CN116064558A

CN116064558A - Construction method and application of TROP2 gene humanized non-human animal

Info

Publication number: CN116064558A
Application number: CN202211163615.7A
Authority: CN
Inventors: 黄德萱; 沈志远; 李冲
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Priority date: 2021-09-24
Filing date: 2022-09-23
Publication date: 2023-05-05
Also published as: WO2023046061A1

Abstract

The invention provides a humanized TROP2 protein, a humanized TROP2 gene, a target vector of the TROP2 gene, a TROP2 gene humanized non-human animal, a construction method thereof and application thereof in the field of biological medicine, and a nucleotide sequence for encoding the human TROP2 protein is introduced into a genome of the non-human animal by utilizing a homologous recombination mode, so that the human or humanized TROP2 protein can be normally expressed in the animal body, and the humanized TROP2 protein can be used as an animal model for human TROP2 signal mechanism research, tumor and immune disease drug screening, and has important application value for developing new drugs of immune targets.

Description

Construction method and application of TROP2 gene humanized non-human animal

Technical Field

The invention belongs to the fields of animal genetic engineering and genetic modification, and particularly relates to a construction method of a TROP2 gene modified non-human animal model and application thereof in the field of biological medicine.

Background

TROP2 is a human trophoblast cell surface glycoprotein antigen, also known as a tumor associated calcium signal transducer (TACSTD 2), belonging to the GA733 family of members, about 35KD, which is a type I transmembrane protein, comprising a large extracellular region, a single transmembrane domain, and a smaller intracellular region. The TROP2 gene is over-expressed in human trophoblasts in embryogenesis, expressed in layered squamous, columnar and cubic epithelial cells, and differentially expressed in various organs and tissues such as cornea, breast, cervix, secretory glandular epithelial tissue, heart, esophagus, kidney, laryngeal tissue, lung, liver, pancreas, prostate, skin, thymus, tonsil, trachea, bladder epithelium, uterus, etc. However, TROP2 has low expression level in normal tissues and is overexpressed in various malignant tumors, is a signal transduction molecule capable of regulating the growth of tumor cells, can promote the processes of proliferation, invasion, metastasis and diffusion of the tumor cells, and is closely related to the reduction of the progression-free survival time and poor prognosis of patients. TROP2 promotes tumor cell growth, proliferation and metastasis mainly by modulating calcium ion signaling pathway, cyclin expression and reducing fibronectin adhesion; can also interact with beta-catenin in the Wnt signaling cascade, and thus act on transcription of nuclear oncogenes and proliferation of cells. The amino acid sequence identity of the human and mouse TROP2 proteins was 79%.

TROP2 is highly expressed in most human epithelial solid tumors, such as oral squamous carcinoma, head and neck tumor, lung cancer, thyroid tumor, angioma, gastric cancer, colorectal tumor, pancreatic cancer, breast cancer, renal tumor, cervical tumor, ovarian cancer, glioma and the like, so that the tumor is a potential target point of medicines for treating various tumor diseases.

In the process of drug development, the discovery and screening of human drugs, particularly antibody drugs, by using an experimental animal model is a very important research process; meanwhile, the pre-clinical research such as pharmacological and pharmacodynamic effects of human medicaments by using an experimental animal model is an indispensable development step. However, due to the difference of physiological structures and metabolic systems of animals and humans, the traditional animal model can not well reflect the real condition of medicines in human bodies, and the establishment of a humanized animal model which is closer to the physiological characteristics of humans is an urgent need of the biomedical industry. However, due to the physiological and pathological differences between animals and humans, coupled with the complexity of the genes, the construction of "efficient" humanized animal models for new drug development remains a great challenge.

In view of the great application potential of TROP2 in the field of tumor disease treatment, in order to further explore related biological characteristics, improve the effectiveness of a TROP2 target related drug preclinical test and reduce clinical research risks, the field is in urgent need of developing a non-human animal model of a TROP2 related signal pathway.

Disclosure of Invention

The humanized non-human animal of TROP2 gene prepared by the invention can improve and promote cell or tissue transplantation, and more importantly, because of the insertion of human gene fragments, the human TROP2 protein can be expressed or partially expressed in the non-human animal body, can be used as a target spot of a medicament capable of only recognizing the amino acid sequence of the human TROP2 protein, and provides possibility for screening human antibodies and other medicaments at animal level.

In a first aspect of the invention, there is provided a humanised TROP2 gene.

Preferably, the humanised TROP2 gene comprises part of a human TROP2 gene.

Preferably, the portion of the human TROP2 gene comprises all or part of exon 1 of the human TROP2 gene. Wherein the portion of exon 1 comprises a contiguous nucleotide sequence of at least 200bp to at least 1820bp, e.g., 200, 500, 700, 900, 950, 970, 971, 972, 973, 974, 975, 1000, 1500, 1800, or 1820 bp; preferably, the portion of exon 1 comprises a nucleotide sequence from the start codon to the stop codon, and more preferably, the humanized TROP2 gene comprises the CDS sequence of a human TROP2 gene.

In a specific embodiment of the invention, the portion of the human TROP2 gene comprises the amino acid sequence of SEQ ID NO: 7; alternatively, comprising a sequence identical to SEQ ID NO:7 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; alternatively, comprising a sequence identical to SEQ ID NO:7 does not differ by more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or not more than 1 nucleotide; alternatively, it comprises a polypeptide having the sequence of SEQ ID NO:7, including substitutions, deletions and/or insertions of one or more nucleotides.

Preferably, the portion of the human TROP2 gene comprises a nucleotide sequence encoding all or part of a human TROP2 protein, more preferably all or part of a nucleotide sequence encoding a signal peptide, extracellular region, transmembrane region and/or intracellular region of a human TROP2 protein, more preferably a nucleotide sequence encoding at least 10 to at least 323, for example 10, 20, 23, 26, 30, 50, 100, 150, 200, 248, 250, 300, 310, 320 or 323 consecutive amino acids of a human TROP2 protein.

In one embodiment of the invention, the portion of the human TROP2 gene comprises a sequence encoding SEQ ID NO:2, a nucleotide sequence of seq id no; alternatively, it comprises a sequence identical to the sequence encoding SEQ ID NO:2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; alternatively, it comprises a sequence identical to the sequence encoding SEQ ID NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; alternatively, it comprises a polypeptide having a sequence encoding SEQ ID NO:2, including nucleotide sequences in which one or more nucleotides are substituted, deleted and/or inserted.

Preferably, the humanised TROP2 gene further comprises all or part of a non-human animal TROP2 gene.

Preferably comprises a nucleotide sequence encoding a non-human animal TROP2 protein, further preferably comprises all or part of a nucleotide sequence encoding an extracellular region, a transmembrane region, a cytoplasmic region and/or a signal peptide of a non-human animal TROP2 protein.

Preferably all or part of exon 1 of the non-human animal TROP2 gene, more preferably part of exon 1 of the non-human animal TROP2 gene. In one embodiment of the invention, the humanized TROP2 gene comprises a non-coding region of exon 1 of a non-human animal TROP2 gene, or comprises a 3'utr and/or a 5' utr of a non-human animal TROP2 gene.

Preferably, the humanized TROP2 gene further comprises SEQ ID NO:8 and/or 9.

In one embodiment of the invention, the humanised TROP2 gene comprises, in order from the 5 'end to the 3' end, the 5'utr of the non-human animal TROP2 gene, a portion of the human TROP2 gene (preferably a portion comprising exon 1 of the human TROP2 gene), the 3' utr of the non-human animal TROP2 gene.

In one embodiment of the present invention, the humanized TROP2 gene comprises, in order from the 5' end to the 3' end, the 5' utr of the non-human animal TROP2 gene, SEQ ID NO:7 or encodes SEQ ID NO:2, 3' utr of a non-human animal TROP2 gene.

Preferably, the humanized TROP2 gene encodes a human or humanized TROP2 protein.

In a specific embodiment of the invention, the nucleotide sequence of the mRNA transcribed from the humanized TROP2 gene comprises any one of the following groups:

a) SEQ ID NO:10, a nucleotide sequence shown in seq id no;

b) And SEQ ID NO:10 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

c) And SEQ ID NO:10 of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or alternatively, the first and second heat exchangers may be,

d) Has the sequence of SEQ ID NO:10, comprising substitution, deletion and/or insertion of one or more nucleotides.

Preferably, the humanized TROP2 gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a substance that is conventionally inducible or repressible. In one embodiment of the invention, the specific inducer is selected from the group consisting of the tetracycline System (Tet-Off System/Tet-On System) and the Tamoxifen System (Tamoxifen System).

Preferably, the non-human animal is selected from any non-human animal that can be genetically edited to produce a humanized gene, such as rodents, zebra fish, pigs, chickens, rabbits, monkeys, etc.

Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Still more preferably, the rodent is a rat or mouse.

Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferred, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Still further preferred, the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag1 ^-/- -IL2rg ^-/- Mouse, rag 2 ^-/- -IL2rg ^-/- Mice, NOD/SCID mice or nude mice.

In a second aspect of the invention, there is provided a humanised TROP2 protein.

Preferably, the humanised TROP2 protein comprises all or part of a human TROP2 protein.

Preferably, the humanized TROP2 protein is encoded by a humanized TROP2 gene as described above.

Preferably, the humanised TROP2 protein comprises all or part of the signal peptide, extracellular region, transmembrane region and/or cytoplasmic region of a human TROP2 protein.

Preferably, the humanised TROP2 protein comprises an amino acid sequence encoded wholly or partially by exon 1 of the human TROP2 gene. Wherein the portion of exon 1 comprises a contiguous nucleotide sequence of at least 200bp to at least 1820bp, preferably 200, 500, 700, 900, 950, 970, 971, 972, 973, 974, 975, 1000, 1500, 1800, 1820 bp; preferably, the portion of exon 1 comprises a nucleotide sequence from the start codon to the stop codon.

In a specific embodiment of the invention, the humanised TROP2 protein comprises all or part of the extracellular region of a human TROP2 protein, preferably the part of the extracellular region of a human TROP2 protein comprises at least 100 to at least 248, preferably 100, 150, 200, 210, 220, 230, 240, 245, 246, 247, 248 consecutive amino acids; preferably, the extracellular region of the humanized TROP2 protein comprises the amino acid sequence of SEQ ID NO:2 from position 27 to 274; alternatively, comprising a sequence identical to SEQ ID NO: the amino acid sequence identity shown at positions 27-274 of 2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; alternatively, comprising a sequence identical to SEQ ID NO: the amino acid sequence shown at positions 27-274 of 2 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; alternatively, comprising a sequence identical to SEQ ID NO:2, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a specific embodiment of the invention, the humanized TROP2 protein further comprises all or part of a transmembrane region of a human TROP2 protein, preferably the human TROP2 protein transmembrane region comprises at least 10 to at least 23, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 consecutive amino acids; preferably, the humanized TROP2 protein transmembrane region comprises SEQ ID NO:2 from position 275 to 297; alternatively, comprising a sequence identical to SEQ ID NO: amino acid sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% shown at positions 275-297 of 2; alternatively, comprising a sequence identical to SEQ ID NO: the amino acid sequences shown at positions 275-297 of 2 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; alternatively, comprising a sequence identical to SEQ ID NO:2, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a specific embodiment of the invention, the humanized TROP2 protein further comprises all or part of a cytoplasmic region of a human TROP2 protein, preferably the cytoplasmic region of a human TROP2 protein comprises at least 10 to at least 26, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 contiguous amino acids; preferably, the humanized TROP2 protein cytoplasmic region comprises SEQ ID NO:2 from position 298 to 323; alternatively, comprising a sequence identical to SEQ ID NO: the amino acid sequence identity shown at positions 298-323 of 2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; alternatively, comprising a sequence identical to SEQ ID NO: the amino acid sequence shown at positions 298-323 of 2 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; alternatively, comprising a sequence identical to SEQ ID NO:2, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a specific embodiment of the invention, the humanized TROP2 protein further comprises all or part of a signal peptide of a human TROP2 protein, preferably the human TROP2 protein signal peptide comprises at least 10 to at least 26, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 consecutive amino acids; preferably, the humanized TROP2 protein signal peptide comprises SEQ ID NO:2 from position 1 to position 26; alternatively, comprising a sequence identical to SEQ ID NO: amino acid sequence identity shown at positions 1-26 of 2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; alternatively, comprising a sequence identical to SEQ ID NO: the amino acid sequence shown at positions 1-26 of 2 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; alternatively, comprising a sequence identical to SEQ ID NO:2, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a specific embodiment of the invention, the humanized TROP2 protein comprises at least 10 to at least 323, preferably 10, 20, 23, 26, 30, 50, 100, 150, 200, 248, 250, 300, 310, 320 or 323 consecutive amino acids of a human TROP2 protein.

In a specific embodiment of the invention, the amino acid sequence of the humanized TROP2 protein comprises any one of the group consisting of:

a) SEQ ID NO:2, an amino acid sequence shown in the formula 2;

b) And SEQ ID NO:2 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

c) And SEQ ID NO:2 of the sequence of amino acids of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or (b)

D) And SEQ ID NO:2, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a third aspect of the invention, there is provided a targeting vector comprising one of the group:

a) A nucleotide sequence encoding all or part of a human TROP2 protein; preferably all or part of a nucleotide sequence comprising a signal peptide encoding a human TROP2 protein, an extracellular region, a transmembrane region and/or a cytoplasmic region; further preferred comprises a nucleotide sequence encoding at least 10 to at least 323, for example 10, 20, 23, 26, 30, 50, 100, 150, 200, 248, 250, 300, 310, 320 or 323 consecutive amino acids of a human TROP2 protein; more preferably comprises a sequence encoding SEQ ID NO:2 or, alternatively, comprises a nucleotide sequence that encodes an amino acid sequence set forth in SEQ ID NO:2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% nucleotide sequence identity to the amino acid sequence set forth in seq id no; alternatively, it comprises a sequence identical to the sequence encoding SEQ ID NO:2, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; alternatively, it comprises a sequence identical to the sequence encoding SEQ ID NO:2, including substitution, deletion and/or insertion of one or more nucleotides;

B) A portion of a human TROP2 gene, preferably all or a portion of exon 1 of a human TROP2 gene, wherein the portion of exon 1 of the human TROP2 gene comprises a contiguous nucleotide sequence of at least 200bp to at least 1820bp, e.g. 200, 500, 700, 900, 950, 970, 971, 972, 973, 974, 975, 1000, 1500, 1800, 1820 bp; preferably, the portion of exon 1 comprises a nucleotide sequence from the start codon to the stop codon, preferably the CDS sequence of the human TROP2 gene; further preferred comprises SEQ ID NO: 7; alternatively, comprising a sequence identical to SEQ ID NO:7 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; alternatively, comprising a sequence identical to SEQ ID NO:7 does not differ by more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or not more than 1 nucleotide; alternatively, it comprises a polypeptide having the sequence of SEQ ID NO:7, a nucleotide sequence comprising one or more substitutions, deletions and/or insertions of nucleotides;

c) The above mentioned humanized TROP2 gene; or alternatively, the first and second heat exchangers may be,

d) Nucleotide sequences encoding the above mentioned humanized TROP2 proteins.

Preferably, the targeting vector further comprises a 5 'arm (5' homology arm) and/or a 3 'arm (3' homology arm).

The 5 'arm is a DNA fragment homologous to the 5' end of the transition region to be changed, which is selected from 100-10000 nucleotides in length of the genomic DNA of the TROP2 gene of the non-human animal. Preferably, the 5' arm has at least 90% homology to NCBI accession number NC_ 000072.7. Further preferred, the 5' arm sequence comprises SEQ ID NO:3 or 5, more preferably as set forth in SEQ ID NO:3 or 5.

The 3 'arm is a DNA fragment homologous to the 3' end of the transition region to be changed, which is selected from 100-10000 nucleotides in length of the genomic DNA of the TROP2 gene of the non-human animal. Preferably, the 3' arm has at least 90% homology to NCBI accession nc_ 000072.7; further preferred, the 3' arm sequence comprises SEQ ID NO:4 or 6, more preferably as set forth in SEQ ID NO:4 or 6.

Preferably, the transition region to be altered is located on exon 1 of the non-human animal TROP2 gene.

In one embodiment of the present invention, the targeting vector comprises a 5 'arm, the nucleotide sequence of any one of the above A) to D), and a 3' arm in order from the 5 'end to the 3' end.

Preferably, the targeting vector further comprises a marker gene. Further preferably, the marker gene is a gene encoding a negative selection marker. Still more preferably, the gene encoding the negative selection marker is the diphtheria toxin A subunit encoding gene (DTA).

In one embodiment of the invention, the targeting vector further comprises a resistance gene selected from positive clones. Further preferably, the resistance gene screened by the positive clone is neomycin phosphotransferase coding sequence Neo. At this time, the targeting vector further comprises SEQ ID NO:8 and/or 9.

In one embodiment of the present invention, the targeting vector further comprises a specific recombination system. Further preferably, the specific recombination system is a Frt recombination site (conventional LoxP recombination systems may also be selected). The number of the specific recombination systems is 2, and the specific recombination systems are respectively arranged at two sides of the resistance gene in the same direction.

Preferably, the non-human animal is an immunodeficient non-human mammal. Advancing onePreferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Still further preferred, the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag1 ^-/- -IL2rg ^-/- Mouse, rag 2 ^-/- -IL2rg ^-/- Mice, NOD/SCID mice or nude mice.

In a fourth aspect of the invention there is provided an sgRNA which targets a non-human animal TROP2 gene.

Preferably, the sequence of the sgRNA is at a target sequence on the TROP2 gene to be altered.

Preferably, the target site of the sgRNA is located on exon 1 of the TROP2 gene.

Preferably, the sgRNA targeting target site sequence comprises SEQ ID NO:13 and/or 14.

In a fifth aspect of the invention there is provided a DNA molecule encoding the sgRNA described above.

Preferably, the double strand of the DNA molecule is the upstream and downstream sequence of the sgRNA, or the forward oligonucleotide sequence or the reverse oligonucleotide sequence after adding the cleavage site.

In a sixth aspect of the invention there is provided an sgRNA vector comprising the above sgRNA or the above DNA molecule.

In a seventh aspect of the invention, there is provided a cell comprising the targeting vector described above, the sgRNA described above, the DNA molecule described above and/or the sgRNA vector described above.

In an eighth aspect of the invention there is provided the use of the targeting vector described above, the sgRNA described above, the DNA molecule described above, the sgRNA vector described above and/or the cell described above in the modification of the TROP2 gene.

Preferably, the application includes, but is not limited to, knockout, insertion, or replacement.

In a ninth aspect of the invention there is provided a non-human animal humanized with a TROP2 gene.

Preferably, the non-human animal expresses human or humanized TROP2 protein in vivo and/or the genome of the non-human animal comprises human or humanized TROP2 gene.

Preferably, the humanized TROP2 protein is expressed in the non-human animal.

Preferably, the non-human animal genome comprises the humanized TROP2 gene described above.

Preferably, the non-human animal has reduced or absent expression of endogenous TROP2 protein.

Preferably, the non-human animal genome comprises a nucleotide sequence encoding all or part of a human TROP2 protein, more preferably comprises all or part of a signal peptide, extracellular region, transmembrane region and/or cytoplasmic region encoding a human TROP2 protein, more preferably comprises a nucleotide sequence encoding at least 10 to at least 323, preferably 10, 20, 23, 26, 30, 50, 100, 150, 200, 248, 250, 300, 310, 320 or 323 consecutive amino acids of a human TROP2 protein.

In one embodiment of the invention, the non-human animal genome comprises a sequence encoding SEQ ID NO:2, a nucleotide sequence of the amino acid sequence shown in seq id no; or, comprises a sequence identical to the sequence encoding SEQ ID NO:2 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, with a sequence encoding SEQ ID NO:2, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, with a sequence encoding SEQ ID NO:2, including nucleotide sequences in which one or more nucleotides are substituted, deleted and/or inserted.

Preferably, the non-human animal genome comprises all or part of exon 1 of the human TROP2 gene. Wherein the portion of exon 1 comprises a contiguous nucleotide sequence of at least 200bp to at least 1820bp, e.g., 200, 500, 700, 900, 950, 970, 971, 972, 973, 974, 975, 1000, 1500, 1800, or 1820 bp; preferably, the portion of exon 1 comprises a nucleotide sequence from the start codon to the stop codon, and more preferably, the genome of the non-human animal comprises the CDS sequence of the human TROP2 gene.

In one embodiment of the present invention, the genome of the non-human animal comprises the amino acid sequence of SEQ ID NO: 7; alternatively, comprising a sequence identical to SEQ ID NO:7 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; alternatively, comprising a sequence identical to SEQ ID NO:7 does not differ by more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or not more than 1 nucleotide; alternatively, it comprises a polypeptide having the sequence of SEQ ID NO:7, including substitutions, deletions and/or insertions of one or more nucleotides.

Preferably, the non-human animal genome comprises a portion of a non-human animal endogenous TROP2 gene.

Further preferred, the non-human animal genome comprises the 5'UTR and/or the 3' UTR of the endogenous TROP2 gene.

Preferably, the genome of at least one cell of said non-human animal comprises a nucleotide sequence encoding a human or humanized TROP2 protein, and/or a human or humanized TROP2 gene.

Preferably, the nucleotide sequence of the human or humanized TROP2 gene, and/or the nucleotide sequence encoding a human or humanized TROP2 protein, is operably linked to endogenous regulatory elements at an endogenous TROP2 locus in at least one chromosome.

Preferably, the non-human animal is constructed by introducing into the non-human animal TROP2 locus any one of the following donor nucleotide sequences:

d) Nucleotide sequences encoding the above mentioned humanized TROP2 proteins.

Preferably, the non-human animal is constructed by targeting vectors as described above.

Preferably, the non-human animal further comprises additional genetic modifications selected from at least one of HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL, CD3, CD28, and CD 38.

Preferably, the non-human animal further expresses at least one of human or humanized HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL, CD3, CD28, and CD38 proteins.

Preferably, the human or humanized TROP2 gene and/or the other gene is homozygous for the endogenous modified (preferably replacement) locus.

Preferably, the human or humanized TROP2 gene and/or the other gene is heterozygous for the endogenous modified (preferably replaced) locus.

Preferably, at least one chromosome of the genome of the non-human animal comprises a human or humanized TROP2 gene, more preferably comprises a humanized TROP2 gene as described above.

Preferably, at least one cell in said non-human animal expresses a human or humanized TROP2 protein, more preferably expresses the above humanized TROP2 protein.

In a tenth aspect of the invention, there is provided a method of constructing a non-human animal humanized with a TROP2 gene.

Preferably, the non-human animal expresses human or humanized TROP2 protein in vivo and/or the non-human animal genome comprises a portion of a human TROP2 gene or a humanized TROP2 gene.

Preferably, the humanized TROP2 protein is expressed in the non-human animal.

Preferably, the non-human animal genome comprises a nucleotide sequence encoding all or part of a human TROP2 protein, more preferably comprises a nucleotide sequence encoding all or part of a signal peptide, extracellular region, transmembrane region and/or cytoplasmic region of a human TROP2 protein, more preferably comprises a nucleotide sequence encoding at least 10 to at least 323, for example 10, 20, 23, 26, 30, 50, 100, 150, 200, 248, 250, 300, 310, 320 or 323 consecutive amino acids of a human TROP2 protein.

Preferably, the non-human animal genome comprises all or part of exon 1 of the human TROP2 gene. Wherein the portion of exon 1 comprises a contiguous nucleotide sequence of at least 200bp to at least 1820bp, e.g., 200, 500, 700, 900, 950, 970, 971, 972, 973, 974, 975, 1000, 1500, 1800, or 1820 bp; preferably, the portion of exon 1 comprises a nucleotide sequence from the start codon to the stop codon, and more preferably, the non-human animal genome comprises the CDS sequence of the human TROP2 gene.

Preferably, the non-human animal is a non-human animal humanized with the TROP2 gene.

Preferably, the construction method comprises introducing a donor nucleotide sequence into a non-human animal TROP2 locus.

Preferably, the donor nucleotide sequence comprises any one of the following groups:

d) Nucleotide sequences encoding the above mentioned humanized TROP2 proteins.

Preferably, the donor nucleotide sequence of any of the above A) -D) is expressed on a plasmid or on a chromosome.

Preferably, the introduction is insertion or substitution or transgene.

The insertion is to place the target fragment between two adjacent bases without deleting the nucleotide, wherein the target fragment is a nucleotide sequence obtained by splicing a human TROP2 gene, a humanized TROP2 gene, a nucleotide sequence encoding a human or humanized TROP2 protein, a human TROP2 gene and a non-human animal TROP2 gene. Of course, it may also be part of the nucleotide sequence of the human TROP2 gene.

In one embodiment of the invention, the construction method comprises modifying the coding box of the non-human animal TROP2 gene, inserting a nucleotide sequence encoding a human or humanized TROP2 protein or a nucleotide sequence of a humanized TROP2 gene into the endogenous regulatory element of the non-human animal TROP2 gene. Wherein, the coding frame of the modified non-human animal TROP2 gene can be a functional region of the non-human animal TROP2 gene or a sequence inserted into the functional region, so that the non-human animal TROP2 protein is not expressed or the expression is reduced or the expressed protein is not functional. Further preferably, the coding box of the modified non-human animal TROP2 gene may be the whole or part of the nucleotide sequence of exon 1 of the non-human animal TROP2 gene.

Preferably, the insertion may also include disruption of the coding box of the endogenous TROP2 gene of the non-human animal or disruption of the coding box of the endogenous TROP2 gene following insertion sequence, as desired in the particular embodiment, followed by an insertion procedure. Alternatively, the insertion step may be performed by either frameshift mutation of the endogenous TROP2 gene or by inserting a sequence of human origin.

In one embodiment of the invention, the construction method comprises inserting a nucleotide sequence encoding a human or humanized TROP2 protein or a nucleotide sequence and/or auxiliary sequences of a humanized TROP2 gene into endogenous regulatory elements of a non-human animal TROP2 gene. Preferably, the auxiliary sequence may be a sequence having a termination function such that the TROP2 gene is humanized in an animal model to express human or humanized TROP2 protein in vivo and not to express non-human animal TROP2 protein. Further preferably, the helper sequence may be a WPRE and/or STOP sequence.

Preferably, the insertion site is after an endogenous regulatory element of the TROP2 gene.

Wherein the substitution includes substitution of corresponding position or substitution of non-corresponding position, the substitution of corresponding position not only represents mechanical substitution of direct correspondence of human and non-human animal TROP2 gene base site, but also includes substitution of corresponding functional region.

Preferably, the introduced non-human animal TROP2 locus is a replacement for a corresponding region of a non-human animal.

It is further preferred to replace all or part of the nucleotide sequence encoding a non-human animal TROP2 protein in the genome of the non-human animal.

More preferably, all or part of exon 1 of the non-human animal TROP2 gene is replaced.

Still more preferably, the non-human animal genome encodes SEQ ID NO:1 is replaced with the nucleotide sequence of the amino acid sequence shown in 1.

Preferably, a portion of the human TROP2 gene or the humanized TROP2 gene is regulated in a non-human animal by regulatory elements. Further preferably, the regulatory element may be endogenous or exogenous.

Preferably, the regulatory elements include, but are not limited to, promoters.

In a specific embodiment of the invention, the endogenous regulatory element is derived from a non-human animal TROP2 gene. The exogenous regulatory element is derived from the human TROP2 gene.

In one embodiment of the invention, the construction method comprises inserting or replacing all or part of the nucleotide sequence of the non-human animal TROP2 gene with a part of the nucleotide sequence comprising the human TROP2 gene.

In one embodiment of the invention, the construction method comprises inserting or replacing all or part of the nucleotide sequence of exon 1 of a non-human animal TROP2 gene with all or part of exon 1 comprising a human TROP2 gene.

In one embodiment of the invention, the construction method comprises inserting or replacing a nucleotide sequence encoding a human or humanized TROP2 protein into the genome of a non-human animal with a nucleotide sequence comprising a human or humanized TROP2 gene or a nucleotide sequence encoding a human or humanized TROP2 protein encoding SEQ ID NO:1, and a nucleotide sequence of the amino acid sequence shown in 1.

In one embodiment of the invention, the construction method comprises inserting or replacing a genomic DNA, cDNA sequence or CDS sequence of the human TROP2 gene in the genome of a non-human animal encoding the sequence of SEQ ID NO:1, and a nucleotide sequence of the amino acid sequence shown in 1.

In one embodiment of the invention, the construction method comprises using a nucleic acid sequence comprising a sequence encoding SEQ ID NO:2 into or replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1, and a nucleotide sequence of the amino acid sequence shown in 1.

In one embodiment of the invention, the construction method comprises using a nucleic acid sequence comprising SEQ ID NO:7 into or replacing the nucleotide sequence encoding SEQ ID NO:1, and a nucleotide sequence of the amino acid sequence shown in 1.

Preferably, the construction of a non-human animal humanized with a TROP2 gene is performed using gene editing techniques including gene targeting techniques using embryonic stem cells, CRISPR/Cas9 techniques, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases or other molecular biology techniques. Preferably, the construction of the non-human animal is performed using the targeting vector described above.

In one embodiment of the present invention, the construction method comprises introducing the targeting vector into a non-human animal cell (preferably an embryonic stem cell), introducing a positive clone cell into the isolated blastocyst, transplanting the blastocyst into a female non-human animal oviduct, allowing the female non-human animal to develop, and identifying and screening the non-human animal to obtain the TROP2 gene humanized animal.

Preferably, to increase recombination efficiency, the targeting vector described above can also be used in conjunction with the sgrnas described above to construct non-human animals.

In one embodiment of the present invention, the construction method comprises introducing the targeting vector, the sgRNA and Cas9 into a non-human animal cell, culturing the cell (preferably a fertilized egg), transplanting the cultured cell into a oviduct of a female non-human animal, allowing the female non-human animal to develop, and identifying and screening the non-human animal to obtain the humanized TROP2 gene.

According to some embodiments of the invention, the method of constructing further comprises: mating the TROP2 gene humanized non-human animal with other non-human animals modified by genes, inseminating in vitro or directly carrying out gene editing, and screening to obtain the non-human animals modified by multiple genes.

Preferably, the additional gene is selected from at least one of HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL17, CD3, CD28 and CD 38.

Preferably, the human or humanized TROP2 gene and/or other genes are homozygous for the endogenous modified (preferably replacement) locus.

Preferably, the human or humanized TROP2 gene and/or other genes are heterozygous with respect to the endogenous modified (preferably alternative) locus.

Preferably, each of the plurality of genes modified in the genome of the polygenously modified non-human animal is homozygous for the endogenous modified (preferably replacement) locus.

Preferably, each of the plurality of genes modified in the genome of the polygenously modified non-human animal is heterozygous for the endogenous modified (preferably alternative) locus.

Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferred, said immunodeficiencyIs an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Still further preferred, the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag1 ^-/- -IL2rg ^-/- Mouse, rag 2 ^-/- -IL2rg ^-/- Mice, NOD/SCID mice or nude mice.

In an eleventh aspect of the invention there is provided a non-human animal having a deletion of the TROP2 gene, said non-human animal having a deletion of all or part of exon 1 of the endogenous TROP2 gene.

In a twelfth aspect of the invention, there is provided a method of constructing a non-human animal with a TROP2 gene deletion, the method comprising preparing the non-human animal using the targeting vector and/or the sgRNA described above.

In a thirteenth aspect of the present invention, there is provided a TROP2 gene-deleted cell which lacks all or part of exon 1 of a TROP2 gene.

In a fourteenth aspect of the present invention, there is provided a method for constructing a TROP2 gene-deleted cell, comprising constructing a TROP2 gene-deleted cell using the targeting vector and/or the sgRNA described above.

In a fifteenth aspect of the present invention, there is provided a method of constructing a polygenically modified non-human animal comprising the steps of:

1) Providing the non-human animal described above, or a non-human animal obtained using the above construction method;

2) Mating the non-human animal provided in the step 1) with other non-human animals modified by genes, inseminating in vitro or directly carrying out gene editing, and screening to obtain the non-human animal modified by multiple genes.

Preferably, the additional genetically modified non-human animal comprises at least one of HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL17, CD3, CD28, and CD38 genetically modified non-human animals.

Preferably, the polygene modified non-human animal is a double-gene humanized non-human animal, a three-gene humanized non-human animal, a four-gene humanized non-human animal, a five-gene humanized non-human animal, a six-gene humanized non-human animal, a seven-gene humanized non-human animal, an eight-gene humanized non-human animal or a nine-gene humanized non-human animal.

In a sixteenth aspect of the invention, there is provided a non-human animal or progeny thereof obtained by any of the above construction methods.

In a seventeenth aspect of the invention there is provided a cell, tissue or organ which expresses a human or humanized TROP2 protein as defined above, or which comprises a humanized TROP2 gene as defined above in its genome, or which is derived from a non-human animal as defined above, or which is obtained by a method of construction as defined above.

In an eighteenth aspect of the invention, there is provided a tumor tissue following tumor bearing, said tumor tissue expressing the above mentioned humanized TROP2 protein or alternatively, said tumor tissue genome comprising the above mentioned humanized TROP2 gene. Alternatively, the tumor tissue after tumor loading is derived from the non-human animal, or the non-human animal obtained by the construction method.

In a nineteenth aspect of the present invention, there is provided an animal model derived from the above-described non-human animal or the non-human animal obtained by the above-described construction method. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

In a twentieth aspect of the present invention, there is provided a method of constructing an animal model, the method comprising using the above-described non-human animal or progeny thereof, the non-human animal obtained by the above-described method of constructing, and progeny thereof. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

In a twenty-first aspect of the present invention, there is provided a non-human animal as described above, and the use of a non-human animal obtained by the above construction method in constructing an animal model. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

In a twenty-second aspect, the invention provides a use of the above-described non-human animal, the non-human animal obtained by the above-described construction method, or the above-described animal model in the preparation of a medicament for treating a tumor, an inflammation, or an immune-related disorder.

In a twenty-third aspect of the invention there is provided a cell humanised with a TROP2 gene, said cell expressing a human or humanised TROP2 protein and/or comprising a portion of a human TROP2 gene or a humanised TROP2 gene in the genome of said cell.

Preferably, the cells express a humanized TROP2 protein as described above.

Preferably, the genome of the cell comprises the above-described humanised TROP2 gene.

In a twenty-fourth aspect of the invention, there is provided a genome of a non-human animal humanized with a TROP2 gene.

Preferably, the genome comprises all or part of a human or humanized TROP2 gene and/or all or part of a nucleotide sequence encoding a human or humanized TROP2 protein.

Preferably, the humanized TROP2 gene is the humanized TROP2 gene described above.

Preferably, the humanized TROP2 protein is a humanized TROP2 protein as described above.

Preferably, the genome comprises replacing a genomic fragment of a human TROP2 gene at a non-human animal endogenous TROP2 locus (preferably all or part of the CDS sequence of the human TROP2 gene or all or part of exon 1 of the human TROP2 gene or the start codon to the stop codon of the human TROP2 gene) with a genomic fragment of a human TROP2 gene to form a modified TROP2 gene.

Preferably, the genomic fragment of the substituted non-human animal TROP2 gene comprises all or part of exon 1 of the non-human animal TROP2 gene.

Preferably, the modified TROP2 gene encodes a humanized TROP2 protein.

Preferably, the expression of the modified TROP2 gene is controlled by regulatory elements endogenous to the non-human animal.

Preferably, the genome comprises a humanised endogenous TROP2 locus in which a fragment of the endogenous TROP2 locus has been deleted and replaced with a corresponding human TROP2 sequence.

Preferably, the humanised TROP2 locus comprises an endogenous TROP2 promoter, wherein the human TROP2 sequence is operably linked to the endogenous TROP2 promoter.

Preferably, all or part of exon 1 of the endogenous TROP2 locus has been deleted and replaced with the corresponding human TROP2 sequence.

In a specific embodiment of the invention, the entire TROP2 coding sequence of the endogenous TROP2 locus has been deleted and replaced with the corresponding human TROP2 sequence.

In a specific embodiment of the invention, the region of the endogenous TROP2 locus from the start codon to the stop codon has been deleted and replaced with the corresponding human TROP2 sequence.

In a specific embodiment of the invention, the 3'UTR and/or the 5' UTR of the endogenous TROP2 gene has not been deleted and has not been replaced with the corresponding human TROP2 sequence.

In a twenty-fifth aspect of the present invention there is provided the use of a humanized TROP2 protein as defined above, a humanized TROP2 gene as defined above, a non-human animal as defined above or as defined above, a cell, tissue or organ as defined above or a tumour tissue or animal model as defined above, wherein said use comprises:

a) Use in the product development of a TROP 2-related immune process involving human cells;

b) Use in a TROP 2-related model system as pharmacological, immunological, microbiological and medical study;

c) To the use of animal experimental disease models for the production and use in the study of etiology associated with TROP2 and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

d) The application of the TROP2 signal path regulator in screening, drug effect detection, efficacy evaluation, verification or evaluation of in-vivo researches is carried out; or alternatively, the process may be performed,

e) The TROP2 gene function is researched, the medicine and the medicine effect aiming at the target site of the human TROP2 are researched, and the application of the TROP 2-related immune-related disease medicine and the anti-tumor medicine is researched.

In a twenty-sixth aspect of the present invention there is provided a non-human animal derived from the above described non-human animal, the above described non-human animal obtained by the above described construction method or the above described animal model for use in screening for a human TROP 2-specific modulator.

In a twenty-seventh aspect of the present invention, there is provided a method of screening for a human TROP 2-specific modulator, said method comprising applying the modulator to an individual in which tumor cells are implanted, and detecting tumor suppression; wherein the individual is selected from the non-human animals described above or the non-human animals constructed by the methods described above or the tumor-bearing animal models described above.

Preferably, the modulator is selected from CAR-T, a drug. Further preferably, the drug is an antibody, in particular, the drug may be an anti-TROP 2 antibody.

Preferably, the regulator is monoclonal antibody or bispecific antibody or the combination of two or more drugs.

Preferably, the detection comprises determining the size and/or proliferation rate of the tumour cells.

Preferably, the method of detection comprises vernier caliper measurement, flow cytometry detection and/or animal live imaging detection.

Preferably, the detecting comprises assessing an individual's weight, fat mass, activation pathway, neuroprotective activity, or metabolic change, including a change in food consumption or water consumption.

Preferably, the tumor cells are derived from a human or non-human animal.

Preferably, the screening method may be for therapeutic or non-therapeutic purposes. The screening method detects and evaluates the effect of the modulator to determine whether the modulator has a therapeutic effect, i.e., the therapeutic effect is not necessarily, but is merely a possibility.

In a twenty-eighth aspect of the present invention, there is provided a method for screening or evaluating human drugs, the method comprising constructing an individual animal model of a disease, administering a drug candidate to the individual animal model of a disease, and detecting and/or comparing the efficacy of the drug candidate administered individual. Wherein the individual is selected from the group consisting of the non-human animal obtained by the above-described construction method, the non-human animal or its progeny or the animal model of tumor or inflammation.

Preferably, the method of drug screening or evaluation may be for therapeutic or non-therapeutic purposes. The method is used for screening or evaluating medicines, detecting and comparing the medicine effects of candidate medicines to determine which candidate medicines can be taken as medicines and which can not be taken as medicines, or comparing the medicine effect sensitivity degree of different medicines, namely that the treatment effect is not necessarily the same, but is only one possibility.

Preferably, the drug candidate comprises a targeted drug. Further preferred, the targeted drug is an antigen binding protein. In one embodiment of the invention, the antigen binding protein is an antibody.

Preferably, the candidate drug is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

Preferably, the detection comprises determining the size and/or proliferation rate of tumor cells; preferably, the method of detection comprises vernier caliper measurement, flow cytometry detection and/or animal live imaging detection.

Preferably, the non-human animal of any of the above is a non-human mammal. Further preferably, the non-human mammal is a rodent. Still more preferably, the rodent is a rat or mouse.

Preferably, the non-human animal of any of the above may also be selected from any non-human animal that can be genetically edited to produce a humanized gene, such as pigs, rabbits, monkeys, etc.

The "immune-related diseases" described herein include, but are not limited to, allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, and the like.

"tumor" as used herein includes, but is not limited to, oral squamous carcinoma, glioma, lymphoma, non-small cell lung carcinoma, cervical carcinoma, leukemia, ovarian carcinoma, nasopharyngeal carcinoma, breast carcinoma, endometrial carcinoma, colon carcinoma, rectal carcinoma, gastric carcinoma, bladder carcinoma, glioma, lung carcinoma, bronchial carcinoma, bone carcinoma, prostate carcinoma, pancreatic carcinoma, liver and bile duct carcinoma, esophageal carcinoma, renal carcinoma, thyroid carcinoma, head and neck carcinoma, testicular carcinoma, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcomas. Wherein the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; the lymphoma is selected from hodgkin's lymphoma and non-hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In one embodiment of the invention, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, liver cancer.

"inflammation" as used herein includes acute inflammation as well as chronic inflammation. In particular, including but not limited to, degenerative inflammation, exudative inflammation (serositis, cellulitis, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, jatropha, lymphogranuloma, etc.).

The term "treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily refer to the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

The "locus" as used herein refers broadly to the location of a gene on a chromosome, and in a narrow sense to a DNA fragment on a gene, either a gene or a portion of a gene. For example, the "TROP2 locus" refers to a DNA fragment of an optional stretch on exon 1 of the TROP2 gene. In a specific embodiment of the invention, the replaced TROP2 locus may be a DNA fragment of an optional stretch on exon 1 of the TROP2 gene.

The "nucleotide sequence" as used herein includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences. Preferably DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, tRNA.

The invention relates to all or part of the whole, the whole is the whole, the part is the part of the whole or the whole individual.

The "humanized TROP2 protein" of the present invention comprises a portion derived from a human TROP2 protein. Wherein, the human TROP2 protein is identical to the whole human TROP2 protein, namely the amino acid sequence of the human TROP2 protein is identical to the full-length amino acid sequence of the human TROP2 protein. The "part of human TROP2 protein" is a continuous or intermittent 5-323 (preferably 10-323, for example, 5, 10, 15, 20, 50, 100, 150, 200, 250, 300, 310, 320 or 323) amino acid sequence which is identical to the amino acid sequence of human TROP2 protein or has more than 70% homology with the amino acid sequence of human TROP2 protein.

The "humanized TROP2 gene" described in the present invention includes a portion derived from a human TROP2 gene. Wherein, the human TROP2 gene is identical to the whole human TROP2 gene, namely the nucleotide sequence is identical to the full-length nucleotide sequence of the human TROP2 gene. The "portion of human TROP2 gene" is a contiguous or spaced 20-1820bp (preferably 20-972bp, e.g., 20, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 950, 970, 971, 972, 973, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1820 bp) nucleotide sequence that is identical to a human TROP2 nucleotide sequence or has more than 70% homology to a human TROP2 nucleotide sequence.

"part of an exon" as used herein means that several, tens or hundreds of nucleotide sequences are identical to all exon nucleotide sequences, either consecutively or at intervals. For example, the portion of exon 1 of the human TROP2 gene comprises a contiguous or spaced 5-1820bp, preferably 10-972bp, nucleotide sequence that is identical to the nucleotide sequence of exon 1 of the human TROP2 gene.

In one embodiment of the present invention, the "portion of exon 1" included in the "humanized TROP2 gene" includes at least a nucleotide sequence from a start codon to a stop codon.

The "cells" as described herein may be fertilized egg cells or other somatic cells, preferably including, but not limited to, platelets, monocytes, microglial cells and endothelial cells, neutrophils, activated macrophages, B cell precursors, dendritic cells, natural killer cells, late B cells or plasma cells, and the like. Thus, depending on the source of the cell, a portion of the cell described herein may develop into an individual animal and a portion may not develop into an individual animal.

"TROP2 proteins", such as "human TROP2 proteins", "non-human animal TROP2 proteins" or "humanized TROP2 proteins", as described herein, each comprise a signal peptide, an extracellular region, an intracellular region and/or a transmembrane region.

The terms "comprises" and "comprising" as used herein are intended to be inclusive and open-ended as described above, and to exclude the presence of any other specified elements or steps. However, when used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, yet still have the activity described herein.

"homology" as used herein means that a person skilled in the art can adjust the sequence according to actual work requirements, using sequences that are 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%,26%,27%,28%,29%,30%,31%,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,70%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%, 99.1%,99.2%,99.3%,99.4%, 99.6%,99.7%, 99.9% and the like, as compared with sequences obtained by the prior art.

One skilled in the art can determine and compare sequence elements or degrees of identity to distinguish additional mouse and human sequences.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology. These techniques are explained in detail in the following documents. For example: molecular Cloning A Laboratory Manual,2ndEd., by Sambrook, fritschand Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, volumes I and II (D.N.Glcovered., 1985); oligonucleotide Synthesis (m.j. Gaited., 1984); mullisetal, u.s.stop.no. 4, 683, 195; nucleic Acid Hybridization (B.D.Hames & S.J.Higginseds.1984); transcription And Translation (B.D.Hames & S.J.Higginseds.1984); culture Of Animal Cells (R.I.Freshney, alanR.Liss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); perbal, A Practical Guide To Molecular Cloning (1984); the services, methods In ENZYMOLOGY (j. Abelson and m. Simon, eds. Inch, academic Press, inc., new York), special, vols.154and 155 (wuetal. Eds.) and vol.185, "Gene Expression Technology" (d. Goeddel, ed.); gene Transfer Vectors For Mammalian Cells (j.h.miller and M.P.Caloseds.,1987,Cold Spring Harbor Laboratory); immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., academic Press, london, 1987); handbook Of Experimental Immunology, volumes V (d.m. weir and c.c. blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y., 1986).

The foregoing is merely illustrative of some aspects of the present invention and is not, nor should it be construed as limiting the invention in any respect.

All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. It will be appreciated by those skilled in the art that certain changes may be made thereto without departing from the spirit or scope of the invention.

The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

The humanized animal model of the gene, namely, the humanized animal model of the gene can be established by replacing homologous genes of animal genome with human normal or mutant genes by utilizing the gene editing technology, and the animal model of the normal or mutant genes which are more similar to the physiological or disease characteristics of human can be established. The humanized animal has important application value, such as the humanized animal model transplanted by cells or tissues can be improved and promoted by gene humanized, and more importantly, the humanized protein can be expressed or partially expressed in the animal body due to the insertion of human gene fragments, can be used as a target spot of a medicament capable of only recognizing human protein sequences, and provides possibility for screening anti-human antibodies and other medicaments at animal level.

In addition, the non-human animal obtained by the method can also be mated with other humanized non-human animals to obtain a polygenic humanized animal model, which is used for screening and evaluating the study of the drug effect of the human drug and the combined drug aiming at the signal path. The invention has wide application prospect in academic and clinical research.

Drawings

Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1: schematic representation of the comparison of the mouse TROP2 locus and the human TROP2 locus (not to scale);

fig. 2: schematic representation (not to scale) of the humanization of the mouse TROP2 gene;

fig. 3: TROP2 gene targeting strategy and targeting vector V1 design schematic (not to scale);

fig. 4: f1 generation genotype identification result, wherein WT is wild type control, PC is positive control, H ₂ O is water control;

fig. 5: TROP2 gene targeting strategy and targeting vector V2 design schematic (not to scale);

fig. 6: genotyping results in F0 mice, wherein WT is wild type control, H ₂ O is water control;

fig. 7: genotyping results in F1 mice, wherein WT is wild type control, H ₂ O is water control;

fig. 8: southern blot detection results, wherein WT is wild-type control;

fig. 9: RT-PCR detection result, wherein +/+ is wild type C57BL/6 mice, H/H is TROP2 gene humanized homozygote mice, H ₂ O is water control, GAPDH is glyceraldehyde-3-phosphate dehydrogenase reference;

fig. 10: western blot detection results, wherein M is Marker, +/+ is wild type contrast, and H/H is TROP2 gene humanized homozygote mouse;

fig. 11: rat tail PCR identification result of gene knockout mouse, wherein WT is wild type, H ₂ O is water control;

fig. 12: flow detection result of leukocyte subpopulation ratio in spleen, wherein +/+ is wild type control, H/H is TROP2 gene humanized homozygote mouse;

fig. 13: flow detection result of T cell subgroup ratio in spleen, wherein +/-is wild control, H/H is TROP2 gene humanized homozygote mouse;

fig. 14: flow detection results of leukocyte subpopulations in lymph nodes, wherein +/+ is wild-type control and H/H is TROP2 gene humanized homozygous mice;

fig. 15: flow detection results of T cell subpopulations in lymph nodes, wherein +/+ is wild type control and H/H is TROP2 gene humanized homozygous mice;

fig. 16: flow detection result of leukocyte subpopulation ratio in peripheral blood, wherein +/+ is wild type control, H/H is TROP2 gene humanized homozygote mouse;

fig. 17: flow detection result of T cell subgroup ratio in peripheral blood, wherein +/- + is wild type control, and H/H is TROP2 gene humanized homozygote mouse;

Fig. 18: average body weight of mice of different groups after injection of normal saline, MMAE or Ab 1;

fig. 19: mean weight change in mice of different groups after injection of saline, MMAE or Ab 1.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

In each of the following examples, the devices and materials were obtained from several companies as indicated below:

BbsI, ecoRI, bamHI, ecoRV, bclI enzyme was purchased from NEB under the accession number R0539L, R0101M, R0136M, R0195L, R3160L, respectively;

c57BL/6 mice were purchased from national rodent laboratory animal seed center of China food and drug verification institute;

ambion in vitro transcription kit was purchased from Ambion under the trade designation AM1354;

cas9mRNA source SIGMA, cat No. Cas9mRNA-1EA;

UCA kit is from Baiocigram company, with the product number BCG-DX-001;

The Human TROP-2 antibody (hTROP 2) was purchased from R & D, cat# AF650-SP;

the Mouse TROP-2 antibody (mTROP 2) is available from R & D under the accession number AF1122-SP;

purified anti-mouse CD16/32 anti-body available from Biolegend under accession number 101302;

Zombie NIR ^TM fixable Viability Kit from Biolegend, cat 423106;

Brilliant Violet 510 ^TM anti-mouse CD45 was purchased from Biolegend, cat 103138;

PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) anti-body available from bioleged under the accession number 108426;

Brilliant Violet 421 ^TM anti-mouse CD4 was purchased from Biolegend, cat 100438;

FITC anti-mouse F4/80 was purchased from Biolegend, cat# 123108;

PE anti-mouse CD8a anti-body is available from Biolegend under accession number 100708;

FITC anti-Mouse CD19 from Biolegend, cat# 115506;

PerCP/Cy5.5 anti-mouse TCR beta chain is available from Biolegend under accession number 109228;

Brilliant Violet 605 ^TM anti-mouse CD11c was purchased from Biolegend, cat 117334;

PE anti-mouse/human CD11b is available from Biolegend under accession number 101208;

PE/Cy ^TM 7Mouse anti-Mouse NK1.1 fromBD Pharmingen, cat No. 552878;

APC Hamster Anti-Mouse TCR beta Chain is available from BD Pharmingen under the accession number 553174;

APC anti-mouse/rate Foxp3 was purchased from eBioscience under the accession number 17-5773-82.

EXAMPLE 1 TROP2 Gene humanized mice

The alignment of the mouse TROP2 Gene (NCBI Gene ID:56753,Primary source:MGI:1861606,UniProt:Q8BGV3, located at positions 67511043 to 67512806 of chromosome 6 NC-000072.7, based on transcript NM-020047.3 and its encoded protein NP-064431.2 (SEQ ID NO: 1)) with the human TROP2 Gene (NCBI Gene ID:4070,Primary source:HGNC:11530,UniProt ID:P09758, located at positions 58575433 to 58577252 of chromosome 1 NC-000001.11, based on transcript NM-002353.3 and its encoded protein NP-002344.2 (SEQ ID NO: 2)) is shown in FIG. 1.

For the purposes of the present invention, a nucleotide sequence encoding a human TROP2 protein may be introduced at the endogenous TROP2 locus of a mouse so that the mouse expresses the human or humanized TROP2 protein. Specifically, by using gene editing technology, under the control of the mouse TROP2 gene regulatory element, the nucleotide sequence encoding human TROP2 protein is used to replace the corresponding sequence of the mouse, so that the humanized TROP2 locus is schematically shown in figure 2, and the humanized modification of the mouse TROP2 gene is realized.

The targeting strategy is further schematically shown in FIG. 3, which shows targeting vector V1 containing homologous arm sequences upstream and downstream of the mouse TROP2 gene, and fragment A comprising the nucleotide sequence encoding human TROP2 protein. Wherein the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 3) is identical to the 67512691-67516273 nucleotide sequence of NCBI accession NC_000072.7 and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) is identical to the 67506222-67510723 nucleotide sequence of NCBI accession NC_ 000072.7; the human TROP2 sequence (SEQ ID NO: 7) contained on the A fragment is identical to the nucleotide sequence at positions 97 to 1068 of NCBI accession No. NM-002353.3.

The targeting vector V1 also comprises a resistance gene for positive clone screening, namely neomycin phosphate transgene The enzyme coding sequence Neo is transferred, and two site-specific recombination systems Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo box (Neo cassette). Wherein the connection of the 5 '-end of Neo box and the mouse gene is designed to be 5' -tattaacaggcacaccttcctttgtgggttttaaaccacggaccattgtcaagcttgatatcgaattccgaagttcctat-3' (SEQ ID NO: 8) in which the sequence "ccacg"g" in "is the last nucleotide of the mouse, and" g "in the sequence" gacca "is the first nucleotide of the Neo cassette; the connection between the 3 '-end of Neo box and mouse gene is designed to be 5' -gtataggaacttcatcagtcaggtacataatggtggatcctgaaggcgcaaagcccacccccaccccccacccccagcag-3' (SEQ ID NO: 9), wherein the last "c" in the sequence "gatcc" is the last nucleotide of the Neo cassette and "t" in the sequence "tgaag" is the first nucleotide of the mouse. In addition, a coding gene (coding gene for diphtheria toxin A subunit (DTA)) with a negative selection marker was also constructed downstream of the targeting vector 3' homology arm. The mRNA sequence of the modified humanized mouse TROP2 is shown as SEQ ID NO:10, the expressed protein sequence is shown as SEQ ID NO: 2.

Targeting vector construction can be performed by conventional methods, such as enzyme digestion ligation, and the like. After the constructed targeting vector is subjected to primary verification through enzyme digestion, the targeting vector is sent to a sequencing company for sequencing verification. And (3) carrying out electroporation transfection of the targeting vector with correct sequencing verification into embryonic stem cells of a C57BL/6 mouse, screening the obtained cells by utilizing a positive clone screening marker gene, detecting and confirming the integration condition of exogenous genes by utilizing PCR and Southern Blot technology, and screening correct positive cloned cells. The correctly positive cloned cells (black mice) are introduced into the isolated blasts (white mice) according to the known technique in the art, and the obtained chimeric blasts are transferred to a culture solution for short culture and then transplanted into oviducts of recipient mice (white mice), so that F0 generation chimeric mice (black-white interphase) can be produced. And backcrossing the F0 generation chimeric mice and the wild mice to obtain F1 generation mice, and then mating the F1 generation heterozygous mice to obtain F2 generation homozygous mice. The positive mice and the Flp tool mice can also be mated to remove the positive clone screening marker genes, and then the TROP2 gene humanized homozygote mice can be obtained through the mutual mating. The genotype of the somatic cells of the offspring mice can be identified by PCR (primer sequences and fragment lengths of interest are shown in Table 1), and the identification results of exemplary F1-generation mice (from which the Neo marker gene has been deleted) are shown in FIG. 4, wherein 3 mice numbered F1-1, F1-2, and F1-3 are positive heterozygous mice. This shows that the TROP2 gene humanized mice which can be stably passaged can be constructed by using the method.

TABLE 1 F1 Generation genotype PCR detection primer sequences and recombinant fragment sizes

In addition, a CRISPR/Cas9 system can be introduced for gene editing, and a targeting strategy shown in figure 5 is designed, wherein the targeting vector V2 contains homologous arm sequences upstream and downstream of a mouse TROP2 gene and human TROP2 nucleotide sequences. Wherein the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 5) is identical to nucleotide sequence 67512691 to 67514056 of NCBI accession No. NC_000072.7, the downstream homology arm sequence (3' homology arm, SEQ ID NO: 6) is identical to nucleotide sequence 67510349 to 67511736 of NCBI accession No. NC_000072.7, and the human TROP2 nucleotide sequence is as shown in SEQ ID NO: shown at 7.

The targeting vector construction can be carried out by conventional methods, such as enzyme digestion, ligation, direct synthesis and the like. After the constructed targeting vector is subjected to primary verification through enzyme digestion, the targeting vector is sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification was used for subsequent experiments.

The target sequence determines the targeting specificity of the sgrnas and the efficiency of inducing Cas9 cleavage of the gene of interest. Therefore, efficient and specific target sequence selection and design are a prerequisite for construction of sgRNA expression vectors. And (3) designing and synthesizing sgRNA sequences for identifying target sites of the 5 'end and the 3' end, and screening sgRNA with better activity and higher sequence specificity for subsequent experiments. Exemplary target sequences for sgrnas on the TROP2 gene are shown below:

sgRNA1 target site (SEQ ID NO: 13): 5'-ATGACGGTCTGCGACACAAATGG-3'

sgRNA2 target site (SEQ ID NO: 14): 5'-CATCGCTGTCGTCTCGGTAGCGG-3'

And respectively adding enzyme cutting sites on the 5' end and the complementary strand of the sgRNA to obtain forward oligonucleotide sequences and reverse oligonucleotide sequences, annealing, and connecting annealing products to pT7-sgRNA plasmids (the plasmids are linearized by BbsI) to obtain expression vectors pT7-TROP2-1 and pT7-TROP2-2.pT7-sgRNA vector A fragment DNA (SEQ ID NO: 15) containing the T7 promoter and sgRNA scaffold was synthesized by plasmid synthesis company and ligated to a backbone vector (source Takara, cat. No. 3299) by cleavage (EcoRI and BamHI) in sequence, and the results were verified by sequencing by a professional sequencing company, and the result showed that the objective plasmid was obtained.

The mouse prokaryotic fertilized eggs, such as C57BL/6 mice, are taken, and the in vitro transcription products of pT7-TROP2-1 and pT7-TROP2-2 plasmids (transcribed according to the instruction method using an Ambion in vitro transcription kit), the targeting vector and Cas9 mRNA are premixed by a microinjection instrument and injected into the cytoplasm or nucleus of the mouse fertilized eggs. Microinjection of fertilized eggs was performed according to the method of the "mouse embryo handling laboratory Manual (third edition)" (andela, nagel, chemical industry Press, 2006), the fertilized eggs after injection were transferred into a culture medium for short-term culture, then transplanted into oviducts of recipient mice for development, and the obtained mice (F0 generation) were subjected to hybridization and selfing to expand population numbers and establish stable TROP2 gene humanized mouse strains.

The genotype of the somatic cells of the F0 mice can be identified by conventional detection methods (e.g., PCR analysis), and exemplary identification results for some F0 mice are shown in FIG. 6. The mice numbered F0-01, F0-02 and F0-03 in FIG. 6 are positive mice by combining the detection result of the 5 '-end primer and the detection result of the 3' -end primer and further verifying by sequencing. The PCR primers are shown in Table 2.

TABLE 2 F0 Generation genotyping PCR detection primer sequences and recombinant fragment sizes

Wherein the primer L-GT-F is positioned at the left side of the 5 'homology arm, the R-GT-R is positioned at the right side of the 3' homology arm, and both the L-GT-R and the R-GT-F are positioned on the human TROP2 sequence.

The TROP2 gene humanized mice identified as positive for F0 were mated with wild-type mice to obtain F1-generation mice. The same PCR method (primer sequences are shown in Table 2) can be used to genotype F1-generation mice, and exemplary test results are shown in FIG. 7, which shows 3 mice numbered F1-01, F1-02, and F1-03 as positive mice.

Southern blot detection was performed on mice identified as positive by F1 PCR to confirm the presence of random insertions. Cutting rat tail to extract genome DNA, digesting the genome with EcoRV enzyme or BclI enzyme, transferring film and hybridizing. The 5 'probe and the 3' probe are respectively positioned on the 5 'homology arm and outside the 3' homology arm, and the lengths of the specific probes and the target fragment are shown in Table 3. The Southern blot results are shown in FIG. 8, and the results of the combination of the 3 'probe and the 5' probe indicate that 3 mice numbered F1-01, F1-02 and F1-03 have no random insertions. This shows that the method can be used for constructing TROP2 gene humanized mice which can be stably passaged and have no random insertion.

TABLE 3 lengths of specific probes and fragments of interest

Restriction enzyme	Probe with a probe tip	Wild fragment size	Recombinant sequence fragment size
				EcoRV	5’Probe	12.3kb	6.2kb
BclI	3’Probe	8.3kb	6.9kb

The probe synthesis primers were as follows:

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’；

and mating the heterozygote mice identified as positive in the F1 generation with each other to obtain the humanized homozygous mice of the F2 generation TROP2 gene.

The expression of humanized TROP2 in positive mice can be confirmed by conventional detection methods, for example, using RT-PCR or flow cytometry. Specifically, 1 male wild type C57BL/6 mice at 6 weeks old and 1 TROP2 gene humanized homozygote prepared by the implementation are respectively selected, skin tissues are taken after neck removal and euthanasia, RT-PCR detection is carried out by using primer sequences shown in Table 4, and the detection results are shown in FIG. 9. As can be seen from the figure, only murine TROP2 mRNA was detected in wild type C57BL/6 mouse skin tissue, and no humanized TROP2 mRNA was detected; only humanized TROP2 mRNA was detected in the skin of the mice homozygous for the TROP2 gene, and no murine TROP2 mRNA was detected.

TABLE 4 RT PCR primer sequences and fragment sizes of interest

Western Blot was further used to detect the expression of TROP2 protein in mice. Specifically, 1 male wild type C57BL/6 mice of 6 weeks old and 1 humanized homozygote of TROP2 gene prepared in this embodiment were selected, and Skin (Skin) tissue and Kidney (Kidney) tissue were taken after neck removal and euthanization, western Blot detection was performed using an anti-human TROP2 antibody (hTROP 2) and an anti-mouse TROP2 antibody (mTROP 2), and the detection results are shown in FIG. 10. As can be seen, the expression of murine TROP2 was detected in wild type C57BL/6 mice skin and kidney tissue, and no human TROP2 was detected; detecting the expression of human TROP2 protein in both skin and kidney tissue of a TROP2 gene humanized homozygous mouse; because the anti-mouse TROP2 antibody has a certain degree of human-mouse cross-binding, a weak mTROP2 band is detected in the kidney tissue of the humanized homozygote of the TROP2 gene. The TROP2 gene humanized homozygote mice prepared in the embodiment can successfully express human TROP2 protein in vivo.

In addition, since cleavage of Cas9 causes double strand break of genomic DNA, insertion/deletion mutation is randomly generated by repair means of chromosome homologous recombination, and thus a knockout mouse with loss of function of TROP2 protein may be obtained. For this purpose, a pair of primers was designed for detecting knockout mice, the detection results are shown in FIG. 11, and the mice with numbers KO-1, KO-2 and KO-3 were further verified to be TROP2 knockout mice by sequencing. The primers were located to the left of the 5 'end target site and to the right of the 3' end target site, respectively, and the primer sequences and recombinant fragment sizes are shown in Table 5.

TABLE 5 TROP2 Gene knockout mouse genotype identification PCR primer sequence and recombinant fragment size

The spleen, lymph node and peripheral blood tissues of C57BL/6 wild-type mice (+/+) and TROP2 gene humanized homozygous mice (H/H) were subjected to immunophenotyping assays using flow cytometry, and the results of the white blood cell subtype and T cell subtype assays in spleen are shown in FIGS. 12 and 13, respectively, and the results of the white blood cell subtype and T cell subtype assays in peripheral blood are shown in FIGS. 16 and 17, respectively, as can be seen from the figures, the white blood cell subtype such as TROP2 gene humanized homozygous mice spleen and peripheral blood B Cells (B Cells), T Cells (T Cells), NK Cells (NK Cells), granulocytes (Granulocytes), DC Cells (Dendriticcells), macrophages (Macrophages) and Monocytes (Monocytes) are substantially identical to those of C57 BL/6-type mice, and the CD4+ T Cells (CD4+ T Cells), CD8+ T Cells (CD 8+ T Cells) and Tregcells (T Cells) are substantially identical to those of wild-type mice (Tregcells) and the wild-type mice (Tregcells) are substantially identical to those of C57 BL/6-type mice (Tregcells).

The results of the detection of the leukocyte subtypes in lymph nodes and the T cell subtypes are shown in FIG. 14 and FIG. 15, respectively, and it can be seen from the figures that the leukocyte subtypes such as B cells, T cells, NK cells in lymph nodes of mice homozygous for TROP2 gene are substantially identical to those of wild-type mice of C57BL/6 (FIG. 14), and the percentage of the T cell subtypes such as CD4+ T cells, CD8+ T cells, and Tregs cells are substantially identical to those of wild-type mice of C57BL/6 (FIG. 15).

It was shown that humanisation of the TROP2 gene did not affect the differentiation, development and distribution of leukocytes and T cells in mice.

In addition, 6 female wild-type C57BL/6 mice (+/+) and TROP2 humanized homozygous mice (H/H) were selected at 9 weeks of age, and peripheral blood was taken for routine blood and biochemical blood tests. Blood routine detection indexes comprise: white Blood Count (WBC), red blood cell count (RBC), hematocrit (HCT), hemoglobin (HGB), mean volume of red blood cells (MCV), mean hemoglobin of red blood cells (MCH), mean hemoglobin concentration of red blood cells (MCHC), platelet count (PLT), lymphocytes (LYMPH), monocytes (MONO), neutrophils (NEUT). Blood biochemical detection indexes comprise glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), albumin (ALB), blood sugar (GLU), UREA (UREA), serum Creatinine (CREA), serum Total Cholesterol (TC) and Triglyceride (TG). The blood routine test results (mean) are shown in Table 6, and the blood biochemical test results are shown in Table 7.

TABLE 6 blood routine test results

TABLE 7 Biochemical blood test results

From tables 6 and 7, it can be seen that the humanized modification of the TROP2 gene did not affect the composition and morphology of blood cells in mice, and the liver function status of the modified mice was substantially identical to that of wild type mice.

EXAMPLE 2 preparation of double or multiple humanized mice

The TROP2 gene humanized mice prepared by the method can also be used for preparing double-humanized or multi-humanized mouse models. For example, in example 1, embryonic stem cells used for blastocyst microinjection can be selected from other genetically modified mice containing HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL17, CD3, CD28, CD38, etc., or can be obtained from a double-or multiple-genetically modified mouse model of TROP2 and other genetically modified mice by using isolated mouse ES embryonic stem cells and a genetic recombination targeting technique based on humanized TROP2 mice. The homozygote or heterozygote of the TROP2 mouse obtained by the method can be mated with other genetically modified homozygote or heterozygote mice, the offspring thereof are screened, the humanized TROP2 and other genetically modified double-gene or polygenic modified heterozygote mice can be obtained with a certain probability according to the Mendelian genetic rule, and the homozygote of double-gene or polygenic modification can be obtained by mating heterozygote with each other, and the in vivo efficacy verification of targeted human TROP2 and other gene regulators can be carried out by utilizing the double-gene or polygenic modified mice.

EXAMPLE 3 drug efficacy

The TROP2 humanized mice prepared by the method can be used for evaluating the drug effect of a regulator targeting human TROP 2. For example, homozygotes of TROP 2-humanized mice are subcutaneously inoculated with TROP 2-humanized MC38 cells until tumor volumes grow to about 100-150mm ³ Then, according to the tumor volume fraction, the tumor volume fraction is taken as a control group or a treatment group, the treatment group randomly selects the drug targeting the human TROP2, and the control group is injected with the physiological saline with the same volume. Tumor volumes are measured periodically and the body weight of the mice is weighed, so that the in vivo safety and in vivo efficacy of the compounds can be effectively evaluated by comparing the body weight change of the mice with the tumor size.

Example 4 toxicity detection

The TROP2 humanized mice prepared by the method can be used for evaluating the toxicity of a regulator targeting human TROP 2. For example, HER2/TROP2 dual anti-ADC drug (Ab 1, conjugated MMAE) toxicity was assessed using HER2/TROP2 dual gene humanized mice. HER2/TROP2 double-gene humanized mice (HER 2/TROP 2) and C57BL/6 wild type mice (C57 BL/6) were divided into different groups according to body weight, and the control group was intravenous injected with physiological saline, and the administration groups were intravenous injected with different concentrations of Ab1 and MMAE, with the administration frequency of specific groups once a week (1 total administration), and the survival of the mice were shown in table 8. The body weight of the mice was measured daily in the experiment until the end of the experiment after 7 days, and the body weight and body weight change of the mice during the experiment are shown in fig. 18 and 19.

Table 8 grouping and dosing of mice

The results show that the mice in the groups G1-G6 and G9-G10 are all in a survival state and have no death condition, but the mice in the groups G7, G8 and G11 all have death condition, which proves that the medicament has certain toxicity. The results of fig. 18 and 19 show that the weight change was not significant after C57BL/6 wild-type mice were injected with different doses of Ab1, whereas the weight change was more significant after HER2/TROP2 double-gene humanized mice were injected with high doses of Ab1, demonstrating that HER2/TROP2 double-gene humanized mice can be used to evaluate toxicity of modulators targeting human HER2 and TROP 2.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Claims

1. A humanized TROP2 gene, wherein said humanized TROP2 gene comprises a portion of a human TROP2 gene.

2. The humanized TROP2 gene according to claim 1, characterized in that said part of the human TROP2 gene comprises all or part of exon 1 of the human TROP2 gene, wherein part of exon 1 comprises at least a nucleotide sequence of 200bp, preferably part of exon 1 comprises a nucleotide sequence from start codon to stop codon; further preferred, said portion of the human TROP2 gene comprises SEQ ID NO: 7; alternatively, comprising a sequence identical to SEQ ID NO:7 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

3. A humanised TROP2 gene according to claim 1 or 2, wherein said portion of the human TROP2 gene comprises a nucleotide sequence encoding all or part of a human TROP2 protein, preferably wherein said portion of the human TROP2 gene comprises a nucleotide sequence encoding SEQ ID NO:2 or, alternatively, comprises a nucleotide sequence encoding SEQ ID NO:2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

4. A humanised TROP2 gene according to any one of claims 1 to 3, wherein said humanised TROP2 gene further comprises a portion of a non-human animal TROP2 gene, preferably, a 5'utr and/or a 3' utr of a non-human animal TROP2 gene.

5. The humanized TROP2 gene according to any one of claims 1 to 4, wherein the nucleotide sequence of the mRNA transcribed from the humanized TROP2 gene comprises any one of the group of:

a) SEQ ID NO:10, a nucleotide sequence shown in seq id no;

6. A targeting vector comprising one of the group consisting of:

a) A nucleotide sequence encoding all or part of a human TROP2 protein; preferably all or part of a nucleotide sequence comprising a signal peptide encoding a human TROP2 protein, an extracellular region, a transmembrane region and/or a cytoplasmic region; further preferred comprises a sequence encoding SEQ ID NO:2 or, alternatively, comprises a nucleotide sequence that encodes an amino acid sequence set forth in SEQ ID NO:2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% nucleotide sequence identity to the amino acid sequence set forth in seq id no;

B) A portion of a human TROP2 gene, preferably all or part of exon 1 of a human TROP2 gene, wherein the portion of exon 1 of the human TROP2 gene comprises at least a nucleotide sequence of 200bp, preferably the portion of exon 1 comprises a nucleotide sequence from a start codon to a stop codon, further preferably the nucleotide sequence comprising SEQ ID NO: 7; alternatively, comprising a sequence identical to SEQ ID NO:7 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

c) The humanised TROP2 gene of any one of claims 1 to 5.

7. The targeting vector according to claim 6, characterized in that the targeting vector further comprises a 5 'arm and/or a 3' arm, said 5 'arm having at least 90% homology to NCBI accession nc_000072.7, preferably, said 5' arm sequence is as set forth in SEQ ID NO:3 or 5; the 3' arm has at least 90% homology to NCBI accession nc_ 000072.7; preferably, the 3' arm sequence is as set forth in SEQ ID NO:4 or 6.

8. A method for constructing a humanized non-human animal with a TROP2 gene, characterized in that the non-human animal expresses human TROP2 protein in vivo and/or the genome of the non-human animal contains a part of the human TROP2 gene or the humanized TROP2 gene;

Preferably, the humanized TROP2 gene is a humanized TROP2 gene according to any one of claims 1 to 5.

9. The method of claim 8, wherein the non-human animal has reduced or absent expression of endogenous TROP2 protein.

10. The method of any one of claims 8 to 9, comprising introducing a donor nucleotide sequence into a non-human animal TROP2 locus, said donor nucleotide sequence comprising any one of the group consisting of:

C) The humanised TROP2 gene of any one of claims 1 to 5;

preferably, the donor nucleotide sequence is regulated in a non-human animal by endogenous regulatory elements.

11. The method of claim 10, wherein the introducing is a substitution or insertion, preferably wherein the introducing is a substitution of a corresponding region of the non-human animal TROP2 locus; more preferably, a portion of exon 1 of the non-human animal TROP2 gene is replaced.

12. Construction method according to any one of claims 8-11, characterized in that the targeting vector according to any one of claims 6-7 is used for construction of non-human animals.

13. The method according to any one of claims 8-12, further comprising mating, inseminating in vitro or directly editing genes of a TROP 2-humanized non-human animal with other genetically modified non-human animals and screening to obtain a polygenic modified non-human animal;

preferably, the other gene is selected from at least one of HER2, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, IL4R, IL6R, IL17, CD3, CD28 and CD 38;

preferably, the human or humanized TROP2 gene and/or the other gene is homozygous or heterozygous for the endogenous modified locus.

14. The humanized TROP2 gene according to any one of claims 4 to 5, the construction method according to any one of claims 8 to 13, wherein said non-human animal is a rat or a mouse.

15. A cell, tissue or organ comprising the humanized TROP2 gene of any one of claims 1 to 5 in its genome, or wherein said cell, tissue or organ expresses a human TROP2 protein, or wherein said cell, tissue or organ is derived from a non-human animal obtained by the construction method of any one of claims 8 to 13;

preferably, the tissue is a neoplastic tissue.

16. Use of a humanized TROP2 gene according to any one of claims 1 to 5, a non-human animal obtained by a construction method according to any one of claims 8 to 13, a cell, tissue or organ according to claim 15, characterized in that said use comprises: