WO2011084664A1 - HUMANIZED FCγ R MICE - Google Patents

HUMANIZED FCγ R MICE Download PDF

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Publication number
WO2011084664A1
WO2011084664A1 PCT/US2010/060925 US2010060925W WO2011084664A1 WO 2011084664 A1 WO2011084664 A1 WO 2011084664A1 US 2010060925 W US2010060925 W US 2010060925W WO 2011084664 A1 WO2011084664 A1 WO 2011084664A1
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WIPO (PCT)
Prior art keywords
human
mouse
fcyr
cell
chain
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Ceased
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PCT/US2010/060925
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English (en)
French (fr)
Inventor
Lynn Macdonald
Naxin Tu
Cagan Gurer
Sean Stevens
Andrew J. Murphy
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=43601078&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2011084664(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to EP18154933.8A priority Critical patent/EP3336102A3/en
Priority to SG2012043683A priority patent/SG181690A1/en
Priority to DK10791039.0T priority patent/DK2516457T3/en
Priority to KR1020127019435A priority patent/KR101862277B1/ko
Priority to EP10791039.0A priority patent/EP2516457B9/en
Priority to LTEP10791039.0T priority patent/LT2516457T/lt
Priority to JP2012544863A priority patent/JP5595517B2/ja
Priority to RU2012131296/10A priority patent/RU2549695C2/ru
Priority to ES10791039.0T priority patent/ES2671188T3/es
Priority to AU2010339863A priority patent/AU2010339863B2/en
Priority to PL10791039T priority patent/PL2516457T3/pl
Priority to NO10791039A priority patent/NO2516457T3/no
Priority to EP19154880.9A priority patent/EP3498730B1/en
Priority to KR1020207007903A priority patent/KR102178064B1/ko
Priority to SM20180296T priority patent/SMT201800296T1/it
Priority to CA2784953A priority patent/CA2784953C/en
Priority to KR1020197011653A priority patent/KR102092744B1/ko
Priority to SI201031700T priority patent/SI2516457T1/en
Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Priority to KR1020187014568A priority patent/KR101973524B1/ko
Priority to CN201080064262.4A priority patent/CN102762590B/zh
Priority to RS20180653A priority patent/RS57280B1/sr
Priority to HRP20180911TT priority patent/HRP20180911T1/hr
Publication of WO2011084664A1 publication Critical patent/WO2011084664A1/en
Priority to IL220365A priority patent/IL220365A/en
Anticipated expiration legal-status Critical
Priority to IL242719A priority patent/IL242719A/en
Priority to CY20181100608T priority patent/CY1120316T1/el
Priority to IL261257A priority patent/IL261257B/en
Priority to IL271083A priority patent/IL271083A/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0387—Animal model for diseases of the immune system

Definitions

  • the field of invention is genetically modified non-human animals that lack endogenous murine FcyR genes, including genetically modified animals that comprise a replacement of endogenous FcyR genes with human Fc R genes, and including mice that are capable of expressing at least two, three, four, or five functional human low affinity FcyR genes, and including genetically modified mice comprising immune cells that do not express endogenous low affinity FcyR genes.
  • Fc receptors are proteins found on the surface of cells of the immune system that carry out a variety of functions of the immune system in mammals. FcRs exist in a variety of types, on a variety of cells, and mediate a variety of immune functions such as, for example, binding to antibodies that are attached to infected cells or invading pathogens, stimulating phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity
  • ADCC is a process whereby effector ceils of the immune system lyse a target cell bound by antibodies. This process depends on prior exposure to a foreign antigen or cell, resulting in an antibody response.
  • ADCC can be mediated through effector cells such as, for example, natural killer (NK) cells, by binding of FcR expressed on the surface of the effector cell to the Fc portion of the antibody which itself is bound to the foreign antigen or cell.
  • effector cells such as, for example, natural killer (NK) cells
  • Figure 1 is a schematic depiction of a wild type low affinity FcyR locus in a mouse, showing mouse FcyRIIB, FcyRIV and FcyRIII genes and a mouse FcyR targeting vector used for a targeted deletion of these genes, which includes a neomycin cassette flanked by site-specific recombination sites.
  • Figure 2 shows histograms of splenocytes gated for B ceils (anti-CD19), NK cells (anti-NKp46) and macrophages (anti-F4/80) including expression of endogenous mFcyRII and mFcyRIII genes for wild type and low affinity FcyR -chain gene-deficient mice (mFcyR KO).
  • Figures 3A-3D show in vivo depletion of B cells with a human anti-human CD20 antibody with mouse Fc (Ab 168) or human Fc (Ab 735) in humanized CD20 mice (hCD20) and humanized CD20 mice bred to FcyR knockout mice (hCD20/FcyR KO) in several lymphocyte compartments: bone marrow ( Figure 3A), blood ( Figure 3B), lymph node (Figure 3C) and spleen (Figure 3D),
  • the y-axis shows the percent of gated B cells (B2207lgM + or B220 + /CD19 + ) and the x-axis shows the antibody dose for each animal group: 10 mg/kg Control antibody (C), 2 mg/kg human anti-human CD20 antibody (2 Ab) and 0 mg/kg human anti-human CD20 antibody (10 Ab).
  • FIG 4 is a schematic depiction of a neomycin-targeted deletion of the low-affinity mouse FcyR locus and a second targeting vector for inserting two human low affinity FcyR genes (hFcyRIIIA and hFcyRIIA) into the deleted mouse locus, which includes a hygromycin cassette flanked by site-specific recombination sites.
  • hFcyRIIA For expression of hFcyRIIA on platelets, an extended promoter region operably linked to the hFcyRIIA gene of the Human FcyRIIIA-IIA Targeting Vector is employed; to prevent expression of hFcyRIIA on platelets, the promoter region is omitted or substantially omitted.
  • Figure 5A shows histograms of splenocytes gated for NK cells (anti- NKp46) and macrophages (anti-F4/80) including expression of human FcyRIIIA for wild type and human FcyRIIIA-IIA homozygote mice (Human FcyRIIIA FcyRIIA HO).
  • Figure 5B shows histograms of splenocytes gated for neutrophils (anti- Ly6G) and macrophages (anti-F4/80) including expression of human FcyRIIA for wild type and human FcyRIIIA-IIA homozygote mice (Human FcyRIIIA/FcyRIIA HO).
  • FIG. 6 is a schematic depiction of a hygromycin-targeted deletion of the low affinity mouse FcyR locus including an insertion of two low affinity human FcyR genes (hFcyRIIIA and hFcyRIIA) and a third targeting vector for inserting three additional low affinity human FcyR genes (hFcyRIIB, hFcyRIIIB and hFcyRIIC) and a neomycin cassette flanked by site-specific recombination sites.
  • Figure 7 shows histograms of splenocytes gated for B cells (anti-CD19) and neutrophils (anti-Ly6G) including expression of human FcyRIIB and human FcyRIIIB for wild type and human FcyRIIIA-IIIB-IIA-IIB-IIC homozygote mice (Human FcyRIIIA/FcyRIIIB/FcyRIIA/FcyRIIB/FcyRIIC HO).
  • non-human animals comprise a human FcyR receptor, a deletion of an endogenous low affinity FcyR receptor, and/or a replacement of an endogenous FcyR receptor with a human FcyR receptor at an endogenous mouse low affinity FcyR locus.
  • genetically modified cells, non-human embryos, and non- human animals comprise a functional FcR ⁇ -chain, wherein the cells, embryos, and animals comprise a further modification comprising a replacement of the low affinity endogenous non-human FcyR gene sequences (e.g., FcyRIIB, FcyRIV and FcyRIII) with one or more low affinity human FcyR gene sequences (e.g., selected from FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA, FcyRIIIB, and a combination thereof).
  • the low affinity endogenous non-human FcyR gene sequences e.g., FcyRIIB, FcyRIV and FcyRIII
  • low affinity human FcyR gene sequences e.g., selected from FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA, FcyRIIIB, and a combination thereof.
  • the cells, non-human embryos, and non-human animals are murine.
  • the functional FcR ⁇ -chain is a mouse FcR ⁇ -chain.
  • the mouse FcR ⁇ -chain is an FcR y-chain endogenous to the mouse, the cell, or the embryo.
  • the cells, embryos, and animals are mice, and the mice express a functional a-chain of a human low affinity FcyR receptor and a functional endogenous mouse y-chain.
  • a genetically modified mouse wherein the mouse does not express an endogenous a-chain selected from an FcyRIIB a-chain, an FcyRIV a-chain, an FcyRIII a-chain, and a combination thereof; wherein the mouse expresses a functional endogenous mouse ⁇ -chain.
  • the mouse does not express a functional FcyRIIB a-chain, does not express a functional FcyRIV a-chain, and does not express a functional FcyRIII a-chain.
  • the mouse genome comprises a deletion of an endogenous FcyRIIB a-chain, a deletion of an endogenous FcyRIV a-chain, and a deletion of an endogenous FcyRIII a-chain.
  • the mouse comprises a deletion of an endogenous FcyRIIB a-chain, a deletion of an endogenous FcyRIV a-chain, and a deletion of an endogenous FcyRIII a-chain, and further comprises a reduced ability to make an immune response to an antigen as compared with a wild type mouse's ability with respect to the same antigen.
  • the reduced immune response includes a decreased antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the reduced immune response includes a reduced ability in a cell killing assay to achieve antibody-dependent NK cell killing.
  • the reduction in ADCC or antibody-dependent NK cell killing is at least 50%, in one embodiment at least 75%, in one embodiment at least 90%.
  • the mouse comprises a deletion of an endogenous FcyRIIB a-chain, a deletion of an endogenous FcyRIV a-chain, and a deletion of an endogenous FC7RIII a-chain, and further comprises an increased humoral antibody response upon immunization with an antigen as compared to a wild type mouse, e.g., a mouse of the same or similar strain that does not comprise the deletion.
  • the increased humoral antibody response is 2-fold as compared to a wild type mouse.
  • the increased humoral antibody response is 3-fold as compared to a wild type mouse.
  • the increased humoral antibody response is 5-fold as compared to a wild type mouse.
  • the increased humoral antibody response is 7-fold as compared to a wild type mouse. In one embodiment, the increased humoral antibody response is 10-fold as compared to a wild type mouse. In a specific embodiment, humoral antibody response is measured by micrograms of antibody that specifically binds an antigen (with which the mouse has been immunized) per microgram of serum protein from the mouse. In one embodiment, the increased humoral antibody response is with respect to an antigen to which a wild type mouse exhibits tolerance, or to an antigen which in a wild type mouse exhibits a poor or minimal humoral immune response. In a specific embodiment, the antigen is a mouse antigen. In a specific embodiment, the antigen is a human antigen that exhibits an identity with a mouse protein of at least about 95%, 96%, 97%, 98%, or 99%.
  • a genetically modified mouse comprising a replacement of a low affinity mouse FcyR a-chain gene with a low affinity human FcyR a-chain gene, wherein the replacement is at the endogenous mouse FcyR a- chain gene locus.
  • the low affinity mouse FcyR a-chain gene is selected from an FcyRIIB, FcyRIV and an FcyRIII a-chain gene.
  • a genetically modified mouse is provided, wherein the mouse expresses an endogenous FcR ⁇ -chain, and wherein the low affinity human FcyR a- chain gene is FcyRIIIA a-chain.
  • the genetically modified mouse expresses an endogenous FcR ⁇ -chain and a functional human FcyRIIIA a-chain on NK cells.
  • the functionality of FcyRIIIA ⁇ -chain on NK cells is reflected by human antibody-mediated NK killing (e.g., ADCC mediated by a human antibody).
  • a genetically modified cell, non-human embryo, or non- human animal wherein the genetic modification comprises a replacement of at least one endogenous low affinity FcyR -chain gene with a human FcyR cc- chain gene, and the cell, embryo, or animal expresses a functional FcR ⁇ -chain.
  • the functional FcRy-chain is an endogenous FcR ⁇ -chain.
  • the low affinity human FcyR a-chain gene is selected from an FcyRIIA a-chain gene, an FcyRIIIA a-chain gene, and a combination thereof.
  • the human FcyRIIA gene comprises a polymorphism, wherein the polymorphism is selected from a 131 His low responder polymorphism and a 131Arg high responder polymorphism. In a specific embodiment, the FcyRIIA polymorphism is the 131 His low responder polymorphism.
  • the FcyRI IIA gene is a specific allelic variant, wherein the allelic variant is selected from a 158Val variant and a 158Phe variant. In a specific embodiment, the FcyRIIIA allelic variant is the 158Val variant.
  • the low affinity human FcyR gene is selected from an FcyRIIB, FcyRIIC, an FcyRIIIB gene, and a combination thereof.
  • the human FcyRIIB gene comprises an amino acid substitution, wherein the substitution is selected from an 232lle or a 232Thr substitution.
  • amino acid substitution is a 232lle substitution.
  • the FcyRIIIB gene is a specific allelic variant, wherein the allelic variant is selected from a NA1 variant and a NA2 variant.
  • the FcyRIIIB allelic variant is a NA2 variant.
  • the low-affinity human FcyR a-chain gene is selected from a FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA, FcyRIIIB a-chain gene, and a
  • the low affinity mouse FcyRIV a-chain gene and the FcyRIII a-chain gene are replaced with at least one low affinity human FcyR a-chain gene.
  • the low affinity mouse FcyRIV a-chain gene and the FcyRIIB a-chain gene are replaced with at least one low affinity human FcyR a-chain gene.
  • the low affinity mouse FcyRIIB a-chain gene and the FcyRIII a-chain gene are replaced with at least one low affinity human FcyR a-chain gene.
  • the at least one low affinity human FcyR a-chain gene is selected from an FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA , FcyRIIIB a-chain gene, and a combination thereof.
  • the at least one low affinity human FcyR a-chain gene is selected from an FcyRIIA a-chain gene, an FcyRIIIA a-chain gene, and a combination thereof.
  • the at least one low affinity human FcyR a-chain gene is selected from an FcyRIIB, FcyRIIC, FcyRIIIB a-chain gene, and a combination thereof.
  • the low affinity mouse FcyR genes are replaced with a human FcyRIIA a-chain gene and a human FcyRIIIA a-chain gene.
  • the low affinity human FcyRIIA and FcyRIIIA a-chain genes comprise variants, wherein the FcyRIIA a-chain gene comprises a 131 His variant and the FcyRIIIA a- chain gene comprises a 58Val variant.
  • the low affinity mouse FcyR a-chain genes are replaced with the following low affinity human FcyR a-chain genes: FcyRIIB, FcyRIIC and FcyRIIIB.
  • the low affinity human FcyRIIB a-chain gene and FcyRIIIB a-chain gene comprise variants, wherein the FcyRIIB a-chain gene comprises a 232lle variant and the FcyRIIIB a-chain gene comprises an NA2 variant.
  • the genetic modifications comprise a replacement of syntenic genomic sequences of mouse and human chromosome 1.
  • the genetic modifications comprise a replacement of a genomic fragment comprising endogenous low affinity mouse FcyR genes with a genomic fragment comprising low affinity human FcyR genes.
  • the mouse genome from chromosome 1 :172,889,983 to
  • chromosomel :172,989,91 1 is replaced with a human genomic fragment comprising human chromosome 1 : 161 ,474,729 to chromosomel .161 ,620,458.
  • a genetically modified cell, non-human embryo, or non- human animal wherein the genetic modification comprises a knockout of one or more endogenous low affinity receptor a-chain genes, and the presence of an episome comprising one or more human FcyR a-chain genes.
  • the cell, embryo, or animal expresses a functional FcR y-chain.
  • the episome is a mini chromosome.
  • the functional FcR ⁇ -chain is endogenous to the cell, embryo, or animal.
  • a genetically modified mouse comprising a replacement of a low affinity mouse FcyR a-chain gene with a low affinity human FcyR a-chain gene, the mouse comprises a mouse FcRy-chain gene, and the mouse expresses a functional human low affinity FcyR receptor.
  • the functional low affinity FcyR receptor is expressed on a cell type in which the low affinity FcyR receptor is expressed in humans.
  • the functional human low affinity FcyR receptor is FcyRIIIA and the FcyRIIIA is expressed on NK cells.
  • the mouse comprises a deletion of two mouse FcyR a-chain genes. In another embodiment, the mouse comprises a deletion of three mouse FcyR a-chain genes.
  • the mouse comprises a replacement of three mouse FcyR a-chain genes with at least one human FcyR a-chain gene. In another embodiment, the mouse comprises a replacement of two mouse FcyR a-chain genes with at least one human FcyR a-chain gene. In a specific embodiment, the mouse comprises a replacement of three mouse FcyR a-chain genes with at least two human FcyR a-chain genes. In another specific embodiment, the three mouse FcyR a-chain genes are replaced with three human FcyR a-chain genes. In another specific embodiment, the mouse comprises a replacement of two mouse FcyR a- chain genes with at least two human FcyR a-chain genes. In yet another specific embodiment, the two mouse FcyR a-chain genes are replaced with at least three human FcyR a-chain genes.
  • the low affinity mouse FcyR a-chain gene is selected from an FcyRIIB, FcyRIV, FcyRIII a-chain gene, and a combination thereof.
  • the low affinity human FcyR a-chain gene is selected from an FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA, FcyRIIIB a-chain gene, and a combination thereof. In one embodiment, the low affinity human FcyR a-chain gene is selected from an FcyRIIA, an FcyRIIIA a-chain gene, and a combination thereof. In one embodiment, the low affinity human FcyR a-chain gene is selected from an FcyRIIB, FcyRIIC, an FcyRIIIB a-chain gene, and a combination thereof.
  • the low affinity mouse FcyRIV a-chain gene and the FcyRIII a-chain gene are replaced with at least one human FcyR a-chain gene.
  • the low-affinity mouse FcyRIV a-chain gene and the FcyRIIB a- chain gene are replaced with at least one human FcyR a-chain gene.
  • the low affinity mouse FcyRIIB a-chain gene and the FcyRIIIB a-chain gene are replaced with at least one human FcyR a-chain gene.
  • the at least one human FcyR a-chain gene is selected from an FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA, FcyRIIIB a-chain gene, and a combination thereof.
  • the at least one human FcyR a-chain gene is selected from an FcyRIIA, an FcyRIIIA a-chain gene, and a combination thereof.
  • the at least one human FcyR a-chain gene is selected from an FcyRIIB, FcyRIIC, FcyRIIIB a-chain gene, and a combination thereof.
  • mouse a-chain genes are replaced with the following human FcyR a-chain genes: FcyRIIA and FcyRIIIA.
  • the mouse a-chain genes are replaced with the following human FcyR a-chain genes: FcyRIIB, FcyRIIC and FcyRIIIB.
  • a genetically modified mouse comprising a low affinity human FcyR a-chain and a mouse FcR ⁇ -chain subunit, wherein the mouse expresses the human FcyR a-chain on a cell selected from a neutrophil, an eosinophil, a basophil, a monocyte, a macrophage, a platelet, a Langerhans cell, a dendritic cell, an NK cell, a mast cell, a B cell, a T cell, and a combination thereof.
  • the mouse expresses a human FcyRIIA a-chain on a cell selected from a neturophil, a macrophage, an eosinophil, a platelet, a dendritic cell, a
  • the mouse is capable of phagocytosis, ADCC and cellular activation initiated or mediated through the expressed human FcyRIIA a-chain.
  • the mouse expresses a human FcyRIIIA a-chain on a cell selected from a macrophage, an NK cell, a monocyte, a mast cell, an eosinophil, a dendritic cell, a Langerhans cell, at least one T cell type, and a combination thereof.
  • the mouse is capable of ADCC mediated through the human FcyRIIIA a-chain expressed on NK cells.
  • the mouse exhibits hFcyRIIIA-mediated ADCC in response to an antibody comprising a human Fc.
  • the mouse expresses both a human FcyRIIA a-chain and a human FcyRIIIA a-chain.
  • the human FcyRIIA a-chain is expressed on platelets and the human FcyRIIIA a-chain is expressed on NK cells.
  • the mouse is capable of ADCC mediated by an antibody comprising a human Fc, wherein the mediation is through either the human FcyRIIA a-chain or through the human FcyRIIIA a-chain expressed on the surface of accessory cells.
  • the human FcyRIIA a-chain is not expressed on platelets.
  • the mouse lacks or substantially lacks a human promoter sequence that operably linked to the human FcyRIIA a-chain in a human genome.
  • the mouse expresses a human FcyRIIB a-chain on a cell selected from a B cell, a mast cell, a basophil, a macrophage, an eosinophil, a neutrophil, a dendritic cell, a Langerhans cell, and a combination thereof.
  • the mouse expresses a human FcyRIIB a-chain on a B cell and a mast cell.
  • the mouse is capable of endocytosis of immune complexes mediated through the expressed human FCYRIIB a-chain.
  • the mouse expresses a human FcyRIIC a-chain on a cell selected from a neutrophil, a macrophage, an eosinophil, a platelet, a dendritic cell, a Langerhans cell, and a combination thereof.
  • the mouse is capable of phagocytosis, ADCC and cellular activation initiated through the expressed human FCYRI IC a-chain.
  • the mouse expresses a human FcyRIIIB a-chain on neutrophils and eosinophils.
  • the mouse is capable of cellular activation, phagocytosis, ADCC and degranulation, wherein the activation, phagocytosis, ADCC, and degranulation are mediated through the expressed human FcyRIIIB a-chain.
  • a mouse comprises a deletion of the endogenous FCYRI IB, FcyRIV and FcyRII I genes and insertion of human FCYRI IA, FCYRIIB, FCYRIIC, FCYRI IIA, and FCYRIIIB genes, and wherein the mouse comprises a functional mouse FcR ⁇ -chain gene.
  • the mouse comprises a deletion of the a-chains encoded by endogenous FCYRI IB, FCYRIV and FcyRIII genes and insertion of the a- chains encoded by human FCYRI IA, FCYRI IB, FCYRI IC, FCYRI I IA, and FCYRI II B genes.
  • the insertion of the human FCYRI IA, FCYRI IB, FCYRI IC, FCYRII IA, and FCYRI I IB a-chain genes is at a random location within the mouse genome.
  • the insertion of the human FCYRI IA, FCYRI IB, FCYRIIC, FCYRI IIA, and FCYRI I IB a-chain genes is at the endogenous mouse low affinity Fc R a-chain locus.
  • the mouse expresses human FCYRI I IA on NK cells and macrophages.
  • all or substantially all NK cells from a splenocyte sample of the mouse express human FCYRIIIA.
  • all or substantially all macrophages from a splenocyte sample of the mouse express human FCYRI IIA.
  • the mouse expresses a human FcyR selected from human FCYRI IA, human FCYRI I IA, and a combination thereof, on a cell type selected from neutrophils, macrophages, and a combination thereof.
  • a human FcyR selected from human FCYRI IA, human FCYRI I IA, and a combination thereof, on a cell type selected from neutrophils, macrophages, and a combination thereof.
  • the mouse expresses human FCYRI IA and human FCYRI IIA on all or substantially all neutrophils and macrophages of a splenocyte sample from the mouse.
  • the mouse expresses human FcyRI IB and human FcyRII IB on B cells and neutrophils of B cells from a B cell-gated splenocyte sample from the mouse.
  • the mouse expresses FcyRIIIB and FcyRIIB on all or substantially all B cells and neutrophils from a B cell-gated splenocyte sample from the mouse.
  • the mouse further comprises a humanized CD20 gene.
  • the mouse that further comprises the humanized CD20 gene following treatment with an anti-CD20 binding protein that comprises an Fc exhibits depletion (in vivo) of B cells.
  • the depletion is in a compartment selected from bone marrow, blood, lymph node, spleen, and a combination thereof.
  • the Fc is a human Fc.
  • the Fc is a mouse Fc.
  • the anti-CD20 binding protein is an anti-CD20 antibody.
  • a cell comprising a genetic modification as described herein.
  • the cell is selected from an embryonic stem (ES) cell, a pluripotent cell, an induced pluripotent cell, and a totipotent cell.
  • the cell is selected from a mouse cell and a rat cell.
  • the cell is an ES cell.
  • the cell is a mouse ES cell.
  • a non-human embryo comprising a genetic modification as described herein.
  • the non-human embryo is selected from a mouse embryo and a rat embryo.
  • a method for determining efficacy of a therapeutic.
  • the therapeutic is an antibody (e.g. , mono-, bi-, tri-, multispecific) comprising a human Fc.
  • the therapeutic is a human antibody.
  • the efficacy is efficacy of therapeutic-mediated cell killing (e.g. , ADCC).
  • the human therapeutic is a fusion protein comprising an Fc of a human immunoglobulin heavy chain.
  • the therapeutic is administered to a mouse as described herein and a level of therapeutic-dependent ADCC is measured.
  • the mouse is used to assess the ADCC activity of a therapeutic by administering the therapeutic to the mouse and then detecting (e.g. , in vitro from a sample (e.g., blood) taken from the animal) binding of the therapeutic to a human low affinity FcyR on an FcyR- expressing cell.
  • accessory cells of the mouse are isolated from the mouse and tested for the ability, in the presence and absence of the therapeutic, to mediate therapeutic-dependent ADCC.
  • a method for determining whether a low affinity FcyR is associated with a human disease or disorder comprising a step of determining a trait associated with the human disease or disorder in a mouse according to the invention.
  • the trait is a phenotype associated with the absence or loss of a function of one or more low affinity FcyRs.
  • the disease or disorder is an autoimmune disease or disorder.
  • the autoimmune disease or disorder is selected from
  • the mouse comprises a polymorphism in a low affinity FcyR, and the trait is selected from an enhanced ability to mediate ADCC in comparison to the majority of the human population that does not bear the polymorphism, and a reduced ability to mediate ADCC in comparison to the majority of the human population that does not bear the polymorphism.
  • a method for making an anti-human FcR a-chain antibody in a mouse comprising exposing a mouse according to the invention to a human FcR as described herein.
  • an antibody that recognizes the human FcR is isolated from the mouse.
  • a nucleic acid sequence that encodes all or part of a variable region of an antibody that recognizes the human FcR is identified and cloned.
  • a method for determining ability of anti-human FcR antibodies to target molecules to FcR-expressing cells for phagocytosis of the target molecule comprising exposing a mouse as described herein to an agent comprising an anti-human FcR antibody, and measuring phagocytosis of the target molecule.
  • a method for making an antibody, in a mouse, to an antigen that is poorly immunogenic in a mouse that is wild type with respect to one or more FcyRs, comprising exposing a mouse as described herein that lacks a mouse low affinity FcR but expresses an FcyR ⁇ -chain to the antigen that is poorly immunogenic in the mouse that is wild type with respect to one or more FcyRs, and identifying an antibody that recognizes the poorly antigenic antigen.
  • the method comprises isolating the antibody from the mouse.
  • a nucleic acid sequence that encodes all or part of a variable region of the antibody is identified and cloned.
  • a method for making a mouse capable of making antibodies comprising human variable regions comprising a step of breeding a first mouse as described herein with a second mouse that comprises (a) one or more human immunoglobulin variable region gene segments and one or more human constant region genes; or, (b) one or more human immunoglobulin variable region gene segments operably linked to a mouse constant region gene, wherein the human gene segments replace variable region gene segments at the mouse variable region gene segment locus.
  • the second mouse (a) comprises a transgene that comprises one or more human immunoglobulin light chain variable region gene segments and a human light chain constant gene, and a transgene that comprises one or more human immunoglobulin heavy chain variable region gene segments and one or more human heavy chain constant genes.
  • the transgene that comprises one or more human immunoglobulin heavy chain variable region gene segments comprises two or more heavy chain constant genes and is capable of class switching.
  • the mouse comprises an inactivated endogenous light chain locus and/or an inactivated endogenous heavy chain locus.
  • the mouse comprises a deletion of an endogenous light chain locus and/or a deletion of an endogenous heavy chain locus.
  • the second mouse (b) comprises human heavy and human light variable region gene segments, at the heavy an light mouse loci, respectively.
  • a method for selecting an anti-tumor antibody comprising a step of determining the ability of an antibody to mediate ADCC, wherein the ability of the antibody to mediate ADCC is tested by determining ADCC mediated by a cell of a mouse as described herein, and the antibody is selected if it mediates ADCC employing a cell of a genetically modified mouse as described herein.
  • binding of the antibody to the cell of the genetically modified mouse is determined, and the anti-tumor antibody is selected for its ability to bind a human FcyR on the cell.
  • the human FcyR is a low affinity FcyR.
  • the anti-tumor antibody is identified by its enhanced ability to mediate ADCC through a cell of the mouse as compared to ability of the anti-tumor antibody to mediate ADCC through a cell of a wild type mouse.
  • the anti-tumor antibody is identified by its ability to mediate ADCC through NK cells.
  • the NK cells express human FcyRIIIA.
  • a method for selecting an anti-tumor agent comprising a step of administering an agent comprising a human Fc or a modified human Fc to a first non-human animal wherein the first non-human animal is genetically modified in accordance with the invention and comprises a human tumor; a step of administering the agent to a second non-human animal comprising the tumor; and determining the ability of the first non-human animal and the second non-human animal to retard growth of the human tumor following administration of the agent, wherein the agent is selected as an anti-tumor agent if it exhibits an enhanced ability to retard growth of the human tumor in the first non-human animal but not in the second non-human animal.
  • the first non-human animal is modified to comprise a deletion of an endogenous FcR a-subunit, and is modified to comprise a human FcR a-subunit selected from the group consisting of an FCYRI IA a-subunit, an FcyRIIB a- subunit, an FcyRIIC a-subunit, an FcyRIIIA a-subunit, an FcyRIIIB a-subunit, and a combination thereof.
  • the second animal is a wild type animal.
  • the first non-human animal expresses an endogenous FcR y- chain.
  • the first non-human animal expresses a functional endogenous FcyRI.
  • a first step comprises deleting the a-chains of the endogenous FcyRIIB, FcyRIV and FcyRIII genes and inserting the a-chains of the human FcyRIIA and FcyRIIIA genes;
  • a second step comprises inserting the a-chains of the human FcyRIIB, FcyRIIC and FcyRIIIB genes into the mouse genome that results from the first step; wherein the mouse comprises a functional mouse FcR y-chain gene.
  • the a-chains of the human FcyRIIB, FcyRIIC and FcyRIIIB genes of the second step are inserted 5' relative to the a- chains of the human FcyRIIA and FcyRIIIA genes of the first step.
  • a method for determining cell killing by a human therapeutic in a non-primate comprising a step of exposing a cell, non- human embryo, or non-human animal to a human therapeutic that comprises a human Fc, wherein the cell, embryo, or animal comprises a functional FcR ⁇ -chain and comprises a replacement of one or more endogenous low affinity FcyR a-chain genes with one or more human FcyR a-chains, and determining the ability of the human therapeutic to mediate cell killing through a low affinity human FcyR of the cell, embryo, or animal.
  • the non-primate is a mouse.
  • endogenous mouse Fc R a-chain genes FcyRIIB, FcyRIV and FCYRI I I are replaced with human FcyR a-chain genes FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA, and FcyRIIIB.
  • the cell is selected from a B cell, a mast cell, a basophil, a macrophage, an eosinophil, a neutrophil, a dendritic cell, a Langerhans cell, and a combination thereof.
  • the cell is an NK cell and NK cell-mediated ADCC by a human or a humanized antibody is determined.
  • the low affinity human FcyR is a human FcyRIIIA.
  • a method for determining therapeutic-dependent thrombosis comprising exposing a first non-human animal that expresses a human FcyRI IA on a platelet to a therapeutic; exposing a second non- human animal that does not express the human FcyRIIA on a platelet to said therapeutic; measuring in the first non-human animal and in the second non-human animal an amount of therapeutic-dependent thrombosis; and, determining a difference in therapeutic-dependent thrombosis.
  • the non-human animal is selected from a mouse and a rat.
  • the determined difference in therapeutic-dependent thrombosis is employed to identify a risk associated with administering the therapeutic to a human. In one embodiment, the determined difference results in a change of administration of the therapeutic to a human patient in need thereof.
  • targeting construct includes a polynucleotide molecule that comprises a targeting region.
  • a targeting region comprises a sequence that is substantially homologous to a sequence in a target cell, tissue or animal and provides for integration of the targeting construct into a position within the genome of the cell, tissue or animal.
  • the targeting construct further comprises a nucleic acid sequence or gene of particular interest, a selectable marker, control and or regulatory sequences, and other nucleic acid sequences that allow for recombination mediated through the exogenous addition of proteins that aid in or facilitate recombination involving such sequences.
  • the targeting construct further comprises a gene of interest, wherein the gene of interest is a heterologous gene that encodes a protein that has a similar function as a protein encoded by the endogenous sequence.
  • the term "replacement” includes wherein a DNA sequence is placed into a genome of a cell in such a way as to replace a sequence within a genome, at the locus of the genomic sequence, with a heterologous sequence (e.g., a human sequence in a mouse), unless otherwise indicated.
  • the DNA sequence so placed may include one or more regulatory sequences that are part of source DNA used to obtain the sequence so placed (e.g., promoters, enhancers, 5'- or 3'-untranslated regions, efc.).
  • the replacement is a substitution of an endogenous sequence for a heterologous sequence that results in the production of a gene product from the DNA sequence so placed (comprising the heterologous sequence), but not expression of the endogenous sequence;
  • the replacement is of an endogenous genomic sequence with a DNA sequence that encodes a protein that has a similar function as a protein encoded by the
  • the endogenous genomic sequence encodes a low affinity mouse FcyR receptor
  • the DNA fragment encodes one or more human low affinity FcyR receptors, such as, e.g., a human FcyRIIC and/or an FcyRIIIB).
  • FcyR includes a receptor for an Fc, e.g. , an Fc portion of an IgG immunoglobulin.
  • the FcyR genes include an a-chain that is expressed on the surface of the cell and serves as a Iigand-binding domain, and associates with either a homodimer of the FcR ⁇ -chain or a heterodimer of the FcR ⁇ -chain and the ⁇ - chain.
  • Low affinity FcyR genes in humans include FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA and FcyRIIIB and within most of these genes naturally occurring genetic differences, or polymorphisms, have been described in human subjects with autoimmune diseases.
  • Persons of skill upon reading this disclosure will recognize that one or more endogenous low affinity FcyR genes in a genome (or all) can be replaced by one or more heterologous low affinity FcyR genes (e.g., variants or polymorphisms such as allelic forms, genes from another species, chimeric forms, efc).
  • allelic variants includes variations of a normal sequence of a gene resulting in a series of different forms of the same gene.
  • the different forms may comprise differences of up to, e.g., 20 amino acids in the sequence of a protein from a gene.
  • alleles can be understood to be alternative DNA sequences at the same physical gene locus, which may or may not result in different traits (e.g., heritable phenotypic characteristics) such as susceptibility to certain diseases or conditions that do not result in other alleles for the same gene or result in varying degrees in the other alleles.
  • An "accessory cell” includes an immune cell that is involved in the effector functions of the immune response.
  • exemplary immune cells include a cell of lymphoid or myeloid origin, e.g., lymphocytes, natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils, basophils, platelets, Langerhans cells, dendritic cells, mast cells etc.
  • Accessory cells carry out specific functions of the immune system through receptors, e.g., FcRs, expressed on their surfaces.
  • an accessory cell is capable of triggering ADCC mediated through an FcR, e.g., a low affinity FcyR, expressed on the cell surface.
  • macrophages expressing FcRs are involved in phagocytosis and destruction of antibody-coated bacteria.
  • Accessory celts might also be capable of releasing an agent that mediates other immune processes.
  • mast cells can be activated by antibody bound to FcRs to release granules, e.g., inflammatory molecules (e.g., cytokines) at a site of infection.
  • inflammatory molecules e.g., cytokines
  • the expression of FcRs on accessory cells can be regulated by other factors (e.g., cytokines).
  • FcyRI and FcyRIII expression can be inducted by stimulation with interferon- ⁇ (IFN- ⁇ ).
  • IFN- ⁇ interferon- ⁇
  • FcRs immunoglobulins
  • FcRs are present on accessory cells of the host's immune system to effectively dispose of foreign antigens bound by an antibody.
  • FcRs also play important roles in balancing both activating and inhibitory responses of the accessory cells of the immune system.
  • FcRs are involved in phagocytosis by macrophages, degranulation of mast cells, uptake of antibody-antigen complexes and modulation of the immune response, as well as other immune system processes.
  • FcyRs In mice and humans, distinct FcRs are differentially expressed on the surface of different accessory cells that are each specific for the immunoglobulin isotypes present in the expressed antibody repertoire.
  • immunoglobulin G (IgG) antibodies mediate effector functions through IgG receptors (FcyRs).
  • FcyRs have been classified into three groups: high affinity activating FcyRI (CD64), low affinity inhibitory FcyRII (CD32) and low affinity activating FcyRIII (CD16). Although each group is present in both mice and humans, the number of isoforms and subsets of immune cells on which they are present are different.
  • FcyRIIA and FcyRIIIB are expressed on accessory cells in humans but are reportedly absent from mice. Further, affinities of the different IgG isotypes (e.g., lgG1 ) for each FcyR is different in mice and humans.
  • Activation or inhibition of cell signaling through FcyRs and the effector functions associated with antibody binding to FcyRs are believed to be mediated by specific sequence motifs of intracellular domains of FcyRs, or of the subunits of co- receptors.
  • Activating receptors are most commonly associated with the common y- chain (FcR y-chain) which contains an immunoreceptor tyrosine-based activation motif (ITA ).
  • ITAMs contain a specific sequence of about 9-12 amino acids that include tyrosine residues that are phosphorlyated in response to antibody binding to an FcR. Phosphorylation leads to a signal transduction cascade.
  • the FcR ⁇ -chain is reportedly essential for proper surface expression and function (e.g. , signal transduction, phagocytosis, etc.) of most of the FcRs; FcR ⁇ -chain KO mice lack FcyRI according to some reports. However, other reports reveal that FcR ⁇ -chain KO mice indeed express FcyRI on the surface of certain accessory cells, and the FcyRI expressed reportedly appears functional in that it binds IgG in mice in the absence of expressed FcR ⁇ -chain (Barnes et al. (2002) FcyRI-Deficient Mice Show Multiple Alterations to Inflammatory and Immune Responses, Immunity 16:379-389).
  • FcyRIIB is an inhibitory receptor that contains an
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • ITAMs immunoreceptor tyrosine-based inhibitory motif
  • ITIMs are sequence motifs that include phosphorylatable tyrosine residues.
  • downstream events following phosphorylation of an ITM lead to inhibition, not activation, of immune cell functions.
  • Mice deficient in FcyRIIB reportedly exhibit an increased antibody response in comparison to wild type mice (Takai et al. (1996) Augmented humoral and anaphylactic responses in FcgRII- deficient mice, Nature 379:346-349), an observation that supports the role of FcyRIIB as a downregulator of the B cell antibody response.
  • FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA and FcyRIIIB are considered the classical low affinity FcyR genes and are located together on the same chromosome (Su et al. (2002) Genomic organization of classical human low- affinity Fey receptor genes, Genes and Immunity 3 (Supple 1 ):S51 -S56). These genes exhibit several polymorphisms associated with distinct phenotypes, e.g., an alteration of ligand binding and function of the receptor.
  • autoimmune diseases e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS).
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • Transgenic mice for different human FcyRs have been developed and used as disease models, generating high affinity antibodies, testing therapeutic antibodies for ability to elicit specific cellular responses, screening compounds that ameliorate aberrant immune responses, efc. (e.g. , see Heijnen et al. (1996) A Human FcgRI/CD64 Transgenic Model for In Vivo Analysis of (Bispecific) Antibody Therapeutics, J. Hematother.
  • Such a mouse would serve as a vital tool in the engineering, analysis and evaluation of therapeutic antibodies for treatment of human diseases such as, e.g., RA, type I diabetes, SLE, and autoimmunity, by providing an animal model capable of achieving a more accurate assessment of immunological processes in humans, particularly in the context of testing human antibody therapeutics.
  • the mouse will also be a valuable source of cells bearing the low affinity receptors, which cells can be used in in vitro assays for assessing therapeutic-dependent cell killing for therapeutics that bind the low affinity receptors, and thus for identifying useful human therapeutics.
  • Genetically modified non-human animals are provided that do not express endogenous low affinity mouse FcyR genes, but that express an endogenous mouse FcR y-chain.
  • the FcR ⁇ -chain is expressed in a distribution (i.e. , in cell types) and at a level in the mouse that is the same or substantially the same as in a wild type mouse.
  • Endogenous low affinity FcyR genes can be expressed either on the surface of immune cells or in a soluble manner in the periphery of the animals. Genetic modifications for making a non-human animal that does not express endogenous low affinity mouse FcyR genes are conveniently described by using the mouse as an illustration.
  • a genetically modified mouse according to the invention can be made in a variety of ways, particular embodiments of which are discussed herein.
  • FIG. 1 A schematic illustration (not to scale) of low affinity mouse FcyR gene locus is provided in Figure 1 (top) to show FcyR gene arrangement at the
  • low affinity mouse FcyR genes FcyRIIB, FcyRIV and FcyRIII are present together in close proximity on one chromosome. Each of these genes comprise the a-chain or ligand binding domain responsible for the binding the Fc portion of an antibody molecule.
  • a genetically modified mouse lacking a nucleotide sequence encoding an a-chain of the endogenous low affinity FcyR genes can be made by any method known in the art.
  • a targeting vector can be made that deletes the low affinity mouse FcyR a-chain genes with selectable marker gene.
  • Figure 1 illustrates a mouse genome (bottom) targeted by a targeting construct having a 5' homology arm containing sequence upstream of the endogenous low affinity FcyR a-chain locus, followed by a drug selection cassette (e.g. a neomycin resistance gene flanked by loxP sequences), and a 3' homology arm containing sequence
  • a drug selection cassette e.g. a neomycin resistance gene flanked by loxP sequences
  • the endogenous low affinity FcyR a-chain locus Upon homologous recombination at the locus, the endogenous low affinity FcyR a-chain locus is replaced by a drug selection cassette (bottom of Figure 1).
  • the endogenous low affinity FcyR a-chain gene locus is thereby deleted resulting in a cell or non-human animal that does not express endogenous low-affinity mouse FcyR a-chain genes.
  • the drug selection cassette may optionally be removed by the subsequent addition of a recombinase (e.g., by Cre treatment).
  • modifying the a- chains of the endogenous low-affinity mouse FcyR genes, but not the FcR ⁇ -chain avoids a potential reduction of other endogenous FcR genes (e.g., high affinity FcyRI) that require the FcR ⁇ -chain for surface expression and function, thus maintaining various other immunological functions and processes mediated through ⁇ -chain-dependent processes.
  • endogenous FcR genes e.g., high affinity FcyRI
  • FcR ⁇ -chain deficient mice lack surface expression of FcyRI II and FcyRI.
  • FcyRI has reportedly been detected on the cell surface in FcR ⁇ -chain deficient mice and is reportedly at least partially functional.
  • mice according to the present invention contain unmodified endogenous FcR ⁇ -chain, which preserves natural cell surface expression patterns and cellular functions of other FcR genes that require FcR ⁇ -chain.
  • mice of the present invention present an advantage over other FcyR gene-deficient mice in that the genetic modifications that they bear result in the maintenance of other genes necessary for other
  • Low affinity human FcyR genes can be expressed either on the surface of accessory cells of the animal's immune system or in a soluble manner in the periphery of the animals.
  • the genetic modification in various embodiments, comprises a deletion of a functional a-chain of one or more low-affinity mouse FcyR genes, and in some embodiments a further modification comprising a replacement with two or more, with three or more, with four or more, or with five low-affinity human FcyR a-subunit genes, wherein the non-human animal expresses a functional mouse FcR ⁇ -chain gene.
  • Genetically modified non-human embryos, cells, and targeting constructs for making the non-human animals, non-human embryos, and cells are also provided.
  • compositions and methods for making a mouse that expresses a human FcyR gene including specific polymorphic forms or allelic variants (e.g., single amino acid differences), are provided, including compositions and method for making a mouse that expresses such genes from a human promoter and a human regulatory sequence.
  • the methods include selectively rendering an endogenous low affinity mouse FcyR gene nonfunctional (e.g., by a deletion of its a-chain), and employing an a-chain of a low affinity human FcyR gene at the endogenous low affinity mouse FcyR gene locus to express a low affinity human FcyR a-subunit gene in a mouse.
  • the deletion of the low affinity mouse FcyR gene is made by deletion of one or more a-chain genes, but not an FcRy-chain gene.
  • the approach selectively renders one or more endogenous low affinity FcyR a-chain genes nonfunctional while retaining a functional endogenous FcRy-chain.
  • the endogenous FcyR a-chain replacement approach employs a relatively minimal disruption in natural FcyR-mediated signal transduction in the animal, in various embodiments, because the genomic sequence of the FcyR a- chains are replaced in a single fragment and therefore retain normal functionality by including necessary regulatory sequences. Thus, in such embodiments, the FcyR a- chain modification does not affect other endogenous FcRs dependent upon functional FcRy-chain molecules.
  • the modification does not affect the assembly of a functional receptor complex involving an FcyR a- chain and the endogenous FcR y-chain, which is believed to be required for proper expression of some FcyR a-chains on the cell surface and for downstream signaling resulting from an activated receptor. Because the FcR y-chain is not deleted, in various embodiments animals containing a replacement of endogenous FcyR - chain genes with human FcyR a-chain genes should be able to process normal effector functions from antibodies through binding of the Fc portion of IgG
  • FIG. 4 A schematic illustration (not to scale) of a deleted endogenous low affinity mouse FcyR gene is provided in Figure 4 (top).
  • low affinity human FcyR genes FcyRIIA and FcyRIIIA are inserted into the deleted endogenous low affinity mouse FcyR gene locus by a targeting construct (Human FcyRlllA-IIA Targeting Vector) with a genomic fragment containing the human low affinity human FcyRIIA and FcyRIIIA genes.
  • a targeting construct Human FcyRlllA-IIA Targeting Vector
  • Each of these genes comprise the a-chain or ligand- binding domain of the human FcyR genes responsible for the binding the Fc portion of an antibody molecule.
  • a genetically modified mouse that expresses low affinity human FcyR genes at the endogenous low affinity mouse FcyR locus can be made by any method known in the art.
  • a targeting vector can be made that introduces low affinity human FcyR genes (e.g., FcyRIIA and FcyRIIIA) with a selectable marker gene.
  • Figure 4 illustrates a mouse genome comprising a deletion of the endogenous low affinity FcgR locus (top).
  • the targeting construct contains a 5' homology arm containing sequence upstream of the endogenous low affinity mouse FcyR locus, followed by a drug selection cassette (e.g.
  • the drug selection cassette is replaced by the sequence contained in the targeting vector (bottom of Figure 4).
  • the endogenous low affinity FcyR gene locus is thus replaced with low affinity human FcyR genes resulting in a cell or animal that expresses low- affinity human FcyR genes.
  • the drug selection cassette may optionally be removed by the subsequent addition of a recombinase (e.g., by Cre treatment).
  • the targeting construct Human hFcyRIIA -IIA Targeting Vector comprises an extended sequence that includes, e.g. , all or substantially all of the human promoter region operably linked to the hFcgRIIA gene in a human genome.
  • the targeting construct lacks all or substantially all of the human promoter region operably linked to the hFcyRIIA gene in a human.
  • FIG. 6 a schematic illustration (not to scale) of an endogenous low affinity FcyR locus replaced with two human low affinity FcyR genes is provided in Figure 6 (top).
  • low affinity human FcyR genes FcyRIIB, FcyRIIC and FcyRIIIB are inserted into the modified endogenous low affinity mouse FcyR gene locus by another targeting construct (Human FcyRIIB-IIIB-IIC Targeting Vector) with a genomic fragment containing the low affinity human FcyRIIB, FcyRIIC and FcyRIIIB genes.
  • Each of these genes comprise the a-chain or ligand-binding domain of the human FcyR genes responsible for the binding the Fc portion of an antibody molecule.
  • a genetically modified mouse that expresses five low affinity human FcyR genes at the endogenous low affinity mouse FcyR locus can be made by any method known in the art.
  • a targeting vector can be made that introduces low affinity human FcyR genes (e.g., FcyRIIB, FcyRIIC and FcyRIIIB) with a selectable marker gene.
  • Figure 6 illustrates a mouse genome comprising a replacement of the endogenous low affinity FcyR locus with two low affinity human FcyR genes (top).
  • the targeting construct contains a 5' homology arm containing sequence upstream of the endogenous low affinity mouse FcyR locus, followed by a drug selection cassette (e.g., a neomycin resistance gene flanked on both sides by loxP sequences), a genomic fragment containing a human FcyRIIB gene, a human FcyRIIIB, a human HSP77 gene, a human FcyRIIC gene, followed by a 3' homology arm containing sequence upstream of the low affinity human FcyRIIIA gene present at the endogenous locus.
  • a drug selection cassette e.g., a neomycin resistance gene flanked on both sides by loxP sequences
  • a genomic fragment containing a human FcyRIIB gene, a human FcyRIIIB, a human HSP77 gene, a human FcyRIIC gene followed by a 3' homology arm containing sequence upstream of the low affinity human FcyRIIIA gene present at
  • a human FcyRIIB, FcyRIIIB and FcyRIIC gene are inserted 5' to the human FcyRIIIA and FcyRIIA genes previously present at the endogenous low affinity FcyR gene locus by the sequence contained in the targeting vector (bottom of Figure 6).
  • the modified endogenous low affinity FcyR gene locus is thus further modified to incorporate three additional low affinity human FcyR genes resulting in a cell or animal that expresses five low-affinity human FcyR genes.
  • the drug selection cassette may optionally be removed by the subsequent addition of a recombinase (e.g., by Cre treatment).
  • Figure 6 (bottom) shows the structure of the resulting locus, which will express five low affinity human FcyR genes that can be detected on the surface of accessory cells of the animal's immune system and independently associate, as appropriate, with an endogenous FcRy-chain.
  • Genetically modified non-human animals that do not express endogenous low affinity mouse FcyR genes are useful, e.g., to elucidate the various functions of the individual low affinity FcyR genes in the immune response, to measure the efficacy of a human therapeutic antibody via cell-mediated immunity (e.g. , ADCC), to determine an FcyR's role in immune diseases or disorder, to serve as models of immune diseases or disorders, to generate antibodies against one or more FcyR proteins, and to serve as breeding mates to generate other genetically modified mice of interest.
  • cell-mediated immunity e.g. , ADCC
  • a mouse according to the invention can be used to determine a cytotoxic effect lost (in comparison to a wild type mouse) by a mouse that does not express low affinity FcyR genes by administering an agent to such a mouse, where the agent is known to trigger an FcyR-dependent cytotoxic effect in wild type mice.
  • a mouse of the present invention is implanted with tumor cells and, after a subsequent period of time, injected with an antibody specific for an antigen expressed on the surface of the tumor cells. The isotype of the antibody is known prior to injection and the animals are analyzed for impairment of FcyR-dependent ADCC by comparison to ADCC observed in wild type animals.
  • mice deficient in endogenous low affinity receptors could be combined (e.g., by breeding) with other immune deficient mice to develop in vivo models of autoimmune disease.
  • SCID mice are routinely used in the art as model organisms for studying the immune system. SCID mice have an impaired ability to make T or B lymphocytes, or activate some components of the complement system, and cannot efficiently fight infections, reject tumors, and reject transplants.
  • Low affinity FcyR a- subunit gene-deficient mice of the present invention may be bred to SCID mice to ascertain cell depletion in a host animal in response to administration of an antibody therapeutic (e.g. , an anti-tumor antibody), which would determine the roles of ADCC and complement-dependent cytotoxicity (CDC) in tumor cell depletion in vivo.
  • an antibody therapeutic e.g. , an anti-tumor antibody
  • genetically modified non-human animals comprising a replacement of the endogenous low affinity FcyR genes with low-affinity human FcyR genes are provided. Such animals are useful for studying the pharmacokinetics of fully human antibodies and hFcyR-mediated ADCC.
  • human FcyR genes have been shown to exhibit polymorphisms or allelic variants associated with disease (e.g., SLE, RA, Wegener's granulomatosis, Guillain-Barre syndrome and Multiple Sclerosis).
  • genetically modified non-human animals that comprise a replacement of the endogenous low affinity FcyR genes with specific allelic or polymorphic forms of human FcyR genes can be used to study human autoimmune diseases, and traits associated with the polymorphisms, in the animal.
  • allelic forms of human FcyR genes are associated with enhanced efficacy for human IgG.
  • the affect of a human low affinity FcyR polymorphism on the efficacy of a human antibody therapeutic is determined.
  • an anti-tumor antibody is administered to a first humanized mouse comprising a first polymorphism of a human FcyR and also to a second humanized mouse comprising a second polymorphism of a human FcyR, wherein the first and the second mice each comprise a human tumor cell; and the anti-tumor activity of the anti-tumor antibody is assessed in the first mouse and in the second mouse.
  • a treatment option is selected by a physician with respect to treating a human having the first or the second polymorphism and having a tumor corresponding to the human tumor cell, based on the assessment of efficacy of the anti-tumor antibody in the first mouse and in the second mouse.
  • Suitable polymorphisms of human FcyR genes include all those known in the art.
  • polymorphisms include, e.g., the high responder and low responder phenotype reported by the ability of T cells to proliferate in response to IgG.
  • the high responder polymorphism is characterized by an arginine residue at position 131 (131Arg) while the low responder is characterized by a histidine residue at position 131 (131 His).
  • the human FcyRIIA sequence comprises the 131 His polymorphism.
  • a representative protein sequence of the human FcyRIIA -chain is shown in SEQ ID NO:32.
  • Single-nucleotide substitutions of the human FcyRIIB gene result in mis- sense substitutions in the ligand-binding domain (a-chain) and putatively affect the binding ability of an Fc portion of an IgG to bind to the a-chain of FcyRIIB on the cell surface.
  • substitution of a threonine residue for an isoleucine at position 232 (lle232Thr) within the transmembrane domain of the FcyRIIB gene in mice has been shown to impair the signaling ability of the receptor.
  • the human FcyRIIB gene comprises the isoleucine variant (232lle).
  • a representative protein sequence of the human FcyRIIB a-chain is shown in SEQ ID NO:33.
  • Allelic variants of the human FcyRIIIA gene are proposed to be involved in susceptibility to SLE and RA. This allelic variant includes a phenylalanine substitution for valine at position 158 (Val158Phe).
  • the valine allelic variant (158Val) is characterized to have a higher affinity for lgG1 and lgG3 than the phenylalanine allelic variant (158Phe).
  • the 158Phe allelic variant has been proposed to lead to a reduced clearance of immune complexes.
  • the human FcyRIIIA gene comprises the 158Val allelic variant.
  • a representative protein sequence of the human FcyRIIIA a-chain is shown in SEQ ID NO:35.
  • Allelic variants of the human FcyRIIIB gene include the neutrophil antigen 1 (NA1) and neutrophil antigen 2 (NA2) alleles. These allelic variants have been proposed to be involved in blood-transfusion reactions, alloimmune neutropaenia, SLE and Wegener's granulomatosis.
  • the NA2 allelic variant is characterized by a diminished ability to mediate phagocytosis.
  • the human FcyRIIIB gene comprises the NA2 allelic variant.
  • a representative protein sequence of the human FcyRIIIB a-chain is shown in SEQ ID NO:36.
  • the genetically modified non-human animals are useful for optimizing FcyR-mediated functions triggered by the Fc portion of therapeutic antibodies.
  • the Fc regions of antibodies can be modified by any method known in the art. For example, amino acid residues within the Fc portion (e.g., CH2 and CH3 domains) can be modified to selectively enhance the binding affinity to human FcyRIIIA. Thus, the resulting antibody should have enhanced FcyRIIIA-dependent ADCC.
  • an animal expressing human FcyRIIIA of the present invention is used to evaluate the enhanced ADCC ability of a modified human antibody by administering a modified human antibody to the animal, detecting (e.g., in vitro) antibody binding to FcyRIIIA-expressing cells and comparing the ADCC activity observed to the ADCC activity observed from that determined in a wild type animal.
  • a targeting construct for introducing a deletion of the endogenous low affinity mouse FcyR locus was constructed ( Figure 1).
  • the targeting construct was made using VELOCIGENE® technology (see, e.g., US Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21 (6):652-659) to modify the Bacterial Artificial Chromosome (BAC) RP23-395f6 (Invitrogen).
  • BAC Bacterial Artificial Chromosome
  • RP23-395f6 BAC DNA was modified to delete the endogenous low affinity FcyRIIB, FcyRIV and FcyRIII genes comprising the -chain of each of the FcyRs.
  • upstream and downstream homology arms were made employing primers mFcR 5-up-1 (5'-ACCAGGATAT GACCTGTAGA G; SEQ ID NO: 1) and mFcR 3-up-1 a (GTCCATGGGT AAGTAGAAAC A; SEQ ID NO:2), and mFcR 5-DN (ATGCGAGCTC ATGCATCTATG TCGGGTGCGG AGAAAGAGGT AATGCATTCT TGCCCAATAC TTAC; SEQ ID NO:3) and mFcR 3-DN (ACTCATGGAG
  • the targeting construct included a loxed neomycin resistance gene comprising homology arms comprising sequence homologous to a 5' and a 3' region with respect to the endogenous locus. Genes and/or sequences upstream of the endogenous FcyRIIB gene and downstream of the endogenous FcyRIII gene (see Figure 1) were unmodified by the targeting construct.
  • the targeted deletion was confirmed by polymerase chain reaction (PCR) using primers outside the deleted region and within the targeting construct.
  • the upstream region of the deleted locus was confirmed by PCR using primers to mFcR- up-detect (ATCCTGAGTA TACTATGACA AGA; SEQ ID NO:5) and PGK-up-detect (ACTAGTGAGA CGTGCTACTT C; SEQ ID NO:6), whereas the downstream region of the deleted locus was confirmed using primers pA-DN-detect (CTCCCACTCA TGATCTATAG A; SEQ ID NO:7) and mFcR-DN-detect (TGGAGCCTCA
  • the nucleotide sequence across the upstream deletion point included the following, which indicates endogenous mouse sequence downstream of the FcyRIIB gene (contained within the parentheses below) linked contiguously to cassette sequence present at the deletion point: (GTCCATGGGT AAGTAGAAAC A)TTCGCTACC TTAGGACCGT TA (SEQ ID NO:9).
  • the nucleotide sequence across the downstream deletion point included the following, which indicates cassette sequence contiguous with endogenous mouse sequence upstream of the FcyRIII gene (contained within the parentheses below):
  • Spleens were harvested from FcyR deficient and wild type mice and perfused with 10 mL Collagenase-D in sterile disposable bags. Each bag containing a single spleen was then placed into a Stomacher® (Seward) and homogenized at a medium setting for 30 seconds. Homogenized spleens were transferred to 10 cm petri dishes and incubated for 25 minutes at 37°C. Cells were separated with a pipette using a 1 :50 dilution of 0.5 M EDTA, followed by another incubation for five minutes at 37°C.
  • Lymphocyte cell populations were identified by FACs on the BD LSR II System (BD Bioscience) with the following flourochrome
  • Lymphocytes were gated for specific cell lineages and analyzed for expression of endogenous FcyRIII and FcyRIIB with a rat anti-mouse FcyRIII/ll antibody (clone 2.4G2, BD Biosciences). Clone 2.4G2 recognizes a common polymorphic epitope on the extracellular domains of murine FCYRII I and FcyRII. The results show that there was no detectable murine low affinity FcyRIII or FcyRII on B-cells, NK cells and macrophages in mFcyR KO mice ( Figure 2).
  • ADCC Assay Splenocytes isolated from FcyR gene deficient and wild type mice were analyzed for their ability to perform ADCC in a cell-killing assay. Cell populations were isolated and separated using MACS® Technology (Miltenyi Biotec). Briefly, T-cells were depleted from splenocytes using magnetically labeled anti- mouse CD3 beads. The T-cell depleted splenocytes were then enriched for NK cells using magnetically labeled anti-mouse CD49B beads. Separately, Raji cells
  • Luminescence signal is derived from lysed cells and proportional to the number of dead cells.
  • Luminescence from controls was determined for background dead cell count for each ratio and subtracted from measurements for wild type and KO mice. Average cell death was calculated and percent decrease in cell killing (% ADCC) was determined by comparison to wild type. Results are shown in Table 1.
  • mice that expressed human CD20 and had a deletion of the endogenous low affinity FcyR genes were administered the control and human anti-hCD20 antibodies (described above).
  • mice in each group were administered the antibodies by intra-peritoneal injections. Seven days post-injection, animals were euthanized and the remaining B cell contents of bone marrow (B220 + /lgM + ), peripheral blood (B220 + /CD19 + ), lymph node (B220 + /CD19 + ) and spleen (B220 + /CD19 + ) were identified by multi-color FACS performed on a LSR-II flow cytometer and analyzed using Flow- Jo software (as described above). The results of the B cell depletion experiments are shown in Figures 3A-3D.
  • a targeting construct for introducing two low affinity human FcyR genes into a deleted endogenous low affinity mouse FcyR locus was constructed ( Figure 4).
  • a targeting construct comprising human FcyRIIA and FcyRIIIA genes was made using similar methods (see Example 1) through modification of BAC RP23- 395f6 and CTD-2514j12 (Invitrogen).
  • BAC DNA of both BACs was modified to introduce a deletion of the a-chains of the low affinity human FcyRIIA and FcyRIIIA genes into the deleted endogenous low affinity FcyR locus.
  • upstream and downstream homology arms were made employing primers h14 (GCCAGCCACA AAGGAGATAA TC; SEQ ID NO: 1 1 ) and h15 (GCAACATTTA GGACAACTCG GG; SEQ ID NO:12), and h4
  • the targeting construct included a 5' homology arm including sequence 5' to the deleted endogenous low affinity FcyR locus, a FRT'ed hygromycin resistance gene, followed by a human genomic fragment from BAC CTD-2514j12 comprising low affinity human FcyRIIA and FcyRIIIA a-chain genes, and a 3' homology arm comprising mouse sequence 3' to the deleted endogenous low affinity FcyR locus (middle of Figure 4).
  • a targeting construct was made in a similar manner (using the same BACs) except that the construct comprises an extended promoter sequence operably linked to the human FcyRIIA gene in the human genome, e.g., up to about 18 kb or more, using a hygromycin cassette that is flanked on both sides by lox2372 sites, wherein the junction of the promoter region and the first lox 2372 site is ATCGGGGATA
  • Targeted insertion of the human FcyRIIA and FcyRIIIA -chain genes was confirmed by PCR (as described above).
  • the upstream region of the partially humanized locus was confirmed by PCR using primers h16 (CCCAGGTAAG TCGTGATGAA ACAG; SEQ ID NO: 15) and pA-DN-detect (CTCCCACTCA
  • the upstream junction includes two novel sequences.
  • One point of the upstream junction includes the following, which indicates nucleotide sequence of the hygromycin cassette contiguous with human genomic sequence (contained within the parentheses below) that comprises the upstream region of the inserted hFcyRIIIA gene: TAAACCCGCG GTGGAGCTC(G CCAGCCACAA AGGAGATAAT CA) (SEQ ID NO:20).
  • the second point of the upstream junction includes the following, which indicates a nucleotide sequence of an endogenous mouse sequence (contained within the parentheses below) from the upstream region of the deleted low affinity FcyR locus contiguous with a nucleotide sequence within the hygromycin cassette: (CCATGGGTAA GTAGAAAC)TC TAGACCCCCG GGCTCGATAA CT (SEQ ID NO:21).
  • mice containing two low affinity human FcyR genes (hFcyRIIA, lacking extended promoter region, and hFcyRIIIA) in place of the endogenous low affinity mouse FcyR locus were generated through electroporation of the targeted BAC DNA (described above) into mouse ES cells. Positive ES cells clones were confirmed by TaqmanTM screening and karyotyping. Positive ES cell clones were then used to implant female mice using the VELOCIMOUSE® method (described below) to generate a litter of pups containing a replacement of the endogenous low affinity FcyR genes with the two human low affinity FcyR genes.
  • Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g. , US Pat. No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25(1 ):91-99.
  • VELOCIMICE® F0 mice fully derived from the donor ES cell bearing hFcyRIIA and hFcyRIIIA were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detected the presence of the hFcyR genes.
  • mice bearing the hFcyR genes can be bred to a Cre deleter mouse strain (see, e.g., International Patent Application Publication No. WO 2009/114400) in order to remove any loxed neo cassette introduced by the targeting construct that is not removed, e.g., at the ES cell stage or in the embryo.
  • the neomycin cassette is retained in the mice.
  • Pups are genotyped and a pup heterozygous for the hFcyR genes is selected for characterizing FcyRIIA and FcyRIIIA humanizations.
  • Lymphocytes were gated for specific cell lineages and analyzed for expression of hFcyRII and hFcyRIII using a mouse anti-human FcyRII antibody (Clone FLI8.26; BD Biosciences) and a mouse anti-human FcyRIII antibody (Clone 3G8; BD Biosciences), respectively. Relative expression (++, +) or no expression (-) observed for each lymphocyte subpopulation is shown in Table 2.
  • FCYRIIIA/FCYRI IA homozygotes, lacking the extended FcyRIIA promoter region
  • Results are shown in Figures 5A and 5B.
  • Percent of separate lymphocyte cell populations expressing human FcyRIIIA, FcyRIIA or both in FCYRI IIA/FCYRI IA homozygote mice is shown in Table 3.
  • mice both heterozygote and homozygote genotypes generated in accordance with Example 3 expressed human FCYRI I IA on NK cells and macrophages; and human FcyRIIA on neutrophils and macrophages, but not platelets.
  • Human FCYRI IIA was highly expressed on NK cells.
  • the expression pattern of human FcyR genes shown in this Example is consistent with the expression patterns of these genes in human accessory cells.
  • a targeting construct for introducing three additional low affinity human FcyR genes into a partially humanized endogenous low affinity FcyR locus was constructed ( Figure 6).
  • a targeting construct comprising human FcyRIIB, FCYRI IIB and FcyRIIC genes was made using similar methods (see Example 1 ) through modification of BAC RP-23 395f6 and RP-1 1 697e5 (Invitrogen). BAC DNA of both BACs was modified to introduce the a-chains of the low affinity human FcyRIIB, FCYRII IB and FcyRIIC genes into the partially humanized endogenous low affinity FcyR locus containing two human low affinity FcyR genes.
  • upstream and downstream homology arms were made employing primers mFcR up-1 (ACCAGGATAT GACCTGTAGA G; SEQ ID NO:22) and mFcR2b Nhel-2 (GTTTCTACTT ACCCATGGAC; SEQ ID NO:23), and M O (AAATACACAC TGCCACAGAC AG; SEQ ID NO:24) and h1 1 (CCTCTTTTGT GAGTTTCCTG TG; SEQ ID NO:25), respectively.
  • These homology arms were used to make a cassette that introduced DNA sequences encoding the -chains of low affinity human FcyRIIB, FcyRIIIB and FcyRIIC.
  • the targeting construct included a 5' homology arm including mouse sequence 5' to the deleted endogenous low affinity FcyR locus, a loxed neomycin resistance gene, followed by a human genomic fragment from BAC RP-1 1 697e5 comprising low affinity human FcyRIIB, FcyRIIIB and FcyRIIC a-chain genes, and a 3' homology arm comprising human sequence 5' to the low affinity human FcyRIIIA a-chain gene (middle of Figure 6).
  • the nucleotide sequence across the downstream junction included the same human genomic sequence upstream of the hFcyRIIA a-chain gene (see Example 3; SEQ ID NO: 19).
  • the nucleotide sequence across the upstream junction included the following, which indicates two novel junctions of mouse and cassette sequences and cassette and human genomic sequences at the insertion point. The junction of genomic mouse sequence
  • TTAGGACCGT TA (SEQ ID NO:30).
  • the second novel junction includes the joining of the 3' end of neo cassette (contained within the parentheses below) and a human genomic sequence downstream of the hFcgRIIB a-chain gene: (GCTTATCGAT ACCGTCGAC)A AATACACACT GCCACAGACA GG; SEQ ID NO:31). These junctions are show in Figure 6 (middle) within the targeting construct. The resulting modified genome of the fully humanized low affinity FcyR locus is shown in Figure 6 (bottom).
  • mice containing five low affinity human FcyR genes in place of the endogenous low affinity mouse FcyR locus were generated through electroporation of the targeted BAC DNA (described above) into mouse ES cells. Positive ES cells clones were confirmed by TaqmanTM screening and karyotyping. Positive ES cell clones were then used to implant female mice (as described above) to give rise to a litter of pups containing a replacement of the endogenous low affinity FcyR genes for five human low affinity FcyR genes.
  • Spleens were harvested from fully humanized FcyR (heterozygotes) and wild type mice and prepared for FACs (as described above).
  • Lymphocytes were gated for specific cell lineages and analyzed for expression of human FcyRIIA and FcyRIIIA using a mouse anti-human FcyRII antibody (Clone FLI8.26; BD Biosciences) and a mouse anti-human FcyRIII antibody (Clone 3G8; BD Biosciences), respectively. Relative expression (++, +) or no expression (-) observed for each lymphocyte subpopulation is shown in Table 4.
  • spleens were harvested from fully humanized FcyR (homozygotes) and wild type mice and prepared for FACs (as described above). Results are shown in Figure 7. Percent of separate lymphocyte cell populations expressing human FCYRI I IA, human FcyRIIIB, human FcyRIIA, human FcyRIIB, human FcyRIIC or a combination thereof in fully humanized FcyR homozygote mice is shown in Table 5.
  • mice both heterzyogote and homozygote genotypes generated in accordance with Example 5 expressed human FcyRIIIA on NK cells and macrophages, human FcyRIIIB on neutrophils, human FcyRIIA on neutrophils and macrophages, human FcyRIIB on B cells, and human FcyRIIC on NK cells.
  • human FcyR genes shown in this Example is consistent with the expression patterns of these genes in human accessory cells.
  • FcyRIIIA/FcYRIIA (homozygotes), FCYRI I IA/FCYRI I IB/FCYRI IA/FCYRI IB/FCYRI IC (homozygotes) and wild type mice were analyzed for their ability to perform ADCC in a cell-killing assay (as described above in Example 2).

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013064443A1 (en) * 2011-11-01 2013-05-10 Roche Glycart Ag In vivo adcc model
CN104039821A (zh) * 2011-10-28 2014-09-10 瑞泽恩制药公司 人源化il-6和il-6受体
CN111372449A (zh) * 2018-09-27 2020-07-03 公益财团法人实验动物中央研究所 免疫缺陷小鼠
US11020495B2 (en) 2009-12-21 2021-06-01 Regeneron Pharmaceuticals, Inc. Humanized low affinity FCγR mouse cells
EP3861854A4 (en) * 2018-09-27 2022-01-26 Central Institute For Experimental Animals IMMUNODEFICIENCY MOUSE

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3939423B1 (en) * 2012-11-05 2024-03-06 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
EP3842528A1 (en) 2013-09-18 2021-06-30 Kymab Limited Methods, cells and organisms
RU2674910C2 (ru) * 2013-10-15 2018-12-13 Регенерон Фармасьютикалс, Инк. Животные с гуманизированным геном il-15
MX388127B (es) 2013-12-11 2025-03-19 Regeneron Pharma Metodos y composiciones para la modificacion dirigida de un genoma.
KR102846903B1 (ko) * 2014-03-21 2025-08-20 리제너론 파마슈티칼스 인코포레이티드 단일 도메인 결합 단백질을 생산하는 비-인간 동물
HUE051088T2 (hu) 2014-04-08 2021-03-01 Regeneron Pharma Humanizált FC-gamma receptorokkal rendelkezõ nem humán élõlények
CN111118047B (zh) 2014-05-30 2023-09-22 再生元制药公司 人源化二肽基肽酶iv(dpp4)动物
LT3230320T (lt) * 2014-12-09 2021-01-11 Regeneron Pharmaceuticals, Inc. Gyvūnai, išskyrus žmogų, turintys humanizuotą diferenciacijos klasterio 274 geną
FI3282835T3 (fi) * 2015-04-13 2023-07-20 Regeneron Pharma Humanisoituja sirpa-il15-poistogeenisiä hiiriä ja niiden käyttömenetelmiä
CN107529739B (zh) * 2015-04-30 2021-01-08 株式会社特殊免疫研究所 表达人特定分子和人Fcγ受体家族的转基因非人动物
CN113831407B (zh) 2016-05-20 2024-06-11 瑞泽恩制药公司 用于使用多个引导rna来破坏免疫耐受性的方法
US11879014B2 (en) * 2017-03-17 2024-01-23 Tusk Therapeutics Ltd. Method of treating cancer or depleting regulatory T cells in a subject by administering a human IGG1 anti-CD25 antibody
KR20250121150A (ko) 2018-03-26 2025-08-11 리제너론 파마슈티칼스 인코포레이티드 치료제를 시험하기 위한 인간화된 설치류
SG11202107589PA (en) 2019-02-22 2021-08-30 Regeneron Pharma Rodents having genetically modified sodium channels and methods of use thereof
CN113614238B (zh) * 2019-03-28 2024-10-22 公益财团法人实中研 免疫缺陷小鼠
KR20230147048A (ko) 2020-12-16 2023-10-20 리제너론 파마슈티칼스 인코포레이티드 인간화 Fc 알파 수용체를 발현하는 마우스
CN114410643B (zh) * 2021-02-02 2024-08-02 百奥赛图(北京)医药科技股份有限公司 FcRn基因人源化的非人动物及其构建方法和应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6111166A (en) 1994-09-19 2000-08-29 Medarex, Incorporated Transgenic mice expressing human Fcα and β receptors
US6586251B2 (en) 2000-10-31 2003-07-01 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
US6676927B1 (en) 1999-01-20 2004-01-13 The Rockefeller University Animal model and methods for its use in the selection of cytotoxic antibodies
WO1995028959A9 (en) * 1994-04-22 2004-03-04 Sloan Kettering Inst Cancer MICE MUTANT FOR Fc RECEPTORS AND METHOD OF TREATING AUTOIMMUNE DISEASE
US7294754B2 (en) 2004-10-19 2007-11-13 Regeneron Pharmaceuticals, Inc. Method for generating an animal homozygous for a genetic modification
US7351875B2 (en) 2002-06-07 2008-04-01 Trillium Therapeutics, Inc. FcγRIIa transgenic animal model for autoimmune disease
US7402728B2 (en) 2002-12-16 2008-07-22 Genentech, Inc. Transgenic mice expressing human CD20 and/or CD16
US7416726B2 (en) 2000-04-13 2008-08-26 The Rockefeller University Enhancement of antibody-mediated immune responses
US20090035322A1 (en) 2007-07-31 2009-02-05 Regeneron Pharmaceuticals, Inc. Human Antibodies to Human CD20 and Method of Using Thereof
WO2009114400A1 (en) 2008-03-07 2009-09-17 Regeneron Pharmaceuticals, Inc. Es cell-derived mice from diploid host embryo injection

Family Cites Families (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE68929256T2 (de) 1988-05-27 2001-05-17 Applied Research Systems Ars Holding N.V., Curacao Humaner Rezeptor Fc(gamma)RIII
JP2938569B2 (ja) * 1990-08-29 1999-08-23 ジェンファーム インターナショナル，インコーポレイティド 異種免疫グロブリンを作る方法及びトランスジェニックマウス
US5258359A (en) * 1991-08-02 1993-11-02 Monsanto Company Glyphosant-containing herbicidal compositions comprising acetylenic diol rainfastness enhancing agents
WO1994006908A1 (en) 1992-09-11 1994-03-31 The Regents Of The University Of California Transgenic non-human animals having targeted lymphocyte transduction genes
CA2273194C (en) 1996-12-03 2011-02-01 Abgenix, Inc. Transgenic mammals having human ig loci including plural vh and vk regions and antibodies produced therefrom
US5899312A (en) * 1997-07-22 1999-05-04 Lucent Technologies Inc Anti-fraud string grabbing device
FR2807767B1 (fr) 2000-04-12 2005-01-14 Lab Francais Du Fractionnement Anticorps monoclonaux anti-d
CN101498731A (zh) * 2000-05-18 2009-08-05 日本烟草产业株式会社 抗协同刺激信号转导分子ailim的人单克隆抗体及其药物用途
US7413031B2 (en) 2000-07-18 2008-08-19 The Charles Machine Works, Inc. Apparatus and method for maintaining control of a drilling machine
US6596541B2 (en) 2000-10-31 2003-07-22 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
FR2822161B1 (fr) 2001-03-15 2003-09-19 Genoway Cellule et animal transgenique modelisant les reponses humaines allergiques mediees par les ige, et leurs utilisations
JP4653915B2 (ja) 2001-10-29 2011-03-16 独立行政法人科学技術振興機構 全身性エリテマトーデスモデル非ヒト動物
US20040132101A1 (en) * 2002-09-27 2004-07-08 Xencor Optimized Fc variants and methods for their generation
JP2004147535A (ja) 2002-10-29 2004-05-27 Japan Science & Technology Agency ギラン・バレー症候群及び／又はフィッシャー症候群発症モデル非ヒト動物
FR2858235B1 (fr) 2003-07-31 2006-02-17 Lab Francais Du Fractionnement Utilisation d'anticorps optimises en adcc pour traiter les patients faibles repondeurs
BRPI0506771A (pt) * 2004-01-12 2007-05-22 Applied Molecular Evolution anticorpo, e, composição farmacêutica
WO2007041635A2 (en) * 2005-10-03 2007-04-12 Xencor, Inc. Fc variants with optimized fc receptor binding properties
SG170110A1 (en) 2006-03-10 2011-04-29 Macrogenics Inc Identification and engineering of antibodies with variant heavy chains and methods of using same
US20080003225A1 (en) 2006-06-29 2008-01-03 Henri Vie Method for enhancing the antibody-dependent cellular cytotoxicity (ADCC) and uses of T cells expressing CD16 receptors
EP2066349B1 (en) * 2006-09-08 2012-03-28 MedImmune, LLC Humanized anti-cd19 antibodies and their use in treatment of tumors, transplantation and autoimmune diseases
HU0700534D0 (en) * 2006-11-24 2007-10-29 Mezoegazdasagi Biotechnologiai Transgenic animal with enhanced immune response and method for the preparation thereof
EP2245155A4 (en) * 2007-12-10 2011-05-25 Aliva Biopharmaceuticals Inc METHODS FOR SEQUENTIALLY REPLACING A TARGETED AREA BY HOMOLOGOUS RECOMBINATION
EP2318439A2 (en) * 2008-07-23 2011-05-11 Rappaport Family Institute for Research in the Medical Sciences Methods of altering peripheral b cell populations and uses thereof
NO2516457T3 (enExample) * 2009-12-21 2018-08-11
US8883496B2 (en) * 2009-12-21 2014-11-11 Regeneron Phamaceuticals, Inc. Humanized FcgR mice

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028959A9 (en) * 1994-04-22 2004-03-04 Sloan Kettering Inst Cancer MICE MUTANT FOR Fc RECEPTORS AND METHOD OF TREATING AUTOIMMUNE DISEASE
US6111166A (en) 1994-09-19 2000-08-29 Medarex, Incorporated Transgenic mice expressing human Fcα and β receptors
US6676927B1 (en) 1999-01-20 2004-01-13 The Rockefeller University Animal model and methods for its use in the selection of cytotoxic antibodies
US7416726B2 (en) 2000-04-13 2008-08-26 The Rockefeller University Enhancement of antibody-mediated immune responses
US6586251B2 (en) 2000-10-31 2003-07-01 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
US7351875B2 (en) 2002-06-07 2008-04-01 Trillium Therapeutics, Inc. FcγRIIa transgenic animal model for autoimmune disease
US7402728B2 (en) 2002-12-16 2008-07-22 Genentech, Inc. Transgenic mice expressing human CD20 and/or CD16
US7294754B2 (en) 2004-10-19 2007-11-13 Regeneron Pharmaceuticals, Inc. Method for generating an animal homozygous for a genetic modification
US20090035322A1 (en) 2007-07-31 2009-02-05 Regeneron Pharmaceuticals, Inc. Human Antibodies to Human CD20 and Method of Using Thereof
WO2009114400A1 (en) 2008-03-07 2009-09-17 Regeneron Pharmaceuticals, Inc. Es cell-derived mice from diploid host embryo injection

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
BARNES ET AL.: "FcyRl-Deficient Mice Show Multiple Alterations to Inflammatory and Immune Responses", IMMUNITY, vol. 16, 2002, pages 379 - 389
GESSNER J E ET AL: "The IgG Fc receptor family", ANNALS OF HEMATOLOGY, BERLIN, DE, vol. 76, no. 6, 1 June 1998 (1998-06-01), pages 231 - 248, XP002350430, ISSN: 0939-5555, DOI: DOI:10.1007/S002770050396 *
HEIJNEN ET AL.: "A Human FcgRl/CD64 Transgenic Model for In Vivo Analysis of (Bispecific) Antibody Therapeutics", J. HEMATOTHER, vol. 4, 1996, pages 351 - 356
HEIJNEN; VAN DE WINKEL: "Antigen Targeting to Myeloid-specific Human FcgRl/CD64 Triggers Enhanced Antibody Responses in Transgenic", J. CLIN. INVEST., vol. 97, no. 2, 1996, pages 331 - 338, XP002345168, DOI: doi:10.1172/JCI118420
LEIBSON PAUL J: "Signal transduction during natural killer cell activation: Inside the mind of a killer", IMMUNITY, vol. 6, no. 6, 1997, pages 655 - 661, XP002625480, ISSN: 1074-7613 *
NIMMERJAHN F ET AL: "Fc gamma receptors: Old friends and new family members", IMMUNITY, CELL PRESS, US, vol. 24, no. 1, 1 January 2006 (2006-01-01), pages 19 - 28, XP002498113, ISSN: 1074-7613, DOI: DOI:10.1016/J.IMMUNI.2005.11.010 *
PARK ET AL.: "Resistance of Fc Receptor-deficient Mice to Fatal Glomerulonephritis", J. CLIN. INVEST., vol. 102, no. 6, 1998, pages 1229 - 1238
POUEYMIROU ET AL.: "FO generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses", NATURE BIOTECH., vol. 25, no. 1, 2007, pages 91 - 99
SU ET AL.: "Genomic organization of classical human low-affinity Fcy receptor genes", GENES AND IMMUNITY, vol. 3, no. 1, 2002, pages S51 - S56, XP002430252, DOI: doi:10.1038/sj.gene.6363879
TAKAI ET AL.: "Augmented humoral and anaphylactic responses in FcgRll- deficient mice", NATURE, vol. 379, 1996, pages 346 - 349, XP002933124, DOI: doi:10.1038/379346a0
TAKAI ET AL.: "FcR y Chain Depletion Results in Pleiotrophic Effector Cell Defects", CELL, vol. 76, 1994, pages 519 - 529
VALENZUELA: "High-throughput engineering of the mouse genome coupled with high-resolution expression analysis", NATURE BIOTECH., vol. 21, no. 6, 2003, pages 652 - 659
VAN VUGT ET AL.: "FcR y-Chain Is Essential for Both Surface Expression and Function of Human FcyRl (CD64) In Vivo", BLOOD, vol. 87, no. 9, 1996, pages 3593 - 3599, XP000199686

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Publication number Priority date Publication date Assignee Title
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US10004211B2 (en) 2011-10-28 2018-06-26 Regeneron Pharmaceuticals, Inc. Humanized IL-6 and IL-6 receptor
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US12426579B2 (en) 2018-09-27 2025-09-30 Central Institute For Experimental Animals Immunodeficient mouse

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