WO2023020612A1 - Animal non humain génétiquement modifié comportant un her2 humain ou chimérique - Google Patents
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Definitions
- This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) HER2, 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 HER2 or chimeric HER2.
- the animal model can express human HER2 or chimeric HER2 (e.g., humanized HER2) protein in its body. It can be used in the studies on the function of HER2 gene, and can be used in the screening and evaluation of anti-human HER2 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 HER2 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 HER2 protein and a platform for screening cancer (e.g., breast 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 HER2 (receptor tyrosine-protein kinase erbB-2) .
- the sequence encoding the human or chimeric HER2 is operably linked to an endogenous regulatory element at the endogenous HER2 gene locus in the at least one chromosome.
- the sequence encoding a human or chimeric HER2 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human HER2 (NP_004439.2 (SEQ ID NO: 2) ) .
- the sequence encoding a human or chimeric HER2 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: 9.
- the sequence encoding a human or chimeric HER2 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 67-678 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 HER2 or expresses a decreased level of endogenous HER2.
- the animal has one or more cells expressing human or chimeric HER2.
- the animal has one or more cells expressing human or chimeric HER2, and the expressed human or chimeric HER2 can interact with a human EGFR family member protein (e.g., HER1, HER3, or HER4) , forming a heterodimer.
- the animal has one or more cells expressing human or chimeric HER2, and the expressed human or chimeric HER2 can interact with an endogenous EGFR family member protein (e.g., HER1, HER3, or HER4) , forming a heterodimer.
- 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 HER2 with a sequence encoding a corresponding region of human HER2 at an endogenous HER2 gene locus.
- the sequence encoding the corresponding region of human HER2 is operably linked to an endogenous regulatory element at the endogenous HER2 locus, and one or more cells of the animal expresses a human or chimeric HER2.
- the animal does not express endogenous HER2 or expresses a decreased level of endogenous HER2.
- the replaced locus comprises all or part of the extracellular region, all or part of the transmembrane region, and/or all or part of the cytoplasmic region of HER2.
- the animal has one or more cells expressing a chimeric HER2 having an extracellular region, a transmembrane region, and a cytoplasmic region.
- the extracellular region of the chimeric HER2 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, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 600, 610, 620, or 630 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human HER2 (e.g., amino acids 67-652 of SEQ ID
- the transmembrane region of the chimeric HER2 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, 20, 21, 22, or 23 contiguous amino acids that are identical to a contiguous sequence present in the transmembrane region of human HER2 (e.g., amino acids 653-675 of SEQ ID NO: 2) .
- the cytoplasmic region of the chimeric HRE2 has a sequence that is at least 1, 2, 3, 4, 5, 6, 7, or 8 contiguous amino acids that are identical to a contiguous sequence present in the cytoplasmic region of human HER2 (e.g., amino acids 676-678 of SEQ ID NO: 2) .
- the sequence encoding a region of endogenous HER2 comprises exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and/or exon 17, or a part thereof, of the endogenous HER2 gene.
- the animal is a mouse.
- the animal is heterozygous with respect to the replacement at the endogenous HER2 gene locus.
- the animal is homozygous with respect to the replacement at the endogenous HER2 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 HER2 gene locus, a sequence encoding a region of endogenous HER2 with a sequence encoding a corresponding region of human HER2.
- the sequence encoding the corresponding region of human HER2 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, and/or exon 27, or a part thereof, of a human HER2 gene.
- the sequence encoding the corresponding region of human HER2 comprises a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and a portion of exon 17, of a human HER2 gene.
- the sequence encoding the corresponding region of human HER2 encodes amino acids 67-678 of SEQ ID NO: 2.
- the region comprises a portion of the extracellular region, the transmembrane region, and/or a portion of the cytoplasmic region of HER2.
- the sequence encoding a region of endogenous HER2 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, and/or exon 27, or a part thereof, of the endogenous HER2 gene.
- the animal is a mouse
- the sequence encoding a region of endogenous HER2 comprises a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and a portion of exon 17 of the endogenous HER2 gene.
- the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric HER2 polypeptide
- the chimeric HER2 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human HER2, in some embodiments, the animal expresses the chimeric HER2 polypeptide.
- the chimeric HER2 polypeptide has at least 50, at least 80, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 560, at least 570, at least 580, or at least 586 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human HER2 extracellular region (e.g., amino acids 67-652 of SEQ ID NO: 2) .
- the chimeric HER2 polypeptide comprises the entire human HER2 transmembrane region (e.g., amino acids 653-675 of SEQ ID NO: 2) .
- the chimeric HER2 polypeptide has at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human HER2 cytoplasmic region (e.g., amino acids 676-678 of SEQ ID NO: 2) .
- the chimeric HER2 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 67-678 of SEQ ID NO: 2.
- the nucleotide sequence is operably linked to an endogenous HER2 regulatory element of the animal.
- the chimeric HER2 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 23-66 and 680-1256 of SEQ ID NO: 1.
- the chimeric HER2 polypeptide comprises an endogenous HER2 signal peptide (e.g., amino acids 1-22 of SEQ ID NO: 1) .
- the nucleotide sequence is integrated to an endogenous HER2 gene locus of the animal.
- the chimeric HER2 polypeptide has at least one mouse HER2 activity and/or at least one human HER2 activity.
- the disclosure is related to a method of making a genetically-modified animal cell that expresses a chimeric HER2, the method comprising: replacing at an endogenous HER2 gene locus, a nucleotide sequence encoding a region of endogenous HER2 with a nucleotide sequence encoding a corresponding region of human HER2, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the chimeric HER2, in some embodiments, the animal cell expresses the chimeric HER2. In some embodiments, the animal is a mouse.
- the chimeric HER2 comprises a humanized HER2 extracellular region comprising at least 500, at least 550, at least 560, at least 570, at least 580, at least 581, at least 582, at least 583, at least 584, at least 585, or at least 586 contiguous amino acids of a human HER2 extracellular region; and a human HER2 transmembrane region.
- the chimeric HER2 further comprises an endogenous HER2 cytoplasmic region, optionally at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 contiguous amino acids of the endogenous HER2 cytoplasmic region are replaced with corresponding sequences of a human HER2 cytoplasmic region.
- the nucleotide sequence encoding the chimeric HER2 is operably linked to an endogenous HER2 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 programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , or CD73.
- PD-1 programmed cell death protein 1
- PD-L1 programmed cell death ligand 1
- CTLA4 cytotoxic T-lymphocyte-associated protein 4
- OX40 tumor necrosis factor receptor superfamily
- LAG3 lymphocyte-activation gene 3
- TIM3 T-cell immunoglobulin and mucin-domain containing-3
- 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-HER2 antibody.
- the tumor comprises one or more cells that express HER2.
- the tumor comprises one or more cancer cells that are injected into the animal.
- determining inhibitory effects of the anti-HER2 antibody to the tumor involves measuring the tumor volume in the animal.
- the cancer is breast cancer, gastric cancer, ovarian cancer, uterine serous endometrial carcinoma, colon cancer, bladder cancer, lung cancer, cervical cancer, head and neck cancer, endometrial cancer, or esophageal cancer.
- the disclosure is related to a method of determining effectiveness of an anti-HER2 antibody and an additional therapeutic agent for the treatment of cancer, comprising a) administering the anti-HER2 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 programmed cell death protein 1 (PD-1) .
- the animal further comprises a sequence encoding a human or chimeric programmed death-ligand 1 (PD-L1) .
- the additional therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.
- the tumor comprises one or more tumor cells that express HER2 or PD-L1.
- the tumor is caused by injection of one or more cancer cells into the animal.
- determining inhibitory effects of the treatment involves measuring the tumor volume in the animal.
- the animal has breast cancer, gastric cancer, ovarian cancer, uterine serous endometrial carcinoma, colon cancer, bladder cancer, lung cancer, cervical cancer, head and neck cancer, endometrial cancer, or esophageal 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-HER2 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
- amino acid sequence is one of the following:
- 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 9.
- the disclosure is related to a nucleic acid comprising a nucleotide sequence
- the nucleotide sequence is one of the following:
- the disclosure is related to a cell comprising the protein and/or the nucleic acid as described herein.
- the disclosure is related to an animal comprising the protein and/or the nucleic acid as described herein.
- the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous HER2 gene, wherein the disruption of the endogenous HER2 gene comprises deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, and/or exon 27, or part thereof of the endogenous HER2 gene.
- the disruption of the endogenous HER2 gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and exon 17 of the endogenous HER2 gene.
- the disruption of the endogenous HER2 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, and intron 16 of the endogenous HER2 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 or more nucleotides.
- the disruption of the endogenous HER2 gene comprises the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, and/or exon 27 (e.g., deletion of at least 10 nucleotides from exon 2, exons 3-16, and at least 50 nucleotides from exon 17) .
- the disclosure further relates to a HER2 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 HER2 gene function, human HER2 antibodies, the drugs or efficacies for human HER2 targeting sites, and the drugs for immune-related diseases and antitumor drugs.
- FIG. 1 is a schematic diagram showing mouse and human HER2 gene loci.
- FIG. 2 is a schematic diagram showing humanized HER2 gene locus.
- FIG. 3 is a schematic diagram showing a HER2 gene targeting strategy.
- FIG. 4 shows Southem Blot results of cells after recombination using the 5’ Probe, Neo Probe, and 3’ Probe.
- WT is a wild-type control.
- FIG. 5 is a schematic diagram showing the Frt recombination process in HER2 gene humanized mice.
- FIGS. 6A-6D show mouse tail PCR identification results off 1 generation mice by primer pairs WT-F/WT-R, Frt-F/Frt-R, WT-F/Mut-R, and Flp-F2/Flp-R2, respectively.
- M is a marker.
- PC is a positive control.
- WT is a wild-type control.
- H 2 O is a water control.
- FIG. 7 shows RT-PCR detection results of mouse HER2 (mHER2) mRNA, humanized HER2 (hHER2) mRNA, and GAPDH mRNA, respectively, in colon cells of wild-type C57BL/6 mice (+/+) and HER2 gene humanized heterozygous mice (H/+) .
- M is a marker.
- H 2 O is a water control.
- GAPDH is an internal reference.
- FIGS. 8A-8I show microscopic examination images by IHC.
- FIG. 8A is a negative control showing the C57BL/6 mouse mammary gland tissue without treatment of the recombinant Anti-ErbB2 /HER2 antibody.
- FIG. 9 shows the percentages of leukocyte subtypes in the spleen of C57BL/6 wild-type mice and HER2 gene humanized homozygous mice (B-hHER2) .
- FIG. 10 shows the percentages of T cell subtypes in the spleen of C57BL/6 wild-type mice and HER2 gene humanized homozygous mice (B-hHER2) .
- FIG. 11 shows the percentages of leukocyte subtypes in the peripheral blood of C57BL/6 wild-type mice and HER2 gene humanized homozygous mice (B-hHER2) .
- FIG. 12 shows the percentages ofT cell subtypes in the peripheral blood of C57BL/6 wild-type mice and HER2 gene humanized homozygous mice (B-hHER2) .
- FIG. 13 shows the percentages of leukocyte subtypes in the lymph nodes of C57BL/6 wild-type mice and HER2 gene humanized homozygous mice (B-hHER2) .
- FIG. 14 shows the percentages of T cell subtypes in the lymph nodes of C57BL/6 wild-type mice and HER2 gene humanized homozygous mice (B-hHER2) .
- FIG. 15 shows the alignment between human HER2 amino acid sequence (NP_004439.2; SEQ ID NO: 2) and mouse HER2 amino acid sequence (NP_001003817.1; SEQ ID NO: 1) .
- FIG. 16 shows the alignment between human HER2 amino acid sequence (NP_004439.2; SEQ ID NO: 2) and rat HER2 amino acid sequence (NP_058699.2; SEQ ID NO: 33) .
- This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) HER2, and methods of use thereof.
- HER2 (also known as ERBB2) is a transmembrane receptor belonging to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases. HER2 is overexpressed in various cancer types such as breast cancer and gastric cancer and has been reported to be a negative prognostic factor in breast cancer. In about 1 of every 5 breast cancers, the cancer cells have extra copies of the gene that makes the HER2 protein. HER2-positive breast cancers tend to be more aggressive than other types of breast cancer. Treatments that specifically target HER2 are very effective. Therefore, HER2 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-HER2 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.
- HER2 human epidermal growth factor receptor 2
- receptor tyrosine-protein kinase erbB-2 CD340, proto-oncogene Neu, or ERBB2
- HER/EGFR/ERBB human epidermal growth factor receptor
- the ErbB family consists of four plasma membrane-bound receptor tyrosine kinases. One of which is ErbB-2 (HER2) , and the other members being ErbB-1 (EGFR or HER1) , ErbB-3 (HER3) , and ErbB-4 (HER4) .
- All four contain an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain that can interact with a multitude of signaling molecules and exhibit both ligand-dependent and ligand-independent activity. Notably, no ligands for HER2 have yet been identified.
- HER2 activation results from heterodimerization with another ERBB member or by homodimerization when HER2 concentration are high, for instance in cancer.
- Dimerisation results in the autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates a variety of signaling pathways. Amplification or over-expression of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. In recent years the protein has become an important biomarker and target of therapy for approximately 30%of breast cancer patients.
- the current model of ligand-induced ErbB dimerization is proposed to be facilitated by the shift between a ‘tethered’ intramolecular conformation and a dimerization-competent conformation.
- a protruding of domain II or the “dimerization arm”
- the dimerization arm and an adjacent loop are exposed to allow contact between the ECDs of two monomers.
- ErbB2 is not likely to have a ligand, and the ‘tethered’ intramolecular conformation is absent.
- HER2 HER2 and its function can be found, e.g., in Hu, S., et al. "Molecular architecture of the ErbB2 extracellular domain homodimer. " Oncotarget 6.3 (2015) : 1695; Gutierrez, C., et al. "HER2: biology, detection, and clinical implications. " Archives of pathology & laboratory medicine 135.1 (2011) : 55-62; Arkhipov, A., et al. "Her2 activation mechanism reflects evolutionary preservation of asymmetric ectodomain dimers in the human EGFR family. " Elife 2 (2013) : e00708; each of which is incorporated by reference in its entirety.
- HER2 gene locus has twenty-seven exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, and exon 27 (FIG. 1) .
- the HER2 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 HER2 mRNA is NM_004448.3, and the amino acid sequence for human HER2 is NP_004439.2 (SEQ ID NO: 2) .
- the location for each exon and each region in human HER2 nucleotide sequence and amino acid sequence is listed below:
- the human HER2 gene (Gene ID: 2064) is located in Chromosome 17 of the human genome, which is located from 39688094 to 39728660 of NC_000017.11.
- the 5’ UTR is from 39700064 to 39700236, exon 1 is from 39700064 to 39700311, the first intron is from 397000312 to 39706989; exon 2 is from 39706990 to 39707141, the second intron is from 39707142 to 39708320; exon 3 is from 39708321 to 39708534, the third intron is from 39708535 to 39709317; exon 4 is from 39709318 to 39709452, the fourth intron is from 39709453 to 39709812; exon 5 is from 39709813 to 39709881, the fifth intron is from 39709882 to 39710085; exon 6 is from 39710086 to 39710201, the sixth intron is from 39710202 to 39710339; exon 7 is from 3
- the extracellular region of human HER2 protein includes four domains, from N-terminus to C-terminus: a first Recep_L_domain (domain I) that corresponds to amino acids 52-172 of SEQ ID NO: 2; a Furin-like domain (domain II) that corresponds to amino acids 190-343 of SEQ ID NO: 2; a second Recep_L_domain (domain III) that corresponds to amino acids 366-484 of SEQ ID NO: 2; and a GF_recep_IV domain (domain IV) that corresponds to amino acids 511-632 of SEQ ID NO: 2.
- HER2 gene locus has twenty-seven exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, and exon 27 (FIG. 1) .
- the mouse HER2 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 HER2 mRNA is NM_001003817.1, the amino acid sequence for mouse HER2 is NP_001003817.1 (SEQ ID NO: 1) .
- the location for each exon and each region in the mouse HER2 nucleotide sequence and amino acid sequence is listed below:
- the mouse HER2 gene (Gene ID: 13866) is located in Chromosome 11 of the mouse genome, which is located from 98, 303, 296 to 98328542 of NC_000077.7.
- the 5’ UTR is from 98303310 to 98303488, exon 1 is from 98303296 to 98303561, the first intron is from 98303562 to 98310914; exon 2 is from 98310915 to 98311066, the second intron is from 98311067 to 98311690; exon 3 is from 98311691 to 98311907, the third intron is from 98311908 to 98312880; exon 4 is from 98312881 to 98313015, the fourth intron is from 98313016 to 98313349; exon 5 is from 98313350 to 98313418, the fifth intron is from 98313419 to 98313580; exon 6 is from 98313581 to 98313696, the sixth intron is
- FIG. 15 shows the alignment between human HER2 amino acid sequence (NP_004439.2; SEQ ID NO: 2) and mouse HER2 amino acid sequence (NP_001003817.1; SEQ ID NO: 1) .
- NP_004439.2 SEQ ID NO: 2
- mouse HER2 amino acid sequence NP_001003817.1; SEQ ID NO: 1
- HER2 genes, proteins, and locus of the other species are also known in the art.
- the gene ID for HER2 (ERBB2) in Rattus norvegicus (rat) is 24337
- the gene ID for HER2 in Macaca mulatta (Rhesus monkey) is 697573
- the gene ID for HER2 in Canis lupus familiaris (dog) is 403883
- the gene ID for HER2 in Equus caballus (horse) is 100054739.
- 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 HER2 amino acid sequence (NP_004439.2; SEQ ID NO: 2) and rat HER2 amino acid sequence (NP_058699.2; SEQ ID NO: 33.
- NP_004439.2 SEQ ID NO: 2
- rat HER2 amino acid sequence NP_058699.2; SEQ ID NO: 33.
- the present disclosure provides human or chimeric (e.g., humanized) HER2 nucleotide sequence and/or amino acid sequences.
- human or chimeric (e.g., humanized) HER2 nucleotide sequence and/or amino acid sequences In some embodiments, the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence.
- a “region” or “portion” of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, 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, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 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, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,
- the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, signal peptide, extracellular region, transmembrane region, or cytoplasmic region.
- a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, and/or exon 27 are replaced by a region, a portion, or the entire sequence of the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, and/or exon 27 (e.g., a portion of exon 2, exons 3-16, and a
- the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a chimeric (e.g., humanized ) HER2 nucleotide sequence.
- the chimeric (e.g., humanized ) HER2 nucleotide sequence encodes a HER2 protein comprising an extracellular region, a transmembrane region, and a cytoplasmic region.
- the extracellular region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 67-652 of SEQ ID NO: 2.
- the extracellular region comprises all or part of human HER2 extracellular region.
- the transmembrane region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 653-675 of SEQ ID NO: 2. In some embodiments, the transmembrane region comprises all or part of human HER2 transmembrane region. In some embodiments, the cytoplasmic region comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids immediately connected to the human HER2 transmembrane region, e.g., amino acids 676-678 of SEQ ID NO: 2. 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, 7, or 8.
- the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized HER2 protein.
- the HER2 protein comprises, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region.
- the humanized HER2 protein comprises a human or humanized extracellular region.
- the humanized HER2 protein comprises an endogenous extracellular region.
- the humanized HER2 protein comprises a human or humanized transmembrane region.
- the humanized HER2 protein comprises an endogenous transmembrane region.
- the humanized HER2 protein comprises a human or humanized cytoplasmic region. In some embodiments, the humanized HER2 protein comprises an endogenous cytoplasmic region. In some embodiments, the humanized HER2 protein comprises a human or humanized signal peptide. In some embodiments, the humanized HER2 protein comprises an endogenous signal peptide.
- the humanized HER2 protein comprises a human or humanized first Recep_L_domain. In some embodiments, the humanized HER2 protein comprises an endogenous first Recep_L_domain. In some embodiments, the humanized HER2 protein comprises a human or humanized Furin-like domain. In some embodiments, the humanized HER2 protein comprises an endogenous Furin-like domain. In some embodiments, the humanized HER2 protein comprises a human or humanized second Recep_L_domain. In some embodiments, the humanized HER2 protein comprises an endogenous second Recep_L_domain. In some embodiments, the humanized HER2 protein comprises a human or humanized GF_recep_IV domain.
- the humanized HER2 protein comprises an endogenous GF_recep_IV domain.
- the humanized HER2 protein comprises a human or humanized first Recep_L_domain, a human or humanized Furin-like domain, a human or humanized second Recep_L_domain, and a human or humanized GF_recep_IV domain.
- the humanized HER2 protein comprises an endogenous sequence that corresponds to amino acids 1-66 or 23-66 of SEQ ID NO: 2.
- the genetically-modified non-human animal described herein comprises a human or humanized HER2 gene.
- the humanized HER2 gene comprises 27 exons.
- the humanized HER2 gene comprises endogenous exon 1, humanized exon 2, human exon 3, human exon 4, human exon 5, human exon 6, human exon 7, human exon 8, human exon 9, human exon 10, human exon 11, human exon 12, human exon 13, human exon 14, human exon 15, human exon 16, humanized exon 17, endogenous exon 18, endogenous exon 19, endogenous exon 20, endogenous exon 21, endogenous exon 22, endogenous exon 23, endogenous exon 24, endogenous exon 25, endogenous exon 26, and/or endogenous exon 27.
- the humanized HER2 gene comprises 26 introns.
- the humanized HER2 gene comprises endogenous intron 1, human intron 2, human intron 3, human intron 4, human intron 5, human intron 6, human intron 7, human intron 8, human intron 9, human intron 10, human intron 11, human intron 12, human intron 13, human intron 14, human intron 15, human intron 16, endogenous intron 17, endogenous intron 18, endogenous intron 19, endogenous intron 20, endogenous intron 21, endogenous intron 22, endogenous intron 23, endogenous intron 23, endogenous intron 25, and/or endogenous intron 26.
- the humanized HER2 gene comprises human or humanized 5’ UTR.
- the humanized HER2 gene comprises human or humanized 3’ UTR. In some embodiments, the humanized HER2 gene comprises endogenous 5’ UTR. In some embodiments, the humanized HER2 gene comprises endogenous 3’ UTR.
- the present disclosure also provides a chimeric (e.g., humanized) HER2 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 HER2 mRNA sequence (e.g., NM_001003817.1) , mouse HER2 amino acid sequence (e.g., SEQ ID NO: 1) , or a portion thereof (e.g., 5’ UTR, exon 1, a portion of exon 2, a portion of exon 17, exons 18-27, and 3’ UTR) ; and in some embodiments
- sequence encoding amino acids 67-679 of mouse HER2 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human HER2 (e.g., amino acids 67-678 of human HER2 (SEQ ID NO: 2) ) .
- sequence encoding amino acids 67-674 of mouse HER2 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human HER2 (e.g., amino acids 67-675 of human HER2 (SEQ ID NO: 2) ) .
- sequence encoding amino acids 67-653 of mouse HER2 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human HER2 (e.g., amino acids 67-652 of human HER2 (SEQ ID NO: 2) ) .
- sequence encoding amino acids 23-653 of mouse HER2 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human HER2 (e.g., amino acids 23-652 of human HER2 (SEQ ID NO: 2) ) .
- sequence encoding amino acids 1-653 of mouse HER2 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human HER2 (e.g., amino acids 1-652 of human HER2 (SEQ ID NO: 2) ) .
- the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse HER2 promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.
- a promotor or regulatory element e.g., an endogenous mouse HER2 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 HER2 nucleotide sequence (e.g., a portion of exon 2, exons 3-16, and a portion of exon 17 NM_001003817.1) .
- 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 HER2 nucleotide sequence (e.g., 5’ UTR, exon 1, a portion of exon 2, a portion of exon 17, exons 18-27, and 3’ UTR of NM_001003817.1) .
- 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 HER2 nucleotide sequence (e.g., 5’ UTR, exon 1, a portion of exon 2, a portion of exon 17, exons 18-27, and 3’ UTR of NM_004448.3) .
- 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 HER2 nucleotide sequence (e.g., a portion (at least 10 bp) of exon 2, exons 3-16, and a portion (at least 50 bp) of exon 17 of NM_004448.3) .
- 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 HER2 amino acid sequence (e.g., amino acids 67-679 of NP_001003817.1 (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 HER2 amino acid sequence (e.g., amino acids 1-66 and 680-1256 of NP_001003817.1 (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 HER2 amino acid sequence (e.g., amino acids 1-66 and 679-1255 of NP_004439.2 (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 HER2 amino acid sequence (e.g., amino acids 67-678 of NP_004439.2 (SEQ ID NO: 2) ) .
- the present disclosure also provides a humanized HER2 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 9 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 9;
- amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 1, 2, or 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or
- 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 9.
- the present disclosure also provides a humanized HER2 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 67-678 of SEQ ID NO: 2;
- amino acid sequence that is different from amino acids 67-678 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 67-678 of SEQ ID NO: 2.
- the present disclosure also relates to a HER2 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, 7, or 8 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, 7, or 8;
- 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 9;
- 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 9;
- 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 9 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 9.
- the present disclosure further relates to a HER2 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 8.
- 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 9, and has protein activity.
- the homology with the sequence shown in SEQ ID NO: 1, 2, or 9 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 9 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 8, and encodes a polypeptide that has protein activity.
- the homology with the sequence shown in SEQ ID NO: 5 or 8 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, 7, or 8 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) HER2 from an endogenous non-human HER2 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 HER2 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 HER2 gene or a humanized HER2 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human HER2 gene, at least one or more portions of the gene or the nucleic acid is from a non-human HER2 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an HER2 protein. The encoded HER2 protein is functional or has at least one activity of the human HER2 protein or the non-human HER2 protein, e.g., heterodimerise with other ERBB family receptors and initiate downstream signaling pathways.
- the chimeric protein or the chimeric polypeptide is a humanized HER2 protein or a humanized HER2 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 HER2 protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human HER2 protein.
- the humanized HER2 protein or the humanized HER2 polypeptide is functional or has at least one activity of the human HER2 protein or the non-human HER2 protein.
- the cytoplasmic region is human or humanized.
- the transmembrane region is human or humanized.
- the extracellular region is human or humanized.
- the first Recep_L_domain is human or humanized.
- the Furin-like domain is human or humanized.
- the second Recep_L_domain is human or humanized.
- the GF_recep_IV domain is human or humanized.
- the sequence corresponding to amino acids 1-66 or 23-66 of SEQ ID NO: 2 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 HER2 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 HER2 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 HER2 coding sequence with human mature HER2 coding sequence.
- Genetically modified non-human animals can comprise a modification at an endogenous non-human HER2 locus.
- the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature HER2 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the mature HER2 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 HER2 locus in the germline of the animal.
- Genetically modified animals can express a human HER2 and/or a chimeric (e.g., humanized) HER2 from endogenous mouse loci, wherein the endogenous mouse HER2 gene has been replaced with a human HER2 gene and/or a nucleotide sequence that encodes a region of human HER2 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 HER2 sequence.
- an endogenous non-human HER2 locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature HER2 protein.
- the genetically modified mice can express the human HER2 and/or chimeric HER2 (e.g., humanized HER2) 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 HER2 or chimeric HER2 (e.g., humanized HER2) 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 HER2 or the chimeric HER2 (e.g., humanized HER2) expressed in animal can maintain one or more functions of the wild-type mouse or human HER2 in the animal.
- the expressed HER2 can bind to human or non-human HER1, HER3, or HER4.
- the animal does not express endogenous HER2.
- the animal expresses a decreased level of endogenous HER2 as compared to a wild-type animal.
- endogenous HER2 refers to HER2 protein that is expressed from an endogenous HER2 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 HER2 (NP_004439.2) (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: 9.
- the genome of the genetically modified animal can comprise a replacement at an endogenous HER2 gene locus of a sequence encoding a region of endogenous HER2 with a sequence encoding a corresponding region of human HER2.
- the sequence that is replaced is any sequence within the endogenous HER2 gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, 5’-UTR, 3’-UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, intron 19, intron 20, intron 21, intron 22, intron 23, intron 24, in
- the sequence that is replaced is within the regulatory region of the endogenous HER2 gene. In some embodiments, the sequence that is replaced is exon 2, exon 3, exon 4, exon 5, exon 6, exon 6, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, or a portion thereof, of an endogenous mouse HER2 gene locus.
- the genetically modified animal can have one or more cells expressing a human or chimeric HER2 (e.g., humanized HER2) having, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region.
- a human or chimeric HER2 e.g., humanized HER2
- the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human HER2.
- the extracellular region of the humanized HER2 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, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 600, 610, 620, or 630 amino acids (e.g., contiguously or non-contiguously) that are identical to the extracellular region of human HER2.
- amino acids e.g., contiguously
- human HER2 and non-human HER2 e.g., mouse HER2 sequences, in many cases, are different, antibodies that bind to human HER2 will not necessarily have the same binding affinity with non-human HER2 or have the same effects to non-human HER2. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human HER2 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 human HER2.
- the transmembrane region of the humanized HER2 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, 21, 22, or 23 amino acids (contiguously or non-contiguously) that are identical to the transmembrane region of human HER2.
- the cytoplasmic comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the cytoplasmic of human HER2.
- the cytoplasmic region of the humanized HER2 has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acids (contiguously or non-contiguously) that are identical to the cytoplasmic region of human HER2.
- the entire transmembrane of the humanized HER2 described herein are derived from human sequence.
- a few cytoplasmic amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) that are close to the transmembrane region of HER2 are also derived from human sequence.
- nucleotides at the border of the exon 17 can affect mRNA splicing of the chimeric HER2 gene. For example, replacing nucleotides at the border of exon 17 with certain other nucleotides may affect splicing, thus, in some embodiments, the nucleotides encoding the entire transmembrane region are replaced.
- 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 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and/or exon 17 of human HER2; a portion or the entire sequence of the extracellular region, a portion or the entire sequence of the transmembrane region, and/or a portion or the entire sequence of the cytoplasmic region of human HER2; or a portion or the entire sequence of amino acids 67-678 of SEQ ID NO: 2.
- the genome of the genetically modified animal comprises a portion of exon 2, exons 3-16, and a portion of exon 17 of human HER2 gene.
- the portion of exon 2 includes at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 70, 90, 100, 110, 130, 150, or 152 nucleotides.
- the portion of exon 2 includes 27 nucleotides.
- the portion of exon 2 includes a nucleotide of at least 10 bp.
- the portion of exon 2 includes nucleotide sequences encoding the C-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of a polypeptide encoded by exon 2 of human HER2 gene.
- the portion of exon 17 includes at least 10, 20, 30, 40, 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 110, 120, 130, or 139 nucleotides.
- the portion of exon 17 includes 88 nucleotides.
- the portion of exon 2 includes a nucleotide of at least 50 bp.
- the portion of exon 2 includes a nucleotide sequence starting from the first nucleotide in exon 17 to the nucleotides encoding the N-terminal 1, 2, 3, 4, or 5 amino acids of the cytoplasmic region.
- the non-human animal can have, at an endogenous HER2 gene locus, a nucleotide sequence encoding a chimeric human/non-human HER2 polypeptide, wherein a human portion of the chimeric human/non-human HER2 polypeptide comprises a portion of human HER2 extracellular domain, the entire human HER2 transmembrane region, and a few human HER2 cytoplasmic amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) close to the transmembrane region, and wherein the animal expresses a functional HER2 on a surface of a cell of the animal.
- a nucleotide sequence encoding a chimeric human/non-human HER2 polypeptide
- a human portion of the chimeric human/non-human HER2 polypeptide comprises a portion of human HER2 extracellular domain, the entire human HER2 transmembrane region, and a few human HER2 cytoplasm
- the human portion of the chimeric human/non-human HER2 polypeptide can comprise an amino acid sequence encoded by a portion of exon 2, exons 3-16, and/or a portion of exon 17 of human HER2.
- the human portion of the chimeric human/non-human HER2 polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 67-678 of SEQ ID NO: 2.
- the extracellular region includes a sequence corresponding to the entire or part of amino acids 23-66 of SEQ ID NO: 1.
- the cytoplasmic region includes a sequence corresponding to the entire or part of amino acids 680-1256 of SEQ ID NO: 1.
- the chimeric human/non-human HER2 polypeptide comprises a signal peptide, which includes a sequence corresponding to the entire or part of amino acids 1-22 of SEQ ID NO: 1.
- the non-human portion of the chimeric human/non-human HER2 polypeptide comprises a portion of extracellular and cytoplasmic regions of an endogenous non-human HER2 polypeptide.
- the genetically modified animal can be heterozygous with respect to the replacement at the endogenous HER2 locus, or homozygous with respect to the replacement at the endogenous HER2 locus.
- the humanized HER2 locus lacks a human HER2 5’-UTR.
- the humanized HER2 locus comprises an endogenous (e.g., mouse) 5’-UTR.
- the humanization comprises an endogenous (e.g., mouse) 3’-UTR.
- mouse and human HER2 genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence.
- humanized HER2 mice that comprise a replacement at an endogenous mouse HER2 locus which retain mouse regulatory elements but comprise a humanization of HER2 encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized HER2 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 HER2 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 HER2 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, and 5) .
- a non-human mammal expressing human or humanized HER2 is provided.
- the tissue-specific expression of human or humanized HER2 protein is provided.
- the expression of human or humanized HER2 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 HER2 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 HER2 protein.
- the present disclosure relates to a targeting vector, comprising: a) a DNA fragment homologous to the 5’ end of a region to be altered (5’ arm) , which is selected from the HER2 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 HER2 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_000077.7; 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_000077.7.
- 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 98307108 to the position 98311039 of the NCBI accession number NC_000077.7; 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 98323825 to the position 98328580 of the NCBI accession number NC_000077.7.
- the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 10 kb, about 11 kb, about 12 kb, about 13 kb, about 14 kb, about 15 kb, about 16 kb, about 17 kb, about 18 kb, about 19 kb, or about 20 kb.
- the region to be altered is exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and/or exon 17 of HER2 gene (e.g., a portion of exon 2, exons 3-16, and a portion of exon 17 of mouse HER2 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.
- the sequence is derived from human (e.g., 39707115-39723406 of NC_000017.11) .
- the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human HER2, preferably exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and/or exon 17 of the human HER2.
- the nucleotide sequence of the humanized HER2 encodes the entire or the part of human HER2 protein with the NCBI accession number NP_004439.2 (SEQ ID NO: 2) .
- 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 HER2 gene locus, a sequence encoding a region of an endogenous HER2 with a sequence encoding a corresponding region of human or chimeric HER2.
- the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc.
- the nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.
- FIG. 3 shows a humanization strategy for a mouse HER2 locus.
- the targeting strategy involves a vector comprising the 5’ end homologous arm, human HER2 gene fragment, 3’ homologous arm.
- the process can involve replacing endogenous HER2 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 HER2 sequence with human HER2 sequence.
- the methods for making a genetically modified, humanized animal can include the step of replacing at an endogenous HER2 locus (or site) , a nucleic acid encoding a region of endogenous HER2 with a sequence encoding a corresponding region of human HER2.
- the sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, and/or exon 27 of a human HER2 gene.
- the sequence includes a portion of exon 2, exons 3-16, and a portion of exon 17 of a human HER2 gene (e.g., nucleic acids 460-2295 of NM_004448.3) .
- the region includes a portion of the extracellular region of human HER2 (e.g., amino acids 67-652 of SEQ ID NO: 2) , the entire transmembrane of human HER2 (e.g., amino acids 653-675 of SEQ ID NO: 2, and a few (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cytoplasmic amino acids of human HER2 (e.g., amino acids 676-678 of SEQ ID NO: 2) ) .
- human HER2 e.g., amino acids 67-652 of SEQ ID NO: 2
- the entire transmembrane of human HER2 e.g., amino acids 653-675 of SEQ ID NO: 2
- a few e.g., 1, 2, 3, 4, 5, 6, 7, or 8
- cytoplasmic amino acids of human HER2 e.g., amino acids 676-678 of SEQ ID NO: 2
- the endogenous HER2 locus is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, and/or exon 27 of mouse HER2.
- the sequence includes a portion of exon 2, exons 3-16, and a portion of exon 17 of mouse HER2 gene (e.g., nucleic acids 378-2216 of NM_001003817.1) .
- the methods of modifying a HER2 locus of a mouse to express a chimeric human/mouse HER2 peptide can include the steps of replacing at the endogenous mouse HER2 locus a nucleotide sequence encoding a mouse HER2 with a nucleotide sequence encoding a human HER2, thereby generating a sequence encoding a chimeric human/mouse HER2.
- the nucleotide sequence encoding the chimeric human/mouse HER2 can include a first nucleotide sequence encoding the signal peptide and a portion of the extracellular region of mouse HER2; a second nucleotide sequence encoding a portion of the extracellular region, the entire transmembrane region, and a few (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cytoplasmic amino acids of human HER2; and/or a third nucleotide sequence encoding all or a portion of the cytoplasmic region of mouse HER2.
- 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 HER2 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 HER2 gene, e.g., by inserting a nucleotide sequence (e.g., DNA or cDNA sequence) encoding human or humanized HER2 protein immediately after the endogenous regulatory element of the non-human animal’s HER2 gene.
- a nucleotide sequence e.g., DNA or cDNA sequence
- one or more functional region sequences of the non-human animal’s HER2 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 HER2 protein.
- the coding frame of the modified non-human animal’s HER2 gene can be all or part of the nucleotide sequence from exon 1 to exon 27 of the non-human animal’s HER2 gene.
- methods of making the genetically modified animal comprises inserting a nucleotide sequence encoding human or humanized HER2 protein and/or an auxiliary sequence after the endogenous regulatory element of the non-human animal’s HER2 gene.
- the auxiliary sequence can be a stop codon, such that the HER2 gene humanized animal model can express human or humanized HER2 protein in vivo, but does not express non-human animal’s HER2 protein.
- the auxiliary sequence includes WPRE (WHP Posttranscriptional Response Element) and/or polyA.
- 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 HER2 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 HER2, which are useful for testing agents that can decrease or block the interaction between the interaction between HER2 and anti-human HER2 antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an HER2 agonist or antagonist.
- the genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (aknock-in or knockout) .
- the genetically modified non-human animals further comprise an impaired immune system, e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor) .
- an impaired immune system e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor) .
- the anti-HER2 antibody blocks or inhibits the HER2-related signaling pathway.
- the anti-HER2 antibody described herein can block the interaction between HER2 and a human EGFR family member protein (e.g., HER1, HER3, or HER4) , thereby inhibiting formation of a heterodimer.
- a human EGFR family member protein e.g., HER1, HER3, or HER4
- the genetically modified animals can be used for determining effectiveness of a therapeutic agent (e.g., an anti-HER2 antibody or a HER2-targeting drug) for the treatment of cancer.
- the methods involve administering the therapeutic agent (e.g., an anti-human HER2 antibody or a HER2-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 HER2 is also expressed on many other cells.
- the humanized HER2 functions in a largely similar way as compared to the endogenous HER2, 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-HER2 antibody can directly target cancer cells expressing HER2, e.g., by inducing complement mediated cytotoxicity (CMC) or antibody dependent cellular cytoxicity (ADCC) to kill the cancer cells.
- CMC complement mediated cytotoxicity
- ADCC antibody dependent cellular cytoxicity
- the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) that are injected into the animal.
- the anti-HER2 antibody inhibits HER2 signaling pathways. In some embodiments, the anti-HER2 antibody does not inhibit HER2 signaling pathways.
- the genetically modified animals can be used for determining whether an anti-HER2 antibody is a HER2 agonist or antagonist.
- the methods as described herein are also designed to determine the effects of the agent (e.g., anti- HER2 antibodies) on HER2, e.g., whether the agent can reduce the amplification signal provided by other HER family receptors by forming heterodimers, whether the agent can induce HER2 down-modulation, whether the agent can inhibit critical signaling pathways (e.g., ras-Raf-MAPK and PI3K/Akt) , whether the agent can block cell cycle progression, e.g., by inducing the formation of p27/Cdk2 complexes, whether the agent can inhibit HER2 cleavage, whether the agent can inhibit angiogenesis, 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 (CMC) or antibody dependent
- 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-HER2 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 breast cancer (e.g., HER2-positive breast cancer) .
- the cancer described herein is metastatic breast cancer, or early and metastatic breast cancer.
- the cancer described herein is gastric cancer, ovarian cancer, uterine serous endometrial carcinoma, colon cancer, bladder cancer, lung cancer, cervical cancer, head and neck cancer, endometrial cancer, or esophageal cancer.
- the therapeutic agent described herein is Trastuzumab, Pertuzumab, Margetuximab, Ado-Trastuzumab Emtansine (T-DM1) , Trastuzumab deruxtecan (fam-trastuzumab deruxtecan) , Lapatinib, Tucatinib, Neratinib, or Afatinib.
- the therapeutic agent is a monoclonal antibody, e.g., Herceptin (chemical name: trastuzumab) , Margenza (chemical name: margetuximab-cmkb) , and Perjeta (chemical name: pertuzumab) .
- Herceptin chemical name: trastuzumab
- Margenza chemical name: margetuximab-cmkb
- Perjeta chemical name: pertuzumab
- the therapeutic agent is an antibody-drug conjugate, e.g., Enhertu (chemical name: am-trastuzumab-deruxtecan-nxki) , Kadcyla (chemical name: T-DM1 or ado-trastuzumab emtansine) , and Phesgo (chemical name: pertuzumab, trastuzumab, and hyaluronidase-zzxf) .
- the therapeutic agent is a pan-HER inhibitor, e.g., Nerlynx (chemical name: neratinib) .
- the therapeutic agent is a signal transduction inhibitor, e.g., Tykerb (chemical name: lapatinib) .
- the therapeutic agent is a tyrosine kinase inhibitor, e.g., Tukysa (chemical name: tucatinib) .
- Tukysa chemical name: tucatinib
- the anti-HER2 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-HER2 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-HER2 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-HER2 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 HER2 gene function, human HER2 antibodies, drugs for human HER2 targeting sites, the drugs or efficacies for human HER2 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 HER2 gene humanized non-human animal prepared by the methods described herein, the HER2 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 HER2 protein, or the tumor-bearing or inflammatory animal models described herein.
- the TCR-T, CAR-T, and/or other immunotherapies can treat the HER2-associated diseases described herein (e.g., breast cancer) .
- the TCA-T, CAR-T, and/or other immunotherapies provides an evaluation method for treating the HER2-associated diseases described herein (e.g., breast cancer) .
- 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 HER2 gene and a sequence encoding an additional human or chimeric protein.
- the additional human or chimeric protein can be programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , CD73, tumor necrosis factor alpha (TNF ⁇ ) , B And T Lymphocyte Associated (BTLA) , CD27, CD28, CD47, CD137, CD154, CD226, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , and/or Signal regulatory protein ⁇ (SIRP ⁇ ) .
- PD-1 programmed cell death protein 1
- PD-L1 programmed cell death ligand 1
- CTLA4 tumor
- the methods of generating genetically modified animal model with two or more human or chimeric genes can include the following steps:
- the genetically modified animal in step (b) of the method, can be mated with a genetically modified non-human animal with human or chimeric PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, TNF ⁇ , BTLA, CD27, CD28, CD47, CD137, CD154, CD226, TIGIT, GITR, and/or SIRP ⁇ .
- the HER2 humanization is directly performed on a genetically modified animal having a human or chimeric PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, TNF ⁇ , BTLA, CD27, CD28, CD47, CD137, CD154, CD226, TIGIT, GITR, and/or SIRP ⁇ gene.
- a combination therapy that targets two or more of these proteins thereof may be a more effective treatment.
- many related clinical trials are in progress and have shown a good effect.
- 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-HER2 antibody and an additional therapeutic agent for the treatment of cancer.
- the methods include administering the anti-HER2 antibody and the additional therapeutic agent to the animal, wherein the animal has a tumor; and determining the inhibitory effects of the combined treatment to the tumor.
- the additional therapeutic agent is an antibody that specifically binds to PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, TNF ⁇ , BTLA, CD27, CD28, CD47, CD137, CD154, CD226, TIGIT, 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 HER2, CD80, CD86, PD-L1, and/or PD-L2.
- the combination treatment is designed for treating various cancers as described herein, e.g., breast cancer (e.g., HER2-positive breast cancer) .
- breast cancer e.g., HER2-positive breast cancer
- the cancer described herein is metastatic breast cancer, or early and metastatic breast cancer.
- the cancer described herein is gastric cancer, ovarian cancer, uterine serous endometrial carcinoma, colon cancer, bladder cancer, lung cancer, cervical cancer, head and neck cancer, endometrial cancer, or esophageal 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
- C57BL/6 mice and Flp transgenic mice were purchased from the China Food and Drugs Research Institute National Rodent Experimental Animal Center.
- ScaI, SspI and AseI restriction enzymes were purchased from NEB (Catalog numbers: R3122S, R0132S, and R0526S, respectively) .
- Recombinant Anti-ErbB2/HER2 antibody was purchased from Abcam (Catalog number: ab214275) .
- EXAMPLE 1 Mice with humanized HER2 gene
- a non-human animal e.g., a mouse
- a non-human animal was modified to include a nucleotide sequence encoding human HER2 protein, and the obtained genetically-modified non-human animal can express a human or humanized HER2 protein in vivo.
- the mouse HER2 gene (NCBI Gene ID: 13866, Primary source: MGI: 95410, UniProt ID: P70424) is located at 98303310 to 98328542 of chromosome 11 (NC_000077.7)
- the human HER2 gene (NCBI Gene ID: 2064, Primary source: HGNC: 3430, UniProt ID: P04626) is located at 39688094 to 39728660 of chromosome 17 (NC_000017.11) .
- the mouse HER2 transcript is NM_001003817.1, and the corresponding protein sequence NP_001003817.1 is set forth in SEQ ID NO: 1.
- the human HER2 transcript is NM_004448.3, and the corresponding protein sequence NP_004439.2 is set forth in SEQ ID NO: 2.
- Mouse and human HER2 gene loci are shown in FIG. 1.
- nucleotide sequences encoding human HER2 protein can be introduced into the mouse endogenous HER2 locus, so that the mouse expresses human or humanized HER2 protein.
- a nucleotide sequence e.g., DNA or cDNA sequence
- a sequence starting from within exon 2 and ending within exon 17 of mouse HER2 gene was replaced with a corresponding sequence starting from within exon 2 and ending within exon 17 of human HER2 gene, to obtain a humanized HER2 gene locus as shown in FIG. 2, thereby humanizing mouse HER2 gene.
- the targeting vector contains homologous arm sequences upstream and downstream of the mouse HER2 gene, and an “A Fragment” containing DNA sequences of human HER2 gene.
- sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 3) is identical to nucleotide sequence of 98307108-98311039 of NCBI accession number NC_000077.7
- sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 4) is identical to nucleotide sequence of 98323825-98328580 of NCBI accession number NC_000077.7.
- the A Fragment contains a human genomic DNA sequence from HER2 genes (SEQ ID NO: 5) , which is identical to nucleotide sequence of 39707115-39723406 of NCBI accession number NC_000017.11.
- 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: (SEQ ID NO: 6) , wherein the last “C” in sequence “ CAATC ” is the last nucleotide of the human sequence, and the first “A” in sequence 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: (SEQ ID NO: 7) , wherein the last “T” in sequence “ GATCT ” is the last nucleotide of the Neo cassette, and the “T” in sequence 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 o f 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. PCR primers are shown in the table below.
- mice embryonic stem cells were transfected with targeting vectors, the clones identified as positive by PCR were then verified by Southern Blot (cell DNA was digested with AseI, ScaI, or SspI; and hybridized with three probes) to screen out correct positive clone cells.
- the restriction enzymes, probes, and the size of target fragments are shown in the table below.
- the Southern Blot detection results are shown in FIG. 4. The results indicate that all twelve PCR-positive embryonic stem cells (ES-1 to ES-12) were verified as positive clones without random insertions.
- Neo Probe-F 5’-GGATCGGCCATTGAACAAGAT-3’ (SEQ ID NO: 18) ,
- Neo Probe-R 5’-CAGAAGAACTCGTCAAGAAGGC-3’ (SEQ ID NO: 19) .
- 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.
- FIGS. 6A-6D The identification results of exemplary F1 generation mice (Neo cassette removed) are shown in FIGS. 6A-6D, wherein four mice numbered F1-1, F1-2, F1-3, and F1-4 were identified as positive heterozygous mice.
- the genotype of the HER2 gene humanized mice were verified by PCR using primers shown in the table below.
- RT-PCR was further used to detect the expression of HER2 protein in HER2 gene humanized mice.
- Total RNA from colon cells of wild-type C57BL/6 mice and HER2 gene humanized heterozygous mice was extracted and reverse-transcribed into cDNA using a reverse transcription kit. The following primer sequences were used.
- mice HER2 can be detected in the colon cells of wild-type C57BL/6 mice, and the mRNA expression of both human and mouse HER2 can be detected in the colon cells of HER2 gene humanized heterozygous mice.
- the heterozygous mice identified as positive in the F1 generation were bred with each other to obtain the F2 generation HER2 gene humanized homozygous mice, and the expression of humanized HER2 protein was detected by Immunohistochemistry (IHC) .
- IHC Immunohistochemistry
- a formalin-fixed mammary gland tissue, gastric tissue, and colon tissue from HER2 gene humanized homozygous mice and C57BL/6 mice; a dog spleen tissue; and a HER2-positive tumor tissue were paraffin-embedded. After tissue dewaxing and blocking, recombinant Anti-ErbB2 /HER2 antibody and Goat Anti-Rabbit IgG Antibody (H+L) , Biotinylated were added for color development. Microscopic examination images are shown in FIGS. 8A-8I, and the staining results are summarized in the table below.
- the staining intensity was scored as follows: 0 points for no color, 1 point for light yellow, 2 points for brownish yellow, and 3 points for dark brown;
- the percentage of positive cells was scored as follows: negative for 0 points, ⁇ 10%for 1 point, 11%-50%for 2 points, 51%-75%for 3 points, >75%for 4 points;
- the total score was multiplied by the score of the staining intensity and the score of the percentage of positive cells, 0 is no positive staining (-) , 1-3 for suspected positive ( ⁇ ) , 4-6 for weakly positive (+) , 7-9 for moderately positive (++) , and 10-12 for strongly positive (+++) .
- the recombinant Anti-ErbB2/HER2 antibody was a human-mouse cross-reactive monoclonal antibody, it can recognize both human HER2 protein and mouse HER2 protein.
- the expression of HER2 mRNA in the HER2 gene humanized homozygous mice was detected by RT-PCR, using methods described above. The test results showed that only mouse HER2 mRNA was detected, and no human HER2 mRNA was detected, in C57BL/6 wild-type mice. By contrast, human HER2 mRNA was only detected in the HER2 gene humanized homozygous mice. Combined with the IHC detection results, it was concluded that only humanized HER2 protein can be detected in HER2 gene humanized homozygous mice.
- the spleen, lymph nodes, and peripheral blood from C57BL/6 wild-type mice and HER2 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. 9 and FIG. 10, respectively.
- the detection results of leukocyte subtypes and T cell subtypes in peripheral blood are shown in FIG. 11 and FIG. 12, 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 HER2 gene humanized homozygous mice were basically the same as those in C57BL/6 wild-type mice (FIG. 9 and FIG. 11) .
- 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. 10 and FIG. 12) .
- the detection results of leukocyte subtypes and T cell subtypes in lymph nodes are shown in FIG. 13 and FIG. 14, respectively.
- the results showed that the leukocyte subtypes, e.g., B cells, T cells, NK cells, and other leukocyte subtypes in the lymph nodes of HER2 gene humanized homozygous mice were basically the same as those of C57BL/6 wild-type mice (FIG. 13) .
- 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. 14) .
- WBC white blood cell count
- RBC red blood cell count
- HCT hemoglobin
- HGB hemoglobin
- MCV mean corpuscular volume
- MHC mean corpuscular hemoglobin
- PHT lymphocytes
- LYMPH lymphocytes
- MONO monocytes
- NEUT neutrophils
- Blood biochemical test indicators included: alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , albumin (ALB) , blood glucose (GLU) , urea (UREA) , serum creatinine (CREA) , serum total cholesterol (TC) , and triglyceride (TG) .
- ALT alanine aminotransferase
- AS aspartate aminotransferase
- ALB albumin
- GLU blood glucose
- urea UREA
- CREA serum creatinine
- TC serum total cholesterol
- TG triglyceride
- mice had similar liver function as the wild type mice.
- the HER2 gene humanized mice prepared using the methods described herein can be used to construct a tumor model, which is useful for testing the efficacy of modulators (e.g., HER2-targeting drugs and antibodies) targeting human HER2.
- modulators e.g., HER2-targeting drugs and antibodies
- the HER2 gene humanized mice prepared by the methods described herein can be used to evaluate the efficacy of modulators targeting human HER2 (e.g., anti-human HER2 antibodies) .
- HER2 gene humanized homozygous mice can be subcutaneously inoculated with HER2 gene humanized MC38 cells. After the tumor volume reaches about 100 mm 3 , the mice can be placed into a control group and several treatment groups according to tumor volume. The treatment group mice can be administered with randomly selected drugs targeting human HER2 (e.g., anti-human HER2 antibodies) , and the control group mice can be administered with an equal volume of saline. Tumor volume and body weight of the mice can be measured regularly. By comparing changes in mouse body weight and tumor size, one can effectively assess the in vivo safety and efficacy of the human HER2-targeting drugs.
- the HER2 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) PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3 and/or CD73 genes.
- embryonic stem cells from humanized HER2 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 HER2 and other gene modifications.
- homozygous or heterozygous HER2 gene humanized mice obtained by the methods described herein with other genetically modified homozygous or heterozygous mice, and the offspring can be screened.
- Mendel it is possible to generate double-gene or multi-gene modified heterozygous mice comprising modified (e.g., human or humanized) HER2 gene and other genetic modifications.
- 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 HER2 and other genes.
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Abstract
La présente invention concerne des animaux non humains génétiquement modifiés exprimant un HER2 humain ou chimérique (par exemple, humanisé), ainsi que des procédés d'utilisation associés.
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CN111088287A (zh) * | 2019-12-20 | 2020-05-01 | 江苏浦珠生物医药科技有限公司 | 一种her2基因人源化小鼠肿瘤细胞模型、构建方法以及用途 |
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