[0231] According to exemplary embodiment 12, the Specified Spectrum provides a mouse according to any one of embodiments 1 to 11, wherein the mouse expresses the FcαR polypeptide on mouse neutrophils, monocytes, macrophages, eosinophils, and plasmacytoid dendritic cells.

[0232] According to exemplary embodiment 13, the Specified Specification provides a mouse which is heterozygous for the FcαR locus, and is one of any of embodiments 1 to 12.

[0233] According to exemplary embodiment 14, the Specified Specification provides a mouse according to any one of embodiments 1 to 12, wherein the mouse is homozygous for the FcαR locus. According to exemplary embodiment 15, the Specified herein provides any one of embodiments 1 to 14, wherein the genome further comprises a human or humanized Fc gamma receptor (FcγR) locus, a human or humanized IgH locus, a human or humanized Igκ locus, a human or humanized Igλ locus, a human or humanized FcRn locus, a human or humanized β2M locus, and / or a human or humanized FcεR1α locus.

[0234] According to an exemplary embodiment 16, the Specified herein provides a mouse of Embodiment 15, wherein the mouse comprises a human or humanized FcγR locus in its genome that includes a nucleic acid sequence encoding human or humanized FcγR.

[0235] According to exemplary embodiment 17, the Specified herein provides a mouse of embodiment 16 in which the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more low-affinity FcγR selected from Fc-gamma receptor 2a (FcγR2a), Fc-gamma receptor 2b (FcγR2b), Fc-gamma receptor 3a (FcγR3a), Fc-gamma receptor 3b (FcγR3b), and / or Fc-gamma receptor 2c (FcγR2c).

[0236] According to exemplary embodiment 18, the Specified herein provides a mouse of embodiment 16 in which the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more FcγR selected from human or humanized Fc-gamma receptor 1-alpha (FcγR1a), Fc-gamma receptor 2a (FcγR2a), Fc-gamma receptor 2b (FcγR2b), Fc-gamma receptor 3a (FcγR3a), Fc-gamma receptor 3b (FcγR3b), and / or Fc-gamma receptor 2c (FcγR2c).

[0237] According to exemplary embodiment 19, the Specified herein provides a mouse in any one of embodiments 16-18, wherein the human or humanized FcγR comprises a human extracellular domain. According to exemplary embodiment 20, the Specified Specification provides a mouse in any one of embodiments 16 to 19, wherein the human or humanized FcγR comprises a mouse transmembrane domain.

[0238] According to exemplary embodiment 21, the Specified Specification provides a mouse in any one of embodiments 16 to 19, wherein the human or humanized FcγR comprises a human transmembrane domain. According to exemplary embodiment 22, the Specified herein provides a mouse from any one of embodiments 16 to 21, wherein the human or humanized FcγR comprises a mouse cytoplasmic domain.

[0239] According to exemplary embodiment 23, the Specified herein provides a mouse from any one of embodiments 16 to 21 in which the human or humanized FcγR comprises a human cytoplasmic domain. According to exemplary embodiment 24, the Specified herein provides a mouse in any one of embodiments 16 to 23, wherein the human or humanized FcγR locus is located at the endogenous mouse FcγR locus.

[0240] According to exemplary embodiment 25, the Specified herein provides a mouse of embodiment 24 in which the nucleic acid sequence encoding human or humanized FcγR replaces all or part of the endogenous mouse FcγR gene.

[0241] According to exemplary embodiment 26, the Specified herein provides a mouse of embodiment 25 in which the nucleic acid sequence encoding human or humanized FcγR comprises a nucleic acid sequence encoding a human FcγR extracellular domain that replaces an endogenous nucleic acid sequence encoding a mouse FcγR extracellular domain.

[0242] According to exemplary embodiment 27, the Specified Specification provides any one of embodiments 15 to 26, wherein the mouse does not express mouse FcγR. According to exemplary embodiment 28, the Specified Specified Mouse is provided which is heterozygous for any one of embodiments 15 to 27, wherein the mouse is heterozygous for the human or humanized FcγR locus, human or humanized IgH locus, human or humanized Igκ locus, human or humanized Igλ locus, human or humanized FcRn locus, human or humanized β2M locus, and / or human or humanized FcεR1α locus.

[0243] According to exemplary embodiment 29, the Specified Specified Mouse is provided which is homozygous for any one of embodiments 15 to 27, wherein the mouse is homozygous for the human or humanized FcγR locus, human or humanized IgH locus, human or humanized Igκ locus, human or humanized Igλ locus, human or humanized FcRn locus, human or humanized β2M locus, and / or human or humanized FcεR1α locus.

[0244] According to an exemplary embodiment 30, the Specified herein provides mouse embryonic stem cells (ES cells) having an Fc alpha receptor (FcαR) locus located in the leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain.

[0245] According to exemplary embodiment 31, the mouse ES cells of embodiment 30 are provided, wherein the FcαR locus is located in the intergenetic region between the Tthy1 protein locus and the Rdh13 protein locus.

[0246] According to exemplary embodiment 32, the mouse ES cells of embodiment 31 or 32 are provided, wherein the FcαR locus is located in the intergenetic region between the Lilra5 protein locus and the Gp6 protein locus.

[0247] According to exemplary embodiment 33, the Specified herein provides a mouse ES cell in any one of embodiments 30-32, wherein the Fc alpha receptor (FcαR) locus is located in an intergenetic region between the Pira6 protein locus and the Ncr1 protein locus, such as between the nucleic acid sequences encoding the Pira6 protein and the Ncr1 protein.

[0248] According to exemplary embodiment 34, the mouse ES cells of embodiment 33 are provided herein, wherein the intergenetic region is a 54kb region between the Pira6 locus and the Ncr1 locus.

[0249] According to exemplary embodiment 35, the Specified herein provides a mouse ES cell of any one of embodiments 30 to 34, wherein the FcαR locus is located between coordinates 4,303,905 and 4,312,280 (+ strand, GRCm38 assembly) on mouse chromosome 7.

[0250] According to exemplary embodiment 36, this specification provides a mouse ES cell from any one of embodiments 30 to 35, wherein the FcαR locus comprises a nucleic acid sequence encoding a human FcαR polypeptide.

[0251] According to exemplary embodiment 37, this specification provides a mouse ES cell from any one of embodiments 30 to 36, wherein the FcαR locus comprises human exons 1 to 5 of the human Fc alpha receptor gene.

[0252] According to exemplary embodiment 38, the Spec herein provides a mouse ES cell of any one of embodiments 30 to 35, wherein the FcαR locus comprises the non-coding portion of non-mouse rodent FcαR exon 1, human FcαR exons 1 and 2, human FcαR exons 3 and 4, and the coding portion of non-mouse rodent FcαR exon 5.

[0253] According to exemplary embodiment 39, the Specified herein provides mouse ES cells of embodiment 36 or 37, wherein the human or humanized FcαR receptor locus comprises a genomic sequence (+ strand, GRCh38 assembly) located between coordinates 54,862,297–54,906,185 on human chromosome 19.

[0254] According to exemplary embodiment 40a, the mouse ES cells of embodiment 36 or 37 are provided, wherein the FcαR locus further comprises a nucleic acid sequence present in the human KIR3DL2 gene. According to exemplary embodiment 40b, the mouse ES cells of embodiment 36, 37, or 40a are provided, wherein the FcαR locus further comprises a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

[0255] According to exemplary embodiment 41, the Specified herein provides mouse ES cells from any one of embodiments 30 to 40, wherein the ES cells are heterozygous for the FcαR locus.

[0256] According to exemplary embodiment 42, the Specified herein provides mouse ES cells from any one of embodiments 30 to 40, wherein the ES cells are homozygous for the FcαR locus.

[0257] According to exemplary embodiment 43, the Specified herein provides a mouse ES cell from any one of embodiments 30 to 42, further comprising in the genome a human or humanized Fc gamma receptor (FcγR) locus, a human or humanized IgH locus, a human or humanized Igκ locus, a human or humanized Igλ locus, a human or humanized FcRn locus, a human or humanized β2M locus, and / or a human or humanized FcεR1α locus.

[0258] According to an exemplary embodiment 44, the mouse ES cells of embodiment 43 are provided herein, wherein the mouse ES cells include a human or humanized FcγR locus in the genome that contains a nucleic acid sequence encoding human or humanized FcγR.

[0259] According to exemplary embodiment 45, the Specified herein provides mouse ES cells of embodiment 44, wherein the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more low-affinity FcγR selected from Fc-gamma receptor 2a (FcγR2a), Fc-gamma receptor 2b (FcγR2b), Fc-gamma receptor 3a (FcγR3a), Fc-gamma receptor 3b (FcγR3b), and / or Fc-gamma receptor 2c (FcγR2c).

[0260] According to an exemplary embodiment 46, the Specified herein provides mouse ES cells of Embodiment 44, wherein the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more FcγR selected from human or humanized Fc-gamma receptor 1-alpha (FcγR1a), Fc-gamma receptor 2a (FcγR2a), Fc-gamma receptor 2b (FcγR2b), Fc-gamma receptor 3a (FcγR3a), Fc-gamma receptor 3b (FcγR3b), and / or Fc-gamma receptor 2c (FcγR2c).

[0261] According to exemplary embodiment 47, the Specified herein provides a mouse ES cell from any one of embodiments 44 to 46, wherein the human or humanized FcγR comprises a human extracellular domain.

[0262] According to exemplary embodiment 48, the Specified herein provides a mouse ES cell in any one of embodiments 44-47, wherein the human or humanized FcγR comprises a mouse transmembrane domain.

[0263] According to exemplary embodiment 49, the Specified herein provides a mouse ES cell from any one of embodiments 44 to 47, wherein the human or humanized FcγR comprises a human transmembrane domain.

[0264] According to exemplary embodiment 50, the Specified herein provides a mouse ES cell from any one of embodiments 44 to 49, wherein the human or humanized FcγR comprises a mouse cytoplasmic domain.

[0265] According to exemplary embodiment 51, the Specified herein provides a mouse ES cell from any one of embodiments 44 to 49, wherein the human or humanized FcγR comprises a human cytoplasmic domain.

[0266] According to exemplary embodiment 52, the Specified herein provides mouse ES cells in any one of embodiments 44 to 51, wherein the human or humanized FcγR locus is located at the endogenous mouse FcγR locus.

[0267] According to exemplary embodiment 53, the mouse ES cells of embodiment 52 are provided wherein the nucleic acid sequence encoding the human or humanized FcγR replaces all or part of the endogenous mouse FcγR gene.

[0268] According to an exemplary embodiment 54, the Specified herein provides mouse ES cells of Embodiment 53, wherein the nucleic acid sequence encoding human or humanized FcγR comprises a nucleic acid sequence encoding a human FcγR extracellular domain that replaces an endogenous nucleic acid sequence encoding the mouse FcγR extracellular domain.

[0269] According to exemplary embodiment 55, the Specified herein provides mouse ES cells from any one of embodiments 43 to 54, wherein the mouse is heterozygous for the human or humanized FcγR locus, human or humanized IgH locus, human or humanized Igκ locus, human or humanized Igλ locus, human or humanized FcRn locus, human or humanized β2M locus, and / or human or humanized FcεR1α locus.

[0270] According to exemplary embodiment 56, the Specified herein provides mouse ES cells from any one of embodiments 43 to 54, wherein the mouse is homozygous for the human or humanized FcγR locus, human or humanized IgH locus, human or humanized Igκ locus, human or humanized Igλ locus, human or humanized FcRn locus, human or humanized β2M locus, and / or human or humanized FcεR1α locus.

[0271] According to exemplary embodiment 57, this specification provides a method for testing a human IgA antibody or Fcα fusion polypeptide, the method comprising administering the IgA antibody or Fcα fusion polypeptide to a mouse from any one of embodiments 1 to 29.

[0272] According to exemplary embodiment 58, the method of embodiment 57 is provided, further comprising measuring one or more pharmacokinetic properties of the administered human IgA antibody or Fcα fusion polypeptide.

[0273] According to an exemplary embodiment 59, the Specified Method of Embodiment 58 is provided, wherein the one or more pharmacokinetic properties are selected from one or more of the following: plasma concentration-to-area-time (AUC), in vivo recovery rate (IVR), clearance rate (CL), mean residence time (MRT), drug half-life (t1 / 2), and / or steady-state volume of distribution (Vss).

[0274] According to exemplary embodiment 60, the Specified herein provides one of any one of embodiments 57 to 59, further comprising measuring the therapeutic efficacy of the administered human antibody or Fcα fusion polypeptide.

[0275] According to exemplary embodiment 61, the Specified herein provides one of the methods of embodiments 57 to 60, further comprising administering a plurality of doses of the human antibody or Fcα fusion polypeptide and determining the therapeutic efficacy of each dose of the human antibody or Fcα fusion polypeptide.

[0276] According to exemplary embodiment 62, the Spectrum provides any one of embodiments 57 to 61, further comprising administering a plurality of doses of the human antibody or Fcα fusion polypeptide and determining the safety of each dose of the human antibody or Fcα fusion polypeptide.

[0277] According to exemplary embodiment 63, the Specified herein provides one of the methods of embodiments 57 to 62, further comprising administering a plurality of doses of the human antibody or Fcα fusion polypeptide and determining the tolerance of each dose of the human antibody or Fcα fusion polypeptide.

[0278] According to exemplary embodiment 64, the Specified herein provides any one of embodiments 57 to 63, further comprising measuring one or more Fc receptor-mediated responses in the mouse.

[0279] According to exemplary embodiment 65, the method of embodiment 64 is provided herein, wherein the one or more Fc receptor-mediated reactions are antibody-dependent cell-mediated cytotoxicity (ADCC) reactions.

[0280] According to an exemplary embodiment 66, the Specified Method provides any one of embodiments 57 to 65, wherein the human antibody binds to the target cells of the mouse, and the Method further comprises measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against the target cells and comparing the amount of ADCC to a control, wherein an increase in target cell death indicates an increased ability of the drug to mediate ADCC.

[0281] According to exemplary embodiment 67, the Specified herein provides any one of embodiments 57 to 66, further comprising measuring the immune response produced by the mouse against the human antibody.

[0282] According to an exemplary embodiment 68, the Specified Method provides a method for modifying a mouse genome, comprising inserting an Fc alpha receptor (FcαR) locus into a leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR locus comprises a nucleic acid sequence encoding an FcαR polypeptide including a human extracellular domain and a human or rodent cytoplasmic domain.

[0283] According to exemplary embodiment 69, the method of embodiment 68 is provided herein, wherein the FcαR locus comprises a nucleic acid sequence encoding a human or humanized FcαR polypeptide.

[0284] According to exemplary embodiment 70, the method of embodiment 68 or 69 is provided herein, wherein the FcαR locus is located in the intergenetic region between the Tthy1 protein locus and the Rdh13 protein locus.

[0285] According to exemplary embodiment 71, the Specified herein provides any one of embodiments 68 to 70, wherein the FcαR locus is located in the intergenetic region between the Lilra5 protein locus and the Gp6 protein locus.

[0286] According to exemplary embodiment 72, the Spectrum provides any one of embodiments 68 to 71, wherein the FcαR locus is located in an intergenetic region between the Pira6 protein locus and the Ncr1 protein locus, such as between the nucleic acid sequences encoding the Pira6 protein and the Ncr1 protein.

[0287] According to exemplary embodiment 73, the method of embodiment 72 is provided herein, wherein the intergenetic region is a 54kb region between the Pira6 locus and the Ncr1 locus.

[0288] According to exemplary embodiment 74, the Specified Method of Any One of Embodiments 68 to 73 is provided, wherein the FcαR locus is located between coordinates 4,303,905 and 4,312,280 (+ strand, GRCm38 assembly) on mouse chromosome 7.

[0289] According to exemplary embodiment 75, this specification provides a method for producing a mouse containing the Fc alpha receptor (FcαR) gene locus, comprising producing mouse ES cells of any one of embodiments 30 to 56, and producing a mouse from the ES cells. [Examples]

[0290] Example 1.1: Genetic engineering of mice containing human or humanized FcαR Targeted vectors containing nucleic acid sequences encoding human FcαR (MAID20277 and MAID20278) cassettes were generated. MAID20277 and MAID20278 were identical except that MAID20277 retained a selection marker (i.e., neomycin resistance). Briefly, the human FCAR genes were introduced into the mouse genome using VELOCIGENE® technology (see, e.g., U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) Nat. Biotech. 21(6):652-659, the contents of both publications are incorporated herein by reference in their entirety). The 44kb genomic sequence of a human FCAR, including the promoter, was inserted into the mouse LRC locus located between the mouse Pira6 gene and the Ncr1 gene on mouse chromosome 7, at coordinates chr7:4,303,905~4,312,280 (+ strand, GRCm38 assembly) (Figures 1A and 1B).

[0291] More specifically, mouse homology arms were prepared by PCR amplification using BAC clone RP23-458i16 as a template, and are shown in Table 1.

[0292] [Table 1]

[0293] The mouse homologous arms were assembled from 5' to 3' into a construct (construct A) containing a 5' arm, a spectinomycin-resistant cassette with adjacent I-CeuI and PI-SceI homing endonuclease sites, a 3' arm, and a chloramphenicol-resistant cassette. In a post-processing step of the genetic engineering, the spectinomycin-resistant cassette in construct A was replaced with an insert containing a human FCAR.

[0294] A human insert containing the entire FCAR gene and adjacent sequences was created by two consecutive bacterial homologous recombination (BHR) modifications of the BAC clone CTD-3161p22. In the first BHR step, the spectinomycin resistance cassette and I-CeuI site were inserted into the 5' end of the human BAC. In the second BHR step, a sequence of approximately 44kb containing the NCR1 gene was deleted from the 3' end of the human BAC, and the mutant lox site was replaced with the adjacent neomycin resistance cassette (lox2372-Ub-neo-lox2372) and 3'PI-SceI site. At this point, this construct (construct B) contained, from 5' to 3', a spectinomycin-resistant cassette, an I-CeuI site, a 44,887 bp human genome sequence (GRCh38 coordinates chr19: 54,862,297~54,906,185), a lox2372-Ub-neo-lox2372 cassette, and a PI-SceI site. The human sequence included the last approximately 5kb of the KIR3DL2 gene (starting at intron 6), the entire FCAR gene (approximately 17kb), and the 5'UTR of the NCR1 gene.

[0295] To construct the final targeting vector (designated MAID20277), constructs A and B were both digested with I-CeuI and PI-SceI restriction enzymes and ligated together. The final targeting vector contained, from 5' to 3', a 5' mouse homology arm, an I-CeuI site, a 44,887 bp human genome insert containing the FCAR gene, a lox2372-Ub-Neo-lox2372 cassette, a PI-SceI site, a 3' mouse homology arm, and a chloramphenicol resistance cassette. The final clone was selected based on CM / kanamycin resistance (Figure 2). Various junctions of the final clone are shown in Table 2.

[0296] [Table 2]

[0297] The MAID20277 targeted vector was electroporated into mouse embryonic stem (ES) cells containing the human low-affinity Fcγ receptor. Genetic modification of the low-affinity Fcγ receptor locus is described in U.S. Patent No. 8,658,154 and is schematically shown in Figure 3. Targeted homologous recombination resulted in the deletion of approximately 8.4 kb of mouse sequence (GRCm38 coordinates chr7: 4,303,905~4,312,280) and the insertion of approximately 44 kb of human sequence containing the FCAR gene. The success of this integration is documented, for example, in the work of Valenzuela et al. As described in al., this was confirmed by a modal allele (MOA) assay. The primers (forward (F) primers and reverse (R) primers) and probes used in the MOA assay to detect the presence of human FCAR sequences and the loss of mouse LRC sequences are shown in Table 3 below, and their locations are shown in Figure 1A. Subsequently, the antibody resistance cassette was removed by transient expression of CRE recombinase in ES cell clones (Figure 1B).

[0298] [Table 3]

[0299] Positively targeted ES cells (including the gene encoding the human low-affinity Fcγ receptor described above) were used as donor ES cells and microinjected into premorlar (8-cell) stage mouse embryos using the VELOCIMOUSE® method (see, e.g., US7,576,259, US7,659,442, US7,294,754, and US2008-0078000A1, the contents of which are incorporated herein by reference in their entirety). Mouse embryos containing donor ES cells were incubated in vitro and transplanted into surrogate mothers to produce F0 mice entirely derived from donor ES cells. Mice possessing the human FCAR gene (and the gene encoding the human low-affinity Fcγ receptor) were identified by genotyping using the MOA assay described above. Heterozygous mice for the human FCAR gene were bred until homozygous.

[0300] Alternatively, instead of the above strategy, a targeted vector is electroporated into wild-type ES cells (containing endogenous low-affinity Fcγ receptors), and mice are generated using the VELOCIMOUSE® method. Mice containing human FcαR are bred until they contain human low-affinity FcγR (having the locus shown in Figure 3). Mice containing human FcαR (and optionally human low-affinity FcγR) can also be bred using techniques well known in the art until they contain human or humanized high-affinity FcγR, FcεR, FcRn, β2M, and mice containing humanized Ig heavy and / or light chain loci.

[0301] Example 1.2: Phenotypic analysis of genetically modified mice containing human or humanized FcαR Mice described in Example 1.1 above, containing the human FCAR gene and the low-affinity FCGR gene, were analyzed for FcαR expression. All mice were housed and bred under conditions free of specific pathogens. Mice were sacrificed and their spleens and blood were collected. Blood was collected in BD microtenor tubes containing EDTA (catalog no. 365973). Red blood cells and blood preparations from the spleen were lysed in ACK lysis buffer (ThermoFisher, catalog no. A1049201) and subsequently washed in complete RPMI medium.

[0302] Both spleen cells and blood cells were incubated in Live Dead Aqua (ThermoFisher) to eliminate unviable cells. Before staining, the cells were incubated on ice for 10 minutes with anti-mouse CD16 / 32 (2.4G2, BD). Next, the cells were stained on ice for 30 minutes with fluorescent dye-conjugated anti-human CD89 (BioLegend, clone number A59) and the following anti-mouse antibodies: CD45 (BioLegend, clone numbers 30-F11), B220 (BioLegend, clone numbers RA3-6B2), CD11c (BioLegend, clone number N418), Ly6G (BioLegend, clone number 1A8), Ly6C (BioLegend, clone number HK1.4), SiglecF (BD Biosciences clone numbers E50-2440), NKp46 (BioLegend, clone number 29A1.4), CD11b (BioLegend, clone number M1 / 70), F4 / 80 (BD Biosciences clone numbers T45-2342), and NK1.1 (BioLegend, clone number PK136).

[0303] Lymphocyte and bone marrow cell populations were identified by flow cytometry using a BD Symphony A3 instrument (BD Biosciences). First, viable single cells were gated with CD45+. Next, the following subsets were identified: B cells (B220+), cDCs (CD11c++), pDCs (CD11cint, Ly6C+ B220+), PMNs (Ly6G+ Ly6C+), eosinophils (Ly6G-, SiglecF+ CD11b+), macrophages / monocytes (Ly6G-, SiglecF-, F4 / 80+ CD11b+), and NK cells (Ly6G-, SiglecF-, F4 / 80-CD11b-, NK1.1+). CD89(FcαR) expression was measured in each cell type.

[0304] Mice genetically engineered with the MAID20278 cassette showed increased hFcαR expression in neutrophils, eosinophils, some blood monocytes / macrophages, and plasmacytoid dendritic cells in the spleen and blood. No hFcαR expression was observed in splenic monocytes or macrophages (Figures 4A-4B).

[0305] Embedding by reference All publications, patents, and patent applications described herein are incorporated herein by reference in whole, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. In the event of any conflict, this application shall prevail, including all definitions herein.

[0306] Equal portions Those skilled in the art will recognize, or can confirm by mere ordinary experimentation, many equivalents to the particular embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. A mouse whose genome contains an Fc alpha receptor (FcαR) locus located in the mouse leukocyte receptor complex (LRC), wherein the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain.

2. The mouse according to claim 1, wherein the FcαR gene locus is located in the intergenetic region between the Tthy1 protein locus and the Rdh13 protein locus.

3. The mouse according to claim 1 or 2, wherein the FcαR gene locus is located in the intergenetic region between the Lilra5 protein locus and the Gp6 protein locus.

4. The mouse according to any one of claims 1 to 3, wherein the FcαR locus is located in an intergenetic region between the Pira6 protein locus and the Ncr1 protein locus, such as between nucleic acid sequences encoding the Pira6 protein and the Ncr1 protein.

5. The mouse according to claim 4, wherein the intergenetic region is a 54 kb region between the Pira6 locus and the Ncr1 locus.

6. The mouse according to any one of claims 1 to 5, wherein the FcαR gene locus is located between coordinates 4,303,905 and 4,312,280 (+ strand, GRCm38 assembly) on mouse chromosome 7.

7. The mouse according to any one of claims 1 to 6, wherein the FcαR gene locus comprises a nucleic acid sequence encoding a human FcαR polypeptide.

8. The mouse according to any one of claims 1 to 7, wherein the FcαR gene locus comprises human exons 1 to 5 of the human Fc alpha receptor gene.

9. The mouse according to any one of claims 1 to 6, wherein the FcαR gene locus includes the non-coding portion of non-mouse rodent FcαR exon 1, human FcαR exons 1 and 2, human FcαR exons 3 and 4, and the coding portion of non-mouse rodent FcαR exon 5.

10. The mouse according to claim 7 or 8, wherein the human or humanized FcαR receptor locus comprises a genomic sequence (+ strand, GRCh38 assembly) found between coordinates 54,862,297 and 54,906,185 on human chromosome 19.

11. The mouse according to claim 7 or 8, wherein the FcαR gene locus further comprises a nucleic acid sequence present in the human KIR3DL2 gene and / or a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

12. The mouse according to any one of claims 1 to 11, wherein the mouse expresses the FcαR polypeptide on mouse neutrophils, monocytes, macrophages, eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells).

13. The mouse according to any one of claims 1 to 12, wherein the mouse is heterozygous or homozygous with respect to the FcαR gene locus.

14. A mouse according to any one of claims 1 to 13, further comprising in its genome a human or humanized Fc gamma receptor (FcγR) locus, a human or humanized IgH locus, a human or humanized Igκ locus, a human or humanized Igλ locus, a human or humanized FcRn locus, a human or humanized β2M locus, and / or a human or humanized FcεR1α locus.

15. Mouse embryonic stem cells (ES cells) having an Fc alpha receptor (FcαR) gene locus located in the leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR gene locus comprises a nucleic acid sequence encoding an FcαR polypeptide including a human extracellular domain and a human or rodent cytoplasmic domain.

16. The mouse ES cell according to claim 15, wherein the FcαR gene locus is located in the intergenetic region between the Tthy1 protein locus and the Rdh13 protein locus.

17. The mouse ES cell according to claim 15 or 16, wherein the FcαR gene locus is located in the intergenetic region between the Lilra5 protein locus and the Gp6 protein locus.

18. The mouse ES cell according to any one of claims 15 to 17, wherein the Fc alpha receptor (FcαR) gene locus is located in an intergenetic region between the Pira6 protein locus and the Ncr1 protein locus, such as between nucleic acid sequences encoding the Pira6 protein and the Ncr1 protein.

19. The mouse ES cell according to claim 18, wherein the intergenetic region is a 54 kb region between the Pira6 locus and the Ncr1 locus.

20. The mouse ES cell according to any one of claims 15 to 19, wherein the FcαR gene locus is located between coordinates 4,303,905 and 4,312,280 (+ strand, GRCm38 assembly) on mouse chromosome 7.

21. The mouse ES cell according to any one of claims 15 to 20, wherein the FcαR gene locus comprises a nucleic acid sequence encoding a human FcαR polypeptide.

22. The mouse ES cell according to any one of claims 15 to 21, wherein the FcαR gene locus contains human exons 1 to 5 of the human Fc alpha receptor gene.

23. The mouse ES cell according to any one of claims 15 to 20, wherein the FcαR gene locus includes the non-coding portion of non-mouse rodent FcαR exon 1, human FcαR exons 1 and 2, human FcαR exons 3 and 4, and the coding portion of non-mouse rodent FcαR exon 5.

24. The mouse ES cell according to claim 21 or 22, wherein the human or humanized FcαR receptor locus comprises a genomic sequence (+ strand, GRCh38 assembly) located between coordinates 54,862,297 and 54,906,185 on human chromosome 19.

25. The mouse ES cell according to claim 21 or 22, wherein the FcαR gene locus further comprises a nucleic acid sequence present in the human KIR3DL2 gene and / or a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

26. The mouse ES cells according to any one of claims 15 to 25, wherein the ES cells are heterozygous or homozygous with respect to the FcαR gene locus.

27. Mouse ES cells according to any one of claims 15 to 26, further comprising a human or humanized Fc gamma receptor (FcγR) locus, a human or humanized IgH locus, a human or humanized Igκ locus, a human or humanized Igλ locus, a human or humanized FcRn locus, a human or humanized β2M locus, and / or a human or humanized FcεR1α locus in the genome.

28. A method for testing a human IgA antibody or an Fcα fusion polypeptide, comprising administering the IgA antibody or Fcα fusion polypeptide to a mouse according to any one of claims 1 to 14.

29. The method according to claim 28, further comprising measuring one or more pharmacokinetic properties of the administered human IgA antibody or Fcα fusion polypeptide, wherein the one or more pharmacokinetic properties are optionally selected from one or more of the following: plasma concentration-to-time area (AUC), in vivo recovery rate (IVR), clearance rate (CL), mean residence time (MRT), drug half-life (t1 / 2), and / or steady-state volume of distribution (Vss).

30. The method according to claim 28 or 29, further comprising measuring the therapeutic efficacy of the administered human antibody or Fcα fusion polypeptide.

31. The method according to any one of claims 28 to 30, further comprising administering multiple doses of the human antibody or Fcα fusion polypeptide and determining the therapeutic efficacy, safety, and / or tolerability of each dose of the human antibody or Fcα fusion polypeptide.

32. The method according to any one of claims 28 to 31, further comprising measuring one or more Fc receptor-mediated responses in the mouse, wherein the one or more Fc receptor-mediated responses are antibody-dependent cell-mediated cytotoxicity (ADCC) responses.

33. The method according to any one of claims 28 to 32, wherein the human antibody binds to the target cells of the mouse, and the method further comprises measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against the target cells and comparing the amount of ADCC with a control, wherein an increase in target cell death indicates an increased ability of the drug to mediate ADCC.

34. The method according to any one of claims 28 to 33, further comprising measuring the immune response produced by the mouse against the human antibody.

35. A method for modifying the mouse genome, The method comprises modifying a mouse genome by inserting an Fc alpha receptor (FcαR) locus into the leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR locus comprises a nucleic acid sequence encoding an FcαR polypeptide including a human extracellular domain and a human or rodent cytoplasmic domain.

36. The method according to claim 35, wherein the FcαR gene locus comprises a nucleic acid sequence encoding a human FcαR polypeptide.

37. The method according to claim 35 or 36, wherein the FcαR locus is located in the intergenetic region between the Tthy1 protein locus and the Rdh13 protein locus.

38. The method according to any one of claims 35 to 37, wherein the FcαR gene locus is located in the intergenetic region between the Lilra5 protein locus and the Gp6 protein locus.

39. The method according to any one of claims 35 to 38, wherein the FcαR locus is located in an intergenetic region between the Pira6 protein locus and the Ncr1 protein locus, such as between nucleic acid sequences encoding the Pira6 protein and the Ncr1 protein.

40. The method according to claim 39, wherein the intergene region is a 54 kb region between the Pira6 locus and the Ncr1 locus.

41. The method according to any one of claims 35 to 40, wherein the FcαR gene locus is located between coordinates 4,303,905 and 4,312,280 (+ strand, GRCm38 assembly) on mouse chromosome 7.

42. A method for producing mice containing the Fc alpha receptor (Fc αR) gene locus, To produce mouse ES cells according to any one of claims 15 to 27, To generate mice from the aforementioned ES cells and The method, including the method described above.

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