WO2025067231A1 - Animaux non humains génétiquement modifiés ayant un locus d'immunoglobuline humanisée - Google Patents

Animaux non humains génétiquement modifiés ayant un locus d'immunoglobuline humanisée Download PDF

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WO2025067231A1
WO2025067231A1 PCT/CN2024/121081 CN2024121081W WO2025067231A1 WO 2025067231 A1 WO2025067231 A1 WO 2025067231A1 CN 2024121081 W CN2024121081 W CN 2024121081W WO 2025067231 A1 WO2025067231 A1 WO 2025067231A1
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human
genes
animal
mouse
endogenous
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Lijun Zhang
Yabo ZHANG
Yiqing HU
Huizhen ZHAO
Jiawei Yao
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Biocytogen Pharmaceuticals Beijing Co Ltd
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Biocytogen Pharmaceuticals Beijing Co Ltd
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • 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
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element

Definitions

  • This disclosure relates to genetically modified animals and cells with humanized light chain immunoglobulin locus and/or humanized heavy chain immunoglobulin locus.
  • Therapeutic antibodies are one of the fastest growing classes of therapeutic compounds, rapidly outpacing the growth of small-molecule drugs. These therapeutic antibodies are usually human or humanized antibodies.
  • the human or humanized antibodies can be generated by humanization of a rodent antibody (e.g., a mouse antibody) or by using phage libraries.
  • the antibodies that are generated by these methods often have suboptimal binding affinities and biophysical attributes, leading to difficulties in manufacture and poor pharmacokinetics.
  • the humanization process may adversely affect the binding affinity and introduce immunogenic epitopes to the antibodies, and antibodies discovered using phage libraries show limited diversity and non-native pairing of immunoglobulin heavy and light chains. Iterative and time-consuming experiments are often required to improve the properties. And in some cases, these antibodies can also be immunogenic in patients, leading to attenuation of their efficacy over time.
  • transgenic animals engineered to express a human antibody repertoire.
  • the generation of transgenic animals has allowed the use of such transgenic animals in various research and development applications, e.g., in drug discovery and basic research into various biological systems.
  • Many of the early generation transgenic animals had incomplete human antibody repertoires, had antibody production below the normal rates due to less efficient V (D) J recombination, had endogenous antibody repertoires which may introduce immunogenic epitopes, and various other issues.
  • antibody light chains are encoded by one of two separate loci: kappa ( ⁇ ) and lambda ( ⁇ ) .
  • the majority of mouse antibody light chains are kappa type.
  • the ratio of kappa to lambda light chains used in humans is about 60: 40. In mice, however, the ratio is about 95: 5.
  • Preferential use of kappa light chains in mice has been reported to be preserved in genetically modified mice capable of expressing fully or partially human antibodies. Thus, mice expressing fully or partially human antibodies showed limited use of the lambda variable regions.
  • the human IGLV genes, the human IGLJ genes, and the human IGLC genes are operably linked and can undergo rearrangement.
  • the animal comprises at least 50 human IGLV genes in Table 1, at least 5 human IGLJ genes in Table 2, and at least 5 human IGLC genes in Table 3.
  • the animal comprises all human IGLV genes, all human IGLJ genes, and all human IGLC genes at the endogenous lambda light chain immunoglobulin gene locus of human chromosome 22 of a human subject.
  • the animal comprises all human IGLV genes, all human IGLJ genes, and all human IGLC genes at the endogenous lambda light chain immunoglobulin gene locus of human chromosome 22 of a human cell.
  • the animal comprises an unmodified human sequence derived from a human lambda light chain immunoglobulin gene locus, in some embodiments, the unmodified human sequence is at least 800 kb, at least 850 kb, at least 855 kb, at least 895 kb, at least 900 kb, at least 950 kb, or at least 1000 kb. In some embodiments, the animal comprises an unmodified sequence derived from a human lambda light chain immunoglobulin gene locus starting from human IGLV (I) -70 to human IGLV3-1.
  • the animal comprises an unmodified sequence derived from a human lambda light chain immunoglobulin gene locus starting from human IGLV (I) -70 to human IGLC7.
  • the animal is homozygous with respect to the lambda light chain immunoglobulin gene locus and/or the safe harbor locus.
  • the animal is heterozygous with respect to the lambda light chain immunoglobulin gene locus and/or the safe harbor locus.
  • the animal is a rodent (e.g., a mouse) .
  • the animal further comprises at an endogenous heavy chain immunoglobulin gene locus, one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes.
  • the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked and can undergo VDJ rearrangement.
  • the animal comprises at least 150 human IGHV genes selected from Table 7, at least 20 human IGHD genes selected from Table 8, and at least 5 human IGHJ genes selected from Table 9.
  • the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin gene locus of human chromosome 14 of a human subject or cell.
  • the animal comprises a disruption in the animal’s endogenous heavy chain immunoglobulin gene locus.
  • the animal is a mouse and the disruption in the animal’s endogenous heavy chain immunoglobulin gene locus comprises a deletion of one or more mouse IGHV genes in Table 4, one or more mouse IGHD genes in Table 5, and/or one or more mouse IGHJ genes in Table 6.
  • the animal is a mouse and the disruption in the animal’s endogenous heavy chain immunoglobulin gene locus comprises a deletion of a contiguous sequence starting from mouse IGHV1-85 to mouse IGHJ4.
  • the animal comprises one or more endogenous IGHM, IGH ⁇ , IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE, and IGHA genes.
  • the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus, in some embodiments, the unmodified human sequence is at least 800 kb.
  • the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus
  • the unmodified human sequence has one of the following features: (1) starting from human IGHV (III) -82 to human IGHV1-2; (2) starting from human IGHV (III) -82 to human IGHV6-1; (3) starting from human IGHD1-1 to human IGHJ6; and (4) starting from human IGHV (III) -82 to human IGHJ6.
  • the animal is homozygous or heterozygous with respect to the heavy chain immunoglobulin gene locus.
  • the animal further comprises at an endogenous kappa light chain immunoglobulin gene locus, one or more human IGKV genes, and one or more human IGKJ genes.
  • the animal comprises all human IGKV genes in Table 13, and all human IGKJ genes in Table 14.
  • the animal comprises all human IGKV genes, and all human IGKJ genes at the endogenous kappa light chain immunoglobulin gene locus of human chromosome 2 of a human subject or cell.
  • the animal comprises a disruption in the animal’s endogenous kappa light chain immunoglobulin gene locus.
  • the animal is a mouse and the disruption in the animal’s endogenous light chain immunoglobulin gene locus comprises a deletion of one or more mouse IGKV genes in Table 15 and one or more mouse IGKJ genes in Table 16.
  • the animal is a mouse and the disruption in the animal’s endogenous kappa light chain immunoglobulin gene locus comprises a deletion of a sequence starting from mouse IGKV2-137 to mouse IGKJ5.
  • the animal comprises an unmodified sequence derived from a human kappa light chain immunoglobulin gene locus starting from human IGKV3D-7 to human IGKJ5.
  • the animal comprises an endogenous IGKC.
  • the animal is homozygous or heterozygous with respect to the kappa light chain immunoglobulin gene locus.
  • the disclosure is related to a genetically-modified, non-human animal whose genome comprises an endogenous light chain immunoglobulin locus comprising: a replacement of one or more endogenous IGLV, endogenous IGLJ, and endogenous IGLC genes with one or more human IGLV, human IGLJ, and human IGLC genes, in some embodiments, the human IGLV, human IGLJ, and human IGLC genes are operably linked. In some embodiments, one or more endogenous IGLV, endogenous IGLJ, and endogenous IGLC genes are replaced by all human IGLV genes in Table 1, all human IGLJ genes in Table 2, and all human IGLC genes in Table 3. In some embodiments, the animal is a mouse, and all mouse IGLV genes in Table 4, all mouse IGLJ genes in Table 5, and all mouse IGLC genes in Table 6 are replaced.
  • the disclosure is related to a genetically-modified, non-human animal whose genome comprises an endogenous safe harbor locus comprising: an insertion of one or more human IGLV, human IGLJ, and human IGLC genes, wherein the human IGLV, human IGLJ, and human IGLC genes are operably linked, optionally wherein the animal comprises a disruption in the animal’s endogenous lambda light chain immunoglobulin gene locus.
  • all human IGLV genes in Table 1, all human IGLJ genes in Table 2, and all human IGLC genes in Table 3 are inserted at the endogenous safe harbor locus.
  • the endogenous safe harbor locus is an endogenous Hipp11 locus or an endogenous Rosa26 locus.
  • the animal lacks an endogenous immunoglobulin lambda light chain variable region locus that is capable of rearranging and forming a nucleic acid sequence that encodes an endogenous lambda light chain variable domain (e.g., a mouse lambda light chain variable domain) .
  • the animal lacks an endogenous immunoglobulin heavy chain variable region locus that is capable of rearranging and forming a nucleic acid sequence that encodes an endogenous heavy chain variable domain (e.g., a mouse heavy chain variable domain) .
  • the animal lacks an endogenous immunoglobulin kappa light chain variable region locus that is capable of rearranging and forming a nucleic acid sequence that encodes an endogenous kappa light chain variable domain (e.g., a mouse kappa light chain variable domain) .
  • an endogenous immunoglobulin kappa light chain variable region locus that is capable of rearranging and forming a nucleic acid sequence that encodes an endogenous kappa light chain variable domain (e.g., a mouse kappa light chain variable domain) .
  • the animal can produce a humanized antibody.
  • the disclosure is related to a cell obtained from the animal described herein.
  • the cell is a B cell that expresses an immunoglobulin lambda light chain that is derived from a rearrangement of one or more human IGLV genes, one or more human IGLJ genes, and one or more human IGLC genes.
  • the cell is a B cell that expresses a chimeric immunoglobulin heavy chain comprising an immunoglobulin heavy chain variable domain that is derived from a rearrangement of one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, in some embodiments, the immunoglobulin heavy chain variable domain is operably linked to a non-human heavy chain constant region.
  • the cell is a B cell that expresses a chimeric immunoglobulin kappa light chain comprising an immunoglobulin kappa light chain variable domain that is derived from a rearrangement of one or more human IGKV genes, and one or more human IGKJ genes, in some embodiments, the immunoglobulin kappa light chain variable domain is operably linked to a non-human kappa light chain constant region.
  • the cell is an embryonic stem (ES) cell.
  • the disclosure is related to a method of making a chimeric antibody that specifically binds to an antigen, the method comprising exposing the animal described herein to the antigen; producing a hybridoma from a cell collected from the animal; and collecting the chimeric antibody produced by the hybridoma.
  • the method further comprises sequencing the genome of the hybridoma.
  • the disclosure is related to a method of modifying genome of a cell, the method comprising modifying one or more human chromosomes; introducing the modified one or more human chromosomes into a cell of the animal; and inducing recombination between the modified one or more human chromosomes and one or more endogenous chromosomes, in some embodiments, at least 150 human IGHV genes selected from Table 7, at least 20 human IGHD genes selected from Table 8, and at least 5 human IGHJ genes selected from Table 9 are integrated into the one or more endogenous chromosomes (e.g., mouse chromosome 12) by recombination; and/or at least 50 human IGLV genes in Table 1, at least 5 human IGLJ genes in Table 2, and at least 5 human IGLC genes in Table 3 are integrated into the one or more endogenous chromosomes (e.g., mouse chromosome 16 or 11) by recombination.
  • at least 150 human IGHV genes selected from Table 7 at
  • the disclosure is related to a method of modifying genome of a cell, the method comprising modifying one or more human chromosomes; introducing the modified one or more human chromosomes into a cell of the animal; and inducing recombination between the modified one or more human chromosomes and one or more endogenous chromosomes, in some embodiments, at least 150 human IGHV genes selected from Table 7, at least 20 human IGHD genes selected from Table 8, and at least 5 human IGHJ genes selected from Table 9 are integrated into the one or more endogenous chromosomes (e.g., mouse chromosome 12) by recombination; at least 50 human IGKV genes in Table 13, and at least 3 human IGKJ genes in Table 14 are integrated into the one or more endogenous chromosomes (e.g., mouse chromosome 6) by recombination; and/or at least 50 human IGLV genes in Table 1, at least 5 human IGLJ genes in Table 2, and
  • the disclosure is related to a method of making an antibody that specifically binds to an antigen, the method comprising exposing the animal described herein to the antigen; sequencing nucleic acids encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a hybrid antibody that specifically binds to the antigen; and expressing in a cell a nucleic acid encoding the human heavy chain immunoglobulin variable region and a human heavy chain immunoglobulin constant region and a nucleic acid encoding the human light chain immunoglobulin variable region and a human light chain immunoglobulin constant region.
  • the disclosure is related to a method of making an antibody that specifically binds to an antigen, the method comprising obtaining a nucleic acid sequence encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a hybrid antibody that specifically binds to the antigen, in some embodiments, the cell is obtained by exposing the animal described herein to the antigen; making a first nucleic acid, in some embodiments, the nucleic acid encoding the human heavy chain immunoglobulin variable region is operably linked with a nucleic acid encoding a human heavy chain immunoglobulin constant region in the first nucleic acid, and making a second nucleic acid, in some embodiments, the nucleic acid encoding the human light chain immunoglobulin variable region is operably linked with a nucleic acid encoding a human light chain immunoglobulin constant region in the second nucleic acid; and expressing the first and the second nucleic acids in a cell, thereby obtaining the antibody.
  • the disclosure is related to a method of obtaining a nucleic acid that encodes an antibody binding domain that specifically binds to an antigen, the method comprising exposing the animal described herein to the antigen; and sequencing nucleic acids encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a hybrid antibody that specifically binds to the antigen.
  • the disclosure is related to a method of obtaining a sample, the method comprising exposing the animal described herein to the antigen; and collecting the sample from the animal.
  • the sample is a spleen tissue, a spleen cell, or a B cell.
  • the disclosure also relates to an offspring of the non-human mammal.
  • the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.
  • the disclosure also provides to a cell including the targeting vector as described herein.
  • the disclosure also relates to a cell (e.g., a stem cell, an embryonic stem cell, an immune cell, a B cell, a T cell, or a hybridoma) or a cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof.
  • a cell e.g., a stem cell, an embryonic stem cell, an immune cell, a B cell, a T cell, or a hybridoma
  • a cell line e.g., a primary cell culture thereof derived from the non-human mammal or an offspring thereof.
  • the disclosure further relates to the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof.
  • the disclosure further relates to the use of the non-human mammal or an offspring thereof, the animal model generated through the method as described herein in the development of a product related to an immunization processes, the manufacture of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.
  • FIG. 1 is a schematic diagram showing human lambda light chain immunoglobulin locus on chromosome 22.
  • FIG. 2 is a schematic diagram showing mouse lambda light chain immunoglobulin locus on chromosome 16.
  • FIG. 3 is a flow chart of a method of introducing immunoglobulin genes into the mouse genome.
  • FIG. 4A shows a targeting strategy for modifying the mouse heavy chain immunoglobulin locus.
  • FIG. 4B shows a targeting strategy for modifying the mouse kappa light chain immunoglobulin locus.
  • FIG. 5 is a schematic diagram showing the mouse lambda light chain immunoglobulin locus (not drawn to scale) .
  • FIG. 6A is a schematic diagram showing the mouse lambda light chain immunoglobulin locus after two recombination sites were introduced to the genome.
  • FIG. 6B is a schematic diagram showing the mouse Hipp11 locus after two recombination sites were introduced to the locus.
  • FIG. 7A shows a targeting strategy at the mouse lambda light chain immunoglobulin locus on mouse chromosome 16 with a targeting vector.
  • the targeting vector does not include mouse IGLV, IGLJ, or IGLC genes.
  • FIG. 7B shows a targeting strategy at the mouse Hipp11 locus on mouse chromosome 11 with a targeting vector.
  • the mouse Hipp11 locus is located in cytoband A1 at ⁇ 3 cM between the Eif4enif1 and Drg1 genes.
  • FIG. 8 is a schematic diagram of the human chromosome 22 highlighting the lambda light chain immunoglobulin locus (not drawn to scale) .
  • the genetically modified animals can be made by introducing human immunoglobulin genes into the genome of non-human animals to produce animals that can express humanized antibodies or chimeric antibodies.
  • FIG. 3 shows the methods of making the humanized mice.
  • the methods first involve modifying the human immunoglobulin region on the human chromosome.
  • the modified human chromosomes are then introduced into the mouse recipient cell.
  • the human immunoglobulin variable region is then introduced into the corresponding region of the mouse genome by direct replacement (e.g., in one step replacement) , or inserted into an endogenous safe harbor locus.
  • the recipient cells are then screened, preferably for the cells that do not contain the human chromosomes.
  • the cells are then injected to blastocysts to prepare chimeric animals (e.g., mice) . Subsequent breeding can be performed to obtain animals containing intact humanized immunoglobulin locus.
  • humanized antibody refers to a non-human antibody which contains sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin.
  • polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
  • nucleic acid molecule As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.
  • an unmodified human sequence refers to a sequence that is derived from a human subject, a human cell, a cultured human cell or a human cell line, wherein the sequence is identical to the genetic sequence of a human subject, a human cell, a cultured human cell or a human cell line.
  • mice having a humanized lambda light chain immunoglobulin locus or a modified locus containing human lambda light chain immunoglobulin genes
  • Lambda light chain immunoglobulin locus (also known as IGL or immunoglobulin lambda locus) is a region on the chromosome (e.g., human chromosome 22) that contains genes for the light chains of human antibodies (or immunoglobulins) .
  • the immunoglobulin light chain genes can also undergo a series rearrangement that lead to the production of a mature immunoglobulin light chain nucleic acid (e.g., a lambda light chain) .
  • the animal comprises a human or humanized lambda light chain immunoglobulin locus.
  • the animal comprises a modified locus (e.g., a modified safe harbor locus) containing human lambda light chain immunoglobulin genes.
  • Lambda ( ⁇ ) light chain immunoglobulin locus (also known as IGL locus or immunoglobulin Lambda locus) is located on human chromosome 22 (Chr22q11.2) .
  • Table 1 lists IGLV genes and its relative orders in this locus. These genes and the order of these genes are also shown in FIG. 1.
  • These IGL genes on human chromosome 22 form three clusters, i.e., cluster A, cluster B, and cluster C (from proximal to distal) .
  • cluster A starts from ZNF280B and ends at the Enhancer downstream of IGLC7
  • cluster B starts from IGLV5-52 and ends at IGLV7-35
  • cluster C starts from BCRL2 and ends at VPREB1.
  • Table 2 lists all IGLJ genes and its relative orders on human chromosome 22. These genes and the order of these genes are also shown in FIG. 1.
  • Table 3 lists all immunoglobulin lambda constant (IGLC) genes. These genes and the order of these genes are also shown in FIG. 1. There is an Enhancer downstream of IGLC7 at the human lambda light chain immunoglobulin locus, and the relative order of the Enhancer is 8 in Table 3.
  • the genetically-modified, non-human animal described herein comprises this human Enhancer at the endogenous lambda light chain immunoglobulin locus or the modified locus (e.g., the modified safe harbor locus) described herein.
  • Lambda light chain immunoglobulin locus is located on mouse chromosome 16.
  • the IGLV genes, IGLJ genes, and IGLC genes are listed in Tables 4-6, along with their relative orders in this locus. These genes and the order of these genes are also shown in FIG. 2.
  • Enhancer 2-4 and Enhancer 3-1 are also located in the mouse lambda light chain immunoglobulin locus. There is also an Enhancer downstream of IGLC1 at the mouse lambda light chain immunoglobulin locus. The relative order of these enhancers can be found in FIG. 2.
  • the genetically-modified, non-human animal described herein does not comprise one or more of the enhancers selected from the mouse Enhancer following IGLC1, Enhancer 2-4, and Enhancer 3-1.
  • the present disclosure provides genetically-modified, non-human animal comprising one or more human IGLV genes, one or more human IGLJ, and/or one or more human IGLC genes.
  • the human IGLV genes, the human IGLJ genes, and the human IGLC genes are operably linked together.
  • the human IGLV genes and the human IGLJ genes can undergo VJ rearrangement.
  • the human IGLV genes, the human IGLJ genes, and the human IGLC genes are at an endogenous lambda light chain immunoglobulin gene locus.
  • the animal comprises an unmodified human sequence comprising a sequence starting from a gene selected from IGLV (I) -70, IGLV4-69, IGLV (I) -68, IGLV10-67, IGLV (IV) -66-1, IGLV (V) -66, IGLV (IV) -65, IGLV (IV) -64, IGLV (I) -63, and IGLV1-62, and ending at a gene selected from IGLC1, IGLC2, IGLC3, IGLC4, IGLC5, IGLC6, and IGLC7.
  • the unmodified human sequence derived from a human lambda light chain immunoglobulin gene locus starting from human IGLV (I) -70 to human IGLC7 In some embodiments, the unmodified human sequence derived from a human lambda light chain immunoglobulin gene locus starting from human IGLV (I) -70 to human IGLJ7. In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGLV (I) -70 to human IGLV3-1. In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGLV (I) -70 to the human Enhancer immediately downstream of human IGLC7.
  • the animal can have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences derived from a human lambda chain immunoglobulin gene locus.
  • the unmodified human sequence has a length of about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 810, 820, 830, 840, 850, 855, 860, 870, 880, 890, 895, 896, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, or 1050 kb.
  • the IGLV genes and/or the IGLJ genes are operably linked together.
  • the VJ recombination can occur among these genes and produce functional antibodies.
  • these genes are arranged in an order that is similar to the order in human lambda light chain immunoglobulin locus. This arrangement offers various advantages, e.g., the arrangement of these genes allow the production of light chain variable domains with a diversity that is very similar to the diversity of the lambda light chain variable domains in human.
  • the IGLV genes and/or the IGLJ genes are operably linked together to the IGLC gene (e.g., human IGLC gene) .
  • the animal is a mouse, which does not comprise Enhancer 2-4 at the endogenous lambda light chain immunoglobulin locus. In some embodiments, the animal is a mouse, which comprises Enhancer 3-1 at the endogenous lambda light chain immunoglobulin locus.
  • the animal comprises a disruption in the animal’s endogenous lambda light chain immunoglobulin gene locus.
  • the disruption in the animal’s endogenous light chain immunoglobulin gene locus comprises a deletion of one or more endogenous IGLV genes, one or more endogenous IGLJ genes, and/or one or more immunoglobulin lambda constant (IGLC) genes.
  • the animal is a mouse.
  • the disruption in the animal’s endogenous lambda light chain immunoglobulin gene locus comprises a deletion of at least or about 1, 2, or 3 mouse IGLV genes (e.g., genes as shown in Table 4) .
  • the disruption comprises a deletion of about or at least 1, 2, or 3 mouse IGLV genes selected from IGLV1, IGLV2, and IGLV3.
  • the mouse still comprises about or at least 1, 2, or 3 mouse IGLV genes selected from IGLV1, IGLV2, and IGLV3.
  • the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, or 6 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ2P, IGLJ3, IGLJ3P, and IGLJ4. In some embodiments, the disruption still comprises about or at least 1, 2, 3, 4, 5, or 6 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ2P, IGLJ3, IGLJ3P, and IGLJ4.
  • the disruption comprises a deletion of about or at least 1, 2, 3, or 4 mouse IGLC genes selected from IGLC1, IGLC2, IGLC3, and IGLC4. In some embodiments, the disruption still comprises about or at least 1, 2, 3, or 4 mouse IGLC genes selected from IGLC1, IGLC2, IGLC3, and IGLC4.
  • the disruption in the animal’s endogenous lambda light chain immunoglobulin gene locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mouse IGLV, IGLJ, and IGLC genes.
  • the deletion comprises about or at least 1, 2, 3, or 4 mouse IGKC genes selected from IGLC1, IGLC2, IGLC3, and IGLC4.
  • the disruption comprises a deletion of about or at least 1, 2, or 3 mouse IGLV genes selected from IGLV1, IGLV2, and IGLV3.
  • the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, or 6 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ2P, IGLJ3, IGLJ3P, and IGLJ4.
  • the deleted sequence starts from IGLV2 to IGLC1. In some embodiments, the deleted sequence starts from IGLV3 to IGLC1. In some embodiments, the deleted sequence starts from IGLJ2 to IGLC1.
  • the human IGLV genes, human IGLJ genes, and human IGLC genes are operably linked together and can undergo rearrangement.
  • the modified mouse has complete human IGLV, IGLJ, and IGLC gene repertoires (e.g., including all non-pseudo human IGLV, IGLJ, and IGLC genes) .
  • the modified mouse can produce an antibody having a human lambda light chain.
  • one human IGLV gene e.g., IGLV4-69, IGLV8-61, IGLV4-60, or IGLV6-57
  • one human IGLJ gene e.g., IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, or IGLJ7
  • one human IGLC gene e.g., IGLC1, IGLC2, IGLC3, IGLC4, IGLC5, IGLC6, or IGLC7 contributes to the sequence that encodes an antibody light chain constant region.
  • the entire mouse IGLV genes, IGLJ genes, and IGLC genes are knocked out, and the light chain will not have any sequence that is encoded by a sequence derived from the mouse, thereby minimizing immunogenicity in humans.
  • the human cluster A IGL genes are included in the modified chromosome.
  • the human cluster B IGL genes are included in the modified chromosome.
  • the human cluster C IGL genes are included in the modified chromosome.
  • at least 1, at least 2, or at least 3 of the human cluster A-C genes are included in the modified chromosome.
  • the human IGLV genes, human IGLJ genes, and human IGLC genes are inserted into one or more endogenous “safe harbor” loci (e.g., an endogenous Hipp11 locus or an endogenous Rosa26 locus) .
  • endogenous “safe harbor” loci e.g., an endogenous Hipp11 locus or an endogenous Rosa26 locus
  • Heavy chain immunoglobulin locus is a region on the chromosome (e.g., human chromosome 14) that contains genes for the heavy chains of human antibodies (or immunoglobulins) .
  • This region represents the germline organization of the heavy chain locus.
  • the locus includes V (variable) , D (diversity) , J (joining) , and C (constant) segments.
  • the genes in the V region form a V gene cluster (also known as IGHV gene cluster) .
  • the genes in the D region form a D gene cluster (also known as IGHD gene cluster) .
  • the genes in the J region form a J gene cluster (also known as IGHJ gene cluster) .
  • RPS8P1, ADAM6, and KIAA0125 are also located in this locus.
  • the relative order of RPS8P1 is 160
  • the relative order of ADAM6 is 161
  • the relative order of KIAA0125 is 164.
  • Table 8 lists all IGHD genes and its relative orders on human chromosome 14.
  • Table 9 lists all IGHJ genes and its relative orders on human chromosome 14. The genes for immunoglobulin constant domains are located after the IGHV, IGHD, and IGHJ genes.
  • the animal can have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences.
  • the unmodified human sequence has a length of about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb.
  • the IGHV genes, the IGHD genes, and/or the IGHJ genes are operably linked together to one or more genes (e.g., all genes) selected from IGHM, IGH ⁇ , IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE, and IGHA genes.
  • the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV1-86, IGHV1-85, IGHV1-84, IGHV1-83, IGHV1-82, IGHV1-81, IGHV1-80, IGHV1-79, IGHV1-78, and IGHV1-77 (e.g., IGHV1-86) .
  • the disruption comprises a deletion of about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4. In some embodiments, the mouse still comprises about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4.
  • the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a sequence in the human heavy chain immunoglobulin gene locus.
  • the sequence has a length of about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or 3500 kb.
  • the sequence starts from human IGHV (III) -82 to IGHV1-2. In some embodiments, the sequence starts from human IGHV7-81 to IGHV1-2. In some embodiments, the sequence starts from human IGHV (II) -1-1 to IGHVJ6. In some embodiments, the sequence starts from human IGHV6-1 to IGHVJ6.
  • the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked together and can undergo VDJ rearrangement.
  • the modified mouse has complete human IGHV, IGHD, and IGHJ gene repertoires (e.g., including all non-pseudo human IGHV, IGHD, and IGHJ genes) .
  • the modified mouse can produce a complete human antibody repertory.
  • one IGHV gene e.g., IGHV3-21 or IGHV3-74
  • One IGHD gene contributes to the sequence that encodes an antibody heavy chain variable region.
  • one IGHJ gene contributes to the sequence that encodes an antibody heavy chain variable region.
  • the IGHV gene is IGHV3-21 or IGHV3-74.
  • the entire mouse IGHV genes, IGHD genes, and IGHJ genes are knocked out, and the heavy chain variable region will not have any sequence that is encoded by a sequence derived from the mouse, thereby minimizing immunogenicity in human.
  • Kappa chain immunoglobulin locus (also known as IGK or immunoglobulin kappa locus) is a region on the chromosome (e.g., human chromosome 2) that contains genes for the kappa light chains of human antibodies (or immunoglobulins) .
  • the immunoglobulin kappa light chain genes can also undergo a series rearrangement that lead to the production of a mature immunoglobulin kappa light-chain nucleic acid (e.g., a kappa chain) .
  • V segment also known as an IGKV gene
  • J segment also known as an IGKJ gene
  • the human kappa light chain immunoglobulin locus is located on human chromosome 2.
  • Table 13 lists IGKV genes and its relative orders in this locus.
  • human IGKV genes including IGKV1 genes (including all IGKV genes starting with IGKV1, also known as V ⁇ I) , IGKV2 genes (including all IGKV genes starting with IGKV2, also known as V ⁇ II) , IGKV3 genes (including all IGKV genes starting with IGKV3, also known as V ⁇ III) , IGKV4 genes (including all IGKV genes starting with IGKV4, also known as V ⁇ IV) , IGKV5 genes (including all IGKV genes starting with IGKV5, also known as V ⁇ V) , IGKV6 genes (including all IGKV genes starting with IGKV6, also known as V ⁇ VI) , and IGKV7 genes (including all IGKV genes starting with IGKV1,
  • IGKV genes in human chromosome 2 also form two clusters, the proximal V ⁇ cluster and the distal V ⁇ cluster.
  • the sequences in the two clusters are similar but are not identical. This large segmental duplication of the sequence occurred since the divergence of the human lineage from the most recent shared ancestor with other great apes.
  • Table 14 lists all IGKJ genes and its relative orders on human chromosome 2.
  • the immunoglobulin kappa constant (IGKC) gene which encodes the kappa light chain immunoglobulin constant domains is located after the IGKV and IGKJ genes.
  • mice kappa light chain immunoglobulin locus is located on mouse chromosome 6.
  • Table 15 lists IGKV genes and its relative orders in this locus.
  • Gm9728 and Amd-ps2 are also located in this locus.
  • the relative order of Gm9728 is 4, and the relative order of Amd-ps2 is 134.
  • Table 16 lists all IGKJ genes and its relative orders on mouse chromosome 6.
  • the IGKC gene, which encodes the kappa light chain immunoglobulin constant domains are after the IGKV and IGKJ genes.
  • the present disclosure provides genetically-modified, non-human animal comprising one or more human IGKV genes and/or one or more human IGKJ genes.
  • the human IGKV genes and the human IGKJ genes are operably linked together and can undergo VJ rearrangement.
  • the human IGKV genes and the human IGKJ genes are at endogenous kappa light chain immunoglobulin gene locus.
  • the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 human IGKV genes (e.g., genes as shown in Table 13) .
  • human IGKV genes e.g., genes as shown in Table 13
  • the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGKV3D-7, IGKV1D-8, IGKV1D-43, IGKV1D-42, IGKV2D-10, IGKV3D-11, IGKV1D-12, IGKV1D-13, IGKV2D-14, and IGKV3D-15.
  • the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGKV2-10, IGKV1-9, IGKV1-8, IGKV3-7, IGKV1-6, IGKV1-5, IGKV2-4, IGKV7-3, IGKV5-2, and IGKV4-1.
  • the animal comprises about or at least 1, 2, 3, 4, or 5 human IGKJ genes (e.g., genes as shown in Table 14) . In some embodiments, the animal comprises 1, 2, 3, 4, or 5 human IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.
  • human IGKJ genes e.g., genes as shown in Table 14
  • the animal comprises 1, 2, 3, 4, or 5 human IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.
  • the animal comprises an endogenous IGKC.
  • the IGKV genes and/or the IGKJ genes are operably linked together. The VJ recombination can occur among these genes and produce functional antibodies.
  • these genes are arranged in an order that is similar to the order in human kappa light chain immunoglobulin locus. This arrangement offers various advantages, e.g., the arrangement of these genes allow the production of kappa light chain variable domains with a diversity that is very similar to the diversity of the kappa light chain variable domains in human.
  • the IGKV genes and/or the IGKJ genes are operably linked together to the IGKC gene (e.g., endogenous IGKC gene) .
  • the animal comprises a disruption in the animal’s endogenous kappa light chain immunoglobulin gene locus.
  • the disruption in the animal’s endogenous kappa light chain immunoglobulin gene locus comprises a deletion of one or more endogenous IGKV genes, and one or more endogenous IGKJ genes.
  • the animal is a mouse.
  • the disruption in the animal’s endogenous heavy chain immunoglobulin gene locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, or 163 mouse IGKV genes (e.g., genes as shown in Table 15) .
  • the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV2-137, IGKV1-136, IGKV1-135, IGKV14-134-1, IGKV17-134, IGKV1-133, IGKV1-132, IGKV1-131, IGKV14-130, and IGKV9-129.
  • the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV2-137, IGKV1-136, IGKV1-135, IGKV14-134-1, IGKV17-134, IGKV1-133, IGKV1-132, IGKV1-131, IGKV14-130, and IGKV9-129.
  • the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV3-10, IGKV3-9, IGKV3-8, IGKV3-7, IGKV3-6, IGKV3-5, IGKV3-4, IGKV3-3, IGKV3-2, and IGKV3-1.
  • the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV3-10, IGKV3-9, IGKV3-8, IGKV3-7, IGKV3-6, IGKV3-5, IGKV3-4, IGKV3-3, IGKV3-2, and IGKV3-1.
  • the disruption comprises a deletion of about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.
  • the mouse still comprises about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5 (e.g., IGKJ5) .
  • the disruption in the animal’s endogenous kappa light chain immunoglobulin gene locus comprises a deletion of about or at least 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb or 3500 kb of an endogenous sequence.
  • the deleted sequence starts from IGKV2-137 to IGKJ4, from IGKV1-136 to IGKJ4, from IGKV1-135 to IGKJ4, from IGKV2-137 to IGKJ5, from IGKV1-136 to IGKJ5, or from IGKV1-135 to IGKJ5 (e.g., from IGKV2-137 to IGKJ5) .
  • the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a sequence in the human kappa light chain immunoglobulin gene locus.
  • the sequence has a length of about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or 3500 kb.
  • the animal can have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences.
  • the unmodified human sequence has a length of about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or 3500 kb.
  • the human chromosome can be obtained from human cell lines, cancer cells, primary cell culture, and/or human fibroblasts.
  • the human cell is introduced with a first vector and is then fused with a recipient cell.
  • the modified chromosome is then separated and introduced into another appropriate recipient cell. Cells with the desired resistance are selected to obtain cells containing only one human chromosome.
  • a second vector is introduced into the cells, and the cells are selected by resistance.
  • a third vector, and/or a fourth vector can be introduced.
  • the recipient cell can be a mammalian cell, a human cell, or a mouse cell.
  • the recipient cell is a CHO cell, or preferably an A9 cell.
  • the endogenous locus e.g., any of the any of the safe harbor loci described herein
  • the endogenous locus can be modified to insert a nucleic acid, as shown in FIG. 7B.
  • a vector is used to insert the nucleic acid at the endogenous locus.
  • the 5’ end homology arm and/or the 3’ end homology arm can have a desired length to facilitate homologous recombination.
  • the homology arm is about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 kb (e.g., about 3 kb) .
  • the homology arm is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 kb.
  • the cell is a stem cell, an embryonic stem cell, or a fertilized egg cell.
  • step (d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .
  • the non-human mammal in the foregoing method is a mouse (e.g., a C57 mouse, a BALB/c mouse, or a C57BL/6 mouse) .
  • the non-human mammal in step (c) is a female with pseudo pregnancy (or false pregnancy) .
  • the fertilized eggs for the methods described above are C57BL/6 fertilized eggs.
  • Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.
  • Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein.
  • the fertilized egg cells are derived from rodents.
  • the genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the methods described above.
  • Cells, tissues, and animals are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express humanized or chimeric antibodies from an endogenous non-human locus.
  • the present disclosure also provides various targeting vectors (e.g., vectors that are useful for making the genetically modified animals) .
  • the vector can comprise: a) a DNA fragment homologous to the 5’ end of a region to be altered (5’ homology arm) ; b) a sequence comprising desired genetic elements (e.g., LoxP recognition site, drug resistance genes, and/or reporter genes etc. ) ; and c) a second DNA fragment homologous to the 3’ end of the region to be altered (3’ homology arm) .
  • the disclosure also relates to a cell comprising the targeting vectors as described herein.
  • the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.
  • the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.
  • the present disclosure further relates to methods for generating genetically modified animal model with two or more human or chimeric genes.
  • the animal can comprise one or more human or humanized immunoglobulin locus and a sequence encoding an additional human or chimeric protein.
  • the additional human or chimeric protein can be programmed cell death protein 1 (PD-1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, CD137, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , or TNF Receptor Superfamily Member 4 (TNFRSF4
  • the methods of generating genetically modified animal model with additional human or chimeric genes can include the following steps:
  • the genetically modified animal in step (b) of the method, can be mated with a genetically modified non-human animal with human or chimeric PD-1, CTLA-4, OX40, TIGIT, PD-L1, BTLA, TIM-3, LAG-3, CD137, CD47, SIRPa, CD27, CD28, CD154, TIGIT, GITR, , or.
  • the genetically modified animals can have a human ADAM6 gene, an endogenous ADAM6 gene or a modified ADAM6 gene.
  • the ADAM6 protein is a member of the ADAM family of proteins, where ADAM is an acronym for A Disintegrin And Metalloprotease.
  • the human ADAM6 gene normally found between human IGHV genes IGHV1-2 and IGHV6-1, is a pseudogene.
  • mice there are two ADAM6 genes, ADAM6a and ADAM6b. They are located in an intergenic region between mouse IGHV and IGHD gene clusters.
  • the mouse ADAM6a is located between mouse IGHV5-1 and mouse IGHD5-1.
  • the mouse ADAM6b is located between mouse IGHD3-1 and mouse IGHD1-1.
  • the genetically modified animals can have a human ADAM6 gene.
  • the genetically modified animals do not have an endogenous ADAM6 gene.
  • the genetically modified animals are mice.
  • the mice are modified to include a nucleotide sequence that encodes an ADAM6 protein (e.g., ADAM6a or ADAM6b) .
  • the sequence is placed at any suitable position. It can be placed in the intergenic region, or in any suitable position in the genome.
  • the nucleic acid encodes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a mouse ADAM6a gene (e.g., 113539230-113547024 of NC_000078.6; SEQ ID NO: 53) or a mouse ADAM6b gene (e.g., 113486188-113492125 of NC_000078.6; SEQ ID NO: 54) .
  • the nucleic acid additionally includes the regulatory elements for the ADAM6a gene and ADAM6b gene (e.g., promoters) .
  • a functional mouse ADAM6 locus can be placed in the midst of human IGHV gene cluster.
  • the mouse ADAM6 locus is between two human IGVH genes.
  • the human ADAM6 pseudogene between human VH1-2 and human VH (II) -1-1 is replaced with the mouse ADAM6 locus.
  • the ADAM6a gene and the ADAM6b gene are located between human IGHV1-2 and human VH (II) -1-1 in the genome of the animal.
  • the location of the mouse ADAM6 sequence within the human gene sequence can approximate the position of the human ADAM6 pseudogene or can approximate the position of the mouse ADAM6 sequence (e.g., within the V-D intergenic region) .
  • the genetic modified mice has a humanized heavy chain immunoglobulin locus.
  • the mouse ADAM6a and the mouse ADAM6b are located between human IGHV1-2 and IGHV6-1 genes. Placing the mouse ADAM6a and the mouse ADAM6b between human IGHV1-2 and IGHV6-1 genes can have various advantages.
  • the disclosure provides a genetically-modified animal comprising at an endogenous heavy chain immunoglobulin gene locus, a first sequence comprising one or more human IGHV genes; a second sequence comprising a ADAM6 gene; and a third sequence comprising one or more human IGHD genes, and one or more human IGHJ genes.
  • the first sequence, the second sequence, and the third sequence are operably linked.
  • the first sequence comprises all human IGHV genes in Table 7 except IGHV2-10, IGHV3-9, IGHV1-8, IGHV (II) -1-1, and IGHV6-1. In some embodiments, the first sequence comprises all human IGHV genes in Table 7 except IGHV5-10-1 and IGHV3-64D, IGHV (II) -1-1, and IGHV6-1. In some embodiments, the first sequence is an unmodified sequence derived from a human heavy chain immunoglobulin gene locus.
  • the second sequence comprises either one or both of a mouse ADAM6a gene and a mouse ADAM6b gene.
  • the animal is a fertile male mouse.
  • the second sequence does not have a mouse ADAM6a gene or a mouse ADAM6b gene.
  • the third sequence comprises all human IGHD genes in Table 8, and all human IGHJ genes in Table 9. In some embodiments, the third sequence comprises human IGHV6-1. In some embodiments, the third sequence comprises human IGHV (II) -1-1. In some embodiments, the third sequence is an unmodified sequence derived from a human heavy chain immunoglobulin gene locus.
  • the AMAM6a and/or ADAM6b are endogenous sequences. In some embodiments, the AMAM6a and/or ADAM6b are not replaced, and/or located in its endogenous or native position.
  • the mouse IGHV genes before mouse IGHV1-2 in the heavy chain variable region locus are replaced with human IGHV genes. In some embodiments, the mouse IGHV, IGHD and IGHJ genes after mouse IGHV6-1 in the heavy chain variable region locus are replaced with one or more human IGHV genes, IGHD and /or IGHJ genes.
  • the mouse IGHV, IGHD and IGHJ genes can be replaced with human IGHV, IGHD and IGHJ by more than one replacements.
  • a selected number of mouse IGHV genes on the 5’s ide of the ADAM6a e.g., all mouse IGHV genes in Table 10
  • human IGHV genes e.g., all mouse IGHV genes in Table 10.
  • a selected number of mouse IGHD and IGHJ genes on the 3’s ide of the ADAM6b e.g., all mouse IGHD genes in Table 11 except IGHD5-1 and IGHD3-1 and all IGHJ genes in Table 12
  • the replacement can be performed by homologous recombination or Cre-mediated recombination.
  • the mice do not have mouse ADAM6a or ADAM6b genes. In some embodiments, the mice have human ADAM6 genes.
  • mice with superovulation can be used in mating.
  • in vitro fertilization can be used.
  • Superovulation can be induced by injecting serum gonadotropin and chorionic gonadotropin (e.g., human or mouse CG) into a mature female mouse.
  • serum gonadotropin and chorionic gonadotropin e.g., human or mouse CG
  • a mature male mouse can be sacrificed and its cauda epididymides can be isolated. The duct of cauda epididymis is cut open to release sperm.
  • a superovulating mature female mouse can be sacrificed and the oviducts can be isolated. Cumulus-oocyte-complexes (COCs) can be released from the oviduct.
  • COCs Cumulus-oocyte-complexes
  • sperm suspension can be added to the COCs and incubated for insemination.
  • Pathenogenic oocytes containing only one pronucleus can be removed.
  • embryos at 2-cell stage can be transferred to recipient females. Methods of increasing mouse fertility are known in the art.
  • the disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein.
  • the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein.
  • the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides.
  • the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.
  • the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.
  • the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology” ) .
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percentage of residues conserved with similar physicochemical properties can also be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the homology percentage in many cases, is higher than the identity percentage.
  • the present disclosure also provides an amino acid sequence that has at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%homology percentage to any amino acid sequence as described herein, or a nucleic acid encoding these amino acid sequences.
  • the genetic modified animals can be used to generate humanized or chimeric antibodies that can bind specifically to a target.
  • the target e.g., a protein or a fragment of the protein
  • the genetic modified animal is exposed to a selected antigen for a time and under conditions which permit the animal to produce antibody specific for the antigen.
  • Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein.
  • the antigenic peptide or protein is injected with at least one adjuvant.
  • the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times) .
  • the full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens.
  • the antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
  • An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., the genetically modified animal as described herein) .
  • An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide (e.g., a fragment of the protein) .
  • the preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide, or an antigenic peptide thereof (e.g., part of the protein) as an immunogen.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized polypeptide or peptide.
  • ELISA enzyme-linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) , or trioma techniques.
  • standard techniques such as the hybridoma technique originally described by Kohler et al. (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Lis
  • Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.
  • the disclosure provides a mouse that comprises a modification of an endogenous immunoglobulin heavy chain locus, wherein the mouse produces a B cell that comprises a rearranged immunoglobulin sequence operably linked to a heavy chain constant region gene sequence.
  • the rearranged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises a human heavy chain V, D, and/or J sequence.
  • the heavy chain constant region gene sequence comprises a human or a mouse heavy chain sequence selected from the group consisting of a CH1, a hinge, a CH2, a CH3, and a combination thereof.
  • the disclosure provides a mouse that comprises a modification of an endogenous immunoglobulin light chain (e.g., kappa or lambda) locus, and/or a modified locus (e.g., any of the safe harbor loci described herein) containing human lambda light chain immunoglobulin genes, wherein the mouse produces a B cell that comprises a rearranged immunoglobulin sequence operably linked to a light chain constant region gene sequence.
  • the rearranged immunoglobulin sequence operably linked to the light chain constant region gene sequence comprises a human light chain V and/or J sequence.
  • the light chain constant region gene sequence comprises a human or a mouse light chain constant region.
  • the mouse B cells or spleen cells can comprise a rearranged non-mouse immunoglobulin variable gene sequence, e.g., operably linked to a mouse immunoglobulin constant region gene.
  • the sequences for encoding human heavy chain variable region and human light chain variable region are determined.
  • the sequences can be determined by e.g., sequencing the hybridoma of interest or B cells.
  • single B cell screening is used. It can screen the natural antibody repertoire without the need for hybridoma fusion and combinatorial display.
  • B cells can be mixed with a panel of DNA-barcoded antigens, such that both the antigen barcode (s) and B-cell receptor (BCR) sequences of individual B cells are recovered via single-cell sequencing protocols.
  • the antibodies can be further modified to obtain a humanized antibody or a human antibody, e.g., by operably linking the sequence encoding human heavy chain variable region to a sequence encoding a human heavy chain constant region, and/or operably linking the sequence encoding human light chain variable region to a sequence encoding a human light chain constant region.
  • the mouse if the mouse expresses a protein that is very similar to the antigen of interest, it can be difficult to elicit an immune response in the mouse. This is because during immune cell development, B-cells and T-cells that recognize MHC molecules bound to peptides of self-origin are deleted from the repertoire of immune cells. In those cases, the humanized mouse can be further modified. The corresponding gene in the mouse can be knocked out, and the mouse is then exposed to the antigen of interest. Because the mouse does not go through negative selection for the gene product, the mouse can generate an antibody that can specifically bind to the target easily.
  • the disclosure also provides methods of making antibodies, nucleic acids, cells, tissues (e.g., spleen tissue) .
  • the methods involve exposing the animal as described herein to the antigen.
  • Antibodies e.g., hybrid antibodies
  • nucleic acids encoding the antibodies, cells, and/or tissues e.g., spleen tissue
  • the nucleic acids encoding human heavy and light chain immunoglobulin variable regions are determined, e.g., by sequencing.
  • the nucleic acid encoding the human heavy chain immunoglobulin variable region can be operably linked with a nucleic acid encoding a human heavy chain immunoglobulin constant region.
  • the nucleic acid encoding the human light chain immunoglobulin variable region can be operably linked with a nucleic acid encoding a human light chain immunoglobulin constant region.
  • the cells containing the nucleic acids as described herein are cultured and the antibodies are collected.
  • no mouse immunoglobulin V, D, J genes contributes to the heavy chain and/or light chain variable region sequence.
  • the heavy chain and/or light chain variable region sequence produced by the animal are fully human, and are completely contributed by human immunoglobulin V, D, J genes (e.g., human IGHV, IGHD, IGHJ, IGKV, IGKJ, IGLV, and/or IGLJ genes) .
  • Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis.
  • Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen-binding domain.
  • some antibodies or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target.
  • the amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell) , or introducing new glycosylation sites.
  • Antibodies disclosed herein can be derived from any species of animal, including mammals.
  • Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas) , chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits) , including transgenic rodents genetically engineered to produce human antibodies.
  • Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) , for example in the CDRs.
  • a cysteine residue can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo.
  • Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al. (Cancer Res. 53: 2560-2565, 1993) .
  • an antibody can be engineered which has dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3: 219-230, 1989) .
  • a covalent modification can be made to the antibody or antigen-binding fragment thereof.
  • These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage.
  • Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N-or C-terminal residues.
  • FIG. 3 shows methods of making the humanized mice. The methods first involve modifying the human immunoglobulin region on the human chromosome. The modified human chromosomes were then introduced into the mouse recipient cell.
  • the mouse immunoglobulin region was replaced by the human immunoglobulin region by direct replacement (e.g., homologous recombination, or Cre-mediated recombination) .
  • the human immunoglobulin region can be introduced into the mouse genome by a stepwise approach. Then, the recipient cells were screened for the correct replacement. The cells were then injected to blastocysts to prepare chimeric mice. Subsequent breeding was performed to obtain mice containing intact human immunoglobulin regions.
  • EXAMPLE 2 Modification of the mouse heavy chain immunoglobulin locus and kappa light chain immunoglobulin locus
  • hVH/hVK mice have both a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus.
  • the heavy chain immunoglobulin locus is a region on the chromosome that contains genes for the heavy chains of antibodies.
  • the locus includes IGHV (variable) , IGHD (diversity) , IGHJ (joining) , and heavy chain constant domain genes (FIG. 4A) .
  • the kappa chain immunoglobulin locus is a region on the chromosome that contains genes that encode the light chains of antibodies (kappa chain) .
  • the kappa chain immunoglobulin locus includes IGKV (variable) , IGKJ (joining) , and light chain constant domain genes (FIG. 4B) .
  • IGKV variable
  • IGKJ joining
  • FOG. 4B light chain constant domain genes
  • the immunoglobulin kappa light chain locus is located on mouse chromosome 6.
  • the mouse chromosome 6 was modified by knocking out the entire sequence of the immunoglobulin kappa light chain variable region locus.
  • Detailed knock-out methods can be found, e.g., in WO2020169022A1 and US20200390073A1; Zou, X., et al. "Subtle differences in antibody responses and hypermutation of lambda light chains in mice with a disrupted ⁇ constant region. " European Journal of Immunology 25.8 (1995) : 2154-2162; Zou, Y. R., et al.
  • the vector (V01) has the following elements from the 5’ to 3’ end: a DNA homology arm sequence upstream of the insertion site, Flp recognition target (FRT) , a mammalian expression promoter (EF-1a) from human elongation factor 1 alpha (EF-1a promoter) , a hygromycin resistance gene (apartial sequence of hygromycin phosphotransferase; “5’ HygR” ) , LoxP (Rec01) , 5’PB (piggyBac) transposon sequence (PB5’) , a first Core insulator, a Ubiquitin C promoter, a sequence encoding the DT receptor (DTR) , a sequence encoding the FMDV self-cleaving peptide (2A) , a neomycin resistance gene sequence (Neo) , a transcription termination/polyadenylation signal sequence (PolyA; “PA” ) , a second Core insulator
  • connection between the 3’ end of the human IGL gene and the mouse sequence was designed as: wherein the sequence in italic at the 3’ end is the mouse sequence, the sequences in bold are the restriction site sequences or additional sequences, the sequences with underlines are lox sites, and the sequence with the normal front at the 5’ end is the human sequence.
  • the leukocytes included: B cells (e.g., characterized by CD45+, CD19+, TCR ⁇ -) , T cells, macrophages (e.g., characterized by CD45+, CD11b+, F4/80+) , monocytes (e.g., characterized by CD45+, CD11b+, F4/80-, Ly-6C high, Ly6G-) , neutrophils (e.g., characterized by CD45+, CD11b+, F4/80-, Ly-6C+, Ly6G+) , DC cells (e.g., characterized by CD45+, CD11b+, F4/80-, Ly-6C+, Ly6G+) and natural killer (NK) cells (e.g., characterized by CD45+, TCR ⁇ -, CD19-, NK1.1+) .
  • B cells e.g., characterized by CD45+, CD19+, TCR ⁇ -
  • T cells e.g., TCR ⁇
  • B cell progenitor cells in bone marrow can be divided into 3 cell populations: pro-B-cells (characterized by B220 low CD43 high IgM low ) , pre-B-cells (characterized by B220 low CD43 int IgM low ) , and immature-B-cells (characterized by B220 high CD43 low IgM high ) .
  • T1 Transitional type 1 B cell, characterized by B220 + IgM + IgD -
  • T2 Transitional type 2 B cell, characterized by B220 + IgM + IgD +
  • mature B cells characterized by B220 + IgM low IgD +
  • B cell development was also evaluated in lymph nodes by flow cytometry to selectively stain plasma cells (B220 low IgM - IgD - CD138 + ) and memory B cells (B220 + IgM + IgD - CD38 + ) . The results are shown in the tables below.
  • Antigen A is a member of the tumor necrosis factor receptor superfamily. Five hVH H/H /hVK H/H /hVL H/H mice and five hVH H/H /hVK -/- /hVL H/H mice were immunized by Antigen A for a total of five times. Freund’s complete adjuvant (CFA) and 20 ⁇ g of Antigen A were used for the first immunization and Freund’s incomplete adjuvant (IFA) , CpG oligonucleotide (CpG) and 20 ⁇ g of Antigen A were used for the second, third, fourth and fifth immunizations. A total of five immunizations were performed.
  • Antigen B is a sialoadhesin molecule and a member of the immunoglobulin supergene family. Antigen B was used to immunize of hVH H/H /hVK -/- /hVL H/H mice. BiacoreTM was then used to detect the affinity of 8 human common light chain antibodies produced against the antigen B.

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Abstract

L'invention concerne des animaux et des cellules génétiquement modifiés avec un locus d'immunoglobuline à chaîne légère lambda humanisé et/ou un locus d'immunoglobuline à chaîne lourde humanisé. L'invention concerne également des animaux et des cellules génétiquement modifiés avec un locus modifié contenant des gènes d'immunoglobuline de chaîne légère lambda humaine et/ou un locus d'immunoglobuline de chaîne lourde humanisé. Dans certains modes de réalisation, les animaux ont en outre un locus d'immunoglobuline à chaîne légère kappa humanisé.
PCT/CN2024/121081 2023-09-26 2024-09-25 Animaux non humains génétiquement modifiés ayant un locus d'immunoglobuline humanisée Pending WO2025067231A1 (fr)

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WO2005104835A2 (fr) * 2004-04-22 2005-11-10 Kirin Beer Kabushiki Kaisha Animaux transgeniques et leurs utilisations
US20120124686A1 (en) * 2010-11-12 2012-05-17 Liqun Luo Site-Directed Integration of Transgenes in Mammals
CN104334732A (zh) * 2012-03-28 2015-02-04 科马布有限公司 表达人λ免疫球蛋白轻链可变结构域的动物
US20190292263A1 (en) * 2018-03-24 2019-09-26 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals for generating therapeutic antibodies against peptide-mhc complexes, methods of making and uses thereof
US20200390073A1 (en) * 2019-02-18 2020-12-17 Beijing Biocytogen Co., Ltd. Genetically modified non-human animals with humanized immunoglobulin locus
US20220330532A1 (en) * 2019-06-05 2022-10-20 Regeneron Pharmaceuticals, Inc. Non-human animals having a limited lambda light chain repertoire expressed from the kappa locus and uses thereof
US20230128645A1 (en) * 2020-06-02 2023-04-27 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animals with common light chain immunoglobulin locus

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005104835A2 (fr) * 2004-04-22 2005-11-10 Kirin Beer Kabushiki Kaisha Animaux transgeniques et leurs utilisations
US20120124686A1 (en) * 2010-11-12 2012-05-17 Liqun Luo Site-Directed Integration of Transgenes in Mammals
CN104334732A (zh) * 2012-03-28 2015-02-04 科马布有限公司 表达人λ免疫球蛋白轻链可变结构域的动物
US20190292263A1 (en) * 2018-03-24 2019-09-26 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals for generating therapeutic antibodies against peptide-mhc complexes, methods of making and uses thereof
US20200390073A1 (en) * 2019-02-18 2020-12-17 Beijing Biocytogen Co., Ltd. Genetically modified non-human animals with humanized immunoglobulin locus
US20220330532A1 (en) * 2019-06-05 2022-10-20 Regeneron Pharmaceuticals, Inc. Non-human animals having a limited lambda light chain repertoire expressed from the kappa locus and uses thereof
US20230128645A1 (en) * 2020-06-02 2023-04-27 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animals with common light chain immunoglobulin locus

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