Mice expressing humanized fealpha receptors

Abstract

This document provides methods and compositions relating to mice that express human or humanized Fcα receptors (FcαR) from the FcαR locus located in the mouse leukocyte receptor complex (LRC). In some embodiments, such mice can be used for in vivo testing of therapeutic agents containing human IgA Fc (e.g., testing of the pharmacokinetic and / or pharmacodynamic properties and dosing regimens of such therapeutic agents). This document also provides methods for using such mice, cells derived from such mice, methods for preparing such mice, and ES cells containing the same genetic modifications as such mice.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 126,326, filed December 16, 2020, which is incorporated herein by reference in its entirety. Background Technology

[0003] Mice are a fundamental in vivo model for preclinical testing of therapeutics due to their small size, well-defined physiological characteristics, and relatively low maintenance costs compared to larger mammalian models such as primates. Mouse models are commonly used to assess the toxicity and pharmacokinetics of therapeutics before initiating human clinical studies. Despite these advantages, in vivo testing of antibodies and Fc fusion proteins has significant drawbacks because these therapeutics behave differently in mice than in humans. This is particularly true for testing IgA-based therapies in mice, as they do not express the Fc receptors required for binding IgA antibodies. Consequently, mouse models have not been widely characterized or used to predict the safety, efficacy, and optimal dosage of IgA-based therapeutics. Novel mouse models and methods are needed that allow for precise preclinical testing of IgA-based therapeutics that can predict their properties in human patients. Summary of the Invention

[0004] This document provides methods and compositions relating to mice expressing human or humanized Fcα receptors (FcαR), and methods and compositions relating to in vivo testing of therapeutic agents containing human IgA Fc in such mice (e.g., testing the pharmacokinetic and / or pharmacodynamic properties of such therapeutic agents and dosing regimens). As described herein, mice are convenient animal models for testing therapeutic antibodies and Fc fusion proteins due to their small size, well-defined physiological characteristics, and ease of genetic modification. Unfortunately, because wild-type mice lack FcαR, drugs containing the human IgA Fc region (such as IgA antibodies and Fcα fusion proteins) typically exhibit very different pharmacokinetic and pharmacodynamic properties when administered to such mice compared to when administered to humans. Therefore, the mice provided herein can be used as in vivo systems for developing, screening, and testing human IgA antibodies and Fcα fusion proteins for therapeutic purposes.

[0005] In some aspects, this document provides a mouse whose genome includes an Fcα receptor (FcαR) locus located in the mouse leukocyte receptor complex (LRC), wherein the FcαR locus (such as a human or humanized FcαR locus) contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain. In some embodiments, the mouse expresses the FcαR polypeptide on mouse neutrophils, monocytes, macrophages, eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells). In some embodiments, the neutrophils, monocytes, macrophages, eosinophils, and / or dendritic cells (e.g., plasmacytoid dendritic cells) are derived from mouse blood. In some embodiments, the neutrophils, monocytes, macrophages, eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells) are derived from mouse spleen.

[0006] In some respects, this article provides a mouse embryonic stem cell (ES cell) whose genome contains an Fcα receptor (FcαR) locus in the leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain.

[0007] In some embodiments, the FcαR locus is located in the intergenic region between the Tthy1 and Rdh13 protein loci. In some embodiments, the FcαR locus is located in the intergenic region between the Lilara5 and Gp6 protein loci. In some embodiments, the FcαR locus is located in the intergenic region between the Pira6 and Gp6 protein loci. In some embodiments, the FcαR locus is located in the intergenic region between the Pira6 and Ncr1 protein loci. In some embodiments, the FcαR locus is located in the intergenic region between the coding nucleic acid sequences of the Pira6 and Ncr1 proteins. In some embodiments, the intergenic region is a 54kb region between the Pira6 and Ncr1 loci. In some embodiments, the FcαR locus is located on mouse chromosome 7 (+ strand, GRCm38 assembly) at coordinates 4,303,905–4,312,280.

[0008] In some embodiments, the FcαR locus contains a nucleic acid sequence encoding the human FcαR polypeptide. In some embodiments, the FcαR locus contains human exons 1-5 of the human Fcα receptor gene. In some embodiments, the FcαR locus contains 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 contains a genomic sequence located between coordinates 54,862,297 and 54,906,185 on human chromosome 19 (+ strand, GRCh38 assembly). In some embodiments, the FcαR locus also contains a nucleic acid sequence present in the human KIR3DL2 gene. In some embodiments, the locus also contains a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

[0009] In some embodiments, the mouse or mouse ES cells are heterozygous for the FcαR locus. In some embodiments, the mouse or mouse ES cells are homozygous for the FcαR locus.

[0010] In some embodiments, the mouse or mouse ES cells described in any of the foregoing aspects or embodiments further contain in their genome a human or humanized Fcγ 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 cells are heterozygous for the human or humanized FcγR locus, the human or humanized IgH locus, the human or humanized Igκ locus, the human or humanized Igλ locus, the human or humanized FcRn locus, the human or humanized β2M locus, and / or the human or humanized FcεR1α locus. In some implementations, the mouse or mouse ES cells are homozygous for the human or humanized FcγR locus, the human or humanized IgH locus, the human or humanized Igκ locus, the human or humanized Igλ locus, the human or humanized FcRn locus, the human or humanized β2M locus, and / or the human or humanized FcεR1α locus.

[0011] In some embodiments, mouse or mouse ES cells comprising the FcαR locus described in the various embodiments herein contain a human or humanized FcγR locus in their genome, the locus containing a nucleic acid sequence encoding a human or humanized FcγR. In some embodiments, the human or humanized FcγR locus contains a nucleic acid sequence encoding one or more low-affinity FcγRs selected from Fcγ receptor 2a (FcγR2a), Fcγ receptor 2b (FcγR2b), Fcγ receptor 3a (FcγR3a), Fcγ receptor 3b (FcγR3b), and / or Fcγ 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 Fcγ receptor 1α (FcγR1a), Fcγ receptor 2a (FcγR2a), Fcγ receptor 2b (FcγR2b), Fcγ receptor 3a (FcγR3a), Fcγ receptor 3b (FcγR3b), and / or Fcγ receptor 2c (FcγR2c). In some embodiments, the human or humanized FcγR comprises a human extracellular domain. In some embodiments, the human or humanized FcγR comprises a mouse transmembrane domain. In some embodiments, the human or humanized FcγR comprises a human transmembrane domain. In some embodiments, the human or humanized FcγR comprises a mouse cytoplasmic domain. In some embodiments, the human or humanized FcγR comprises a human cytoplasmic domain. In some embodiments, the human or humanized FcγR locus is located at the endogenous mouse FcγR locus. In some embodiments, the nucleic acid sequence encoding human or humanized FcγR replaces all or part of the endogenous mouse FcγR gene. In some embodiments, the nucleic acid sequence encoding human or humanized FcγR includes a nucleic acid sequence encoding the extracellular domain of human FcγR, which replaces the endogenous nucleic acid sequence encoding the extracellular domain of mouse FcγR. In some embodiments, the mice described in any of the above embodiments do not express mouse FcγR.

[0012] In some respects, this document provides a method for testing human IgA antibodies or Fcα fusion peptides, the method comprising administering an IgA antibody or Fcα fusion peptide to mice as described in any of the above embodiments.

[0013] In some embodiments, the method further includes measuring one or more pharmacokinetic properties of the administered human IgA antibody or Fcα fusion peptide. In some embodiments, the one or more pharmacokinetic properties are selected from one or more of the following: area under the plasma concentration-time curve (AUC), in vivo recovery rate (IVR), clearance rate (CL), mean residence time (MRT), drug half-life (t1 / 2), and / or steady-state volume of distribution (Vss). In some embodiments, the method further includes measuring the therapeutic efficacy of the administered human antibody or Fcα fusion peptide. In some embodiments, the method further includes administering multiple doses of the human antibody or Fcα fusion peptide and determining the therapeutic efficacy of each dose. In some embodiments, the method further includes administering multiple doses of the human antibody or Fcα fusion peptide and determining the safety of each dose. In some embodiments, the method further includes administering multiple doses of the human antibody or Fcα fusion peptide and determining the tolerability of each dose. In some embodiments, the method further includes measuring one or more Fc receptor-mediated responses in mice. In some embodiments, the response mediated by one or more Fc receptors is an antibody-dependent cell-mediated cytotoxicity (ADCC) response. In some embodiments, the human antibody binds to target cells in a mouse, and the method further includes 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. In some embodiments, the method further includes measuring the immune response in mice against the human antibody.

[0014] In some respects, this article provides a method for preparing mice containing the Fcα receptor (FcαR) locus, the method comprising: generating mouse ES cells containing the Fcα receptor (FcαR) locus in their genome, the locus being located in the leukocyte receptor complex (LRC) of the mouse genome provided herein; and generating mice from said ES cells.

[0015] In some aspects, this document provides a method for modifying the mouse genome, the method comprising: inserting an Fcα receptor (FcαR) locus into the leukocyte receptor complex (LRC) of the mouse genome, thereby modifying the mouse genome, wherein the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain. In some embodiments, the FcαR locus contains a nucleic acid sequence encoding a human or humanized FcαR polypeptide. In some embodiments, the FcαR locus is located in an intergenic region between the gene loci of the Tthy1 protein and the Rdh13 protein. In some embodiments, the FcαR locus is located in an intergenic region between the gene loci of the Lilara5 protein and the Gp6 protein. In some embodiments, the FcαR locus is located in an intergenic region between the gene loci of the Pira6 protein and the Gp6 protein. In some embodiments, the FcαR locus is located in an intergenic region between the gene loci of the Pira6 protein and the Ncr1 protein. In some implementations, the intergenic region is a 54kb region between the Pira6 and Ncr1 loci. In some implementations, the FcαR locus is located on mouse chromosome 7 (+ strand, GRCm38 assembly) at coordinates 4,303,905–4,312,280. Attached Figure Description

[0016] Unless otherwise specified, human gene sequences in the diagram are represented by hollow boxes, mouse gene sequences by solid boxes, mouse gene sequences by single lines, human gene sequences by double lines, and selection boxes by hollow boxes containing text (e.g., Lox, CM).

[0017] Figure 1A This is a diagram (not to scale) illustrating the insertion of the MAID20277 box into the mouse genome to generate heterozygous mice that can breed homozygous. “LRC” stands for leukocyte receptor complex, and “EP” stands for electroporation. The names and locations of the primers and probes used in the allele modification (MOA) assay (described in the examples) are indicated by short lines above the diagram.

[0018] Figure 1B This is a graph (not to scale) showing heterozygous ES cells resulting from the insertion of the MAID20277 cassette into the mouse genome using CRE recombinase, which causes the loss of a selective marker, thus producing MAID20278 cassette heterozygous ES cells. The names and locations of the primers and probes used in the allele modification (MOA) assay (described in the examples) are indicated by short lines above the graph.

[0019] Figure 2This is a schematic diagram (not to scale) of a targeting vector containing a sequence homologous to the mouse genome (“5' mouse homologous arm”), the I-CeuI site, a 44,887 bp human genome insertion segment including the FCAR gene, the lox2372-Ub-Neo-lox2372 box, the PI-SceI site, a second sequence homologous to the mouse genome, and a chloramphenicol (denoted as “CM”) resistance box.

[0020] Figure 3 This is a diagram (not to scale) of the humanized low-affinity FcγR locus on mouse chromosome 1 containing the human FcγR2B, FcγR3B, FcγR2C, FcγR3A, and FcγR2A genes. The detailed steps of humanizing this locus are described in U.S. Patent No. 8,658,154, which is incorporated herein by reference.

[0021] Figure 4A This includes flow cytometry images of intracellular FcαR expression in blood samples obtained from mice genetically engineered with MAID20278 kits.

[0022] Figure 4B This includes flow cytometry images of FcαR expression in spleen cells obtained from mice genetically engineered with MAID20278 kits. Detailed Implementation

[0023] General Principles

[0024] This document provides methods and compositions relating to mice expressing human, humanized, or partially humanized Fcα receptors (FcαR), and methods and compositions relating to in vivo testing of therapeutics containing human IgA Fc in such mice (e.g., testing the pharmacokinetic and / or pharmacodynamic properties and dosing regimens of such therapeutics). Specifically, the various embodiments described herein relate to mice, ES cells, and methods comprising genes encoding and / or expressing fully human or partially human FcαR1 / CD89 genes / receptors (referred to herein as “FcαR”). The genetically modified mice contain a nucleic acid sequence encoding a human or humanized Fcα receptor (FcαR) protein in their genome. In some embodiments, the sequence encoding the human or humanized Fcα receptor (FcαR) protein is located in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, the nucleic acid sequence encoding FcαR is located in an intergenetic region between gene loci of Tthy1 and Rdh13 proteins, Lilara5 and Gp6 peptides, and / or Pira6 and Ncr1 proteins (e.g., between the nucleic acid sequences encoding Pira6 and Ncr1 proteins). In some embodiments, the intergenetic region is a 54 kb region between the Pira6 and Ncr1 gene loci. In some embodiments, the mice provided herein express human FcαR from a locus located at coordinates chr7:4,303,905–4,312,280 in the mouse genome (+ strand, GRCm38 assembly). In some embodiments, FcαR contains a human extracellular domain. The transmembrane and / or cytoplasmic domains of such receptors can be human or non-human (e.g., rat).

[0025] Therapeutic agents containing human IgA Fc, such as therapeutic human antibodies and human Fcα fusion proteins, are typically tested in non-human species before being administered to humans. While such agents are frequently tested in non-human primates or other relatively large mammals, this testing is expensive and places a significant financial burden on drug developers. Furthermore, non-human primates and other relatively large mammals are generally not cooperative with genetic modification, which limits the availability of disease models in such organisms.

[0026] In contrast, mice are a convenient animal model for testing therapeutic antibodies and Fc fusion proteins due to their small size, well-defined physiological characteristics, and ease of genetic modification. Unfortunately, because wild-type mice lack FcαR, drugs containing the human IgA Fc region typically exhibit very different pharmacokinetic and pharmacodynamic properties when administered to existing-technology mice compared to when administered to humans. Therefore, the mice described herein can be used as an in vivo system for developing, screening, and testing human IgA antibodies and Fcα fusion proteins for therapeutic purposes.

[0027] The location of the transgene in the mouse genome has a significant impact on its expression. For example, mice containing the human FcαR transgene have been reported to express FcαR on neutrophils, but only on a subset of monocytes. For instance, macrophages and non-myeloid cells (such as lymphocytes, endothelial cells, and hepatocytes) isolated from the peritoneum show no FcαR expression in such mice. (van Egmond et al., Blood 93(12):4387-4394(1999). In contrast, in humans, FcαR is typically expressed on neutrophils, monocytes, macrophages (e.g., Kupffer cells), eosinophils, and dendritic cells. Therefore, in some embodiments, the mice provided herein express human FcαR from a location in the mouse genome corresponding to the human endogenous FcαR locus or from a location in the genome corresponding to a locus located near the human endogenous FcαR locus in the human genome. Specifically, in some embodiments, the sequence encoding the human or humanized Fcα receptor (FcαR) protein is located in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, the mice provided herein express human FcαR from a locus in an intergenic region located between gene loci of Tthy1 and Rdh13 proteins, Lilara5 and Gp6 peptides, and / or Pira6 and Ncr1 proteins (e.g., between the coding nucleic acid sequences of Pira6 and Ncr1 proteins). In some embodiments, the intergenic region is a 54 kb region between the Pira6 and Ncr1 loci. In some embodiments, the mice provided herein express human FcαR from a locus located at coordinates chr7: 4,303,905–4,312,280 in the mouse genome (+ strand, GRCm38 assembly). In some embodiments provided herein, the mice described herein express FcαR on the cell surfaces of their neutrophils, monocytes / macrophages (e.g., blood monocytes / macrophages), eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells).

[0028] In various embodiments of the mice, ES cells, and methods provided herein, the genome contains and / or the mice express human or humanized FcαRs described herein that are associated with the wild-type mouse FcRγ chain. Therefore, in some embodiments, human or humanized FcαRs are expressed on the surface of mouse cells and are associated with the endogenous mouse FcRγ chain.

[0029] In some embodiments, the mice provided herein also include a reduced mouse anti-human immune response following administration of a therapeutic agent containing human Fcα. This can be achieved by using genetically modified mice that express human Fc that matches the Fc present in the administered antibody or Fc fusion protein. For example, such mice can be prepared by inserting all or part of a nucleic acid sequence encoding the constant region of the human immunoglobulin heavy chain into the sequence encoding the corresponding portion of the gene segment encoding the constant region of the endogenous non-human immunoglobulin heavy chain. Such animals recognize human Fc as their “self” protein and are therefore less likely to develop an immune response to the administered therapeutic agent containing human Fc.

[0030] Furthermore, in some embodiments, the mice provided herein express an Fcα receptor (FcαR) that interacts with human Fcα, similar to the FcαR expressed by human patients. For example, in some embodiments, the genetically modified mice provided herein express an FcαR having at least a human extracellular domain (e.g., the transmembrane and cytoplasmic domains may be human or rat). In some embodiments, in addition to expressing human or partially human FcαR, the mice provided herein also 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. Therefore, such mice are able to more accurately mimic the human Fc response of human patients compared to mice with completely non-human Fc receptors.

[0031] Therefore, in some embodiments, the mice provided herein are novel in vivo systems for developing, selecting, and testing therapeutic human IgA antibodies and Fc fusion proteins, based not only on specificity and / or affinity for antigens but also on the relevant overall biological function of the antibodies selected by assessing effector functions of the immune system. In this way, human therapeutic candidates can be developed and selected based on the therapeutic potential assessed at the entire molecular level with relevant biological responses (e.g., cellular responses), rather than solely on predictions of individual components assessed separately. Therefore, the mice disclosed herein specifically provide a suitable system for predicting and characterizing the function of human therapeutic antibodies in vivo.

[0032] The transgenic mice expressing human FcαR disclosed in this paper can be used to study the functional role of this receptor in the immune response at a lower cost compared to larger animal models or humans. Furthermore, mice are a more suitable model for determining essential biological functions that can be confirmed in larger animals or humans. For example, mice expressing human FcαR have previously been used to study the role of the receptor in antibody-dependent cytotoxicity (ADCT) and to identify other components necessary for FcαR-mediated responses. See van Egmond et al. (1999) Blood, 93(12):4387-94.

[0033] IgA antibodies play an important role in the development and progression of certain diseases. Breedveld and van Egmond discuss several diseases and disorders in which IgA antibodies exert their effects ((2019) Frontiers in Immunology, 10:553). For example, some autoimmune diseases are characterized by increased abundance of IgA antibodies (e.g., rheumatoid arthritis and IgA nephropathy). In inflammatory bowel disease, bacteria conditioned by IgA can lead to FcαR crosslinking and neutrophil activation, resulting in tissue damage. Low IgA levels are also associated with certain diseases. A potential link has been found between low IgA levels and the severity of allergic asthma, while high IgA levels have been found in subjects with allergic rhinitis. The mice described in this article can be used to identify the role of FcαR in disease severity or the effect of FcαR inhibitors on symptom relief.

[0034] IgA deficiency can lead to increased susceptibility to infections, particularly mucosal infections. Excessive IgA administration can result in enhanced FcαR activation, which can lead to unexpected complications. Therefore, the mice described herein can be used to determine suitable dosing regimens that adequately overcome IgA deficiency without inducing side effects. The mice described herein can also be used to evaluate the efficacy of FcαR inhibitors in treating inflammation that may be IgA-mediated.

[0035] In some implementations, the mice described herein can be treated with anti-tumor (or pathogen) candidate IgA therapeutics or IgA Fc fusion proteins, or bispecific antibodies targeting FcαR and tumor antigens. In other words, the mice described herein can be used for preclinical evaluation of therapies involving IgA and / or FcαR.

[0036] definition

[0037] As used in this article, the articles “a” and “a kind” refer to one or more (i.e., referring to at least one) of the grammatical objects of the article. For example, “element” means one element or more elements.

[0038] The term "amino acid" is intended to include all molecules, whether natural or synthetic, that include both amino and acid functional groups, and can be included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives, and homologs; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing.

[0039] As used herein, the term "antibody" can refer to both the complete antibody and its antigen-binding fragment. A complete antibody is a glycoprotein consisting of two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain includes a heavy chain variable domain and a heavy chain constant domain. Each light chain includes a light chain variable domain and a light chain constant domain. The heavy chain variable domain and light chain variable domain can be further subdivided into hypervariable domains called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each heavy chain variable region and light chain variable region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable domains of the heavy and light chains contain binding domains that interact with the antigen.

[0040] As used herein, the terms “antigen-binding fragment” and “antigen-binding moiety” of an antibody refer to one or more fragments of the antibody that retain the ability to bind antigens. Examples of binding segments encompassed in 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 complete antibody variable domain. These antibody fragments can be obtained using conventional recombinant and / or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.

[0041] As used herein, the term "area under the curve of plasma concentration relative to time" or "AUC" refers to the rate and extent of elimination of the therapeutic agent after administration. In some embodiments, the AUC is determined over specific time periods (such as 12, 18, 24, 36, 48, or 72 hours), or at infinity using extrapolation based on the slope of the curve. Unless otherwise stated herein, the AUC is determined at infinity. INF AUC can also be calculated per dose. Like many other PK parameters, AUC can be determined in a single animal or in a population of animals from which the average value is calculated.

[0042] As used herein, the term "clearance" or "CL" refers to a measure of the body's ability to clear drugs and is expressed as the plasma volume that clears drugs over time.

[0043] When the phrase "derives from" is used with respect to rearranged variable region genes, "derived from" unrearranged variable regions and / or unrearranged variable region gene segments refers to the ability to trace the sequence of a rearranged variable region gene back to a set of unrearranged variable region gene segments that have been rearranged to form a gene expressing a variable domain (where applicable, splicing differences and somatic mutations are considered). For example, a rearranged variable region gene that has undergone somatic mutations still originates from an unrearranged variable region gene segment. In some embodiments, when an endogenous locus is replaced by a universal light or heavy chain locus, the term "derives from" indicates the ability to trace the origin of a sequence to the rearranged locus, even if the sequence may have undergone somatic mutations.

[0044] 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 the 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 an engineered organism. In some embodiments, the reference organism is a laboratory-grown organism (whether wild-type or engineered).

[0045] The term "in vivo recovery" or "IVR" refers to incremental recovery (K-value), which is the observed peak activity minus the pre-dose level, then divided by the dose. IVR can also be calculated as a percentage. Average IVR can be determined in a population of animals, or individual IVR can be determined in a single animal.

[0046] As used herein, the term "locus" refers to a location on a chromosome. In some embodiments, a locus contains a group 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. A mouse locus homologous to the human LRC locus is located on mouse chromosome 7 and contains several genes homologous to those in the human LRC (e.g., Ncr1, Gp6, and Rdh13 genes). Locuses can be endogenous or non-endogenous. The term "endogenous locus" refers to the location of a specific genetic element that is naturally present on a chromosome. 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.

[0047] The terms “polynucleotide” and “nucleic acid” are used interchangeably. They refer to polymeric forms of nucleotides of any length, which can be deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene segments, one or more loci defined by 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, the nucleotide structure can be modified before or after polymer assembly. Polynucleotides can be further modified, such as by conjugation with labeled components. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0048] As used herein, the term "steady-state distribution volume" or "Vss" refers to the apparent space (volume) of drug distribution. More specifically, Vss represents the amount of drug in an animal divided by the steady-state plasma concentration.

[0049] As used herein, the term "CH gene segment" (e.g., Cγ gene segment, Cγ2a gene segment, Cγ2c gene segment, Cμ gene segment, Cγ2b gene segment, Cγ3 gene segment, Cδ gene segment, Cε gene segment, Cα gene segment, etc.) refers to a segment of DNA sequence encoding the constant region of the immunoglobulin heavy chain, and can be associated with C... H Genes (e.g., Cγ gene, C) γ2a Gene, C γ2c Gene, C γ3 Gene, C γ2b Gene, Cμ gene, C δ Gene, Cε gene, C α The terms "C" and "gene" are used interchangeably. For example, the Cγ1 gene segment or Cγ1 gene refers to the segment of DNA sequence encoding the IgG1 constant region. H "Gene segment locus" refers to a naturally occurring C locus on a chromosome. H Gene segment or C H The location of the gene.

[0050] Genetic modification sites

[0051] In some aspects, the genetically modified mice and cells (e.g., ES cells) used for in vivo testing of the therapeutic agent described herein contain human IgA antibodies or Fcα fusion proteins (e.g., for testing the pharmacokinetic and / or pharmacodynamic properties of such therapeutic agents). The genetically modified mice disclosed herein contain at least one locus in their genome that contains a nucleic acid sequence encoding a human or humanized FcαR, and this locus corresponds to a location in the human endogenous FcαR locus, or a location in the mouse genome corresponding to a locus in the human genome located near the human endogenous FcαR locus. For example, in some embodiments, the sequence encoding a human or humanized Fcα receptor (FcαR) protein is located in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, the sequence encoding a human or humanized Fcα receptor (FcαR) protein is located in an intergenic region between the gene loci of Tthy1 and Rdh13 proteins, Lilara5 and Gp6 peptides, Pira6 and Gp6 proteins, and / or Pira6 and Ncr1 proteins. In some embodiments, the sequence encoding the human or humanized Fcα receptor (FcαR) protein is located at coordinates chr7:4,303,905–4,312,280 in the mouse genome (+ strand, GRCm38 assembly). In some embodiments, the intergenic region is a 54 kb region between the Pira6 and Ncr1 loci. In some embodiments, the nucleic acid sequence encoding FcαR also includes all or part of the nucleic acid sequence encoding the human KIR3DL2 gene, and / or also includes all or part of the nucleic acid sequence of the human NCR1 gene, such as the nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

[0052] In some embodiments, the genetically modified mice contain genetically modified loci encoding antibody heavy chains comprising human Fc (e.g., human IgA1 Fc, human IgA2 Fc). Such loci are disclosed in WO2019 / 190990, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the mice and / or ES cells contain genetically modified loci encoding, wholly or partially, human light chains (e.g., encoding λ or κ light chains). Such loci are disclosed in WO2019 / 190990 and U.S. Patent No. 10,820,582, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse and ES cells also contain one or more loci encoding Fc receptors having human extracellular domains (e.g., neonatal Fc receptor (FcRn) α-chain, β-2-microglobulin polypeptide (β2M), Fcε receptor 1α (FcεR1α) α-chain, Fcγ receptor 1α (FcγR1a) α-chain, Fcγ receptor 2a (Fcγr2a) α-chain, Fcγ receptor 2b (Fcγr2b) α-chain, Fcγ receptor 3a (Fcγr3a) α-chain, Fcγ receptor 3b (Fcγr3b) α-chain, Fcγ 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., rodents, such as mice). Such loci are disclosed in WO2019 / 190990 and U.S. Patent Nos. 9,474,255 and 8,658,154, the contents of which are incorporated herein by reference in their entirety.

[0053] Humanized Fcα receptor

[0054] The mouse or embryonic stem (ES) cells described herein express and / or contain humanized or human Fcα receptor 1 (FcαR1) in their genome. Mice do not have FcαR homologs. Therefore, in some embodiments, the mouse or ES cells contain at least one locus in their genome that contains a nucleic acid sequence encoding human or humanized FcαR, and this locus corresponds to a location in the human endogenous FcαR locus, or a location in the mouse genome corresponding to a locus in the human genome located near the human endogenous FcαR locus. For example, in some embodiments, the sequence encoding the human or humanized Fcα receptor (FcαR) protein is located in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, the sequence encoding the human or humanized Fcα receptor (FcαR) protein is located in an intergenic region between the gene loci of Tthy1 and Rdh13 proteins, Lilara5 and Gp6 peptides, and / or Pira6 and Ncr1 proteins. In some embodiments, the sequence encoding the human or humanized Fcα receptor (FcαR) protein is located at coordinates chr7:4,303,905–4,312,280 in the mouse genome (+ strand, GRCm38 assembly). In some embodiments, the intergenic region is a 54 kb region between the Pira6 and Ncr1 loci. In some embodiments, the nucleic acid sequence encoding FcαR also comprises all or part of the nucleic acid sequence encoding the human KIR3DL2 gene, and / or also comprises all or part of the nucleic acid sequence of the human NCR1 gene, such as the nucleic acid sequence present in the 5'UTR of the human NCR1 gene. In some embodiments, the mice provided herein express FcαR on neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells. In some embodiments, neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells are blood neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells. In some embodiments, neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells are splenic neutrophils, monocytes, macrophages (e.g., induced macrophages), eosinophils, and / or dendritic cells.

[0055] FcαR is an activated receptor that complexes with the human FcRγ chain and has moderate affinity for human IgA1, IgA2, and secretory IgA (K). a ~5x10 6 M -1 It has a high affinity for the complexed IgA (K). a ~5x10 6 M -1The binding of FcαR to IgA-coated particles triggers the release of inflammatory mediators, phagocytosis, and antibody-dependent cell-mediated cytotoxicity. IgA-based antitumor therapy, by directing neutrophils to tumor cells, can effectively lyse tumor cells. In addition to the full-length receptor, neutrophils and eosinophils express selectively spliced ​​forms of the receptor that can bind secreted IgA. In vivo, two FcαR molecules dimerize and interact with IgA monomers.

[0056] In some embodiments, the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain, a rodent (not a mouse, but a rat) transmembrane domain, and a rodent (not a mouse, but a rat) cytoplasmic domain. In some embodiments, the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain, a human transmembrane domain, and a rodent (not a mouse, but a rat) cytoplasmic domain. In some embodiments, the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain.

[0057] In some embodiments, the FcαR locus contains a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer). In other embodiments, the FcαR locus contains a human regulatory element (e.g., a human promoter and / or enhancer).

[0058] As described in the art (see, for example, Monteiro and van de Winkel, (2003) Annu. Rev. Immunol. 21: 177-204), the human FcαR gene consists of five exons: exon 1 includes the 5' UTR, the ATG translation start codon, and part of the leader peptide coding sequence; exon 2 encodes the remainder of the leader peptide; exons 3 and 4 encode two extracellular domains of FcαR, EC1 and EC2, wherein EC1 is the domain that binds to IgA Fc; and exon 5 encodes the cytoplasmic and transmembrane domains of the protein.

[0059] In some embodiments, the nucleic acid encoding the human or humanized FcαR polypeptide provided herein encodes the complete human FcαR polypeptide. In some embodiments, this nucleic acid comprises humanized exon 1 (e.g., containing a rodent (non-mouse, e.g., rat) 5' UTR and a human coding sequence), human coding exons 2 to 5 to a stop codon, and a human or rodent (non-mouse, e.g., rat) 3' UTR and polyA. In some embodiments, the nucleic acid encoding the human or humanized FcαR polypeptide comprises exons 1 to 5 of the human FcαR gene.

[0060] In some embodiments, the nucleic acid is used to generate a targeting vector inserted into the genome of mouse ES cells. In some embodiments, the nucleic acid encoding a human or humanized FcαR polypeptide comprises approximately 44 kb of human genome sequence, including the FCAR gene. In some embodiments, the human genome sequence is a genome sequence located on human chromosome 19 at coordinates chr19:54,862,297-54,906,185 (+ strand, GRCh38 assembly), which includes the 3' end of the KIR3DL2 gene and the entire human FCAR gene.

[0061] In some embodiments, the nucleic acid encoding the FcαR polypeptide is a chimeric rodent (non-mouse, e.g., rat) / human sequence. In some embodiments, the chimeric rodent (non-mouse, e.g., rat) / human sequence comprises human, rat, or chimeric rat / human exon 1 (such that the nucleic acid sequence encoding the leader sequence is human or rat); human or rat exon 2, human exons 3 to 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., promoters and UTRs) are rodent (e.g., rat) regulatory regions.

[0062] NCBI reference sequence NW_016107304.1 is a representative source sequence of the human FcαR gene. NCBI reference sequences NM_002000.4 and NP_001991.1, NM_133269.4 and NP_579803.1, NM_133271.4 and NP_579805.1, NM_133272.4 and NP_579806.1, NM_133273.4 and NP_579807.1, NM_133274.4 and NP_579808.1, and NM_133277 are also relevant. 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 FcαRcDNA and peptides from which the desired human fraction can be obtained.

[0063] The NCBI reference sequence number NC_005100.4 provides a representative source sequence of the brown rat FcαR gene, while the NCBI reference sequence number NM_201992.1→NP_973721.1 provides representative source sequences of brown rat FcαR cDNA and polypeptide, from which the desired brown rat portion and / or homologous arms for the design of targeting vectors can be obtained.

[0064] In some embodiments, the mice are heterozygous for the genetically modified FcαR locus. In some embodiments, the mice are homozygous for the genetically modified FcαR locus.

[0065] In various embodiments, the human or humanized FcαR peptide described herein is expressed on the cell surface and is associated with the wild-type mouse FcRγ chain. In various embodiments, the human or humanized FcαR peptide described herein is expressed on the cell surface and is associated with the endogenous mouse FcRγ chain.

[0066] Humanized low-affinity Fcγ receptor

[0067] In some embodiments, mouse and ES cells genetically modified to include a humanized or human FcαR1 locus in their genome also contain a locus encoding a human low-affinity Fcγ receptor (FcγR) polypeptide (e.g., human FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, or FcγRIIIb polypeptide; see, for example, U.S. Patent No. 8,658,154, the contents of which are incorporated herein by reference in their entirety).

[0068] In some embodiments, the low-affinity FcγR locus contains a nucleic acid sequence encoding the human FcγRIIa polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIa polypeptide is located at the endogenous mouse low-affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding the FcγRIIa polypeptide replaces all or part of the endogenous mouse low-affinity FcγR locus. In some embodiments, the human FcγRIIa gene contains a polymorphism, wherein the polymorphism is selected from the 131His low-responder polymorphism and the 131Arg high-responder polymorphism. In some embodiments, the FcγRIIa polymorphism is the 131His low-responder polymorphism. In some embodiments, the mouse does not express the mouse low-affinity FcγR polypeptide (e.g., does not express mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides, or does not express functional mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides). In some embodiments, the FcγRIIa locus contains 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).

[0069] In some embodiments, the low-affinity FcγR locus contains a nucleic acid sequence encoding the human FcγRIIb polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIb polypeptide is located at the endogenous mouse low-affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding the FcγRIIb polypeptide replaces all or part of the endogenous mouse low-affinity FcγR locus. In some embodiments, the human FcγRIIb gene contains amino acid substitutions, wherein the substitutions are selected from 187Ile or 187Thr substitutions. In some embodiments, the mouse does not express the mouse low-affinity FcγR polypeptide (e.g., does not express mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides, or does not express functional mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides). In some embodiments, the FcγRIIb locus contains non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some implementations, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0070] In some embodiments, the low-affinity FcγR locus contains a nucleic acid sequence encoding the human FcγRIIc polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIc polypeptide is located at the endogenous mouse low-affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding the FcγRIIc polypeptide replaces all or part of the endogenous mouse low-affinity FcγR locus. In one embodiment, the FcγRIIc gene is a specific allelic variant selected from the 57Stop and 57Q variants. In some embodiments, the mouse does not express the mouse low-affinity FcγR polypeptide (e.g., does not express mouse FcγRIIB, FcγRIV, and / or FcγRIII polypeptides). In some embodiments, the FcγRIIc locus contains 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).

[0071] In some embodiments, the low-affinity FcγR locus contains a nucleic acid sequence encoding the human FcγRIIIa polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIIa polypeptide is located at the endogenous mouse low-affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding the FcγRIIIa polypeptide replaces all or part of the endogenous mouse low-affinity FcγR locus. In some embodiments, the mouse does not express the mouse low-affinity FcγR polypeptide (e.g., does not express mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides, or does not express functional mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides). In one embodiment, the FcγRIIIa gene is a specific allelic variant selected from the 176Val and 176Phe variants. In some embodiments, the FcγRIIIa allelic variant is the 176Val variant. In some embodiments, the FcγRIIIa locus contains 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).

[0072] In some embodiments, the low-affinity FcγR locus contains a nucleic acid sequence encoding the human FcγRIIIb polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcγRIIIb polypeptide is located at the endogenous mouse low-affinity FcγR locus. In some embodiments, the nucleic acid sequence encoding the FcγRIIIb polypeptide replaces all or part of the endogenous mouse low-affinity FcγR locus. In some embodiments, the FcγRIIIb gene is a specific allelic variant selected from the NA1 and NA2 variants. In another specific embodiment, the FcγRIIIb allelic variant is the NA2 variant. In some embodiments, the mouse does not express the mouse low-affinity FcγR polypeptide (e.g., does not express mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides, or does not express functional mouse FcγRIIb, FcγRIV, and / or FcγRIII polypeptides). In some embodiments, the FcγRIIIb locus contains a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a mouse regulatory element (e.g., a rat or mouse promoter or enhancer).

[0073] In some embodiments, the mice provided herein comprise one or more human low-affinity FcγR genes, as described in U.S. Patent Nos. 9,221,894, 9,056,130, 9,089,599, 8,658,154, 8,883,496, or 8,658,853, which are incorporated herein by reference. In some embodiments, the mice provided herein comprise at least two low-affinity human FcγR genes and an endogenous mouse Fcγ chain gene, wherein the low-affinity human FcγR genes are selected from the group consisting of human FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the mice provided herein comprise human FcγRIIa and FcγRIIIb, and an endogenous mouse Fcγ chain gene. In some embodiments, the mice provided herein comprise the FcγRIIa, FcγRIIIa, FcγRIIb, FcγRIIc, and FcγRIIId genes, as well as endogenous mouse Fcγ chain genes. In various embodiments, mice comprising one or more human FcγRs also comprise homozygous breaks in the endogenous mouse FcγRIIB, FcγRIV, and FcγRIII genes (i.e., the coding sequences of the endogenous mouse FcγRIIb, FcγRIV, and FcγRIIIα chains). In various embodiments, mice comprising one or more of the human low-affinity FcγRs described herein do not detectably express endogenous mouse low-affinity FcγR peptides (e.g., endogenous low-affinity FcγRα chain peptides).

[0074] In some embodiments, the mice are heterozygous for the genetically modified low-affinity FcγR locus. In some embodiments, the mice are homozygous for the genetically modified low-affinity FcγR locus.

[0075] Humanized neonatal Fc receptor locus

[0076] In some embodiments, mouse and ES cells are genetically modified to include a humanized or human FcαR1 locus in their genome, and in some embodiments, a human or humanized FcγR locus (as described above), as well as 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 β-2-microglobulin (β2M) and binds to the Fc domains of IgG antibodies and serum albumin. FcRn prolongs the half-life of IgG and serum albumin. Specifically, by binding to IgG and serum albumin in a pH-dependent manner, FcRn is able to rescue these serum proteins from lysosomal degradation by endothelial cells, thereby increasing the serum half-life of such proteins.

[0077] In some embodiments, the FcRn locus contains 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 contains 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 contains 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.

[0078] In some embodiments, the nucleic acid sequence encoding the FcRn polypeptide is located at the endogenous mouse FcRn locus. In some 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 the endogenous FcRn at the endogenous FcRn locus is replaced by a nucleic acid sequence encoding the extracellular domain of the human FcRn, such that mice containing this locus express FcRn having both human extracellular domains and rodent (e.g., rat or mouse) transmembrane and cytoplasmic domains. In some embodiments, the mice do not express mouse FcRn, or do not express functional mouse FcRn. In some embodiments, the FcRn locus contains 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).

[0079] In some embodiments, mouse exons (exons 3, 4, and 5, which are the first three coding exons) encoding the α1, α2, and α3 domains of the mouse FcRn gene are replaced with human exons (exons 3, 4, and 5) encoding the α1, α2, and α3 domains of the human FcRn gene. 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 through 6, and mouse exons 6 and 7 (encoding transmembrane and cytoplasmic domains). Exemplary humanized FcRn loci are described in WO2019 / 190990 (e.g., in Figure 4), which is incorporated herein by reference.

[0080] GenBank accessions NC_000019.10 (49512279-49526428), NM_001136019.1, and NP_001129491.1 provide representative source sequences of the human FcRn gene, cDNA, and peptide, from which the desired human fraction can be obtained. GenBank accessions NC_000073.6 (45092992-45103846), NM_010189.1, and NP_034319.1 provide representative source sequences of the mouse FcRn gene, cDNA, and peptide, from which the desired mouse fraction and / or its homologous arms for designing targeting vectors can be obtained.

[0081] In some implementations, the mice are heterozygous for the genetically modified FcRn locus. In some implementations, the mice are homozygous for the genetically modified FcRn locus.

[0082] Humanized Fcε receptor 1α

[0083] In some embodiments, mouse and ES cells are genetically modified to include a humanized or human FcαR1 locus in their genome, and in some embodiments, the inclusion of a human or humanized FcγR locus (as described above) also includes a humanized or human Fcε receptor 1α (FcεR1α) locus. FcεR1α associates with FcεR1β and FcεR1γ to form FcεR1, a receptor with high affinity for IgE, expressed on epidermal Langerhans cells, eosinophils, mast cells, and basophils. The IgE binding site of FcεR1 is located in the FcεR1α subunit.

[0084] In some embodiments, the FcεR1α locus comprises a nucleic acid sequence encoding a 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 a 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 a FcεR1α polypeptide comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain. Exemplary embodiments of engineered FcεR1α loci are described in WO2019 / 190990, which are incorporated herein by reference.

[0085] In some embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide is located at the endogenous mouse FcεR1α gene locus. In some 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 gene locus is replaced by a nucleic acid sequence encoding the extracellular domain of human FcεR1α, such that mice containing this locus express FcεR1α having a human extracellular domain and rodent (e.g., rat or mouse) transmembrane and cytoplasmic domains. 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 gene locus. In some embodiments, the nucleic acid sequence encoding the FcεR1α polypeptide, comprising the human extracellular domain, human transmembrane domain, and human cytoplasmic domain, replaces all or part of the endogenous mouse FcεR1α gene. In some embodiments, the mouse does not express mouse FcεR1α, or does not express functional mouse FcεR1α. In some embodiments, the FcεR1α locus contains a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0086] In some embodiments, portions of mouse-encoding exons 1, 2, 3, 4, and 5 of the mouse FcεRIα gene are replaced by portions of human-encoding exons 1, 2, 3, 4, and 5 of the human FcεRIα gene. In some embodiments, the FcεRIα gene comprises a chimeric mouse / human exon 1 (containing the mouse promoter and 5'UTR), human-encoding exons 2 through 5 to the stop codon, the human 3'UTR and polyA, followed by the mouse 3'UTR and polyA. In some embodiments, chimeric gene exons 1 (partially) and 2 encode a signal peptide, exons 3 and 4 encode two Ig-like domains of FcεRIα believed to interact with IgE, and exon 5 encodes the cytoplasmic and transmembrane domains of the protein (see Figure 9 of WO2019 / 190990).

[0087] GenBank accessions NC_000001.11 (159283888-159308224), NM_002001.3, and NP_001992.1 provide representative source sequences for the human FcεR1α gene, cDNA, and peptide, from which the desired human fraction can be obtained. GenBank accessions NC_000067.6 (173221269-173227232), NM_010184.1, and NP_034314.1 provide representative source sequences for the mouse FcεR1α gene, cDNA, and peptide, from which the desired mouse fraction and / or its homologous arms for designing targeting vectors can be obtained.

[0088] In some embodiments, the mice are heterozygous for the genetically modified FcεR1α locus. In some embodiments, the mice are homozygous for the genetically modified FcεR1α locus.

[0089] Humanized Fcγ receptor 1a

[0090] In some embodiments, mouse and ES cells that have been genetically modified to include a humanized or human FcαR1 locus in their genome also contain a humanized or human Fcγ receptor 1a (FcγR1a) locus. FcγR1a is a high-affinity FcγR protein expressed on monocytes that binds to the Fc portion of IgG and induces host cell activation.

[0091] In some embodiments, the FcγR1a locus contains a nucleic acid sequence encoding a FcγR1a polypeptide, said 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 contains a nucleic acid sequence encoding a FcγR1a polypeptide, said 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 contains a nucleic acid sequence encoding a FcγR1a polypeptide, said 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.

[0092] In some embodiments, the nucleic acid sequence encoding the FcγR1α polypeptide is located at the endogenous mouse FcγR1a gene locus. In some 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 gene locus is replaced by a nucleic acid sequence encoding the extracellular domain of human FcγR1a, such that mice containing this locus express FcγR1a having both human extracellular domains and rodent (e.g., rat or mouse) transmembrane and cytoplasmic domains. In some embodiments, the mice do not express mouse FcγR1a, or do not express functional mouse FcγR1a. In some embodiments, the FcγR1a gene locus contains 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).

[0093] U.S. Patent Publication Nos. 9,474,255 and 2017 / 0086432 describe humanized FcγR1a peptide, loci encoding humanized FcγR1a peptide, and mice expressing humanized FcγR1a peptide, each of which is incorporated herein by reference.

[0094] In some embodiments, the mice are heterozygous for the genetically modified FcγR1a locus. In some embodiments, the mice are homozygous for the genetically modified FcγR1a locus.

[0095] Humanized Immunoglobulin Heavy Chain Locus

[0096] As described above, in some embodiments, the mice expressing human FcαR provided herein are used to test agents containing human Fc (e.g., agents containing human Fcα, such as therapeutic human IgA antibodies and human Fcα fusion proteins). However, when therapeutic agents with human Fc regions are administered to wild-type mice, the human sequence in the Fc region is typically identified as foreign by the mouse immune system. Therefore, the mice may develop an immune response to the administered therapeutic agent (referred to as 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 therapeutic agents containing human Fc in mice with human immunoglobulin locus regions can reduce or eliminate the MAHA response because human Fc is less likely to be identified as a foreign substance in such mice.

[0097] In some embodiments, mouse and ES cells genetically modified to include a humanized or human FcαR1 locus in their genome, and optionally including 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, also include genetically modified immunoglobulin (Ig) heavy chain loci. Such loci typically contain variable and constant regions. The variable region includes Ig heavy chain variable region gene segments (e.g., at least V...). H Gene segments, D H Gene segments and J H Gene segments). Constant region loci include one or more Ig heavy chain constant region gene segments (C). H In some implementations, the immunoglobulin heavy chain variable region is operatively linked to the immunoglobulin heavy chain constant region, causing mice to produce proteins containing V... H Gene segments, D H Gene segments and J H Variable domains of gene segments and those derived from C H Antibodies against the heavy chain constant domain of gene segments.

[0098] The ability of genetically modified animals to produce antibodies has been used to generate therapeutic antibodies against human targets. Exemplary genetically modified mice containing the human V(D)J gene segment 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, and 8,907,157. The mouse described in U.S. Patent Publications 9,145,588 (each of which is incorporated herein by reference in its entirety), and in U.S. Patent Publications 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) and U.S. Patent Publications 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) The mice described in PCT Publications WO2007117410, WO2008151081, WO2009157771, WO2010039900, WO2011004192, WO2011123708, WO2014093908, WO2014093908, WO2006008548, WO2010109165, and WO2016 (incorporated in their entirety in this paper) and PCT Publications WO2007117410, WO2008151081, WO2009157771, WO2010039900, WO2011004192, WO2011123708, WO2014093908, WO2014093908, WO2006008548, WO2010109165, and WO2016 (incorporated in their entirety in this paper) are all mice. Mice in WO2018039180, WO2011158009, WO2013041844, WO2013041846, WO2013079953, WO2013061098, WO2013144567, WO2013144566, WO2013171505 and WO2019008123 (each of these references is incorporated herein by reference in its entirety).Other exemplary genetically modified mice containing the human V(D)J gene segment for generating therapeutic antibodies are mice described in U.S. Patent 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, 10,130,081, 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), and mice described in U.S. Patent Publications 2013 / 0212719, 2015 / 0289489, 2017 / 0347633, 2 Mice described in PCT patents WO2013138680, WO2013138712, WO2013138681, WO2015042250, WO2012148873, WO2013134263, WO2013184761, WO2014160179, WO2017214089, WO2016149678 and WO2017123808 and Murphy, A., “Veloc Immune: Immunoglobulin” "Variable Region Humanized Mouse," in Recombinant Antibodies for Immunotherapy, New York, NY, Cambridge University Press, 101-107 (2009) (each of these articles is incorporated herein by reference in its entirety).

[0099] In some embodiments, the Ig heavy chain variable region locus contains an unrearranged human Ig heavy chain variable region gene segment. In some embodiments, the unrearranged human Ig variable region gene segment contains one or more human V... H Section, one or more people D H Section and one or more people J H Segment. In some implementations, the unrearranged human Ig variable region gene segment contains at least 3 V... H Gene segments, at least 18 V H Gene segments, at least 20 V H Gene segments, at least 30 V H Gene segments, at least 40 V H Gene segments, at least 50 VH Gene segments, at least 60 V H Gene segments, at least 70 V H Gene segment, or at least 80 V H Gene segment. In some embodiments, the engineered IgH locus (or allele) contains the human V gene segment from the naturally occurring human IgH locus. H 3-74 and Person V H All or substantially all functional human V genes found between (including both ends) of the 6-1 gene region. H Gene segment. In some implementations, the engineered IgH locus (or allele) contains at least human V. H Gene segment V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H 3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H1-3, V H 1-2 and V H 6-1. In some embodiments, the mice provided herein possess a restriction immunoglobulin heavy chain gene locus, characterized by a single polymorphism of human V. H Gene segments, multiple D H Gene segments and multiple J H Genetic segments (e.g., those described in U.S. Patent Publication No. 2013 / 0096287, which are incorporated herein by reference). In some embodiments, V H The gene segment is V H 1-2 or V H 1-69. In some embodiments, the mice provided herein have rearranged heavy chain variable regions (universal heavy chain variable regions or common heavy chain coding sequences, such as those described in U.S. Patent Publications 20140245468 and 9,204,624 and 9,930,871, each of which is incorporated herein by reference in its entirety). In some embodiments, the mice provided herein include human unrearranged immunoglobulin light chain (e.g., κ) gene segments operatively linked to heavy chain constant regions at immunoglobulin heavy chain loci (e.g., U.S. Patent No. 9,516,868, which is incorporated herein by reference in its entirety).

[0100] In other embodiments, mice may include a heavy chain immunoglobulin locus in their germline and / or genome, the locus comprising the insertion and / or substitution of a histidine codon designed to introduce pH-dependent binding properties into antibodies produced in such mice. In some such embodiments, the histidine codon is inserted and / or substituted in a nucleic acid sequence encoding CDR3. Various 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, 20140013456, which are incorporated herein by reference.

[0101] In some implementations, the engineered IgH locus (or allele) contains 5, 10, 15, 20, 25 or more (e.g., 26, 27, etc.) human D H Gene segment. In some embodiments, the engineered IgH locus (or allele) contains human D gene segments from naturally occurring human IgH loci. H 1-1 and Person D H All or substantially all functional human D was found between (including both ends) gene segments 7-27. H Gene segment. In some embodiments, the engineered IgH locus (or allele) contains at least human D. H Gene segment D H 1-1, DH 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25, D H 1-26 and D H 7-27. In some implementations, the unrearranged human Ig gene segment includes all human D... H Gene segments.

[0102] In some implementations, the engineered IgH locus (or allele) contains 1, 2, 3, 4, 5, 6 or more functional human J. H Gene segment. In some embodiments, the engineered IgH locus (or allele) is contained within the naturally occurring human IgH locus in human J. H 1 and person J H All or substantially all functional human J genes found between (including both ends) of the 6 gene segments. H Gene segment. In some embodiments, the engineered IgH locus (or allele) contains at least human J. H Gene segment J H 1. J H 2. J H 3. J H 4. J H 5 and J H 6. In some implementations, the unrearranged human Ig gene segment includes all human J... H Gene segments.

[0103] In some embodiments, the engineered IgH locus described herein does not contain the endogenous Adam6 gene. In some embodiments, the engineered IgH locus described herein does not contain the endogenous Adam6 gene (or Adam6 coding sequence) at the same phylogenetic location found in the germline genome of a wild-type nonhuman animal of the same species. In some embodiments, the engineered IgH locus described herein does not contain the human Adam6 pseudogene. In some embodiments, the engineered IgH locus described herein comprises an insertion of at least one nucleotide sequence encoding one or more nonhuman (e.g., mouse) Adam6 polypeptides. This insertion may be outside of the engineered immunoglobulin heavy chain locus described herein (e.g., 5' V). H Upstream of the 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.

[0104] In some embodiments, the engineered endogenous immunoglobulin heavy chain locus lacks the functional endogenous mouse Adam6 gene. In some embodiments, the mouse germline genome containing the engineered heavy chain locus includes one or more nucleotide sequences encoding one or more mouse ADAM6 peptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, one or more mouse ADAM6 peptides, functional orthologs, functional homologs, or functional fragments thereof are expressed (e.g., in cells of the male reproductive system, such as testicular cells).

[0105] In some embodiments, one or more nucleotide sequences encoding one or more mouse ADAM6 peptides, functional orthologs, functional homologs, or functional fragments thereof are located on the same chromosome as the engineered endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more mouse ADAM6 peptides, functional orthologs, functional homologs, or functional fragments thereof are located in an engineered endogenous immunoglobulin heavy chain locus. In some embodiments, one or more nucleotide sequences encoding one or more mouse ADAM6 peptides, functional orthologs, functional homologs, or functional fragments thereof are located in a first-person V... H Gene segments and second person V H Between gene segments. In some implementations, the first person V H The gene segment is V H 1-2, Second person V H The gene segment is V H6-1. In some embodiments, one or more nucleotide sequences encoding one or more mouse ADAM6 peptides, functional orthologs, functional homologs, or functional fragments thereof replace the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more mouse ADAM6 peptides, functional orthologs, functional homologs, or functional fragments thereof replace the human Adam6 pseudogene. In some embodiments, one or more nucleotide sequences encoding one or more mouse ADAM6 peptides, functional orthologs, functional homologs, or functional fragments thereof are located in human V H Gene segments and human D H Between gene segments.

[0106] Exemplary Ig variable regions containing 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.

[0107] In some embodiments, the Ig heavy chain variable locus containing the unrearranged human Ig heavy chain variable region gene segment also includes a human Ig heavy chain variable region intergenic sequence. In some embodiments, the Ig heavy chain variable locus includes a non-human (e.g., rodent, rat, mouse) Ig heavy chain variable region intergenic sequence. In some embodiments, the IgH locus contains a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer). In some embodiments, the IgH locus contains 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).

[0108] In some embodiments, the Ig heavy chain variable region is a rearranged variable region containing an Ig heavy chain variable region gene (universal heavy chain variable region). In some embodiments, the rearranged Ig heavy chain variable region gene is a human rearranged Ig heavy chain variable region gene. An exemplary rearranged Ig heavy chain variable region is provided in U.S. Patent Publication No. 2014 / 0245468, which is incorporated herein by reference.

[0109] In some embodiments, the mice provided herein contain the human immunoglobulin constant region (e.g., human C60). α The constant region enables them to express antibodies containing human Fc (e.g., human Fcα). For example, mice containing the constant region of human immunoglobulins are disclosed in WO2019 / 190990, which is incorporated herein by reference in its entirety.

[0110] In some implementations, the immunoglobulin constant region includes a C-type region encoding the IgG constant domain. H Gene segment, the constant structural domain containing human C H 1. Structural domain, human hinge region, human C H 2. Structural domain, human C H 3. The IgG transmembrane domain and IgG cytoplasmic domain are defined as follows: In some embodiments, the IgG transmembrane domain is a rodent IgG transmembrane domain (e.g., a mouse or rat transmembrane domain). In some 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 linker region is a rodent IgG linker region (e.g., a mouse or rat linker domain). In some embodiments, the IgG linker region is a human IgG linker region.

[0111] In some implementations, human C H 1. Structural domain, human hinge region, human C H 2. Structural Domains and Human C H Domain 3 is the IgG1 domain. In some embodiments, this IgG1 domain is encoded by alleles selected from IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04, and IGHG1*05.

[0112] In some implementations, human C H 1. Structural domain, human hinge region, human C H 2. Structural Domains and Human C H Domain 3 is the IgG2 domain. In some embodiments, this IgG2 domain is encoded by an allele selected from IGHG2*01, IGHG2*02, IGHG2*03, IGHG2*04, IGHG2*05, and IGHG2*06.

[0113] In some implementations, human C H 1. Structural domain, human hinge region, human C H 2. Structural Domains and Human C HThe 3-domain is the IgG3 domain. In some embodiments, this IgG3 domain is encoded by alleles 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.

[0114] In some implementations, human C H 1. Structural domain, human hinge region, human C H 2. Structural Domains and Human C H Domain 3 is the IgG4 domain. In some embodiments, this IgG4 domain is encoded by alleles selected from IGHG4*01, IGHG4*02, IGHG4*03, and IGHG4*04.

[0115] In some implementation schemes, C H A gene segment encoding a variant of the human immunoglobulin heavy chain constant region sequence (i.e., including one or more human immunoglobulin heavy chain constant region sequences with additions, deletions, and / or substitutions relative to a suitable reference human immunoglobulin heavy chain constant region sequence), characterized in that, relative to the reference human immunoglobulin heavy chain constant region, the effector function and / or affinity for FcR are enhanced or weakened.

[0116] In some implementation schemes, C H The gene segment encodes the constant region of the human immunoglobulin heavy chain, characterized by altered affinity for activating and / or inhibiting receptors. In some embodiments, C H The gene segment encodes the constant region of the human immunoglobulin heavy chain, characterized for example, by enhanced or weakened binding to the FcRn receptor at acidic pH compared to neutral pH. In some embodiments, C HThe gene segment encodes the human immunoglobulin heavy chain constant region, and encodes the human immunoglobulin heavy chain constant region in whole or in part with one or more amino acid modifications. Exemplary amino acid modifications include, but are not limited to, substitutions 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 307A), position 308 (e.g., 308F or 308V), position 428 (e.g., 428L or 428F), position 433 (e.g., 433H, 433Lm, 433R, 433S, 433P, 433Q or 433K), position 434 (e.g., 434A, 434W, 434H, 434F or 434Y), and combinations thereof. In some implementation schemes, C H The gene segment encodes the constant region of the human immunoglobulin heavy chain, which has one or more pairs of amino acid modifications selected from the following groups: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F).

[0117] In some implementation schemes, C H Gene segments encode chimeric immunoglobulin heavy chain constant regions, which include segments or portions derived from (or present in) more than one human immunoglobulin isotype. For example, this chimeric C H The region may contain C molecules derived from human IgG1, human IgG2, or human IgG4 molecules. H 2. The structural domain, along with the C1360-C ... H 3. Domain Combination. In some implementations, the chimera C HThe region also includes a chimeric hinge region. For example, the chimeric hinge may comprise an “upper hinge” amino acid sequence (amino acid residues 216 to 227, according to EU numbering) derived from the hinge region of human IgG1, human IgG2, or human IgG4, and a “lower hinge” sequence (amino acid residues 228 to 236, according to EU numbering) derived from the hinge region of human IgG1, human IgG2, or human IgG4. In some embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2.

[0118] In some implementations, the modified C H The gene segment is located in endogenous C H Gene segment locus. In some implementations, the modified C H The gene segment is located in endogenous C γ1 Gene segment locus, endogenous C γ2a Gene segment locus, endogenous C γ2b Gene segment locus, endogenous C γ2c Gene segment locus or endogenous C γ3 Gene segment locus.

[0119] In some implementations, the modified C H The gene segment is human C γ1 Gene segment (or at least C encoding the constant domain of IgG1) H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ1 (part of the gene segment), and it is located in endogenous C. γ2a At the gene locus in the gene segment. In some implementations, C encodes the IgG1 constant domain. H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ1 Part of the gene segment is related to endogenous mouse C-type IgG2a transmembrane domain and / or cytoplasmic domain. γ2a Partially operable connections of gene segments.

[0120] In some implementations, the modified C H The gene segment is human C γ1 Gene segment (or at least C encoding the constant domain of IgG1) H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ1 (part of the gene segment), and it is located in endogenous C. γ2cAt the gene locus in the gene segment. In some implementations, C encodes the IgG1 constant domain. H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ1 Part of the gene segment is related to endogenous mouse C-type IgG2c transmembrane domain and / or cytoplasmic domain. γ2c Partially operable connections of gene segments.

[0121] In some implementations, the modified C H The gene segment is human C γ4 Gene segment (or at least C encoding the constant domain of IgG4) H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ4 (part of the gene segment), and it is located in endogenous C. γ1 At the gene locus in the gene segment. In some implementations, C encodes the IgG4 constant domain. H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ4 Part of the gene segment is related to endogenous mouse C-cells encoding the transmembrane domain and / or cytoplasmic domain of IgG1. γ1 Partially operable connections of gene segments.

[0122] In some implementations, the modified C H Gene segment replaced endogenous C H All or part of the gene segment. In some implementations, the modified C H Gene segment replaced endogenous C γ1 Gene segments, endogenous C γ2a Gene segments, endogenous C γ2b Gene segments, endogenous C γ2c Gene segment or endogenous C γ3 All or part of a gene segment.

[0123] In some implementations, the modified C H The gene segment is human C γ1 Gene segment (or at least C encoding the constant domain of IgG1) H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ1 (a portion of the gene segment), and it replaced endogenous C. γ2a All or part of the gene segment locus. In some implementations, C encodes the IgG1 constant domain.H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ1 A portion of the gene segment replaced the C gene encoding the IgG2a constant domain. H 1. Structural domain, hinge region, C H 2. Structural Domains and C H Endogenous mouse C3 domain γ2a Part of the gene segment makes the C encoding the constant domain of IgG1... H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ1 A portion of the gene segment is operatively linked to an endogenous mouse C gene encoding the transmembrane and / or cytoplasmic domain of IgG2a. γ2a Part of the gene segment.

[0124] In some implementations, the modified C H The gene segment is human C γ4 Gene segment (or at least C encoding the constant domain of IgG4) H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ4 (a portion of the gene segment), and it replaced endogenous C. γ1 All or part of the gene segment locus. In some implementations, C encodes the IgG4 constant domain. H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ4 A portion of the gene segment replaced the C gene encoding the IgG1 constant domain. H 1. Structural domain, hinge region, C H 2. Structural Domains and C H Endogenous mouse C3 domain γ1 Part of the gene segment makes the C encoding the constant domain of IgG4... H 1. Structural domain, hinge region, C H 2. Structural Domains and C H 3. Structural domain of human C γ4 A portion of the gene segment is operatively linked to an endogenous mouse C1 gene encoding the transmembrane and / or cytoplasmic domain of IgG1. γ1 Part of the gene segment.

[0125] In some implementations, the Ig heavy chain constant region includes one or more rodent (e.g., rat or mouse) C HGene segments. In some implementations, the Ig constant region includes rodent (e.g., rat or mouse) C... μ Gene segments. In some implementations, the Ig constant region includes rodent (e.g., rat or mouse) C... δ Gene segments. In some implementations, the Ig constant region includes rodent (e.g., rat or mouse) C... γ1 Gene segments. In some implementations, the Ig constant region includes rodent (e.g., rat or mouse) C... γ2a Gene segments. In some implementations, the Ig constant region includes rodent (e.g., rat or mouse) C... γ2b Gene segments. In some implementations, the Ig constant region includes rodent (e.g., rat or mouse) C... γ2c Gene segments. In some implementations, the Ig constant region includes rodent (e.g., mouse) C... γ3 Gene segments. In some implementations, the Ig constant region includes rodent (e.g., rat or mouse) C... ε Gene segments. In some implementations, the Ig constant region includes rodent (e.g., rat or mouse) C... ε Gene segments. In some embodiments, one or more rodent homeostatic gene segments are endogenous homeostatic gene segments. In some embodiments, the C modified above... H The gene segment is the only C-modified region in the Ig heavy chain constant region. H Gene segment. In some implementations, the above-modified C H Gene segments are C-terminal regions of multiple modifications in the constant region of the Ig heavy chain. H One of the gene segments (e.g., one of the 2, 3, 4, 5, 6, 7, or 8 modified gene segments that are partially or fully humanized in the Ig heavy chain constant region). In some embodiments, the Ig heavy chain constant region includes human or partially human C. μ Gene segments. In some implementations, the Ig heavy chain constant region includes human or partial human C... δ Gene segments. In some implementations, the Ig heavy chain constant region includes human or partial human C... γ1 Gene segments. In some implementations, the Ig heavy chain constant region includes human or partial human C... γ2 Gene segments. In some implementations, the Ig heavy chain constant region includes human or partial human C... γ3 Gene segments. In some implementations, the Ig heavy chain constant region includes human or partial human C... γ4 Gene segments. In some implementations, the Ig heavy chain constant region includes human or partial human C... ε Gene segments. In some implementations, the Ig heavy chain constant region includes human or partial human C... αGene segment. In some implementations, the Ig heavy chain constant region contains human C. μ Gene segments, human C δ Gene segments, human C γ1 Gene segments and human C γ3 Gene segment. In some implementations, the Ig heavy chain constant region also contains human C. γ2 Gene segments and human C γ4 Gene segment. In some implementations, the Ig heavy chain constant region also contains human C. α Gene segment. In some implementations, the Ig heavy chain constant region also contains human C. ε Gene segments.

[0126] In some embodiments, the IgH locus contains a human or rodent (e.g., rat or mouse) regulatory element. In some embodiments, the regulatory element is an endogenous regulatory element. In some embodiments, the IgH locus contains a rodent (e.g., rat or mouse) or human in-vivo enhancer (E) element. i In some implementations, the IgH locus contains a rodent (e.g., rat or mouse) or human 3′ regulatory region (3′RR).

[0127] In some embodiments, the modified immunoglobulin heavy chain locus is located at the endogenous immunoglobulin heavy chain locus. In some embodiments, the immunoglobulin heavy chain locus replaces all or part of the endogenous immunoglobulin heavy chain locus. In some embodiments, the modified IgH locus is located on a transgene outside the endogenous locus. In some embodiments, the endogenous IgH locus is inactivated (e.g., through deletion, relocation, and / or inversion of all or part of the endogenous Ig heavy chain locus).

[0128] Therefore, in some embodiments, one or more immunoglobulin heavy chain constant regions (or portions thereof) of the immunoglobulin heavy chain locus are not missing (i.e., intact). In some embodiments, one or more C... H Gene segments are defined by the immunoglobulin heavy chain constant region sequences described herein (e.g., those encoding human IgG C). H 1-HC H 2-C H 3. The sequence of the polypeptide is altered, disrupted, deleted, or replaced, and is operatively linked to one or more transmembrane and cytoplasmic coding sequences (e.g., M1 and / or M2 coding sequences) of a non-human immunoglobulin heavy chain IgG constant region gene, and in some embodiments, the immunoglobulin heavy chain constant region sequence (e.g., encoding human IgE C) is modified, disrupted, deleted, or replaced. H 1-C H 2-C H 3-C HThe sequence of the 4-peptide is operatively linked to one or more transmembrane and cytoplasmic coding sequences of an IgE constant region gene. In some embodiments, all or substantially all of the immunoglobulin heavy chain constant region is replaced by a heterologous immunoglobulin heavy chain constant region. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operatively linked to one or more transmembrane and cytoplasmic coding sequences (e.g., exons M1 and M2) of an IgG constant region gene. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operatively linked to the transmembrane and cytoplasmic coding sequences (e.g., exons M1 and M2) of an IgE constant region gene. In some embodiments, the heterologous immunoglobulin heavy chain constant region sequence is operatively linked to the transmembrane and cytoplasmic coding sequences (e.g., exon M) of an IgA constant region gene. In some embodiments, one or more C H Gene segments (e.g., C) μ C δ (e.g., IgG) are not deleted or replaced in the immunoglobulin heavy chain constant region, which includes a heterologous immunoglobulin heavy chain constant region sequence operatively linked to 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 altered, disrupted, deleted, replaced, or engineered with one or more heterologous immunoglobulin heavy chain constant region sequences is a mouse immunoglobulin heavy chain constant region. In some embodiments, a heterologous immunoglobulin heavy chain constant region sequence is inserted into two copies of the IgG constant region gene (e.g., C15) of the immunoglobulin heavy chain constant region gene. γ1 C γ2a C γ2b C γ2c Or C γ3 A heterozygous mouse relative to the heterologous immunoglobulin heavy chain constant region sequence is produced from one copy (i.e., allele) of the immunoglobulin heavy chain. In some embodiments, a homozygous immunoglobulin heavy chain constant region mouse is provided, which includes the heterologous immunoglobulin heavy chain constant region sequence described herein.

[0129] In some implementations, the engineered immunoglobulin heavy chain constant region described herein contains one or more IgG encoding C H Gene segments, each containing human extracellular domain coding sequences (e.g., human IgG C) operably linked to nonhuman transmembrane and cytoplasmic domain coding sequences (e.g., nonhuman IgG M1-M2) of the same or different IgG subclasses. H 1-HC H 2-C H 3).

[0130] In some implementations, the engineered immunoglobulin heavy chain constant region, as described herein, contains one or more encoding C as described herein. H Engineered IgG of gene segments, and also containing wild-type (e.g., unmodified non-human, such as rat or mouse) C μ Constant region genes.

[0131] In some implementations, the engineered immunoglobulin heavy chain constant region described herein comprises one or more encoding C as described herein. H Engineered IgG of gene segments, and also containing wild-type (e.g., unmodified non-human, such as rat or mouse) C μ and C δ Constant region genes.

[0132] In various implementations, engineered IgG encoding C, comprising the human immunoglobulin heavy chain constant region sequence described herein, is used. H The gene segment is selected from C γ1 C γ2a C γ2b C γ2c Or C γ3 Engineered IgG subclasses of IgG encode C H Gene segments.

[0133] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein includes engineered C γ2a Gene segment containing the encoding human IgG1 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to non-human (e.g., rat or mouse) C γ2a The M1 and M2 exons of the gene segment.

[0134] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein includes engineered C γ2a Gene segment containing the encoding human IgG1 C H 1-HC H 2-C H The sequence 3-M1-M2 replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to non-human (e.g., rat or mouse) C γ2a The transition region of a gene segment.

[0135] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein includes engineered C γ2c Gene segment containing the encoding human IgG1 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to non-human (e.g., rat or mouse) C γ2c The M1 and M2 exons of the gene segment.

[0136] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein includes engineered C γ2c Gene segment containing the encoding human IgG1 C H 1-HC H 2-C H The sequence 3-M1-M2 replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to non-human (e.g., rat or mouse) C γ2c The transition region of a gene segment.

[0137] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein includes engineered C γ1 Gene segment containing the encoding human IgG4 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to non-human (e.g., rat or mouse) C γ1 The M1 and M2 exons of the gene segment.

[0138] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein includes engineered C γ1 Gene segment containing the encoding human IgG4 C H 1-HC H 2-C H The sequence 3-M1-M2 replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to non-human (e.g., rat or mouse) C γ1 The transition region of a gene segment.

[0139] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein includes engineered C γ2a Gene segments and engineered C γ1 Gene segments, engineered C γ2a The gene segment contains the encoding human IgG1 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to non-human (e.g., rat or mouse) C γ2a M1 and M2 exons of the gene segment; engineered C γ1 The gene segment contains the encoding human IgG4 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to non-human (e.g., rat or mouse) C γ1 The M1 and M2 exons of the gene segment.

[0140] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein includes engineered C γ2a Gene segment containing the encoding human IgG1 C H 1-HC H 2-C H The sequence 3-M1-M2 replaces C H 1-HC H 2-C H The 3-M1-M2 exon is operably linked to non-human (e.g., rat or mouse) C γ2a The conversion region of the gene segment; and the engineered C γ1 Gene segment containing the encoding human IgG4 C H 1-HC H 2-C H The sequence 3-M1-M2 replaces C H 1-HC H 2-C H The 3-M1-M2 exon is operably linked to non-human (e.g., rat or mouse) C γ1 The transition region of a gene segment.

[0141] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein includes engineered C γ2c Gene segment containing the encoding human IgG1 CH 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons and operably linked to non-human (e.g., rat or mouse) C γ2c The conversion region of the gene segment; and the engineered C γ1 Gene segment containing the encoding human IgG4 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons and operably linked to non-human (e.g., rat or mouse) C γ1 The M1 and M2 exons of the gene segment.

[0142] In some embodiments, the engineered immunoglobulin heavy chain constant region described herein includes engineered C γ2c Gene segment containing the encoding human IgG1 C H 1-HC H 2-C H The sequence 3-M1-M2 replaces C H 1-HC H 2-C H The 3-M1-M2 exon is operably linked to non-human (e.g., rat or mouse) C γ2c The conversion region of the gene segment; and the engineered C γ1 Gene segment containing the encoding human IgG4 C H 1-HC H 2-C H The sequence 3-M1-M2 replaces C H 1-HC H 2-C H The 3-M1-M2 exon is operably linked to non-human (e.g., rat or mouse) C γ1 The transition region of a gene segment.

[0143] In various embodiments, the engineered immunoglobulin heavy chain constant region, as described herein, includes one or more additional modifications, including making it, in addition to containing the region encoding human IgG C... H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C HOne or more IgG constant region genes (i.e., isotypes) outside the IgG constant region of the 3-M1-M2 sequence (e.g., IgG1 and / or IgG2a) are nonfunctional, for example, by means of complete or partial deletion, complete or partial alteration, complete or partial destruction, or complete or partial replacement of one or more immunoglobulin constant region genes encoding IgD, IgE, IgA, and IgG, and do not themselves contain the encoding of human IgG C as described herein. H 1-HC H 2-C H 3 or human IgG C H 1-HC H 2-C H Sequences of 3-M1-M2 (e.g., IgG2b and / or IgG3) are also provided. Engineered non-human embryos, cells, and targeting vectors for the preparation of such mice, embryos, and cells are also provided.

[0144] In some embodiments, the engineered immunoglobulin heavy chain constant region provided herein contains wild-type (e.g., unmodified non-human, such as rat or mouse) C μ Gene segment, C γ1 Gene segment containing the encoding human IgG4 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to the C γ1 The M1-M2 exons of the gene segment are missing, and C is deleted. δ C γ2a、 C γ2c C γ2b C γ3 C ε and C α Gene segments.

[0145] In some embodiments, the engineered immunoglobulin heavy chain constant region provided herein contains wild-type (e.g., unmodified non-human, such as rat or mouse) C μ Gene segment, C γ1 Gene segment containing the encoding human IgG4 C H 1-HC H 2-C H The sequence 3-M1-M2 replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to the C γ1 The transition region of the gene segment, and the deletion of C δ Cγ2a C γ2c C γ2b C γ3 C ε and C α Gene segments.

[0146] In some embodiments, the engineered immunoglobulin heavy chain constant region provided herein contains wild-type (e.g., unmodified non-human, such as rat or mouse) C μ Gene segment, C γ2a Gene segment containing the encoding human IgG1 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to the C γ2a The M1-M2 exons of the gene segment are missing, and C is deleted. δ C γ1 C γ2b C γ2c C γ3 C ε and C α Gene segments.

[0147] In some embodiments, the engineered immunoglobulin heavy chain constant region provided herein contains wild-type (e.g., unmodified non-human, such as rat or mouse) C μ Gene segment, C γ2a Gene segment containing the encoding human IgG1 C H 1-HC H 2-C H The sequence 3-M1-M2 replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to the C γ2a The transition region of the gene segment, and the deletion of C δ C γ1 C γ2b C γ2c C γ3 C ε and C α Gene segments.

[0148] In some embodiments, the engineered immunoglobulin heavy chain constant region provided herein contains wild-type (e.g., unmodified non-human, such as rat or mouse) C μ Gene segment, C γ2c Gene segment containing the encoding human IgG1 CH 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to the C γ2c The M1-M2 exons of the gene segment are missing, and C is deleted. δ C γ1 C γ2a C γ2b C γ3 C ε and C α Gene segments.

[0149] In some embodiments, the engineered immunoglobulin heavy chain constant region provided herein contains wild-type (e.g., unmodified non-human, such as rat or mouse) C μ Gene segment, C γ2c Gene segment containing the encoding human IgG1 C H 1-HC H 2-C H The sequence 3-M1-M2 replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to the C γ2c The transition region of the gene segment, and the deletion of C δ C γ1 C γ2a C γ2b C γ3、 C ε and C α Gene segments.

[0150] In various embodiments, the engineered immunoglobulin heavy chain constant region described herein includes one or more additional modifications, including making it, in addition to containing the region encoding human IgG C... H 1-HC H2 -C H 3 or human IgG C H 1-HC H 2-C H The sequence 3-M1-M2 (e.g., IgG1 and / or IgG2a) is altered, modified, replaced, or engineered by inserting one or more engineered constant region genes (i.e., isotypes) outside the IgG constant region, via the insertion of the human immunoglobulin heavy chain constant region sequence described herein into one or more immunoglobulin constant region genes of IgD, IgE, IgA, and IgG, wherein the constant region itself does not contain the encoding of human IgG C as described herein. H 1-HCH 2-C H 3 or human IgG C H 1-HC H 2-C H The sequence of 3-M1-M2 (e.g., IgG2b and / or IgG3).

[0151] Also provided are engineered mouse embryos, cells, and targeting vectors for preparing mice, embryos, and cells containing immunoglobulin loci with the engineered constant regions described herein.

[0152] In some implementations, the engineered immunoglobulin heavy chain constant region provided herein contains wild-type C μ Gene segment, wild-type C δ Gene segments, C γ3 Gene segment containing the encoding human IgG3 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to the C γ3 The M1-M2 exons of the gene segment, C γ1 The gene segment contains the encoding human IgG4 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to the C γ1 The M1-M2 exons of the gene segment, C γ2b The gene segment contains the encoding human IgG2 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to the C γ2b M1-M2 exons of the gene sequence, C γ2a Contains encoding human IgG1 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to the C γ2a M1-M2 exons (and / or C) of the gene segmentγ2c The gene segment contains the encoding human IgG1 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to the C γ2c (M1-M2 exons of the gene segment), C ε The gene segment contains the encoding human IgE C H 1-C H 2-C H 3-C H Section 4, replacing C H 1-C H 2-C H 3-C H 4 exons, and operably linked to the C ε The M1-M2 exons of the gene segment, and C α The gene segment contains the encoding of human IgA1 or IgA2 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to the C α One or more M exons in a gene segment.

[0153] In some implementations, the engineered immunoglobulin heavy chain constant region provided herein contains wild-type C μ Gene segment, wild-type C δ Gene segments, C γ3 Gene segment containing the encoding human IgG3 C H 1-HC H 2-C H The 3-M1-M2 sequence replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to the C γ3 The transition region of gene segments, C γ1 The gene segment contains the encoding human IgG4 C H 1-HC H 2-C H The 3-M1-M2 sequence replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to the Cγ1 The transition region of gene segments, C γ2b The gene segment contains the encoding human IgG2 C H 1-HC H 2-C H The 3-M1-M2 sequence replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to the C γ2b The transition region of gene segments, C γ2a The gene segment contains the encoding human IgG1 C H 1-HC H 2-C H The 3-M1-M2 sequence replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to the C γ2a Transition regions of gene segments (and / or C) γ2c The gene segment contains the encoding human IgG1 C H 1-HC H 2-C H The 3-M1-M2 sequence replaces C H 1-HC H 2-C H 3-M1-M2 exons, and operably linked to the C γ2c (Transition region of gene segment), C ε The gene segment contains the encoding human IgE C H 1-C H 2-C H 3-C H The sequence 4 replaces C H 1-C H 2-C H 3-C H 4 exons, and operably linked to the C ε The M1-M2 exons of the gene segment, and C α The gene segment contains the encoding of human IgA1 or IgA2 C H 1-HC H 2-C H The sequence 3 replaces C H 1-HC H 2-C H 3 exons, and operably linked to the C α One or more M exons in a gene segment.

[0154] In some embodiments, the mice are heterozygous for the modified immunoglobulin heavy chain locus described herein. In some embodiments, the mice are homozygous for the modified immunoglobulin heavy chain locus described herein.

[0155] Therapeutic agents containing human Fc regions (e.g., those containing human Fcα, such as therapeutic human IgA antibodies) are typically tested in non-human species (e.g., mice) before being administered to humans. Unfortunately, drugs containing human Fc regions often exhibit very different pharmacokinetic and pharmacodynamic properties when administered to prior art mice compared to when administered to humans. For example, when a therapeutic agent containing a human Fc region is administered to conventional mice, the human sequence in the Fc region is often identified as foreign by the mouse immune system. Consequently, the mice may develop an immune response to the administered therapeutic agent (referred to as a mouse anti-human response or MAHA), which affects the pharmacokinetic and pharmacodynamic properties of the administered drug. Therefore, conventional mouse models are generally not good predictors of human therapeutic responses to therapeutic agents containing human Fc regions. In some embodiments, the mice disclosed herein exhibit a reduced MAHA response after administration of a therapeutic agent containing human Fc regions. Therefore, the mice disclosed herein containing human immunoglobulin locus regions contribute to a reduced or eliminated MAHA response.

[0156] Therefore, this paper provides an expression for human or humanized FcαR that includes the human constant region (e.g., human C). α Various mice (constant region) can be used to test MAHA responses to administration of agents containing human Fc (e.g., agents containing human Fcα, such as therapeutic human IgA antibodies), as disclosed in, for example, WO2019 / 190990, which is incorporated herein by reference in its entirety.

[0157] Humanized immunoglobulin κ locus

[0158] In some implementations, the mice described herein also contain a humanized Igκ chain locus. As disclosed in WO2019 / 190990, which is incorporated herein by reference in its entirety, testing therapeutics containing the human κ chain in mice with the humanized Igκ chain locus can reduce or eliminate the MAHA response because the human κ chain is unlikely to be identified as a foreign substance in such mice.

[0159] In some implementations, mouse and ES cells genetically modified to include the humanized or human FcαR1 locus in their genome also contain a genetically modified Igκ chain locus. Such loci contain a κ variable region and a κ constant region. The κ variable region includes the Igκ chain variable region gene segment (i.e., at least V...). κ Gene segments and J κ Gene segments). Constant regions include the Igκ chain constant region (C). κGene segments. In some implementations, the variable region of the immunoglobulin κ chain, such as the human Igκ variable region, is operatively linked to the constant region of the immunoglobulin κ chain, causing mice to produce genes containing V derived from human Igκ. κ Gene segments and human J κ Light chain variable domains of gene segments and C κ Antibodies containing the light chain constant domain of a gene segment. In some embodiments, the Igκ variable region will be an unrearranged Igκ variable region, and therefore will contain an unrearranged Igκ variable region gene segment. In some embodiments, the Igκ variable region will be a rearranged Igκ variable region, and therefore will contain a rearranged Igκ variable region gene. In some embodiments, the Igκ variable region gene segment is a human Igκ variable region gene segment. In some 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 contains 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.

[0160] In some embodiments, the Igκ variable region contains an unrearranged human Igκ variable region gene segment. In some embodiments, the engineered Igκ light chain locus (or allele) contains at least human V. κ A gene segment that appears in the distal variable cluster (or distal arm or distal repeat) of a naturally occurring human Igκ light chain locus. In some embodiments, the engineered Igκ light chain locus (or allele) contains at least human V. κ Gene segments that appear in the proximal variable clusters (or proximal arms or proximal repeats) of naturally occurring human Igκ light chain loci. In some embodiments, engineered Igκ light chain loci (or alleles) contain human V κ Gene segments that appear in the distal and proximal variable clusters of the naturally occurring human Igκ light chain locus. In some embodiments, the engineered Igκ light chain locus (or allele) is contained within the human V gene at the naturally occurring human Igκ light chain locus. κ 2-40 (or V) κ 3D-7) and Human V κ All or substantially all functional human V genes found between (including both ends) of the 4-1 gene region. κ Gene segments.

[0161] In some implementations, the unrearranged human immunoglobulin variable region gene segment contains multiple human V... κ Section and one or more people J κ Segment. In some implementations, the immunoglobulin variable region gene segment contains four functional V...κ Section and all J κ Segment. In some implementations, the immunoglobulin variable region gene segment contains 16 functional V... κ Section and all J κ Segment. In some implementations, the unrearranged human immunoglobulin variable region gene segment contains all V... κ Section and all J κ Section.

[0162] In some implementations, the engineered Igκ light chain locus (or allele) contains 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 Gene segment. In some embodiments, the engineered Igκ light chain locus (or allele) contains 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, Vκ 1-8, V κ 1-6, V κ 1-5, V κ 5-2 and V κ 4-1. In some embodiments, the engineered Igκ light chain locus (or allele) contains at least human V. κ Gene segment V κ 3D-7, V κ 1D-8, V κ 1D-43, V κ 3D-11, V κ 1D-12, V κ 1D-13, V κ 3D-15, V κ 1D-16, V κ 1D-17, V κ 3D-20, V κ 6D-21, V κ 2D-26, V κ 2D-28, V κ 2D-29, V κ 2D-30, V κ 1D-33, V κ 1D-39 and V κ 2D-40. In some embodiments, the engineered Igκ light chain locus (or allele) contains at least human V. κ Gene segment V κ 2-40, V κ 1-39, V κ 1-33, V κ 2-30, V κ 2-28, V κ 1-27, V κ 2-24, V κ 6-21, V κ 3-20, V κ 1-17, V κ 1-16, V κ 3-15, V κ 1-12, V κ 3-11, V κ 1-9, V κ 1-8, V κ 1-6, V κ 1-5, V κ 5-2 and V κ 4-1.

[0163] In some embodiments, the mice provided herein possess a restriction immunoglobulin light chain gene locus, characterized by having a gene sequence of no more than two individuals.L Gene segments and multiple J L Genetic segments (e.g., double light chain mice or DLC, as described in U.S. Patent Publication No. 2013 / 0198880, which is incorporated herein by reference). In some embodiments, V L The gene segment is V κ Gene segment. In some implementations, V L The gene segment is V λ Gene segment. In some implementations, V κ The gene segment is V κ 3-20 and V κ 1-39.

[0164] In some implementations, the engineered Igκ light chain locus (or allele) contains 1, 2, 3, 4, 5 or more functional human J. κ Gene segment. In some embodiments, the engineered Igκ light chain locus (or allele) is contained within the naturally occurring human Igκ light chain locus in human J. κ 1 and person J κ All or substantially all functional human J genes found between (including both ends) of the 5 gene segments κ Gene segment. In some implementations, the engineered Igκ light chain locus (or allele) contains at least human J. κ Gene segment J κ 1. J κ 2. J κ 3. J κ 4 and J κ 5.

[0165] In other embodiments, a non-human organism may contain a light chain immunoglobulin locus in its germline and / or genome, the locus comprising the insertion and / or substitution of a histidine codon designed to introduce pH-dependent binding properties into antibodies generated in such a non-human organism. In some such embodiments, the histidine codon is inserted and / or substituted in a nucleic acid sequence encoding CDR3. Various such light chain immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, and U.S. Patent Application Publication Nos. 2013 / 0247236, 2014 / 0013456, which are incorporated herein by reference.

[0166] Exemplary variable regions containing 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 segment contains all human Jκ segments.

[0167] In some embodiments, the Igκ variable locus containing the unrearranged human Igκ variable region gene segment also includes a human Igκ variable region intergenic sequence. In some embodiments, the Igκ variable locus includes a non-human (e.g., rodent, rat, mouse) Igκ variable region intergenic sequence. In some embodiments, the Igκ locus contains a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0168] In some embodiments, the Igκ variable region locus is a rearranged variable region locus containing an Igκ variable region gene (universal light chain variable region). In some embodiments, the rearranged Igκ variable region gene is a human rearranged Igκ variable region gene (e.g., a human rearranged Igκ variable region containing human Vκ1-39Jκ or human Vκ3-20Jκ sequences (such as human Vκ1-39Jκ5 or human Vκ3-20Jκ1 sequences)). The use of the universal light chain variable region facilitates the generation of bispecific antibodies. An exemplary rearranged Ig light chain variable region is provided in U.S. Patent Publication No. 2013 / 0185821, which is incorporated herein by reference.

[0169] In some embodiments, the Igκ chain locus contains a human or rodent (e.g., rat or mouse) regulatory element. In some embodiments, the regulatory element is an endogenous regulatory element. In some embodiments, the Igκ chain locus contains a rodent (e.g., rat or mouse) or human intron κ enhancer (E... κi In some implementations, the IgH locus contains a rodent (e.g., rat or mouse) or human 3′κ enhancer (E). κ3′ ).

[0170] 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 located upstream of the Cλ gene (e.g., operatively linked). Rodents comprising such a genetically modified immunoglobulin κ chain locus are illustrated, for example, in U.S. Patent No. 11,051,498, which is incorporated herein by reference in its entirety. In some embodiments, the modified immunoglobulin κ chain locus in the genetically modified rodent comprises a limited library of human λ light chain variable regions, wherein the limited library of human λ light chain variable regions 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 that is conjugated to a human Jλ gene segment and is operatively linked to a Cκ or Cλ gene segment (e.g., a rodent Cκ or Cλ gene segment (e.g., a mouse Cλ1 gene segment)). WO2020 / 247623 provides an example of rodents that include this genetically modified immunoglobulin κ chain locus, which is incorporated herein by reference in its entirety.

[0171] In some embodiments, the modified immunoglobulin κ chain locus is located at the endogenous immunoglobulin κ chain locus. In some embodiments, the immunoglobulin κ chain locus replaces all or part of the endogenous immunoglobulin κ chain locus. In some embodiments, the modified Igκ chain locus is located on a transgene outside the endogenous locus. In some embodiments, the endogenous Igκ chain locus is inactivated (e.g., through deletion, relocation, and / or inversion of all or part of the endogenous Igκ chain locus).

[0172] In some embodiments, the mice are heterozygous for the modified immunoglobulin κ chain locus described herein. In some embodiments, the mice are homozygous for the modified immunoglobulin κ chain locus described herein.

[0173] Therefore, this paper provides an expression of human or humanized FcαR and includes a human light chain constant region (and in some embodiments, also includes human C). α Various mice (constant region) can be used to test MAHA responses to administration of agents containing human Fc (e.g., agents containing human Fcα, such as therapeutic human IgA antibodies), as disclosed in, for example, WO2019 / 190990, which is incorporated herein by reference in its entirety.

[0174] Humanized immunoglobulin λ locus

[0175] In some implementations, the mice described herein also contain a humanized Igλ chain locus. As disclosed in WO2019 / 190990, which is incorporated herein by reference in its entirety, testing therapeutics containing the human κ chain in mice with the humanized Igλ chain locus can reduce or eliminate the MAHA response because the human λ chain is unlikely to be identified as a foreign substance in such mice.

[0176] In some implementations, mouse and ES cells genetically modified to include a humanized or human FcαR1 locus in their genome also contain a genetically modified Igλ chain locus. Such loci contain Igλ chain variable region gene segments (i.e., at least V...). λ Gene segments and J λ Gene segment). The modified λ locus also includes at least one Igλ chain constant region (C λ ) gene segment. In some implementations, V λ Gene segments and J λ Gene segments, such as human V λ Gene segments and human J λ Gene segments are operatively linked to C λ This caused mice to produce substances containing human vitamin V. λ Gene segments and human J λ Light chain variable domains of gene segments and C λ Antibodies targeting the light chain constant domains of gene segments. In some implementations, V λ Gene segments and J λ The gene segment will be the unrearranged V λ and J λ Gene segment. In some implementations, V λ Gene segments and J λ The gene segment will be rearranged V λ and J λ Gene segments, and therefore will be in the form of rearranged variable regions. In some implementations, Ig λ The variable region gene segment is the human variable region gene segment. In some implementations, Ig... λ Variable region gene segments are variable region gene segments in rodents (e.g., variable region gene segments in rats or mice). In some implementations, Ig... λThe constant region locus contains a portion or a complete human λ constant region gene segment. In some embodiments, the Igλ chain locus described herein is located at an endogenous Igλ chain locus. Exemplary variable regions containing Igλ gene segments are provided, such as U.S. Patent Publications 2012 / 0073004 and 2002 / 0088016 and U.S. Patent Application No. 15 / 803,513 (filed November 3, 2017; published in US2018 / 0125043), each of which is incorporated herein by reference.

[0177] In some embodiments, the Igλ variable locus containing the unrearranged human Igλ variable region gene segment also includes an intergenic sequence of the human Igλ variable region. In some embodiments, the Igλ variable locus includes an intergenic sequence of a non-human (e.g., rodent, rat, mouse) Igλ variable region gene. In some embodiments, the Igλ locus contains a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0178] In some embodiments, the human Igλ light chain locus contains genetic material derived from the human Igλ light chain locus. In some embodiments, the human Igλ light chain locus described herein contains at least one human V... λ Gene segment, at least one person J λ Gene segment, at least one human C λ Gene segments and one or more promoting the at least one human V λ Gene segment and at least one human J λ Rearrangement of gene segments (e.g., one or more recombination signal sequences) to form the human V gene. λ Human V, a functional rearrangement of structural domains λ -J λ The sequence required for the sequence. In many embodiments, the human Igλ light chain sequence contains multiple human V... λ Gene segments and promoting the aforementioned human V λ Gene segment and at least one person J λ One or more sequences necessary for gene segment rearrangement. In some embodiments, the human Igλ light chain sequence described herein is the genomic sequence of the human Igλ light chain locus (e.g., isolated and / or cloned from a bacterial artificial chromosome) and contains multiple germline configurations of human V λ Gene segment. In some implementations, the human Igλ light chain sequence contains the germline human V gene segment. λ J λ and C λ Sequence (i.e., because the person V) λ J λ and Cλ The sequence appears at the Igλ light chain locus in human cells; in other words, J λ and C λ Sequence with J λ C λ (Cluster appearance). In some embodiments, the human Igλ light chain sequence encodes a complete or partial Igλ light chain polypeptide that appears in immunoglobulins, particularly those expressed by human B cells. Mice, embryos, cells, and targeted constructs for preparing mice, non-human embryos, and cells are also provided, wherein the human Igλ light chain sequence replaces the corresponding non-human Igλ light chain sequence (e.g., an endogenous mouse Igλ light chain locus).

[0179] In some embodiments, a human Igλ light chain sequence is inserted to replace the corresponding non-human Igλ light chain sequence in the non-human animal phylogenetic genome. In some embodiments, the human Igλ light chain sequence is inserted upstream of the non-human Igλ light chain sequence (e.g., a non-human Igλ light chain constant region sequence). In some embodiments, the human Igλ light chain sequence is inserted into one or more non-human Igλ light chain sequences such that the human Igλ light chain sequence is parallel to the non-human Igλ light chain sequence.

[0180] In some implementations, the Igλ chain locus contains at least 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, or 40 functional V atoms. λ Gene segment. In some implementations, the locus contains human V. λ Gene segment V λ 3-10, V λ 3-9, V λ 2-8, V λ 4-3 and V λ 3-1. In some implementations, the locus contains V λ 2-11, V λ 3-12, V λ 2-14, V λ 3-16, V λ 3-19, V λ 3-21, V λ 3-22, V λ 2-23, V λ 3-25 and V λ 3-27. In some implementations, the locus contains V λ 3-27, V λ 1-36, V λ 5-37, V λ 5-39, V λ 1-40, V λ 7-43, Vλ 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 some implementations, the locus contains V λ 10-54, V λ 6-57, V λ 4-60, V λ 8-61 and V λ 4-69. In some implementations, the Igλ chain locus contains one or more human J... λ -C λ Yes. For example, in some implementations, the Igλ chain locus contains human V. λ Human J gene segment 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. Downstream. In some implementations, Ig λ The chain locus contains human V λ Human J gene segment λ 1-C λ 1. J λ 2-C λ 2. J λ 3-C λ 3. J λ 6-C λ 6. Person J λ 7 and Mouse C λ 1. Downstream.

[0181] In some embodiments, the Igλ locus is a rearranged locus containing an Igλ variable region gene (universal light chain variable region). In some embodiments, the rearranged Igλ variable region gene is a human rearranged Igλ variable region gene. The use of the universal light chain variable region facilitates the generation of bispecific antibodies in which at least one antigen-binding domain has been bound. An exemplary rearranged Ig light chain variable region is provided in U.S. Patent Publication No. 2013 / 0185821, which is incorporated herein by reference.

[0182] In some embodiments, the Igλ chain locus contains a human or rodent (e.g., rat or mouse) regulatory element. In some embodiments, the regulatory element is an endogenous regulatory element. In some embodiments, the Igλ chain locus contains a rodent (e.g., rat or mouse) λ enhancer 2.4. In some embodiments, the Igλ chain locus contains a human or rodent (e.g., rat or mouse) 3′λ enhancer. In some embodiments, the Igλ chain locus contains a rodent (e.g., rat or mouse) λ enhancer 3.1.

[0183] In some embodiments, the modified immunoglobulin λ chain locus is located at the endogenous immunoglobulin λ chain locus. In some embodiments, the immunoglobulin λ chain locus replaces all or part of the endogenous immunoglobulin λ chain locus. In some embodiments, the modified Igλ chain locus is located on a transgene outside the endogenous locus. In some embodiments, the modified Igλ chain locus is located at the endogenous immunoglobulin κ chain locus. In some embodiments, the endogenous Igλ chain locus is inactivated (e.g., through deletion, relocation, and / or inversion of all or part of the endogenous Igλ chain locus).

[0184] In some embodiments, the mice are heterozygous for the modified immunoglobulin λ chain locus. In other embodiments, the mice are homozygous for the modified immunoglobulin λ chain locus.

[0185] In some embodiments, mice contain a light chain immunoglobulin locus in their germline and / or genome that contains a limited library of light chain variable gene segments (e.g., a dual light chain variable region containing two light chain variable gene segments). In some embodiments, the light chain variable gene segments within the limited library of light chain variable gene segments are human light chain gene segments. An exemplary dual light chain variable region is provided in U.S. Patent Publication No. 2013 / 0198880, which is incorporated herein by reference. In some embodiments, mice containing dual light chain variable regions are used to generate bispecific antibodies.

[0186] Therefore, this paper provides an expression of human or humanized FcαR and includes a human light chain constant region (and in some embodiments, also includes human C). α Various mice (constant region) can be used to test MAHA responses to administration of agents containing human Fc (e.g., agents containing human Fcα, such as therapeutic human IgA antibodies), as disclosed in, for example, WO2019 / 190990, which is incorporated herein by reference in its entirety.

[0187] Humanized CD79a and CD79b loci

[0188] In some embodiments, mice and ES cells genetically modified to include the humanized or human FcαR1 locus in their genome also contain the human or humanized B-cell antigen receptor complex-associated protein α chain (CD79a or Igα) and / or B-cell antigen receptor complex-associated protein β chain (CD79b or Igβ) loci. Mice containing the human or humanized CD79a and CD79b genes express human or humanized CD79a and CD79b polypeptides in heterodimeric form on the surface of B cells, which non-covalently associate with membrane-expressed immunoglobulins to form B-cell receptors (BCRs). BCRs associate with antigens and play a role in signal transduction and internalization after antigen binding. In some embodiments, the mice described herein contain the human or humanized CD79a and CD79b genes. In some embodiments, the mice described herein further comprise CD79a and CD79b genes, the CD79a gene comprising a rodent CD79a portion and a human CD79a portion, and the CD79b gene comprising a rodent CD79b portion and a human CD79b portion, wherein the human CD79a portion encodes substantially all extracellular domains of the human CD79a polypeptide (e.g., amino acids corresponding to residues 33-143 of the human CD79a polypeptide), and the human CD79b portion encodes substantially all extracellular domains of the human CD79b polypeptide (e.g., amino acids corresponding to residues 29-159 of the human CD79b polypeptide). In some embodiments, the rodent CD79a and CD79b portions each encode at least the intracellular domains of the endogenous CD79a and CD79b polypeptides, respectively; in some embodiments, they encode at least the transmembrane domains and intracellular domains of the endogenous CD79a and CD79b polypeptides, respectively. In some implementations, the human and endogenous portions are operatively connected to the endogenous CD79a or CD79b promoters, respectively.

[0189] In some embodiments, the mice described herein further comprise a chimeric CD79a gene comprising a rodent CD79a portion and a human CD79a portion, wherein the human CD79a portion encodes a sequence containing amino acids 33-116 corresponding to the human CD79a polypeptide; in one embodiment, it encodes a sequence containing amino acids 33-119 of the human CD79a polypeptide; in another embodiment, it encodes a sequence containing amino acids 33-143 of the human CD79a polypeptide; and in yet another embodiment, it encodes a sequence containing amino acids 33-165 of the human CD79a polypeptide. In some embodiments, the chimeric CD79a polypeptide comprises a human IgC2-like domain; in some embodiments, the chimeric CD79a polypeptide further comprises a human stalk region; in some embodiments, the chimeric CD79a polypeptide further comprises a human transmembrane domain; and in some embodiments, the chimeric CD79a polypeptide further comprises a rodent (e.g., mouse) cytoplasmic domain. In some embodiments, the mouse contains a chimeric CD79a gene that includes the human region portion described herein and a sequence encoding a human or rodent (e.g., mouse) CD79a signal peptide; in one embodiment, the sequence encoding the signal peptide is a mouse CD79a sequence encoding amino acids 1 to 28 of mouse CD79a.

[0190] In some embodiments, the mice described herein further comprise a chimeric CD79b gene comprising a rodent CD79b portion and a human CD79b portion, wherein the human CD79b portion encodes a sequence containing amino acids 29-135 corresponding to the human CD79b polypeptide; in one embodiment, it encodes a sequence containing amino acids 29-159 of the human CD79b polypeptide; and in another embodiment, it encodes a sequence containing amino acids 29-184 of the human CD79b polypeptide. In some embodiments, the chimeric CD79b polypeptide comprises a human IgV-like domain; in some embodiments, the chimeric CD79b polypeptide further comprises a human stalk region; in some embodiments, the chimeric CD79b polypeptide further comprises a human transmembrane domain; and in some embodiments, the chimeric CD79b polypeptide further comprises a rodent (e.g., mouse) cytoplasmic domain. In some embodiments, the mouse contains a chimeric CD79b gene that includes the human region portion described herein and a sequence encoding a human or rodent (e.g., mouse) CD79b signal peptide; in one embodiment, the sequence encoding the signal peptide is a mouse CD79b sequence encoding amino acids 1 to 25 of mouse CD79b.

[0191] GenBank accessions NP_001774.1, NM_001783.3, NP_067612.1, and NM_021601.3, along with UniProt ID P11912, provide representative source sequences for the human CD79A gene and human CD79A peptide, from which the desired human fraction can be obtained. GenBank accessions NP_000617.1, NM_000626.2, NP_001035022.1, NM_001039933.1, NP_067613.1, and NM_021602.2, along with UniProt ID P40259, provide representative source sequences for the human CD79B gene and human CD79B peptide, from which the desired human fraction can be obtained.

[0192] In some embodiments, the mice provided herein also contain one or more human CD79A and CD79B genes, as described in U.S. Patent Application Publication Nos. 2011-0093963A1 and 2009-0053210A1; International Patent Application Publication No. WO 2008 / 027986; and European Patent No. 2064 325B1, each of which is incorporated herein by reference. In some embodiments, the mice provided herein comprise a humanized CD79a gene and a humanized CD79b gene, the humanized CD79a gene comprising an endogenous CD79a portion and a human CD79a portion, and the humanized CD79b gene comprising an endogenous CD79b portion and a human CD79b portion, wherein the human CD79a portion encodes substantially all of the extracellular domains of the human CD79a polypeptide (e.g., amino acids corresponding to residues 33-143 of the human CD79a polypeptide), and the human CD79b portion encodes substantially all of the extracellular domains of the human CD79b polypeptide (e.g., amino acids corresponding to residues 29-159 of the human CD79b polypeptide). In some embodiments, the mice provided herein comprise a humanized CD79a gene and a humanized CD79b gene. The humanized CD79a gene comprises an endogenous CD79a portion and a human CD79a portion, and the humanized CD79b gene comprises an endogenous CD79b portion and a human CD79b portion. The human CD79a portion encodes a sequence comprising amino acids 33-116 (e.g., amino acid sequence comprising amino acids 33-119, amino acid sequence comprising amino acids 33-143, or amino acid sequence comprising amino acids 33-165 of the human CD79a polypeptide), and the human CD79b portion encodes a sequence comprising amino acids 29-135 (e.g., amino acid sequence comprising amino acids 29-159, or amino acid sequence comprising amino acids 29-184 of the human CD79a polypeptide). In some embodiments, the endogenous CD79a and CD79b portions each encode at least the intracellular domains of the endogenous CD79a and CD79b peptides, respectively; in some embodiments, they each encode at least the transmembrane domain and the intracellular domain of the endogenous CD79a and CD79b peptides, respectively.

[0193] Humanized β-2-microglobulin

[0194] In some embodiments, mouse and ES cells genetically modified to include the humanized or human FcαR1 locus in their genome also contain a locus encoding a humanized β-2-microglobulin (β2M) polypeptide. β2M is a polypeptide lacking a transmembrane region and associated with FcRn and MHC class I molecules.

[0195] In some embodiments, the β2M locus contains a nucleic acid sequence encoding a human β2M polypeptide. In some embodiments, the nucleic acid sequence encoding the human β2M polypeptide is located at an endogenous mouse β2M locus. In some embodiments, the nucleic acid sequence encoding the β2M polypeptide replaces all or part of the endogenous mouse β2M gene. In some embodiments, the mouse does not express mouse β2M, or does not express a functional mouse β2M polypeptide. In some embodiments, the β2M locus contains a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer).

[0196] U.S. Patent Publications 2013 / 0111617 and 2013 / 0185819 describe a humanized β2M peptide, a locus encoding the humanized β2M peptide, and mice expressing the humanized β2M peptide, each of which is incorporated herein by reference. Thus, as described in U.S. Patent Publications 2013 / 0111617 and 2013 / 0185819, in some embodiments, the mouse comprises a humanized β2M gene, wherein the gene 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.

[0197] Genetically modified mice and ES cells

[0198] In some respects, this document provides genetically modified mice and ES cells containing one or more humanized loci disclosed herein, as well as genetically modified mouse ES cells for preparing such mice.

[0199] In some aspects, this document provides mice and mouse ES cells that contain genetically modified humanized or human FcαR1 loci in their germline and / or genome. For example, in some embodiments, the mouse or ES cells contain the FcαR locus provided herein in their germline and / or genome. In some embodiments, the mouse or ES cells also contain the IgH locus provided herein. In some embodiments, the mouse or ES cells also contain the Igκ and / or Igλ loci provided herein. In some embodiments, the mouse or ES cells contain the CD79a and / or CD79b loci provided herein in their germline and / or genome. In some embodiments, the mouse or ES cells contain the FcRn locus provided herein in their germline and / or genome. In some embodiments, the mouse or ES cells contain the β2M locus provided herein in their germline and / or genome. In some embodiments, the mouse or ES cells contain the FcεR1α locus provided herein in their germline and / or genome. In some embodiments, the mouse or ES cells contain the FcγR1a locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR2a locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR2b locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR3a locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR3b locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells contain the FcγR2c locus provided herein in their germline and / or genome. In some embodiments, mouse or ES cells are heterozygous for one or more loci provided herein (e.g., genetically engineered loci). In some embodiments, mouse or ES cells are homozygous for one or more loci provided herein (e.g., genetically engineered loci).

[0200] In some embodiments, the mice are C57BL strain mice. 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 mice are 129 strain mice. In some embodiments, the 129 strain is selected from the group consisting of the following strains: 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. In some embodiments, the genetically modified mice are a mixture of the 129 strain and the C57BL strain. In some embodiments, the mice are a mixture of the 129 strain and / or a mixture of the C57BL / 6 strain. In some embodiments, the mixed 129 strain is the 129S6 (129 / SvEvTac) strain. In some embodiments, the mice are the BALB strain (e.g., BALB / c). In some embodiments, the mice are a mixture of the BALB strain and another strain (e.g., the C57BL strain and / or the 129 strain). In some embodiments, the mice provided herein may be mice derived from any combination of the above-mentioned strains.

[0201] Genetically modified mice and ES cells can be produced using any suitable method known in the art. For example, such genetically modified mouse ES cells can be produced using... The technology is 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 mous e genome coupled with high-resolution expression analysis” Nat. Biotech. 21(6):652-659, each of which is incorporated herein by reference. Modification can also be performed using genome-targeting nuclease systems, such as CRISPR / Cas systems, transcription activator-like effector nuclease (TALEN) systems, or zinc finger nuclease (ZFN) systems. In some embodiments, modification is performed using CRISPR / Cas systems, as described, for example, 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. An exemplary method for preparing such genetically modified mice and ES cells is also provided in Example 1 of this document.

[0202] 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... The method produces genetically modified mice, as described in U.S. Patent No. 7,294,754 and Poueymir ou et al., Nature Biotech 25:91-99 (2007), each of which is incorporated herein by reference. The resulting mice can be bred to homozygosity.

[0203] Methods for testing therapeutic agents containing human Fcα

[0204] In some respects, this document provides methods for testing therapeutic proteins containing a human Fcα domain (e.g., a human antibody or an Fcα fusion protein), methods comprising administering the therapeutic protein to mice as provided herein. In some embodiments, this document provides animal models for performing such methods.

[0205] In some embodiments, the administered human antibody or Fc fusion protein has a genetically modified IgH locus in the mouse genome as described herein, derived from human C… H The encoded Fc domain is an isotype and / or allotype that matches the isotype and / or allotype. For example, in some embodiments, the agent is a human IgA1 antibody, and the mouse contains a genetically modified IgH locus containing a C-type encoding the human IgA1 CH1, hinge, CH2, and CH3 domains. HGene segment. In some embodiments, the agent is a human IgA2 antibody, and the mouse contains a genetically modified IgH locus that includes C1, hinge, CH2, and CH3 domains encoding human IgA2. H Gene segment. In some implementations, the agent is an antibody containing a variable domain of the human heavy chain (e.g., an IgA1 or IgA2 antibody).

[0206] In some embodiments, the method includes measuring one or more pharmacokinetic properties of the administered therapeutic protein. In some embodiments, the animal model used to determine the pharmacokinetic properties of the administered human antibody or fusion protein is a genetically modified mouse containing the human FcαR locus, as provided herein.

[0207] In some embodiments, one or more pharmacokinetic parameters include, but are not limited to, area under time (AUC), in vivo recovery (IVR), clearance (CL), mean residence time (MRT), and drug half-life (t). 1 / 2 The pharmacokinetic characteristics of the administered 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 / 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) are determined by administering a selected dose of the therapeutic agent, and then how the plasma concentration of the therapeutic agent changes over time (e.g., 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or more) is determined.

[0208] In some embodiments, the method further includes measuring the therapeutic efficacy of the administered therapeutic protein (e.g., the ability of an administered dose of therapeutic protein to reduce or eliminate one or more disease symptoms in an animal model). In some embodiments, the animal model is a cancer model, and disease symptoms may include, for example, tumor size, tumor metastasis, and / or animal survival. In some embodiments, the animal model is an autoimmune or inflammatory model, and disease symptoms may include, for example, cytokine expression levels, immune cell proliferation, tissue damage, and / or animal survival. In some embodiments, the animal model is an infectious disease model, and disease symptoms may include, for example, levels of infectious agents, tissue damage, and / or animal survival.

[0209] In some embodiments, the method further includes measuring the safety and dosage of the administered therapeutic protein (e.g., the extent to which the administered dose of the therapeutic protein produces one or more side effects in an animal model). Side effects include, but are not limited to, allergic reactions, hair loss, allergic reactions, anemia, loss of appetite, imbalance, bleeding, blood clots, dyspnea, bronchitis, bruising, low white blood cell count, low red blood cell count, low platelet count, cardiotoxicity, conjunctivitis, constipation, cough, dehydration, diarrhea, electrolyte imbalance, infertility, fever, hair loss, heart failure, infection, injection site reaction, iron deficiency, renal failure, leukopenia, liver dysfunction, pneumonia, tachycardia, rectal bleeding, seizures, weight loss, and weight gain. In some embodiments, this document provides methods for measuring allergic reactions induced by the therapeutic agent using passive cutaneous allergy (PCA) and / or passive systemic allergy (PSA) models.

[0210] In some embodiments, the method further includes measuring the extent to which the therapeutic protein induces one or more FcαR-mediated responses in mice (e.g., the extent to which the therapeutic protein induces antibody-dependent cell-mediated cytotoxicity (ADCC)). For example, in some embodiments, this document provides a method for screening therapeutic agents containing the human Fcα region of a human antibody, the method comprising: (a) administering an agent containing the Fcα region of a human antibody to mice provided herein, wherein the agent binds to target cells in the mice; (b) measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against the target cells; and (c) comparing the amount of ADCC in step (b) with a control, wherein increased target cell killing indicates an increased ability of the agent to mediate ADCC.

[0211] In some implementations, the method also includes measuring the extent to which the administration of the therapeutic protein induces an anti-human Fcα immune response in mice.

[0212] In some embodiments, the antibody tested in the methods provided herein has a human light chain variable domain. In some embodiments, the light chain variable domain is a λ light chain variable domain. In some embodiments, the light chain variable domain is a κ light chain variable domain. In some embodiments, the antibody has a human light chain constant domain. In some embodiments, the light chain constant domain is a λ light chain constant domain. In some embodiments, the light chain constant domain is a κ light chain constant domain. The sequences of the human light chain constant regions are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and the IMGT database available at www.imgt.org).

[0213] In some embodiments, the therapeutic agent is administered to mice provided herein as part of a pharmaceutical composition (e.g., a pharmaceutical composition containing a human IgA antibody or Fc fusion protein formulated with a pharmaceutically acceptable carrier).

[0214] The pharmaceutical compositions provided herein can be specifically formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, for example, oral enemas (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for oral, sublingual and systemic absorption), pills, powders, granules, pastes for tongue administration; or (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection, as, for example, sterile solutions or suspensions or sustained-release formulations.

[0215] Pharmaceutical compositions suitable for parenteral administration contain human IgA antibodies or Fc fusion proteins, combined with one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions, emulsions, or sterile powders, which can be reconstituted into sterile injectable solutions or dispersions prior to use. These may contain sugars, alcohols, antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient or a suspension or thickener.

[0216] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (ethyl oleate). Appropriate flowability can be maintained, for example, by using coating materials (such as lecithin), in the case of dispersions by maintaining the desired particle size, and by using surfactants.

[0217] In some embodiments, the composition comprises a human antibody or Fc fusion protein at a concentration that results in a w / v suitable for the desired dose. The antibody may be present in the composition at concentrations of 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, or at least 80 mg / mL. mg / mL, at least 85 mg / mL, at least 90 mg / mL, at least 95 mg / mL, at least 100 mg / mL, at least 105 mg / mL, at least 110 mg / mL, at least 115 mg / mL, at least 120 mg / mL, at least 125 mg / mL, at least 130 mg / mL, at least 135 mg / mL, at least 140 mg / mL, at least 150 mg / mL, at least 200 mg / mL, at least 250 mg / mL, or at least 300 mg / mL.

[0218] In some embodiments, the composition is prepared by mixing a human IgA antibody or Fc fusion protein with an optional physiologically acceptable carrier, excipient, or stabilizer, including but not limited to buffers, sugars, salts, surfactants, solubilizers, polyols, diluents, binders, stabilizers, salts, lipophilic solvents, amino acids, chelating agents, preservatives, etc. (Goodman and Gilman, The Pharmacological Basis of Therapeutics, 12th edition, L. Brunton, et al. and Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1999)), in the form of a lyophilized composition or an aqueous solution at the desired final concentration. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as histidine, phosphate, citrate, glycine, acetate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butyl or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residuals). Polypeptides (based on hydroxyl groups); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including trehalose, glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium ions; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN, polysorbate 80, etc. Or polyethylene glycol (PEG).

[0219] 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.

[0220] In some embodiments, the composition comprises a buffer or pH adjuster to provide improved pH control. The composition may have a pH between about 3.0 and about 9.0, between about 4.0 and about 8.0, between about 5.0 and about 8.0, between about 5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.5 and about 8.0, between about 5.5 and about 7.0, or between about 5.5 and about 6.5. In another embodiment, the composition may 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 composition has a pH of about 6.0. Those skilled in the art will understand that the pH of the composition should generally not be equal to 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 buffers include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. In addition, amino acid components can also act as buffers. Representative amino acid components that can be used as buffers in the composition include, but are not limited to, glycine and histidine. In some embodiments, the buffer is selected from histidine, citrate, phosphate, glycine, and acetate. In some embodiments, the buffer is histidine. In another specific embodiment, the buffer is citrate. In yet another specific embodiment, the buffer is glycine. The purity of the buffer should be at least 98%, or at least 99%, or at least 99.5%. As used herein, the term "purity" in the context of histidine and glycine refers to the chemical purity of histidine or glycine as understood in the art, for example, as described in The Merck Index, 13th edition, edited by O'Neil et al. (Merck & Co., 2001).

[0221] In some embodiments, the composition comprises histidine as a buffer. In some embodiments, the concentration of histidine in the composition is at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 150 mM, or at least about 200 mM. In another embodiment, the composition comprises histidine between about 1 mM and about 200 mM, between about 1 mM and about 150 mM, between about 1 mM and about 100 mM, between about 1 mM and about 75 mM, between about 10 mM and about 200 mM, between about 10 mM and about 150 mM, between about 10 mM and about 100 mM, between about 10 mM and about 75 mM, between about 10 mM and about 50 mM, between about 10 mM and about 40 mM, between about 10 mM and about 30 mM, between about 20 mM and about 75 mM, between about 20 mM and about 50 mM, between about 20 mM and about 40 mM, or between about 20 mM and about 30 mM. In another embodiment, the composition comprises about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM, or about 200 mM of histidine. In some embodiments, the composition may comprise about 10 mM, about 25 mM of histidine, or may not contain histidine.

[0222] In some embodiments, the composition comprises a carbohydrate excipient. The carbohydrate excipient may be, for example, a viscosity enhancer, stabilizer, filler, solubilizer, and / or the like. The carbohydrate excipient is typically present in amounts from about 1% to about 99% by weight or volume, for example, between about 0.1% and about 20%, between about 0.1% and about 15%, between about 0.1% and about 5%, between about 1% and about 20%, between about 5% and about 15%, between about 8% and about 10%, between about 10% and about 15%, between about 15% and about 20%, between 0.1% and about 20%, between 5% and about 15%, between 8% and about 10%, between 10% and about 15%, between 15% and about 20%, between about 0.1% and about 5%, between about 5% and about 10%, or between about 15% and about 20%. In other specific embodiments, the content of the carbohydrate excipient is 1%, or 1.5%, or 2%, or 2.5%, or 3%, or 4%, or 5%, or 10%, or 15%, or 20%.

[0223] In some embodiments, the composition comprises a carbohydrate excipient. Suitable carbohydrate excipients for the composition include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melitriose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucosol), etc. In some embodiments, the carbohydrate excipient used in the compositions 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 still 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%.

[0224] 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% of trehalose. In another embodiment, the composition comprises between about 1% and about 40%, between about 1% and about 30%, between about 1% and about 20%, between about 2% and about 40%, between about 2% and about 30%, between about 2% and about 20%, between about 4% and about 40%, between about 4% and about 30%, or between about 4% and about 20% of trehalose. In yet another embodiment, the composition comprises about 1%, about 2%, about 4%, about 6%, about 8%, about 15%, about 20%, about 30%, or about 40% of trehalose. In some embodiments, the composition comprises about 4%, about 6%, or about 15% of trehalose.

[0225] In some embodiments, the composition comprises an excipient. In some embodiments, the composition comprises at least one excipient selected from the group consisting of 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.

[0226] 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 comprise an amino acid between about 1 mM and about 100 mM, between about 10 mM and about 150 mM, between about 25 mM and about 250 mM, between about 25 mM and about 300 mM, between about 25 mM and about 350 mM, between about 25 mM and about 400 mM, between about 50 mM and about 250 mM, between about 50 mM and about 300 mM, between about 50 mM and about 350 mM, between about 50 mM and about 400 mM, between about 100 mM and about 250 mM, between about 100 mM and about 300 mM, between about 100 mM and about 400 mM, between about 150 mM and about 250 mM, between about 150 mM and about 300 mM, or between about 150 mM and about 400 mM. In a further embodiment, the composition comprises about 1 mM, 1.6 mM, 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about 400 mM of amino acids.

[0227] In some embodiments, the composition comprises a surfactant. As used herein, the term "surfactant" refers to an organic substance having an amphiphilic structure; that is, it consists of groups with opposite solubility, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Based on the charge of the surface-active moiety, surfactants can be classified as anionic, cationic, and nonionic surfactants. Surfactants are commonly used as wetting agents, emulsifiers, solubilizers, and dispersants in various pharmaceutical compositions and biomaterial formulations. Pharmaceutically acceptable surfactants include polysorbates (e.g., polysorbate 20 or 80); poloxamer (e.g., poloxamer 188); Triton; sodium octyl glycoside; lauroyl sulfobetaine, myristoyl sulfobetaine, linoleyl sulfobetaine, or stearoyl sulfobetaine; lauroyl sarcosine, myristoyl sarcosine, linoleyl sarcosine, or stearoyl sarcosine; linoleyl betaine, myristoyl betaine, or hexadecyl sulfobetaine. Betaine; lauramidopropyl betaine, capsicumamide propyl betaine, linoleamide propyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, or isostearamidopropyl betaine (e.g., lauramidopropyl betaine); myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methylcocoyl taurate or disodium methyl oleyl taurate; and the MONAQUA series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., ... PF68, etc., 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 if a pump or plastic container is used to administer the composition. The presence of pharmaceutically acceptable surfactants reduces the tendency for protein aggregation. The composition may contain polysorbate in concentrations ranging from about 0.001% to about 1%, or from about 0.001% to about 0.1%, or from about 0.01% to about 0.1%. In other specific embodiments, the composition comprises polysorbate in concentrations 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%.

[0228] In some embodiments, the composition includes other excipients and / or additives, including but not limited to diluents, binders, stabilizers, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutically acceptable excipients and / or additives may be used in the compositions provided herein. Commonly used excipients / additives, such as pharmaceutically acceptable chelating agents (e.g., but not limited to EDTA, DTPA, or EGTA), may optionally be added to the composition to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the composition.

[0229] In some embodiments, the composition comprises 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 (methylparaben, ethylparaben, propylparaben, butylparaben, etc.), benzalkonium chloride, benzyl chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, may optionally be added to the composition at any suitable concentration, such as between about 0.001% and about 5%, or any range or value thereof. The concentration of the preservative used in the composition is sufficient to produce a microbial effect. Such concentrations depend on the preservative selected and are readily determined by a person skilled in the art.

[0230] In some embodiments, the composition is isotonic with human blood, wherein the composition has a substantially the same osmotic pressure as human blood. Such isotonic compositions typically have an osmotic pressure of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, using a vapor pressure or cryo-osmometer. The tension of the composition is adjusted by using a tension modifier. A “tension modifier” is a pharmaceutically acceptable inert substance that can be added to a composition to provide isotonicity. Tension modifiers suitable for the compositions provided herein include, but are not limited to, sugars, salts, and amino acids.

[0231] In some embodiments, the composition is a pyrogen-free composition that is substantially free of endotoxins and / or associated pyrogenic substances. Endotoxins include toxins confined within microorganisms and released only when the microorganisms are broken down or die. Pyrogenic substances also include heat-stable substances from the outer membranes of bacteria and other microorganisms that cause fever. Both of these substances can cause fever, hypotension, and shock if administered to a human. Due to their potentially harmful effects, even small amounts of endotoxins must be removed from intravenously administered drug solutions. The U.S. Food and Drug Administration (“FDA”) has set a limit of 5 endotoxin units (EUs) per kilogram of body weight per dose for intravenous drug administration (The United States Pharmacopeial Convention, Pharmacopeial Forum 26(1):223(2000)). When therapeutic proteins are administered in doses of hundreds or thousands of milligrams per kilogram of body weight (as in the case of proteins of interest, such as antibodies), even trace amounts of harmful and dangerous endotoxins must be removed. In some embodiments, the endotoxin and pyrogen levels in the composition are less than 10 EU / mg, or less than 5 EU / mg, or less than 1 EU / mg, or less than 0.1 EU / mg, or less than 0.01 EU / mg, or less than 0.001 EU / mg.

[0232] When intended for in vivo administration, the compositions described herein should be sterile. The compositions can be sterilized by various sterilization methods, including sterile filtration, irradiation, etc. In some embodiments, the sterilized composition is filtered through a pre-sterilized 0.22-micron filter. Sterile compositions for injection may be formulated according to standard pharmaceutical practices described in Remington: The Science & Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, (2005). Compositions containing proteins of interest (e.g., antibodies), such as those disclosed herein, will typically be stored in lyophilized or solution form. Sterile compositions containing proteins of interest (e.g., antibodies) are intended to be placed in containers with sterile inlets, such as intravenous bags or vials with connectors that allow retrieval of the composition, such as stoppers that can be punctured by a hypodermic needle. In some embodiments, the compositions are provided as pre-filled syringes.

[0233] In some embodiments, the composition is a lyophilized formulation. The terms "lyophilized" or "freeze-dried" include the state of a substance that has undergone a drying process such as lyophilization, in which at least 50% of the moisture has been removed.

[0234] Regardless of the chosen route of administration, the pharmaceutical agents and / or pharmaceutical compositions provided herein that can be used in a suitable hydrated form shall be formulated into a pharmaceutically acceptable dosage form using conventional methods known to those skilled in the art.

[0235] In the methods provided herein, human antibodies, Fc fusion proteins, and / or pharmaceutical compositions may be delivered via any suitable route of administration, including oral, nasal (e.g., by spray), rectal, vaginal, parenteral, intracerebrospinal, and topical (e.g., by powder, ointment, or drops), including oral and sublingual administration. In some embodiments, common methods are used to deliver the pharmaceutical composition (e.g., via oral or parenteral administration).

[0236] In some embodiments, the actual dose level of the active ingredient in the pharmaceutical composition described herein may be varied to determine the amount of active ingredient that effectively achieves the desired therapeutic response in animal models, compositions, and administration methods and is non-toxic in animal models.

[0237] For example, in some embodiments, the mice described herein are used to determine the pharmacokinetic characteristics of one or more human antibody candidates. In various embodiments, one or more mice and one or more control or reference mice described herein are each exposed to different doses of one or more human antibody candidates (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 antibody can be administered via any desired route of administration, including parenteral and non-parenteral routes. Parenteral routes include, for example, intravenous, intra-arterial, intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intraventricular, intracranial, intrapleural, or other injection routes. Non-parenteral routes include, for example, oral, nasal, transdermal, pulmonary, rectal, oral, vaginal, and ocular administration. Administration can also be achieved through continuous infusion, local application, sustained release from implants (gels, membranes, etc.), and / or intravenous injection. Blood is isolated from mice (humanized and control) at different 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). Various assays can be performed to determine the pharmacokinetic characteristics of the administered candidate therapeutic antibody or Fc fusion peptide using samples obtained from mice as described herein, including but not limited to total IgA, anti-therapeutic antibody response, agglutination, etc.

[0238] In various embodiments, the mice described herein are used to measure the therapeutic effect of blocking or modulating the activity of the peptide of interest, as well as the effect on gene expression due to cellular changes, or, in the case of a receptor peptide, the density of the receptor peptide on the mouse cell surface. In various embodiments, the mice described herein, or cells isolated therefrom, are exposed to a candidate therapeutic agent that binds to the peptide of interest, and after a subsequent period of time, the effect on specific cellular processes associated with the peptide of interest, such as ligand-receptor interactions or signal transduction, is analyzed.

[0239] The mice described herein provide an improved in vivo system for developing and selecting human IgA antibodies or Fc fusion peptides for oncology and / or infectious diseases. In various embodiments, tumors (or tumor cells) can be implanted into the mice described herein and control mice (e.g., those with genetic modifications different from those described herein or those without genetic modifications, i.e., wild-type), or these mice can be infected with a virus (e.g., influenza virus, HIV, HCV, HPV, etc.). Following implantation or infection, a candidate therapeutic agent can be administered to the mice. Sufficient time can be allowed to establish the tumor or virus at one or more sites within the mouse before administration of the candidate therapeutic agent. Optionally and / or additionally, the immune response can be monitored in such mice to characterize and select potential human antibodies that can be developed into therapeutic agents.

[0240] Methods for preparing genetically modified mice and ES cells

[0241] In some aspects, this document provides methods for preparing mouse and ES cells comprising one or more of the genetically modified loci provided herein. Exemplary methods for preparing the genetically modified mouse and ES cells provided herein are described in the specification, examples, and / or figures herein. For example, in some embodiments, this document provides methods for preparing mouse and ES cells comprising the FcαR locus provided herein. In some embodiments, the FcαR locus is inserted between the Pira6 gene and the Ncr1 gene on mouse chromosome 7. In some embodiments, the FcαR locus is inserted between nucleotide residues 4,303,905–4,312,280 on mouse chromosome 7 (GRCm38 assembly).

[0242] In some embodiments, this document provides methods for preparing mouse and ES cells, the mouse and ES cells further comprising the humanized CD79a locus and / or the humanized CD79b locus provided herein. In some embodiments, this document provides methods for preparing mouse and ES cells, the mouse and ES cells further comprising the human or humanized FcRn locus and / or the human or humanized β2M locus provided herein. In some embodiments, this document provides methods for preparing mouse and ES cells, the mouse and ES cells further comprising the human or humanized FcεR1α locus provided herein. In some embodiments, this document provides methods for preparing mouse and ES cells that also comprise the human or humanized FcγR1a locus provided herein. In some embodiments, this document provides methods for preparing mouse and ES cells, the methods further comprising the human or humanized FcγR2a locus, the human or humanized FcγR2b locus, the human or humanized FcγR2c locus, the human or humanized FcγR3a locus, and / or the human or humanized FcγR3b locus provided herein. In some embodiments, this document provides methods for preparing mouse and ES cells that further comprise the human or humanized heavy chain and / or light chain loci provided herein. Exemplary methods for preparing the genetically modified mouse and ES cells provided herein are described in the specification, examples, and / or figures herein.

[0243] Reagent test kit

[0244] This document provides packaging or kits comprising one or more containers containing at least one mouse, mouse cells, DNA fragment, and / or targeting vector as described in the instructions, examples, and / or figures herein. The kit can be used with any applicable method (e.g., research methods). Optionally, associated with such a container may be instructions for manufacture, use, or sale of a drug or biological product as prescribed by a government agency, reflecting (a) agency-approved manufacture, use, or sale for human administration, (b) instructions for use, and (c) protocols 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.

[0245] Exemplary Implementation

[0246] According to exemplary embodiment 1, this document provides a mouse whose genome includes an Fcα receptor (FcαR) locus located in the mouse leukocyte receptor complex (LRC), wherein the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide, the FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain.

[0247] According to exemplary embodiment 2, this document provides a mouse according to embodiment 1, wherein the FcαR locus is located in an intergenic region between the gene loci of the Tthy1 protein and the Rdh13 protein.

[0248] According to exemplary embodiment 3, this document provides a mouse according to embodiment 1 or 2, wherein the FcαR locus is located in the intergenic region between the gene loci of the Lilra5 protein and the Gp6 protein.

[0249] According to exemplary embodiment 4, this document provides a mouse according to any one of embodiments 1 to 3, wherein the FcαR locus is located in an intergenetic region between gene loci of Pira6 protein and Ncr1 protein, such as an intergenetic region between the coding nucleic acid sequences of Pira6 protein and Ncr1 protein.

[0250] According to exemplary embodiment 5, this document provides a mouse according to embodiment 4, wherein the intergenetic region is a 54kb region between the Pira6 and Ncr1 loci.

[0251] According to exemplary embodiment 6, this document provides a mouse according to any one of embodiments 1 to 5, wherein the FcαR locus is located on mouse chromosome 7 (+ strand, GRCm38 assembly) at coordinates 4,303,905–4,312,280.

[0252] According to exemplary embodiment 7, this document provides a mouse according to any one of embodiments 1 to 6, wherein the FcαR locus contains a nucleic acid sequence encoding a human FcαR polypeptide.

[0253] According to exemplary embodiment 8, this document provides a mouse according to any one of embodiments 1 to 7, wherein the FcαR locus contains human exons 1 to 5 of the human Fcα receptor gene.

[0254] According to exemplary embodiment 9, this document provides a mouse according to any one of embodiments 1 to 6, wherein the FcαR locus comprises the non-coding portion of non-mouse rodent FcαR exon 1, the coding portions of human FcαR exons 1 and 2, human FcαR exons 3 and 4, and non-mouse rodent FcαR exon 5.

[0255] According to exemplary embodiment 10, this document provides a mouse according to 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).

[0256] According to exemplary embodiment 11a, this document provides a mouse according to embodiment 7 or 8, wherein the FcαR locus further comprises a nucleic acid sequence present in the human KIR3DL2 gene. Furthermore, according to exemplary embodiment 11b, this document provides a mouse according to embodiment 7, 8, or 11a, wherein the FcαR locus further comprises a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

[0257] According to exemplary embodiment 12, this document provides a mouse according to any one of embodiments 1 to 11, wherein the mouse expresses the FcαR polypeptide on mouse neutrophils, monocytes, macrophages, eosinophils and plasmacytoid dendritic cells.

[0258] According to exemplary embodiment 13, this document provides a mouse according to any one of embodiments 1 to 12, wherein the mouse is heterozygous for the FcαR locus.

[0259] According to exemplary embodiment 14, this document provides a mouse according to any one of embodiments 1 to 12, wherein the mouse is homozygous for the FcαR locus.

[0260] According to exemplary embodiment 15, this document provides a mouse according to any one of embodiments 1 to 14, whose genome further includes a human or humanized Fcγ 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.

[0261] According to exemplary embodiment 16, this document provides a mouse according to embodiment 15, wherein the mouse contains a human or humanized FcγR locus in its genome, the human or humanized FcγR locus containing a nucleic acid sequence encoding a human or humanized FcγR.

[0262] According to exemplary embodiment 17, this document provides a mouse according to embodiment 16, wherein the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more low-affinity FcγRs selected from Fcγ receptor 2a (FcγR2a), Fcγ receptor 2b (FcγR2b), Fcγ receptor 3a (FcγR3a), Fcγ receptor 3b (FcγR3b), and / or Fcγ receptor 2c (FcγR2c).

[0263] According to exemplary embodiment 18, this document provides a mouse according to embodiment 16, wherein the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more FcγRs selected from human or humanized Fcγ receptor 1α (FcγR1a), Fcγ receptor 2a (FcγR2a), Fcγ receptor 2b (FcγR2b), Fcγ receptor 3a (FcγR3a), Fcγ receptor 3b (FcγR3b) and / or Fcγ receptor 2c (FcγR2c).

[0264] According to exemplary embodiment 19, this document provides a mouse according to any one of embodiments 16 to 18, wherein the human or humanized FcγR comprises a human extracellular domain.

[0265] According to exemplary embodiment 20, this document provides a mouse according to any one of embodiments 16 to 19, wherein the human or humanized FcγR comprises a mouse transmembrane domain.

[0266] According to exemplary embodiment 21, this document provides a mouse according to any one of embodiments 16 to 19, wherein the human or humanized FcγR comprises a human transmembrane domain.

[0267] According to exemplary embodiment 22, this document provides a mouse according to any one of embodiments 16 to 21, wherein the human or humanized FcγR comprises a mouse cytoplasmic domain.

[0268] According to exemplary embodiment 23, this document provides a mouse according to any one of embodiments 16 to 21, wherein the human or humanized FcγR comprises a human cytoplasmic domain.

[0269] According to exemplary embodiment 24, this document provides a mouse according to any one of embodiments 16 to 23, wherein the human or humanized FcγR locus is located at the endogenous mouse FcγR locus.

[0270] According to exemplary embodiment 25, this document provides a mouse according to embodiment 24, wherein the nucleic acid sequence encoding human or humanized FcγR replaces all or part of the endogenous mouse FcγR gene.

[0271] According to exemplary embodiment 26, this document provides a mouse according to embodiment 25, wherein the nucleic acid sequence encoding human or humanized FcγR comprises a nucleic acid sequence encoding the extracellular domain of human FcγR, which replaces the endogenous nucleic acid sequence encoding the extracellular domain of mouse FcγR.

[0272] According to exemplary embodiment 27, this document provides a mouse according to any one of embodiments 15 to 26, wherein the mouse does not express mouse FcγR.

[0273] According to exemplary embodiment 28, this document provides a mouse according to any one of embodiments 15 to 27, wherein the mouse is heterozygous for 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.

[0274] According to exemplary embodiment 29, this document provides a mouse according to any one of embodiments 15 to 27, wherein the mouse is homozygous for 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.

[0275] According to exemplary embodiment 30, this document provides mouse embryonic stem cells (ES cells) containing an Fcα receptor (FcαR) locus in the leukocyte receptor complex (LRC) of the mouse genome, wherein the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain.

[0276] According to exemplary embodiment 31, this document provides mouse ES cells according to embodiment 30, wherein the FcαR locus is located in an intergenic region between the gene loci of the Tthy1 protein and the Rdh13 protein.

[0277] According to exemplary embodiment 32, this document provides mouse ES cells according to embodiment 31 or 32, wherein the FcαR locus is located in an intergenic region between the gene loci of the Lilra5 protein and the Gp6 protein.

[0278] According to exemplary embodiment 33, this document provides mouse ES cells according to any one of embodiments 30 to 32, wherein the Fcα receptor (FcαR) locus is located in an intergenic region between gene loci of Pira6 protein and Ncr1 protein, such as an intergenic region between the coding nucleic acid sequences of Pira6 protein and Ncr1 protein.

[0279] According to exemplary embodiment 34, this document provides mouse ES cells according to embodiment 33, wherein the intergenetic region is a 54kb region between the Pira6 and Ncr1 loci.

[0280] According to exemplary embodiment 35, this document provides mouse ES cells according to any one of embodiments 30 to 34, wherein the FcαR locus is located on mouse chromosome 7 (+ strand, GRCm38 assembly) at coordinates 4,303,905–4,312,280.

[0281] According to exemplary embodiment 36, this document provides mouse ES cells according to any one of embodiments 30 to 35, wherein the FcαR locus contains a nucleic acid sequence encoding a human FcαR polypeptide.

[0282] According to exemplary embodiment 37, this document provides mouse ES cells according to any one of embodiments 30 to 36, wherein the FcαR locus contains human exons 1 to 5 of the human Fcα receptor gene.

[0283] According to exemplary embodiment 38, this document provides mouse ES cells according to any one of embodiments 30 to 35, wherein the FcαR locus comprises the non-coding portion of non-mouse rodent FcαR exon 1, the coding portions of human FcαR exons 1 and 2, human FcαR exons 3 and 4, and non-mouse rodent FcαR exon 5.

[0284] According to exemplary embodiment 39, this document provides mouse ES cells according to embodiment 36 or 37, wherein the human or humanized FcαR receptor locus contains a genomic sequence found between coordinates 54,862,297 and 54,906,185 on human chromosome 19 (+ strand, GRCh38 assembly).

[0285] According to exemplary embodiment 40a, this document provides mouse ES cells according to embodiment 36 or 37, wherein the FcαR locus further comprises a nucleic acid sequence present in the human KIR3DL2 gene. Furthermore, according to exemplary embodiment 40b, this document provides mouse ES cells according to embodiment 36, 37, or 40a, wherein the FcαR locus further comprises a nucleic acid sequence present in the 5'UTR of the human NCR1 gene.

[0286] According to exemplary embodiment 41, this document provides mouse ES cells according to any one of embodiments 30 to 40, wherein the ES cells are heterozygous for the FcαR locus.

[0287] According to exemplary embodiment 42, this document provides mouse ES cells according to any one of embodiments 30 to 40, wherein the ES cells are homozygous for the FcαR locus.

[0288] According to exemplary embodiment 43, this document provides mouse ES cells according to any one of embodiments 30 to 42, which further include in their genome human or humanized Fcγ receptor (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.

[0289] According to exemplary embodiment 44, this document provides mouse ES cells according to embodiment 43, wherein the mouse ES cells contain a human or humanized FcγR locus in their genome, the human or humanized FcγR locus containing a nucleic acid sequence encoding a human or humanized FcγR.

[0290] According to exemplary embodiment 45, this document provides mouse ES cells according to embodiment 44, wherein the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more low-affinity FcγRs selected from Fcγ receptor 2a (FcγR2a), Fcγ receptor 2b (FcγR2b), Fcγ receptor 3a (FcγR3a), Fcγ receptor 3b (FcγR3b), and / or Fcγ receptor 2c (FcγR2c).

[0291] According to exemplary embodiment 46, this document provides mouse ES cells according to embodiment 44, wherein the human or humanized FcγR locus comprises a nucleic acid sequence encoding one or more FcγRs selected from human or humanized Fcγ receptor 1α (FcγR1a), Fcγ receptor 2a (FcγR2a), Fcγ receptor 2b (FcγR2b), Fcγ receptor 3a (FcγR3a), Fcγ receptor 3b (FcγR3b), and / or Fcγ receptor 2c (FcγR2c).

[0292] According to exemplary embodiment 47, this document provides mouse ES cells according to any one of embodiments 44 to 46, wherein the human or humanized FcγR contains a human extracellular domain.

[0293] According to exemplary embodiment 48, this document provides mouse ES cells according to any one of embodiments 44 to 47, wherein the human or humanized FcγR comprises a mouse transmembrane domain.

[0294] According to exemplary embodiment 49, this document provides mouse ES cells according to any one of embodiments 44 to 47, wherein the human or humanized FcγR comprises a human transmembrane domain.

[0295] According to exemplary embodiment 50, this document provides mouse ES cells according to any one of embodiments 44 to 49, wherein the human or humanized FcγR comprises a mouse cytoplasmic domain.

[0296] According to exemplary embodiment 51, this document provides mouse ES cells according to any one of embodiments 44 to 49, wherein the human or humanized FcγR comprises a human cytoplasmic domain.

[0297] According to exemplary embodiment 52, this document provides mouse ES cells according to any one of embodiments 44 to 51, wherein the human or humanized FcγR locus is located at an endogenous mouse FcγR locus.

[0298] According to exemplary embodiment 53, this document provides mouse ES cells according to embodiment 52, wherein the nucleic acid sequence encoding human or humanized FcγR replaces all or part of the endogenous mouse FcγR gene.

[0299] According to exemplary embodiment 54, this document provides mouse ES cells according to embodiment 53, wherein the nucleic acid sequence encoding human or humanized FcγR comprises a nucleic acid sequence encoding the extracellular domain of human FcγR, which replaces the endogenous nucleic acid sequence encoding the extracellular domain of mouse FcγR.

[0300] According to exemplary embodiment 55, this document provides mouse ES cells according to any one of embodiments 43 to 54, wherein the mouse is heterozygous for 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.

[0301] According to exemplary embodiment 56, this document provides mouse ES cells according to any one of embodiments 43 to 54, wherein the mouse is homozygous for 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.

[0302] According to exemplary embodiment 57, this document provides a method for testing a human IgA antibody or an Fcα fusion peptide, the method comprising administering an IgA antibody or an Fcα fusion peptide to a mouse according to any one of embodiments 1 to 29.

[0303] According to exemplary embodiment 58, this document provides a method according to embodiment 57, the method further comprising measuring one or more pharmacokinetic properties of an administered human IgA antibody or Fcα fusion peptide.

[0304] According to exemplary embodiment 59, this document provides a method according to embodiment 58, wherein one or more pharmacokinetic properties are selected from plasma concentration-time area under the curve (AUC), in vivo recovery rate (IVR), clearance rate (CL), mean residence time (MRT), and drug half-life (t). 1 / 2 One or more of the following: ) and / or steady-state distributed volume (Vss).

[0305] According to exemplary embodiment 60, this document provides a method according to any one of embodiments 57 to 59, the method further comprising measuring the therapeutic efficacy of the applied human antibody or Fcα fusion peptide.

[0306] According to exemplary embodiment 61, this document provides a method according to any one of embodiments 57 to 60, the method further comprising administering multiple doses of the human antibody or Fcα fusion peptide, and determining the therapeutic efficacy of each dose of the human antibody or Fcα fusion peptide.

[0307] According to exemplary embodiment 62, this document provides a method according to any one of embodiments 57 to 61, the method further comprising administering multiple doses of the human antibody or Fcα fusion peptide, and determining the safety of each dose of the human antibody or Fcα fusion peptide.

[0308] According to exemplary embodiment 63, this document provides a method according to any one of embodiments 57 to 62, the method further comprising administering multiple doses of the human antibody or Fcα fusion peptide, and determining the tolerability of each dose of the human antibody or Fcα fusion peptide.

[0309] According to exemplary embodiment 64, this document provides a method according to any one of embodiments 57 to 63, the method further comprising measuring one or more Fc receptor-mediated responses in mice.

[0310] According to exemplary embodiment 65, this document provides a method according to embodiment 64, wherein the one or more Fc receptor-mediated responses are antibody-dependent cell-mediated cytotoxic (ADCC) responses.

[0311] According to exemplary embodiment 66, this document provides a method according to any one of embodiments 57 to 65, wherein the human antibody binds to target cells in a mouse, and the method further includes measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against the target cells and comparing the amount of ADCC with a control, wherein increased target cell killing indicates an increased ability of the agent to mediate ADCC.

[0312] According to exemplary embodiment 67, this document provides a method according to any one of embodiments 57 to 66, the method further comprising measuring the immune response of the mouse to the human antibody.

[0313] According to exemplary embodiment 68, this document provides a method for modifying a mouse genome, the method comprising: inserting an Fcα receptor (FcαR) locus into a leukocyte receptor complex (LRC) of a mouse genome to modify the mouse genome, wherein the FcαR locus contains a nucleic acid sequence encoding an FcαR polypeptide, the FcαR polypeptide containing a human extracellular domain and a human or rodent cytoplasmic domain.

[0314] According to exemplary embodiment 69, this document provides a method according to embodiment 68, wherein the FcαR locus comprises a nucleic acid sequence encoding a human or humanized FcαR polypeptide.

[0315] According to exemplary embodiment 70, this document provides a method according to embodiment 68 or 69, wherein the FcαR locus is located in an intergenic region between the gene loci of the Tthy1 protein and the Rdh13 protein.

[0316] According to exemplary embodiment 71, this document provides a method according to any one of embodiments 68 to 70, wherein the FcαR locus is located in an intergenic region between gene loci of the Lilra5 protein and the Gp6 protein.

[0317] According to exemplary embodiment 72, this document provides a method according to any one of embodiments 68 to 71, wherein the FcαR locus is located in an intergenetic region between gene loci of Pira6 protein and Ncr1 protein, such as an intergenetic region between the coding nucleic acid sequences of Pira6 protein and Ncr1 protein.

[0318] According to exemplary embodiment 73, this document provides a method according to embodiment 72, wherein the intergenetic region is a 54kb region between the Pira6 and Ncr1 loci.

[0319] According to exemplary embodiment 74, this document provides a method according to any one of embodiments 68 to 73, wherein the FcαR locus is located on mouse chromosome 7 (+ strand, GRCm38 assembly) at coordinates 4,303,905–4,312,280.

[0320] According to exemplary embodiment 75, this document provides a method for preparing mice containing the Fcα receptor (FcαR) gene locus, the method comprising: generating mouse ES cells according to any one of embodiments 30 to 56; and generating mice from said ES cells.

[0321] Example

[0322] Example 1.1: Genetic engineering of mice containing human or humanized FcαR

[0323] Targeting vectors containing nucleic acid sequences encoding human FcαR (MAID20277 and MAID20278) cassettes were generated. MAID20277 and MAID20278 are identical, except that MAID20277 retains the selection marker (i.e., neomycin resistance). In short, using... The technique involves introducing the human FCAR gene into the mouse genome (see, for example, US Patent No. 6,586,251 and Valenzuela et al. (2003) Nat. Biotech. 21(6):652-659, the contents of which are incorporated herein by reference in their entirety). A 44kb genomic sequence of the human FCAR gene (including the promoter) is inserted into the mouse LRC locus located on mouse chromosome 7 between the mouse Pira6 and Ncr1 genes, coordinates chr7:4,303,905-4,312,280 (+ chain; GRCm38 assembly) ( Figure 1A and 1B ).

[0324] More specifically, mouse homologous arms were prepared by PCR amplification using BAC clone RP23-458i16 as a template, as shown in Table 1.

[0325] Table 1

[0326]

[0327]

[0328] Mouse homologous arms were assembled into a construct containing, from 5' to 3': a 5' arm, a spectinomycin resistance cassette (with I-CeuI and PI-SceI homing endonuclease sites attached laterally), a 3' arm, and a chloramphenicol resistance cassette (construct A). In subsequent steps of genetic engineering, the spectinomycin resistance cassette in construct A was replaced with an insert containing human FCAR.

[0329] The human insert containing the complete FCAR gene and a flanked sequence was prepared via two consecutive 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 into the 5' end of the human BAC. In the second BHR step, an approximately 44 kb sequence containing the NCR1 gene was deleted from the 3' end of the human BAC and replaced with a neomycin resistance cassette flanked by a mutant lox site (lox2372-Ub-neo-lox2372) and a 3' PI-SceI site. This construct (construct B) now contains, from 5' to 3': a spectinomycin resistance cassette, an I-CeuI site, a 44,887 bp human genome sequence (GRCh38 coordinates chr19:54,862,297-54,906,185), a lox2372-Ub-neo-lox2372 cassette, and a PI-SceI site. The human sequence includes the last approximately 5 kb of the KIR3DL2 gene (starting from intron 6), the entire FCAR gene (approximately 17 kb), and the 5' UTR of the NCR1 gene.

[0330] To prepare the final targeting vector (named MAID20277), constructs A and B were digested with I-CeuI and PI-SceI restriction enzymes and ligated together. The final targeting vector, from 5' to 3', contains: a 5' mouse homologous arm, an I-CeuI site, a 44,887 bp human genome insertion segment including the FCAR gene, a lox2372-Ub-Neo-lox2372 cassette, a PI-SceI site, a 3' mouse homologous arm, and a chloramphenicol resistance cassette; the final clone was selected based on CM / kanamycin resistance. Figure 2 Table 2 describes the various connections in the final clone.

[0331] Table 2

[0332]

[0333]

[0334] The MAID20277 targeting vector was electroporated into mouse embryonic stem cells (ES) containing a human low-affinity Fcγ receptor. Genetic modification of the low-affinity Fcγ receptor locus is described in U.S. Patent No. 8,658,154. Figure 3 The diagram is illustrated below. Targeted homologous recombination resulted in the deletion of approximately 8.4 kb of mouse sequence (GRCm38 coordinates chr7: 4,303,905–4,312,280) and the insertion of approximately 44 kb of human sequence (including the FCAR gene). Successful integration was confirmed by allele modification (MOA) assays, as described above, for example, by Valenzuela. Primers (forward (F) and reverse (R) primers) and probes used for MOA assays to detect the presence of the human FCAR sequence and the loss of the mouse LRC sequence are described in Table 3 below, and their positions are shown in Table 3. Figure 1A The antibody resistance cassette was subsequently removed by transiently expressing CRE recombinase in ES cell clones. Figure 1B .

[0335] Table 3

[0336]

[0337]

[0338] Positively targeted ES cells (which also contain genes encoding the human low-affinity Fcγ receptor, as described above) were used as donor ES cells, and through... Methods: Microinjection was performed into pre-morula (8-cell) mouse embryos (see, for example, US 7,576,259; US 7,659,442; US 7,294,754; and US 2008-0078000 A1, the contents of which are incorporated herein by reference in their entirety). Mouse embryos containing donor ES cells were incubated in vitro and then implanted into surrogate mothers to produce 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. Heterozygous mice carrying the human FCAR gene were bred to homozygous mice.

[0339] As an alternative to the above strategy, the targeting vector is electroporated into wild-type ES cells (containing endogenous low-affinity Fcγ receptors), and then... The method produces mice. Mice containing human FcαR are bred into mice containing human low-affinity FcγR (with... Figure 3 Mice containing human FcαR (and optionally human low-affinity FcγR) can also be bred into mice containing human or humanized high-affinity FcγR, FcεR, FcRn, β2M using techniques well known in the art; and mice containing humanized Ig heavy chain and / or light chain loci.

[0340] Example 1.2: Phenotypic analysis of genetically engineered mice containing human or humanized FcαR

[0341] FcαR expression in mice containing the human FCAR and low-affinity FCGR genes described in Example 1.1 above was analyzed. All mice were housed and bred under specific pathogen-free conditions. Mice were euthanized, and spleens and blood were collected. Blood was collected into BD microblood collection tubes containing EDTA (catalog number 365973). Red blood cells from the spleen and blood products were lysed with ACK lysis buffer (ThermoFisher, catalog number A1049201) and then washed with complete RPMI medium.

[0342] Both spleen and blood cells were incubated with Live Dead Aqua (ThermoFisher) to exclude non-viable cells. Prior to staining, cells were incubated with anti-mouse CD16 / 32 (2.4G2;BD) on ice for 10 minutes. Cells were then stained on ice for 30 minutes with fluorescently conjugated anti-human CD89 (BioLegend, clone A59) and the following anti-mouse antibodies: CD45 (BioLegend, clone 30-F11), B220 (BioLegend, clone RA3-6B2), CD11c (BioLegend, clone N418), Ly6G (BioLegend, clone 1A8), Ly6C (BioLegend, clone HK1.4), SiglecF (BD Biosciences, clone E50-2440), NKp46 (BioLegend, clone 29A1.4), CD11b (BioLegend, clone M1 / 70), F4 / 80 (BD Biosciences, clone T45-2342), and NK1.1 (BioLegend, clone PK136).

[0343] Lymphocyte and myeloid cell populations were identified by flow cytometry on a BD Symphony A3 instrument (BD Biosciences). First, live single cells were gated for CD45+. The following subsets were then identified: B cells (B220+), cDCs (CD11c++), pDCs (CD11cint, Ly6C+B220+), PMNs (Ly6G+Ly6C+), eosinophils (Ly6G-, SiglecF+CD11b+), macrophages / monocytes (Ly6G-, SiglecF-, F4 / 80+CD11b+), and NK cells (Ly6G-, SiglecF-, F4 / 80-CD11b-, NK1.1+). CD89 (FcαR) expression was determined in each cell type.

[0344] Mice genetically engineered using the MAID20278 kit showed increased hFcαR expression in spleen and blood neutrophils, eosinophils, some blood monocytes / macrophages, and splenic plasmacytoid dendritic cells. No hFcαR expression was observed in splenic monocytes or macrophages. Figures 4A-4B ).

[0345] By incorporating via reference

[0346] All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety, as if specifically and separately each individual publication, patent, or patent application were incorporated by reference. In case of any conflict, this application (including any definitions herein) shall prevail.

[0347] Equivalent solution

[0348] Those skilled in the art will recognize or be able to determine, using only conventional testing, numerous equivalents to specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. A method of making a mouse comprising genetically modifying the mouse to comprise in its genome: an Fc alpha receptor (FcaR) locus in a mouse leukocyte receptor complex (LRC), wherein the FcaR locus comprises a nucleic acid sequence encoding an FcaR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain.

2. The method of claim 1, wherein the FcaR locus is located in an intergenic region between the genetic loci of a Tthy 1 protein and a Rdh 13 protein.

3. The method of claim 1, wherein the FcaR locus is located in an intergenic region between the genetic loci of a Lilra 5 protein and a Gp6 protein.

4. The method of claim 1, wherein the FcaR locus is located in an intergenic region between the genetic loci of a Pira 6 protein and a Ncr 1 protein.

5. The method of claim 1, wherein the FcaR locus is located between coordinates 4,303,905 - 4,312,280 on the + strand of mouse chromosome 7 according to the GRCm38 assembly.

6. The method of claim 1, wherein the FcaR polypeptide comprises a human or rodent transmembrane domain.

7. The method of claim 6, wherein the nucleic acid sequence encodes a full-length human FcaR polypeptide.

8. The method of claim 1, wherein the FcaR locus comprises human exons 1-5 of a human Fca receptor gene.

9. The method of claim 1, wherein the FcaR locus comprises a non-coding portion of a non-mouse rodent FcaR exon 1, a human FcaR exon 1 and 2, a human FcaR exon 3 and 4, and a coding portion of a non-mouse rodent FcaR exon 5.

10. The method of claim 7, wherein the FcaR locus comprises genomic sequences present between coordinates 54,862,297 and 54,906,185 on the + strand of human chromosome 19 according to the GRCh38 assembly.

11. The method of claim 7, wherein the FcaR locus further comprises nucleic acid sequences present in a human KIR3DL2 gene, and / or nucleic acid sequences present in the 5’ UTR of a human NCR1 gene.

12. The method of any one of claims 1-11, wherein the mouse expresses the FcaR polypeptide on mouse neutrophils, monocytes, macrophages, eosinophils, and dendritic cells.

13. The method of any one of claims 1-11, wherein the mouse is heterozygous for the FcaR locus.

14. The method of any one of claims 1-11, wherein the mouse is homozygous for the FcaR locus.

15. The method of any one of claims 1-11, further comprising in its genome a human or humanized Fc gamma receptor (FcyR) locus, a human or humanized IgH locus, a human or humanized IgK locus, a human or humanized IgA locus, a human or humanized FcRn locus, a human or humanized b2M locus, and / or a human or humanized FcsRl a locus.

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

17. The method of claim 16, wherein the FcaR locus is located in an intergenic region between the genetic loci of the Tthy1 protein and the Rdh13 protein.

18. The method of claim 16, wherein the FcaR locus is located in an intergenic region between the genetic loci of the Lilra5 protein and the Gp6 protein.

19. The method of claim 16, wherein the Fc alpha receptor (FcaR) locus is located in an intergenic region between the genetic loci of the Pira6 protein and the Ncr1 protein.

20. The method of claim 16, wherein the FcaR locus is located between coordinates 4,303,905 - 4,312,280 on the + strand of mouse chromosome 7 according to the GRCm38 assembly.

21. The method of claim 16, wherein the FcaR polypeptide comprises a human or rodent transmembrane domain.

22. The method of claim 16, wherein the nucleic acid sequence encodes a full-length human FcaR polypeptide.

23. The method of claim 16, wherein the FcaR locus comprises human exons 1-5 of the human Fca receptor gene.

24. The method of claim 16, wherein the FcaR locus comprises a non-coding portion of non-mouse rodent FcaR exon 1, human FcaR exons 1 and 2, human FcaR exons 3 and 4, and a coding portion of non-mouse rodent FcaR exon 5.

25. The method of claim 22, wherein the FcaR locus comprises genomic sequences present between coordinates 54,862,297 and 54,906,185 on the + strand of human chromosome 19 according to the GRCh38 assembly.

26. The method of claim 22, wherein the FcaR locus further comprises nucleic acid sequences present in the human KIR3DL2 gene, and / or nucleic acid sequences present in the 5’ UTR of the human NCR1 gene.

27. The method of any one of claims 16-26, wherein the ES cell is heterozygous for the FcaR locus.

28. The method of any one of claims 16-26, wherein the ES cell is homozygous for the FcαR locus.

29. The method of any one of claims 16-26, further comprising in its genome a human or humanized Fc gamma receptor (FcγR) locus, a human or humanized IgH locus, a human or humanized IgK locus, a human or humanized IgA locus, a human or humanized FcRn locus, a human or humanized β2M locus, and / or a human or humanized FcεRl a locus.

30. A method of making a mouse comprising an Fc alpha receptor (FcαR) locus, the method comprising: generating a mouse ES cell made by the method of any one of claims 16-29; and generating a mouse from the ES cell.

31. A method of testing a human IgA antibody or Fcα fusion polypeptide, the method comprising administering the IgA antibody or Fcα fusion polypeptide to a mouse made by the method of any one of claims 1 to 15, 30.

32. The method of claim 31, 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 the plasma concentration versus time curve (AUC), in vivo recovery (IVR), clearance (CL), mean residence time (MRT), drug half-life (t 1 / 2), and / or steady state volume of distribution (Vss).

33. The method of claim 31, further comprising measuring the therapeutic efficacy of the administered human IgA antibody or Fcα fusion polypeptide.

34. The method of any one of claims 31-33, further comprising administering a plurality of 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 antibody or Fcα fusion polypeptide.

35. The method of any one of claims 31-33, further comprising measuring one or more Fc receptor-mediated responses in the mouse, optionally wherein the one or more Fc receptor-mediated responses comprise an antibody-dependent cell-mediated cytotoxicity (ADCC) response.

36. The method of any one of claims 31-33, wherein the human IgA antibody binds to a target cell in the mouse, and the method further comprises measuring antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer (NK) cells against the target cell, and comparing the amount of ADCC to a control, wherein increased target cell killing indicates an increased ability of the ADCC.

37. The method of any one of claims 31-33, further comprising measuring an immune response generated by the mouse against the human IgA antibody.

38. A method of modifying a mouse genome, the method comprising: inserting an Fc alpha receptor (FcαR) locus into a leukocyte receptor complex (LRC) of a mouse genome, thereby modifying 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.

39. The method of claim 38, wherein the FcαR polypeptide comprises a human or rodent transmembrane domain.

40. The method of claim 38, wherein the nucleic acid sequence encodes a full-length human FcαR polypeptide.

41. The method of any one of claims 38-40, wherein the FcαR locus is located in an intergenic region between the gene loci of Tthy1 protein and Rdh13 protein.

42. The method of any one of claims 38-40, wherein the FcαR locus is located in an intergenic region between the gene loci of Lilra5 protein and Gp6 protein.

43. The method of any one of claims 38-40, wherein the FcαR locus is located in an intergenic region between the gene loci of Pira6 protein and Ncr1 protein.

44. The method of any one of claims 38-40, wherein the FcαR locus is between coordinates 4,303,905 - 4,312,280 on the + strand of mouse chromosome 7 assembled according to GRCm38.

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