WO2023046061A1

WO2023046061A1 - Genetically modified non-human animal with human or chimeric trop2

Info

Publication number: WO2023046061A1
Application number: PCT/CN2022/120819
Authority: WO
Inventors: Dexuan HUANG; Zhiyuan Shen; Chong Li
Original assignee: Biocytogen Pharmaceuticals Beijing Co Ltd
Current assignee: Biocytogen Pharmaceuticals Beijing Co Ltd
Priority date: 2021-09-24
Filing date: 2022-09-23
Publication date: 2023-03-30
Anticipated expiration: 2024-03-24
Also published as: CN116064558A

Abstract

Provided are genetically modified non-human animals that express a human or chimeric (e.g., humanized) TROP2, and methods of use thereof.

Description

GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC TROP2

CLAIM OF PRIORITY

This application claims the benefit of Chinese Patent Application App. No. 202111119814.3, filed on September 24, 2022. The entire contents of the foregoing application are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) TROP2, and methods of use thereof.

BACKGROUND

The traditional drug research and development typically use in vitro screening approaches. However, these screening approaches cannot provide the body environment (such as tumor microenvironment, stromal cells, extracellular matrix components and immune cell interaction, etc. ) , resulting in a higher rate of failure in drug development. In addition, in view of the differences between humans and animals, the 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.

Therefore, the development of humanized animal models that are suitable for human antibody screening and evaluation will significantly improve the efficiency of new drug development and reduce the cost for drug research and development.

SUMMARY

This disclosure is related to an animal model with human TROP2 or chimeric TROP2. The animal model can express human TROP2 or chimeric TROP2 (e.g., humanized TROP2) protein in its body. It can be used in the studies on the function of TROP2 gene, and can be used in the screening and evaluation of anti-human TROP2 antibodies or ADCs. In addition, 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 TROP2 target sites; they can also be used to facilitate the development and design of new drugs, and save time and cost. In summary, this disclosure provides a powerful tool for studying the function of TROP2 protein and a platform for screening cancer (e.g., breast cancer) drugs.

In one aspect, 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 TROP2 (trophoblast cell-surface antigen 2) . In some embodiments, the sequence encoding the human or chimeric TROP2 is operably linked to an endogenous regulatory element at the endogenous TROP2 gene locus in the at least one chromosome. In some embodiments, the sequence encoding a human or chimeric TROP2 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human TROP2 (NP_002344.2 (SEQ ID NO: 2) ) . In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a mt. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous TROP2 or expresses a decreased level of endogenous TROP2. In some embodiments, the animal has one or more cells expressing human or chimeric TROP2. In some embodiments, the animal has one or more cells expressing human or chimeric TROP2, and the expressed human or chimeric TROP2 can transduce an intracellular calcium signal. In some embodiments, the animal has one or more cells expressing human or chimeric TROP2, and the expressed human or chimeric TROP2 can interact with endogenous β-catenin, Claudin 1 and 7, Occludin, α5β1 integrin//Talin complex, IGF-1, MDK, and/or NRG-1. In some embodiments, the animal has one or more cells expressing human or chimeric TROP2, and the expressed human or chimeric TROP2 can interact with human β-catenin, Claudin 1 and 7, Occludin, α5β1 integrin//Talin complex, IGF-1, MDK, and/or NRG-1.

In one aspect, the disclosure is related to a genetically-modified, non-human animal, in some embodiments, the genome of the animal comprises a replacement of a sequence encoding a region of endogenous TROP2 with a sequence encoding a corresponding region of human TROP2 at an endogenous TROP2 gene locus. In some embodiments, the sequence encoding the corresponding region of human TROP2 is operably linked to an endogenous regulatory element at the endogenous TROP2 locus, and one or more cells of the animal expresses a human or chimeric TROP2. In some embodiments, the animal does not express endogenous TROP2 or expresses a decreased level of endogenous TROP2. In some embodiments, the animal has one or more cells expressing a human TROP2. In some embodiments, the animal has one or more cells expressing a chimeric TROP2 having all or part of the signal peptide, all or part of the extracellular region, all or part of the transmembrane region, and/or all or part of the cytoplasmic region of human TROP2. In some embodiments, the signal peptide of the chimeric TROP2 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, 23, 24, 25, or 26 contiguous amino acids that are identical to a contiguous sequence present in the signal peptide of human TROP2 (e.g., amino acids 1-26 of SEQ ID NO: 2) . In some embodiments, the extracellular region of the chimeric TROP2 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, 245, 246, 247, or 248 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human TROP2 (e.g., amino acids 27-274 of SEQ ID NO: 2) . In some embodiments, the transmembrane region of the chimeric TROP2 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 TROP2 (e.g., amino acids 275-297 of SEQ ID NO: 2) . In some embodiments, the cytoplasmic region of the chimeric TROP2 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, 23, 24, 25, or 26 contiguous amino acids that are identical to a contiguous sequence present in the cytoplasmic region of human TROP2 (e.g., amino acids 298-323 of SEQ ID NO: 2) . In some embodiments, the sequence encoding a region of endogenous TROP2 comprises exon 1 or a part thereof of the endogenous TROP2 gene. In some embodiments, the animal is a mouse. In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous TROP2 gene locus. In some embodiments, the animal is homozygous with respect to the replacement at the endogenous TROP2 gene locus.

In one aspect, 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 TROP2 gene locus, a sequence encoding a region of endogenous TROP2 with a sequence encoding a corresponding region of human TROP2. In some embodiments, the sequence encoding the corresponding region of human TROP2 comprises exon 1 or a part thereof of a human TROP2 gene. In some embodiments, the sequence encoding the corresponding region of human TROP2 comprises at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 910, 920, 930, 940, 950, 960, 970, 971, or 972 nucleotides of a human TROP2 gene exon 1. In some embodiments, the sequence encoding the corresponding region of human TROP2 encodes SEQ ID NO: 2. In some embodiments, the sequence encoding a region of endogenous TROP2 comprises exon 1 or a part thereof of the endogenous TROP2 gene. In some embodiments, the animal is a mouse, and the sequence encoding a region of endogenous TROP2 comprises at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 910, 920, 930, 940, 950, 951, 952, 953, or 954 nucleotides of the endogenous mouse TROP2 gene exon 1.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric TROP2 polypeptide, in some embodiments, the chimeric TROP2 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human TROP2, in some embodiments, the animal expresses the chimeric TROP2 polypeptide. In some embodiments, the chimeric TROP2 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 310, at least 315, at least 316, or at least 317 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human TROP2. In some embodiments, the chimeric TROP2 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to SEQ ID NO: 2. In some embodiments, the nucleotide sequence is operably linked to an endogenous TROP2 regulatory element (e.g., 5’ UTR and/or 3’ UTR) of the animal. In some embodiments, the nucleotide sequence is integrated to an endogenous TROP2 gene locus of the animal. In some embodiments, the chimeric TROP2 polypeptide has at least one mouse TROP2 activity and/or at least one human TROP2 activity.

In one aspect, the disclosure is related to a method of making a genetically-modified animal cell that expresses a human or chimeric TROP2, the method comprising: replacing at an endogenous TROP2 gene locus, a nucleotide sequence encoding a region of endogenous TROP2 with a nucleotide sequence encoding a corresponding region of human TROP2, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the human or chimeric TROP2, in some embodiments, the animal cell expresses the human or chimeric TROP2. In some embodiments, the animal is a mouse. In some embodiments, the nucleotide sequence encoding the human or chimeric TROP2 comprises: an endogenous 5’ UTR, a sequence encoding the human or chimeric TROP2, and an endogenous 3’ UTR. In some embodiments, the nucleotide sequence encoding the human or chimeric TROP2 is operably linked to an endogenous TROP2 regulatory region, e.g., promoter.

In some embodiments, the animal described herein further comprises a sequence encoding an additional human or chimeric protein. In some embodiments, the additional human or chimeric protein is erb-b2 receptor tyrosine kinase 2 (HER2) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L 1) , lymphocyte-activation gene 3 (LAG3) , TNF receptor superfamily member 9 (4-1BB) , TNF receptor superfamily Member 5 (CD40) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , interleukin 4 receptor (IL4R) , interleukin 6 receptor (IL6R) , interleukin 17A (IL17) , CD3, CD28 or CD38.

In one aspect, 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 described herein, in some embodiments, the animal has a tumor; and b) determining inhibitory effects of the therapeutic agent to the tumor. In some embodiments, the therapeutic agent is an anti-TROP2 antibody or an antibody-drug conjugate targeting TROP2. In some embodiments, the tumor comprises one or more cells that express TROP2. In some embodiments, the tumor comprises one or more cancer cells that are injected into the animal. In some embodiments, determining inhibitory effects of the anti-TROP2 antibody to the tumor involves measuring the tumor volume in the animal. In some embodiments, the cancer is breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancers, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, bladder cancer, or uterine cancer.

In one aspect, the disclosure is related to a method of determining effectiveness of a therapeutic agent targeting TROP2 and an additional therapeutic agent for the treatment of cancer, comprising a) administering the therapeutic agent targeting TROP2 and the additional therapeutic agent to the animal described herein, in some embodiments, the animal has a tumor; and b) determining inhibitory effects on the tumor. In some embodiments, the therapeutic agent targeting TROP2 is an anti-TROP2 antibody or an antibody-drug conjugate targeting TROP2. In some embodiments, the animal further comprises a sequence encoding a human or chimeric erb-b2 receptor tyrosine kinase 2 (HER2) . In some embodiments, the animal further comprises a sequence encoding a human or chimeric programmed cell death protein 1 (PD-1) . In some embodiments, the animal further comprises a sequence encoding a human or chimeric programmed death-ligand 1 (PD-L 1) . In some embodiments, the additional therapeutic agent is an HER2 antibody, an anti-PD-1 antibody or an anti-PD-L 1 antibody. In some embodiments, the tumor comprises one or more tumor cells that express TROP2 or HER2. In some embodiments, the tumor comprises one or more tumor cells that express TROP2 or PD-L1. In some embodiments, the tumor is caused by injection of one or more cancer cells into the animal. In some embodiments, determining inhibitory effects of the treatment involves measuring the tumor volume in the animal. In some embodiments, the animal has breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancers, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, bladder cancer, or uterine cancer.

In one aspect, the disclosure is related to a method of determining toxicity of a therapeutic agent comprising: a) administering the therapeutic agent to the animal described herein; and b) determining effects of the therapeutic agent to the animal. In some embodiments, the therapeutic agent is an anti-TROP2 antibody or an antibody-drug conjugate targeting TROP2. In some embodiments, 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.

In one aspect, the disclosure is related to a protein comprising an amino acid sequence, in some embodiments, the amino acid sequence is one of the following: (a) an amino acid sequence set forth in SEQ ID NO: 1 or 2; (b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 1 or 2; (c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1 or 2; (d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and (e) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 1 or 2.

In one aspect, the disclosure is related to a nucleic acid comprising a nucleotide sequence, in some embodiments, the nucleotide sequence is one of the following: (a) a sequence that encodes the protein described herein; (b) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10; (c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10; and (d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10.

In one aspect, the disclosure is related to a cell comprising the protein and/or the nucleic acid as described herein.

In one aspect, the disclosure is related to an animal comprising the protein and/or the nucleic acid as described herein.

In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous TROP2 gene, wherein the disruption of the endogenous TROP2 gene comprises deletion of exon 1 or part thereof of the endogenous TROP2 gene.

In some embodiments, the disruption of the endogenous TROP2 gene comprises deletion of a portion of exon 1 (e.g., coding sequence) of the endogenous TROP2 gene.

In some embodiments, wherein the 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, 950, 951, 952, 953, 954 or more nucleotides from exon 1 of the endogenous TROP2 gene.

The disclosure further relates to a TROP2 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 TROP2 gene function, human TROP2 antibodies, the drugs or efficacies for human TROP2 targeting sites, and the drugs for immune-related diseases and antitumor drugs.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing mouse and human TROP2 gene loci.

FIG. 2 is a schematic diagram showing humanized TROP2 gene locus.

FIG. 3 is a schematic diagram showing a TROP2 gene targeting strategy using targeting vector V1.

FIG. 4 shows genotyping results of F 1 generation mice by primer pairs Mut-F and WT-R. PC is a positive control. WT is a wild-type control. H2O is a water control.

FIG. 5 is a schematic diagram showing a TROP2 gene targeting strategy using targeting vector V2.

FIG. 6 shows genotyping results of F0 generation mice by primer pairs L-GT-F/L-GT-R and R-GT-F/R-GT-R, respectively. WT is a wild-type control. H2O is a water control.

FIG. 7 shows genotyping results of F 1 generation mice by primer pairs L-GT-F/L-GT-R and R-GT-F/R-GT-R, respectively. WT is a wild-type control. H2O is a water control.

FIG. 8 shows Southern Blot results of cells after recombination using the 5’ Probe and 3’ Probe. WT is a wild-type control.

FIGS. 9A-9C show RT-PCR detection results of mouse TROP2 (mTROP2) mRNA, humanized TROP2 (hTROP2) mRNA, and GAPDH mRNA, respectively, in skin tissues of a wild-type C57BL/6 mouse (+/+) and a TROP2 gene humanized homozygous mouse (H/H) . H2O is a water control. GAPDH is an internal reference.

FIG. 10 shows Western Blot results of mouse TROP2 (mTROP2) protein, human TROP2 (hTROP2) protein, and β-actin in skin and kidney tissues of a wild-type C57BL/6 mouse (+/+) and a TROP2 gene humanized homozygous mouse (H/H) . M is a marker.

FIG. 11 shows mouse tail PCR detection results of TROP2 gene knockout mice. WT is a wild-type control. H2O is a water control.

FIG. 12 shows the percentages of leukocyte subtypes in the spleen of C57BL/6 wild-type mice (+/+) and TROP2 gene humanized homozygous mice (H/H) .

FIG. 13 shows the percentages of T cell subtypes in the spleen of C57BL/6 wild-type mice (+/+) and TROP2 gene humanized homozygous mice (H/H) .

FIG. 14 shows the percentages of leukocyte subtypes in the lymph nodes of C57BL/6 wild-type mice (+/+) and TROP2 gene humanized homozygous mice (H/H) .

FIG. 15 shows the percentages oft cell subtypes in the lymph nodes of C57BL/6 wild-type mice (+/+) and TROP2 gene humanized homozygous mice (H/H) .

FIG. 16 shows the percentages of leukocyte subtypes in the peripheral blood of C57BL/6 wild-type mice (+/+) and TROP2 gene humanized homozygous mice (H/H) .

FIG. 17 shows the percentages of T cell subtypes in the peripheral blood of C57BL/6 wild-type mice (+/+) and TROP2 gene humanized homozygous mice (H/H) .

FIG. 18 shows the alignment between human TROP2 amino acid sequence (NP_002344.2; SEQ ID NO: 2) and mouse TROP2 amino acid sequence (NP_064431.2; SEQ ID NO: 1) .

FIG. 19 shows the alignment between human TROP2 amino acid sequence (NP_002344.2; SEQ ID NO: 2) and rat TROP2 amino acid sequence (NP_001009540.2; SEQ ID NO: 32.

FIG. 20 shows average body weight in different groups of mice that were administered with physiological saline, MMAE or Ab1.

FIG. 21 shows the average body weight changes in different groups of mice that were administered with physiological saline, MMAE or Ab1.

DETAILED DESCRIPTION

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) TROP2, and methods of use thereof.

TROP2 is a protein closely related to tumors. It is an intracellular calcium signal transducer that is differentially expressed in many cancers. It signals cells for self-renewal, proliferation, invasion, and survival. It has stem cell-like qualities. TROP2 is expressed in many normal tissues, though in contrast, it is overexpressed in many cancers and the overexpression of TROP2 is of prognostic significance. Therefore, TROP2 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-TROP2 antibodies) . Common experimental animals include mice, rats, guinea pigs, hamsters, rabbits, dogs, monkeys, pigs, fish and so on. However, 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. With the continuous development and maturation of genetic engineering technologies, 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. In this context, 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.

TROP2

Trophoblast cell-surface antigen 2 (TROP2) , also known as Tumor-associated calcium signal transducer 2 (TACSTD2) , is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene. It has high structural sequence similarity with epithelial adhesion molecule Epcam. TROP2 is a protein closely related to tumors. It mainly promotes tumor cell growth, proliferation and metastasis by regulating calcium ion signaling pathways, cyclin expression, and reducing fibronectin adhesion. Studies have found that TROP2 protein is highly expressed in breast cancer, colon cancer, bladder cancer, gastric cancer, oral squamous cell carcinoma and ovarian cancer. The protein can promote tumor cell proliferation, invasion, metastasis, spread and other processes. In addition, in breast cancer and other cancers, the high expression of TROP2 has also been found to be closely related to more aggressive diseases and poor clinical prognosis of tumors.

TROP2 has been implicated in numerous intracellular signaling pathways. TROP2 transduces an intracellular calcium signal. Trop2-induced signal transduction can occur without extracellular Ca2+, suggesting a mobilization of Ca2+ from internal stores. Specific antibodies have been used for cross-linking TROP2. TROP2 provides crucial signals for cells with requirements for proliferation, survival, self-renewal, and invasion. TROP2 has several ligands, inlcluding claudin-1, claudin-7, cyclin D1, and IGF-1. Trop2 has stem cell-like qualities and regulates cell growth, transformation, regeneration, and proliferation, which explains why its overexpression can lead to tumor progression. It is expressed on the surface of many stem/progenitor cells and has a role in maintaining tight junction integrity.

A detailed description of TROP2 and its function can be found, e.g., in Lenárt, S., et al. "Trop2: Jack of all trades, master of none. " Cancers 12.11 (2020) : 3328; Goldenberg, D M., et al. "The emergence of trophoblast cell-surface antigen 2 (TROP-2) as a novel cancer target. " Oncotarget 9.48 (2018) : 28989; and Shvartsur, A., et al. "Trop2 and its overexpression in cancers: regulation and clinical/therapeutic implications. " Genes &Cancer 6.3-4 (2015) : 84; each of which is incorporated by reference in its entirety.

In human genomes, TROP2 gene (Gene ID: 4070) locus has one exon: exon 1 (FIG. 1) . The TROP2 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 TROP2 mRNA is NM_002353.3, and the amino acid sequence for human TROP2 is NP_002344.2 (SEQ ID NO: 2) . The location for exon 1 and each region in human TROP2 nucleotide sequence and amino acid sequence is listed below:

Table 1

The human TROP2 gene (Gene ID: 4070) is located in Chromosome 1 of the human genome, which is located from 58575433 to 58577252 (GRCh38. p13 (GCF_000001405.39) ) . The 5’-UTR is from 58577157 to 58577252, exon 1 is from 58,577,252 to 58,575,433, and the 3’-UTR is from 58575433 to 58576184, based on transcript NM_002353.3. All relevant information for human TROP2 locus can be found in the NCBI website with Gene ID: 4070, which is incorporated by reference herein in its entirety. According to UniProt ID: P09758, the extracellular region of human TROP2 includes a thyroglobulin type-1 domain, which corresponds to amino acids 70-145 of SEQ ID NO: 2.

In mice, TROP2 gene locus has one exon: exon 1 (FIG. 1) . The mouse TROP2 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 TROP2 mRNA is NM_020047.3, the amino acid sequence for mouse TROP2 is NP_064431.2 (SEQ ID NO: 1) . The location for exon 1 and each region in the mouse TROP2 nucleotide sequence and amino acid sequence is listed below:

Table 2

The mouse TROP2 gene (Gene ID: 56753) is located in Chromosome 6 of the mouse genome, which is located from 67511043 to 67512806 (GRCm39 (GCF_000001635.27) ) . The 5’-UTR is from 67512691 to 67512780, exon 1 is from 67,511,046 to 67,512,780, and the 3’-UTR is from 67511046 to 67511736, based on transcript NM_020047.3. All relevant information for mouse TROP2 locus can be found in the NCBI website with Gene ID: 56753, which is incorporated by reference herein in its entirety.

FIG. 18 shows the alignment between human TROP2 amino acid sequence (NP_002344.2; SEQ ID NO: 2) and mouse TROP2 amino acid sequence (NP_064431.2; SEQ ID NO: 1) . Thus, the corresponding amino acid residue or region between human and mouse TROP2 can be found in FIG. 18.

TROP2 genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for TROP2 (TACSTD2) in Rattus norvegicus (rat) is 494343, the gene ID for TROP2 in Macaca mulatta (Rhesus monkey) is 716334, the gene ID for TROP2 in Canis lupus familiaris (dog) is 610286, and the gene ID for TROP2 in Sus scrofa (pig) is 100510966. The relevant information for these genes (e.g., intron sequences, exon sequences, amino acid residues of these proteins) can be found, e.g., in NCBI database, which is incorporated by reference herein in its entirety. FIG. 19 shows the alignment between human TROP2 amino acid sequence (NP_002344.2; SEQ ID NO: 2) and rat TROP2 amino acid sequence (NP_001009540.2; SEQ ID NO: 32. Thus, the corresponding amino acid residue or region between human and rodent TROP2 can be found in FIG. 19.

The present disclosure provides human or chimeric (e.g., humanized) TROP2 nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence. In some embodiments, a “region” or “portion” of mouse exon 1, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence. The term “region” or “portion” 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, or 950 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, or 310 amino acid residues. In some embodiments, the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, signal peptide, extracellular region, transmembrane region, or cytoplasmic region. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1 (e.g., the coding sequence) are replaced by a region, a portion, or the entire sequence of the human exon 1 (e.g., the coding sequence) .

In some embodiments, a “region” or “portion” of the signal peptide, extracellular region, transmembrane region, cytoplasmic region, or exon 1 (e.g., the coding sequence) is deleted.

In some embodiments, the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a chimeric (e.g., humanized ) TROP2 nucleotide sequence. In some embodiments, the chimeric (e.g., humanized ) TROP2 nucleotide sequence encodes a TROP2 protein comprising a signal peptide, an extracellular region, a transmembrane region, and/or a cytoplasmic region. In some embodiments, the signal peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-26 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises all or part of human TROP2 signal peptide. In some embodiments, the extracellular region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 27-274 of SEQ ID NO: 2. In some embodiments, the extracellular region comprises all or part of human TROP2 extracellular region. In some embodiments, the transmembrane region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 275-297 of SEQ ID NO: 2. In some embodiments, the transmembrane region comprises all or part of human TROP2 transmembrane region. In some embodiments, the cytoplasmic region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids amino acids 298-323 of SEQ ID NO: 2. In some embodiments, the cytoplasmic region comprises all or part of human TROP2 cytoplasmic region. In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized TROP2 protein. In some embodiments, the TROP2 protein comprises, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized TROP2 protein comprises a human or humanized extracellular region. In some embodiments, the humanized TROP2 protein comprises an endogenous extracellular region. In some embodiments, the humanized TROP2 protein comprises a human or humanized transmembrane region. In some embodiments, the humanized TROP2 protein comprises an endogenous transmembrane region. In some embodiments, the humanized TROP2 protein comprises a human or humanized cytoplasmic region. In some embodiments, the humanized TROP2 protein comprises an endogenous cytoplasmic region. In some embodiments, the humanized TROP2 protein comprises a human or humanized signal peptide. In some embodiments, the humanized TROP2 protein comprises an endogenous signal peptide.

In some embodiments, the humanized TROP2 protein comprises a human or humanized thyroglobulin type-1 domain. In some embodiments, the humanized TROP2 protein comprises an endogenous thyroglobulin type-1 domain.

In some embodiments, the genetically-modified non-human animal described herein comprises a human or humanized TROP2 gene. In some embodiments, the humanized TROP2 gene comprises human or humanized TROP2 exon 1. In some embodiments, the humanized TROP2 gene comprises endogenous TROP2 exon 1. In some embodiments, the humanized TROP2 gene comprises human or humanized 5’ UTR. In some embodiments, the humanized TROP2 gene comprises human or humanized 3’ UTR. In some embodiments, the humanized TROP2 gene comprises endogenous 5’ UTR. In some embodiments, the humanized TROP2 gene comprises endogenous 3’ UTR.

Thus, in some embodiments, the present disclosure also provides a chimeric (e.g., humanized) TROP2 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 TROP2 mRNA sequence (e.g., NM_020047.3) , mouse TROP2 amino acid sequence (e.g., SEQ ID NO: 1) , or a portion thereof (e.g., 5’ UTR and 3’ UTR) ; and 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 human TROP2 mRNA sequence (e.g., NM_002353.3) , human TROP2 amino acid sequence (e.g., SEQ ID NO: 2) , or a portion thereof (e.g., the coding sequence within exon 1) .

In some embodiments, the sequence encoding amino acids 1-317 of mouse TROP2 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human TROP2 (e.g., amino acids 1-323 of human TROP2 (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding amino acids 25-317 of mouse TROP2 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human TROP2 (e.g., amino acids 27-323 of human TROP2 (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding amino acids 25-270 of mouse TROP2 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human TROP2 (e.g., amino acids 27-274 of human TROP2 (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding amino acids 1-270 of mouse TROP2 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human TROP2 (e.g., amino acids 1-274 of human TROP2 (SEQ ID NO: 2) ) .

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse TROP2 promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, 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 TROP2 nucleotide sequence (e.g., the coding sequence of exon 1 in NM_020047.3) .

In some embodiments, 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 TROP2 nucleotide sequence (e.g., 5’ UTR and 3’ UTR of exon 1 in NM_020047.3) .

In some embodiments, 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 TROP2 nucleotide sequence (e.g., 5’ UTR and 3’ UTR of exon 1 in NM_002353.3) .

In some embodiments, 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 TROP2 nucleotide sequence (e.g., the coding sequence of exon 1 in NM_002353.3) .

In some embodiments, 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 TROP2 amino acid sequence (e.g., NP_064431.2 (SEQ ID NO: 1) ) .

In some embodiments, 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 TROP2 amino acid sequence (e.g., NP_064431.2 (SEQ ID NO: 1) ) .

In some embodiments, 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 TROP2 amino acid sequence (e.g., NP_002344.2 (SEQ ID NO: 2) ) .

In some embodiments, 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 TROP2 amino acid sequence (e.g., NP_002344.2 (SEQ ID NO: 2) ) .

The present disclosure also provides a humanized TROP2 mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) an amino acid sequence shown in SEQ ID NO: 1 or 2;

b) an amino acid sequence having a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

c) 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 or 2 under a low stringency condition or a strict stringency condition;

d) an 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 or 2;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

f) an 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 or 2.

The present disclosure also provides a humanized TROP2 amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) all or part of SEQ ID NO: 2;

b) an amino acid sequence have a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%to SEQ ID NO: 2;

c) an amino acid sequence that is different from SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and

d) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one or more amino acids to SEQ ID NO: 2.

The present disclosure also relates to a TROP2 nucleic acid (e.g., DNA or RNA) sequence, wherein the nucleic acid sequence can be selected from the group consisting of:

a) a nucleic acid sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10, or a nucleic acid sequence encoding a homologous TROP2 amino acid sequence of a humanized mouse TROP2;

b) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10 under a low stringency condition or a strict stringency condition;

c) a 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, 8, 9, or 10;

d) a 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 or 2;

e) a 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 or 2;

f) a 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 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

g) a 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 or 2.

The present disclosure further relates to a TROP2 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: 7 or 10.

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 or 2, and has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 1 or 2 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, 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%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 1 or 2 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: 7 or 10, and encodes a polypeptide that has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 7 or 10 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, 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. For example, 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 (percent homology) , e.g. leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include 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) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . The homology percentage, in many cases, is higher than the identity percentage.

Cells, tissues, and animals (e.g., mouse) 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) TROP2 from an endogenous non-human TROP2 locus.

Genetically modified animals

As used herein, 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. In some embodiments, 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. In some embodiments, genetically-modified non-human animals are provided that comprise a modified endogenous TROP2 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.

As used herein, the term “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. In some embodiments, 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. In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized gene or humanized nucleic acid.

As used herein, the term “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. In some embodiments, 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. In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized protein or a humanized polypeptide.

As used herein, the term “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.

As used herein, the term “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.

In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized TROP2 gene or a humanized TROP2 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human TROP2 gene, at least one or more portions of the gene or the nucleic acid is from a non-human TROP2 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an TROP2 protein. In some embodiments, the encoded TROP2 protein is functional or has at least one activity of the human TROP2 protein or the non-human TROP2 protein, e.g., binding to factors such as IGF-1, claudin-1 and 7, cyclin D1, or PKC; regulating normal fetal lung growth; activating CREB1 (cyclic AMP-responsive element binding protein) , Jun, NF-κB, Rb, STAT 1 and STAT3 through induction of the cyclin D1 and the ERK (extracellular signal regulated kinase) /MEK (MAPK/ERK kinase) pathways; activating ERK1/3-MAPK pathways; deregulating characteristic stem cell proliferation and differentiation pathways such as Notch, hedgehog, and Wnt; stimulating tumor cell proliferation and growth; and promoting the metastasis of tumors.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized TROP2 protein or a humanized TROP2 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 TROP2 protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human TROP2 protein. The humanized TROP2 protein or the humanized TROP2 polypeptide is functional or has at least one activity of the human TROP2 protein or the non-human TROP2 protein.

In some embodiments, the cytoplasmic region is human or humanized. In some embodiments, the transmembrane region is human or humanized. In some embodiments, the extracellular region is human or humanized. In some embodiments, the thyroglobulin type-1 domain is human or humanized.

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) . For the non-human animals where suitable genetically modifiable embryonic stem (ES) cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. 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. These methods are known in the art, and are described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003, which is incorporated by reference herein in its entirety.

In one aspect, the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiments, 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) . In some embodiments, the genetically modified rodent is selected from a true mouse or rat (family Muridae) , a gerbil, a spiny mouse, and a crested rat. In some embodiments, the non-human animal is a mouse.

In some embodiments, 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. In some embodiments, 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. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999) ; Auerbach et al., Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000) , both of which are incorporated herein by reference in the entirety. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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 TROP2 animal is made. For example, suitable mice for maintaining a xenograft (e.g., a human cancer or tumor) , 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, IL2Rγknockout mice, NOD/SCID/γc ^null mice (Ito, M. et al., NOD/SCID/γc ^null mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100 (9) : 3175-3182, 2002) , nude mice, and Rag1 and/or Rag2 knockout mice. These mice can optionally be irradiated, or otherwise treated to destroy one or more immune cell type. Thus, in various embodiments, a genetically modified mouse is provided that can include a humanization of at least a portion of an endogenous non-human TROP2 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. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD mice, SCID mice, NOD/SCID mice, IL-2Rγ knockout mice, NOD/SCID/γc ^null mice, nude mice, Ragl and/or Rag2 knockout mice, NOD-Prkdc ^scid IL-2rγ ^null mice, NOD-Rag 1 ^-/--IL2rg ^-/- (NRG) mice, Rag 2 ^-/--IL2rg ^-/- (RG) mice, and a combination thereof. These genetically modified animals are described, e.g., in US20150106961, which is incorporated herein by reference in its entirety. In some embodiments, the mouse can include a replacement of all or part of mature TROP2 coding sequence with human mature TROP2 coding sequence.

Genetically modified non-human animals can comprise a modification at an endogenous non-human TROP2 locus. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature TROP2 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the mature TROP2 protein sequence) . Although genetically modified cells are also provided that can comprise the modifications described herein (e.g., ES cells, somatic cells) , in many embodiments, the genetically modified non-human animals comprise the modification of the endogenous TROP2 locus in the germline of the animal.

Genetically modified animals can express a human TROP2 and/or a chimeric (e.g., humanized) TROP2 from endogenous mouse loci, wherein the endogenous mouse TROP2 gene has been replaced with a human TROP2 gene and/or a nucleotide sequence that encodes a region of human TROP2 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 TROP2 sequence. In various embodiments, an endogenous non-human TROP2 locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature TROP2 protein.

In some embodiments, the genetically modified mice can express the human TROP2 and/or chimeric TROP2 (e.g., humanized TROP2) 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 TROP2 or chimeric TROP2 (e.g., humanized TROP2) 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 TROP2 or the chimeric TROP2 (e.g., humanized TROP2) expressed in animal can maintain one or more functions of the wild-type mouse or human TROP2 in the animal. For example, the expressed TROP2 can transduce an intracellular calcium signal. Furthermore, in some embodiments, the animal does not express endogenous TROP2. In some embodiments, the animal expresses a decreased level of endogenous TROP2 as compared to a wild-type animal. As used herein, the term “endogenousTROP2” referstoTROP2protein that is expressed from an endogenous TROP2 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 TROP2 (NP_002344.2) (SEQ ID NO: 2) . In some embodiments, the genome comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 7 or 10.

The genome of the genetically modified animal can comprise a replacement at an endogenous TROP2 gene locus of a sequence encoding a region of endogenous TROP2 with a sequence encoding a corresponding region of human TROP2. In some embodiments, the sequence that is replaced is any sequence within the endogenous TROP2 gene locus, e.g., exon 1, 5’-UTR, 3’-UTR, or any combination thereof. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous TROP2 gene. In some embodiments, the sequence that is replaced is the coding sequence (CDS) , or a portion thereof, of an endogenous mouse TROP2 gene locus.

The genetically modified animal can have one or more cells expressing a human or chimeric TROP2 (e.g., humanized TROP2) having, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human TROP2. In some embodiments, the extracellular region of the humanized TROP2 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, 245, 246, 247, or 248 amino acids (e.g., contiguously or non-contiguously) that are identical to the extracellular region of human TROP2. In some embodiments, the extracellular region comprises at least 100 amino acids that are identical to the extracellular region of human TROP2.

In some embodiments, the transmembrane comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the transmembrane region of human TROP2. In some embodiments, the transmembrane region of the humanized TROP2 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 TROP2. In some embodiments, the transmembrane region comprises at least 10 amino acids that are identical to the transmembrane region of human TROP2. In some embodiments, the cytoplasmic comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the cytoplasmic of human TROP2. In some embodiments, the cytoplasmic region of the humanized TROP2 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, 23, 24, 25, or 26 amino acids (contiguously or non-contiguously) that are identical to the cytoplasmic region of human TROP2. In some embodiments, the cytoplasmic region of the humanized TROP2 has a sequence that has at least 10 amino acids that are identical to the cytoplasmic region of human TROP2. In some embodiments, the signal peptide comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the signal peptide of human TROP2. In some embodiments, the signal peptide of the humanized TROP2 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, 23, 24, 25, or 26 amino acids (contiguously or non-contiguously) that are identical to the signal peptide of human TROP2. In some embodiments, the signal peptide of the humanized TROP2 has a sequence that has at least 10 amino acids that are identical to the signal peptide of human TROP2. In some embodiments, the humanized TROP2 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, 321, 322, or 323 amino acids (e.g., contiguously or non-contiguously) that are identical to human TROP2 (e.g., SEQ ID NO: 2) .

In some embodiments, the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to a portion or the entire sequence of exon 1 of human TROP2 gene; a portion or the entire sequence of the signal peptide, 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 TROP2; or a portion or the entire sequence of amino acids 1-323 of SEQ ID NO: 2.

In some embodiments, the genome of the genetically modified animal comprises a portion of exon 1 (e.g., the coding sequence (CDS) ) of human TROP2 gene. In some embodiments, the portion of exon 1 includes at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 910, 920, 930, 940, 950, 960, 970, 971, 972, 973, 974, 975, 1000, 1500, 1800, or 1820 nucleotides. In some embodiments, the portion of exon 1 includes at least 200 nucleotides from human TROP2 gene exon 1. In some embodiments, the portion of exon 1 includes 972 nucleotides (including STOP codon) .

In some embodiments, the non-human animal can have, at an endogenous TROP2 gene locus, a nucleotide sequence encoding a chimeric human/non-human TROP2 polypeptide, and the animal expresses a functional TROP2 on a surface of a cell of the animal. The human portion of the chimeric human/non-human TROP2 polypeptide can comprise an amino acid sequence encoded by a portion of exon 1 of human TROP2. In some embodiments, the chimeric human/non-human TROP2 polypeptide comprises a signal peptide, which includes a sequence corresponding to the entire or part of amino acids 1-24 of SEQ ID NO: 1. In some embodiments, the non-human portion of the chimeric human/non-human TROP2 polypeptide comprises a transmembrane region and a cytoplasmic region of an endogenous non-human TROP2 polypeptide.

In some embodiments, the non-human animal can have, at an endogenous TROP2 gene locus, a chimeric human/non-human TROP2 gene encoding a human TROP2 polypeptide, and the animal expresses the human TROP2 polypeptide on a surface of a cell of the animal.

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous TROP2 locus, or homozygous with respect to the replacement at the endogenous TROP2 locus.

In some embodiments, the humanized TROP2 locus lacks a human TROP2 5’-UTR. In some embodiment, the humanized TROP2 locus comprises an endogenous (e.g., mouse) 5’-UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3’-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human TROP2 genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence. As shown in the present disclosure, humanized TROP2 mice that comprise a replacement at an endogenous mouse TROP2 locus, which retain mouse regulatory elements but comprise a humanization of TROP2 encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized TROP2 are grossly normal.

The present disclosure further relates to a non-human mammal generated through the method mentioned above. In some embodiments, the genome thereof contains human gene (s) .

In some embodiments, the non-human mammal is a rodent, and preferably, the non-human mammal is a mouse.

In some embodiments, the non-human mammal expresses a protein encoded by a humanized TROP2 gene.

In addition, the present disclosure also relates to a tumor beating non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein. In some embodiments, 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 beating 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 beating non-human mammal.

The present disclosure also provides non-human mammals produced by any of the methods described herein. In some embodiments, a non-human mammal is provided; and the genetically modified animal contains the DNA encoding human or humanized TROP2 in the genome of the animal.

In some embodiments, the non-human mammal comprises the genetic construct as described herein (e.g., gene construct as shown in FIGS. 2, 3, and 5) . In some embodiments, a non-human mammal expressing human or humanized TROP2 is provided. In some embodiments, the tissue-specific expression of human or humanized TROP2 protein is provided.

In some embodiments, the expression of human or humanized TROP2 in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance. In some embodiments, 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) . In some embodiments, the non-human mammal is a mouse.

Genetic, molecular and behavioral analyses for the non-human mammals described above can performed. 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 TROP2 protein can be detected by a variety of methods.

There are many analytical methods that can be used to detect exogenous DNA, including methods at the level of nucleic acid (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies) . In addition, 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 TROP2 protein.

Vectors

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 TROP2 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 TROP2 gene genomic DNAs in the length of 100 to 10,000 nucleotides.

In some embodiments, a) the DNA fragment homologous to the 5’ end ofa 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_000072.7; 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_000072.7.

In some embodiments, 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 67512691 to the position 67516273 of the NCBI accession number NC_000072.7; 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 67506222 to the position 67510723 of the NCBI accession number NC_000072.7.

In some embodiments, 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 67512691 to the position 67514056 of the NCBI accession number NC_000072.7; 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 67510349 to the position 67511736 of the NCBI accession number NC_000072.7.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 910 bp, 920 bp, 930 bp, 940 bp, 950 bp, 960 bp, 970 bp, 971 bp, or 972 bp.

In some embodiments, the region to be altered is exon 1 of TROP2 gene (e.g., the coding sequence of mouse TROP2 gene) .

The targeting vector can further include one or more selectable markers, e.g., positive or negative selectable markers. In some embodiments, the positive selectable marker is a Neo gene or Neo cassette. In some embodiments, the negative selectable marker is a DTA gene.

In some embodiments, the 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. In some embodiments, the sequence of the 5’ arm is shown in SEQ ID NO: 5; and the sequence of the 3’ arm is shown in SEQ ID NO: 6.

In some embodiments, the sequence is derived from human (e.g., 97-1068 of NM_002353.3) . For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human TROP2 gene exon 1. In some embodiments, the nucleotide sequence of the humanized TROP2 encodes the entire or the part of human TROP2 protein with the NCBI accession number NP_002344.2 (SEQ ID NO: 2) .

The disclosure also provides vectors for constructing a humanized animal model or a knock-out model. In some embodiments, the vectors comprise sgRNA sequence, wherein the sgRNA sequence target TROP2 gene, and the sgRNA is unique on the target sequence of the gene to be altered, and meets the sequence arrangement rule of 5’-NNN (20) -NGG3’ or 5’-CCN-N (20) -3’; and in some embodiments, the targeting site of the sgRNA in the mouse TROP2 gene is located on the exon 1, upstream of exon 1, or downstream of exon 1 of the mouse TROP2 gene. In some embodiments, the sgRNAs target exon 1.

In some embodiments, the targeting sequences are shown as SEQ ID NO: 13 and SEQ ID NO: 14. Thus, the disclosure provides sgRNA sequences for constructing a genetic modified animal model. In some embodiments, the oligonucleotide sgRNA sequences are set forth in SEQ ID NOS: 13 and 14. In some embodiments, the oligonucleotide sgRNA sequences targeting 5’ end of the endogenous TROP2 gene are set forth in SEQ ID NO: 13. In some embodiments, the oligonucleotide sgRNA sequences targeting 3’ end of the endogenous TROP2 gene are set forth in SEQ ID NO: 14.

In some embodiments, the disclosure relates to a plasmid construct (e.g., pT7-sgRNA) including the sgRNA sequence, and/or a cell including the construct.

The disclosure also relates to a cell comprising the targeting vectors as described above.

In addition, 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. In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.

In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.

In some embodiments, 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.

Methods of making genetically modified animals

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. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these 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.

Thus, in some embodiments, the disclosure provides replacing in at least one cell of the animal, at an endogenous TROP2 gene locus, a sequence encoding a region of an endogenous TROP2 with a sequence encoding a corresponding region of human TROP2. In some embodiments, the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.

FIG. 3 and FIG. 5 show a humanization strategy for a mouse TROP2 locus. In FIG. 3 and FIG. 5, the targeting strategy involves a vector comprising the 5’ end homologous arm, human TROP2 gene fragment, 3’ homologous arm. The process can involve replacing endogenous TROP2 sequence with human sequence by homologous recombination. In some embodiments, the cleavage at the upstream and the downstream of the target site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strands break, and the homologous recombination is used to replace endogenous TROP2 sequence with human TROP2 sequence.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous TROP2 locus (or site) , a nucleic acid encoding a region of endogenous TROP2 with a sequence encoding a corresponding region of human TROP2. The sequence can include a region (e.g., a part or the entire region) of exon 1 of a human TROP2 gene. In some embodiments, the sequence includes the coding sequence of a human TROP2 gene (e.g., nucleic acids 97-1068 of NM_002353.3) . In some embodiments, the endogenous TROP2 locus is exon 1 of mouse TROP2. In some embodiments, the sequence includes a portion of exon 1 of mouse TROP2 gene (e.g., nucleic acids 117-1070 of NM_020047.3) .

In some embodiments, the methods of modifying a TROP2 locus of a mouse to express a human TROP2 or a chimeric human/mouse TROP2 peptide can include the steps of replacing at the endogenous mouse TROP2 locus a nucleotide sequence encoding a mouse TROP2 with a nucleotide sequence encoding a human TROP2, thereby generating a sequence encoding human TROP2 or a chimeric human/mouse TROP2.

In some embodiments, the nucleotide sequence encoding the human TROP2 can include a first nucleotide sequence (e.gr5’ UTR) of mouse TROP2; a second nucleotide sequence (e.g., CDS) encoding human TROP2; and/or a third nucleotide sequence (e.g., 3’ UTR) of mouse TROP2.

In some embodiments, 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) . In some embodiments, the amino acid sequences as described herein do not overlap with each other.

The present disclosure further provides a method for establishing a TROP2 gene humanized animal model, involving the following steps:

(a) providing the cell (e.g. a fertilized egg cell) based on the methods described herein;

(b) culturing the cell in a liquid culture medium;

(c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;

(d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .

In some embodiments, the non-human mammal in the foregoing method is a mouse (e.g., a C57BL/6 mouse) .

In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or false pregnancy) .

In some embodiments, 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. In some embodiments, 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.

In some embodiments, methods of making the genetically modified animal comprises modifying the coding frame of the non-human animal’s TROP2 gene, e.g., by inserting a nucleotide sequence (e.g., DNA or cDNA sequence) encoding human or humanized TROP2 protein immediately after the endogenous regulatory element of the non-human animal’s TROP2 gene. For example, one or more functional region sequences of the non-human animal’s TROP2 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 TROP2 protein. In some embodiments, the coding frame of the modified non-human animal’s TROP2 gene can be all or part of the nucleotide sequence (e.g., exon 1) of the non-human animal’s TROP2 gene.

In some embodiments, methods of making the genetically modified animal comprises inserting a nucleotide sequence encoding human or humanized TROP2 protein and/or an auxiliary sequence after the endogenous regulatory element of the non-human animal’s TROP2 gene. In some embodiments, the auxiliary sequence can be a stop codon, such that the TROP2 gene humanized animal model can express human or humanized TROP2 protein in vivo, but does not express non-human animal’s TROP2 protein. In some embodiments, the auxiliary sequence includes WPRE (WHP Posttranscriptional Response Element) , STOP, and/or polyA (e.g., SV40 polyA, or BGH polyA) . In some embodiments, the auxiliary sequence is a sequence that can terminate transcription and/or translation of the inserted nucleotide sequence.

In some embodiments, the method for making the genetically modified animal comprises:

(1) providing a plasmid comprising a human TROP2 gene fragment, flanked by a 5’ homologous arm and a 3’ homologous arm, wherein the 5’ and 3’ homology arms target an endogenous TROP2 gene;

(2) providing two small guide RNAs (sgRNAs) that target the endogenous TROP2 gene;

(3) modifying genome of a fertilized egg or an embryonic stem cell by using the plasmid of step (1) , the sgRNAs of step (2) , and Cas9;

(4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudopregnant female mouse or transplanting the embryonic stem cell obtained in step (3) into a blastocyst which is then transplanted into the oviduct of a pseudopregnant female mouse to produce a child mouse that functionally expresses a humanized TROP2 protein; and

(5) mating the child mouse obtained in step (2) to obtain a homozygote mouse,

In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs that target a 5’-terminal targeting site and a 3’-terminal targeting site.

In some embodiments, the sequence encoding the humanized TROP2 protein is operably linked to an endogenous regulatory element at the endogenous TROP2 gene locus.

In some embodiments, the genetically-modified animal does not express an endogenous TROP2 protein.

(1) providing a plasmid comprising a human or chimeric TROP2 gene fragment, flanked by a 5’ homologous arm and a 3’ homologous arm, wherein the 5’ and 3’ homology arms target an endogenous TROP2 gene;

(2) providing one small guide RNAs (sgRNAs) that target the endogenous TROP2 gene; and

(3) modifying genome of a fertilized egg or an embryonic stem cell by inserting the human or chimeric TROP2 gene fragment into the genome.

Methods of using genetically modified animals

Replacement of non-human genes in a non-human animal with homologous or orthologous human genes or human sequences, at the endogenous non-human locus and under control of endogenous promoters and/or regulatory elements, can result in a non-human animal with qualities and characteristics that may be substantially different from a typical knockout-plus-transgene animal. In the typical knockout-plus-transgene animal, an endogenous locus is removed or damaged and a fully human transgene is inserted into the animal's genome and presumably integrates at random into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of the human gene and/or protein assay and/or functional assay. Inclusion in the human transgene of upstream and/or downstream human sequences are apparently presumed to be sufficient to provide suitable support for expression and/or regulation of the transgene.

In some cases, 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 TROP2 protein, e.g., in a physiologically appropriate manner, 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.

In various aspects, genetically modified animals are provided that express human or humanized TROP2, which are useful for testing agents that can decrease or block the interaction between the interaction between TROP2 and anti-human TROP2 antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an TROP2 agonist or antagonist. The genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (a knock-in or knockout) . In various embodiments, 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) . In some embodiments, the anti-TROP2 antibody blocks or inhibits the TROP2-related signaling pathway.

In some embodiments, the anti-TROP2 antibody described herein can block the interaction between TROP2 and β-catenin,

Claudin

1 and 7, Occluding, α5β1 integrin//Talin complex, IGF-1, MDK, and/or NRG-1.

In some embodiments, the genetically modified animals can be used for determining effectiveness of a therapeutic agent (e.g., an anti-TROP2 antibody, ADC, or a TROP2-targeting drug) for the treatment of cancer. In some embodiments, the methods involve administering the therapeutic agent (e.g., an anti-human TROP2 antibody, ADC, or a TROP2-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 therapeutic agents, as TROP2 is also expressed on many other cells. Thus, it is important that the humanized TROP2 functions in a largely similar way as compared to the endogenous TROP2, so that the results in the humanized animals can be used to predict the efficacy or toxicity of these therapeutic agents in the human. In some embodiments, the anti-TROP2 antibody can directly target cancer cells expressing TROP2, e.g., by inducing complement mediated cytotoxicity (CMC) or antibody dependent cellular cytotoxicity (ADCC) to kill the cancer cells.

In some embodiments, the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) that are injected into the animal. In some embodiments, the anti-TROP2 antibody or ADC inhibits TROP2 signaling pathways. In some embodiments, the anti-TROP2 antibody or ADC does not inhibit TROP2 signaling pathways.

In some embodiments, the genetically modified animals can be used for determining whether an anti-TROP2 antibody is a TROP2 agonist or antagonist. In some embodiments, the methods as described herein are also designed to determine the effects of the agent (e.g., anti-TROP2 antibodies or ADCs) on TROP2, e.g., whether the agent can upregulate the immune response or downregulate immune response, and/or whether the agent can induce complement mediated cytotoxicity (CMC) or antibody dependent cellular cytotoxicity (ADCC) . In some embodiments, the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., cancer.

The inhibitory effects on tumors can also be determined by methods known in the art, e.g., measuring the tumor volume in the animal, and/or determining tumor (volume) inhibition rate (TGITv) . The tumor growth inhibition rate can be calculated using the formula TGI _TV (%) =(1 -TVt/TVc) x 100, where TVt and TVc are the mean tumor volume (or weight) of treated and control groups.

In some embodiments, the therapeutic agent (e.g., an anti-TROP2 antibody, ADC, or a TROP2-targeting drug) is designed for treating various cancers. As used herein, the term “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. The term “tumor” as used herein 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. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “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. In some embodiments, 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. The term also includes 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In a specific embodiment, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, or liver cancer.

In some embodiments, the cancer described herein is breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancers, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, bladder cancer, or uterine cancer. In some embodiments, the cancer described herein is endometrioid endometrial cancer (EEC) , glioma, Hilar cholangiocarcinoma, chronic lymphocytic lymphoma (CLL) , extranodal NK/T-cell lymphoma, nasal type (ENKTL) , Non-Hodgkin's lymphoma (NHL) , small-sized pulmonary adenocarcinoma, or squamos cell carcinoma of the oral cavity.

In some embodiments, the cancer described herein is a solid tumor (e.g., an epithelial solid tumor) .

In some embodiments, the therapeutic agent described herein is an antibody-drug conjugate (ADC) targeting TROP2, e.g., an ADC comprising the anti-TROP2 antibody described herein. In some embodiments, the ADC is sacituzumab govitecan (IMMU-132; brand name:

) , PF-06664178, RN927C, Datopotamab deruxtecan (dato-DXd) , FDA018-ADC, JS108, SKB264, DS-1062a, BAT8003, or STI-3258. More details can be found, e.g., in Zaman, S., et al. "Targeting Trop-2 in solid tumors: future prospects. " OncoTargets and therapy 12 (2019) : 1781; and Nagayama, A., et al. "Antibody-drug conjugates for the treatment of solid tumors: clinical experience and latest developments. " Targeted oncology 12.6 (2017) : 719-739; each of which is incorporated herein by reference in its entirety.

In some embodiments, the therapeutic agent is a monoclonal antibody. In some embodiments, the therapeutic agent is a multi-specific antibody, e.g., a bispecific antibody targeting TROP2 and a second antigen. In some embodiments, the second antigen is HER2 or CD3.

In some embodiments, the anti-TROP2 antibody is designed for treating various autoimmunediseases, includingrheumatoidarthritis, Crohn’s disease, systemiclupus erythematosus, ankylosing spondylitis, inflammatory bowel diseases (IBD) , ulcerative colitis, or scleroderma. In some embodiments, the anti-TROP2 antibody is designed for treating various immune disorders, including allergy, asthma, and/or atopic dermatitis. Thus, the methods as described herein can be used to determine the effectiveness of an anti-TROP2 antibody in inhibiting immune response. In some embodiments, 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-TROP2 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. In some embodiments, the antibody can decrease the red blood cells (RBC) , hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, 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.

In some embodiments, 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 TROP2 gene function, human TROP2 antibodies, drugs for human TROP2 targeting sites, the drugs or efficacies for human TROP2 targeting sites, the drugs for immune-related diseases and antitumor drugs.

In some embodiments, 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) . For example, 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. In some embodiments, the animal is selected from the TROP2 gene humanized non-human animal prepared by the methods described herein, the TROP2 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 TROP2 protein, or the tumor-bearing or inflammatory animal models described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies can treat the TROP2-associated diseases described herein (e.g., breast cancer) . In some embodiments, the TCA-T, CAR-T, and/or other immunotherapies provides an evaluation method for treating the TROP2-associated diseases described herein (e.g., breast cancer) .

Genetically modified animal model with two or more human or chimeric genes

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 TROP2 gene and a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein can be erb-b2 receptor tyrosine kinase 2 (HER2) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , lymphocyte-activation gene 3 (LAG3) , TNF receptor superfamily member 9 (4-1BB) , TNF receptor superfamily Member 5 (CD40) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , interleukin 4 receptor (IL4R) , interleukin 6 receptor (IL6R) , interleukin 17A (IL17) , CD3, CD28, CD38, tumor necrosis factor receptor superfamily, member 4 (OX40) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , CD73, tumor necrosis factor alpha (TNFα) , B And T Lymphocyte Associated (BTLA) , CD27, CD47, CD137, CD154, CD226, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , and/or Signal regulatory protein α (SIRPα) .

The methods of generating genetically modified animal model with two or more human or chimeric genes (e.g., humanized genes) can include the following steps:

(a) using the methods of introducing human TROP2 gene or chimeric TROP2 gene as described herein to obtain a genetically modified non-human animal;

(b) mating the genetically modified non-human animal with another genetically modified non-human animal, and then screening the progeny to obtain a genetically modified non-human animal with two or more human or chimeric genes.

In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal with human or chimeric HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL17, CD3, CD28 CD38, OX40, TIM3, CD73, TNFα, BTLA, CD27, CD47, CD137, CD154, CD226, TIGIT, GITR, and/or SIRPα. Some of these genetically modified non-human animal are described, e.g., in PCT/CN2017/090320, PCT/CN2017/099577, PCT/CN2017/099575, PCT/CN2017/099576, PCT/CN2017/099574, PCT/CN2017/106024, PCT/CN2017/110494, PCT/CN2017/110435, PCT/CN2017/120388, PCT/CN2018/081628, PCT/CN2018/081629; each of which is incorporated herein by reference in its entirety.

In some embodiments, the TROP2 humanization is directly performed on a genetically modified animal having a human or chimeric HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL17, CD3, CD28 CD38, OX40, TIM3, CD73, TNFα, BTLA, CD27, CD47, CD137, CD154, CD226, TIGIT, GITR, and/or SIRPα gene.

As these proteins may involve different mechanisms, a combination therapy that targets two or more of these proteins thereof may be a more effective treatment. In fact, 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-TROP2 antibody and an additional therapeutic agent for the treatment of cancer. The methods include administering the anti-TROP2 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. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL17, CD3, CD28 CD38, OX40, TIM3, CD73, TNFα, BTLA, CD27, CD47, CD137, CD154, CD226, TIGIT, GITR, and/or SIRPα. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab) , an anti-HER2 antibody (e.g., Trastuzumab) , an anti-PD-1 antibody (e.g., nivolumab) , or an anti-PD-L1 antibody.

In some embodiments, the animal further comprises a sequence encoding a human or humanized HER2, 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. In some embodiments, the additional therapeutic agent is an anti-HER2 antibody, an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab) , an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the tumor comprises one or more tumor cells that express TROP2, HER2, CD80, CD86, PD-L1, and/or PD-L2.

In some embodiments, the combination treatment is designed for treating various cancers as described herein, e.g., breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancers, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, bladder cancer, or uterine cancer.

In some embodiments, 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. Alternatively or in addition, the methods can include performing surgery on the subject to remove at least a portion of the cancer, e.g., to remove a portion of or all of a tumor (s) , from the patient.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

The following materials were used in the following examples.

BbsI, EcoRI, BamHI, EcoRV, and BclI restriction enzymes were purchased from NEB (Catalog numbers: R0539L, R0101 M, R0136M, R0195L, and R3160L, respectively) .

C57BL/6 mice were purchased from the China Food and Drugs Research Institute National Rodent Experimental Animal Center.

Ambion ^TM in vitro transcription kit (MEGAshortscript ^TM T7 Transcription Kit) was purchased from Thermo Fisher Scientific (Catalog number: AM1354) .

Cas9mRNA was purchased from SIGMA (Catalog number: CAS9MRNA-1EA) .

UCA kit was obtained from Biocytogen Pharmaceuticals (Beijing) Co., Ltd. The catalog number is BCG-DX-001.

Human TROP-2 antibody (hTROP2) was purchased from R&D (Catalog number: AF650-SP) .

Mouse TROP-2 antibody (mTROP2) was purchased from R&D (Catalog number: AF1122-SP) .

Purified anti-mouse CD16/32 Antibody was purchased from BioLegend (Catalog number: 101302) .

Zombie NIR ^TM Fixable Viability Kit was purchased from BioLegend (Catalog number: 423106) .

Brilliant Violet 510 ^TM anti-mouse CD45 was purchased from BioLegend (Catalog number: 103138) .

PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody was purchased from BioLegend (Catalog number: 108426) .

Brilliant Violet 421 ^TM anti-mouse CD4 Antibody was purchased from BioLegend (Catalog number: 100438) .

FITC anti-mouse F4/80 was purchased from BioLegend (Catalog number: 123108) .

PE anti-mouse CD8a Antibody was purchased from BioLegend (Catalog number: 100708) .

FITC anti-Mouse CD19 Antibody was purchased from BioLegend (Catalog number: 115506) .

PerCP/Cy5.5 anti-mouse TCRβ chain Antibody was purchased from BioLegend (Catalog number: 109228) .

Brilliant Violet 605 ^TM anti-mouse CD 11 c Antibody was purchased from BioLegend (Catalog number: 117334) .

PE anti-mouse/human CD11b Antibody was purchased from BioLegend (Catalog number: 101208) .

PE/Cy ^TM 7 Mouse anti-mouse NK1.1 Antibody (BD Pharmingen ^TM) was purchased from BD Biosciences (Catalog number: 552878) .

APC Hamster Anti-Mouse TCR β Chain (BD Pharmingen ^TM) was purchased from BD Bioscience (Catalog number: 553174) .

APC anti-mouse/rat Foxp3 was purchased from eBioscience (Catalog number: 17-5773-82) .

EXAMPLE 1: Mice with humanized TROP2 gene

In this example, a non-human animal (e.g., a mouse) was modified to include a nucleotide sequence encoding human TROP2 protein, and the obtained genetically-modified non-human animal can express a human or humanized TROP2 protein in vivo. The mouse TROP2 gene (NCBI Gene ID: 56753, Primary source: MGI: 1861606, UniProt ID: Q8BGV3) is located at 67511043 to 67512806 of chromosome 6 (NC_000072.7) , and the human TROP2 gene (NCBI Gene ID: 4070, Primary source: HGNC: 11530, UniProt ID: P09758) is located at 58575433 to 58577252 of chromosome 1 (NC_000001.11) . The mouse TROP2 transcript is NM_020047.3, and the corresponding protein sequence NP_064431.2 is set forth in SEQ ID NO: 1. The human TROP2 transcript is NM_002353.3, and the corresponding protein sequence NP_002344.2 is set forth in SEQ ID NO: 2. Mouse and human TROP2 gene loci are shown in FIG. 1.

All or part ofnucleotide sequences encoding human TROP2 protein can be introduced into the mouse endogenous TROP2 locus, so that the mouse expresses human or humanized TROP2 protein. Specifically, using gene-editing techniques and under the control of regulatory elements of the mouse TROP2 gene, a nucleotide sequence (e.g., DNA or cDNA sequence) of the human TROP2 gene can be used to replace the corresponding mouse sequence at the mouse endogenous TROP2 locus, to obtain a humanized TROP2 gene locus as shown in FIG. 2, thereby humanizing mouse TROP2 gene.

As shown in the schematic diagram of the targeting strategy in FIG. 3, the targeting vector V1 contains homologous arm sequences upstream and downstream of the mouse TROP2 gene, and an “A Fragment” containing DNA sequences of human TROP2 gene. Specifically, sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 3) is identical to nucleotide sequence of 67512691-67516273 of NCBI accession number NC_000072.7, and sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 4) is identical to nucleotide sequence of 67506222-67510723 of NCBI accession number NC_000072.7. The A Fragment contains a human genomic DNA sequence from TROP2 genes (SEQ ID NO: 7) , which is identical to nucleotide sequence of 97-1068 of NCBI accession number NM_002353.3.

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) . The connection between the 5’ end of the Neo cassette and the mouse sequence was designed as: 5’-TATTAACAGGCACACCTTCCTTTGTGGGTTTTAAA CCACG

TTGTCAAGCTTGATATCGAATTCCGAAGTTCCTAT -3’ (SEQ ID NO: 8) , wherein the “G” in sequence “ CCACG” is the last nucleotide of the mouse sequence, and the first “G” in sequence

is the first nucleotide of the Neo cassette. The connection between the 3’ end of the Neo cassette and the mouse sequence was designed as: 5’-GTATAGGAACTTCATCAGTCAGGTACATAATGGTG GATCC

GCGCAAAGCCCACCCCCACCCCCCACCCCCAGCAG -3’ (SEQ ID NO: 9) , wherein the last “C” in sequence “ GATCC” is the last nucleotide of the Neo cassette, and the “T” in sequence

is the first nucleotide of the mouse sequence. In addition, a coding gene with a negative selectable marker (a gene encoding diphtheria toxin A subunit (DTA) ) was also constructed downstream of the 3' homologous arm of the targeting vector. The mRNA sequence of the engineered mouse TROP2 after humanization and its encoded protein sequence are shown in SEQ ID NO: 10 and SEQ ID NO: 2, respectively.

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. Correct positive clone cells were screened. The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice) according to techniques known in the art, 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 F 1 generation mice, and then breeding the F 1 generation heterozygous mice with each other. The positive mice were also bred with the Flp transgenic mice to remove the positive selectable marker gene, and then the humanized TROP2 homozygous mice were obtained by breeding with each other. The genotype of the offspring mouse somatic cells can be identified by PCR (see the table below for primer sequences and target fragment sizes) . The identification results of exemplary F 1 generation mice (Neo cassette removed) are shown in FIG. 4, wherein three mice numbered F1-1, F1-2, and F1-3 were identified as positive heterozygous mice. The results indicate that the TROP2 gene humanized mice prepared using the methods described herein can be stably passaged.

Table 3. F1 generation genotype PCR primer sequences and recombinant fragment sizes

In addition, CRISPR/Cas gene editing technology was also used to obtain the TROP2 gene humanized mice. A targeting strategy was designed as shown in FIG. 5. The targeting vector V2 has an upstream homologous arm sequence (5’ homologous arm; SEQ ID NO: 5) , a downstream homologous ann sequence (3’ homologous ann; SEQ ID NO: 6) , and a fragment comprising a nucleotide sequence encoding human TROP2 protein. Specifically, the 5’ homologous arm is identical to nucleotide sequence of 67512691-67514056 of NCBI accession number NC_000072.7, and the 3’ homologous arm is identical to nucleotide sequence of 67510349-67511736 of NCBI accession number NC_000072.7. The human TROP2 nucleotidesequence is set forth in SEQ ID NO: 7.

The targeting vector was constructed, e.g., by restriction enzyme digestion and ligation, or synthesized directly. The constructed targeting vector sequence was preliminarily verified by restriction enzyme digestion, then verified by sequencing. The correct targeting vector verified by sequencing was used for subsequent experiments.

The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Specific sgRNA sequences were designed and synthesized that recognize the 5’ end targeting sites and 3’ end targeting sites, sgRNAs with better activity and higher sequence specificity were selected for subsequent experiments. Exemplary target sequences for sgRNAs on the TROP2 gene are shown below:

sgRNA1 targeting site (SEQ ID NO: 13) : 5’-ATGACGGTCTGCGACACAAATGG -3’

sgRNA2 targeting site (SEQ ID NO: 14) : 5’-CATCGCTGTCGTCTCGGTAGCGG -3’

Oligonucleotides were added to the 5’ end of the sgRNA and a complementary strand to obtain a forward oligonucleotide and a reverse oligonucleotide. After annealing, the products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) , respectively, to obtain expression vectors PT7-TROP2-1 and pT7-TROP2-2. The pT7-sgRNA vector was synthesized, which included a DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 15) , and was ligated to the backbone vector (Takara, Catalog number: 3299) after restriction enzyme digestion (EcoRI and BamHI) . The resulting plasmid was confirmed by sequencing.

The pre-mixed Cas9 mRNA, the targeting vector, and in vitro transcription products of the pT7-TROP2-1, pT7-TROP2-2 plasmids (using Ambion ^TM in vitro transcription kit to carry out the transcription according to the method provided in the product instruction) were injected into the cytoplasm or nucleus of mouse fertilized eggs with a microinjection instrument. The embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2006. The injected fertilized eggs were then transferred to a culture medium to culture for a short time and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified mice (F0 generation) . The mouse population was further expanded by cross-breeding and self-breeding to establish stable homozygous mouse lines with genetically-modified TROP2 gene locus.

The genotype of somatic cells of F0 generation mice can be identified, e.g., by PCR analysis. The identification results of some F0 generation mice are shown in FIG. 6. In view of the 5' end primer detection result, the 3' end primer detection result, and further sequencing verification result, three mice numbered F0-01, F0-02, and F0-03 were identified as positive mice. The PCR primers are shown in the table below.

Table 4. F0 generation genotype PCR primer sequences and recombinant fragment sizes

Primer L-GT-F is located upstream of the 5’ homologous arm, R-GT-R is located downstream of the 3’ homologous arm, and both L-GT-R and R-GT-F are located on the human TROP2 sequence.

The positive F0 generation TROP2 gene humanized mice generated were bred with wild-type mice to generate F1 generation mice. The same method (e.g., PCR) was used to genotype the F1 generation mice. As shown in FIG. 7, three mice numbered F1-01, F1-02, and F1-03 were identified as positive mice.

The F1 generation mice were further analyzed by Southern Blot to confirm whether random insertions were introduced. Specifically, mouse tail genomic DNA was extracted, digested with EcoRV or BclI restriction enzyme, transferred to a membrane, and then hybridized with the respective probe. The 5’ Probe and 3’ Probe are located on the 5’ homologous arm and downstream of the 3’ homologous arm, respectively. The specific probes and target fragment sizes are shown in the table below. The Southern blot results are shown in FIG. 8. In view of the 5' Probe and 3' Probe detection results, no random insertions were detected in the three mice numbered F1-01, F1-02, and F1-03. The results indicate that the TROP2 gene humanized mice generated using the methods described herein can be stably passaged and free of random insertions.

Table 5. Specific probes and target fragment sizes

Restriction enzyme	Probe	Wildtype fragment size	Recombinant fragment size
EcoRV	5’Probe	12.3 kb	6.2 kb
BclI	3’Probe	8.3 kb	6.9 kb

The following primers were used to synthesize probes used in Southern Blot assays:

5’ Probe-F (SEQ ID NO: 20) : 5’-TAACACTAACCCCCAAACCATAACT-3’.

5’ Probe-R (SEQ ID NO: 21) : 5’-ATGGTCAGGATTAAGGTTAGATGTAAAT-3’;

3’ Probe-F (SEQ ID NO: 22) : 5’-TTAAGGACGCATTTCTGAAATTCCTT-3’,

3’ Probe-R (SEQ ID NO: 23) : 5’-CATCAAGGACAAGCAGAAAAATAGATGC-3’.

The heterozygous mice identified as positive in the F1 generation were bred with each other to obtain the F2 generation TROP2 gene humanized homozygous mice.

The expression of humanized TROP2 gene in positive mice can be confirmed, e.g., by RT-PCR or flow cytometry. Specifically, one 6-week-old male wild-type C57BL/6 mouse and one 6-week-old male TROP2 gene humanized homozygous mouse prepared using the methods described herein were selected. Mouse skin tissues were collected after euthanasia by cervical dislocation, and the primer sequences shown in the table below were used for RT-PCR detection. The detection results are shown in FIGS. 9A-9C. In the skin tissues of the wild-type C57BL/6 mouse, only mouse TROP2 mRNA was detected, and no humanized TROP2 mRNA was detected. In the skin tissues of the TROP2 gene humanized homozygous mouse, only humanized TROP2 mRNA was detected, and no mouse TROP2 mRNA was detected.

Table 6. RT-PCR primer sequences and target fragment sizes

Further, Western Blot was used to detect the expression of TROP2 protein in mice. Specifically, one 6-week-old male wild-type C57BL/6 mouse and one 6-week-old male TROP2 gene humanized homozygous mouse prepared using the methods described herein were selected. Mouse skin and kidney tissues were collected after euthanasia by cervical dislocation, and Western Blot was performed using anti-human TROP2 antibody (hTROP2) and anti-mouse TROP2 antibody (mTROP2) . The results are shown in FIG. 10. Expression of mouse TROP2 protein, but not human TROP2 protein, was detected in the skin and kidney tissues of the wild-type C57BL/6 mouse. Expression of human TROP2 protein was detected in the skin and kidney tissues of TROP2 gene humanized homozygous mouse. Because the anti-mouse TROP2 antibody can cross-react with human TROP2 protein, a weak mTROP2 band was detected in the kidney tissues of TROP2 gene humanized homozygous mouse. The results showed that the TROP2 gene humanized homozygous mice prepared using the methods described herein can successfully express human TROP2 protein in vivo.

In addition, due to the double-strand break of genomic DNA caused by Cas9 cleavage, insertion/deletion mutations can be randomly generated through chromosome homologous recombination repair, which may result in knockout mice with loss of TROP2 protein function. A pair of primers were designed to detect the knockout mice. As shown in FIG. 11, three mice numbered KO-1, KO2, and KO3 were identified as TROP2 gene knockout mice. The primers are located upstream of the 5’ end targeting site and downstream of the 3’ end targeting site, respectively, with sequences shown in the table below.

Table 7. Genotype PCR primer sequences and recombinant fragment sizes of TROP2 gene knockout mice

Further, the spleen, lymph nodes, and peripheral blood from C57BL/6 wild-type mice (+/+) and TROP2 gene humanized homozygous mice (H/H) 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. 12 and FIG. 13, respectively. The detection results of leukocyte subtypes and T cell subtypes in peripheral blood are shown in FIG. 16 and FIG. 17, respectively. The results showed that the percentages of B cells, T cells, NK cells, granulocytes, dendritic cells (DC cells) , macrophages, monocytes, and other leukocyte subtypes in the spleen and peripheral blood of TROP2 gene humanized homozygous mice were basically the same as those in C57BL/6 wild-type mice (FIG. 12 and FIG. 16) . In addition, 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. 13 and FIG. 17) .

The detection results of leukocyte subtypes and T cell subtypes in lymph nodes are shown in FIG. 14 and FIG. 15, 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 TROP2 gene humanized homozygous mice were basically the same as those of C57BL/6 wild-type mice (FIG. 14) . In addition, 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. 15) .

The results indicate that the humanization of TROP2 gene did not affect the differentiation, development and distribution of leukocytes and T cells in mice.

In addition, six 9-week-old female wild-type C57BL/6 mice (+/+) and six 9-week-old TROP2 gene humanized homozygous mice (H/H) were selected, and peripheral blood was collected for blood routine and blood biochemical tests. Blood routine test indicators included: white blood cell count (WBC) , red blood cell count (RBC) , hematocrit (HCT) , hemoglobin (HGB) , mean corpuscular volume (MCV) , mean corpuscular hemoglobin (MCH) , mean corpuscular hemoglobin concentration (MCHC) , platelet count (PLT) , lymphocytes (LYMPH) , monocytes (MONO) , and neutrophils (NEUT) . 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) . The blood routine test results (mean) and the blood biochemical test results are shown in the tables below.

Table 8. Blood routine test results

Table 9. Blood biochemical test results

As shown in the tables above, the results showed that humanization of TROP2 gene did not affect the composition and morphology of blood cells in mice, and the TROP2 gene humanized mice had similar liver function as the wild type mice.

EXAMPLE 2. Generation of double-or multi-gene humanized mice

The TROP2 gene humanized mice generated using the methods described herein can also be used to generate double-or multi-gene humanized mouse models. For example, in Example 1, 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) HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL17, CD3, CD28 and/or CD38 genes. Alternatively, embryonic stem cells from humanized TROP2 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 TROP2 and other gene modifications. In addition, it is also possible to breed the homozygous or heterozygous TROP2 gene humanized mice obtained by the methods described herein with other genetically modified homozygous or heterozygous mice, and the offspring can be screened. According to Mendel’s law, it is possible to generate double-gene or multi-gene modified heterozygous mice comprising modified (e.g., human or humanized) TROP2 gene and other genetic modifications. Then the heterozygous mice can be bred with each other to obtain homozygous double-gene or multi-gene modified mice. These double-gene or multi-gene modified mice can be used for in vivo validation of gene regulators targeting human TROP2 and other genes.

EXAMPLE 3: In vivo efficacy verification

The TROP2 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., TROP2-targeting drugs, antibodies, and ADCs) targeting human TROP2.

In addition, the TROP2 gene humanized mice prepared by the methods described herein can be used to evaluate the efficacy of modulators targeting human TROP2 (e.g., anti-human TROP2 antibodies or ADCs targeting TROP2) . For example, TROP2 gene humanized homozygous mice can be subcutaneously inoculated with TROP2 gene humanized MC38 cells. After the tumor volume reaches about 100-150 mm3, 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 TROP2 (e.g., anti-human TROP2 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 TROP2-targeting drugs.

EXAMPLE 4: In vivo toxicity testing

The TROP2 gene humanized mice prepared by the methods described herein can be used to evaluate the toxicity of modulators targeting human TROP2. For example, the toxicity of the anti-HER2/TROP2 bispecific antibody ADC (Ab1) in C57BL/6 mice and hHER2/TROP2 mice were determined. HER2/TROP2 humanized mice was generated by crossing a HER2 humanized mice with a TROP2 humanized mice. The HER2/TROP2 humanized mice and C57BL/6 mice were placed into different groups (6 mice per group) based on the body weight, administered with physiological saline, Ab1 (10 mg/kg, 30 mg/kg, 90 mg/kg) or MMAE (0.19 mg/kg, 0.57 mg/kg, 1.14 mg/kg and 1.71 mg/kg, equimolar amounts of Ab1 at 10 mg/kg, 30 mg/kg, 60 mg/kg and 90 mg/kg, respectively) by intravenous injection. The frequency of administration was once a week (1 administrations in total) . Details of the administration scheme and survival on day 7 are shown in the Table 10. The body weight was measured every day until the end of the experiment after 1 week. The results of body weight and body weight change are shown in FIG. 20 and FIG. 21.

Table 10. Group assignment

The results showed that all mice in the G1-G6 and G9-G10 groups survived. However, treatment groups G7, G8 and G11 showed some toxicity. The results in Figure 20 and Figure 21 show that after C57BL/6 wild-type mice were injected with different doses of Ab1, the body weight changes were not obvious. However, the body weight of HER2/TROP2 humanized mice changed significantly after high-dose Ab1 injection. Results show that HER2/TROP2 humanized mice can be used to detect therapeutic agent toxicity.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric TROP2 (trophoblast cell-surface antigen 2) .
The animal of claim 1, wherein the sequence encoding the human or chimeric TROP2 is operably linked to an endogenous regulatory element at the endogenous TROP2 gene locus in the at least one chromosome.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric TROP2 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human TROP2 (NP_002344.2 (SEQ ID NO: 2) ) .
The animal of any one of claims 1-3, wherein the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.
The animal of any one of claims 1-4, wherein the animal is a mouse.
The animal of any one of claims 1-5, wherein the animal does not express endogenous TROP2 or expresses a decreased level of endogenous TROP2.
The animal of any one of claims 1-6, wherein the animal has one or more cells expressing human or chimeric TROP2.
The animal of any one of claims 1-7, wherein the animal has one or more cells expressing human or chimeric TROP2, and the expressed human or chimeric TROP2 can transduce an intracellular calcium signal.
The animal of any one of claims 1-8, wherein the animal has one or more cells expressing human or chimeric TROP2, and the expressed human or chimeric TROP2 can interact with endogenous β-catenin, Claudin 1 and 7, Occludin, α5β1 integrin//Talin complex, IGF-1, MDK, and/or NRG-1.
The animal of any one of claims 1-9, wherein the animal has one or more cells expressing human or chimeric TROP2, and the expressed human or chimeric TROP2 can interact with human β-catenin, Claudin 1 and 7, Occludin, α5β1 integrin//Talin complex, IGF-1, MDK, and/or NRG-1.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous TROP2 with a sequence encoding a corresponding region of human TROP2 at an endogenous TROP2 gene locus.
The animal of claim 11, wherein the sequence encoding the corresponding region of human TROP2 is operably linked to an endogenous regulatory element at the endogenous TROP2 locus, and one or more cells of the animal expresses a human or chimeric TROP2.
The animal of claim 11 or 12, wherein the animal does not express endogenous TROP2 or expresses a decreased level of endogenous TROP2.
The animal of any one of claims 11-13, wherein the animal has one or more cells expressing a human TROP2.
The animal of any one of claims 11-13, wherein the animal has one or more cells expressing a chimeric TROP2 having all or part of the signal peptide, all or part of the extracellular region, all or part of the transmembrane region, and/or all or part of the cytoplasmic region of human TROP2.
The animal of claim 15, wherein the signal peptide of the chimeric TROP2 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, 23, 24, 25, or 26 contiguous amino acids that are identical to a contiguous sequence present in the signal peptide of human TROP2 (e.g., amino acids 1-26 of SEQ ID NO: 2) .
The animal of claim 15 or 16, wherein the extracellular region of the chimeric TROP2 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, 245, 246, 247, or 248 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human TROP2 (e.g., amino acids 27-274 of SEQ ID NO: 2) .
The animal of any one of claims 15-17, wherein the transmembrane region of the chimeric TROP2 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 TROP2 (e.g., amino acids 275-297 of SEQ ID NO: 2) .
The animal of any one of claims 15-18, wherein the cytoplasmic region of the chimeric TROP2 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, 23, 24, 25, or 26 contiguous amino acids that are identical to a contiguous sequence present in the cytoplasmic region of human TROP2 (e.g., amino acids 298-323 of SEQ ID NO: 2) .
The animal of any one of claims 11-19, wherein the sequence encoding a region of endogenous TROP2 comprises exon 1 or a part thereof of the endogenous TROP2 gene.
The animal of claim 20, wherein the animal is a mouse.
The animal of any one of claims 11-21, wherein the animal is heterozygous with respect to the replacement at the endogenous TROP2 gene locus.
The animal of any one of claims 11-21, wherein the animal is homozygous with respect to the replacement at the endogenous TROP2 gene locus.
A method for making a genetically-modified, non-human animal, comprising:

replacing in at least one cell of the animal, at an endogenous TROP2 gene locus, a sequence encoding a region of endogenous TROP2 with a sequence encoding a corresponding region of human TROP2.
The method of claim 24, wherein the sequence encoding the corresponding region of human TROP2 comprises exon 1 or a part thereof of a human TROP2 gene.
The method of claim 24 or 25, wherein the sequence encoding the corresponding region of human TROP2 comprises at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 910, 920, 930, 940, 950, 960, 970, 971, or 972 nucleotides of a human TROP2 gene exon 1.
The method of any one of claims 24-26, wherein the sequence encoding the corresponding region of human TROP2 encodes SEQ ID NO: 2.
The method of any one of claims 24-27, wherein the sequence encoding a region of endogenous TROP2 comprises exon 1 or a part thereof of the endogenous TROP2 gene.
The method of any one of claims 24-28, wherein the animal is a mouse, and the sequence encoding a region of endogenous TROP2 comprises at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 910, 920, 930, 940, 950, 951, 952, 953, or 954 nucleotides of the endogenous mouse TROP2 gene exon 1.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric TROP2 polypeptide, wherein the chimeric TROP2 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human TROP2, wherein the animal expresses the chimeric TROP2 polypeptide.
The animal of claim 30, wherein the chimeric TROP2 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 310, at least 315, at least 316, or at least 317 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human TROP2.
The animal of claim 30 or 31, wherein the chimeric TROP2 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to SEQ ID NO: 2.
The animal of any one of claims 30-32, wherein the nucleotide sequence is operably linked to an endogenous TROP2 regulatory element (e.g., 5’ UTR and/or 3’ UTR) of the animal.
The animal of any one of claims 30-33, wherein the nucleotide sequence is integrated to an endogenous TROP2 gene locus of the animal.
The animal of any one of claims 30-34, wherein the chimeric TROP2 polypeptide has at least one mouse TROP2 activity and/or at least one human TROP2 activity.
A method of making a genetically-modified animal cell that expresses a human or chimeric TROP2, the method comprising:

replacing at an endogenous TROP2 gene locus, a nucleotide sequence encoding a region of endogenous TROP2 with a nucleotide sequence encoding a corresponding region of human TROP2, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the human or chimeric TROP2, wherein the animal cell expresses the human or chimeric TROP2.
The method of claim 36, wherein the animal is a mouse.
The method of claim 36 or 37, wherein the nucleotide sequence encoding the human or chimeric TROP2 comprises: an endogenous 5’ UTR, a sequence encoding the human or chimeric TROP2, and an endogenous 3’ UTR.
The method of any one of 36-38, wherein the nucleotide sequence encoding the human or chimeric TROP2 is operably linked to an endogenous TROP2 regulatory region, e.g., promoter.
The animal of any one of claims 1-23 and 30-35, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The animal of claim 40, wherein the additional human or chimeric protein is erb-b2 receptor tyrosine kinase 2 (HER2) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , lymphocyte-activation gene 3 (LAG3) , TNF receptor superfamily member 9 (4-1BB) , TNF receptor superfamily Member 5 (CD40) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , interleukin 4 receptor (IL4R) , interleukin 6 receptor (IL6R) , interleukin 17A (IL17) , CD3, CD28 or CD38.
The method of any one of claims 24-29 and 36-39, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein.
The method of claim 42, wherein the additional human or chimeric protein is HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL17, CD3, CD28 or CD38.
A method of determining effectiveness of a therapeutic agent for the treatment of cancer, comprising:

a) administering the therapeutic agent to the animal of any one of claims 1-23, 30-35, 40, and 41, wherein the animal has a tumor; and

b) determining inhibitory effects of the therapeutic agent to the tumor.
The method of claim 44, wherein the therapeutic agent is an anti-TROP2 antibody or an antibody-drug conjugate targeting TROP2.
The method of claim 44 or 45, wherein the tumor comprises one or more cells that express TROP2.
The method of any one of claims 44-46, wherein the tumor comprises one or more cancer cells that are injected into the animal.
The method of any one of claims 44-47, wherein determining inhibitory effects of the anti-TROP2 antibody to the tumor involves measuring the tumor volume in the animal.
The method of any one of claims 44-48, wherein the cancer is breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancers, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, bladder cancer, or uterine cancer.
A method of determining effectiveness of a therapeutic agent targeting TROP2 and an additional therapeutic agent for the treatment of cancer, comprising

a) administering the therapeutic agent targeting TROP2 and the additional therapeutic agent to the animal of any one of claims 1-23, 30-35, 40, and 41, wherein the animal has a tumor; and

b) determining inhibitory effects on the tumor.
The method of claim 50, wherein the therapeutic agent targeting TROP2 is an anti-TROP2 antibody or an antibody-drug conjugate targeting TROP2.
The method of claim 50 or 51, wherein the animal further comprises a sequence encoding a human or chimeric erb-b2 receptor tyrosine kinase 2 (HER2) .
The method of any one of claims 50-52, wherein the animal further comprises a sequence encoding a human or chimeric programmed death-ligand 1 (PD-L1) .
The method of any one of claims 50-53, wherein the additional therapeutic agent is an anti-HER2 antibody or an anti-PD-L1 antibody.
The method of any one of claims 50-54, wherein the tumor comprises one or more tumor cells that express TROP2 or HER2.
The method of any one of claims 50-55, wherein the tumor is caused by injection of one or more cancer cells into the animal.
The method of any one of claims 50-56, wherein determining inhibitory effects of the treatment involves measuring the tumor volume in the animal.
The method of any one of claims 50-57, wherein the animal has breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancers, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, bladder cancer, or uterine cancer.
A method of determining toxicity of a therapeutic agent comprising:

a) administering the therapeutic agent to the animal of any one of claims 1-23, 30-35, 40, and 41; and

b) determining effects of the therapeutic agent to the animal.
The method of claim 59, wherein the therapeutic agent is an anti-TROP2 antibody or an anti-HER2 antibody or an antibody-drug conjugate targeting TROP2.
The method of claim 59 or 60, wherein 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.
A protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following:

(a) an amino acid sequence set forth in SEQ ID NO: 1 or 2;

(b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 1 or 2;

(c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1 or 2;

(d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

(e) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 1 or 2.
A nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following:

(a) a sequence that encodes the protein of claim 62;

(b) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10;

(c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10; and

(d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10.
A cell comprising the protein of claim 62 and/or the nucleic acid of claim 63.
An animal comprising the protein of claim 62 and/or the nucleic acid of claim 63.