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WO2023066250A1 - Animal non humain génétiquement modifié portant un trem1 humain ou chimérique - Google Patents

Animal non humain génétiquement modifié portant un trem1 humain ou chimérique Download PDF

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WO2023066250A1
WO2023066250A1 PCT/CN2022/125929 CN2022125929W WO2023066250A1 WO 2023066250 A1 WO2023066250 A1 WO 2023066250A1 CN 2022125929 W CN2022125929 W CN 2022125929W WO 2023066250 A1 WO2023066250 A1 WO 2023066250A1
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trem1
animal
human
endogenous
sequence
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PCT/CN2022/125929
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English (en)
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Lei Zhao
Zhiyuan Shen
Tao Li
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Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • A01K2217/15—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
    • A01K2267/0306—Animal model for genetic diseases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) TREM1, and methods of use thereof.
  • test results obtained from the use of conventional experimental animals for in vivo pharmacological test may not reflect the real disease state and the interaction at the targeting sites, resulting in that the results in many clinical trials are significantly different from the animal experimental results.
  • This disclosure is related to an animal model with human TREM1 or chimeric TREM1.
  • the animal model can express human TREM1 or chimeric TREM1 (e.g., humanized TREM1) protein in its body. It can be used in the studies on the function of TREM1 gene, and can be used in the screening and evaluation of anti-human TREM1 antibodies.
  • the animal models prepared by the methods described herein can be used in drug screening, pharmacodynamics studies, treatments for immune-related diseases, and cancer therapy for human TREM1 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 TREM1 protein and a platform for screening cancer drugs.
  • the disclosure is related to a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric triggering receptor expressed on myeloid cells 1 (TREM1) .
  • the sequence encoding the human or chimeric TREM1 is operably linked to an endogenous regulatory element at the endogenous TREM1 gene locus in the at least one chromosome.
  • the sequence encoding a human or chimeric TREM1 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human TREM1 (NP_061113.1 (SEQ ID NO: 2) ) .
  • the sequence encoding a human or chimeric TREM1 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: 7.
  • the sequence encoding a human or chimeric TREM1 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 1-205 of SEQ ID NO: 2.
  • the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.
  • the animal is a mouse.
  • the animal does not express endogenous TREM1 or expresses a decreased level of endogenous TREM1 as compared to TREM1 expression level in a wild-type animal.
  • the animal has one or more cells expressing human or chimeric TREM1.
  • the animal has one or more cells expressing human or chimeric TREM1, and the expressed human or chimeric TREM1 can interact with a human TYRO protein tyrosine kinase-binding protein (DAP12) to form a heterodimer, activating downstream signaling pathways.
  • DAP12 human TYRO protein tyrosine kinase-binding protein
  • the animal has one or more cells expressing human or chimeric TREM1, and the expressed human or chimeric TREM1 can interact with an endogenous DAP12 to form a heterodimer, activating downstream signaling pathways.
  • the disclosure is related to a genetically-modified, non-human animal
  • the genome of the animal comprises a replacement of a sequence encoding a region of endogenous TREM1 with a sequence encoding a corresponding region of human TREM1 at an endogenous TREM1 gene locus.
  • the sequence encoding the corresponding region of human TREM1 is operably linked to an endogenous regulatory element at the endogenous TREM1 locus, and one or more cells of the animal expresses a human or chimeric TREM1.
  • the animal does not express endogenous TREM1 or expresses a decreased level of endogenous TREM1 as compared to TREM1 expression level in a wild-type animal.
  • the replaced sequence encodes the extracellular region of TREM1, optionally including the signal peptide.
  • the animal has one or more cells expressing a chimeric TREM1 having a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region, in some embodiments, 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 TREM1 (NP_061113.1 (SEQ ID NO: 2) ) .
  • the extracellular region of the chimeric TREM1 has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 181, 182, 183, 184, or 185 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human TREM1 (e.g., amino acids 21-205 of SEQ ID NO: 2) .
  • the signal peptide of the chimeric TREM1 has a sequence that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids that are identical to a contiguous sequence present in the transmembrane region of human TREM1 (e.g., amino acids 1-20 of SEQ ID NO: 2) .
  • the sequence encoding a region of endogenous TREM1 comprises exon 1, exon 2, exon 3, and/or exon 4, or a part thereof, of the endogenous TREM1 gene.
  • the animal is a mouse.
  • the animal is heterozygous with respect to the replacement at the endogenous TREM1 gene locus.
  • the animal is homozygous with respect to the replacement at the endogenous TREM1 gene locus.
  • the disclosure is related to a method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous TREM1 gene locus, a sequence encoding a region of endogenous TREM1 with a sequence encoding a corresponding region of human TREM1.
  • the sequence encoding the corresponding region of human TREM1 comprises exon 1, exon 2, exon 3, and/or exon 4, or a part thereof, of a human TREM1 gene.
  • the sequence encoding the corresponding region of human TREM1 comprises a portion of exon 1, exon 2, exon 3, and a portion of exon 4, of a human TREM1 gene.
  • the sequence encoding the corresponding region of human TREM1 encodes amino acids 1-205 of SEQ ID NO: 2.
  • the region comprises the extracellular region, optionally the signal peptide, of TREM1.
  • the sequence encoding a region of endogenous TREM1 comprises exon 1, exon 2, exon 3, and/or exon 4, or a part thereof, of the endogenous TREM1 gene.
  • the animal is a mouse, and the sequence encoding a region of endogenous TREM1 comprises a portion of exon 1, exon 2, exon 3, and a portion of exon 4 of the endogenous TREM1 gene.
  • the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a humanized TREM1 polypeptide
  • the humanized TREM1 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human TREM1
  • the animal expresses the humanized TREM1 polypeptide.
  • the humanized TREM1 polypeptide has at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 181, 182, 183, 184, or 185 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of human TREM1 extracellular region (e.g., amino acids 21-205 of SEQ ID NO: 2) .
  • the humanized TREM1 polypeptide has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of human TREM1 signal peptide (e.g., amino acids 1-20 of SEQ ID NO: 2) .
  • the humanized TREM1 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 1-205 of SEQ ID NO: 2.
  • the nucleotide sequence is operably linked to an endogenous TREM1 regulatory element of the animal.
  • the chimeric TREM1 polypeptide comprises an endogenous TREM1 transmembrane region and/or an endogenous TREM1 cytoplasmic region.
  • the nucleotide sequence is integrated to an endogenous TREM1 gene locus of the animal.
  • the humanized TREM1 polypeptide has at least one mouse TREM1 activity and/or at least one human TREM1 activity.
  • the disclosure is related to a method of making a genetically-modified animal cell that expresses a chimeric TREM1, the method comprising: replacing at an endogenous TREM1 gene locus, a nucleotide sequence encoding a region of endogenous TREM1 with a nucleotide sequence encoding a corresponding region of human TREM1, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the chimeric TREM1, in some embodiments, the animal cell expresses the chimeric TREM1. In some embodiments, the animal is a mouse.
  • the chimeric TREM1 comprises a human or humanized TREM1 extracellular region; and a transmembrane and/or a cytoplasmic region of mouse TREM1. In some embodiments, the chimeric TREM1 further comprises a human or humanized TREM1 signal peptide. In some embodiments, the nucleotide sequence encoding the chimeric TREM1 is operably linked to an endogenous TREM1 regulatory region, e.g., promoter.
  • the animal further comprises a sequence encoding an additional human or chimeric protein.
  • the additional human or chimeric protein is interleukin 1 alpha (IL1A) , interleukin 1 beta (IL1B) , interleukin 6 (IL6) , interleukin 15 (IL15) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , T cell immunoreceptor with Ig and ITIM domains (TIGIT) , lymphocyte-activation gene 3 (LAG3) , CD226, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , and/or tumor necrosis factor alpha (TNF- ⁇ ) .
  • IL1A interleukin 1 alpha
  • IL1B interleukin 1 beta
  • IL6 interleukin 6
  • IL15 interleukin 15
  • PD-1 programmed cell death protein 1
  • PD-L1 programmed cell death ligand 1
  • TAGIT
  • the disclosure is related to a method of determining effectiveness of a therapeutic agent for the treatment of cancer, comprising: a) administering the therapeutic agent to the animal as described herein, in some embodiments, the animal has a tumor; and b) determining inhibitory effects of the therapeutic agent to the tumor.
  • the therapeutic agent is an anti-TREM1 antibody.
  • the tumor comprises one or more cancer cells that are injected into the animal.
  • determining inhibitory effects of the anti-TREM1 antibody to the tumor involves measuring the tumor volume in the animal.
  • the cancer is a solid tumor, gynecologic cancer, breast cancer, colorectal cancer, gastric adenocarcinoma, lung adenocarcinoma, pancreatic cancer, or head and neck cancer.
  • the disclosure is related to a method of determining effectiveness of an anti-TREM1 antibody and an additional therapeutic agent for the treatment of cancer, comprising a) administering the anti-TREM1 antibody and the additional therapeutic agent to the animal as described herein, in some embodiments, the animal has a tumor; and b) determining inhibitory effects on the tumor.
  • the animal further comprises a sequence encoding a human or chimeric PD-1, a human or chimeric PD-L1, and/or a human or chimeric CTLA4.
  • the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody.
  • the tumor comprises one or more tumor cells that express PD-L1. In some embodiments, the tumor comprises one or more cancer cells that are injected into the animal. In some embodiments, determining inhibitory effects of the treatment involves measuring the tumor volume in the animal. In some embodiments, the animal has a solid tumor, gynecologic cancer, breast cancer, colorectal cancer, gastric adenocarcinoma, lung adenocarcinoma, pancreatic cancer, or head and neck cancer.
  • the disclosure is related to a method of determining toxicity of a therapeutic agent comprising: a) administering the therapeutic agent to the animal as described herein; and b) determining effects of the therapeutic agent to the animal.
  • the therapeutic agent is an anti-TREM1 antibody.
  • determining effects of the therapeutic agent to the animal involves measuring the body weight, red blood cell count, hematocrit, and/or hemoglobin of the animal.
  • the disclosure is related to a protein comprising an amino acid sequence
  • the amino acid sequence is one of the following: (a) an amino acid sequence set forth in SEQ ID NO: 1, 2, or 7; (b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 1, 2, or 7; (c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1, 2, or 7; (d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1, 2, or 7 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: 1, 2, or 7.
  • the disclosure is related to a nucleic acid comprising a nucleotide sequence
  • the nucleotide sequence is one of the following: (a) a sequence that encodes the protein as described herein; (b) SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 12, or 13; (c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 12, or 13; and (d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 12, or 13.
  • the disclosure is related to a cell comprising the protein and/or the nucleic acid as described herein. In one aspect, the disclosure is related to an animal comprising the protein and/or the nucleic acid as described herein.
  • the disclosure further relates to a TREM1 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 TREM1 gene function, human TREM1 antibodies, the drugs or efficacies for human TREM1 targeting sites, and the drugs for immune-related diseases and antitumor drugs.
  • FIG. 1 is a schematic diagram showing mouse and human TREM1 gene loci.
  • FIG. 2 is a schematic diagram showing humanized TREM1 gene locus.
  • FIG. 3 is a schematic diagram showing a TREM1 gene targeting strategy.
  • FIG. 4 is a schematic diagram showing the Frt recombination process in TREM1 gene humanized mice.
  • FIG. 5 is a schematic diagram showing a TREM1 gene targeting strategy.
  • FIG. 6 shows mouse tail PCR identification results of F1 generation mice by primers F1-F and F1-R.
  • M is a marker.
  • WT is a wild-type control.
  • H 2 O is a water control.
  • FIG. 7 shows Southern Blot results of cells after recombination using the 3’ Probe and A Probe (5’) .
  • WT is a wild-type control.
  • FIG. 8 shows the percentages of leukocyte subtypes in the spleen of C57BL/6 wild-type mice and TREM1 gene humanized homozygous mice (H/H) .
  • FIG. 9 shows the percentages of T cell subtypes in the spleen of C57BL/6 wild-type mice and TREM1 gene humanized homozygous mice (H/H) .
  • FIG. 10 shows the percentages of leukocyte subtypes in the peripheral blood of C57BL/6 wild-type mice and TREM1 gene humanized homozygous mice (H/H) .
  • FIG. 11 shows the percentages of T cell subtypes in the peripheral blood of C57BL/6 wild-type mice and TREM1 gene humanized homozygous mice (H/H) .
  • FIG. 12 shows the percentages of leukocyte subtypes in the lymph nodes of C57BL/6 wild-type mice and TREM1 gene humanized homozygous mice (H/H) .
  • FIG. 13 shows the percentages of T cell subtypes in the lymph nodes of C57BL/6 wild-type mice and TREM1 gene humanized homozygous mice (H/H) .
  • FIG. 14A shows expression levels of mouse soluble TREM1 (sTREM1) in C57BL/6 wild-type mice and TREM1 gene humanized homozygous mice (H/H) , either before or after LPS stimulation. ND stands for not detected.
  • FIG. 14B shows expression levels of human soluble TREM1 (sTREM1) in C57BL/6 wild-type mice and TREM1 gene humanized homozygous mice (H/H) , either before or after LPS stimulation. ND stands for not detected.
  • FIG. 15 shows the alignment between human TREM1 amino acid sequence (NP_061113.1; SEQ ID NO: 2) and mouse TREM1 amino acid sequence (NP_067381.1; SEQ ID NO: 1) .
  • FIG. 16 shows the alignment between human TREM1 amino acid sequence (NP_061113.1; SEQ ID NO: 2) and rat TREM1 amino acid sequence (NP_001100355.1; SEQ ID NO: 36) .
  • This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) TREM1, and methods of use thereof.
  • TREM proteins Triggering receptors expressed on myeloid cells
  • TREM family of protein receptors consists of TREM-1, TREM-2, TREM-3 (mouse) , TREM-like transcript (TLT) -1, and TLT-2.
  • TREM-1 was the first TREM identified and initial studies established TREM-1 as an amplifier of the systemic inflammatory response syndrome and sepsis. Studies have shown that bacterial and viral products can induce expression of TREM-1.
  • TREM-1 The functional consequences of silencing TREM-1 gene in macrophages include an altered availability of key signaling (CD14, I ⁇ B ⁇ , MyD88) , and effector molecules (MCP-1, IL-1 ⁇ , IL-6, IL-23) downstream of TLR activation. Studies have also shown that lipid mediators such as prostaglandins modulate expression of TREM-1. Together these studies have suggested a pivotal role for TREM-1 in amplification of TLR induced responses. Therefore, TREM1 is regarded as a potential biomarker and therapeutic target for cancer.
  • Experimental animal models are an indispensable research tool for studying the effects of these antibodies (e.g., anti-TREM1 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.
  • Triggering receptor expressed on myeloid cells 1 is a transmembrane glycoprotein having a single extracellular immunoglobulin-like domain, a transmembrane region, and a short cytoplasmic tail.
  • TREM-1 is expressed as a transmembrane receptor complex with adaptor protein DNA X-activating protein of 12 kD (DAP12) in myeloid cells. After TREM-1 cross-linking, the phosphorylated DAP12 can recruit and phosphorylate growth receptor binding protein 2 (GRBP-2) and phosphatidylinositol-3 kinase (PI3K) to amplify Toll-like receptors (TLRs) .
  • GRBP-2 phosphorylate growth receptor binding protein 2
  • PI3K phosphatidylinositol-3 kinase
  • TREM-1 can enhance neutrophils or monocytes/macrophages to secret myeloperoxidase, monocyte chemoattractant protein-3 (MCP-3) , IL-8, macrophage inflammatory protein-1 ⁇ (MIP-1 ⁇ ) , monocyte chemoattractant protein-1 (MCP-1) , and granulocyte-monocyte colony–stimulating factor (GM-CSF) .
  • MIP-1 ⁇ macrophage inflammatory protein-1 ⁇
  • MCP-1 monocyte chemoattractant protein-1
  • GM-CSF granulocyte-monocyte colony–stimulating factor
  • TREM-1 can be shed from the membrane of activated phagocytes without the transmembrane and intracellular domains, and can be found as soluble TREM (sTREM-1) in body fluids.
  • sTREM-1 is a diagnostic marker for sepsis, pneumonia, inflammatory bowel diseases, and liver cirrhosis.
  • TREM-1 is highly expressed in colon, hepatocellular and lung carcinoma tissue. Furthermore, TREM-1 expression in patients with NSCLC has been associated with cancer recurrence and poor survival of patients suggesting that TREM-1 may play an important role in cancer progression. However the mechanism by which TREM-1 is induced in tumor tissue has not been defined.
  • TREM-1 should to be an anti-tumor molecule through enhancing immune responses.
  • TREM-1 could promote tumor progression.
  • TREM-1 is expressed by Kupffer cells (KC) and regulates the inflammatory response to induce aseptic inflammation in damaged liver tissues, which lead to carcinogenesis subsequently.
  • KC Kupffer cells
  • tumor cells can promote the up-regulation of TREM-1 expression on TAM in tumor environment, suggesting that TREM-1 could boost carcinogenesis and cancer progression with an unknown mechanism.
  • chronic inflammatory state could contribute to tumor progression through inducing tumor angiogenesis. Therefore, all these evidences indicated that the expression and functions of TREM-1 might be different between pathogen infection status and tumor-bearing status.
  • TREM1 is induced in tumor associated macrophages by cyclo-oxygenase pathway in human non-small cell lung cancer.
  • American Journal of Respiratory and Critical Care Medicine 177.7 (2008) : 763-770; Saurer, L., et al. TREM-1 promotes intestinal tumorigenesis.
  • Scientific Reports 7.1 (2017) : 1-12; and Zhang, G., et al. TREM-1low is a novel characteristic for tumor-associated macrophages in lung cancer.
  • Oncotarget 7.26 (2016) : 40508 each of which is incorporated by reference in its entirety.
  • TREM1 gene (Gene ID: 54210) locus has four exons, exon 1, exon 2, exon 3, and exon 4 (FIG. 1) .
  • the TREM1 protein also has, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region.
  • the nucleotide sequence for human TREM1 mRNA is NM_018643.5
  • the amino acid sequence for human TREM1 is NP_061113.1 (SEQ ID NO: 2) .
  • the location for each exon and each region in human TREM1 nucleotide sequence and amino acid sequence is listed below:
  • the human TREM1 gene (Gene ID: 54210) is located in Chromosome 6 of the human genome, which is located from 41267385 to 41286692 (GRCh38. p13 (GCF_000001405.39) ) .
  • the 5’-UTR is from 41286656 to 41286682, exon 1 is from 41286682 to 41286607, intron 1 is from 41286606 to 41282752, exon 2 is from 41282751 to 41282395, intron 2 is from 41282394 to 41281154, exon 3 is from 41281153 to 41280961, intron 3 is from 41280960 to 41276231, exon 4 is from 41276230 to 41273605, and the 3’-UTR is from 41273605 to 41276124, based on transcript NM_018643.5. All relevant information for human TREM1 locus can be found in the NCBI website with Gene ID: 54210, which is incorporated by reference herein in its entirety.
  • TREM1 gene locus has four exons, exon 1, exon 2, exon 3, and exon 4 (FIG. 1) .
  • the mouse TREM1 protein also has, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region.
  • the nucleotide sequence for mouse TREM1 mRNA is NM_021406.5
  • the amino acid sequence for mouse TREM1 is NP_067381.1 (SEQ ID NO: 1) .
  • the location for each exon and each region in the mouse TREM1 nucleotide sequence and amino acid sequence is listed below:
  • the mouse TREM1 gene (Gene ID: 58217) is located in Chromosome 17 of the mouse genome, which is located from 48539763 to 48553955 (GRCm39 (GCF_000001635.27) ) .
  • the 5’-UTR is from 48539799 to 48539822, exon 1 is from 48539799 to 48539871, intron 1 is from 48539872 to 48544025, exon 2 is from 48544026 to 48544382, intron 2 is from 48544383 to 48548513, exon 3 is from 48548514 to 48548697, intron 3 is from 48548698 to 48551573, exon 4 is from 48551574 to 48553952, and the 3’-UTR is from 48551677 to 48553952, based on transcript NM_021406.5. All relevant information for mouse Trem1 locus can be found in the NCBI website with Gene ID: 58217, which is incorporated by reference herein in its entirety.
  • FIG. 15 shows the alignment between human TREM1 amino acid sequence (NP_061113.1; SEQ ID NO: 2) and mouse TREM1 amino acid sequence (NP_067381.1; SEQ ID NO: 1) .
  • NP_061113.1 human TREM1 amino acid sequence
  • NP_067381.1 mouse TREM1 amino acid sequence
  • TREM1 genes, proteins, and locus of the other species are also known in the art.
  • the gene ID for TREM1 (ERBB2) in Rattus norvegicus (rat) is 301229
  • the gene ID for TREM1 in Macaca mulatta (Rhesus monkey) is 693558
  • the gene ID for TREM1 in Canis lupus familiaris (dog) is 608994
  • the gene ID for TREM1 in Sus scrofa (pig) is 396601.
  • 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.
  • FIG. 16 shows the alignment between human TREM1 amino acid sequence (NP_061113.1; SEQ ID NO: 2) and rat TREM1 amino acid sequence (NP_001100355.1; SEQ ID NO: 36.
  • NP_061113.1 human TREM1 amino acid sequence
  • NP_001100355.1 rat TREM1 amino acid sequence
  • FIG. 16 shows the alignment between human TREM1 amino acid sequence (NP_061113.1; SEQ ID NO: 2) and rat TREM1 amino acid sequence (NP_001100355.1; SEQ ID NO: 36.
  • the present disclosure provides human or chimeric (e.g., humanized) TREM1 nucleotide sequence and/or amino acid sequences.
  • the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, 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, 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, 450, 500, 550, 600, 610, or 620 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, 200, 201, 202, 203, 204, 205, 210, 220, 230, 231, 232, 233, or 234 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, 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, and/or exon 4 are replaced by a region, a portion, or the entire sequence of the human exon 1, exon 2, exon 3, and/or exon 4 (e.g., a portion of exon 1, exon 2, exon 3, and a portion of exon 4) .
  • a “region” or “portion” of the signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, exon 2, exon 3, and/or exon 4 is deleted.
  • the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a chimeric (e.g., humanized ) TREM1 nucleotide sequence.
  • the chimeric (e.g., humanized ) TREM1 nucleotide sequence encodes a TREM1 protein comprising a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region.
  • the signal peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-20 of SEQ ID NO: 2.
  • the signal peptide comprises all or part of human TREM1 signal peptide.
  • the extracellular region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 21-205 of SEQ ID NO: 2. In some embodiments, the extracellular region comprises all or part of human TREM1 extracellular region. In some embodiments, the transmembrane region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 203-223 of SEQ ID NO: 1. In some embodiments, the transmembrane region comprises all or part of endogenous TREM1 transmembrane region.
  • the cytoplasmic region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 224-230 of SEQ ID NO: 1. In some embodiments, the cytoplasmic region comprises all or part of endogenous TREM1 cytoplasmic region. In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 12, or 13.
  • the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized TREM1 protein.
  • the TREM1 protein comprises, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region.
  • the humanized TREM1 protein comprises a human or humanized signal peptide.
  • the humanized TREM1 protein comprises an endogenous signal peptide.
  • the humanized TREM1 protein comprises a human or humanized extracellular region.
  • the humanized TREM1 protein comprises an endogenous extracellular region.
  • the humanized TREM1 protein comprises a human or humanized transmembrane region. In some embodiments, the humanized TREM1 protein comprises an endogenous transmembrane region. In some embodiments, the humanized TREM1 protein comprises a human or humanized cytoplasmic region. In some embodiments, the humanized TREM1 protein comprises an endogenous cytoplasmic region. In some embodiments, the humanized TREM1 protein comprises a human or humanized signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the humanized TREM1 protein comprises an endogenous sequence that corresponds to amino acids 203-230 of SEQ ID NO: 1.
  • the genetically-modified non-human animal described herein comprises a human or humanized TREM1 gene.
  • the humanized TREM1 gene comprises 4 exons.
  • the humanized TREM1 gene comprises humanized exon 1, human exon 2, human exon 3, and/or humanized exon 4.
  • the humanized TREM1 gene comprises 3 introns.
  • the humanized TREM1 gene comprises human intron 1, human intron 2, and/or human intron 3.
  • the humanized TREM1 gene comprises human or humanized 5’ UTR.
  • the humanized TREM1 gene comprises human or humanized 3’ UTR.
  • the humanized TREM1 gene comprises endogenous 5’ UTR.
  • the humanized TREM1 gene comprises endogenous 3’ UTR.
  • the present disclosure also provides a chimeric (e.g., humanized) TREM1 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 TREM1 mRNA sequence (e.g., NM_021406.5) , mouse TREM1 amino acid sequence (e.g., SEQ ID NO: 1) , or a portion thereof (e.g., 5’ UTR, a portion of exon 1, a portion of exon 4, and 3’ UTR) ; and in some embodiments, at least 1%, 2%, 3%,
  • the sequence encoding amino acids 1-202 of mouse TREM1 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human TREM1 (e.g., amino acids 1-205 of human TREM1 (SEQ ID NO: 2) ) .
  • the sequence encoding amino acids 21-202 of mouse TREM1 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human TREM1 (e.g., amino acids 21-205 of human TREM1 (SEQ ID NO: 2) ) .
  • the sequence encoding amino acids 1-230 of mouse TREM1 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human TREM1 (e.g., amino acids 1-234 of human TREM1 (SEQ ID NO: 2) ) .
  • the sequence encoding amino acids 21-230 of mouse TREM1 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human TREM1 (e.g., amino acids 21-234 of human TREM1 (SEQ ID NO: 2) ) .
  • the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse TREM1 promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.
  • a promotor or regulatory element e.g., an endogenous mouse TREM1 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 part of or the entire mouse TREM1 nucleotide sequence (e.g., a portion of exon 1, exons 2-3, and a portion of exon 4 of NM_021406.5) .
  • 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
  • part of or the entire mouse TREM1 nucleotide sequence e.g., a portion of exon 1, exons 2-3, and a portion of exon 4 of NM_021406.5
  • 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 part of or the entire mouse TREM1 nucleotide sequence (e.g., 5’ UTR, a portion of exon 1, a portion of exon 4, and 3’ UTR of NM_021406.5) .
  • 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 part of or the entire human TREM1 nucleotide sequence (e.g., 5’ UTR, a portion of exon 1, a portion of exon 4, and 3’ UTR of NM_018643.5) .
  • 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 part of or the entire human TREM1 nucleotide sequence (e.g., a portion (at least 20 bp) of exon 1, exons 2-3, and a portion (at least 10 bp) of exon 4 of NM_018643.5) .
  • 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
  • the entire human TREM1 nucleotide sequence e.g., a portion (at least 20 bp) of ex
  • 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 part of or the entire mouse TREM1 amino acid sequence (e.g., amino acids 1-202 of NP_067381.1 (SEQ ID NO: 1) ) .
  • 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 part of or the entire mouse TREM1 amino acid sequence (e.g., amino acids 203-230 of NP_067381.1 (SEQ ID NO: 1) ).
  • 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 part of or the entire human TREM1 amino acid sequence (e.g., amino acids 206-234 of NP_061113.1 (SEQ ID NO: 2) ).
  • 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 part of or the entire human TREM1 amino acid sequence (e.g., amino acids 1-205 of NP_061113.1 (SEQ ID NO: 2) ) .
  • the present disclosure also provides a humanized TREM1 mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:
  • nucleic acid sequence an amino acid sequence encoded by 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: 1, 2, or 7 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: 1, 2, or 7;
  • amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 1, 2, or 7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • amino acid sequence that comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1, 2, or 7.
  • the present disclosure also provides a humanized TREM1 amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:
  • an amino acid sequence have a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%to amino acids 1-205 or 21-205 of SEQ ID NO: 2;
  • amino acid sequence that is different from amino acids 1-205 or 21-205 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • amino acid sequence that comprises a substitution, a deletion and /or insertion of one or more amino acids to amino acids 1-205 or 21-205 of SEQ ID NO: 2.
  • the present disclosure also provides a humanized TREM1 amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:
  • an amino acid sequence have a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%to amino acids 203-230 of SEQ ID NO: 1;
  • amino acid sequence that is different from amino acids 203-230 of SEQ ID NO: 1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • amino acid sequence that comprises a substitution, a deletion and /or insertion of one or more amino acids to amino acids 203-230 of SEQ ID NO: 1.
  • the present disclosure also relates to a TREM1 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: 3, 4, 5, 6, 8, 9, 10, 11, 12, or 13 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: 3, 4, 5, 6, 8, 9, 10, 11, 12, or 13;
  • 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: 1, 2, or 7;
  • 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: 1, 2, or 7;
  • 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: 1, 2, or 7 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: 1, 2, or 7.
  • the present disclosure further relates to a TREM1 genomic DNA sequence of a humanized mouse.
  • the DNA sequence is obtained by 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: 5 or 6.
  • 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: 1, 2, or 7, and has protein activity.
  • the homology with the sequence shown in SEQ ID NO: 1, 2, or 7 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: 1, 2, or 7 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: 5 or 6, and encodes a polypeptide that has protein activity.
  • the homology with the sequence shown in SEQ ID NO: 5 or 6 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: 3, 4, 5, 6, 8, 9, 10, 11, 12, or 13 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 amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • 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) TREM1 from an endogenous non-human TREM1 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 TREM1 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 wild-type 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 wild-type 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.
  • humanized protein or “humanized polypeptide” refers to a protein or a polypeptide, wherein at least a portion of the protein or the polypeptide is from the human protein or human polypeptide. In some embodiments, the humanized protein or polypeptide is a human protein or polypeptide.
  • humanized nucleic acid refers to a nucleic acid, wherein at least a portion of the nucleic acid is from the human. In some embodiments, the entire nucleic acid of the humanized nucleic acid is from human. In some embodiments, the humanized nucleic acid is a humanized exon. A humanized exon can be e.g., a human exon or a chimeric exon.
  • the chimeric gene or the chimeric nucleic acid is a humanized TREM1 gene or a humanized TREM1 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human TREM1 gene, at least one or more portions of the gene or the nucleic acid is from a non-human TREM1 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an TREM1 protein.
  • the encoded TREM1 protein is functional or has at least one activity of the human TREM1 protein or the non-human TREM1 protein, e.g., heterodimerise with DAP12 and initiate downstream signaling pathways, e.g., PLC ⁇ , PI3K, and MAPK.
  • the chimeric protein or the chimeric polypeptide is a humanized TREM1 protein or a humanized TREM1 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 TREM1 protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human TREM1 protein.
  • the humanized TREM1 protein or the humanized TREM1 polypeptide is functional or has at least one activity of the human TREM1 protein or the non-human TREM1 protein.
  • the cytoplasmic region is human or humanized.
  • the transmembrane region is human or humanized.
  • the extracellular region is human or humanized.
  • the signal peptide is human or humanized.
  • both the transmembrane and cytoplasmic regions are endogenous.
  • 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) . In some embodiments, the non-human animal is a rodent.
  • the non-human animal is a mouse having a BALB/c, A, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL (C57BL/10Cr and C57BL/Ola) , C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, or CBA/H background.
  • 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 TREM1 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/ ⁇ c null mice (Ito, M.
  • a genetically modified mouse can include a humanization of at least a portion of an endogenous non-human TREM1 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, NOD-Prkdc scid IL-2r ⁇ null mice, NOD-Rag 1 -/- -IL2rg -/- (NRG) mice, Rag 2 -/- -IL2rg -/- (RG) mice, and a combination thereof.
  • NSG NSG
  • RG -/-IL2rg -/-
  • the mouse can include a replacement of all or part of mature TREM1 coding sequence with human mature TREM1 coding sequence.
  • Genetically modified non-human animals can comprise a modification at an endogenous non-human TREM1 locus.
  • the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature TREM1 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the mature TREM1 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 TREM1 locus in the germline of the animal.
  • Genetically modified animals can express a human TREM1 and/or a chimeric (e.g., humanized) TREM1 from endogenous mouse loci, wherein the endogenous mouse TREM1 gene has been replaced with a human TREM1 gene and/or a nucleotide sequence that encodes a region of human TREM1 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 TREM1 sequence.
  • an endogenous non-human TREM1 locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature TREM1 protein.
  • the genetically modified mice can express the human TREM1 and/or chimeric TREM1 (e.g., humanized TREM1) 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 TREM1 or chimeric TREM1 (e.g., humanized TREM1) 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 TREM1 or the chimeric TREM1 (e.g., humanized TREM1) expressed in animal can maintain one or more functions of the wild-type mouse or human TREM1 in the animal.
  • the expressed TREM1 can bind to human or non-human DAP12.
  • the animal does not express endogenous TREM1.
  • the animal expresses a decreased level of endogenous TREM1 as compared to a wild-type animal.
  • endogenous TREM1 refers to TREM1 protein that is expressed from an endogenous TREM1 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 TREM1 (NP_061113.1) (SEQ ID NO: 2) .
  • 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: 7.
  • the genome of the genetically modified animal can comprise a replacement at an endogenous TREM1 gene locus of a sequence encoding a region of endogenous TREM1 with a sequence encoding a corresponding region of human TREM1.
  • the sequence that is replaced is any sequence within the endogenous TREM1 gene locus, e.g., exon 1, exon 2, exon 3, exon 4, 5’-UTR, 3’-UTR, intron 1, intron 2, intron 3, or any combination thereof.
  • the sequence that is replaced is within the regulatory region of the endogenous TREM1 gene.
  • the sequence that is replaced is a portion of exon 1, exon 2, exon 3, and a portion of exon 4, of an endogenous mouse TREM1 gene locus.
  • the genetically modified animal can have one or more cells expressing a human or chimeric TREM1 (e.g., humanized TREM1) having, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region.
  • a human or chimeric TREM1 e.g., humanized TREM1
  • the signal peptide comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the signal peptide of human TREM1.
  • the signal peptide of the humanized TREM1 has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., contiguously or non-contiguously) that are identical to the signal peptide of human TREM1.
  • the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human TREM1.
  • the extracellular region of the humanized TREM1 has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 181, 182, 183, 184, or 185 amino acids (e.g., contiguously or non-contiguously) that are identical to the extracellular region of human TREM1.
  • the extracellular region described herein includes the signal peptide. In some embodiments, the extracellular region described herein does not include the signal peptide.
  • human TREM1 and non-human TREM1 e.g., mouse TREM1 sequences
  • antibodies that bind to human TREM1 will not necessarily have the same binding affinity with non-human TREM1 or have the same effects to non-human TREM1. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human TREM1 antibodies in an animal model.
  • the transmembrane comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the transmembrane region of endogenous TREM1 (e.g., amino acids 203-223 of SEQ ID NO: 1) .
  • the transmembrane region of the humanized TREM1 has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids (contiguously or non-contiguously) that are identical to the transmembrane region of endogenous TREM1 (e.g., mouse TREM1) .
  • the cytoplasmic comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the cytoplasmic of endogenous TREM1 (e.g., amino acids 224-230 of SEQ ID NO: 1) .
  • the cytoplasmic region of the humanized TREM1 has a sequence that has at least 1, 2, 3, 4, 5, 6, or 7 amino acids (contiguously or non-contiguously) that are identical to the cytoplasmic region of endogenous TREM1 (e.g., mouse TREM1) .
  • the entire transmembrane region and the entire cytoplasmic region of the humanized TREM1 described herein are derived from endogenous sequence.
  • the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to a portion or the entire sequence of exon 1, exon 2, exon 3, and/or exon 4 of human TREM1; a portion or the entire sequence of the extracellular region, and/or a portion or the entire sequence of the transmembrane region of human TREM1; or a portion or the entire sequence of amino acids 1-205 of SEQ ID NO: 2.
  • the genome of the genetically modified animal comprises a portion of exon 1, exons 2-3, and a portion of exon 4 of human TREM1 gene.
  • the portion of exon 1 includes at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, or 76 nucleotides.
  • the portion of exon 1 includes 49 nucleotides.
  • the portion of exon 2 includes a nucleotide of at least 20 bp.
  • the portion of exon 4 includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200, 500, 1000, 1500, 2000, 2500, 2600, 2610, 2620, or 2626 nucleotides. In some embodiments, the portion of exon 4 includes 16 nucleotides. In some embodiments, the portion of exon 4 includes a nucleotide of at least 10 bp.
  • the non-human animal can have, at an endogenous TREM1 gene locus, a nucleotide sequence encoding a chimeric human/non-human TREM1 polypeptide, wherein a human portion of the chimeric human/non-human TREM1 polypeptide comprises the entire human TREM1 extracellular region, the entire human TREM1 transmembrane region, and wherein the animal expresses a functional TREM1 on a surface of a cell of the animal.
  • the human portion of the chimeric human/non-human TREM1 polypeptide can comprise an amino acid sequence encoded by a portion of exon 1, exons 2-3, and/or a portion of exon 4 of human TREM1.
  • the human portion of the chimeric human/non-human TREM1 polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 1-205 of SEQ ID NO: 2.
  • the transmembrane region includes a sequence corresponding to the entire or part of amino acids 203-223 of SEQ ID NO: 1.
  • the cytoplasmic region includes a sequence corresponding to the entire or part of amino acids 224-230 of SEQ ID NO: 1.
  • the chimeric human/non-human TREM1 polypeptide comprises a signal peptide, which includes a sequence corresponding to the entire or part of amino acids 1-20 of SEQ ID NO: 2.
  • the non-human portion of the chimeric human/non-human TREM1 polypeptide comprises the entire transmembrane region and/or the entire cytoplasmic region of an endogenous non-human TREM1 polypeptide.
  • the non-human animal described herein can generate a soluble TREM1.
  • the soluble TREM1 is cleaved from any of the TREM1 protein (e.g., humanized TREM1) described herein.
  • the genetically modified animal can be heterozygous with respect to the replacement at the endogenous TREM1 locus, or homozygous with respect to the replacement at the endogenous TREM1 locus.
  • the humanized TREM1 locus lacks a human TREM1 5’-UTR.
  • the humanized TREM1 locus comprises an endogenous (e.g., mouse) 5’-UTR.
  • the humanization comprises an endogenous (e.g., mouse) 3’-UTR.
  • mouse and human TREM1 genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence.
  • humanized TREM1 mice that comprise a replacement at an endogenous mouse TREM1 locus which retain mouse regulatory elements but comprise a humanization of TREM1 encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized TREM1 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 TREM1 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.
  • the present disclosure also provides 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 TREM1 in the genome of the animal.
  • the non-human mammal comprises the genetic construct as described herein (e.g., gene construct as shown in FIGS. 2, 3, 4, and 5) .
  • a non-human mammal expressing human or humanized TREM1 is provided.
  • the tissue-specific expression of human or humanized TREM1 protein is provided.
  • the expression of human or humanized TREM1 in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance.
  • the specific inducer is selected from Tet-Off System/Tet-On System, or Tamoxifen System.
  • 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 cells 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 TREM1 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 TREM1 protein.
  • the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous TREM1 gene, wherein the disruption of the endogenous TREM1 gene comprises deletion of exon 1, exon 2, exon 3, and/or exon 4, or part thereof of the endogenous TREM1 gene.
  • the disruption of the endogenous TREM1 gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, and exon 4 of the endogenous TREM1 gene.
  • the disruption of the endogenous TREM1 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, and intron 3 of the endogenous TREM1 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, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 5000, 10000, 11000, 12000, 13000, 14000, 15000, or more nucleotides.
  • the disruption of the endogenous TREM1 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, and/or exon 4 (e.g., deletion of at least 10 nucleotides from exon 1, exons 2-3, and at least 5 nucleotides from exon 4) .
  • 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 TREM1 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 TREM1 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_000083.7; 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_000083.7.
  • 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 48535775 to the position 48539822 of the NCBI accession number NC_000083.7; 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 48551590 to the position 48555472 of the NCBI accession number NC_000083.7.
  • 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 48538483 to the position 48539822 of the NCBI accession number NC_000083.7; 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 48551590 to the position 48552918 of the NCBI accession number NC_000083.7.
  • the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 8 kb, about 8.5 kb, about 9 kb, about 9.5 kb, about 10 kb, about 10.5 kb, about 11 kb, about 12 kb, or about 13 kb.
  • the region to be altered is exon 1, exon 2, exon 3, and/or exon 4 of TREM1 gene (e.g., a portion of exon 1, exons 2-3, and a portion of exon 4 of mouse TREM1 gene) .
  • the targeting vector can further include one or more selectable markers, e.g., positive or negative selectable markers.
  • the positive selectable marker is a Neo gene or Neo cassette.
  • the negative selectable marker is a DTA gene.
  • sequence of the 5’ arm is shown in SEQ ID NO: 3; and the sequence of the 3’ arm is shown in SEQ ID NO: 4.
  • sequence of the 5’ arm is shown in SEQ ID NO: 8; and the sequence of the 3’ arm is shown in SEQ ID NO: 9.
  • the sequence is derived from human (e.g., 41276215-41286655 of NC_000006.12; or 28-642 of NM_018643.5) .
  • the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human TREM1, preferably exon 1, exon 2, exon 3, and/or exon 4 of the human TREM1.
  • the nucleotide sequence of the humanized TREM1 encodes the entire or the part of human TREM1 protein with the NCBI accession number NP_061113.1 (SEQ ID NO: 2) .
  • the disclosure also provides vectors for constructing a humanized animal model or a knock-out model.
  • the vectors comprise sgRNA sequence, wherein the sgRNA sequence target TREM1 gene, and the sgRNA is unique on the target sequence of the gene to be altered, and meets the sequence arrangement rule of 5’-NNN (20) -NGG3’ or 5’-CCN-N (20) -3’ ; and in some embodiments, the targeting site of the sgRNA in the mouse TREM1 gene is located on the exon 1, exon 2, exon 3, exon 4, intron 1, intron 2, intron 3, upstream of exon 1, or downstream of exon 4 (e.g., exon 1 and exon 4) of the mouse TREM1 gene.
  • the targeting sequences are shown as SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, and 23.
  • the disclosure provides sgRNA sequences for constructing a genetic modified animal model.
  • the oligonucleotide sgRNA sequences are set forth in SEQ ID NO: 16 or 17.
  • the disclosure relates to a plasmid construct (e.g., pT7-sgRNA) including the sgRNA sequence, and/or a cell including the construct.
  • a plasmid construct e.g., pT7-sgRNA
  • 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. In some embodiments, the cell is an embryonic stem 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 TREM1 gene locus, a sequence encoding a region of an endogenous TREM1 with a sequence encoding a corresponding region of human or chimeric TREM1.
  • 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 and FIG. 5 show humanization strategies for a mouse TREM1 locus.
  • the targeting strategies involve a vector comprising a 5’ homologous arm, a human TREM1 gene fragment, and a 3’ homologous arm.
  • the process can involve replacing endogenous TREM1 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 TREM1 sequence with human TREM1 sequence.
  • the methods for making a genetically modified, humanized animal can include the step of replacing at an endogenous TREM1 locus (or site) , a nucleic acid encoding a region of endogenous TREM1 with a sequence encoding a corresponding region of human TREM1.
  • the sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, and/or exon 4 of a human TREM1 gene.
  • the sequence includes a portion of exon 1, exons 2-3, and a portion of exon 4 of a human TREM1 gene (e.g., nucleic acids 28-642 of NM_018643.5) .
  • the region includes the signal peptide of human TREM1 (e.g., amino acids 1-20 of SEQ ID NO: 2) , and/or the extracellular region of human TREM1 (e.g., amino acids 21-205 of SEQ ID NO: 2) .
  • the endogenous TREM1 locus is exon 1, exon 2, exon 3, and/or exon 4 of mouse TREM1.
  • the sequence includes a portion of exon 1, and a portion of exon 4 of mouse TREM1 gene (e.g., nucleic acids 1-56 and 663-3025 of NM_021406.5) .
  • the methods of modifying a TREM1 locus of a mouse to express a chimeric human/mouse TREM1 peptide can include the steps of replacing at the endogenous mouse TREM1 locus a nucleotide sequence encoding a mouse TREM1 with a nucleotide sequence encoding a human TREM1, thereby generating a sequence encoding a chimeric human/mouse TREM1.
  • the nucleotide sequence encoding the chimeric human/mouse TREM1 can include a first nucleotide sequence encoding the signal peptide and the extracellular region of human TREM1; and a second nucleotide sequence encoding the transmembrane region and the cytoplasmic region of mouse TREM1.
  • 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 TREM1 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 pseudopregnancy (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.
  • methods of making the genetically modified animal comprises modifying the coding frame of the non-human animal’s TREM1 gene, e.g., by inserting a nucleotide sequence (e.g., DNA or cDNA sequence) encoding human or humanized TREM1 protein, e.g., immediately after the endogenous regulatory element of the non-human animal’s TREM1 gene.
  • a nucleotide sequence e.g., DNA or cDNA sequence
  • one or more functional region sequences of the non-human animal’s TREM1 gene can be knocked out, or inserted with a sequence, such that the non-human animal cannot express or expresses a decreased level of endogenous TREM1 protein.
  • the coding frame of the modified non-human animal’s TREM1 gene can be all or part of the nucleotide sequence from exon 1 to exon 4 of the non-human animal’s TREM1 gene.
  • methods of making the genetically modified animal comprises inserting a nucleotide sequence encoding human or humanized TREM1 protein and/or an auxiliary sequence after the endogenous regulatory element of the non-human animal’s TREM1 gene.
  • the auxiliary sequence can be a stop codon, such that the TREM1 gene humanized animal model can express human or humanized TREM1 protein in vivo, but does not express non-human animal’s TREM1 protein.
  • the auxiliary sequence includes WPRE (WHP Posttranscriptional Response Element) , loxP, and/or polyA.
  • the method for making the genetically modified animal comprises:
  • plasmid comprising a human TREM1 gene fragment, flanked by a 5’ homologous arm and a 3’ homologous arm, wherein the 5’ and 3’ homologous arms target an endogenous TREM1 gene;
  • sgRNAs small guide RNAs
  • step (3) modifying genome of a fertilized egg or an embryonic stem cell by using the plasmid of step (1) , the sgRNAs of step (2) , and Cas9;
  • step (2) mating the child mouse obtained in step (2) to obtain a homozygote mouse
  • the fertilized egg is modified by CRISPR with sgRNAs that target a 5’-terminal targeting site and a 3’-terminal targeting site.
  • sequence encoding the humanized TREM1 protein is operably linked to an endogenous regulatory element at the endogenous TREM1 gene locus.
  • the genetically-modified animal does not express an endogenous TREM1 protein.
  • the method for making the genetically modified animal comprises:
  • plasmid comprising a human or chimeric TREM1 gene fragment, flanked by a 5’ homologous arm and a 3’ homologous arm, wherein the 5’ and 3’ homologous arms target an endogenous TREM1 gene;
  • sgRNAs small guide RNAs
  • 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 TREM1 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 are provided that express human or humanized TREM1, which are useful for testing agents that can decrease or block the interaction between the interaction between TREM1 and anti-human TREM1 antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an TREM1 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 (a knock-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 (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor) .
  • the anti-TREM1 antibody activates the TREM1-related signaling pathway.
  • the anti-TREM1 antibody described herein can facilitate the interaction between TREM1 and DAP12, to induce signaling through the TREM1-DAP12 complex.
  • the genetically modified animals can be used for determining effectiveness of a therapeutic agent (e.g., an anti-TREM1 antibody or a TREM1-targeting drug) for the treatment of cancer.
  • a therapeutic agent e.g., an anti-TREM1 antibody or a TREM1-targeting drug
  • the methods involve administering the therapeutic agent (e.g., an anti-human TREM1 antibody or a TREM1-targeting drug) to the animal as described herein, wherein the animal has a cancer or tumor; and determining inhibitory effects of the therapeutic agent to the cancer or tumor.
  • 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. In addition, a delicate balance is required for these antibodies, as TREM1 is also expressed on many other cells.
  • the humanized TREM1 functions in a largely similar way as compared to the endogenous TREM1, so that the results in the humanized animals can be used to predict the efficacy or toxicity of these therapeutic agents in the human.
  • the anti-TREM1 antibody can directly target cancer cells or tumor-associated cells expressing TREM1, e.g., by inducing complement mediated cytotoxicity (CMC) or antibody dependent cellular cytotoxicity (ADCC) to kill the cancer cells.
  • CMC complement mediated cytotoxicity
  • ADCC antibody dependent cellular cytotoxicity
  • the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) that are injected into the animal.
  • the anti-TREM1 antibody inhibits TREM1 signaling pathways. In some embodiments, the anti-TREM1 antibody does not inhibit TREM1 signaling pathways. In some embodiments, the anti-TREM1 antibody activates TREM1 signaling pathways.
  • the genetically modified animals can be used for determining whether an anti-TREM1 antibody is a TREM1 agonist or antagonist.
  • the methods as described herein are also designed to determine the effects of the agent (e.g., anti- TREM1 antibodies) on TREM1, e.g., whether the agent can activate TREM1 signaling to repolarize suppressive tumor-associated myeloid populations (e.g., mMDSCs, TANs, and TAMs) , whether the agent can induce a pro-inflammatory phenotype in the tumor microenvironment, whether the agent can upregulate the immune response or downregulate immune response, and/or whether the agent can induce complement mediated cytotoxicity (CMC) or antibody dependent cellular cytotoxicity (ADCC) .
  • 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.
  • 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 therapeutic agent e.g., an anti-TREM1 antibody
  • 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 cancer described herein is lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myeloproliferation abnormal syndromes, and sarcomas.
  • the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia.
  • the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom macroglobulinemia.
  • the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma , and chondrosarcoma.
  • the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, or liver cancer.
  • the cancer described herein is glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, stomach cancer, liver cancer, pancreatic cancer, urothelial cancer, testis cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, carcinoid, renal cancer, prostate cancer, skin cancer, lymphoma, or melanoma.
  • the cancer described herein is a solid tumor, gynecologic cancer, breast cancer, colorectal cancer, gastric adenocarcinoma, lung adenocarcinoma, pancreatic cancer, or head and neck cancer.
  • the cancer described herein is ovarian cancer, pancreatic ductal adenocarcinoma, non-small cell lung cancer, triple-negative breast cancer, hormone receptor-positive, HER2-negative breast cancer, head and neck squamous cell carcinomas, or colorectal cancer.
  • the therapeutic agent described herein is PY159 or its analog thereof.
  • the anti-TREM1 antibody is designed for treating various autoimmune diseases, including rheumatoid arthritis, Crohn’s disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel diseases (IBD) , ulcerative colitis, or scleroderma.
  • the anti-TREM1 antibody is designed for treating various immune disorders, including allergy, asthma, and/or atopic dermatitis.
  • the methods as described herein can be used to determine the effectiveness of an anti-TREM1 antibody in inhibiting immune response.
  • the immune disorders described herein is allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, etc.
  • the present disclosure also provides methods of determining toxicity of an antibody (e.g., anti-TREM1 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 TREM1 gene function, human TREM1 antibodies, drugs for human TREM1 targeting sites, the drugs or efficacies for human TREM1 targeting sites, the drugs for immune-related diseases and antitumor drugs.
  • the disclosure provides a method to verify in vivo efficacy of TCR-T, CAR-T, and/or other immunotherapies (e.g., T-cell adoptive transfer therapies) .
  • the methods include transplanting human tumor cells into the animal described herein, and applying human CAR-T to the animal with human tumor cells. Effectiveness of the CAR-T therapy can be determined and evaluated.
  • the animal is selected from the TREM1 gene humanized non-human animal prepared by the methods described herein, the TREM1 gene humanized non-human animal described herein, the double-or multi-humanized non-human animal generated by the methods described herein (or progeny thereof) , a non-human animal expressing the human or humanized TREM1 protein, or the tumor-bearing or inflammatory animal models described herein.
  • the TCR-T, CAR-T, and/or other immunotherapies can treat the TREM1-associated diseases described herein (e.g., breast cancer) .
  • the TCA-T, CAR-T, and/or other immunotherapies provides an evaluation method for treating the TREM1-associated diseases described herein (e.g., solid tumors) .
  • 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 TREM1 gene and a sequence encoding an additional human or chimeric protein.
  • the additional human or chimeric protein can be interleukin 1 alpha (IL1A) , interleukin 1 beta (IL1B) , interleukin 6 (IL6) , interleukin 15 (IL15) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , T cell immunoreceptor with Ig and ITIM domains (TIGIT) , lymphocyte-activation gene 3 (LAG3) , CD226, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor alpha (TNF- ⁇ ) , tumor necrosis factor receptor superfamily, member 4 (OX40) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , CD73, B And T Lymphocyte Associated (BTLA) , CD27, CD28, CD47, CD137, CD154, Glucocorticoid-Induced TNFR-Related Protein (GITR)
  • 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 IL1A, IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4, TNF- ⁇ , OX40, TIM3, CD73, BTLA, CD27, CD28, CD47, CD137, CD154, GITR, and/or SIRP ⁇ .
  • a genetically modified non-human animal with human or chimeric IL1A, IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4, TNF- ⁇ , OX40, TIM3, CD73, BTLA, CD27, CD28, CD47, CD137, CD154, GITR, and/or SIRP ⁇ .
  • the TREM1 humanization is directly performed on a genetically modified animal having a human or chimeric IL1A, IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4, TNF- ⁇ , OX40, TIM3, CD73, BTLA, CD27, CD28, CD47, CD137, CD154, GITR, and/or SIRP ⁇ 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-TREM1 antibody and an additional therapeutic agent for the treatment of cancer.
  • the methods include administering the anti-TREM1 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 IL1A, IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4, TNF- ⁇ , OX40, TIM3, CD73, BTLA, CD27, CD28, CD47, CD137, CD154, GITR, and/or SIRP ⁇ .
  • 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 cancers as described herein, e.g., a solid tumor, gynecologic cancer, breast cancer, colorectal cancer, gastric adenocarcinoma, lung adenocarcinoma, pancreatic cancer, or head and neck cancer.
  • a solid tumor e.g., a solid tumor, gynecologic cancer, breast cancer, colorectal cancer, gastric adenocarcinoma, lung adenocarcinoma, pancreatic cancer, or head and neck cancer.
  • 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
  • NdeI and BsrGI restriction enzymes were purchased from NEB (Catalog numbers: R0111S and R0575S, respectively) .
  • C57BL/6 mice and Flp transgenic mice were purchased from the China Food and Drugs Research Institute National Rodent Experimental Animal Center.
  • V450 Rat Anti-mouse CD11b was purchased from BD Horizon (Catalog number: 560455) .
  • PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody was purchased from BioLegend (Catalog number: 108426) .
  • FITC anti-mouse F4/80 was purchased from BioLegend (Catalog number: 123108) .
  • PerCP/Cyanine5.5 anti-mouse TCR ⁇ chain Antibody was purchased from BioLegend (Catalog number: 109228) .
  • FITC anti-mouse CD19 Antibody was purchased from BioLegend (Catalog number: 115506) .
  • Mouse TREM-1 PE-conjugated Antibody was purchased from R&D Systems (Catalog number: FAB1187P) .
  • APC anti-human CD354 (TREM-1) Antibody was purchased from BioLegend (Catalog number: 314909) .
  • APC Mouse IgG1, ⁇ Isotype Ctrl (FC) Antibody was purchased from BioLegend (Catalog number: 400122) .
  • Purified anti-mouse CD16/32 was purchased from BioLegend (Catalog number: 101302) .
  • TREM1-PI64062-hIgG1-SI was purchased from Biocytogen (Catalog number: 20210910) .
  • Mouse/Rat TREM-1 Quantikine ELISA Kit was purchased from R&D Systems (Catalog number: MTRM10) .
  • Human TREM -1 Quantikine ELISA Kit was purchased from R&D Systems (Catalog number: DTRM10C) .
  • EXAMPLE 1 Mice with humanized TREM1 gene
  • a non-human animal e.g., a mouse
  • a non-human animal was modified to include a nucleotide sequence encoding human TREM1 protein, and the obtained genetically-modified non-human animal can express a human or humanized TREM1 protein in vivo.
  • the mouse TREM1 gene (NCBI Gene ID: 58217, Primary source: MGI: 1930005, UniProt ID: Q9JKE2) is located at 48539763 to 48553955 of chromosome 17 (NC_000083.7)
  • the human TREM1 gene (NCBI Gene ID: 54210, Primary source: HGNC: 17760, UniProt ID: Q9NP99-1) is located at 41267385 to 41286692 of chromosome 6 (NC_000006.12) .
  • the mouse TREM1 transcript is NM_021406.5, and the corresponding protein sequence NP_067381.1 is set forth in SEQ ID NO: 1.
  • the human TREM1 transcript is NM_018643.5, and the corresponding protein sequence NP_061113.1 is set forth in SEQ ID NO: 2.
  • Mouse and human TREM1 gene loci are shown in FIG. 1.
  • nucleotide sequences encoding human TREM1 protein can be introduced into the mouse endogenous TREM1 locus, so that the mouse expresses human or humanized TREM1 protein.
  • a sequence (about 11.8 kb) starting from within exon 1 and ending within exon 4 of mouse TREM1 gene was replaced with a corresponding sequence (about 10.4 kb) starting from within exon 1 and ending within exon 4 of human TREM1 gene, to obtain a humanized TREM1 gene locus as shown in FIG. 2, thereby humanizing mouse TREM1 gene.
  • the targeting vector contains homologous arm sequences upstream and downstream of the mouse TREM1 gene, and an “A Fragment” containing DNA sequences of human TREM1 gene (SEQ ID NO: 5) .
  • sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 3) is identical to nucleotide sequence of 48535775-48539822 of NCBI accession number NC_000083.7
  • sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 4) is identical to nucleotide sequence of 48551590-48555472 of NCBI accession number NC_000083.7.
  • the genomic DNA sequence from human TREM1 gene (SEQ ID NO: 5) is identical to nucleotide sequence of 41276215-41286655 of NCBI accession number NC_000006.12.
  • the connection between the 5’ end of the A fragment and the mouse sequence was designed as: 5’-GTCGTTGGAGCTGAGCTTG GGAAGACCAGGCTCTGGG-3’ (SEQ ID NO: 10) , wherein the last “G” in sequence is the last nucleotide of the mouse sequence, the first “A” in sequence is the first nucleotide of the human sequence.
  • connection between the 3’ end of the A fragment and the mouse sequence was designed as: 5’-CGCAGGGTTCCGGTGTT GTTACTATTTCAGTCATCTGTGGAC -3’ (SEQ ID NO: 11) , wherein the last “C” in sequence is the last nucleotide of the human sequence, and the “G” in sequence “ GTTA ” is the first nucleotide of the mouse sequence.
  • the targeting vector also includes an antibiotic resistance gene for positive clone screening (neomycin phosphotransferase gene, or Neo) , and two Frt recombination sites flanking the antibiotic resistance gene, that formed a Neo cassette (within the A Fragment) .
  • Neo cassette within the A Fragment
  • the connection between the 5’ end of the Neo cassette and the human sequence was designed as: 5’-GACCATCCCAGTA GTCG ACGGTATCGATAAGCTTGATATCGAATTCCGAAGTT CCTATT-3’ (SEQ ID NO: 12) , wherein the “A” in sequence is the last nucleotide of the human sequence, and the first “G” in sequence “ GTCG ” is the first nucleotide of the Neo cassette.
  • the connection between the 3’ end of the Neo cassette and the human sequence was designed as: 5’-AAGTATAGGAACTTCATCAGTCAGGTACATAATGGTGG GGGTACGGTACTGA GAAT -3’ (SEQ ID NO: 13) , wherein the last “C” in sequence is the last nucleotide of the Neo cassette, and the first “G” in sequence “ GGGT ” is the first nucleotide of the human sequence.
  • a coding gene with a negative selectable marker (a gene encoding diphtheria toxin A subunit (DTA) ) was also constructed downstream of the 3' homologous arm of the targeting vector.
  • DTA diphtheria toxin A subunit
  • the targeting vector was constructed, e.g., by restriction enzyme digestion and ligation.
  • the constructed targeting vector sequences were preliminarily confirmed by restriction enzyme digestion, and then verified by sequencing.
  • Embryonic stem cells of C57BL/6 mice were transfected with the correct targeting vector by electroporation.
  • the positive selectable marker genes were used to screen the cells, and the integration of exogenous genes was confirmed by PCR and Southern Blot.
  • the clones identified as positive by PCR (primers shown in the table below) were then verified by Southern Blot.
  • the clones that were positive in Southern Blot detection were further sequenced. Clones verified to have no random insertion were subjected for subsequent experiments.
  • the positive clones that had been screened were introduced into isolated blastocysts (white mice) , and the resulted chimeric blastocysts were transferred to a culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white) .
  • the F2 generation homozygous mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then breeding the F1 generation heterozygous mice with each other.
  • the positive mice were also bred with the Flp transgenic mice to remove the positive selectable marker genes (schematic diagram shown in FIG. 4) , and then the humanized homozygous mice with a humanized TREM1 gene were obtained by breeding the heterozygous mice with each other.
  • the CRISPR/Cas system can also be used for gene editing, and the targeting strategy shown in FIG. 5 was designed.
  • the targeting vector contains homologous arm sequences upstream and downstream of the mouse TREM1 gene, and a human TREM1 gene fragment.
  • sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 8) is identical to nucleotide sequence of 48538483-48539822 of NCBI accession number NC_000083.7
  • sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 9) is identical to nucleotide sequence of 48551590-48552918 of NCBI accession number NC_000083.7.
  • the human TREM1 gene fragment (SEQ ID NO: 5) is identical to identical to nucleotide sequence of 41276215-41286655 of NCBI accession number NC_000006.12.
  • the targeting vector was constructed, e.g., by restriction enzyme digestion, ligation, or direct synthesis.
  • the constructed targeting vector sequences were preliminarily confirmed by restriction enzyme digestion, and then verified by sequencing. Targeting vectors with verified sequences were used for subsequent experiments.
  • the targeting site sequences of the sgRNAs on the TREM1 gene locus are as follows:
  • sgRNA1 targeting site (SEQ ID NO: 16) : 5’-CAGTATGTAGCCTGTATGGCTGG-3’;
  • sgRNA2 targeting site (SEQ ID NO: 17) : 5’-GGGAAGAATTTTCTACTCCATGG-3’.
  • UCA kit was used to detect the activity of the sgRNAs. After confirming that the sgRNAs can induce efficient Cas9 cleavage, restriction enzyme cleavage sites were added to its 5' end and a complementary strand to obtain a forward oligonucleotide and a reverse oligonucleotide, as shown in the table below. After annealing, the products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) , to obtain expression vector pT7-TREM1-1 and pT7-TREM1-2.
  • the pT7-sgRNA vector was synthesized, which included a DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 24) , and was ligated to the backbone vector (Takara, Catalog number: 3299) after restriction enzyme digestion (EcoRI and BamHI) . The resulting plasmid was confirmed by sequencing.
  • the pre-mixed Cas9 mRNA, the targeting vector, and in vitro transcription products of the pT7-TREM1-1 and pT7-TREM1-2 plasmids were injected into the cytoplasm or nucleus of fertilized eggs of C57BL/6 mice with a microinjection instrument.
  • 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, 2006.
  • the injected fertilized eggs were then transferred to a culture medium to culture for a short time and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified mice (F0 generation) .
  • the mouse population was further expanded by cross-breeding and self-breeding to establish stable homozygous mouse lines.
  • the genotype of the somatic cells of the F0 generation mice can be identified by PCR analysis. The PCR primers are shown in the table below.
  • mice identified as positive by PCR were then subjected to Southern Blot detection. Those mice identified as positive by Southern Blot were further sequenced to confirm there was no random insertion.
  • TREM1 gene humanized mice Many methods can be used to verify the success of generating TREM1 gene humanized mice. For example, the genotype of the somatic cells of the F1 generation mice can be identified by PCR analysis. The PCR primers are shown in the table below.
  • the identification results of some F1 generation mice are shown in FIG. 6. The results indicate that mice numbered F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, F1-07, F1-08, F1-09, F1-10, F1-11, F1-12, F1-13, and F1-14 were verified as positive heterozygous mice.
  • the F1 generation mice identified as positive by PCR were further verified by Southern Blot to confirm whether there was random insertion. Specifically, genomic DNA from the mouse tail was extracted, which was digested with BsrGI or NdeI restriction enzyme. The digested genomic DNA was then transferred to a membrane and hybridized with respective probes. The restriction enzymes, probes, and the size of target fragment sizes are shown in the table below.
  • the Southern Blot detection results are shown in FIG. 7. The results indicate that mice numbered F1-05, F1-06, F1-07, F1-08, F1-09, F1-10, F1-11, F1-12, F1-13, and F1-14 were verified as positive clones without random insertions.
  • a Probe (5’) -F (SEQ ID NO: 29) : 5’-TTCCGAGGTTTCCCTAGAAACTGGA-3’
  • a Probe (5’) -R (SEQ ID NO: 30) : 5’-TGGAGTCCGAACTGGGTTAACC-3’
  • 3’Probe-F (SEQ ID NO: 31) : 5’-GTCAGTTTCTATGTGGGGAAGC-3’
  • 3’Probe-R SEQ ID NO: 32) : 5’-GTTGATTGTTCCTTTAAGGGAG-3’.
  • human or humanized TREM1 protein in positive mice can be confirmed, e.g., by flow cytometry. Specifically, one 6-week-old male C57BL/6 wild-type mouse and one 6-week-old male TREM1 gene humanized heterozygous mouse were selected, and peripheral blood was collected after euthanasia by cervical dislocation.
  • mice identified as positive in the F1 generation were bred with each other to obtain TREM1 gene humanized homozygous mice.
  • Three 6-week-old C57BL/6 wild-type mice and three 6-week-old TREM1 gene humanized homozygous mice (H/H) were selected.
  • the mice were subcutaneously inoculated with mouse colon cancer cells MC38. When the tumor volume reached about 500 mm 3 , the tumor tissues and peripheral blood were collected after euthanasia by cervical dislocation.
  • Expression of human or humanized TREM1 protein in macrophages, monocyte-type MDSCs (M-MDSCs) , neutrophil-type MDSCs (G-MDSC) , T cells, and B cells were detected by flow cytometry as described above. The results are shown in the table below.
  • mice TREM1 protein As shown in the table above, only mouse TREM1 protein, but not human or humanized TREM1 protein, was detected in macrophages, M-MDSCs, and G-MDSCs in tumor tissues and peripheral blood of wild-type C57BL/6 mice.
  • Humanized TREM1 protein can only be detected in macrophages, M-MDSCs, and G-MDSCs in tumor tissues and peripheral blood of TREM1 gene humanized homozygous mice.
  • neither mouse TREM1 protein nor human or humanized TREM1 protein was detected in T cells and B cells in tumor tissues and peripheral blood of wild-type C57BL/6 mice and TREM1 gene humanized homozygous mice.
  • the spleen, peripheral blood, and lymph nodes from C57BL/6 wild-type mice and TREM1 gene humanized homozygous mice were collected for immuno-phenotyping detection by flow cytometry.
  • the detection results of leukocyte subtypes and T cell subtypes in the spleen are shown in FIG. 8 and FIG. 9, respectively.
  • the detection results of leukocyte subtypes and T cell subtypes in peripheral blood are shown in FIG. 10 and FIG. 11, respectively.
  • mice The results showed that the percentages of B cells, T cells, NK cells, CD4+ T cells, CD8+ T cells, granulocytes, dendritic cells (DC cells) , macrophages, monocytes, and other leukocyte subtypes in the spleen and peripheral blood of TREM1 gene humanized homozygous mice were basically the same as those in C57BL/6 wild-type mice (FIG. 8 and FIG. 10) .
  • the percentages of CD4+ T cells, CD8+ T cells, and Treg cells (Tregs) were basically the same as those in C57BL/6 wild-type mice (FIG. 9 and FIG. 11) .
  • the detection results of leukocyte subtypes and T cell subtypes in lymph nodes are shown in FIG. 12 and FIG. 13, respectively.
  • the results showed that the leukocyte subtypes, e.g., T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells, and other leukocyte subtypes in the lymph nodes of TREM1 gene humanized homozygous mice were basically the same as those of C57BL/6 wild-type mice (FIG. 12) .
  • the percentages of T cell subtypes, e.g., CD4+ T cells, CD8+ T cells and Tregs cells were basically the same as those of C57BL/6 wild-type mice (FIG. 13) .
  • TREM1 gene did not affect the differentiation, development and distribution of leukocytes and T cells in mice.
  • spleen tyrosine kinase phosphorylation p-syk+
  • ELISA was also used to detect the expression of soluble TREM1 (sTREM1) in serum to verify whether the signaling pathways can function normally in TREM1 gene humanized mice.
  • sTREM1 soluble TREM1
  • three 6-week-old female wild-type C57BL/6 mice (+/+) and three 9-week-old female TREM1 gene humanized homozygous mice (H/H) were selected. The mice were randomly placed in a control group and an LPS (lipopolysaccharides) group.
  • the LPS group mice were injected intraperitoneally with LPS (500 ⁇ g/mouse) , and the control group mice were injected with an equal volume of PBS or saline. Peritoneal lavage fluid and serum were collected after 12 hours.
  • the spleen tyrosine kinase phosphorylation in peritoneal lavage macrophages was determined by staining cells with: Brilliant Violet 510 TM anti-mouse CD45 (mCD45; an anti-mouse CD45 antibody) , V450 Rat Anti-mouse CD11b (mCD11b; an anti-mouse CD11b antibody) , PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody (mGr-1; an anti-mouse Ly-6G/Ly-6C (Gr-1) antibody) , FITC anti-mouse F4/80 (mF4/80; an anti-mouse F4/80 antibody) , Mouse TREM-1 PE-
  • sTREM1 Expression of sTREM1 in serum was detected using Mouse/Rat TREM-1 Quantikine ELISA Kit and Human TREM-1 Quantikine ELISA Kit. As shown in FIGS. 14A-14B, only mouse sTREM1, but not human sTREM1, was detected in wild-type C57BL/6 mice; whereas only human sTREM1, but not mouse sTREM1, was detected in TREM1 gene humanized homozygous mice. Low expression levels of sTREM1 were detected in the serum of wild-type C57BL/6 mice and TREM1 gene humanized homozygous mice in the absence of LPS stimulation. However, the expression levels of sTREM1 in the serum significantly increased after LPS stimulation for 12 hours. The upregulation of sTREM1 in the serum of TREM1 gene humanized homozygous mice indicates that TREM1 can be activated.
  • TREM1 receptor can be activated; MMP (matrix metalloproteinase) can induce the release of sTREM1 into the supernatant; and the activation of TREM1 can induce rapid phosphorylation of syk.
  • MMP matrix metalloproteinase
  • the TREM1 gene humanized mice prepared by the methods described herein can be used to evaluate the efficacy of drugs targeting human TREM1.
  • the TREM1 gene humanized homozygous mice can be subcutaneously inoculated with mouse colon cancer cells MC38.
  • the mice can be placed into a control group and one or more treatment groups according to the tumor volume.
  • the treatment group mice can be administered with randomly selected drugs targeting human TREM1 (e.g., an anti-human TREM1 antibody) , and the control group mice can be injected with an equal volume of saline.
  • the tumor volume and body weight of the mice can be measured regularly, and the in vivo safety and efficacy of the drugs can be effectively assessed by comparing the changes in the body weight of the mice and the tumor size.
  • the TREM1 gene humanized mice generated using the methods described herein can also be used to generate double-or multi-gene humanized mouse models.
  • the embryonic stem (ES) cells for blastocyst microinjection can be selected from mice comprising other genetic modifications such as modified (e.g., human or humanized) IL1A, IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4, and/or TNF- ⁇ genes.
  • embryonic stem cells from humanized TREM1 mice described herein can be isolated, and gene recombination targeting technology can be used to obtain double-gene or multi-gene-modified mouse models of TREM1 and other gene modifications.
  • Mendel it is possible to generate double-gene or multi-gene modified heterozygous mice comprising modified (e.g., human or humanized) TREM1 gene and other genetic modifications. Then the heterozygous mice can be bred with each other to obtain homozygous double-gene or multi-gene modified mice.
  • These double-gene or multi-gene modified mice can be used for in vivo validation of gene regulators targeting human TREM1 and other genes.

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Abstract

La présente invention concerne des animaux non humains génétiquement modifiés exprimant un TREM1 humain ou chimérique (par exemple, humanisé), ainsi que des procédés d'utilisation associés.
PCT/CN2022/125929 2021-10-18 2022-10-18 Animal non humain génétiquement modifié portant un trem1 humain ou chimérique WO2023066250A1 (fr)

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