WO2018233606A1 - Animal non humain génétiquement modifié avec gitr humain ou chimérique - Google Patents

Animal non humain génétiquement modifié avec gitr humain ou chimérique Download PDF

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WO2018233606A1
WO2018233606A1 PCT/CN2018/091844 CN2018091844W WO2018233606A1 WO 2018233606 A1 WO2018233606 A1 WO 2018233606A1 CN 2018091844 W CN2018091844 W CN 2018091844W WO 2018233606 A1 WO2018233606 A1 WO 2018233606A1
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gitr
animal
human
exon
chimeric
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PCT/CN2018/091844
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English (en)
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Yuelei SHEN
Yanan GUO
yang BAI
Jiawei Yao
Rui Huang
Lei Zhao
Meiling Zhang
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Beijing Biocytogen Co., Ltd
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Priority claimed from CN201810623611.XA external-priority patent/CN109136275B/zh
Application filed by Beijing Biocytogen Co., Ltd filed Critical Beijing Biocytogen Co., Ltd
Publication of WO2018233606A1 publication Critical patent/WO2018233606A1/fr
Priority to US16/435,243 priority Critical patent/US10945419B2/en

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    • 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
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • 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
    • A01K2207/12—Animals modified by administration of exogenous 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
    • 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
    • A01K2267/0331—Animal model for proliferative diseases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2015/8527—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic for producing animal models, e.g. for tests or diseases

Definitions

  • This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) glucocorticoid-induced TNFR-related protein (GITR) , and methods of use thereof.
  • human or chimeric e.g., humanized
  • GITR glucocorticoid-induced TNFR-related protein
  • the immune system has developed multiple mechanisms to prevent deleterious activation of immune cells.
  • One such mechanism is the intricate balance between positive and negative costimulatory signals delivered to immune cells.
  • Targeting the stimulatory or inhibitory pathways for the immune system is considered to be a potential approach for the treatment of various diseases, e.g., cancers and autoimmune diseases.
  • This disclosure is related to an animal model with human GITR or chimeric GITR.
  • the animal model can express human GITR or chimeric GITR (e.g., humanized GITR) protein in its body. It can be used in the studies on the function of GITR gene, and can be used in the screening and evaluation of anti-human GITR antibodies.
  • the animal models prepared by the methods described herein can be used in drug screening, pharmacodynamics studies, treatments for immune-related diseases (e.g., autoimmune disease) , and cancer therapy for human GITR target sites; they can also be used to facilitate the development and design of new drugs, and save time and cost.
  • this disclosure provides a powerful tool for studying the function of GITR protein and a platform for screening cancer drugs.
  • the disclosure relates to genetically-modified, non-human animals whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric GITR.
  • the sequence encoding the human or chimeric GITR is operably linked to an endogenous regulatory element at the endogenous GITR gene locus in the at least one chromosome.
  • the sequence encoding a human or chimeric GITR comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human GITR (NP_004186.1 (SEQ ID NO: 23) ) .
  • the sequence encoding a human or chimeric GITR comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 27. In some embodiments, the sequence encoding a human or chimeric GITR comprises a sequence encoding an amino acid sequence that corresponds to amino acids 1-142 of SEQ ID NO: 23.
  • the animal is a mammal, e.g., a monkey, a rodent or a mouse. In some embodiments, the animal is a C57BL/6 mouse. In some embodiments, the animal does not express endogenous GITR. In some embodiments, the animal has one or more cells expressing human or chimeric GITR. In some embodiments, the expressed human or chimeric GITR can bind to or interact with human protein GITRL (also known as TNFSF18) . In some embodiments, the expressed human or chimeric GITR can bind to or interact with endogenous TNFSF18.
  • human protein GITRL also known as TNFSF18
  • the disclosure relates to genetically-modified, non-human animals, wherein the genome of the animals comprises a replacement, at an endogenous GITR gene locus, of a sequence encoding a region of endogenous GITR with a sequence encoding a corresponding region of human GITR.
  • the sequence encoding the corresponding region of human GITR is operably linked to an endogenous regulatory element at the endogenous GITR locus, and one or more cells of the animal expresses a chimeric GITR.
  • the animal does not express endogenous GITR.
  • the locus of endogenous GITR is the extracellular region of GITR.
  • the animal has one or more cells expressing a chimeric GITR having an extracellular region, a transmembrane region, and a cytoplasmic region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%identical to the extracellular region of human GITR.
  • the extracellular region of the chimeric GITR has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human GITR.
  • the animal is a mouse, and the sequence encoding the region of endogenous GITR is exon 1, exon 2, exon 3, exon 4, and/or exon 5 of the endogenous mouse GITR gene (e.g., exon 1, exon 2, exon 3, and part of exon 4) .
  • the animal is heterozygous with respect to the replacement at the endogenous GITR gene locus. In some embodiments, the animal is homozygous with respect to the replacement at the endogenous GITR gene locus.
  • the disclosure relates to methods for making a genetically-modified, non-human animal.
  • the methods involve replacing in at least one cell of the animal, at an endogenous GITR gene locus, a sequence encoding a region of an endogenous GITR with a sequence encoding a corresponding region of human GITR.
  • the sequence encoding the corresponding region of human GITR comprises exon 1, exon 2, exon 3, exon 4, and/or exon 5 of a human GITR gene.
  • the sequence encoding the corresponding region of GITR comprises exon 1, exon 2, exon 3, and/or exon 4 (or part thereof, e.g., part of exon 1 and/or exon 4) of a human GITR gene.
  • the sequence encoding the corresponding region of human GITR encodes amino acids 1-142 of SEQ ID NO: 23.
  • the region is located within the extracellular region of GITR.
  • the animal is a mouse
  • the sequence encoding the region of the endogenous GITR locus is exon 1, exon 2, exon 3, exon 4, and/or exon 5 of mouse GITR gene (e.g., part of exon 1, exon 2, exon 3, and part of exon 4) .
  • the disclosure relates to non-human animals comprising at least one cell comprising a nucleotide sequence encoding a chimeric GITR polypeptide, wherein the chimeric GITR polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human GITR, wherein the animal expresses the chimeric GITR.
  • the chimeric GITR polypeptide has at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human GITR extracellular region.
  • the chimeric GITR polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 1-142 of SEQ ID NO: 23.
  • the nucleotide sequence is operably linked to an endogenous GITR regulatory element of the animal.
  • the chimeric GITR polypeptide comprises an endogenous GITR transmembrane region and/or an endogenous GITR cytoplasmic region.
  • the nucleotide sequence is integrated to an endogenous GITR gene locus of the animal.
  • the chimeric GITR has at least one mouse GITR activity (e.g., interacting with mouse GITRL (TNFSF18) , and promoting immune responses in mice) and/or at least one human GITR activity (e.g., interacting with human GITRL (TNFSF18) , and promoting immune responses in human) .
  • mouse GITR activity e.g., interacting with mouse GITRL (TNFSF18) , and promoting immune responses in mice
  • human GITR activity e.g., interacting with human GITRL (TNFSF18) , and promoting immune responses in human
  • the disclosure relates to methods of making a genetically-modified mouse cell that expresses a chimeric GITR, the method including: replacing, at an endogenous mouse GITR gene locus, a nucleotide sequence encoding a region of mouse GITR with a nucleotide sequence encoding a corresponding region of human GITR, thereby generating a genetically-modified mouse cell that includes a nucleotide sequence that encodes the chimeric GITR, wherein the mouse cell expresses the chimeric GITR.
  • the chimeric GITR comprises a signal peptide sequence (e.g., a mouse signal peptide sequence or a human signal peptide sequence) , an extracellular region of mouse GITR, an extracellular region of human GITR, a transmembrane and/or a cytoplasmic region of a mouse GITR.
  • the nucleotide sequence encoding the chimeric GITR is operably linked to an endogenous GITR regulatory region, e.g., promoter.
  • the animals further comprise a sequence encoding an additional human or chimeric protein.
  • the additional human or chimeric protein is 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, CD40, CD47, CD137, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , Signal regulatory protein ⁇ (SIRP ⁇ ) , or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40) .
  • PD-1 programmed cell death protein 1
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • LAG-3 Lymphocyte Activating 3
  • BTLA B And T Lymphocyte Associated
  • the disclosure relates to methods of determining effectiveness of an anti-GITR antibody for the treatment of cancer, including: administering the anti-GITR antibody to the animal as described herein, wherein the animal has a tumor, and determining the inhibitory effects of the anti-GITR antibody to the tumor.
  • the animal has one or more cells (e.g., T cells, T regulatory cells or T effector cells) that express GITR.
  • the tumor comprises one or more cancer cells that are injected into the animal.
  • determining the inhibitory effects of the anti-GITR antibody to the tumor involves measuring the tumor volume in the animal.
  • the tumor cells are melanoma cells (e.g., advanced melanoma cells) , non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, bladder cancer cells, non–Hodgkin lymphoma cells, and/or prostate cancer cells (e.g., metastatic hormone-refractory prostate cancer) .
  • the tumor cells are hepatocellular, ovarian, colon, or cervical tumor cells.
  • the tumor cells are breast cancer cells, ovarian cancer cells, and/or solid tumor cells.
  • the tumor cells are lymphoma cells, colorectal cancer cells, or oropharyngeal cancer cells.
  • the animal has metastatic solid tumors, NSCLC, melanoma, lymphoma (e.g., non-Hodgkin lymphoma) , colorectal cancer, or multiple myeloma.
  • the animal has melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies (e.g., Non-Hodgkin’s lymphoma, lymphoma, chronic lymphocytic leukemia) , or solid tumors.
  • melanoma pancreatic carcinoma
  • mesothelioma mesothelioma
  • hematological malignancies e.g., Non-Hodgkin’s lymphoma, lymphoma, chronic lymphocytic leukemia
  • the disclosure relates to methods of determining effectiveness of an anti-GITR antibody for the treatment of various immune-related disorders, e.g., autoimmune diseases.
  • the disclosure relates to methods of determining effectiveness of an anti-GITR antibody and an additional therapeutic agent for the treatment of a tumor, including administering the anti-GITR antibody and the additional therapeutic agent to the animal as described herein, wherein the animal has a tumor, and determining the inhibitory effects on the tumor.
  • the animal or mouse further comprises a sequence encoding an additional human or chimeric protein.
  • the additional human or chimeric protein is PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, SIRP ⁇ , or OX40.
  • the animal further comprises a sequence encoding a human or chimeric PD-1, PD-L1, or CTLA-4.
  • the additional therapeutic agent is an antibody (e.g., human antibody) the specifically binds to PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, SIRP ⁇ , OX40, CD20, EGFR, or CD319.
  • an antibody e.g., human antibody
  • the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab) , an anti-PD-L1 antibody, an anti-CTLA4 antibody (e.g., ipilimumab) , an anti-CD20 antibody (e.g., rituximab) , an anti-EGFR antibody (e.g., cetuximab) , or an anti-CD319 antibody (e.g., elotuzumab) .
  • an anti-PD-1 antibody e.g., nivolumab
  • an anti-PD-L1 antibody e.g., an anti-CTLA4 antibody (e.g., ipilimumab)
  • an anti-CD20 antibody e.g., rituximab
  • an anti-EGFR antibody e.g., cetuximab
  • an anti-CD319 antibody e.g., elotuzumab
  • the animal comprises one or more cells (e.g., immune cells, T cells, Treg, Teff, macrophages, dendritic cells) that express GITR.
  • the animal comprises one or more cells (e.g., antigen presenting cells, dendritic cells, macrophages, B cells) that express GITRL (TNFSF18) .
  • the tumor comprises one or more tumor cells that express PD-L1, PD-L2, CD80 or CD86.
  • the tumor is caused by injection of one or more cancer cells into the animal.
  • determining the inhibitory effects of the treatment involves measuring the tumor volume in the animal.
  • the tumor comprises melanoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, bladder cancer cells, and/or prostate cancer cells (e.g., metastatic hormone-refractory prostate cancer cells) .
  • the animal has metastatic solid tumors, NSCLC, melanoma, lymphoma (e.g., non-Hodgkin lymphoma) , colorectal cancer, or multiple myeloma.
  • the animal has melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies (e.g., Non-Hodgkin’s lymphoma, lymphoma, chronic lymphocytic leukemia) , or solid tumors.
  • melanoma pancreatic carcinoma
  • mesothelioma mesothelioma
  • hematological malignancies e.g., Non-Hodgkin’s lymphoma, lymphoma, chronic lymphocytic leukemia
  • the disclosure relates to proteins comprising an amino acid sequence, wherein the amino acid sequence is one of the following: (a) an amino acid sequence set forth in SEQ ID NO: 27; (b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 27; (c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 27; (d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 27 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and (e) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 27.
  • cells comprising the proteins disclosed herein.
  • provided herein are animals having the proteins disclosed herein.
  • the disclosure relates to nucleic acids comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following: (a) a sequence that encodes the protein as described herein; (b) SEQ ID NO: 25; (c) SEQ ID NO: 26; (d) a sequence that is at least 90%identical to SEQ ID NO: 25 or SEQ ID NO: 26; (e) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 25; and (f) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 26.
  • cells comprising the nucleic acids disclosed herein.
  • provided herein are animals having the nucleic acids disclosed herein.
  • the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous GITR gene, wherein the disruption of the endogenous GITR gene comprises deletion of exon 1, exon 2, exon 3, exon 4, and/or exon 5, or part thereof of the endogenous GITR gene.
  • the disruption of the endogenous GITR gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, and exon 5 of the endogenous GITR gene.
  • the disruption of the endogenous GITR gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, and intron 4 of the endogenous GITR gene.
  • deletion can comprise deleting at least 1, 2, 3, 4, 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, 10, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, or more nucleotides.
  • the disruption of the endogenous GITR gene comprises the deletion of at least 1, 2, 3, 4, 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, 10, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides of exon 1, exon 2, exon 3, exon 4, and/or exon 5 (e.g., deletion of at least 300 nucleotides of exon 1 or exon 4) .
  • mice described in the present disclosure can be mated with the mice containing other human or chimeric genes (e.g., chimeric SIRP ⁇ , chimeric PD-1, chimeric PD-L1, chimeric CTLA-4, or other immunomodulatory factors) , so as to obtain a mouse expressing two or more human or chimeric proteins.
  • the mice can also, e.g., be used for screening antibodies in the case of a combined use of drugs, as well as evaluating the efficacy of the combination therapy.
  • the disclosure further provides methods of determining toxicity of an agent (e.g., a GITR antagonist or agonist) .
  • the methods involve administering the agent to the animal as described herein; and determining weight change of the animal.
  • the methods further involve performing a blood test (e.g., determining red blood cell count) .
  • the disclosure relates to a targeting vector, including a) a DNA fragment homologous to the 5’end of a region to be altered (5’arm) , which is selected from the GITR gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3’end of the region to be altered (3’arm) , which is selected from the GITR gene genomic DNAs in the length of 100 to 10,000 nucleotides.
  • a) the DNA fragment homologous to the 5’end of a region to be altered (5’arm/receptor) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000070.6; c) the DNA fragment homologous to the 3’end of the region to be altered (3’arm/receptor) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000070.6.
  • a) the DNA fragment homologous to the 5’end of a region to be altered (5’arm/receptor) is selected from the nucleotides from the position 156024998 to the position 156026386 of the NCBI accession number NC_000070.6; c) the DNA fragment homologous to the 3’end of the region to be altered (3’arm/receptor) is selected from the nucleotides from the position 156028249 to the position 156029078 of the NCBI accession number NC_000070.6.
  • a length of the selected genomic nucleotide sequence is more than 2kb, 2.3 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, or 6 kb. In some embodiments, the length is about 2363 bp. In some embodiments, the region to be altered is exon1, exon 2, exon 3, exon 4, and/or exon 5 of mouse GITR gene.
  • sequence of the 5’arm is shown in SEQ ID NO: 28. In some embodiments, the sequence of the 3’arm is shown in SEQ ID NO: 29.
  • the targeting vector further includes a selectable gene marker.
  • the target region is derived from human. In some embodiments, the target region is a part or entirety of the nucleotide sequence of a humanized GITR. In some embodiments, the nucleotide sequence is shown as one or more of exon 1, exon 2, exon 3, and exon 4 of the human GITR.
  • the nucleotide sequence of the human GITR encodes the human GITR protein with the NCBI accession number NP_004186.1 (SEQ ID NO: 23) .
  • the nucleotide sequence of the human GITR is selected from the nucleotides from the position 1204209 to the position 1206571 of NC_000001.11 (SEQ ID NO: 30) .
  • the disclosure also relates to a cell including the targeting vector as described herein.
  • the disclosure also relates to a method for establishing a genetically-modified non-human animal expressing two human or chimeric (e.g., humanized) genes.
  • the method includes the steps of
  • step (b) mating the GITR gene genetically modified humanized mouse obtained in step (a) with another humanized mouse, and then screening to obtain a double humanized mouse model.
  • step (b) the GITR gene genetically modified humanized mouse obtained in step (a) is mated with a PD-1 or PD-L1 humanized mouse to obtain a GITR and PD-1 double humanized mouse model or a GITR and PD-L1 double humanized mouse model.
  • the disclosure also relates to non-human mammal generated through the methods as described herein.
  • the genome thereof contains one or more human genes.
  • the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.
  • the non-human mammal expresses a protein encoded by a humanized GITR gene.
  • the disclosure also relates to an offspring of the non-human mammal.
  • the disclosure relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein.
  • the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.
  • the disclosure also relates to a cell (e.g., stem cell or embryonic stem cell) or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.
  • a cell e.g., stem cell or embryonic stem cell
  • a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.
  • the disclosure further relates to the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.
  • the disclosure relates to a tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.
  • the disclosure relates to a GITR amino acid sequence of a humanized mouse, wherein the amino acid sequence is selected from the group consisting of:
  • nucleic acid sequence a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 27 under a low stringency condition or a strict stringency condition;
  • amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%with the amino acid sequence shown in SEQ ID NO: 27;
  • amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 27 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • the disclosure also relates to a GITR nucleic acid sequence of a humanized mouse, wherein the nucleic acid sequence is selected from the group consisting of:
  • nucleic acid sequence that can hybridize to the nucleotide sequence as shown in SEQ ID NO: 25 or SEQ ID NO: 26 under a low stringency condition or a strict stringency condition;
  • nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%with the nucleotide sequence as shown in SEQ ID NO: 25 or SEQ ID NO: 26;
  • nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with the amino acid sequence shown in SEQ ID NO: 27;
  • nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%with the amino acid sequence shown in SEQ ID NO: 27;
  • nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 27 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and /or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acids to the amino acid sequence shown in SEQ ID NO: 27.
  • the disclosure further relates to a GITR genomic DNA sequence of a humanized mouse, a DNA sequence obtained by a reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell comprising the construct thereof; a tissue comprising the cell thereof.
  • the disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the development of a product related to an immunization processes of human cells, the manufacture of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.
  • the disclosure also relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.
  • the disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the methods as described herein, in the screening, verifying, evaluating or studying the GITR gene function, human GITR antibodies, the drugs or efficacies for human GITR targeting sites, and the drugs for immune-related diseases and antitumor drugs.
  • FIG. 1A is a graph showing activity testing results for sgRNA1-sgRNA7 (Con is a negative control; PC is a positive control) .
  • FIG. 1B is a graph showing activity testing results for sgRNA8-sgRNA14 (Con is a negative control; PC is a positive control) .
  • FIG. 2 is a schematic diagram showing the structure of pT7-sgRNA-G2 plasmid.
  • FIG. 3 is a schematic diagram showing a gene targeting strategy.
  • FIG. 4 is a schematic diagram showing a map of an example of humanized GITR gene in mice.
  • FIG. 5 shows the restriction enzymes digestion results of the targeting plasmid pClon-2G-GITR by three sets of restriction enzymes.
  • FIGS. 6A-6D show PCR identification results of samples collected from tails of F0 generation mice. WT is wildtype. Mice labeled with F0-20 and F0-48 are positive.
  • FIGS. 7A-7B show PCR identification results of samples collected from tails of F1 generation mice.
  • WT is wildtype; M is the marker; Mice labeled with F1-1, F1-2, F1-3, F1-4 are humanized GITR mice.
  • FIGS. 8A-8D are flow cytometry results of wildtype mice and heterozygous humanized GITR mice.
  • Anti-mCD3 antibody was used to activate spleen cells.
  • Flow cytometry was performed with 1) antibody against mouse GITR (mGITR PE) and antibody against mouse TCR ⁇ chain (mTcR ⁇ PerCP) ; and 2) antibody against human GITR (hGITR-01-IgG PE) , and antibody against mouse TCR ⁇ chain (mTcR ⁇ PerCP) .
  • spleen cells that express human or humanized GITR were not detected.
  • Humanized GITR was detected on spleen cells in heterozygous humanized GITR mice.
  • FIG. 9 shows PCR results for GITR knockout mice.
  • F1-KO-1, F1-KO-2, F1-KO-3, F1-KO-4, F1-KO-7, F1-KO-8, F1-KO-9, F1-KO-10 were GITR knockout mice.
  • WT indicates wildtype.
  • FIG. 10 The average weight of the different groups of humanized GITR heterozygous mice that were injected with mouse colon cancer cells MC38, and were treated with 3 different anti-human GITR antibodies (10 mg/kg) .
  • FIG. 11 The percentage change of average weight of the different groups of humanized GITR heterozygous mice that were injected with mouse colon cancer cells MC38, and were treated with 3 different anti-human GITR antibodies (10 mg/kg) .
  • FIG. 12 The average tumor volume in different groups of humanized GITR heterozygous mice that were injected with mouse colon cancer cells MC38, and were treated with 3 different anti-human GITR antibodies (10 mg/kg) .
  • FIGS. 13A-13B show PCR identification results. -is wildtype; + is the humanized GITR heterozygous mouse positive control.
  • FIGS. 13C-13D show PCR identification results.
  • WT is wildtype; -/-is the humanized PD-1 homozygous mouse positive control; +/-is the humanized PD-1 heterozygous mouse positive control.
  • FIG. 14 is a schematic diagram showing gene targeting strategy based on embryonic stem cells.
  • FIG. 15 shows the alignment between mouse GITR amino acid sequence (NP_033426.1; SEQ ID NO: 21) and human GITR amino acid sequence (NP_004186.1; SEQ ID NO: 23) .
  • This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) GITR, and methods of use thereof.
  • GITR Glucocorticoid-induced TNFR-related protein
  • TNFRSF18 tumor necrosis factor receptor superfamily member 18
  • AITR activation-inducible TNFR family receptor
  • CD357 GITR is a co-stimulatory immune checkpoint molecule. It is a member of the tumor necrosis factor receptor (TNF-R) superfamily, and has been shown to be involved in inhibiting the suppressive activity of T-regulatory (Treg) cells and extending the survival of T-effector (Teff) cells.
  • TNF-R tumor necrosis factor receptor
  • Agonist anti-GITR antibodies can expand the number of effector CD8+ T cells with increased cytokine production; and at the same time, it can abate T regulatory cell (Treg) -mediated immune suppression or deplete Treg cells.
  • Treg T regulatory cell
  • anti-GITR antibodies can be potentially used to treat cancers and/or autoimmune diseases.
  • Experimental animal models are an indispensable research tool for studying the effects of these antibodies (e.g., anti-GITR antibodies) .
  • Common experimental animals include mice, rats, guinea pigs, hamsters, rabbits, dogs, monkeys, pigs, fish and so on.
  • human and animal genes and protein sequences there are many differences between human and animal genes and protein sequences, and many human proteins cannot bind to the animal’s homologous proteins to produce biological activity, leading to that the results of many clinical trials do not match the results obtained from animal experiments.
  • a large number of clinical studies are in urgent need of better animal models.
  • the use of human cells or genes to replace or substitute an animal’s endogenous similar cells or genes to establish a biological system or disease model closer to human, and establish the humanized experimental animal models (humanized animal model) has provided an important tool for new clinical approaches or means.
  • the genetically engineered animal model that is, the use of genetic manipulation techniques, the use of human normal or mutant genes to replace animal homologous genes, can be used to establish the genetically modified animal models that are closer to human gene systems.
  • the humanized animal models have various important applications. For example, due to the presence of human or humanized genes, the animals can express or express in part of the proteins with human functions, so as to greatly reduce the differences in clinical trials between humans and animals, and provide the possibility of drug screening at animal levels.
  • GITR also known as TNFRSF18, CD357, or AITR
  • T regulatory cells Treg
  • Activation of T cells by a number of different stimuli rapidly increases GITR expression within 24 hours, on both Treg and T effector (Teff) cells.
  • Treg and T effector (Teff) cells Low to moderate levels of GITR are also detected on innate immune cells following activation. Within innate cell types, the highest induction is seen on activated natural killer cells with levels comparable to GITR expression on activated Teff cells. Only intermediate levels are seen on activated macrophage and dendritic cells (DCs) . Of all immune cells, activated Tregs exhibit the highest level of GITR.
  • the ligand of GITR, GITRL Tumor Necrosis Factor Superfamily, Member 18 or TNFSF18
  • GITRL Tumor Necrosis Factor Superfamily, Member 18 or TNFSF18
  • APCs activated antigen presenting cells
  • GITR does not have intrinsic enzymatic activity, the activation of these signaling pathways occurs via recruitment of TRAF family members, most notably TRAF2 and TRAF5.
  • GITR engagement by GITRL enhances T cell proliferation and effector function by upregulating CD25 and inducing cytokine (IL-2/IFN-gamma) expression.
  • IL-2/IFN-gamma cytokine
  • GITR signaling can lower the threshold for CD28 signaling on CD8+ T cells, induce expression of 4-1BB in CD8+ memory T cells, and/or promote survival of bone marrow CD8+ memory T cells.
  • GITR co-stimulation can also lead to TRAF6-dependent NF ⁇ B activation and IL-9 production, thereby enhancing the function of DCs and promoting cytotoxic T lymphocyte responses.
  • GITR modulation shows compelling anti-tumor activity which is attributed to both its costimulatory role on Teff cells (e.g., CD4+ and CD8+ T cells) as well as inhibitory effects of Tregs.
  • anti-GITR antibodies have also been used in clinical trials to treat solid tumors or melanoma.
  • anti-GITR antibodies have been used in combination with anti-PD1 antibodies (e.g., pembrolizumab, nivolumab) and/or anti-CTLA4 antibodies (e.g., ipilimumab) for treating solid tumors (e.g., advanced solid tumors) , melanoma, advanced malignancies, metastatic cancer, etc.
  • anti-PD1 antibodies e.g., pembrolizumab, nivolumab
  • anti-CTLA4 antibodies e.g., ipilimumab
  • GITR gene (Gene ID: 8784) locus has five exons, exon 1, exon 2, exon 3, exon 4, and exon 5.
  • the GITR protein also has an extracellular region, a transmembrane region, and a cytoplasmic region, and the signal peptide is located at the extracellular region of GITR.
  • the nucleotide sequence for human GITR mRNA is NM_004195.2 (SEQ ID NO: 22)
  • amino acid sequence for human GITR is NP_004186.1 (SEQ ID NO: 23) .
  • the location for each exon and each region in human GITR nucleotide sequence and amino acid sequence is listed below:
  • GITR gene locus has five exons, exon 1, exon 2, exon 3, exon 4, and exon 5 (FIG. 3) .
  • the mouse GITR protein also has an extracellular region, a transmembrane region, and a cytoplasmic region, and the signal peptide is located at the extracellular region of GITR.
  • the nucleotide sequence for mouse GITR cDNA is NM_009400.2 (SEQ ID NO: 20)
  • amino acid sequence for mouse GITR is NP_033426.1 (SEQ ID NO: 21) .
  • the location for each exon and each region in the mouse GITR nucleotide sequence and amino acid sequence is listed below:
  • the mouse GITR gene (Gene ID: 21936) is located in Chromosome 4 of the mouse genome, which is located from 156026164 to 156028896, of NC_000070.6 (GRCm38. p4 (GCF_000001635.24) ) .
  • the 5’-UTR is from 156026164 to 156025341, exon 1 is from 156025342 to 156026537, the first intron is from 156026538 to 156027315, exon 2 is from 156027316 to 156027438, the second intron is from 156027439 to 156027961, exon 3 is from 156027962 to 156,028,049, the third intron is from 156028050 to 156028220, exon 4 is from 156028221 to 156,028,423, the fourth intron is from 156028424 to 156028494, exon 5 is from 156028495 to 156028891, the 3’-UTR is from 156028892 to 156028895, based on transcript NM_009400.2. All relevant information for mouse GITR locus can be found in the NCBI website with Gene ID: 21936, which is incorporated by reference herein in its entirety.
  • FIG. 15 shows the alignment between mouse GITR amino acid sequence (NP_033426.1; SEQ ID NO: 21) and human GITR amino acid sequence (NP_004186.1; SEQ ID NO: 23) .
  • mouse GITR amino acid sequence NP_033426.1; SEQ ID NO: 21
  • human GITR amino acid sequence NP_004186.1; SEQ ID NO: 23
  • GITR genes, proteins, and locus of the other species are also known in the art.
  • the gene ID for GITR in Rattus norvegicus is 500598
  • the gene ID for GITR in Macaca mulatta (Rhesus monkey) is 699559
  • the gene ID for GITR in Canis lupus familiaris (dog) is 606971
  • the gene ID for GITR in Sus scrofa (pig) is 100622025.
  • the relevant information for these genes e.g., intron sequences, exon sequences, amino acid residues of these proteins
  • NCBI database which is incorporated by reference herein in its entirety.
  • the present disclosure provides human or chimeric (e.g., humanized) GITR nucleotide sequence and/or amino acid sequences.
  • the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence.
  • a “region” or “portion” of mouse exon 1, exon 2, exon 3, exon 4, exon 5, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence.
  • region can refer to at least 1, 2, 3, 4, 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, 400, 500, or 600 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.
  • the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, exon 2, exon 3, exon 4, exon 5, signal peptide, extracellular region, transmembrane region, or cytoplasmic region.
  • a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, and/or exon 5 e.g., exon 1, exon 2, exon 3, exon 4) are replaced by the human exon 1, exon 2, exon 3, exon 4, and/or exon 5 (e.g., exon 1, exon 2, exon 3, exon 4) sequence.
  • the present disclosure also provides a chimeric (e.g., humanized) GITR nucleotide sequence and/or amino acid sequences, wherein in some embodiments, 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%of the sequence are identical to or derived from mouse GITR mRNA sequence (e.g., SEQ ID NO: 20) , mouse GITR amino acid sequence (e.g., SEQ ID NO: 21) , or a portion thereof (e.g., exon 1, exon 2, exon 3, exon 4) ; and in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,
  • sequence encoding amino acids 1-142 of mouse GITR (SEQ ID NO: 21) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human GITR (e.g., amino acids 1-142 of human GITR (SEQ ID NO: 23) ) .
  • the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse GITR promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.
  • a promotor or regulatory element e.g., an endogenous mouse GITR promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.
  • the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from a portion of or the entire mouse GITR nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, or NM_009400.2 (SEQ ID NO: 20) ) .
  • a portion e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides
  • NM_009400.2 SEQ ID NO: 20
  • the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire mouse GITR nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, or NM_009400.2 (SEQ ID NO: 20) ) .
  • the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from a portion of or the entire human GITR nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, or NM_004195.2 (SEQ ID NO: 22) ) .
  • the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire human GITR nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, or NM_004195.2 (SEQ ID NO: 22) ) .
  • the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire mouse GITR amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, or NP_033426.1 (SEQ ID NO: 21) ) .
  • the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire mouse GITR amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, or NP_033426.1 (SEQ ID NO: 21) ) .
  • the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire human GITR amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, or NP_004186.1 (SEQ ID NO: 23) ) .
  • a portion e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues
  • a portion of or the entire human GITR amino acid sequence e.g., exon 1, exon 2, exon 3, exon 4, or NP_004186.1 (SEQ ID NO: 23)
  • the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire human GITR amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, or NP_004186.1 (SEQ ID NO: 23) ) .
  • the present disclosure also provides a humanized GITR mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:
  • nucleic acid sequence a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 27 under a low stringency condition or a strict stringency condition;
  • amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 27;
  • amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 27 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • the present disclosure also relates to a GITR nucleic acid (e.g., DNA or RNA) sequence, wherein the nucleic acid sequence can be selected from the group consisting of:
  • nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 25 or SEQ ID NO: 26 under a low stringency condition or a strict stringency condition;
  • nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleotide sequence as shown in SEQ ID NO: 25 or SEQ ID NO: 26;
  • nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 27;
  • nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 27;
  • nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 27 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 27.
  • the present disclosure further relates to a GITR genomic DNA sequence of a humanized mouse.
  • the DNA sequence is obtained by a reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 25 or SEQ ID NO: 26.
  • the disclosure also provides an amino acid sequence that has a homology of at least 90%with, or at least 90%identical to the sequence shown in SEQ ID NO: 27, and has protein activity.
  • the homology with the sequence shown in SEQ ID NO: 27 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
  • the foregoing homology is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.
  • the percentage identity with the sequence shown in SEQ ID NO: 27 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.
  • the disclosure also provides a nucleotide sequence that has a homology of at least 90%, or at least 90%identical to the sequence shown in SEQ ID NO: 25 or SEQ ID NO: 26, and encodes a polypeptide that has protein activity.
  • the homology with the sequence shown in SEQ ID NO: 25 or SEQ ID NO: 26 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
  • the foregoing homology is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.
  • the percentage identity with the sequence shown in SEQ ID NO: 25 or SEQ ID NO: 26 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.
  • 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, or 200 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 length of a reference sequence aligned for comparison purposes is at least 80%of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • 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
  • 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 human or chimeric (e.g., humanized) GITR from an endogenous non-human GITR locus.
  • the term “genetically-modified non-human animal” refers to a non-human animal having exogenous DNA in at least one chromosome of the animal’s genome.
  • at least one or more cells e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%of cells of the genetically-modified non-human animal have the exogenous DNA in its genome.
  • the cell having exogenous DNA can be various kinds of cells, e.g., an endogenous cell, a somatic cell, an immune cell, a T cell, a B cell, an antigen presenting cell, a macrophage, a dendritic cell, a germ cell, a blastocyst, or an endogenous tumor cell.
  • genetically-modified non-human animals are provided that comprise a modified endogenous GITR locus that comprises an exogenous sequence (e.g., a human sequence) , e.g., a replacement of one or more non-human sequences with one or more human sequences.
  • the animals are generally able to pass the modification to progeny, i.e., through germline transmission.
  • chimeric gene or “chimeric nucleic acid” refers to a gene or a nucleic acid, wherein two or more portions of the gene or the nucleic acid are from different species, or at least one of the sequences of the gene or the nucleic acid does not correspond to the wildtype nucleic acid in the animal.
  • the chimeric gene or chimeric nucleic acid has at least one portion of the sequence that is derived from two or more different sources, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) protein of two or more different species.
  • the chimeric gene or the chimeric nucleic acid is a humanized gene or humanized nucleic acid.
  • chimeric protein or “chimeric polypeptide” refers to a protein or a polypeptide, wherein two or more portions of the protein or the polypeptide are from different species, or at least one of the sequences of the protein or the polypeptide does not correspond to wildtype amino acid sequence in the animal.
  • the chimeric protein or the chimeric polypeptide has at least one portion of the sequence that is derived from two or more different sources, e.g., same (or homologous) proteins of different species.
  • the chimeric protein or the chimeric polypeptide is a humanized protein or a humanized polypeptide.
  • the chimeric gene or the chimeric nucleic acid is a humanized GITR gene or a humanized GITR nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human GITR gene, at least one or more portions of the gene or the nucleic acid is from a non-human GITR gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes a GITR protein.
  • the encoded GITR protein is functional or has at least one activity of the human GITR protein or the non-human GITR protein, e.g., binding with human or non-human GITRL, activating T effector cells, increasing the level of activation of T effector cells, inhibiting or depleting T regulator cells, inducing activation and proliferation of immune cells (e.g., T cells) , increasing the production of cytokines, and/or upregulating the immune response.
  • the chimeric protein or the chimeric polypeptide is a humanized GITR protein or a humanized GITR polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a human GITR protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human GITR protein.
  • the humanized GITR protein or the humanized GITR polypeptide is functional or has at least one activity of the human GITR protein or the non-human GITR protein.
  • the genetically modified non-human animal can be various animals, e.g., a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo) , deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey) .
  • ES embryonic stem
  • Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo.
  • a suitable cell e.g., an oocyte
  • gestating the modified cell e.g., the modified oocyte
  • the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea.
  • the genetically modified animal is a rodent.
  • the rodent can be selected from a mouse, a rat, and a hamster.
  • the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters) , Cricetidae (e.g., hamster, New World rats and mice, voles) , Muridae (true mice and rats, gerbils, spiny mice, crested rats) , Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice) , Platacanthomyidae (e.g., spiny dormice) , and Spalacidae (e.g., mole rates, bamboo rats, and zokors) .
  • Calomyscidae e.g., mouse-like hamsters
  • Cricetidae e.g., hamster, New World rats and mice, voles
  • Muridae true mice and rats, gerbils, spiny mice, crested rats
  • the genetically modified rodent is selected from a true mouse or rat (family Muridae) , a gerbil, a spiny mouse, and a crested rat.
  • the non-human animal is a mouse.
  • the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola.
  • a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola.
  • the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2.
  • a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2.
  • the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains.
  • the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) .
  • a hybrid line e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129
  • the animal is a rat.
  • the rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti.
  • the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.
  • the animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the humanized GITR animal is made.
  • suitable mice for maintaining a xenograft e.g., a human cancer or tumor
  • mice for maintaining a xenograft can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part.
  • Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) .
  • Non-limiting examples of such mice include, e.g., NOD mice, SCID mice, NOD/SCID mice, IL2R ⁇ knockout mice, NOD/SCID/ ⁇ cnull mice (Ito, M.
  • a genetically modified mouse can include a humanization of at least a portion of an endogenous non-human GITR locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part.
  • modification is, e.g., selected from the group consisting of a modification that results in NOD mice, SCID mice, NOD/SCID mice, IL-2R ⁇ knockout mice, NOD/SCID/ ⁇ c null mice, nude mice, Rag1 and/or Rag2 knockout mice, and a combination thereof.
  • NOD mice SCID mice, NOD/SCID mice, IL-2R ⁇ knockout mice, NOD/SCID/ ⁇ c null mice, nude mice, Rag1 and/or Rag2 knockout mice, and a combination thereof.
  • the mouse can include a replacement of all or part of mature GITR coding sequence with human mature GITR coding sequence.
  • Genetically modified non-human animals that comprise a modification of an endogenous non-human GITR locus.
  • the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature GITR protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the mature GITR protein sequence) .
  • genetically modified cells are also provided that can comprise the modifications described herein (e.g., ES cells, somatic cells)
  • the genetically modified non-human animals comprise the modification of the endogenous GITR locus in the germline of the animal.
  • Genetically modified animals can express a human GITR and/or a chimeric (e.g., humanized) GITR from endogenous mouse loci, wherein the endogenous mouse GITR gene has been replaced with a human GITR gene and/or a nucleotide sequence that encodes a region of human GITR sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the human GITR sequence.
  • an endogenous non-human GITR locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature GITR protein.
  • the genetically modified mice express the human GITR and/or chimeric GITR (e.g., humanized GITR) from endogenous loci that are under control of mouse promoters and/or mouse regulatory elements.
  • the replacement (s) at the endogenous mouse loci provide non-human animals that express human GITR or chimeric GITR (e.g., humanized GITR) in appropriate cell types and in a manner that does not result in the potential pathologies observed in some other transgenic mice known in the art.
  • the human GITR or the chimeric GITR (e.g., humanized GITR) expressed in animal can maintain one or more functions of the wildtype mouse or human GITR in the animal.
  • human or non-human GITR ligands can bind to the expressed GITR, upregulate immune response, e.g., upregulate immune response by at least 10%, 20%, 30%, 40%, or 50%.
  • the animal does not express endogenous GITR.
  • endogenous GITR refers to GITR protein that is expressed from an endogenous GITR nucleotide sequence of the non-human animal (e.g., mouse) before any genetic modification.
  • the genome of the animal can comprise a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human GITR (NP_004186.1) (SEQ ID NO: 23) .
  • the genome comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 27.
  • the genome of the genetically modified animal can comprise a replacement at an endogenous GITR gene locus of a sequence encoding a region of endogenous GITR with a sequence encoding a corresponding region of human GITR.
  • the sequence that is replaced is any sequence within the endogenous GITR gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, 5’-UTR, 3’-UTR, the first intron, the second intron, the third intron, the fourth intron etc.
  • the sequence that is replaced is within the regulatory region of the endogenous GITR gene.
  • the sequence that is replaced is exon1, exon 2, exon 3, exon 4 or part thereof, of an endogenous mouse GITR gene locus.
  • the genetically modified animal can have one or more cells expressing a human or chimeric GITR (e.g., humanized GITR) having an extracellular region and a cytoplasmic region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human GITR.
  • the extracellular region of the humanized GITR has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 amino acids (e.g., contiguously or non-contiguously) that are identical to human GITR.
  • human GITR and non-human GITR e.g., mouse GITR sequences
  • antibodies that bind to human GITR will not necessarily have the same binding affinity with non-human GITR or have the same effects to non-human GITR. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human GITR antibodies in an animal model.
  • the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to part or the entire sequence of exon 1, exon 2, exon 3, and/or exon 4 of human GITR, part or the entire sequence of extracellular region of human GITR (with or without signal peptide) , or part or the entire sequence of amino acids 1-142 of SEQ ID NO: 23.
  • the non-human animal can have, at an endogenous GITR gene locus, a nucleotide sequence encoding a chimeric human/non-human GITR polypeptide, wherein a human portion of the chimeric human/non-human GITR polypeptide comprises a portion of human GITR extracellular domain, and wherein the animal expresses a functional GITR on a surface of a cell of the animal.
  • the human portion of the chimeric human/non-human GITR polypeptide can comprise a portion of exon 1, exon 2, exon 3, and/or exon 4 of human GITR.
  • the human portion of the chimeric human/non-human GITR polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 1-142 of SEQ ID NO: 23.
  • the non-human portion of the chimeric human/non-human GITR polypeptide comprises transmembrane and/or cytoplasmic regions of an endogenous non-human GITR polypeptide.
  • a GITR ligand e.g., GITRL
  • an anti-GITR antibody binds to GITR, they can properly transmit extracellular signals into the cells and initiate the downstream pathway.
  • a human or humanized transmembrane and/or cytoplasmic regions may not function properly in non-human animal cells.
  • a few extracellular amino acids that are close to the transmembrane region of GITR are also derived from endogenous sequence. These amino acids can also be important for transmembrane signal transmission.
  • the genetically modified animal can be heterozygous with respect to the replacement at the endogenous GITR locus, or homozygous with respect to the replacement at the endogenous GITR locus.
  • the humanized GITR locus lacks a human GITR 5’-UTR.
  • the humanized GITR locus comprises a rodent (e.g., mouse) 5’-UTR.
  • the humanization comprises a human 3’-UTR.
  • mouse and human GITR genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence.
  • humanized GITR mice that comprise a replacement at an endogenous mouse GITR locus, which retain mouse regulatory elements but comprise a humanization of GITR encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized GITR are grossly normal.
  • the present disclosure further relates to a non-human mammal generated through the method mentioned above.
  • the genome thereof contains human gene (s) .
  • the non-human mammal is a rodent, and preferably, the non-human mammal is a mouse.
  • the non-human mammal expresses a protein encoded by a humanized GITR gene.
  • the present disclosure also relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein.
  • the non-human mammal is a rodent (e.g., a mouse) .
  • the present disclosure further relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; and the tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.
  • non-human mammals produced by any of the methods described herein.
  • a non-human mammal is provided; and the genetically modified animal contains the DNA encoding human or humanized GITR in the genome of the animal.
  • the non-human mammal comprises the genetic construct as described herein (e.g., gene construct as shown in FIG. 2 or FIG. 3) .
  • a non-human mammal expressing human or humanized GITR is provided.
  • the tissue-specific expression of human or humanized GITR protein is provided.
  • the expression of human or humanized GITR in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance.
  • Non-human mammals can be any non-human animal known in the art and which can be used in the methods as described herein.
  • Preferred non-human mammals are mammals, (e.g., rodents) .
  • the non-human mammal is a mouse.
  • the present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes.
  • the present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof.
  • a model based on cell culture can be prepared, for example, by the following methods.
  • Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cell can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques.
  • the integration of genetic constructs containing DNA sequences encoding human GITR protein can be detected by a variety of methods.
  • RNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization
  • protein level including histochemistry, immunoblot analysis and in vitro binding studies
  • RT-PCR reverse transcriptase polymerase chain reaction
  • protein level including histochemistry, immunoblot analysis and in vitro binding studies
  • the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art.
  • Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human or humanized GITR protein.
  • the present disclosure relates to a targeting vector, comprising: a) a DNA fragment homologous to the 5’end of a region to be altered (5’arm) , which is selected from the GITR gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3’end of the region to be altered (3’arm) , which is selected from the GITR gene genomic DNAs in the length of 100 to 10,000 nucleotides.
  • a) the DNA fragment homologous to the 5’end of a conversion region to be altered (5’arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000070.6; c) the DNA fragment homologous to the 3’end of the region to be altered (3’arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000070.6.
  • a) the DNA fragment homologous to the 5’end of a region to be altered (5’arm) is selected from the nucleotides from the position 156024998 to the position 156026386 of the NCBI accession number NC_000070.6; c) the DNA fragment homologous to the 3’end of the region to be altered (3’arm) is selected from the nucleotides from the position 156028249 to the position 156029078 of the NCBI accession number NC_000070.6.
  • the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 2 kb, about 2.5 kb, about 3 kb, about 3.5 kb, about 4 kb, about 4.5 kb, about 5 kb, about 5.5 kb, or about 6 kb.
  • the region to be altered is exon 1, exon 2, exon 3, exon 4, and/or exon 5 of GITR gene (e.g., exon 1, exon 2, exon 3, and/or exon 4 of mouse GITR gene) .
  • the targeting vector can further include a selected gene marker.
  • sequence of the 5’arm is shown in SEQ ID NO: 28; and the sequence of the 3’arm is shown in SEQ ID NO: 29.
  • the sequence is derived from human (e.g., 1204209 -1206571 of NC_000001.11) .
  • the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human GITR, preferably exon 1, exon 2, exon 3, and/or exon 4 of the human GITR.
  • the nucleotide sequence of the humanized GITR encodes the entire or the part of human GITR protein with the NCBI accession number NP_004186.1 (SEQ ID NO: 23) .
  • the disclosure also relates to a cell comprising the targeting vectors as described above.
  • the present disclosure further relates to a non-human mammalian cell, having any one of the foregoing targeting vectors, and one or more in vitro transcripts of the construct as described herein.
  • the cell includes Cas9 mRNA or an in vitro transcript thereof.
  • 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.
  • Genetically modified animals can be made by several techniques that are known in the art, including, e.g., nonhomologous end-joining (NHEJ) , homologous recombination (HR) , zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR) -Cas system.
  • NHEJ nonhomologous end-joining
  • HR homologous recombination
  • ZFNs zinc finger nucleases
  • TALEN transcription activator-like effector-based nucleases
  • CRISPR clustered regularly interspaced short palindromic repeats
  • homologous recombination is used.
  • CRISPR-Cas9 genome editing is used to generate genetically modified animals.
  • genome editing techniques are known in the art, and is described, e.g., in Yin et al., "Delivery technologies for genome editing, " Nature Reviews Drug Discovery 16.6 (2017) : 387-399, which is incorporated by reference in its entirety.
  • Many other methods are also provided and can be used in genome editing, e.g., micro-injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.
  • the disclosure provides replacing in at least one cell of the animal, at an endogenous GITR gene locus, a sequence encoding a region of an endogenous GITR with a sequence encoding a corresponding region of human or chimeric GITR.
  • the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc.
  • the nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.
  • FIG. 3 shows a humanization strategy for a mouse GITR locus.
  • the targeting strategy involves a vector comprising the 5’end homologous arm, human GITR gene fragment, 3’homologous arm.
  • the process can involve replacing endogenous GITR sequence with human sequence by homologous recombination.
  • the cleavage at the upstream and the downstream of the target site e.g., by zinc finger nucleases, TALEN or CRISPR
  • the homologous recombination is used to replace endogenous GITR sequence with human GITR sequence.
  • the methods for making a genetically modified, humanized animal can include the step of replacing at an endogenous GITR locus (or site) , a nucleic acid encoding a sequence encoding a region of endogenous GITR with a sequence encoding a corresponding region of human GITR.
  • the sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4, and/or exon 5 of a human GITR gene.
  • the sequence includes a region of exon 1, exon 2, exon 3, and exon 4 of a human GITR gene (e.g., amino acids 1-142 of SEQ ID NO: 23) .
  • the region is located within the extracellular region of GITR.
  • the endogenous GITR locus comprises exon 1, exon 2, exon 3, exon 4, and/or exon 5 of mouse GITR.
  • the methods of modifying a GITR locus of a mouse to express a chimeric human/mouse GITR peptide can include the steps of replacing at the endogenous mouse GITR locus a nucleotide sequence encoding a mouse GITR with a nucleotide sequence encoding a human GITR, thereby generating a sequence encoding a chimeric human/mouse GITR.
  • the nucleotide sequence encoding the chimeric human/mouse GITR can include a first nucleotide sequence encoding an extracellular region of mouse GITR (with or without the mouse or human signal peptide sequence) ; a second nucleotide sequence encoding an extracellular region of human GITR; a third nucleotide sequence encoding a transmembrane and a cytoplasmic region of a mouse GITR.
  • the nucleotide sequences as described herein do not overlap with each other (e.g., the first nucleotide sequence, the second nucleotide sequence, and/or the third nucleotide sequence do not overlap) .
  • the amino acid sequences as described herein do not overlap with each other.
  • the present disclosure further provides a method for establishing a GITR gene humanized animal model, involving the following steps:
  • 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 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.
  • the transgene with human regulatory elements expresses in a manner that is unphysiological or otherwise unsatisfactory, and can be actually detrimental to the animal.
  • the disclosure demonstrates that a replacement with human sequence at an endogenous locus under control of endogenous regulatory elements provides a physiologically appropriate expression pattern and level that results in a useful humanized animal whose physiology with respect to the replaced gene are meaningful and appropriate in the context of the humanized animal’s physiology.
  • Genetically modified animals that express human or humanized GITR protein provide a variety of uses that include, but are not limited to, developing therapeutics for human diseases and disorders, and assessing the toxicity and/or the efficacy of these human therapeutics in the animal models.
  • genetically modified animals that express human or humanized GITR, which are useful for testing the interaction between GITR and anti-human GITR antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an GITR agonist or antagonist.
  • the genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (aknock-in or knockout) .
  • the genetically modified non-human animals further comprise an impaired immune system, e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor or a blood cell tumor (e.g., a lymphocyte tumor, e.g., a B or T cell tumor) .
  • an impaired immune system e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor or a blood cell tumor (e.g., a lymphocyte tumor, e.g., a B or T cell tumor) .
  • the genetically modified animals can be used for determining effectiveness of an anti-GITR antibody for the treatment of cancer.
  • the methods involve administering the anti-GITR antibody (e.g., anti-human GITR antibody) to the animal as described herein, wherein the animal has a tumor; and determining the inhibitory effects of the anti-GITR antibody to the tumor.
  • the anti-GITR antibody e.g., anti-human GITR antibody
  • the inhibitory effects that can be determined include, e.g., a decrease of tumor size or tumor volume, a decrease of tumor growth, a reduction of the increase rate of tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or in another subject without such treatment) , a decrease in the risk of developing a metastasis or the risk of developing one or more additional metastasis, an increase of survival rate, and an increase of life expectancy, etc.
  • the tumor volume in a subject can be determined by various methods, e.g., as determined by direct measurement, MRI or CT.
  • the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) that are injected into the animal.
  • the anti-GITR antibody prevents GITRL from binding to GITR. In some embodiments, the anti-GITR antibody does not prevent GITRL from binding to GITR.
  • the genetically modified animals can be used for determining whether an anti-GITR antibody is a GITR agonist or antagonist.
  • the methods as described herein are also designed to determine the effects of the agent (e.g., anti-GITR antibodies) on GITR, e.g., whether the agent can stimulate immune cells or inhibit immune cells (e.g., T cells, T regulatory cells, or T effector cells) , whether the agent can increase or decrease the production of cytokines, whether the agent can activate or deactivate immune cells (e.g., T regulatory cells, or T effector cells) , and/or whether the agent can upregulate the immune response or downregulate immune response.
  • the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., cancer, or autoimmune diseases.
  • the inhibitory effects on tumors can also be determined by methods known in the art, e.g., measuring the tumor volume in the animal, and/or determining tumor (volume) inhibition rate (TGI TV ) .
  • the anti-GITR antibody is designed for treating various cancers.
  • cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • tumor refers to cancerous cells, e.g., a mass of cancerous cells.
  • Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • the agents described herein are designed for treating or diagnosing a carcinoma in a subject.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • the cancer is renal carcinoma or melanoma.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • an “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • the term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
  • the anti-GITR antibody is designed for treating melanoma (e.g., advanced melanoma) , non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , B-cell non–Hodgkin lymphoma, bladder cancer, and/or prostate cancer (e.g., metastatic hormone-refractory prostate cancer) .
  • the anti-GITR antibody is designed for treating hepatocellular, ovarian, colon, or cervical carcinomas.
  • the anti-GITR antibody is designed for treating advanced breast cancer, advanced ovarian cancer, and/or advanced refractory solid tumor.
  • the anti-GITR antibody is designed for treating metastatic solid tumors, NSCLC, melanoma, non-Hodgkin lymphoma, colorectal cancer, and multiple myeloma. In some embodiments, the anti-GITR antibody is designed for treating melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies (e.g., Non-Hodgkin’s lymphoma, lymphoma, chronic lymphocytic leukemia) , or solid tumors (e.g., advanced solid tumors) . In some embodiments, the anti-GITR antibody is designed for treating carcinomas (e.g., nasopharynx carcinoma, bladder carcinoma, cervix carcinoma, kidney carcinoma or ovary carcinoma) .
  • carcinomas e.g., nasopharynx carcinoma, bladder carcinoma, cervix carcinoma, kidney carcinoma or ovary carcinoma
  • the anti-GITR antibody is designed for treating various autoimmune diseases.
  • the methods as described herein can be used to determine the effectiveness of an anti-GITR antibody in inhibiting immune response.
  • the present disclosure also provides methods of determining toxicity of an antibody (e.g., anti-GITR antibody) .
  • the methods involve administering the antibody to the animal as described herein.
  • the animal is then evaluated for its weight change, red blood cell count, hematocrit, and/or hemoglobin.
  • the antibody can decrease the red blood cells (RBC) , hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%.
  • the animals can have a weight that is at least 5%, 10%, 20%, 30%, or 40%smaller than the weight of the control group (e.g., average weight of the animals that are not treated with the antibody) .
  • the present disclosure also relates to the use of the animal model generated through the methods as described herein in the development of a product related to an immunization processes of human cells, the manufacturing of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.
  • the disclosure provides the use of the animal model generated through the methods as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.
  • the disclosure also relates to the use of the animal model generated through the methods as described herein in the screening, verifying, evaluating or studying the GITR gene function, human GITR antibodies, drugs for human GITR targeting sites, the drugs or efficacies for human GITR targeting sites, the drugs for immune-related diseases and antitumor drugs.
  • 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 a human or chimeric GITR gene 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, CD40, CD47, CD137, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Signal regulatory protein ⁇ (SIRP ⁇ ) , or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40) .
  • PD-1 programmed cell death protein 1
  • CTL-4 Lymphocyte Activating 3
  • BTLA B And T Lymphocyte Associated
  • PD-L1 Programmed Cell Death 1 Ligand 1
  • the methods of generating genetically modified animal model with two or more 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, LAG-3, BTLA, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, SIRP ⁇ , or OX40.
  • the GITR humanization is directly performed on a genetically modified animal having a human or chimeric PD-1, CTLA-4, BTLA, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, SIRP ⁇ , or OX40 gene.
  • the genetically modified animal model with two or more human or humanized genes can be used for determining effectiveness of a combination therapy that targets two or more of these proteins, e.g., an anti-GITR antibody and an additional therapeutic agent for the treatment of cancer.
  • the methods include administering the anti-GITR antibody and the additional therapeutic agent to the animal, wherein the animal has a tumor; and determining the inhibitory effects of the combined treatment to the tumor.
  • the additional therapeutic agent is an antibody that specifically binds to PD-1, CTLA-4, BTLA, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, SIRP ⁇ , or OX40.
  • the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab) , an anti-PD-1 antibody (e.g., nivolumab) , or an anti-PD-L1 antibody.
  • the animal further comprises a sequence encoding a human or humanized PD-1, a sequence encoding a human or humanized PD-L1, or a sequence encoding a human or humanized CTLA-4.
  • the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab) , an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.
  • the tumor comprises one or more tumor cells that express CD80, CD86, PD-L1, and/or PD-L2.
  • the combination treatment is designed for treating various cancer as described herein, e.g., melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, prostate cancer (e.g., metastatic hormone-refractory prostate cancer) , advanced breast cancer, advanced ovarian cancer, and/or advanced refractory solid tumor.
  • the combination treatment is designed for treating metastatic solid tumors, NSCLC, melanoma, B-cell non-Hodgkin lymphoma, colorectal cancer, and multiple myeloma.
  • the combination treatment is designed for treating melanoma, carcinomas (e.g., pancreatic carcinoma) , mesothelioma, hematological malignancies (e.g., Non-Hodgkin’s lymphoma, lymphoma, chronic lymphocytic leukemia) , or solid tumors (e.g., advanced solid tumors) .
  • carcinomas e.g., pancreatic carcinoma
  • mesothelioma e.g., mesothelioma
  • hematological malignancies e.g., Non-Hodgkin’s lymphoma, lymphoma, chronic lymphocytic leukemia
  • solid tumors e.g., advanced solid tumors
  • the methods described herein can be used to evaluate the combination treatment with some other methods.
  • the methods of treating a cancer that can be used alone or in combination with methods described herein, include, e.g., treating the subject with chemotherapy, e.g., campothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, transplatinum, 5-flurouracil, vincristin, vinblastin, and/or methotrexate.
  • the methods can include performing surgery on the subject to remove at least a portion of the subject to remove at least
  • C57BL/6 mice were purchased from the China Food and Drugs Research Institute National Rodent Experimental Animal Center.
  • EcoRI, BamHI, BbsI, HindII, XhoI, SacI, NotI restriction enzymes were purchased from NEB (Catalog numbers R3101M, R3136M, R0539L, R3104M, R0146S, R3122M, R3189M) .
  • Ambion in vitro transcription kit was purchased from Ambion (Catalog number: AM1354) .
  • UCA kit was obtained from Beijing Biocytogen Co., Ltd. (Catalog number: BCG-DX-001) .
  • TOP10 competent cells were purchased from the Tiangen Biotech (Beijing) Co. (Catalog number: CB104-02) .
  • Cas9 mRNA was purchased from SIGMA (Catalog number: CAS9MRNA-1EA) .
  • AIO kit was obtained from Beijing Biocytogen Co., Ltd. (Catalog number: BCG-DX-004) .
  • the pHSG299 plasmid was purchased from Takara (Catalog number: 3299) .
  • mCD3 Purified NA/LE hamster anti-mouse CD3e (mCD3) antibody was purchased from BD (Catalog number: 553057) .
  • GITR PE anti-mouse CD357 antibody was purchased from Biolegend (Catalog number: 120208) .
  • EXAMPLE 1 sgRNAs for GITR gene
  • the 5’-terminal targeting sites (sgRNA1 to sgRNA7) and the 3’-terminal targeting sites (sgRNA8 to sgRNA14) were designed and synthesized.
  • the 5’-terminal targeting sites were located in exon 1 of mouse GITR gene.
  • the 3’-terminal targeting sites were located in exon 4 of mouse GITR gene.
  • the targeting site sequences on GITR for each sgRNA are shown below:
  • sgRNA-1 target sequence (SEQ ID NO: 1) : 5’-aggtcagccgagtgtagttgagg-3’
  • sgRNA-2 target sequence SEQ ID NO: 2: 5’-caactacactcggctgacctagg-3’
  • sgRNA-3 target sequence (SEQ ID NO: 3) : 5’-ccaggctcctcaactacactcgg-3’
  • sgRNA-4 target sequence (SEQ ID NO: 4) : 5’-ccgagtgtagttgaggagcctgg-3’
  • sgRNA-5 target sequence (SEQ ID NO: 5) : 5’-ggggcatgggccatgctgtatgg-3’
  • sgRNA-6 target sequence (SEQ ID NO: 6) : 5’-actcaggagaagcactatggggg-3’
  • sgRNA-7 target sequence (SEQ ID NO: 7) : 5’-tgctgcagcctgtatgctccagg-3’
  • sgRNA-8 target sequence (SEQ ID NO: 8) : 5’-cgggcctaggctactcatccagg-3’
  • sgRNA-9 target sequence (SEQ ID NO: 9) : 5’-tccttctagtgtggtccctttgg-3’
  • sgRNA-10 target sequence (SEQ ID NO: 10) : 5’-ttcttctcccaaatggcttaggg-3’
  • sgRNA-11 target sequence (SEQ ID NO: 11) : 5’-ggagaagaatgggggttctctgg-3’
  • sgRNA-12 target sequence (SEQ ID NO: 12) : 5’-tgctgtccctaagccatttggg-3’
  • sgRNA-13 target sequence (SEQ ID NO: 13) : 5’-aatccaaactgagaacagctggg-3’
  • sgRNA-14 target sequence (SEQ ID NO: 14) : 5’-ctgtcaggattggttaccaaagg-3’
  • the UCA kit was used to detect the activities of sgRNAs (FIGS. 1A-1B and Table 4) .
  • the results show that the guide sgRNAs had different activities.
  • Two of them sgRNA1 (SEQ ID NO: 1) and sgRNA11 (SEQ ID NO: 11) were selected for further experiments.
  • the synthesized sgRNA sequences based on sgRNA1 and sgRNA11 target sequences are listed in the following table:
  • the DNA fragment containing T7 promoter and sgRNA scaffold was synthesized, and linked to the backbone vector pHSG299 by restriction enzyme digestion (EcoRI and BamHI) and ligation.
  • the target plasmid sequences were confirmed by the sequencing results.
  • the map of pT7-sgRNA G2 vector is shown in FIG. 2.
  • the DNA fragment containing the T7 promoter and sgRNA scaffold sequence (SEQ ID NO: 19) is shown below:
  • EXAMPLE 4 Constructing recombinant expression vectors pT7-GITR-1 and pT7-GITR-11
  • TAGG was added to the 5’end of SEQ ID NO: 15 and SEQ ID NO: 17 to obtain a forward oligonucleotide sequence
  • AAAC was added to the 5’end of the complementary strand (SEQ ID NO: 16 and SEQ ID NO: 18) to obtain a reverse oligonucleotide sequence.
  • the ligation reaction was carried out at room temperature for 10 to 30 minutes.
  • the ligation product was then transferred to 30 ⁇ L of TOP10 competent cells.
  • the cells were then plated on a petri dish with Kanamycin, and then cultured at 37 °C for at least 12 hours and then two clones were selected and added to LB medium with Kanamycin (5 ml) , and then cultured at 37 °C at 250 rpm for at least 12 hours.
  • Clones were randomly selected and sequenced to verify their sequences. The vectors with correct sequences were selected for subsequent experiments.
  • a partial coding sequence of the mouse GITR gene (Gene ID: 21936) from exons 1-4 (based on the transcript of NCBI accession number NM_009400.2 ⁇ NP_033426.1 whose mRNA sequence is shown in SEQ ID NO: 20, and the corresponding protein sequence is shown in SEQ ID NO: 21) was replaced with a corresponding coding sequence of human homologous GITR gene (Gene ID: 8784) (based on the transcript of NCBI accession number NM_004195.2 ⁇ NP_004186.1, whose mRNA sequence was shown in SEQ ID NO: 22, and the corresponding protein sequence is shown in SEQ ID NO: 23) .
  • the comparison between the mouse GITR and human GITR is shown in FIG. 15, and the finally obtained humanized GITR gene is shown in FIG. 4.
  • the humanized mouse GITR gene DNA sequence (chimeric GITR gene DNA) is shown in SEQ ID NO: 24.
  • SEQ ID NO: 24 shows only the modified portion of DNA sequence, wherein the italicized underlined region is from human GITR gene.
  • the coding region sequence, mRNA sequence and the encoded protein sequence thereof of the modified humanized GITR are respectively shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
  • the 5’homologous arm comprises nucleic acid 156024998-156026386 of NCBI Accession No. NC_000070.6 (SEQ ID NO: 28) .
  • the 3’homologous arm comprises nucleic acid 156028249-156029078 of NCBI Accession No. NC_000070.6 (SEQ ID NO: 29) .
  • the human sequence corresponds to 1204209-1206571 of NCBI Accession No. NC_000001.11 (SEQ ID NO: 30) .
  • LR recombination fragment
  • A1 + A2 fragments correspond to the human GITR sequence.
  • the primers are shown in the table below.
  • the LR and RR fragments were prepared by using C57BL/6 mouse genomic DNA as a template.
  • A1 and A2 fragments were obtained by using human genomic DNA as a template. Fragments LR and A1 were linked by PCR, and A2 and RR were also linked by PCR (reaction conditions are shown in Tables 7 and 8) .
  • the LR+A1 fragment (XhoI+SacI) and the A2+RR (SacI+NotI) fragment were ligated to the pClon-2G plasmid from the AIO kit to obtain the pClon-2G-GITR vector.
  • Primer F in Table 7 was SEQ ID NO: 31
  • Primer R was SEQ ID NO: 34
  • the DNA template was the recovered PCR amplification product of the LR fragment and A1 fragment.
  • Primer F was SEQ ID NO: 35
  • primer R was SEQ ID NO: 38
  • the DNA template was the recovered PCR amplification product of the A2 fragment and RR fragment.
  • Plasmid 1 had the expected results (FIG. 5) . The sequences of Plasmid 1 were further confirmed by sequencing.
  • EXAMPLE 8 Microinjection and embryo transfer using C57BL/6 mice
  • the pre-mixed Cas9 mRNA, pClon-2G-GITR plasmid and in vitro transcription products of pT7-GITR-1, pT7-GITR-11 plasmids were injected into the cytoplasm or nucleus of mouse fertilized eggs (C57BL/6 background) with a microinjection instrument (using Ambion in vitro transcription kit to carry out the transcription according to the method provided in the product instruction) .
  • the embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003.
  • the injected fertilized eggs were then transferred to a culture medium for a short time culture, and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified humanized mice (F0 generation) .
  • the mouse population was further expanded by cross-mating and self-mating to establish stable mouse lines. These humanized mice were named as B-hGITR.
  • PCR analysis was performed using mouse tail genomic DNA of F0 generation mice.
  • Primer L-GT-F is located on the left side of 5’homologous arm
  • Primer R-GT-R is located on the right side of 3’homologous arm
  • R-GT-F and L-GT-R are located within intron 1
  • Mut-R is located within exon 1
  • Mut-F is located within exon 3.
  • the first pair of primers is the first pair of primers:
  • L-GT-F (SEQ ID NO: 39) : 5’-cctgtgtcctttctttcccactatg-3’;
  • R-GT-R (SEQ ID NO: 42) : 5’-tccgaaagcctccttagcttgatgg-3’
  • L-GT-F (SEQ ID NO: 39) : 5’-cctgtgtcctttctttcccactatg-3’;
  • L-GT-R (SEQ ID NO: 43) : 5’-gctaactccatatgcaaccagg-3’
  • R-GT-F (SEQ ID NO: 44) : 5’-cttccagtgtatcgactgtgcctcg-3’;
  • R-GT-R (SEQ ID NO: 42) : 5’-tccgaaagcctccttagcttgatgg-3’
  • PCR experiments using the above primers should generate only one band.
  • the first pair of primers should produce a band of about 1876 bp
  • the second pair of primers should produce a band of about 1343 bp
  • the third pair of primers should produce a band of about 2584 bp
  • the fourth pair of primers should produce a band of about 3018 bp.
  • the results for F0 generation mice are shown in FIGS. 6A-6D. Among these tested mice, F0-20 and F0-48 were positive.
  • One humanized heterozygous mouse was selected.
  • One wildtype mouse with the same background was used as the control.
  • 7.5 ⁇ g of anti-mCD3 antibody was injected intraperitoneally to the mice.
  • the spleens were collected 24 hours after the injection, and the spleen samples were grinded.
  • the samples were then passed through 70 ⁇ m cell mesh.
  • the filtered cell suspensions were centrifuged and the supernatants were discarded.
  • Erythrocyte lysis solution was added to the sample, which was lysed for 5 min and neutralized with PBS solution. The solution was centrifuged again and the supernatants were discarded.
  • the cells were washed with PBS and tested in FACS.
  • the cells were then stained with (1) mouse anti-mouse CD357 (GITR) (mGitr PE) and PerCP/Cy5.5 anti-mouse TCR ⁇ chain (mTcR ⁇ PerCP) , or (2) human antibody anti-hGITR-01-IgG PE (hGITR PE) and PerCP/Cy5.5 anti-mouse TCR ⁇ chain (mTcR ⁇ PerCP) antibody.
  • GITR mouse anti-mouse CD357
  • mTcR ⁇ PerCP PerCP/Cy5.5 anti-mouse TCR ⁇ chain
  • human antibody anti-hGITR-01-IgG PE hGITR PE
  • mTcR ⁇ PerCP PerCP/Cy5.5 anti-mouse TCR ⁇ chain
  • FIGS. 8A-8D The results are shown in FIGS. 8A-8D.
  • FIGS. 8C-8D In the control groups, no spleen cells stained with hGITR PE were detected (FIG. 8C) ; in contrast, spleen cells stained with hGITR PE were observed in heterozygous humanized GITR mice (FIG. 8D) .
  • EXAMPLE 10 GITR knockout mice
  • FIG. 9 shows the PCR results.
  • F1-KO-1, F1-KO-2, F1-KO-3, F1-KO-4, F1-KO-7, F1-KO-8, F1-KO-9, and F1-KO-10 were positive GITR knockout mice.
  • the treatment groups were randomly selected for anti-human GITR antibodies (GITR Ab1, GITR Ab2, GITR Ab3) treatment (10 mg/kg) ; the control group was injected with an equal volume of blank solvent.
  • the frequency of administration was once every three days (6 times of administrations in total) .
  • the tumor volume was measured twice a week and the body weight of the mice was weighed as well.
  • Euthanasia was performed when the tumor volume of the mouse reached 3000 mm 3 .
  • the tested antibodies were generated by immunizing mice with human GITR proteins. A detailed description of these methods can be found in Murphy, et al., Janeway’s immunobiology. Garland Science, 2016 (9 th edition) , which is incorporated herein by reference in its entirety.
  • the animals in each group were healthy, and the body weights of all the treatment and control group mice increased, and were not significantly different from each other (FIGS. 10 and 11) .
  • the tumor in the control group continued growing during the experimental period (FIG. 12) ; when compared with the control group mice, the tumor volumes in the treatment groups were smaller than the control group (FIG. 12) .
  • the anti-GITR antibodies were well tolerated, and the antibodies inhibited the tumor growth in mice.
  • Table 11 shows results for this experiment, including the tumor volumes at the day of grouping, 14 days after the grouping, and at the end of the experiment (day 21) , the survival rate of the mice, the number of tumor free mice, the Tumor Growth Inhibition value (TGI TV ) , and the statistical differences (P value) in mouse body weights and tumor volume between the treatment and control groups.
  • the body weight of each group increased and there was no significant difference between the groups (p> 0.05) , indicating that the animals tolerated the three anti-hGITR antibodies well.
  • the average tumor volume was 1904 ⁇ 294 mm 3 .
  • the average tumor volumes in the treatment groups were 833 ⁇ 135mm 3 (G2) , 1112 ⁇ 105mm 3 (G3) , and 828 ⁇ 214mm 3 (G4) .
  • the tumor volumes in all treatment groups (G2 ⁇ G4) were smaller than those in the control group (G1) .
  • the TGI TV %for each treatment group is 59.0% (G2) , 43.6% (G3) , and 59.2% (G4) .
  • B-hGITR mouse model can be used as an in vivo animal model for screening, evaluating human GITR signaling pathway regulators, and testing the efficacy of anti-human GITR antibodies.
  • EXAMPLE 12 Mice with two or more humanized genes
  • mice with the humanized GITR gene can also be used to prepare an animal model with double-humanized or multi-humanized genes.
  • the embryonic stem cell used in the microinjection and embryo transfer process can be selected from the embryos of other genetically modified mice, so as to obtain double-or multiple-gene modified mouse models.
  • the fertilized eggs of B-hGITR mice can also be further genetically engineered to produce mouse lines with one or more humanized or otherwise genetically modified mouse models.
  • the humanized GITR animal model homozygote or heterozygote can be mated with other genetically modified homozygous or heterozygous animal models (or through IVF) , and the progeny can be screened. According to the Mendelian law, there is a chance to obtain the double-gene or multiple-gene modified heterozygous animals, and then the heterozygous animals can be mated with each other to finally obtain the double-gene or multiple-gene modified homozygotes.
  • the double humanized GITR/PD-1 mouse model was obtained by crossing the GITR humanized mice with PD-1 humanized mice (e.g., B-hPD-1 mice obtained from Beijing Biocytogen Co., Ltd, Catalog number: B-CM-001) .
  • PD-1 humanized mice e.g., B-hPD-1 mice obtained from Beijing Biocytogen Co., Ltd, Catalog number: B-CM-001
  • FIGS. 13A-13D The results for a number of humanized GITR/PD-1 mice are shown in FIGS. 13A-13D, wherein FIGS. 13A and 13B show that the mice numbered 6870 and 6872 were heterozygous for humanized GITR.
  • FIGS. 13C and 13D show that the mice numbered 6865 ⁇ 6872 were heterozygous for humanized PD-1.
  • the results show that the mice numbered 6870 and 6872 were heterozygous for both humanized GITR and humanized PD-1 (GITR H/+ /PD-1 H/+ ) .
  • non-human mammals described herein can also be prepared through other gene editing systems and approaches, including but not limited to: gene homologous recombination techniques based on embryonic stem cells (ES) , zinc finger nuclease (ZFN) techniques, transcriptional activator-like effector factor nuclease (TALEN) technique, homing endonuclease (megakable base ribozyme) , or other techniques.
  • ES embryonic stem cells
  • ZFN zinc finger nuclease
  • TALEN transcriptional activator-like effector factor nuclease
  • homing endonuclease homing endonuclease (megakable base ribozyme) , or other techniques.
  • a targeting strategy for generating the humanized GITR mouse model with Embryonic Stem Cell Technologies is developed (FIG. 14) . Since the objective is to replace exons 1-4 of the mouse GITR gene in whole or in part with the corresponding sequence in human GITR gene, a recombinant vector that contains a 5’homologous arm (4285 bp) , a 3’homologous arm (4581 bp) and a sequence fragment from human GITR (2363 bp) is designed.
  • the vector can also contain a resistance gene for positive clone screening, such as neomycin phosphotransferase coding sequence Neo.
  • a resistance gene for positive clone screening such as neomycin phosphotransferase coding sequence Neo.
  • two site-specific recombination systems in the same orientation such as Frt or LoxP, can be added.
  • a coding gene with a negative screening marker such as the diphtheria toxin A subunit coding gene (DTA) , can be constructed downstream of the recombinant vector 3’homologous arm.
  • DTA diphtheria toxin A subunit coding gene
  • Vector construction can be carried out using methods known in the art, such as enzyme digestion and so on.
  • the recombinant vector with correct sequence can be next transfected into mouse embryonic stem cells, such as C57BL/6 mouse embryonic stem cells, and then the recombinant vector can be screened by positive clone screening gene.
  • the cells transfected with the recombinant vector are next screened by using the positive clone marker gene, and Southern Blot technique can be used for DNA recombination identification.
  • the positive clonal cells black mice
  • the isolated blastocysts white mice
  • the resulting chimeric blastocysts formed following the injection are transferred to the culture medium for a short time culture and then transplanted into the fallopian tubes of the recipient mice (white mice) to produce F0 generation chimeric mice (black and white) .
  • the F0 generation chimeric mice with correct gene recombination are then selected by extracting the mouse tail genome and detecting by PCR for subsequent breeding and identification.
  • the F1 generation mice are obtained by mating the F0 generation chimeric mice with wildtype mice.
  • Stable gene recombination positive F1 heterozygous mice are selected by extracting rat tail genome and PCR detection. Next, the F1 heterozygous mice are mated to each other to obtain genetically recombinant positive F2 generation homozygous mice. In addition, the F1 heterozygous mice can also be mated with Flp or Cre mice to remove the positive clone screening marker gene (e.g., neo) , and then the GITR gene humanized homozygous mice can be obtained by mating these mice with each other.
  • the methods of genotyping and using the F1 heterozygous mice or F2 homozygous mice are similar to the methods as described in the examples above.

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Abstract

La présente invention concerne des animaux non humains génétiquement modifiés qui expriment une protéine apparentée à TNFR induite par les glucocorticoïdes (GITR) humaine ou chimérique (par exemple, humanisée) et des procédés d'utilisation de ceux-ci.
PCT/CN2018/091844 2017-06-19 2018-06-19 Animal non humain génétiquement modifié avec gitr humain ou chimérique WO2018233606A1 (fr)

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CN114134152A (zh) * 2020-09-17 2022-03-04 百奥赛图(北京)医药科技股份有限公司 Glp1r基因人源化的非人动物及其构建方法和应用
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