Mice expressing humanized Fc alpha receptors

Abstract

Provided herein are methods and compositions relating to mice expressing human or humanized Foot receptor (FcaR) from the FcaR locus located in the mouse leukocyte receptor complex (LRC). In certain embodiments, such mice are useful for in vivo testing of therapeutics comprising human IgA Fc (e.g., testing the pharmacokinetic and / or pharmacodynamic properties of such therapeutics, as well as dosing regimens). Also provided herein are methods of using such mice, cells obtained from such mice, methods of making such mice, and ES cells containing similar genetic modifications as such mice.

Description

[Technical field]

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

[0002] Mice are an essential in vivo model for preclinical testing of therapeutics due to their small size, well-characterized physiology, and relatively inexpensive maintenance compared to larger mammalian models (e.g., primates). Mouse models are often used to assess the toxicity and pharmacokinetics of therapeutics before proceeding to human clinical studies. Despite these advantages, in vivo testing of antibodies and Fc fusion proteins has significant drawbacks as these therapeutics may behave differently in mice than in humans. This is especially true when testing IgA-based therapeutics in mice, as mice do not express Fc receptors that bind IgA antibodies. Thus, mouse models have not been extensively characterized or used to predict the safety, efficacy, and optimal dosing of IgA-based therapeutics. There is a need for new mouse models and methods that allow accurate preclinical testing of IgA-based therapeutics that predict the properties of such IgA-based therapeutics in human patients. Summary of the Invention

[0003] Provided herein are methods and compositions related to mice expressing human or humanized Fcα receptors (FcαR), as well as methods and compositions related to in vivo testing of therapeutics comprising human IgA Fc in such mice (e.g., testing of pharmacokinetic and / or pharmacodynamic properties, and dosing regimens of such therapeutics). 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 amenability to genetic modification. Unfortunately, drugs comprising human IgA Fc regions (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, due to the lack of FcαR in such mice. Thus, 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.

[0004] In a particular aspect, the present disclosure provides a mouse comprising in its genome an Fc alpha receptor (FcαR) locus located in the mouse leukocyte receptor complex (LRC), wherein the FcαR locus (such as a human or humanized FcαR locus) comprises 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, the neutrophils, monocytes, macrophages, eosinophils, and / or dendritic cells (e.g., plasmacytoid dendritic cells) are obtained from the blood of the mouse. In some embodiments, the neutrophils, monocytes, macrophages, eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells) are obtained from the spleen of the mouse.

[0005] In some aspects, the present specification provides a mouse embryonic stem cell (ES cell) comprising in its genome an Fc alpha receptor (FcαR) locus located in the leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR locus comprises 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 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 the 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 the 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 the intergenic region between the locus of the Pira6 protein and the locus of the Ncr1 protein. In some embodiments, the FcαR locus is located in the intergenic region between the nucleic acid sequences encoding the Pira6 protein and the Ncr1 protein. In some embodiments, the intergenic region is the 54 kb region between the Pira6 locus and the Ncr1 locus. In some embodiments, the FcαR locus is located between coordinates 4,303,905-4,312,280 (+ strand, GRCm38 assembly) on mouse chromosome 7.

[0007] In some embodiments, the FcαR locus comprises a nucleic acid sequence encoding a human FcαR polypeptide. In some embodiments, the FcαR locus comprises human exons 1-5 of the human Fc alpha receptor gene. In some embodiments, the FcαR locus comprises 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 comprises the genomic sequence found between coordinates 54,862,297-54,906,185 on human chromosome 19 (+ strand, GRCh38 assembly). In some embodiments, the FcαR locus further comprises a nucleic acid sequence present in the human KIR3DL2 gene. In some embodiments, the locus further comprises a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

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

[0009] In some embodiments, the mouse or mouse ES cell of any of the above aspects or embodiments further comprises 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. In some embodiments, the mouse or mouse ES cell is heterozygous for a human or humanized 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 some embodiments, the mouse or mouse ES cell is homozygous for a human or humanized 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.

[0010] In some embodiments, the mouse or mouse ES cell comprising the FcαR locus of various embodiments described herein comprises in its genome a human or humanized FcγR locus comprising a nucleic acid sequence encoding a human or humanized FcγR. In some embodiments, the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more low affinity FcγRs 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 FcγRs 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 the human or humanized FcγR replaces all or part of the endogenous mouse FcγR gene. In some embodiments, the nucleic acid sequence encoding the human or humanized FcγR comprises a nucleic acid sequence encoding a human FcγR extracellular domain that 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 a mouse FcγR.

[0011] In a particular aspect, the present 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 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 area under plasma concentration versus 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 the human antibody or Fcα fusion polypeptide and determining the tolerability 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 the mouse. In some embodiments, the 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 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, where an increase in target cell killing indicates an increased ability of the agent to mediate ADCC. In some embodiments, the method further comprises measuring an immune response generated by the mouse against the human antibody.

[0013] In a particular aspect, the present specification provides a method of producing a mouse comprising an Fc alpha receptor (FcαR) locus, the method comprising generating a mouse ES cell comprising in its genome an 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 cell.

[0014] In certain aspects, the present disclosure provides a method of modifying a mouse genome, comprising inserting an Fc alpha receptor (FcαR) locus into a leukocyte receptor complex (LRC) of the mouse genome to modify the mouse genome, wherein the FcαR locus comprises a nucleic acid sequence encoding an 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 the 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 the 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 the 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 the intergenic region between the locus of the Pira6 protein and the locus of the Ncr1 protein. In some embodiments, the intergenic region is the 54 kb region between the Pira6 and Ncr1 loci. In certain embodiments, the FcαR locus is between coordinates 4,303,905-4,312,280 (+ strand, GRCm38 assembly) on mouse chromosome 7.

[0015] Unless otherwise indicated, the diagram uses white boxes for human gene sequences, black boxes for mouse gene sequences, single lines for mouse intergenic sequences, double lines for human intergenic sequences, and white boxes with text for selection cassettes (e.g., Lox, CM). [Brief description of the drawings]

[0016] [Figure 1A] The insertion of the MAID20277 cassette into the mouse genome to generate heterozygous mice that can be bred to homozygosity is shown (not to scale). "LRC" indicates leukocyte receptor complex and "EP" indicates electroporation. The names and positions of the primers and probes used in the modification of alleles (MOA) assay (described in the Examples) are shown as short lines above the figure. [Figure 1B] Treatment of heterozygous ES cells with CRE recombinase to cause deletion of the selectable marker for insertion of the MAID20277 cassette in the mouse genome, thereby generating ES cells heterozygous for the MAID20278 cassette (not to scale). The names and positions of the primers and probes used in the modification of allele (MOA) assay (described in the Examples) are shown as short lines above the figure. [Diagram 2] The targeting vector is shown (not to scale) containing sequences homologous to the mouse genome ("5' mouse homology arm"), an I-CeuI site, a 44,887 bp human genomic insert containing the FCAR gene, a lox2372-Ub-Neo-lox2372 cassette, a PI-SceI site, a second sequence homologous to the mouse genome, and a chloramphenicol (labeled "CM") resistance cassette. [Diagram 3] Illustrated (not to scale) is the humanized low affinity FcγR locus, including the human FcγR2B, FcγR3B, FcγR2C, FcγR3A, and FcγR2A genes on mouse chromosome 1. The steps for humanizing this locus are described in U.S. Patent No. 8,658,154, which is incorporated herein by reference. [Figure 4A] 1 shows flow cytometry plots detecting expression of FcαR on cells in blood samples obtained from mice genetically engineered with the MAID20278 cassette. [Figure 4B]1 shows flow cytometry plots detecting expression of FcαR in splenocytes obtained from mice genetically engineered with the MAID20278 cassette. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] overview Provided herein are methods and compositions related to mice expressing human, humanized, or partially humanized Fcα receptors (FcαR), as well as methods and compositions related to in vivo testing of therapeutics comprising human IgA Fc in such mice (e.g., testing of pharmacokinetic and / or pharmacodynamic properties of such therapeutics, as well as dosing regimens). Specifically, various embodiments described herein relate to mice, ES cells, and methods that include genes encoding and / or expressing fully human or partially human FcαR1 / CD89 genes / receptors (referred to herein as "FcαR"). The genetically modified mice include in their genomes nucleic acid sequences encoding human or humanized FcαR proteins. In some embodiments, the sequences encoding human or humanized FcαR proteins are located in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, the nucleic acid sequence encoding the FcαR is located in an intergenic region between the locus of the Tthy1 protein and the locus of the Rdh13 protein, between the locus of the Lilra5 polypeptide and the locus of the Gp6 polypeptide, and / or between the locus of the Pira6 protein and the locus of the Ncr1 protein (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 intergenic region is a 54 kb region between the Pira6 locus and the Ncr1 locus. In some embodiments, the mouse provided herein expresses a human FcαR at a locus located between coordinates chr7:4,303,905-4,312,280 of the mouse genome (+ strand, GRCm38 assembly). In some embodiments, the FcαR comprises a human extracellular domain. The transmembrane and / or cytoplasmic domains of such receptors can be human or non-human (e.g., rat).

[0018] Therapeutic human antibodies and therapeutics containing human IgA Fc, such as human Fcα fusion proteins, are typically tested in non-human species before being administered to humans. Such drugs are often tested in non-human primates and other larger mammals, but such testing is expensive and imposes a significant economic burden on drug developers. Furthermore, non-human primates and other larger mammals are often not amenable to genetic modification, limiting the disease models available in such organisms.

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

[0020] The location of the transgene in the mouse genome can have a significant effect on its expression. For example, mice containing a human FcαR transgene have been reported to express FcαR on neutrophils, but only on a subpopulation of monocytes. For example, in such mice, macrophages isolated from the peritoneal cavity and 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. Thus, in certain embodiments, the mice provided herein express human FcαR at a location in the mouse genome that corresponds to the location of the human endogenous FcαR locus, or a locus located near the location of the human endogenous FcαR locus in the human genome. Specifically, in certain embodiments, a sequence encoding a human or humanized Fc alpha receptor (FcαR) protein is located in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, the mouse provided herein expresses human FcαR at a locus located in the intergenic region between the locus of the Tthy1 protein and the locus of the Rdh13 protein, between the locus of the Lilra5 polypeptide and the locus of the Gp6 polypeptide, and / or between the locus of the Pira6 protein and the locus of the Ncr1 protein (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 intergenic region is the 54 kb region between the Pira6 locus and the Ncr1 locus. In some embodiments, the mouse provided herein expresses human FcαR at a locus located between coordinates chr7:4,303,905-4,312,280 of 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 comprises and / or the mice express a human or humanized FcαR described herein associated with a wild-type mouse FcRγ chain. Thus, in some embodiments, the human or humanized FcαR is expressed on the surface of mouse cells in association with an endogenous mouse FcRγ chain.

[0022] In some embodiments, the mice provided herein further comprise a reduced mouse anti-human immune response following administration of a therapeutic agent comprising human Fcα. This can be achieved by using mice genetically modified to express a human Fc that matches the Fc present in the administered antibody or Fc fusion protein. Such mice can be generated, for example, by inserting all or part of a nucleic acid sequence encoding a human immunoglobulin heavy chain constant region in place of a sequence encoding a portion corresponding to an endogenous non-human immunoglobulin heavy chain constant region gene segment. Such animals are less likely to develop an immune response to an administered human Fc-containing therapeutic agent because they recognize the human Fc as a "self" protein.

[0023] Furthermore, in some embodiments, the mice provided herein express an Fcα receptor (FcαR) capable of interacting with human Fcα, similar to the FcαR expressed in a human patient. 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 can be either human or rat). In certain embodiments, in addition to expressing a 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 are able to more accurately mimic the human Fc response of a human patient compared to mice with fully non-human Fc receptors.

[0024] Thus, in certain embodiments, the mouse provided herein is a novel in vivo system for developing, selecting, and testing therapeutic human IgA antibodies and Fc fusion proteins not simply based on specificity and / or affinity for antigen, but also based on the entire biological function associated with the selected antibody through evaluation of immune system effector functions. In this way, human therapeutic candidates can be developed and selected based on therapeutic potential assessed at the whole molecule level with associated biological responses (e.g., cellular responses) rather than predictions based only on individual components evaluated separately. Thus, the mouse disclosed herein provides a suitable system for predicting and characterizing human therapeutic antibody function in vivo, among others.

[0025] The 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 than larger animal models or humans. Furthermore, mice are a more suitable model to identify basic biological functions and then confirm them in larger animals or humans. For example, mice expressing human FcαR have been used previously to study the role of the receptor in antibody-dependent cellular 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 an important role in the development 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, some autoimmune diseases are characterized by increased amounts of IgA antibodies (e.g., rheumatoid arthritis and IgA nephropathy). In inflammatory bowel disease, bacteria opsonized with IgA can cause cross-linking of FcαR and activation of neutrophils, resulting in 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 at the same time high levels are also found in subjects with allergic rhinitis. The mice described herein can be used to identify the role of FcαR on disease severity or the effect of FcαR inhibitors on attenuating symptoms.

[0027] Lack of IgA may lead to increased susceptibility to infections, especially mucosal infections. Administration of excessive amounts of IgA may lead to increased activation of FcαR, which may cause unintended complications. Therefore, the mice described herein may be used to identify appropriate dosing regimens that favorably overcome IgA deficiency without causing side effects. The mice described herein may also be used to evaluate the effectiveness of FcαR inhibitors for the treatment of inflammation that may be mediated by IgA.

[0028] In some embodiments, the mice described herein can be treated with candidate anti-tumor (or pathogen) IgA therapeutics or IgA Fc fusion proteins, or bispecific antibodies against FcαR and tumor antigens. In other words, the mice described herein can be used for preclinical evaluation of therapeutics that involve IgA and / or FcαR.

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

[0030] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both amino and acid functional groups and that can be included in a polymer of naturally occurring amino acids. Exemplary amino acids include the naturally occurring amino acids, their analogs, derivatives and congeners, amino acid analogs having variant side chains, and all stereoisomers of any of the foregoing.

[0031] As used herein, the term "antibody" can refer to both intact antibodies and antigen-binding fragments thereof. An intact antibody is a glycoprotein that contains at least two heavy (H) chains and two light (L) chains inter-connected 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 and light chain variable domains can be further subdivided into hypervariable domains called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). The heavy and light chain variable domains are composed of three CDRs and four FRs, respectively, arranged from the amino terminus to the carboxy 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 antigens.

[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 encompassed by the term "antigen-binding fragment" of an antibody include Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, single-chain antibodies, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable domains of an intact antibody. These antibody fragments can be obtained using conventional recombinant and / or enzymatic techniques and screened for antigen binding in the same manner as intact antibodies.

[0033] As used herein, the term "area under the plasma concentration versus time curve" or "AUC" refers to the rate and extent of elimination of a therapeutic agent following administration. In some embodiments, the AUC is determined over a specific period of time, such as 12, 18, 24, 36, 48, or 72 hours, or to infinity using extrapolation based on the slope of the curve. Unless otherwise specified herein, the AUC is not taken to mean infinity (AUC INF ) The AUC may be calculated for each dose. As with many other PK parameters, AUC determinations may be made in a single animal or in a population of animals where the mean is calculated.

[0034] The term "clearance rate" or "CL" as used herein refers to a measure of the body's ability to eliminate a drug and is expressed as the amount of plasma cleared of the drug over time. The phrase "derived from," when used with respect to an unrearranged variable region and / or a rearranged variable region gene "derived from" an unrearranged variable region gene segment, refers to the fact that the sequence of the rearranged variable region gene can be traced back to the set of unrearranged variable region gene segments that were rearranged to form the gene that expresses the variable domain (accounting for splice divergence and somatic mutations, if applicable). For example, a rearranged variable region gene that is somatically mutated is still derived from an unrearranged variable region gene segment. In some embodiments, when an endogenous locus is replaced with a universal light or heavy chain locus, the term "derived from" indicates that the origin of the sequence can be traced back to said rearranged locus even if the sequence has been somatically mutated.

[0035] As used herein, the phrase "endogenous gene" or "endogenous gene segment" refers to a gene or gene segment found in a parent or reference organism prior to the introduction of a disruption, deletion, substitution, alteration, or modification described herein. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is a genetically engineered organism. In some embodiments, the reference organism is a laboratory-raised organism (either a wild-type organism or a genetically engineered organism).

[0036] The term "in vivo recovery" or "IVR" refers to the incremental recovery (K value) of the peak activity observed minus the pre-dose level, then divided by the dose. IVR may be calculated on a percentage basis. The average IVR can be determined for a population of animals, 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, gene segments, regulatory elements). For example, the human leukocyte receptor complex (LRC) locus is located on chromosome 19 and contains genes including KIR, FcαR, NCRI, NLRP, GP6, and Rdh13, among others. The mouse locus homologous to the human LRC locus is located on mouse chromosome 7 and contains several genes homologous to genes of the human LRC (e.g., Ncr1, Gp6, and Rdh13 genes). A locus may be endogenous or non-endogenous. The term "endogenous locus" refers to the location on a chromosome where a particular genetic element is found in nature. In some embodiments, an endogenous locus has a sequence found in nature. In some embodiments, an endogenous locus is a wild-type locus. In some embodiments, an endogenous locus is an engineered locus.

[0038] The terms "polynucleotide" and "nucleic acid" are used interchangeably. The terms refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and perform any function. Non-limiting examples of polynucleotides are: coding or non-coding regions of a gene or gene fragment, a locus (or 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 contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Polynucleotides may be further modified, for example, by the attachment of a labeling component. In all nucleic acid sequences provided herein, U nucleotides can be substituted for T nucleotides.

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

[0040] As used herein, the term "CH gene segment" (e.g., a Cγ1 gene segment, a Cγ2a gene segment, a Cγ2c gene segment, a Cμ gene segment, a Cγ2b gene segment, a Cγ3 gene segment, a Cδ gene segment, a Cε gene segment, a Cα gene segment, etc.) refers to a segment of a DNA sequence that encodes an immunoglobulin heavy chain constant region, H Genes (e.g., Cγ genes, C γ2a Gene, C γ2c Gene, C γ3 Gene, C γ2b gene, Cμ gene, C δgene, Cε gene, C α For example, a Cγ1 gene segment or a Cγ1 gene refers to a segment of a DNA sequence that encodes an IgG1 constant region. H The term "gene segment locus" refers to H Gene segment or C H Refers to the location on a chromosome where a gene is found in nature.

[0041] Genetically modified loci In some embodiments, the genetically modified mice and cells (e.g., ES cells) useful for in vivo testing of therapeutics (e.g., testing the pharmacokinetic and / or pharmacodynamic properties of such therapeutics) described herein comprise a human IgA antibody or an Fcα fusion protein. The genetically modified mice disclosed herein comprise a nucleic acid sequence encoding a human or humanized FcαR in the genome of the mouse, and comprise at least one locus that corresponds to a location in the mouse genome that corresponds to the location of the human endogenous FcαR locus or 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 intergenic region between the locus of the Tthy1 protein and the locus of the Rdh13 protein, between the locus of the Lilra5 polypeptide and the locus of the Gp6 polypeptide, between the locus of the Pira6 protein and the locus of the Gp6 protein, and / or between the locus of the Pira6 protein and the locus of the Ncr1 protein. 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 of the mouse genome (+ strand, GRCm38 assembly). In some embodiments, the intergenic region is the 54 kb 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 further comprises a nucleic acid sequence encoding all or part of the human NCR1 gene, e.g., a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

[0042] In some embodiments, the genetically modified mouse comprises a genetically modified locus encoding an antibody heavy chain comprising a human Fc (e.g., human IgA1Fc, human IgA2Fc). Such loci are disclosed in WO2019 / 190990, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the mouse and / or ES cells comprise a genetically modified locus encoding a fully or partially human light chain (e.g., encoding a lambda light chain or a kappa light chain). Such loci are disclosed in WO2019 / 190990 and U.S. Pat. No. 10,820,582, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mice and ES cells further comprise one or more loci encoding an Fc receptor having a human extracellular domain (e.g., neonatal Fc receptor (FcRn) α chain, β-2-microglobulin polypeptide (β2M), Fcε receptor 1 α (FcεR1α) α chain, Fcγ receptor 1 alpha (FcγR1a) α chain, Fc gamma receptor 2a (FcγR2a) α chain, Fc gamma receptor 2b (FcγR2b) α chain, Fc gamma receptor 3a (FcγR3a) α chain, Fc gamma receptor 3b (FcγR3b) α chain, Fc gamma receptor 2c (FcγR2c) α chain). In some embodiments, the transmembrane and cytoplasmic domains encoded by such loci can be human or non-human (e.g., rodent, such as mouse). Such loci are disclosed in WO2019 / 190990 and U.S. Patent Nos. 9,474,255 and 8,658,154, each of which is incorporated by reference in its entirety.

[0043] Humanized Fc alpha receptor The mouse or embryonic stem (ES) cell provided herein expresses and / or comprises in its genome a humanized or human Fc alpha receptor 1 (FcαR1). Mice do not have an FcαR homologue. Thus, in some embodiments, the mouse or ES cell comprises in its genome a nucleic acid sequence encoding a human or humanized FcαR and comprises at least one locus corresponding to a position in the mouse genome corresponding to the location of the human endogenous FcαR locus or 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 a human or humanized Fc alpha receptor (FcαR) protein is located in an intergenic region between the locus of the Tthy1 protein and the locus of the Rdh13 protein, between the locus of the Lilra5 polypeptide and the locus of the Gp6 polypeptide, and / or between the locus of the Pira6 protein and the locus of the Ncr1 protein. In some embodiments, the sequence encoding a human or humanized Fc alpha receptor (FcαR) protein is located between coordinates chr7:4,303,905-4,312,280 of the mouse genome (+strand, GRCm38 assembly). In some embodiments, the intergenic region is the 54 kb region between the Pira6 locus and the Ncr1 locus. In some embodiments, the nucleic acid sequence encoding an FcαR further comprises a nucleic acid sequence encoding all or a portion of the human KIR3DL2 gene, and / or further comprises a nucleic acid sequence of all or a portion of the human NCR1 gene, e.g., 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, the neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells are blood neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells.In some embodiments, the neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells are splenic neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells.

[0044] FcαR is an activating receptor that forms a complex with the human FcRγ chain and has intermediate affinity (K a Approximately 5x10 6 M -1 ) and high binding activity for complex IgA (K a Approximately 5x10 6 M -1 ) Binding of FcαR to IgA-coated particles leads to the release of inflammatory mediators, phagocytosis, and antibody-dependent cell-mediated cytotoxicity. IgA antitumor therapy can be used to target neutrophils to tumor cells, resulting in their efficient lysis. In addition to the full-length receptor, neutrophils and eosinophils express an alternatively spliced ​​form of the receptor that can bind secretory IgA. In vivo, two FcαR molecules dimerize and interact with IgA monomers.

[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 rodent (non-mouse, e.g., rat) cytoplasmic domain.

[0046] In some embodiments, the FcαR locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., a rat or mouse promoter or enhancer). In other embodiments, the FcαR locus comprises human regulatory elements (e.g., a human promoter and / or enhancer).

[0047] As described in the art (see, e.g., Monteiro and van de Winkel, (2003) Annu. Rev. Immunol. 21:177-204), the human FcαR gene consists of five exons: exon 1, which contains the 5'UTR, the ATG translation initiation codon, and sequences encoding the leader peptide; exon 2, which encodes the remainder of the leader peptide; exons 3 and 4, which encode the two extracellular domains of FcαR, EC1 and EC2 (EC1 is the domain that binds IgA Fc); and exon 5, which encodes the cytoplasmic and transmembrane domains of the protein.

[0048] In some embodiments, nucleic acids encoding human or humanized FcαR polypeptides provided herein encode a fully human FcαR polypeptide. In certain embodiments, such nucleic acids comprise humanized exon 1 (e.g., including a rodent (non-mouse, e.g., rat) 5′ UTR and human coding sequence), human coding exons 2-5 through to a stop codon, and a human or rodent (non-mouse, e.g., rat) 3′ UTR and polyA. In some embodiments, 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 targeting vector for insertion into a mouse ES cell genome. In some embodiments, the nucleic acid encoding a human or humanized FcαR polypeptide comprises approximately 44 kb of human genomic sequence that includes the FCAR gene. In some embodiments, the human genomic sequence is located on human chromosome 19 and has coordinates chr19:54,862,297 to 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 (wherein 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 regions (e.g., promoter and UTRs) are rodent (e.g., rat) regulatory regions.

[0051] NCBI reference sequence NW_016107304.1 is a representative source sequence of the human FcαR gene. NCBI reference sequence 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 provide representative source sequences of human FcαR cDNA and the polypeptide from which the desired human portion is derived.

[0052] NCBI Reference SEQ ID NO: NC_005100.4 provides a representative source sequence for the Rattus norvegicus FcαR gene, and NCBI Reference SEQ ID NO: NM_201992.1→NP_973721.1 provide representative source sequences for Rattus norvegicus FcαR cDNA and polypeptides from which desired Rattus norvegicus portions can be derived and / or used to design targeting vector homology arms.

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

[0054] In various embodiments, the human or humanized FcαR polypeptides described herein are expressed on the cell surface in association with a wild-type mouse FcRγ chain. In various embodiments, the human or humanized FcαR polypeptides described herein are expressed on the cell surface in association with an endogenous mouse FcRγ chain.

[0055] Humanized low affinity Fc gamma receptor In some embodiments, mice and ES cells that have been genetically modified to contain a humanized or human FcαR1 locus in their genome further contain a locus encoding a human low affinity Fc gamma receptor (FcγR) polypeptide (e.g., a human FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, or FcγRIIIb polypeptide; see, e.g., U.S. Patent No. 8,658,154, the contents of which are incorporated herein by reference in their entirety).

[0056] In some embodiments, the low affinity FcγR locus comprises a nucleic acid sequence encoding a human FcγRIIa polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIa polypeptide is located at an endogenous 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 comprises a polymorphism, the polymorphism being selected from a 131His low reactivity polymorphism and a 131Arg high reactivity polymorphism. In some embodiments, the FcγRIIa polymorphism is a 131His low reactivity polymorphism. In some embodiments, the mouse does not express a mouse low affinity FcγR polypeptide (e.g., does not express mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptide, or does not express a functional mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptide). In some embodiments, the FcγRIIa locus comprises a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

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

[0058] In some embodiments, the low affinity FcγR locus comprises a nucleic acid sequence encoding a human FcγRIIc polypeptide. In some embodiments, the nucleic acid sequence encoding a human FcγRIIc polypeptide is located at an endogenous mouse low affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding an FcγRIIc polypeptide replaces all or part of an endogenous mouse low affinity FcγR locus. In one embodiment, the FcγRIIc gene is a specific allelic variant, the allelic variant being selected from a 57Stop variant and a 57Q variant. In some embodiments, the mouse does not express a mouse low affinity FcγR polypeptide (e.g., does not express a mouse FcγRIIB, FcγRIV, and / or FcγRIII polypeptide). In some embodiments, the FcγRIIc locus comprises a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0059] In some embodiments, the low affinity FcγR locus comprises a nucleic acid sequence encoding a human FcγRIIIa polypeptide. In some embodiments, the nucleic acid sequence encoding a human FcγRIIIa polypeptide is located at an endogenous mouse low affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding an FcγRIIIa polypeptide replaces all or part of an endogenous mouse low affinity FcγR locus. In some embodiments, the mouse does not express a 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 one embodiment, the FcγRIIIa gene is a specific allelic variant, which is selected from a 176Val variant and a 176Phe variant. In some embodiments, the FcγRIIIa allelic variant is a 176Val variant. In some embodiments, the FcγRIIIa locus comprises a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

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

[0061] In some embodiments, the mouse provided herein comprises one or more human low affinity FcγR genes described in U.S. Patent Nos. 9,221,894, 9,056,130, 9,089,599, 8,658,154, 8,883,496, or 8,658,853, which are incorporated herein by reference. In some embodiments, the mouse provided herein comprises at least two low affinity human FcγR genes and an endogenous mouse Fcγ chain gene, and the low affinity human FcγR genes are selected from the group consisting of human FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some specific embodiments, the mouse provided herein comprises human FcγRIIa and FcγRIIIb, and an endogenous mouse Fcγ chain gene. In some specific embodiments, the mice provided herein comprise FcγRIIa, FcγRIIIa, FcγRIIb, FcγRIIc, and FcγRIIId genes, as well as endogenous mouse Fcγ chain genes. In various embodiments, the mice comprising one or more human FcγRs further comprise homozygous disruptions in endogenous mouse FcγRIIB, FcγRIV, and FcγRIII genes (i.e., endogenous mouse FcγRIIb, FcγRIV, and FcγRIII α chain coding sequences). In various embodiments, the mice comprising one or more human low affinity FcγRs described herein do not detectably express endogenous mouse low affinity FcγR polypeptides (e.g., endogenous low affinity FcγR α chain polypeptides).

[0062] In some embodiments, the mouse is heterozygous for the genetically engineered low affinity FcγR locus. In some embodiments, the mouse is homozygous for the genetically engineered low affinity FcγR locus.

[0063] Humanized neonatal Fc receptor locus In some embodiments, mice and ES cells genetically modified to 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 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 both the Fc domain of IgG antibodies and serum albumin. FcRn extends the half-life of IgG and serum albumin. Specifically, FcRn can rescue these serum proteins from lysosomal degradation by endothelial cells by binding to IgG and serum albumin in a pH-dependent manner, 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, such that a mouse comprising such a locus expresses an FcRn having a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domain. In some embodiments, the mouse does not express mouse FcRn or does not express a functional mouse FcRn. In some embodiments, the FcRn locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0066] In certain embodiments, the mouse exons encoding the alpha1, alpha2, and alpha3 domains of the mouse FcRn gene (the first three coding exons, exons 3, 4, and 5) are replaced with human exons encoding the alpha1, alpha2, and alpha3 domains of the human FcRn gene (exons 3, 4, and 5). In some embodiments, the FcRn gene comprises mouse exon 1 (a non-coding exon), mouse exon 2 (containing a nucleic acid sequence encoding a signal peptide), and human exons 3-6, mouse exons 6 and 7 (encoding transmembrane and cytoplasmic domains). An exemplary humanized FcRn locus is described in WO2019 / 190990 (e.g., FIG. 4), which is incorporated herein by reference.

[0067] GenBank Accession Nos. NC_000019.10 (49512279-49526428), NM_001136019.1, and NP_001129491.1 provide representative source sequences of human FcRn genes, cDNAs, and polypeptides from which desired human portions can be derived. GenBank Accession Nos. NC_000073.6 (45092992-45103846), NM_010189.1, and NP_034319.1 provide representative source sequences of mouse FcRn genes, cDNAs, and polypeptides from which desired mouse portions can be derived and / or used to design targeting vector homology arms.

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

[0069] Humanized Fc epsilon receptor 1 alpha In some embodiments, mice and ES cells genetically modified to contain a humanized or human FcαR1 locus, and in some embodiments a human or humanized FcγR locus (as described above) in their genome further contain 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 comprising a human extracellular domain, a rodent (e.g., mouse or rat) transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcεR1α locus comprises a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain, a human transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcεR1α locus comprises a nucleic acid sequence encoding an FcεR1α polypeptide comprising a human extracellular domain, a human transmembrane domain, and a rodent (e.g., mouse or rat) cytoplasmic domain. Exemplary embodiments of engineered FcεR1α loci 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, such that a mouse comprising such a locus expresses an FcεR1α having a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domain. In some embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide comprising 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 an FcεR1α polypeptide comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain replaces all or part of an endogenous mouse FcεR1α gene. In some embodiments, the mouse does not express mouse FcεR1α or does not express a functional mouse FcεR1α. In some embodiments, the FcεR1α locus comprises non-human regulatory elements (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., a rat or mouse promoter or enhancer).

[0072] In certain embodiments, mouse coding exon 1, coding exon 2, coding exon 3, coding exon 4, and part of coding exon 5 of mouse FcεRIα are replaced by human coding exon 1, coding exon 2, coding exon 3, coding exon 4, and part of coding exon 5 of human FcεRIα gene. In some embodiments, FcεRIα gene comprises mouse / human chimeric exon 1 (including mouse promoter and 5′UTR), human coding exons 2-5 to stop codon, human 3′UTR and polyA, followed by mouse 3′UTR and polyA. In some embodiments, chimeric gene exons 1 (part) and 2 code for a signal peptide, exons 3 and 4 code for two Ig-like domains of FcεRIα that are believed to interact with IgE, and exon 5 codes for the cytoplasmic and transmembrane domains of the protein (see FIG. 9 of WO2019 / 190990).

[0073] GenBank Accession Nos. NC_000001.11 (159283888-159308224), NM_002001.3, and NP_001992.1 provide representative source sequences of human FcεR1α genes, cDNAs, and polypeptides from which desired human portions can be derived. GenBank Accession Nos. NC_000067.6 (173221269-173227232), NM_010184.1, and NP_034314.1 provide representative source sequences of mouse FcεR1α genes, cDNAs, and polypeptides from which desired mouse portions can be derived and / or used to design targeting vector homology arms.

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

[0075] Humanized Fc gamma receptor 1a In some embodiments, mice and ES cells genetically engineered to contain a humanized or human FcαR1 locus in their genome further contain a humanized or human Fc gamma receptor 1a (FcγR1a) locus, which is a high affinity FcγR protein expressed on monocytes that binds the Fc portion of IgG and causes 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 a nucleic acid sequence encoding the extracellular domain of human FcγR1α, such that a mouse comprising such a locus expresses 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 comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (eg, a rat or mouse promoter or enhancer).

[0078] Humanized FcγR1a polypeptides, loci encoding humanized FcγR1a polypeptides, and mice expressing humanized FcγR1a polypeptides are described in U.S. Pat. No. 9,474,255 and U.S. Patent Publication No. 2017 / 0086432, each of which is incorporated herein by reference.

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

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

[0081] In some embodiments, mice and ES cells genetically modified to include 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 in their genome further include a genetically modified immunoglobulin (Ig) heavy chain locus. 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 a V H Gene segment, D H Gene segments, and J H A constant region locus contains one or more Ig heavy chain constant region gene segments (C H In certain embodiments, the mouse immunoglobulin heavy chain variable region comprises V H Gene segment, D H Gene segments, and J HVariable domains and C H The gene segment is operably linked to an immunoglobulin heavy chain constant region to produce an antibody that contains a heavy chain constant domain derived from the gene segment.

[0082] The ability to generate antibodies has been exploited in genetically modified animals to generate therapeutic antibodies against human targets. Examples of genetically modified mice containing human V(D)J gene segments for generating therapeutic antibodies are described in U.S. Patent Nos. 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 8,907,157. No. 9,145,588, each of which is incorporated herein by reference in its entirety, as well as those described in U.S. Patent Publication Nos. 2008 / 0098490, 2010 / 0146647, 2013 / 0145484, 2012 / 0167237, 2013 / 0167256, 2013 / 0219535, 2012 / 0207278, and 2015 / 0113668, each of which is incorporated herein by reference in its entirety. Nos. WO2007117410, WO2008151081, WO2009157771, WO2010039900, WO2011004192, WO2011123708, WO2014093908, WO2014093908, WO2006008548, WO2010109165, WO20160629 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 human V(D)J gene segments for generating therapeutic antibodies are described in U.S. Pat. Nos. 6,596,541, 6,586,251, 8,642,835, 9,706,759, 10,238,093, 8,754,287, 10,143,186, 9,796,788, 10,130,080, and 10,230,081. Nos. 9,226,484, 9,012,717, 10,246,509, 9,204,624, and 9,686,970, each of which is incorporated herein by reference in its entirety, as well as those described in U.S. Patent Publication Nos. 2013 / 0212719, 2015 / 0289489, 2017 / 034763, 2018 / 0330363, 2019 / 02262719, 2019 / 02262729, 2019 / 02262730, 2019 / 02262740, 2019 / 02262750, 2019 / 02262760, 2019 / 02262770, 2019 / 02262780, 2019 / 0226279 ... No. 2019 / 0223418, No. 2018 / 0125043, No. 2019 / 0261612, and No. 2019 / 0380316, each of which is incorporated by reference in its entirety, and further including those described in PCT Publication Nos. WO2013138680, WO2013138712, WO2013138681, WO2013138722, WO201313871 ... Nos. 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 by reference in its entirety.

[0083] In certain embodiments, the Ig heavy chain variable region locus comprises an unrearranged human Ig heavy chain variable region gene segment. In some embodiments, the unrearranged human Ig variable region gene segment comprises one or more human VH Segment, one or more human D H segment, and one or more human J H In some embodiments, the unrearranged human Ig variable region gene segment comprises at least three 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 A gene segment, or at least 80 V H In some specific embodiments, the engineered IgH locus (or allele) comprises a human V gene segment of a naturally occurring human IgH locus. H 3-74 gene segment and human V H Functional human V found between (and including) the 6-1 gene segment H In some specific embodiments, the engineered IgH locus (or allele) comprises at least one of the 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 In some embodiments, the mouse provided herein comprises a single polymorphic human V H Gene segment, multiple D H Gene segments and multiple J H In some embodiments, the V gene has a restricted immunoglobulin heavy chain locus characterized by a V gene segment (e.g., as described in U.S. Patent Publication No. 2013 / 0096287, which is incorporated herein by reference). H The gene segment is V H 1-2 or V H1-69. In some embodiments, the mouse provided herein has a rearranged heavy chain variable region (e.g., a universal heavy chain variable region or consensus heavy chain coding sequence described in U.S. Patent Publication No. 20140245468, and U.S. Patent Nos. 9,204,624 and 9,930,871, the entireties of which are incorporated herein by reference). In some embodiments, the mouse provided herein comprises an unrearranged human immunoglobulin light chain, e.g., kappa, gene segment operably linked to a heavy chain constant region gene at an immunoglobulin heavy chain locus (e.g., U.S. Patent No. 9,516,868, the entireties of which are incorporated herein by reference).

[0084] In yet other embodiments, the mouse may comprise in its germline and / or genome a heavy chain immunoglobulin locus that comprises an insertion and / or substitution of a histidine codon designed to introduce pH-dependent binding properties into antibodies generated in such mice. In some such embodiments, the histidine codon is inserted and / or substituted in the nucleic acid sequence encoding CDR3. A variety of such heavy chain immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, U.S. Patent Application Publication Nos. 2013 / 0247236 and 20140013456, which are incorporated herein by reference.

[0085] In some embodiments, the engineered IgH locus (or allele) is selected from 5, 10, 15, 20, 25, or more (e.g., 26, 27, etc.) human D H In some specific embodiments, the engineered IgH locus (or allele) comprises a human D gene segment of the naturally occurring human IgH locus. H 1-1 gene segment and human D H Functional human D found between (and including) the 7-27 gene segment HIn some specific embodiments, the engineered IgH locus (or allele) comprises at least one of 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 In some embodiments, the unrearranged human Ig gene segment comprises a human D H Includes all gene segments.

[0086] In some embodiments, the engineered IgH locus (or allele) comprises one, two, three, four, five, six, or more functional human J H In some specific embodiments, the engineered IgH locus (or allele) comprises a human J gene segment of a naturally occurring human IgH locus. H 1 gene segment and human J H Functional human J found between (and including) six gene segments H In some specific embodiments, the engineered IgH locus (or allele) comprises at least one of the human J gene segments. H Gene segment J H 1. J H 2. J H 3. JH 4. J H 5, and J H In some embodiments, the unrearranged human Ig gene segment comprises a human J H Includes all gene segments.

[0087] In some embodiments, the engineered IgH loci described herein do not contain an endogenous Adam6 gene. In some embodiments, the engineered IgH loci described herein do not contain an endogenous Adam6 gene (or Adam6 coding sequence) in a germline genomic location similar to that found in the germline genome of a wild-type non-human animal of the same species. In some embodiments, the engineered IgH loci described herein do not contain a human Adam6 pseudogene. In some embodiments, the engineered IgH loci described herein include an insertion of at least one nucleotide sequence encoding one or more non-human (e.g., murine) Adam6 polypeptides. The insertion may be located outside (e.g., between the 5'-most V and 5'-most V) of the engineered immunoglobulin heavy chain loci described herein. H The IgH locus may be located upstream of a gene segment, within an engineered IgH locus, or elsewhere in the germline genome of a non-human animal (e.g., a randomly introduced non-human Adam6 coding sequence), cell or tissue.

[0088] In some embodiments, the engineered endogenous immunoglobulin heavy chain locus lacks a functional endogenous mouse Adam6 gene. In some embodiments, the mouse germline genome comprising the engineered heavy chain locus comprises one or more nucleotide sequences encoding one or more mouse ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, one or more mouse ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are expressed (e.g., in cells of the male germline, e.g., testicular cells).

[0089] In some embodiments, one or more nucleotide sequences encoding one or more mouse ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are included on the same chromosome as the engineered endogenous immunoglobulin heavy chain locus. ... H Gene segments and the second human V H In some embodiments, the first human V H The gene segment is V H 1-2, the second human V H The gene segment is V H 6-1. In some embodiments, the one or more nucleotide sequences encoding one or more mouse ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are in place of the human Adam6 pseudogene. In some embodiments, the one or more nucleotide sequences encoding one or more mouse ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof replace the human Adam6 pseudogene. In some embodiments, the one or more nucleotide sequences encoding one or more mouse ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are in place of the human Adam6 pseudogene. H Gene segments and human D H Between gene segments.

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

[0092] In some embodiments, the Ig heavy chain variable region is a rearranged variable region comprising an Ig heavy chain variable region gene (universal heavy chain variable region). In some embodiments, the rearranged Ig heavy chain variable region gene is a rearranged human Ig heavy chain variable region gene. Examples of rearranged Ig heavy chain variable regions include those provided in U.S. Patent Publication No. 2014 / 0245468, which is incorporated herein by reference.

[0093] In certain embodiments, the mice provided herein are engineered to express antibodies that contain a human immunoglobulin constant region (e.g., human C) such that the antibodies contain a human Fc (e.g., human Fcα). αMice comprising a human immunoglobulin constant region 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 C3 domain, which encodes the IgG constant domain including the IgG transmembrane domain, and the IgG cytoplasmic domain H The IgG connecting region comprises a gene segment. In some embodiments, the IgG transmembrane domain is a rodent IgG transmembrane domain (e.g., a mouse or rat transmembrane domain). In certain embodiments, the transmembrane domain is a human IgG transmembrane domain. In some embodiments, the IgG cytoplasmic domain is a rodent IgG cytoplasmic domain (e.g., a mouse or rat cytoplasmic domain). In some embodiments, the IgG cytoplasmic domain is a human IgG cytoplasmic domain. In some embodiments, the IgG connecting region is a rodent IgG connecting region (e.g., a mouse or rat connecting domain). In certain embodiments, the IgG connecting region is a human IgG connecting region.

[0095] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domain, and human C H The three domains are IgG1 domains. In some embodiments, such IgG1 domains are encoded by alleles selected from IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04, and IGHG1*05.

[0096] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domain, and human C HThe three domains are IgG2 domains. In some embodiments, such IgG2 domains are encoded by alleles selected from IGHG2*01, IGHG2*02, IGHG2*03, IGHG2*04, IGHG2*05, and IGHG2*06.

[0097] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domain, and human C H The 3 domain is an IgG3 domain. In some embodiments, such an IgG3 domain is encoded by an allele selected from IGHG3*01, IGHG3*02, IGHG3*03, IGHG3*04, IGHG3*05, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*13, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18, and IGHG3*19.

[0098] In certain embodiments, human C H 1 domain, human hinge region, human C H 2 domain, and human C H The three domains are IgG4 domains. In some embodiments, such IgG4 domains are encoded by alleles selected from IGHG4*01, IGHG4*02, IGHG4*03, and IGHG4*04.

[0099] In some embodiments, C H The gene segments encode variant human immunoglobulin heavy chain constant region sequences (i.e., human immunoglobulin heavy chain constant region sequences that contain one or more additions, deletions, and / or substitutions relative to an appropriate reference human immunoglobulin heavy chain constant region sequence) that are characterized by increased or decreased effector function and / or affinity for FcR compared to a reference human immunoglobulin heavy chain constant region.

[0100] In some embodiments, C H The gene segments encode human immunoglobulin heavy chain constant regions characterized by altered affinities for activating and / or inhibitory receptors. In some embodiments, H The gene segment encodes a human immunoglobulin heavy chain constant region that is characterized by increased or decreased binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H The gene segment encodes, in whole or in part, a human immunoglobulin heavy chain constant region, and encodes a human immunoglobulin heavy chain constant region having one or more amino acid modifications, such as modifications at position 297 (e.g., N297A), position 250 (e.g., 250E or 250Q), position 252 (e.g., 252L, 252Y, 252F, 252W, or 252T), position 254 (e.g., 254S or 254T), position 256 (e.g., 256S, 256R, 256Q, 256E, 256D, or 256T), position 307 (e.g., 307P or 307R), or at position 310 (e.g., 310S, 310R, 310Q, 310E, 310T, or 310T). Substitutions at positions 307A and 308F, 308V, 428L, 428F, 433H, 433Lm, 433R, 433S, 433P, 433Q, or 433K, 434A, 434W, 434H, 434F, or 434Y, and combinations thereof are included, but are not limited to these. HGene segments include 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 N434S); 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 includes segments or portions derived from (or represented by) more than one human immunoglobulin isotype. For example, such a chimeric C H The region is a C H 2 domains from human IgG1, human IgG2, or human IgG4 molecules. H In some specific embodiments, the chimeric C H The region further comprises a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues at positions 228 to 236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. In some specific embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge.

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

[0103] In some embodiments, the modified C H The gene segment is human C γ1 Gene segments (or at least human C γ1 Gene segment, IgG1 constant domain C H 1 domain, hinge region, C H 2 domain, and C H 3 domain) and endogenous C γ2a Located at a gene segment locus. In some embodiments, the human C γ1 Gene segment, IgG1 constant domain C H 1 domain, hinge region, C H 2 domain, and C H The region encoding the 3 domain is endogenous mouse C γ2a It is operably linked to portions of the gene segment encoding the IgG2a transmembrane and / or cytoplasmic domains.

[0104] In some embodiments, the modified C H The gene segment is human C γ1 Gene segments (or at least human C γ1 Gene segment, IgG1 constant domain C H 1 domain, hinge region, C H 2 domain, and C H 3 domain) and endogenous C γ2cLocated at a gene segment locus. In some embodiments, the human C γ1 Gene segment, IgG1 constant domain C H 1 domain, hinge region, C H 2 domain, and C H The region encoding the 3 domain is endogenous mouse C γ2c It is operably linked to portions of the gene segment encoding the IgG2c transmembrane and / or cytoplasmic domains.

[0105] In some embodiments, the modified C H The gene segment is human C γ4 Gene segments (or at least human C γ4 Gene segment, IgG4 constant domain C H 1 domain, hinge region, C H 2 domain, and C H 3 domain) and endogenous C γ1 Located at a gene segment locus. In some embodiments, the human C γ4 Gene segment, IgG4 constant domain C H 1 domain, hinge region, C H 2 domain, and C H The region encoding the 3 domain is endogenous mouse C γ1 It is operably linked to portions of the gene segment encoding the IgG1 transmembrane and / or cytoplasmic domains.

[0106] In certain embodiments, modified C H The gene segment is an endogenous C H In certain embodiments, the modified C H The gene segment is an 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, the modified C H The gene segment is human C γ1 Gene segments (or at least human C γ1 Gene segment, IgG1 constant domain C H 1 domain, hinge region, C H 2 domain, and C H 3 domain) and endogenous C γ2a In some embodiments, the human C γ1 Gene segment, IgG1 constant domain C H 1 domain, hinge region, C H 2 domain, and C H The region encoding the 3 domain is endogenous mouse C γ2a Gene segment, IgG2a constant domain C H 1 domain, hinge region, C H 2 domain, and C H The part encoding the 3 domain was replaced to give human C γ1 Gene segment, IgG1 constant domain C H 1 domain, hinge region, C H 2 domain, and C H The region encoding the 3 domain is endogenous mouse C γ2a It is operably linked to portions of the gene segment encoding the IgG2a transmembrane and / or cytoplasmic domains.

[0108] In some embodiments, the modified C H The gene segment is human C γ4 Gene segments (or at least human C γ4 Gene segment, IgG4 constant domain C H 1 domain, hinge region, C H 2 domain, and C H 3 domain) and endogenous C γ1In some embodiments, the human C γ4 Gene segment, IgG4 constant domain C H 1 domain, hinge region, C H 2 domain, and C H The region encoding the 3 domain is endogenous mouse C γ1 Gene segment, IgG1 constant domain C H 1 domain, hinge region, C H 2 domain, and C H The part encoding the 3 domain was replaced to give human C γ4 Gene segment, IgG4 constant domain C H 1 domain, hinge region, C H 2 domain, and C H The region encoding the 3 domain is endogenous mouse C γ1 It is operably linked to portions of the gene segment encoding the IgG1 transmembrane and / or cytoplasmic domains.

[0109] In certain embodiments, the Ig heavy chain constant region is selected from one or more C H In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C μ In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C δ In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C γ1 In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C γ2a In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C γ2b In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C γ2cIn some embodiments, the Ig constant region comprises a rodent (e.g., mouse) C γ3 In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C ε In some embodiments, the Ig constant region comprises a rodent (e.g., rat or mouse) C ε In some embodiments, the one or more rodent constant region gene segments are endogenous constant region gene segments. In some embodiments, the modified C H The gene segment contains the only modified C in the Ig heavy chain constant region. H In some embodiments, the modified C H The gene segment comprises multiple modified C H In some embodiments, the Ig heavy chain constant region is one of the human or partially human C gene segments (e.g., one of two, three, four, five, six, seven, or eight modified gene segments in the Ig heavy chain constant region that are partially or fully human). μ In some embodiments, the Ig heavy chain constant region comprises a human or partially human C δ In some embodiments, the Ig heavy chain constant region comprises a human or partially human C γ1 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C γ2 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C γ3 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C γ4 In some embodiments, the Ig heavy chain constant region comprises a human or partially human C ε In some embodiments, the Ig heavy chain constant region comprises a human or partially human C αIn some embodiments, the Ig heavy chain constant region comprises a human C μ Gene segment, human C δ Gene segment, human C γ1 Gene segments and human C γ3 In some embodiments, the Ig heavy chain constant region comprises a human C γ2 Gene segments and human C γ4 In some embodiments, the Ig heavy chain constant region further comprises a human C α In some embodiments, the Ig heavy chain constant region further comprises a human C ε It further includes gene segments.

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

[0111] In some embodiments, the modified immunoglobulin heavy chain locus is positioned at an endogenous immunoglobulin heavy chain locus. In some embodiments, the immunoglobulin heavy chain locus replaces all or a portion of the endogenous immunoglobulin heavy chain locus. In certain embodiments, the modified IgH locus is located on a transgene that is positioned outside the endogenous locus. In some embodiments, the endogenous IgH locus is inactivated (e.g., by deletion, rearrangement, and / or inversion of all or a portion of the endogenous Ig heavy chain locus).

[0112] Thus, in some embodiments, one or more immunoglobulin heavy chain constant regions (or portions) of an immunoglobulin heavy chain locus are not deleted (i.e., intact). H The gene segment may comprise, inter alia, an immunoglobulin heavy chain constant region sequence described herein (e.g., a human IgG C-terminal region) operably linked to transmembrane and cytoplasmic coding sequence(s) of a non-human immunoglobulin heavy chain IgG constant region gene (e.g., an M1 and / or M2 coding sequence). H 1-HC H 2-C H and, in some embodiments, an immunoglobulin heavy chain constant region sequence (e.g., human IgE C3 polypeptide-encoding sequence) operably linked to the transmembrane and cytoplasmic coding sequence(s) of an IgE constant region gene. H 1-C H 2-C H 3-C H In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operably linked to transmembrane and cytoplasmic coding sequences (e.g., M1 and M2 exons) of one or more IgG constant region genes. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operably linked to transmembrane and cytoplasmic coding sequences (e.g., M1 and M2 exons) of an IgE constant region gene. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operably linked to transmembrane and cytoplasmic coding sequences (e.g., M exon(s)) of an IgA constant region gene. In some particular embodiments, one or more C H Gene segments (e.g., C μ , C δand the like) are not deleted or replaced in the immunoglobulin heavy chain constant region comprising a heterologous immunoglobulin heavy chain constant region sequence operably linked to the transmembrane and cytoplasmic coding sequences of one or more constant region genes 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 altered, disrupted, deleted, replaced or engineered with one or more heterologous immunoglobulin heavy chain constant region sequences is a murine immunoglobulin heavy chain constant region. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is an IgG constant region gene (e.g., C γ1 , C γ2a , C γ2b , C γ2c , or C γ3 ) into one copy (i.e., allele) of the IgG constant region gene, resulting in a mouse that is heterozygous for the xenogenous immunoglobulin heavy chain constant region sequence. In some embodiments, a mouse is provided that is homozygous for an immunoglobulin heavy chain constant region comprising a xenogenous immunoglobulin heavy chain constant region sequence as described herein.

[0113] In some embodiments, the engineered immunoglobulin heavy chain constant regions described herein comprise a human extracellular domain coding sequence (e.g., human IgG C) operably linked to a non-human transmembrane and cytoplasmic domain coding sequence (e.g., non-human IgG M1-M2) of the same or a different IgG subclass. H 1-HC H 2-C H 3) one or more IgG codes C H Contains gene segments.

[0114] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein comprises one or more engineered IgG coding constant regions described herein. H A gene segment, comprising a wild-type (e.g., unmodified, non-human, such as rat or mouse) Cμ It further comprises a constant region gene.

[0115] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein comprises one or more engineered IgG coding constant regions described herein. H A gene segment, comprising a wild-type (e.g., unmodified, non-human, such as rat or mouse) C μ and C δ It further comprises a constant region gene.

[0116] In various embodiments, an engineered IgG-encoded C-protein comprising a human immunoglobulin heavy chain constant region sequence as described herein. H The gene segment is C γ1 , C γ2a , C γ2b , C γ2c , or C γ3 An engineered IgG code C of an IgG subclass selected from H A gene segment.

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

[0118] In some specific embodiments, the engineered immunoglobulin heavy chain constant region described herein is H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H3-M1-M2 coding sequence, γ2a An engineered C operably linked to a switch region of a gene segment. γ2a Contains gene segments.

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

[0120] In some specific embodiments, the engineered immunoglobulin heavy chain constant region described herein is H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H 3-M1-M2 coding sequence, γ2c An engineered C operably linked to a switch region of a gene segment. γ2c Contains gene segments.

[0121] In some specific embodiments, the engineered immunoglobulin heavy chain constant region described herein is H 1-HC H 2-C H Instead of exon 3, human IgG4 C H 1-HC H 2-C H 3, and a non-human (e.g., rat or mouse) C γ1An engineered C operably linked to the M1 and M2 exons of the gene segment. γ1 Contains gene segments.

[0122] In some specific embodiments, the engineered immunoglobulin heavy chain constant region described herein is H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG4 C H 1-HC H 2-C H 3-M1-M2 coding sequence, γ1 An engineered C operably linked to a switch region of a gene segment. γ1 Contains gene segments.

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

[0124] In some specific embodiments, the engineered immunoglobulin heavy chain constant region described herein is H1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H 3-M1-M2 coding sequence, γ2a An engineered C operably linked to a switch region of a gene segment. γ2a Gene segments and C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG4 C H 1-HC H 2-C H 3-M1-M2 coding sequence, γ1 An engineered C operably linked to a switch region of a gene segment. γ1 and a gene segment.

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

[0126] In some specific embodiments, the engineered immunoglobulin heavy chain constant region described herein is H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H 3-M1-M2 coding sequence, γ2c An engineered C operably linked to a switch region of a gene segment. γ2c Gene segments and C H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG4 C H 1-HC H 2-C H 3-M1-M2 coding sequence, γ1 An engineered C operably linked to a switch region of a gene segment. γ1 and a gene segment.

[0127] In various embodiments, the engineered immunoglobulin heavy chain constant region described herein is a human IgG 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 other than the IgG constant region (i.e., isotypes) including sequences encoding 3-M1-M2 (e.g., IgG1 and / or IgG2a) can be used to encode, for example, IgD, IgE, IgA, and IgG (as such, human IgG C as described herein). H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C Hand one or more further modifications, including rendering non-human mice, including mice that do not contain sequences encoding 3-M1-M2, non-human IgG1b and / or IgG2b, non-human IgG2c, and non-human IgG3b. Engineered non-human embryos, cells, and targeting vectors for producing such mice, embryos, and cells are also provided.

[0128] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ Gene segments and C γ1 A gene segment comprising: H 1-HC H 2-C H Instead of exon 3, human IgG4 C H 1-HC H 2-C H 3, γ1 C operably linked to the M1-M2 exons of the gene segment γ1 Gene segments and C δ , C γ2a , C γ2c , C γ2b , C γ3 , C ε and C α and deletion of a gene segment.

[0129] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ Gene segments and C γ1 A gene segment comprising: H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG4 C H 1-HC H 2-C H3-M1-M2 coding sequence, γ1 C operably linked to the switch region of the gene segment γ1 Gene segments and C δ , C γ2a , C γ2c , C γ2b , C γ3 , C ε and C α and deletion of a gene segment.

[0130] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ Gene segments and C γ2a A gene segment comprising: H 1-HC H 2-C H Instead of exon 3, human IgG1 C H 1-HC H 2-C H 3, γ2a C operably linked to the M1-M2 exons of the gene segment γ2a Gene segments and C δ , C γ1 , C γ2b , C γ2c , C γ3 , C ε and C α and deletion of a gene segment.

[0131] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ Gene segments and C γ2a A gene segment comprising: H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H3-M1-M2 coding sequence, γ2a C operably linked to the switch region of the gene segment γ2a Gene segments and C δ , C γ1 , C γ2b , C γ2c , C γ3 , C ε and C α and deletion of a gene segment.

[0132] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ Gene segments and C γ2c A gene segment comprising: H 1-HC H 2-C H Instead of exon 3, human IgG1 C H 1-HC H 2-C H 3, γ2c C operably linked to the M1-M2 exons of the gene segment γ2c Gene segments and C δ , C γ1 , C γ2a , C γ2b , C γ3 , C ε and C α and deletion of a gene segment.

[0133] In some specific embodiments, the engineered immunoglobulin heavy chain constant regions provided herein are derived from wild-type (e.g., unmodified non-human (such as rat or mouse)) C μ Gene segments and C γ2c A gene segment comprising: H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H3-M1-M2 coding sequence, γ2c C operably linked to the switch region of the gene segment γ2c Gene segments and C δ , C γ1 , C γ2a , C γ2b , C γ3 , C ε and C α and deletion of a gene segment.

[0134] In various embodiments, the engineered immunoglobulin heavy chain constant region described herein is a human IgG H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H 3- One or more constant region genes other than the IgG constant region (i.e., isotypes) containing sequences encoding M1-M2 (e.g., IgG1 and / or IgG2a) are included in the human immunoglobulin heavy chain constant region sequences described herein, including IgD, IgE, IgA, and IgG (which are themselves human IgG C sequences described herein). H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H 3-M1-M2) (e.g., IgG2b and / or IgG3) that does not contain sequences encoding M1-M2, and one or more further modifications, including engineering the resulting gene(s) to be altered, modified, substituted, engineered, etc., via insertion into one or more immunoglobulin constant region genes of the IgG (e.g., IgG2b and / or IgG3) that does not contain sequences encoding M1-M2.

[0135] Also provided are engineered mouse embryos, cells, and targeting vectors for generating mice, embryos, and cells that contain immunoglobulin loci with the engineered constant regions described herein.

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

[0137] In some particular embodiments, the engineered immunoglobulin heavy chain constant region provided herein is a wild-type C μ Gene segments and wild-type C δ Gene segments and C γ3 A gene segment comprising: H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG3 C H 1-HC H 2-C H 3-M1-M2 coding sequence, γ3 C operably linked to the switch region of the gene segment γ3Gene segments and C γ1 A gene segment comprising: H 1-HC H 2-C H Human IgG4 C instead of 3-M1-M2 exons H 1-HC H 2-C H 3-M1-M2 coding sequence, γ1 C operably linked to the switch region of the gene segment γ1 Gene segments and C γ2b A gene segment comprising: H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG2 C H 1-HC H 2-C H 3-M1-M2 coding sequence, γ2b C operably linked to the switch region of the gene segment γ2b Gene segments and C γ2a A gene segment comprising: H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H 3-M1-M2 coding sequence, γ2a C operably linked to the switch region of the gene segment γ2a Gene segment (and / or C γ2c A gene segment comprising: H 1-HC H 2-C H Instead of the 3-M1-M2 exon, human IgG1 C H 1-HC H 2-C H 3-M1-M2 coding sequence, γ2c C operably linked to the switch region of the gene segment γ2c gene segment) and C ε A gene segment comprising: 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 4, ε C operably linked to the M1-M2 exons of the gene segment ε Gene segments and C α A gene segment comprising: H 1-HC H 2-C H Instead of exon 3, human IgA1 or IgA2 C H 1-HC H 2-C H 3, α C operably linked to M exon(s) of a gene segment α and a gene segment.

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

[0139] Therapeutic agents comprising human Fc (e.g., therapeutic agents comprising human Fcα, such as therapeutic human IgA antibodies) are typically tested in non-human species (e.g., mice) before being administered to humans. Unfortunately, agents comprising 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 having a human Fc region is administered to conventional mice, the human sequences in the Fc region are often identified as foreign by the mouse immune system. As a result, the mice may mount an immune response to the administered therapeutic agent (known as a mouse anti-human response or MAHA), which affects the pharmacokinetic and pharmacodynamic properties of the administered agent. Thus, conventional mouse models are often poor predictors of human therapeutic response to therapeutic agents comprising human Fc. In certain embodiments, the mice provided herein exhibit reduced MAHA responses following administration of therapeutic agents comprising human Fc. Thus, the mice disclosed herein having human immunoglobulin locus regions are useful for reducing or eliminating MAHA responses.

[0140] Thus, for example, as disclosed in WO2019 / 190990, which is incorporated herein by reference in its entirety, human or humanized FcαR can be expressed and a human constant region (e.g., human C α Various mice provided herein that contain a human Fcα-containing Fcα constant region can be used to test the MAHA response to administration of an agent that contains a human Fcα (e.g., an agent that contains a human Fcα, such as a therapeutic human IgA antibody).

[0141] Humanized immunoglobulin kappa locus In some embodiments, the mice provided herein further comprise a humanized Ig κ chain locus. As disclosed in WO2019 / 190990, which is incorporated herein by reference in its entirety, testing therapeutic agents comprising human κ chains in mice with a humanized Ig κ chain locus can reduce or eliminate MAHA responses, since the human κ chain is less likely to be identified as foreign in such mice.

[0142] In some embodiments, mice and ES cells genetically modified to contain a humanized or human FcαR1 locus in their genome further comprise a genetically modified Ig κ chain locus. Such a locus comprises a κ variable region and a κ constant region. The κ variable region comprises an Ig κ chain variable region gene segment (i.e., at least the V κ Gene segments and J κ The constant region includes the Ig κ chain constant region (C κ In certain embodiments, the immunoglobulin kappa chain variable region, such as the human Igκ variable region, is derived from a mouse that is a human V κ Gene segments and human J κ a light chain variable domain derived from a gene segment and κ and a light chain constant domain derived from the gene segment. In some embodiments, the Igκ variable region is an unrearranged Igκ variable region and thus will comprise an unrearranged Igκ variable region gene segment. In some embodiments, the Igκ variable region is a rearranged Igκ variable region and thus will comprise a rearranged Igκ variable region gene. In certain embodiments, the Igκ variable region gene segment is a human Igκ variable region gene segment. In certain embodiments, the Igκ variable region gene segment is a rodent Igκ variable region gene segment (e.g., a rat or mouse variable region gene segment). In some embodiments, the Igκ constant region locus comprises a partially or fully human Igκ constant region gene segment. In some embodiments, the Igκ chain locus described herein is located at an endogenous Igκ chain locus.

[0143] In certain embodiments, the Igκ variable region comprises unrearranged human Igκ variable region gene segments. In some specific embodiments, the engineered Igκ light chain locus (or allele) comprises at least one human V that appears in the distal variable cluster (or distal arm or distal duplication) of a naturally occurring human Igκ light chain locus. κ In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises at least a human V κ gene segment that appears in the proximal variable cluster (or proximal arm or proximal duplication) of a naturally occurring human Ig κ light chain locus. κ In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises a human V that appears in the distal and proximal variable clusters of a naturally occurring human Ig κ light chain locus. κ In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises a human V gene segment of a naturally occurring human Ig κ light chain locus. κ 2-40 (or V κ 3D-7) Gene segments and human V κ Functional human V found between (and including) the 4-1 gene segment κ It includes all or substantially all of a gene segment.

[0144] In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises a plurality of human V κ Segment and one or more human J κ In some embodiments, the immunoglobulin variable region gene segment comprises four functional V κ Segment and all human J κ In some embodiments, the immunoglobulin variable region gene segment comprises 16 functional V κ Segment and all human J κ In some embodiments, the unrearranged human immunoglobulin variable region gene segments include all human V κSegment and all human J κ Includes segments.

[0145] In some specific embodiments, the engineered Ig κ light chain locus (or allele) is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or more (e.g., 36, 37, 38, 39, 40, etc.) human V κ In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises a human V κ Gene segment V κ 3D-7, V κ 1D-8, V κ 1D-43, V κ 3D-11, V κ 1D-12, V κ 1D-13, V κ 3D-15, V κ 1D-16, V κ 1D-17, V κ 3D-20, V κ 6D-21, V κ 2D-26, V κ 2D-28, V κ 2D-29, V κ 2D-30, V κ 1D-33, V κ 1D-39, V κ 2D-40, V κ 2-40, V κ 1-39, V κ 1-33, V κ 2-30, V κ 2-28, V κ 1-27, V κ 2-24, V κ 6-21, V κ 3-20, V 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 In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises at least one human V κ Gene segment V κ 3D-7, V κ 1D-8, V κ 1D-43, V κ 3D-11, V κ 1D-12, V κ 1D-13, V κ 3D-15, V κ 1D-16, V κ 1D-17, V κ 3D-20, V κ 6D-21, V κ 2D-26, V κ 2D-28, V κ 2D-29, V κ 2D-30, V κ 1D-33, V κ 1D-39 and V κ In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises at least one 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 Including 4-1.

[0146] In some embodiments, the mouse provided herein has no more than two human V LGene segments and multiple J L In some embodiments, the mouse has a restricted immunoglobulin light chain locus characterized by a dual light chain gene segment (e.g., a dual light chain mouse or DLC, as described in U.S. Patent Publication No. 2013 / 0198880, which is incorporated herein by reference). L The gene segment is V κ In some embodiments, the V L The gene segment is V λ In some embodiments, the V κ The gene segment is V κ 3-20 and V κ The number is 1-39.

[0147] In some embodiments, the engineered Ig κ light chain locus (or allele) comprises one, two, three, four, five, or more functional human J κ In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises a human J gene segment of a naturally occurring human Ig κ light chain locus. κ 1 gene segment and human J κ Functional human J found between (and including) the 5 gene segment κ In some specific embodiments, the engineered Ig κ light chain locus (or allele) comprises at least one 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 other embodiments, a non-human organism may comprise in its germline and / or genome a light chain immunoglobulin locus that comprises an insertion and / or substitution of a histidine codon designed to introduce pH-dependent binding properties into antibodies generated in such non-human organism. In some such embodiments, the histidine codon is inserted and / or substituted in the nucleic acid sequence encoding CDR3. A variety of such light chain immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, U.S. Patent Application Publication Nos. 2013 / 0247236 and 2014 / 0013456, which are incorporated herein by reference.

[0149] Exemplary variable regions comprising Igκ 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. In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all human Jκ segments.

[0150] In some embodiments, the Igκ variable locus comprising unrearranged human Igκ variable region gene segments also comprises a human Igκ variable region intergenic sequence. In some embodiments, the Igκ variable locus comprises a non-human (e.g., rodent, rat, mouse) Igκ variable region intergenic sequence. In some embodiments, the Igκ locus comprises a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0151] In some embodiments, the Igκ variable region locus is a rearranged variable region locus comprising an Igκ variable region gene (universal light chain variable region). In some embodiments, the rearranged Igκ variable region gene is a rearranged human Igκ variable region gene (e.g., a rearranged human Igκ variable region comprising a human Vκ1-39Jκ or a human Vκ3-20Jκ sequence, such as a human Vκ1-39Jκ5 or a human Vκ3-20Jκ1 sequence). The use of a universal light chain variable region facilitates the generation of bispecific antibodies. Examples of rearranged Ig light chain variable regions are provided in U.S. Patent Publication No. 2013 / 0185821, which is incorporated herein by reference.

[0152] In some embodiments, the Ig κ chain locus comprises a human or rodent (e.g., rat or mouse) regulatory element. In some embodiments, the regulatory element is an endogenous regulatory element. In certain embodiments, the Ig κ chain locus comprises a rodent (e.g., rat or mouse) or human intronic κ enhancer (E κi In some embodiments, the IgH locus comprises a rodent (e.g., rat or mouse) or human 3′ kappa enhancer (E κ3’ ).

[0153] In some embodiments, the modified immunoglobulin κ chain locus in the genetically modified rodent comprises one or more unrearranged human Vλ gene segments and one or more unrearranged human Jλ gene segments upstream (e.g., operably linked) to a Cλ gene. Examples of rodents comprising such genetically modified immunoglobulin κ chain loci include those described in U.S. Pat. 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 comprises a limited human λ light chain variable region repertoire, and the limited human λ light chain variable region repertoire comprises a single rearranged human immunoglobulin λ light chain variable region (Vλ / Jλ). The single rearranged human λ light chain variable region comprises a human Vλ gene segment linked to a human Jλ gene segment and operably linked to a Cκ or Cλ gene segment (e.g., a rodent Cκ or Cλ gene segment (e.g., a mouse Cλ1 gene segment). Examples of rodents that contain such genetically modified immunoglobulin κ chain loci include those described in WO2020 / 247623, which is incorporated by reference in its entirety.

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

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

[0156] Thus, for example, as disclosed in WO2019 / 190990, which is incorporated herein by reference in its entirety, human or humanized FcαR can be expressed and a human light chain constant region (and, in some embodiments, also a human C α Various mice provided herein that contain a human Fcα-containing Fcα constant region can be used to test the MAHA response to administration of an agent that contains a human Fc (e.g., an agent that contains a human Fcα, such as a therapeutic human IgA antibody).

[0157] Humanized immunoglobulin lambda locus In some embodiments, the mice provided herein further comprise a humanized Ig λ chain locus. As disclosed in WO2019 / 190990, which is incorporated herein by reference in its entirety, testing therapeutic agents comprising human λ chains in mice with humanized Ig λ chain loci can reduce or eliminate MAHA responses, since human κ chains are less likely to be identified as foreign 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 Ig λ chain locus. Such a locus includes an Ig λ chain variable region gene segment (i.e., at least the V λ Gene segments and J λ The modified λ locus contains at least one Ig λ chain constant region (C λ In certain embodiments, the human V λ Gene segments and human J λ V, such as gene segments λGene segments and J λ The gene segments are: mouse V λ Gene segments and human J λ a light chain variable domain derived from a gene segment and λ and a light chain constant domain derived from the gene segment, λ In some embodiments, V λ Gene segments and J λ The gene segment is unrearranged V λ and J λ In some embodiments, the V λ Gene segments and J λ The gene segment is a rearranged V λ and J λ In certain embodiments, the Ig λ The variable region gene segments are human variable region gene segments. λ The variable region gene segments are rodent variable region gene segments (e.g., rat or mouse variable region gene segments). λ The constant region locus comprises a partially or completely human λ constant region gene segment. In some embodiments, the Ig λ chain locus described herein is located at an endogenous Ig λ chain locus. Examples of variable regions comprising Ig λ gene segments include those provided in, for example, U.S. Patent Publication Nos. 2012 / 0073004 and 2002 / 0088016, and U.S. Patent Application No. 15 / 803,513 (filed November 3, 2017, published as US2018 / 0125043), which are incorporated herein by reference.

[0159] In some embodiments, an Igλ variable locus that comprises an unrearranged human Igλ variable region gene segment also comprises a human Igλ variable region intergenic sequence. In some embodiments, an Igλ variable locus comprises a non-human (e.g., rodent, rat, mouse) Igλ variable region intergenic sequence. In some embodiments, an Igλ locus comprises a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0160] In some embodiments, the human Ig λ light chain locus comprises genetic material derived from a human Ig λ light chain locus. In some embodiments, the human Ig λ light chain locus described herein comprises at least one human V λ Gene segment, at least one human J λ Gene segment, at least one human C λ gene segment and the at least one human V λ A gene segment and at least one human J λ Promotes rearrangement with gene segments and human V λ A functional reconstituted human V encoding domain λ -J λ In many embodiments, the human Ig λ light chain sequence comprises one or more sequences necessary to form the VV ... λ Gene segments, and the human V λ A gene segment and at least one human J λ In some embodiments, the human Ig λ light chain sequences described herein are genomic sequences of the human Ig λ light chain locus (e.g., isolated and / or cloned from a bacterial artificial chromosome) and contain multiple human V sequences in germline configuration. λ In some embodiments, the human Ig λ light chain sequence comprises a human Vλ , J λ , and C λ The sequence includes (i.e., the human V λ , J λ , and C λ In other words, the J λ and C λ The sequence is J λ C λ In some embodiments, the human Ig λ light chain sequence encodes, in whole or in part, an Ig λ light chain polypeptide that is present in immunoglobulins, particularly immunoglobulins expressed by human B cells. Mice, embryos, cells, and targeting constructs for generating mice, non-human embryos, and cells that comprise the human Ig λ light chain sequence in place of a corresponding non-human Ig λ light chain sequence (e.g., an endogenous mouse Ig λ light chain locus) are also provided.

[0161] In some embodiments, the human Igλ light chain sequence is inserted in place of the corresponding non-human Igλ light chain sequence in the germline genome of the non-human animal. In some embodiments, the human Igλ light chain sequence is inserted upstream of the non-human Igλ light chain sequence (e.g., the non-human Igλ light chain constant region sequence). In some embodiments, the human Igλ light chain sequence is inserted in the middle of one or more non-human Igλ light chain sequences such that the human Igλ light chain sequence is juxtaposed with the non-human Igλ light chain sequence.

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

[0163] In some embodiments, the Igλ locus is a rearranged locus that includes an Igλ variable region gene (universal light chain variable region). In some embodiments, the rearranged Igλ variable region gene is a rearranged human Igλ variable region gene. The use of a universal light chain variable region facilitates the generation of bispecific antibodies that bind at least one antigen binding domain. Examples of rearranged Ig light chain variable regions include those provided in U.S. Patent Publication No. 2013 / 0185821, which is incorporated herein by reference.

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

[0165] In some embodiments, the modified immunoglobulin λ chain locus is positioned at an endogenous immunoglobulin λ chain locus. In some embodiments, the immunoglobulin λ chain locus replaces all or a portion of an endogenous immunoglobulin λ chain locus. In particular embodiments, the modified Ig λ chain locus is located on a transgene that is positioned outside the endogenous locus. In particular embodiments, the modified Ig λ chain locus is located at an endogenous immunoglobulin κ chain locus. In some embodiments, the endogenous Ig λ chain locus is inactivated (e.g., by deletion, rearrangement, and / or inversion of all or a portion of the endogenous Ig λ chain locus).

[0166] In some embodiments, the mouse is heterozygous for the modified λ immunoglobulin chain locus. In certain embodiments, the mouse is homozygous for the modified λ immunoglobulin chain locus.

[0167] In some embodiments, the mouse comprises in its germline and / or genome a light chain immunoglobulin locus that comprises a limited repertoire of light chain variable gene segments (e.g., a dual light chain variable region comprising two light chain variable gene segments). In some embodiments, the light chain variable gene segments in the limited repertoire of light chain gene segments are human light chain gene segments. Examples of dual light chain variable regions include those provided in U.S. Patent Publication No. 2013 / 0198880, which is incorporated herein by reference. In some embodiments, the mouse comprising the dual light chain variable regions is used to generate bispecific antibodies.

[0168] Thus, for example, as disclosed in WO2019 / 190990, which is incorporated herein by reference in its entirety, human or humanized FcαR can be expressed and a human light chain constant region (and, in some embodiments, also a human C αVarious mice provided herein that contain a human Fcα-containing Fcα constant region can be used to test the MAHA response to administration of an agent that contains a human Fc (e.g., an agent that contains a human Fcα, such as a therapeutic human IgA antibody).

[0169] Humanized CD79a and CD79b loci In some embodiments, mice and ES cells genetically modified to contain a humanized or human FcαR1 locus in their genome further contain a 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 human or humanized CD79a and CD79b genes express human or humanized CD79a and CD79b polypeptides as heterodimers on the surface of B cells, which non-covalently associate with membrane-expressed immunoglobulin to form the B cell receptor (BCR). The BCR associates with antigen and functions in signal transduction and internalization after antigen binding. In some embodiments, the mice described herein contain human or humanized CD79a and CD79b genes. In some specific embodiments, a 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 a human CD79a polypeptide (e.g., amino acids corresponding to residues 33-143 of the human CD79a polypeptide), and the human CD79b portion encodes substantially all of the extracellular domain of a human CD79b polypeptide (e.g., amino acids corresponding to residues 29-159 of the human CD79b polypeptide). In some embodiments, each of the rodent CD79a and CD79b portions encodes at least the intracellular domain of an endogenous CD79a and CD79b polypeptide, respectively, and in some specific embodiments, encodes the transmembrane and intracellular domains of the endogenous CD79a and CD79b polypeptide, respectively. In some embodiments, the human portion and the endogenous portion are operably linked to the endogenous CD79a promoter or CD79b promoter, respectively.

[0170] In some embodiments, a mouse as described herein further comprises a chimeric CD79a gene comprising a rodent CD79a portion and a human CD79a portion, wherein the human CD79a portion encodes a sequence comprising amino acids corresponding to residues 33-116 of a human CD79a polypeptide, in one embodiment encoding a sequence comprising amino acids 33-119 of a human CD79a polypeptide, in one embodiment encoding a sequence comprising amino acids 33-143 of a human CD79a polypeptide, and in one embodiment encoding a sequence comprising amino acids 33-165 of a human CD79a polypeptide. In some embodiments, the chimeric CD79a polypeptide comprises a human Ig C2-like domain, in some embodiments, the chimeric CD79a polypeptide also comprises a human stalk region, in some embodiments, the chimeric CD79a polypeptide also comprises a human transmembrane domain, and in some embodiments, the chimeric CD79a polypeptide further comprises a rodent (e.g., murine) cytoplasmic domain. In some embodiments, the mouse comprises a chimeric CD79a gene comprising a human region portion as described herein and a sequence encoding a human or rodent (e.g., mouse) CD79a signal peptide, and in one embodiment, the sequence encoding the signal peptide is a mouse CD79a sequence encoding amino acids 1-28 of mouse CD79a.

[0171] In some embodiments, a mouse as described herein further comprises a chimeric CD79b gene comprising a rodent CD79b portion and a human CD79b portion, wherein the human CD79b portion encodes a sequence comprising amino acids corresponding to residues 29-135 of a human CD79b polypeptide, in one embodiment encoding a sequence comprising amino acids 29-159 of a human CD79b polypeptide, and in one embodiment encoding a sequence comprising amino acids 29-184 of a 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., murine) cytoplasmic domain. In some embodiments, the mouse comprises a chimeric CD79b gene comprising a human region portion as described herein and a sequence encoding a human or rodent (e.g., mouse) CD79b signal peptide, and in one embodiment, the sequence encoding the signal peptide is a mouse CD79b sequence encoding amino acids 1-25 of mouse CD79b.

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

[0173] In some embodiments, the mice provided herein further comprise one or more of the human CD79A and CD79B genes described in U.S. Patent Application Publication Nos. 2011-0093963A1 and 2009-0053210A1, International Patent Application Publication No. WO2008 / 027986, and European Patent No. 2 064 325 B1, each of which is incorporated herein by reference. In some specific embodiments, a mouse provided herein comprises a humanized CD79a gene comprising an endogenous CD79a portion and a human CD79a portion, and a humanized CD79b gene comprising an endogenous CD79b portion and a human CD79b portion, wherein the human CD79a portion encodes substantially all of the extracellular domain of a human CD79a polypeptide (e.g., amino acids corresponding to residues 33-143 of the human CD79a polypeptide), and the human CD79b portion encodes substantially all of the extracellular domain of a human CD79b polypeptide (e.g., amino acids corresponding to residues 29-159 of the human CD79b polypeptide). In some embodiments, a mouse provided herein comprises a humanized CD79a gene comprising an endogenous CD79a portion and a human CD79a portion, and a humanized CD79b gene comprising an endogenous CD79b portion and a human CD79b portion, wherein the human CD79a portion encodes a sequence comprising amino acids 33-116 (e.g., a sequence comprising amino acids 33-119, a sequence comprising amino acids 33-143, or a sequence comprising amino acids 33-165 of a human CD79a polypeptide) and the human CD79b portion encodes a sequence comprising amino acids 29-135 (e.g., a sequence comprising amino acids 29-159, or a sequence comprising amino acids 29-184 of a human CD79a polypeptide). In some embodiments, each of the endogenous CD79a and CD79b portions encodes at least the intracellular domains of the endogenous CD79a and CD79b polypeptides, respectively, and in some specific embodiments, the transmembrane and intracellular domains 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, which is a polypeptide that lacks a transmembrane domain and binds to FcRn and MHC class I molecules.

[0175] In some embodiments, the β2M locus comprises a nucleic acid sequence encoding a human β2M polypeptide. In some embodiments, the nucleic acid sequence encoding a human β2M polypeptide is located at the endogenous mouse β2M locus. In certain embodiments, the nucleic acid sequence encoding a 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 comprises a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0176] Humanized β2M polypeptides, loci encoding humanized β2M polypeptides, and mice expressing humanized β2M polypeptides are described in U.S. Patent Publication Nos. 2013 / 0111617 and 2013 / 0185819, each of which is incorporated herein by reference. Thus, as described in U.S. Patent Publication Nos. 2013 / 0111617 and 2013 / 0185819, in some embodiments, the 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 a 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 aspects, provided herein are genetically modified mice and ES cells that comprise one or more of the humanized loci disclosed herein, as well as genetically modified mouse ES cells useful for producing such mice.

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

[0179] In some embodiments, the mouse is a C57BL strain mouse. 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 mouse. In some embodiments, the 129 strain is selected from the group consisting of 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 strains. In some embodiments, the genetically modified mouse is a mix of 129 and C57BL strains. In some embodiments, the mouse is a mix of 129 strains and / or a mix of C57BL / 6 strains. In some embodiments, the mixed 129 strain is a 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse is a BALB strain (e.g., BALB / c). In some embodiments, the mice are a mix of BALB and another strain (e.g., C57BL and / or 129). In some embodiments, the mice provided herein can be mice 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 VELOCIGENE® technology as described in U.S. Patent Nos. 6,586,251, 6,596,541, 7,105,348, and Valenzuela et al. (2003) "High-throughput engineering of the mouse genome coupled with high-resolution expression analysis" Nat. Biotech. 21(6):652-659, each of which is incorporated herein by reference. Modifications can also be made using genome-targeted nuclease systems, such as CRISPR / Cas systems, transcription activator-like effector nuclease (TALEN) systems, or zinc finger nuclease (ZFN) systems. In some embodiments, the modification is carried out using the CRISPR / Cas system, for example as described in U.S. Patent Application Nos. 14 / 314,866, 14 / 515,503, 14 / 747,461, and 14 / 731,914, each of which is incorporated herein by reference.Exemplary methods for producing such genetically modified mice and ES cells are also provided in Example 1 herein.

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

[0182] Methods for testing human Fcα-containing therapeutics In certain aspects, provided herein are methods of testing a therapeutic protein that comprises a human Fcα domain (e.g., a human antibody or an Fcα fusion protein), comprising administering the therapeutic protein to a mouse provided herein. In some embodiments, provided herein are animal models for carrying out such methods.

[0183] In some embodiments, the administered human antibody or Fc fusion protein comprises a human C1 gene at the genetically engineered IgH locus of the mouse provided herein. H For example, in some embodiments, the agent is a human IgA1 antibody and the mouse has an isotype and / or allotype that matches the isotype and / or allotype of the Fc domain encoded by C H In some embodiments, the agent is a human IgA2 antibody and the mouse comprises a genetically modified IgH locus comprising gene segments encoding human IgA2 CH1, hinge, CH2, and CH3 domains. H The agent comprises a genetically modified IgH locus comprising a gene segment, hi some embodiments, the agent is an antibody comprising a human heavy chain variable domain (e.g., an IgA1 or IgA2 antibody).

[0184] In some embodiments, the methods include 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 that contains a human FcαR locus.

[0185] In some embodiments, the one or more pharmacokinetic parameters include, but are not limited to, area under plasma concentration versus time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), drug half-life (t1 / 2), and steady-state volume of distribution (Vss). Generally, the pharmacokinetic properties of an administered therapeutic agent are determined by administering a selected dose of the therapeutic agent (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / mg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg or more) and then determining how the plasma concentration of the therapeutic agent changes over time (e.g., at 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or more).

[0186] In some embodiments, the method further comprises measuring the therapeutic efficacy of the administered therapeutic protein (e.g., the ability of a dose of a therapeutic protein to reduce or eliminate one or more disease symptoms in an animal model). In some embodiments, the animal model is a cancer model, and the 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 the 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 the disease symptoms may include, for example, infectious agent levels, tissue damage, and / or animal survival.

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

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

[0189] In some embodiments, the method further comprises determining the extent to which administration of the therapeutic protein induces an anti-human Fcα immune response in the mouse. In some embodiments, the antibodies tested in the methods provided herein have a human light chain variable domain. In some embodiments, the light chain variable domain is a λ light chain variable domain. In some embodiments, the light chain variable domain is a κ light chain variable domain. In some embodiments, the antibody has a human light chain constant domain. In some embodiments, the light chain constant domain is a λ light chain constant domain. In some embodiments, the light chain constant domain is a κ light chain constant domain. Sequences of human light chain constant domains are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 and the IMGT database (available at www.imgt.org)).

[0190] In some embodiments, a Therapeutic Agent is administered to a mouse provided herein as part of a pharmaceutical composition, e.g., a pharmaceutical composition containing a human IgA antibody or an Fc fusion protein formulated together with a pharma- ceutical acceptable carrier.

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

[0192] Pharmaceutical compositions suitable for parenteral administration comprise human IgA antibodies or Fc fusion proteins in combination with one or more pharma- ceutically acceptable sterile, isotonic, aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately prior to use, and may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the preparation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0193] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions provided herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0194] In some embodiments, the composition comprises a human antibody or Fc fusion protein at a concentration that provides a suitable w / v for a desired dosage. 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, at least 90 mg / mL, at least 100 mg / mL, at least 120 mg / mL, at least 140 mg / mL, at least 160 mg / mL, at least 180 mg / mL, at least 190 mg / mL, at least 210 mg / mL, at least 220 mg / mL, at least 230 mg / mL, at least 240 mg / mL, at least 250 mg / mL, at least 260 mg / mL, at least 270 mg / mL, at least 280 mg / mL, at least 290 mg / mL, at least 300 mg / mL, at least 310 mg / mL, at least 320 mg / mL, at least 330 mg / mL, at least 340 mg / mL, at least 350 mg / mL, at least 350 mg / mL, at least 360 mg / mL, at least 370 mg / mL, at least 380 mg / mL, at least 390 mg / mL, at least 400 mg / mL, at least 400 mg / mL, at least 400 mg / mL, at least 400 mg / mL, at least 400 mg / The compound may be present in the composition at a concentration of 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, compositions are prepared in the form of a lyophilized composition or aqueous solution at a desired final concentration by mixing the human IgA antibody or Fc fusion protein with optional physiologically acceptable carriers, excipients, or stabilizers, such as, but not limited to, buffers, sugars, salts, surfactants, solubilizers, polyols, diluents, binders, stabilizers, salts, lipophilic solvents, amino acids, chelating agents, preservatives, and the like (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 nontoxic to recipients at the dosages and concentrations employed, and include buffers, such as histidine, phosphate, citrate, glycine, acetate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as Examples of suitable surfactants include serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including trehalose, glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN, polysorbate 80, PLURONICS®, or polyethylene glycol (PEG).

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

[0197] In some embodiments, the composition includes a buffer or pH adjuster to provide improved pH control. Such compositions may have a pH of about 3.0 to about 9.0, about 4.0 to about 8.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.5 to about 8.0, about 5.5 to about 7.0, or about 5.5 to about 6.5. In further embodiments, such compositions have a pH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In some embodiments, the pH of the composition is about 6.0. Those skilled in the art will appreciate 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, the buffer is a salt prepared from an organic or inorganic acid or base. Representative buffering agents include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris buffer, tromethamine hydrochloride buffer, or phosphate buffer. In addition, amino acid components may also function as buffering agents. Representative amino acid components that may be utilized in the composition as buffering agents include, but are not limited to, glycine and histidine. In some embodiments, the buffering agent is selected from histidine, citrate, phosphate, glycine, and acetate. In some embodiments, the buffering agent is histidine. In another specific embodiment, the buffering agent is citrate. In yet another embodiment, the buffering agent is glycine. The purity of the buffering agent should be at least 98%, or at least 99%, or at least 99.5%.As used herein, the term "purity" in the context of histidine and glycine refers to the chemical purity of histidine or glycine as understood in the art, e.g., as described in The Merck Index, 13th ed., O'Neil et al. ed. (Merck & Co., 2001).

[0198] In some embodiments, the composition comprises histidine as a buffering agent, hi some embodiments, the histidine is present in the composition at a concentration of at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 150 mM, or at least about 200 mM of histidine. In another embodiment, the composition comprises about 1 mM to about 200 mM, about 1 mM to about 150 mM, about 1 mM to about 100 mM, about 1 mM to about 75 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 10 mM to about 100 mM, about 10 mM to about 75 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 20 mM to about 75 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, or about 20 mM to about 30 mM histidine. In further embodiments, the composition comprises about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM, or about 200 mM histidine. In some embodiments, the composition comprises about 10 mM, about 25 mM histidine, or can be free of histidine.

[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 the like. The carbohydrate excipient is generally present in a weight or volume percentage of about 1% to about 99%, 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 20%, 5% to 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 other specific embodiments, the carbohydrate excipient is present at 1%, or 1.5%, or 2%, or 2.5%, or 3%, or 4%, or 5%, or 10%, or 15%, or 20%.

[0200] In some embodiments, the composition comprises a carbohydrate excipient. Suitable carbohydrate excipients for use in the composition include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols 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 should be at least 98%, or at least 99%, or at least 99.5%.

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

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

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

[0204] In some embodiments, the composition comprises a surfactant. The term "surfactant" as used herein refers to an organic substance with an amphiphilic structure, i.e., consisting of groups with opposite solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Depending on the charge of the surface-active moiety, surfactants can be classified into anionic, cationic, and nonionic surfactants. Surfactants are often used as wetting agents, emulsifiers, solubilizers, and dispersants in various pharmaceutical compositions and preparations of biological materials. Polysorbates (e.g., Polysorbate 20 or 80); poloxamers (e.g., Poloxamer 188); Triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, paImidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, paImidopropyl-, or isostearamidopropyl-dimethylamine; sodium cocoyl methyl taurate or disodium oleyl methyl taurate; and the MONAQUA® series (Mona Pharmaceutically acceptable surfactants such as polysorbate 20, polysorbate 40, polysorbate 60, and copolymers of ethylene and propylene glycol (e.g., PLURONICS® PF68) can optionally be 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 administer the composition. The presence of a pharma- ceutically acceptable surfactant mitigates the tendency of proteins to aggregate.The composition may include a polysorbate at a concentration ranging from about 0.001% to about 1%, or from about 0.001% to about 0.1%, or from about 0.01% to about 0.1%. In other specific embodiments, the composition includes a polysorbate at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or 0.015%, or 0.02%.

[0205] In some embodiments, the composition comprises other excipients and / or additives, including but not limited to diluents, binders, stabilizers, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutically acceptable excipients and / or additives can be used in the compositions provided herein. Commonly used excipients / additives, such as pharma-ceutically acceptable chelating agents (e.g., but not limited to, EDTA, DTPA, or EGTA), can be optionally added to the composition to reduce aggregation. These additives are particularly useful when pumps or plastic containers are used to administer the composition.

[0206] In some embodiments, the composition includes a preservative. Preservatives such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., but not limited to, hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, can be optionally added to the composition at any suitable concentration, such as about 0.001% to about 5%, or any range or value within said range. The concentration of the preservative used in the composition is a concentration sufficient to provide microbial action. Such concentrations will depend on the preservative selected and can be readily determined by one of ordinary skill in the art.

[0207] In some embodiments, the composition is isotonic with human blood, and the composition has essentially the same osmolarity as human blood. Such isotonic compositions generally have an osmolarity of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, by using a vapor pressure or freezing point osmometer. The tonicity of the composition is adjusted using a tonicity adjuster. A "tonicity adjuster" is a pharma- ceutically acceptable inert substance that can be added to a composition to render the composition isotonic. Suitable tonicity adjusters for the compositions provided herein include, but are not limited to, sugars, salts, and amino acids.

[0208] In some embodiments, the composition is a pyrogen-free composition that is substantially free of endotoxins and / or related pyrogens. Endotoxins include toxins trapped inside microorganisms that are released only when the microorganism decomposes or dies. Pyrogens also include heat-generating, heat-stable substances from the outer membrane of bacteria and other microorganisms. Any of these substances can cause fever, low blood pressure, and shock when administered to humans. Due to potential harmful effects, endotoxins must be removed from pharmaceutical solutions administered intravenously, even in small amounts. The United States Food and Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram of 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 proteins of interest (e.g., antibodies), harmful and dangerous endotoxins must be removed, even in trace amounts. In some embodiments, the levels of endotoxins 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 can be sterilized by various sterilization methods, including sterile filtration, radiation, etc. In some embodiments, the compositions are sterile filtered through a sterile 0.22 micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as described in "Remington: The Science & Practice of Pharmacy", 21st ed., Lippincott Williams & Wilkins, (2005). Compositions containing a protein of interest (e.g., an antibody), such as those disclosed herein, are usually stored in lyophilized form or in solution. It is contemplated that the sterile composition containing a protein of interest (e.g., an antibody) is placed in a container with a sterile access port, for example, an intravenous solution bag or vial with an adaptor that allows the composition to be removed, such as a stopper that can be pierced by a hypodermic needle. In some embodiments, the composition is provided as a prefilled syringe.

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

[0211] Regardless of the route of administration selected, the agents provided herein, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions provided herein, are formulated into pharma- ceutical acceptable dosage forms by conventional methods known to those of skill in the art.

[0212] In the methods provided herein, the human antibodies, Fc fusion proteins, and / or pharmaceutical compositions can be delivered by any suitable route of administration, including, for example, oral, nasal, e.g., by spray, rectal, vaginal, parenteral, intracisternal and topical, by powders, ointments or drops, buccal and sublingual administration. In some embodiments, the pharmaceutical compositions are administered systemically (e.g., via oral or parenteral administration).

[0213] In some embodiments, the actual dosage of the active ingredient in the pharmaceutical compositions described herein may be varied to determine the amount of active ingredient that is non-toxic in animal models and effective to achieve a desired therapeutic response in that animal model, composition, and method of administration.

[0214] For example, in certain embodiments, the mice described herein are used to determine the pharmacokinetic profile 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 each exposed to one or more human antibody candidates at various dosages (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg or more). The candidate therapeutic antibodies can be administered via any desired route of administration, including parenteral and non-parenteral routes of administration. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intrathecal, intrathecal, intraventricular, intracranial, intrathoracic or other injection routes. Non-parenteral routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, ocular. Administration may be by continuous infusion, topical administration, sustained release from implants (gels, membranes, 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 more). To determine the pharmacokinetic profile of the administered candidate therapeutic antibody or Fc fusion polypeptide, samples obtained from the mice described herein may be used to perform various assays, including, but not limited to, total IgA, anti-therapeutic antibody response, agglutination, etc.

[0215] In various embodiments, the mice described herein are used to measure the effect of a therapy that blocks or modulates the activity of a polypeptide of interest, and the effect on gene expression as a result of cellular changes, or, in the context of a receptor polypeptide, on the density of the receptor polypeptide on the cell surface of the mouse. In various embodiments, the mice described herein or cells isolated from the mice are exposed to a candidate therapeutic agent that binds to the polypeptide of interest, and after a period of time, are analyzed for the effect on a particular cellular process associated with the polypeptide of interest, such as ligand-receptor interaction or signal transduction.

[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 genetic modifications different from those described herein or without genetic modifications, i.e., wild-type mice) can be implanted with a tumor (or tumor cells) or infected with a virus (e.g., influenza, HIV, HCV, HPV, etc.). After implantation or infection, the mice can be administered a candidate therapeutic agent. The tumor or virus may be allowed sufficient time to become established in one or more locations within the mouse prior to administration of the candidate therapeutic agent. Alternatively and / or additionally, immune responses in such mice can be monitored for characterization and selection of potential human antibodies that can be developed as therapeutic agents.

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

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

[0219] kit Provided herein is a pack or kit comprising one or more containers filled with at least one mouse, mouse cell, DNA fragment, and / or targeting vector as described in the description, examples, and / or figures herein. The kit may be used in any applicable method (e.g., research method). Such container(s) may optionally be accompanied by a document in a format prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which document (a) the agency's approval for manufacture, use, or sale for human administration, (b) instructions for use, and (c) the agreement 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 present specification provides a mouse comprising in its genome an Fc alpha receptor (FcαR) locus located in the mouse leukocyte receptor complex (LRC), wherein the FcαR locus comprises a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain.

[0221] According to exemplary embodiment 2, there is provided herein a mouse of embodiment 1, wherein said FcαR locus is located in the intergenic region between the locus of Tthy1 protein and the locus of Rdh13 protein.

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

[0223] According to exemplary embodiment 4, the present specification provides a mouse of any one of embodiments 1 to 3, wherein the FcαR locus is located in an intergenic 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, there is provided herein the mouse of embodiment 4, wherein the intergenic region is a 54 kb region between the Pira6 locus and the Ncr1 locus.

[0225] According to exemplary embodiment 6, the present specification 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 exemplary embodiment 7, there is provided herein a mouse of any one of embodiments 1 to 6, wherein said FcαR locus comprises a nucleic acid sequence encoding a human FcαR polypeptide.

[0227] According to exemplary embodiment 8, there is provided herein a mouse of any one of embodiments 1-7, wherein said FcαR locus comprises human exons 1-5 of the human Fc alpha receptor gene.

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

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

[0230] According to exemplary embodiment 11a, provided herein is a mouse of embodiment 7 or 8, wherein said FcαR locus further comprises a nucleic acid sequence present in the human KIR3DL2 gene. According to exemplary embodiment 11b, provided herein is a mouse of embodiment 7, 8, or 11a, wherein said 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 present specification provides a mouse of 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, provided herein is the mouse of any one of embodiments 1 to 12, wherein the mouse is heterozygous for the FcαR locus.

[0233] According to exemplary embodiment 14, there is provided herein the mouse of any one of embodiments 1 to 12, wherein the mouse is homozygous for the FcαR locus. According to exemplary embodiment 15, there is provided herein a mouse of any one of embodiments 1 to 14, 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.

[0234] According to exemplary embodiment 16, there is provided herein a mouse of embodiment 15, wherein the mouse comprises in its genome a human or humanized FcγR locus comprising a nucleic acid sequence encoding a human or humanized FcγR.

[0235] According to exemplary embodiment 17, there is provided herein a mouse of embodiment 16, wherein the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more low affinity FcγRs 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, there is provided herein a mouse of embodiment 16, wherein the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more FcγRs 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, provided herein is a mouse of any one of embodiments 16-18, wherein the human or humanized FcγR comprises a human extracellular domain. According to exemplary embodiment 20, provided herein is a mouse of any one of embodiments 16-19, wherein the human or humanized FcγR comprises a mouse transmembrane domain.

[0238] According to exemplary embodiment 21, provided herein is a mouse of any one of embodiments 16-19, wherein the human or humanized FcγR comprises a human transmembrane domain. According to exemplary embodiment 22, provided herein is a mouse of any one of embodiments 16-21, wherein the human or humanized FcγR comprises a mouse cytoplasmic domain.

[0239] According to exemplary embodiment 23, provided herein is a mouse of any one of embodiments 16-21, wherein the human or humanized FcγR comprises a human cytoplasmic domain. According to exemplary embodiment 24, provided herein is the mouse of any one of embodiments 16-23, wherein said human or humanized FcγR locus is located at an endogenous mouse FcγR locus.

[0240] According to exemplary embodiment 25, provided herein is the mouse of embodiment 24, wherein said nucleic acid sequence encoding said human or humanized FcγR replaces all or part of an endogenous mouse FcγR gene.

[0241] According to exemplary embodiment 26, there is provided herein a mouse of embodiment 25, wherein the nucleic acid sequence encoding the 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.

[0242] According to exemplary embodiment 27, provided herein is the mouse of any one of embodiments 15 to 26, wherein the mouse does not express mouse FcγR. According to exemplary embodiment 28, provided herein is a mouse of 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, provided herein is a mouse of 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 exemplary embodiment 30, provided herein is a mouse embryonic stem cell (ES cell) comprising in its genome an Fc alpha receptor (FcαR) locus located in the leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR locus comprises 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, there is provided herein a mouse ES cell of embodiment 30, wherein the FcαR locus is located in the intergenic region between the locus of Tthy1 protein and the locus of Rdh13 protein.

[0246] According to exemplary embodiment 32, the present specification provides a mouse ES cell of embodiment 31 or 32, wherein the FcαR locus is located in the intergenic region between the Lilra5 protein locus and the Gp6 protein locus.

[0247] According to exemplary embodiment 33, the present specification provides a mouse ES cell of any one of embodiments 30 to 32, wherein the Fc alpha receptor (FcαR) locus is located in an intergenic 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.

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

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

[0250] According to exemplary embodiment 36, there is provided herein a mouse ES cell of 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, there is provided herein a mouse ES cell of any one of embodiments 30-36, wherein said FcαR locus comprises human exons 1-5 of the human Fc alpha receptor gene.

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

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

[0254] According to exemplary embodiment 40a, provided herein is a mouse ES cell of embodiment 36 or 37, wherein said FcαR locus further comprises a nucleic acid sequence present in the human KIR3DL2 gene. According to exemplary embodiment 40b, provided herein is a mouse ES cell of embodiment 36, 37, or 40a, wherein said 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 present specification provides the mouse ES cell of any one of embodiments 30 to 40, wherein the ES cell is heterozygous for the FcαR locus.

[0256] According to exemplary embodiment 42, the present specification provides the mouse ES cell of any one of embodiments 30 to 40, wherein the ES cell is homozygous for the FcαR locus.

[0257] According to exemplary embodiment 43, provided herein is a mouse ES cell of any one of embodiments 30 to 42, 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.

[0258] According to exemplary embodiment 44, there is provided herein a mouse ES cell of embodiment 43, wherein the mouse ES cell comprises, in its genome, a human or humanized FcγR locus comprising a nucleic acid sequence encoding a human or humanized FcγR.

[0259] According to exemplary embodiment 45, there is provided herein a mouse ES cell of embodiment 44, wherein the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more low affinity FcγRs 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 exemplary embodiment 46, there is provided herein a mouse ES cell of embodiment 44, wherein the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more FcγRs 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, provided herein is a mouse ES cell of any one of embodiments 44 to 46, wherein the human or humanized FcγR comprises a human extracellular domain.

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

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

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

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

[0266] According to exemplary embodiment 52, provided herein is a mouse ES cell of any one of embodiments 44 to 51, wherein the human or humanized FcγR locus is located at an endogenous mouse FcγR locus.

[0267] According to exemplary embodiment 53, there is provided herein a mouse ES cell of embodiment 52, wherein said nucleic acid sequence encoding said human or humanized FcγR replaces all or part of an endogenous mouse FcγR gene.

[0268] According to exemplary embodiment 54, provided herein is a mouse ES cell of embodiment 53, wherein the nucleic acid sequence encoding the 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.

[0269] According to exemplary embodiment 55, there is provided herein a mouse ES cell of 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, there is provided herein a mouse ES cell of 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, the present 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 of any one of embodiments 1 to 29.

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

[0273] According to exemplary embodiment 59, there is provided herein the method of embodiment 58, wherein the one or more pharmacokinetic properties are selected from one or more of area under plasma concentration versus 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).

[0274] According to exemplary embodiment 60, the present specification provides a method 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, provided herein is a method of any one 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, provided herein is a method of any one of embodiments 57 to 61, further comprising 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.

[0277] According to exemplary embodiment 63, provided herein is any 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 tolerability of each dose of the human antibody or Fcα fusion polypeptide.

[0278] According to exemplary embodiment 64, there is provided herein a method of 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, there is provided herein the method of embodiment 64, wherein said one or more Fc receptor-mediated responses are antibody-dependent cell-mediated cytotoxicity (ADCC) responses.

[0280] According to exemplary embodiment 66, provided herein is a method of any one of embodiments 57 to 65, wherein the human antibody binds to 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 increased target cell killing indicates an increased ability of the agent to mediate ADCC.

[0281] According to exemplary embodiment 67, the present specification provides a method of any one of embodiments 57 to 66, further comprising measuring the immune response generated by the mouse against the human antibody.

[0282] According to exemplary embodiment 68, there is provided herein a method for modifying a mouse genome, comprising modifying the mouse genome by 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 comprising a human extracellular domain and a human or rodent cytoplasmic domain.

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

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

[0285] According to exemplary embodiment 71, the present specification provides a method of any one of embodiments 68 to 70, wherein the FcαR locus is located in an intergenic region between the Lilra5 protein locus and the Gp6 protein locus.

[0286] According to exemplary embodiment 72, the present specification provides a method of any one of embodiments 68 to 71, wherein the FcαR locus is located in an intergenic 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, there is provided herein the method of embodiment 72, wherein the intergenic region is the 54 kb region between the Pira6 locus and the Ncr1 locus.

[0288] According to exemplary embodiment 74, the present specification provides a method of any one of embodiments 68 to 73, wherein the FcαR locus is between coordinates 4,303,905 and 4,312,280 (+ strand, GRCm38 assembly) on mouse chromosome 7.

[0289] According to exemplary embodiment 75, the present specification provides a method for generating a mouse comprising an Fc alpha receptor (FcαR) locus, the method comprising generating a mouse ES cell of any one of embodiments 30 to 56, and generating a mouse from the ES cell. EXAMPLES

[0290] Example 1.1: Genetic Engineering of Mice Containing Human or Humanized FcαR A targeting vector was generated that contained a nucleic acid sequence encoding a human FcαR (MAID20277 and MAID20278) cassette. MAID20277 and MAID20278 were identical except that MAID20277 carried a selection marker (i.e., neomycin resistance). Briefly, the human FCAR gene was introduced into the mouse genome using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) Nat. Biotech. 21(6):652-659, the contents of both of which are incorporated herein by reference in their entirety). A 44 kb genomic sequence of human FCAR, including the promoter, was inserted into the mouse LRC locus, located between the mouse Pira6 and Ncr1 genes on mouse chromosome 7, between coordinates chr7:4,303,905 to 4,312,280 (+ strand, GRCm38 assembly) (Figures 1A and 1B).

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

[0292] [Table 1]

[0293] The mouse homology arms were assembled 5' to 3' into a construct containing a 5' arm, a spectinomycin resistance cassette flanked by I-CeuI and PI-SceI homing endonuclease sites, a 3' arm, and a chloramphenicol resistance cassette (construct A). In a subsequent genetic engineering step, the spectinomycin resistance cassette of construct A was replaced by an insert containing human FCAR.

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

[0295] To generate 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 contains, from 5' to 3', a 5' mouse homology arm, an I-CeuI site, a 44,887 bp human genomic 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, and the final clone was selected based on CM / kanamycin resistance (Figure 2). The various junctions of the final clone are shown in Table 2.

[0296] [Table 2]

[0297] The MAID20277 targeting 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 US Pat. No. 8,658,154 and is shown diagrammatically in FIG. 3. Targeted homologous recombination resulted in the deletion of approximately 8.4 kb of mouse sequence (coordinates chr7:4,303,905-4,312,280 in GRCm38) and the insertion of approximately 44 kb of human sequence containing the FCAR gene. Successful integration was confirmed by modification of allele (MOA) assay, as described, for example, in Valenzuela et al., supra. The primers (forward (F) primer and reverse (R) primer) and probes used in the MOA assay to detect the presence of human FCAR sequence and loss of mouse LRC sequence are shown in Table 3 below and their positions are shown in FIG. 1A. The antibody resistance cassette was subsequently removed by transient expression of CRE recombinase in ES cell clones (Fig. 1B).

[0298] [Table 3]

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

[0300] Alternatively to the above strategy, the targeting 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 to mice containing human low affinity FcγR (having the locus described in Figure 3). Mice containing human FcαR (and optionally human low affinity FcγR) can also be bred to mice containing human or humanized high affinity FcγR, FcεR, FcRn, β2M, and humanized Ig heavy and / or light chain loci using techniques well known in the art.

[0301] Example 1.2: Phenotypic analysis of genetically engineered mice containing human or humanized FcαR Mice containing the human FCAR gene and the low affinity FCGR gene as described in Example 1.1 above were analyzed for FcαR expression. All mice were kept and bred under specific pathogen-free conditions. Mice were sacrificed and spleens and blood were harvested. Blood was collected in BD microtainer tubes containing EDTA (cat. no. 365973). Red blood cells from spleens and blood preparations were lysed with ACK lysis buffer (ThermoFisher, cat. no. A1049201) followed by washing with complete RPMI medium.

[0302] Both spleen and blood cells were incubated with Live Dead Aqua (ThermoFisher) to exclude nonviable cells. Prior to staining, cells were incubated with anti-mouse CD16 / 32 (2.4G2, BD) for 10 min on ice. Cells were then stained for 30 min on ice with fluorochrome-conjugated anti-human CD89 (BioLegend, clone no. A59) and the following anti-mouse antibodies: CD45 (BioLegend, clone no. 30-F11), B220 (BioLegend, clone no. RA3-6B2), CD11c (BioLegend, clone no. N418), Ly6G (BioLegend, clone no. 1A8), Ly6C (BioLegend, clone no. HK1.4), SiglecF (BD Biosciences clone no. E50-2440), NKp46 (BioLegend, clone no. 29A1.4), CD11b (BioLegend, clone no. M1 / 70), F4 / 80 (BD Biosciences clone no. T45-2342), and NK1.1 (BioLegend, clone no. PK136).

[0303] Lymphoid and myeloid cell populations were identified by flow cytometry on a BD Symphony A3 instrument (BD Biosciences). First, viable single cells were gated on CD45+. The following subsets were then identified: B cells (B220+), cDCs (CD11c++), pDCs (CD11cint, Ly6C+ B220+), PMN Ly6G+ Ly6C+, eosinophils (Ly6G-, SiglecF+CD11b+), macrophages / monocytes (Ly6G-, SiglecF-, F4 / 80+ CD11b+), and NK cells (Ly6G-, SiglecF-, F4 / 80-CD11b-, NK1.1+). Expression of CD89 (FcαR) was measured for each cell type.

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

[0305] Incorporation by Reference All publications, patents, and patent applications mentioned herein are herein incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application will control, including any definitions herein.

[0306] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

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

2. The FcαR locus is (a) the intergenic region between the Tthy1 protein locus and the Rdh13 protein locus; (b) the intergenic region between the Lilra5 protein locus and the Gp6 protein locus; (c) the intergenic region between the Pira6 protein locus and the Ncr1 protein locus, or (d) Between coordinates 4,303,905 and 4,312,280 on mouse chromosome 7 (positive strand, GRCm38 assembly) The mouse of claim 1 , wherein the

3. A mouse described in claim 1 or 2, wherein the FcαR polypeptide includes a human or rat transmembrane domain.

4. The mouse of any one of claims 1 to 3, wherein the nucleic acid sequence encodes a fully human FcαR polypeptide.

5. The mouse of any one of claims 1 to 4, wherein the FcαR locus comprises human exons 1 to 5 of the human Fc alpha receptor gene.

6. The mouse of any one of claims 1 to 3, wherein the FcαR locus comprises a non-coding portion of rat FcαR exon 1, human FcαR exons 1 and 2, human FcαR exons 3 and 4, and a coding portion of rat FcαR exon 5.

7. The mouse of claim 4 or 5, wherein the human or humanized FcαR receptor locus comprises the genomic sequence found between coordinates 54,862,297 and 54,906,185 on human chromosome 19 (+ strand, GRCh38 assembly).

8. The mouse of claim 4 or 5, wherein the FcαR 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.

9. The mouse of any one of claims 1 to 8, wherein the mouse expresses the FcαR polypeptide on mouse neutrophils, monocytes, macrophages, eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells).

10. The mouse of any one of claims 1 to 9, wherein the mouse is homozygous for the FcαR locus.

11. 11. The mouse of any one of claims 1 to 10, 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.

12. A mouse embryonic stem cell (ES cell) comprising in its genome an Fc alpha receptor (FcαR) locus located in the leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR locus comprises a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain.

13. The FcαR locus is (a) the intergenic region between the Tthy1 protein locus and the Rdh13 protein locus; (b) the intergenic region between the Lilra5 protein locus and the Gp6 protein locus; (c) the intergenic region between the Pira6 protein locus and the Ncr1 protein locus, or (d) Between coordinates 4,303,905 and 4,312,280 on mouse chromosome 7 (positive strand, GRCm38 assembly) The mouse ES cell of claim 12, located in

14. A mouse ES cell described in claim 12 or 13, wherein the FcαR polypeptide includes a human or rat transmembrane domain.

15. The mouse ES cell of any one of claims 12 to 14, wherein the FcαR locus comprises a nucleic acid sequence encoding a fully human FcαR polypeptide.

16. The mouse ES cell of any one of claims 12 to 15, wherein the FcαR locus comprises human exons 1 to 5 of the human Fc alpha receptor gene.

17. The mouse ES cell of any one of claims 12 to 16, wherein the FcαR locus comprises a non-coding portion of rat FcαR exon 1, human FcαR exons 1 and 2, human FcαR exons 3 and 4, and a coding portion of rat FcαR exon 5.

18. The mouse ES cell of claim 15 or 16, wherein the human or humanized FcαR receptor locus comprises the genomic sequence found between coordinates 54,862,297 and 54,906,185 on human chromosome 19 (+ strand, GRCh38 assembly).

19. The mouse ES cell of claim 15 or 16, wherein the FcαR 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.

20. The mouse ES cell according to any one of claims 12 to 19, wherein the ES cell is homozygous for the FcαR locus.

21. 21. The mouse ES cell of any one of claims 12 to 20, 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.

22. A method for testing a human IgA antibody or an Fcα fusion polypeptide, comprising administering said human IgA antibody or Fcα fusion polypeptide to a mouse according to any one of claims 1 to 11.

23. 23. The method of claim 22, further comprising measuring one or more pharmacokinetic properties of the administered human IgA antibody or Fcα fusion polypeptide, optionally wherein the one or more pharmacokinetic properties are selected from one or more of: area under plasma concentration versus 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).

24. The method of claim 22 or 23, further comprising measuring the therapeutic efficacy of the administered human IgA antibody or Fcα fusion polypeptide.

25. The method of claim 22 or 23, further comprising administering multiple doses of the human IgA antibody or Fcα fusion polypeptide and determining the therapeutic efficacy, safety, and / or tolerability of each dose of the human IgA antibody or Fcα fusion polypeptide.

26. 24. The method of claim 22 or 23, further comprising measuring one or more Fc receptor mediated responses in the mouse, optionally wherein the one or more Fc receptor mediated responses are antibody-dependent cell-mediated cytotoxicity (ADCC) responses.

27. ​​The method of claim 22 or 23, further comprising measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against target cells and comparing the amount of ADCC to a control, wherein the human IgA antibody binds to the target cells in the mouse, and an increase in target cell killing indicates an increased ability of the agent to mediate ADCC.

28. 24. The method of claim 22 or 23, further comprising measuring the immune response generated by the mouse against the human IgA antibodies.

29. 1. A method for modifying a mouse genome, comprising: The method comprises modifying the mouse genome by 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 comprising a human extracellular domain and a human or rodent cytoplasmic domain.

30. The method described in claim 29, wherein the FcαR polypeptide includes a human or rat transmembrane domain.

31. The method of claim 29 or 30, wherein the FcαR locus comprises a nucleic acid sequence encoding a fully human FcαR polypeptide.

32. The method of any one of claims 29 to 31, wherein the FcαR locus comprises human exons 1 to 5 of the human Fc alpha receptor gene.

33. The method of any one of claims 29 to 32, wherein the FcαR locus includes the non-coding portion of rat FcαR exon 1, human FcαR exons 1 and 2, human FcαR exons 3 and 4, and the coding portion of rat FcαR exon 5.

34. The FcαR locus is (a) the intergenic region between the Tthy1 protein locus and the Rdh13 protein locus; (b) the intergenic region between the Lilra5 protein locus and the Gp6 protein locus; (c) the intergenic region between the Pira6 protein locus and the Ncr1 protein locus, or (d) Between coordinates 4,303,905 and 4,312,280 on mouse chromosome 7 (positive strand, GRCm38 assembly) The method according to any one of claims 29 to 33, wherein the

35. 1. A method of producing a mouse comprising an Fc alpha receptor (FcαR) locus, comprising: Producing a mouse ES cell according to any one of claims 12 to 21; Producing a mouse from the ES cells. The method comprising:

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