Non-human animal modified by IL12RB1, IL12RB2 and / or IL23R genes

Abstract

The present invention provides a non-human animal expressing human or chimeric (e.g., humanized) IL12RB1, IL12RB2, and / or IL23R proteins, and methods of use thereof.

Description

Technical Field

[0001] The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) IL12RB1, IL12RB2, and / or IL23R protein and methods of using the same. Background Art

[0002] Traditional drug research and development typically uses in vitro screening methods. However, these screening methods cannot provide the in vivo environment (such as the tumor microenvironment, stromal cells, extracellular matrix components, and immune cell interactions), resulting in a relatively high failure rate in drug development. In addition, due to the differences between humans and non-human animals, the test results obtained from in vivo pharmacological tests using conventional laboratory animals may not reflect the true disease state and the interactions at the target site, leading to significant differences between the results of many clinical trials and those of animal experiments.

[0003] Therefore, the development of humanized animal models suitable for screening and evaluating human drugs will significantly improve the efficiency of new drug development and reduce the cost of drug research and development. Summary of the Invention

[0004] The present application provides an animal model having a human or chimeric IL12RB1, IL12RB2, and / or IL23R protein. The animal model can express a human or chimeric IL12RB1, IL12RB2, and / or IL23R (e.g., humanized IL12RB1, IL12RB2, and / or IL23R) protein. It can be used for studying the functions of the IL12RB1, IL12RB2, and / or IL23R genes, and can also be used for screening and evaluating regulators of the IL12RB1, IL12RB2, and / or IL23R signaling pathway (e.g., therapeutic agents targeting IL12RB1, IL12RB2, and / or IL23R, such as antibodies targeting IL12RB1, IL12RB2, and / or IL23R, nucleic acid drugs targeting IL12RB1, IL12RB2, and / or IL23R, and / or polypeptide drugs). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamic studies, and treatment studies of diseases (such as cancer, inflammation, or immune diseases) targeting the human IL12RB1, IL12RB2, and / or IL23R targets; the animal model can also be used to facilitate the development and design of new drugs, saving time and cost. In summary, the present invention provides a powerful tool for studying the functions of the IL12RB1, IL12RB2, and / or IL23R proteins and provides a platform for screening drugs for the treatment of related diseases.

[0005] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. The genome of the non-human animal comprises at least one chromosome, and the chromosome comprises a nucleotide sequence encoding a human or chimeric interleukin-12 receptor subunit beta 1 (IL12RB1) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB1 protein can be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB1 protein is operably linked to an endogenous regulatory element (such as a promoter, 5'UTR and / or 3'UTR) of the endogenous IL12RB1 locus of at least one chromosome. In some embodiments, the chimeric IL12RB1 protein comprises or does not comprise a signal peptide. In some embodiments, the chimeric IL12RB1 protein comprises a human or humanized signal peptide. In some embodiments, the chimeric IL12RB1 protein comprises a human or humanized transmembrane region. In some embodiments, the chimeric IL12RB1 protein comprises a human or humanized extracellular region. In some embodiments, the chimeric IL12RB1 protein comprises a human or humanized signal peptide, a human or humanized extracellular region and a human or humanized transmembrane region. In some embodiments, the chimeric IL12RB1 protein comprises a human or humanized extracellular region, a human or humanized transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric IL12RB1 protein comprises a human or humanized signal peptide, a human or humanized extracellular region, a human or humanized transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric IL12RB1 protein is a humanized IL12RB1 protein. In some embodiments, the amino acid sequence of the chimeric IL12RB1 protein comprises an amino acid sequence that is identical to at least 50 to 662, such as at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 520, 521, 522, 523, 524, 525, 526, 530, 549, 550, 600, 650 or 662 consecutive or non-consecutive amino acid sequences of the human IL12RB1 protein. In some embodiments, the chimeric IL12RB1 protein comprises all or part of the human IL12RB1 protein. In some embodiments, the chimeric IL12RB1 protein comprises all or part of the extracellular region of the human IL12RB1 protein, preferably further comprises all or part of the transmembrane region of the human IL12RB1 protein. In some embodiments, the chimeric IL12RB1 protein comprises or does not comprise all or part of the signal peptide of the human IL12RB1 protein.In some embodiments, the amino acid sequence of the human or chimeric IL12RB1 protein comprises amino acids 1-549 of SEQ ID NO: 2, amino acids 1-570 of SEQ ID NO: 2, amino acids 24-549 of SEQ ID NO: 2, amino acids 24-570 of SEQ ID NO: 2, amino acids 1-545 of SEQ ID NO: 2, or amino acids 24-545 of SEQ ID NO: 2; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to amino acids 1-549 of SEQ ID NO: 2, amino acids 1-570 of SEQ ID NO: 2, amino acids 24-549 of SEQ ID NO: 2, amino acids 24-570 of SEQ ID NO: 2, amino acids 1-545 of SEQ ID NO: 2, or amino acids 24-545 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the chimeric IL12RB1 protein comprises amino acids 571-738 of SEQ ID NO: 1, amino acids 592-738 of SEQ ID NO: 1, amino acids 1-19 and 571-738 of SEQ ID NO: 1, amino acids 1-19 and 592-738 of SEQ ID NO: 1, amino acids 566-738 of SEQ ID NO: 1, amino acids 1-19 and 566-738 of SEQ ID NO: 1; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to amino acids 571-738 of SEQ ID NO: 1, amino acids 592-738 of SEQ ID NO: 1, amino acids 1-19 and 571-738 of SEQ ID NO: 1, amino acids 1-19 and 592-738 of SEQ ID NO: 1, amino acids 566-738 of SEQ ID NO: 1, amino acids 1-19 and 566-738 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the chimeric IL12RB1 protein comprises SEQ ID NO: 11; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB1 protein comprises the nucleotide sequence shown at positions 18,062,249 to 18,086,823 of NCBI accession number NC_000019.10; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence shown at positions 18,062,249 to 18,086,823 of NCBI accession number NC_000019.10.In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous IL12RB1 protein in the non-human animal is not expressed or has a reduced expression level compared to IL12RB1 in wild-type animals. In some embodiments, one or more cells of the non-human animal express a human or chimeric IL12RB1 protein.

[0006] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises replacing a nucleotide sequence encoding a corresponding region of endogenous IL12RB1 of the non-human animal with a nucleotide sequence encoding a human or chimeric IL12RB1 at the endogenous IL12RB1 locus of the non-human animal. In some embodiments, the nucleotide sequence encoding a human or chimeric IL12RB1 is operably linked to an endogenous regulatory element of the endogenous IL12RB1 locus, and the regulatory element preferably comprises a promoter, 5'UTR and / or 3'UTR. In some embodiments, the endogenous IL12RB1 protein of the non-human animal is not expressed or has a reduced expression level compared to IL12RB1 in a wild-type animal. In some embodiments, the corresponding region of endogenous IL12RB1 of the non-human animal comprises all or part of the endogenous extracellular region and / or all or part of the endogenous transmembrane region. In some embodiments, the corresponding region of endogenous IL12RB1 of the non-human animal comprises or does not comprise an endogenous signal peptide. In some embodiments, one or more cells of the non-human animal express a chimeric IL12RB1, which comprises an extracellular region, a transmembrane region, and a cytoplasmic region (preferably further comprises or does not comprise a signal peptide). In some embodiments, the chimeric IL12RB1 comprises a signal peptide, which is identical to or has an identity of at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% with the signal peptide of the human IL12RB1 protein. In some embodiments, the extracellular region is identical to or has an identity of at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% with the extracellular region of the human IL12RB1 protein. In some embodiments, the transmembrane region is identical to or has an identity of at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% with the transmembrane region of the human IL12RB1 protein. In some embodiments, the signal peptide comprises an amino acid sequence that is identical to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive amino acids of the signal peptide of the human IL12RB1 protein (e.g., positions 1-23 of SEQ ID NO: 2). In some embodiments, the extracellular region comprises an amino acid sequence that is identical to at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 522 consecutive amino acids of the extracellular region of the human IL12RB1 protein (e.g., positions 24-545 of SEQ ID NO: 2). In some embodiments, the transmembrane region comprises an amino acid sequence that is identical to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive amino acids of the transmembrane region of the human IL12RB1 protein (e.g., positions 546-570 of SEQ ID NO: 2).In some embodiments, the nucleotide sequence encoding human or chimeric IL12RB1 comprises a portion of exon 1, all of exons 2-13, and a portion of exon 14 of the human IL12RB1 gene (preferably also comprising intron 1 and / or intron 13). In some embodiments, the nucleotide sequence encoding human or chimeric IL12RB1 comprises a portion of exon 1, all of exons 2-13, and a portion of exon 14 of the human IL12RB1 gene (preferably also comprising intron 1 and / or intron 13) of at least 100-1000, 2000-5000, 8000-15000, 20000-24000 bp or 24000-25000 bp nucleotides. In some embodiments, the nucleotide sequence encoding human or chimeric IL12RB1 comprises the nucleotide sequence shown at positions 18062249 to 18086823 of NCBI accession number NC_000019.10; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown at positions 18062249 to 18086823 of NCBI accession number NC_000019.10. In some embodiments, the nucleotide sequence encoding the corresponding region of non-human animal endogenous IL12RB1 comprises a portion of exon 1, all of exons 2-13, and a portion of exon 14 of the non-human animal endogenous IL12RB1 gene (preferably also comprising intron 1 and / or intron 13). In some embodiments, the modified IL12RB1 gene in the non-human animal genome is homozygous or heterozygous for the endogenous locus being replaced.

[0007] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein at the endogenous IL12RB1 locus of the non-human animal, the nucleotide sequence of the corresponding region of the endogenous IL12RB1 of the non-human animal is replaced with a nucleotide sequence comprising the nucleotide sequence of human IL12RB1. In some embodiments, the nucleotide sequence of human IL12RB1 may be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the nucleotide sequence of human IL12RB1 comprises a nucleotide sequence encoding a human or chimeric IL12RB1 protein. In some embodiments, the nucleotide sequence of human IL12RB1 comprises a nucleotide sequence encoding all or part of the extracellular region of the human IL12RB1 protein, preferably further comprising a nucleotide sequence encoding all or part of the transmembrane region of the human IL12RB1 protein. In some embodiments, the nucleotide sequence of human IL12RB1 comprises a nucleotide sequence encoding all or part of the signal peptide of the human IL12RB1 protein. In some embodiments, the nucleotide sequence of human IL12RB1 comprises a nucleotide sequence encoding all of the signal peptide, all of the extracellular region and part of the transmembrane region of the human IL12RB1 protein. In some embodiments, the nucleotide sequence of human IL12RB1 includes a nucleotide sequence encoding positions 1-549 of SEQ ID NO: 2, positions 1-570 of SEQ ID NO: 2, positions 24-549 of SEQ ID NO: 2, positions 24-570 of SEQ ID NO: 2, positions 1-545 of SEQ ID NO: 2 or positions 24-545 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence of human IL12RB1 comprises all or part of the human IL12RB1 gene. In some embodiments, the nucleotide sequence of human IL12RB1 comprises a part of exon 1 to a part of exon 14 of the human IL12RB1 gene. In some embodiments, the nucleotide sequence of human IL12RB1 comprises the start codon of the human IL12RB1 gene to a part of exon 14. In some embodiments, the part of exon 1 of the human IL12RB1 gene preferably comprises at least 5-175 bp, such as at least 5, 20, 50, 60, 64, 65, 70, 100, 110, 120, 130, 140, 150, 160, 170 or 175 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region, and the part of exon 14 of the human IL12RB1 gene preferably comprises at least 5-97 bp, such as at least 5, 10, 15, 20, 25, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 97 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region.In some embodiments, the nucleotide sequence of the human IL12RB1 comprises the nucleotide sequence shown at positions 18,062,249 to 18,086,823 of NCBI accession number NC_000019.10; or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown at positions 18,062,249 to 18,086,823 of NCBI accession number NC_000019.10. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB1 comprises a nucleotide sequence encoding the non-human animal endogenous IL12RB1 protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB1 comprises a nucleotide sequence encoding all or part of the extracellular region of the non-human animal endogenous IL12RB1 protein, preferably further comprising a nucleotide sequence encoding all or part of the transmembrane region of the non-human animal endogenous IL12RB1 protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB1 comprises a nucleotide sequence encoding all or part of the signal peptide of the non-human animal endogenous IL12RB1 protein. In some embodiments, the corresponding region of the non-human animal endogenous IL12RB1 comprises a nucleotide sequence encoding all of the signal peptide, all of the extracellular region and part of the transmembrane region of the non-human animal endogenous IL12RB1 protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB1 comprises a nucleotide sequence encoding positions 1-570 of SEQ ID NO: 1, positions 1-591 of SEQ ID NO: 1, positions 20-565 of SEQ ID NO: 1, positions 20-570 of SEQ ID NO: 1, positions 20-591 of SEQ ID NO: 1 or positions 1-565 of SEQ ID NO: 1. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB1 comprises all or part of the non-human animal endogenous IL12RB1 gene. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB1 comprises part of exon 1 to part of exon 14 of the non-human animal endogenous IL12RB1 gene. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB1 comprises the start codon of the non-human animal endogenous IL12RB1 gene to part of exon 14.In some embodiments, a portion of exon 1 of the endogenous IL12RB1 gene in a non-human animal preferably comprises at least 5-170 bp, such as at least 5, 20, 50, 60, 65, 67, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 or 170 bp of contiguous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region. A portion of exon 14 of the endogenous IL12RB1 gene in a non-human animal preferably comprises at least 5-97 bp, such as at least 5, 10, 15, 20, 25, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 97 bp of contiguous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region. In some embodiments, the nucleotide sequence encoding a human or chimeric IL12RB1 protein or the nucleotide sequence of human IL12RB1 is operably linked to an endogenous regulatory element of the endogenous IL12RB1 locus. In some embodiments, the endogenous regulatory element includes a promoter, 5'UTR and / or 3'UTR. In some embodiments, the endogenous IL12RB1 protein in the non-human animal is not expressed or has a reduced expression level compared to IL12RB1 in a wild-type animal. In some embodiments, the modified IL12RB1 gene in the non-human animal genome is homozygous or heterozygous for the endogenous locus being replaced. In some embodiments, the non-human animal includes a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the mRNA transcribed from the modified IL12RB1 gene in the non-human animal genome comprises SEQ ID NO: 10; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 10. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding another human or chimeric protein, and the other human or chimeric protein preferably comprises at least one of IL12A, IL12B, IL12RB2, IL23R, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the other human or chimeric protein comprises IL12RB2, IL23R and / or PD-1. The other human or chimeric protein is a humanized IL12RB2 protein, and the amino acid sequence of the humanized IL12RB2 protein preferably comprises SEQ ID NO: 21 or 56; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 21 or 56.In some embodiments, the other human or chimeric protein is a humanized IL23R protein, and the amino acid sequence of the humanized IL23R protein preferably comprises SEQ ID NO: 78; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 78. In some embodiments, the other human or chimeric protein is a humanized PD-1 protein.

[0008] In one aspect, the present invention provides a non-human animal or a method for constructing the same, wherein the non-human animal comprises at least one cell encoding a nucleotide sequence of a human or chimeric IL12RB1 protein. In some embodiments, the chimeric IL12RB1 protein comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 520, 521, 522, 523, 524, 525, 526, 530, 549, 550, 600, 650 or 662 consecutive amino acid sequences identical to the human IL12RB1 protein. In some embodiments, the non-human animal expresses a human or chimeric IL12RB1 protein. In some embodiments, the chimeric IL12RB1 protein comprises an amino acid sequence identical to at least 50 consecutive amino acids in the extracellular region of the human IL12RB1 protein. In some embodiments, the chimeric IL12RB1 protein comprises an amino acid sequence identical to at least 1 consecutive amino acid in the transmembrane region of the human IL12RB1 protein. In some embodiments, the chimeric IL12RB1 protein comprises an amino acid sequence identical to at least 1 consecutive amino acid in the signal peptide of the human IL12RB1 protein. In some embodiments, the human or chimeric IL12RB1 protein comprises an amino acid sequence identical or having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acids at positions 1-549 of SEQ ID NO: 2, positions 1-570 of SEQ ID NO: 2, positions 24-549 of SEQ ID NO: 2, positions 24-570 of SEQ ID NO: 2, positions 1-545 of SEQ ID NO: 2 or positions 24-545 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB1 protein is operably linked to an endogenous IL12RB1 regulatory element (such as a promoter, 5'UTR and / or 3'UTR). In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB1 protein is integrated into the endogenous IL12RB1 locus of the non-human animal. In some embodiments, the chimeric IL12RB1 protein has at least one activity, such as endogenous IL12RB1 activity of the non-human animal and / or human IL12RB1 activity.

[0009] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. In at least one cell of the non-human animal, at the endogenous IL12RB1 locus of the non-human animal, the nucleotide sequence encoding the endogenous IL12RB1 region is replaced with a nucleotide sequence encoding the corresponding region of human or chimeric IL12RB1. In some embodiments, the endogenous IL12RB1 protein of the non-human animal is not expressed or the expression level is reduced compared to IL12RB1 in a wild-type animal. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric IL12RB1 comprises a part of exon 1 of the human IL12RB1 gene, all of exons 2-13, and a part of exon 14 (preferably also comprising intron 1 and / or intron 13). In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric IL12RB1 comprises a part of exon 1 of the human IL12RB1 gene, all of exons 2-13, and a part of exon 14 (preferably also comprising intron 1 and / or intron 13) of at least 100-1000, 2000-5000, 8000-15000, 20000-24000 or 24000-25000 bp nucleotides. In some embodiments, the amino acid sequence of the corresponding region of the human or chimeric IL12RB1 comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acids at positions 1-549 of SEQ ID NO: 2, positions 1-570 of SEQ ID NO: 2, positions 24-549 of SEQ ID NO: 2, positions 24-570 of SEQ ID NO: 2, positions 1-545 of SEQ ID NO: 2 or positions 24-545 of SEQ ID NO: 2. In some embodiments, the endogenous IL12RB1 region is all or part of the endogenous extracellular region and / or all or part of the endogenous transmembrane region (preferably with or without the endogenous signal peptide). In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric IL12RB1 comprises the nucleotide sequence shown at positions 18062249 to 18086823 of NCBI accession number NC_000019.10; or comprises a nucleotide sequence having an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the nucleotide sequence shown at positions 18062249 to 18086823 of NCBI accession number NC_000019.10. In some embodiments, the nucleotide sequence encoding the endogenous IL12RB1 region comprises a part of exon 1 of the endogenous IL12RB1 gene of the non-human animal, all of exons 2-13, and a part of exon 14 (preferably also comprising intron 1 and / or intron 13).In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric IL12RB1 is operably linked to a non-human animal endogenous IL12RB1 regulatory element, such as a promoter, 5'UTR, and / or 3'UTR. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a mouse or a rat).

[0010] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric IL12RB1, the construction method comprising replacing the nucleotide sequence encoding the endogenous IL12RB1 region with the nucleotide sequence encoding the corresponding region of human IL12RB1 at the endogenous IL12RB1 locus of the non-human animal to generate a genetically modified non-human animal cell. In some embodiments, the non-human animal cell expresses human or chimeric IL12RB1 protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL12RB1 comprises a portion of exon 1 of the human IL12RB1 gene, all of exons 2-13, and a portion of exon 14 (preferably also comprising intron 1 and / or intron 13). In some embodiments, the nucleotide sequence encoding the corresponding region of human IL12RB1 comprises a portion of exon 1 of the human IL12RB1 gene, all of exons 2-13, and a portion of exon 14 (preferably also comprising intron 1 and / or intron 13) of at least 100-1000, 2000-5000, 8000-15000, 20000-24000, or 24000-25000 bp nucleotides. In some embodiments, the amino acid sequence of the corresponding region of human IL12RB1 comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% to the amino acid sequence of positions 1-549 of SEQ ID NO: 2, positions 1-570 of SEQ ID NO: 2, positions 24-549 of SEQ ID NO: 2, positions 24-570 of SEQ ID NO: 2, positions 1-545 of SEQ ID NO: 2, or positions 24-545 of SEQ ID NO: 2. In some embodiments, the endogenous IL12RB1 region is all or part of the endogenous extracellular region and / or all or part of the endogenous transmembrane region (preferably also comprising or not comprising the endogenous signal peptide). In some embodiments, the nucleotide sequence encoding the endogenous IL12RB1 region comprises a portion of exon 1 of the non-human animal endogenous IL12RB1 gene, all of exons 2-13, and a portion of exon 14 (preferably also comprising intron 1 and / or intron 13). In some embodiments, the nucleotide sequence encoding the corresponding region of human IL12RB1 is operably linked to a regulatory element of endogenous IL12RB1, such as a promoter, 5'UTR, and / or 3'UTR. In some embodiments, the non-human animal is a mouse.

[0011] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. The genome of the non-human animal comprises at least one chromosome, and the chromosome comprises a nucleotide sequence encoding a human or chimeric interleukin-12 receptor subunit beta 2 (IL12RB2) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB2 protein can be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB2 protein is operably linked to an endogenous regulatory element (such as a promoter, 5'UTR and / or 3'UTR) of the endogenous IL12RB2 locus of at least one chromosome. In some embodiments, the chimeric IL12RB2 protein comprises or does not comprise a signal peptide. In some embodiments, the chimeric IL12RB2 protein comprises a signal peptide, and the signal peptide is endogenous to the non-human animal or a human or humanized signal peptide. In some embodiments, the chimeric IL12RB2 protein comprises a human or humanized extracellular region. In some embodiments, the chimeric IL12RB2 protein comprises a human or humanized transmembrane region. In some embodiments, the chimeric IL12RB2 protein comprises a human or humanized extracellular region, a human or humanized transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric IL12RB2 protein comprises an endogenous signal peptide of the non-human animal, a human or humanized extracellular region, a human or humanized transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric IL12RB2 protein comprises a human or humanized signal peptide, a human or humanized extracellular region, a human or humanized transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric IL12RB2 protein is a humanized IL12RB2 protein. In some embodiments, the chimeric IL12RB2 protein comprises an amino acid sequence that is at least 50 to 862, such as at least 50, 100, 200, 300, 400, 500, 590, 599, 600, 620, 640, 643, 700, 800, 850 or 862 consecutive or non-consecutive amino acids identical to the human IL12RB2 protein. In some embodiments, the chimeric IL12RB2 protein comprises all or part of the human IL12RB2 protein. In some embodiments, the chimeric IL12RB2 protein comprises all or part of the extracellular region of the human IL12RB2 protein, preferably all or part of the transmembrane region of the human IL12RB2 protein. In some embodiments, the chimeric IL12RB2 protein comprises all or part of the signal peptide of the human IL12RB2 protein.In some embodiments, the chimeric IL12RB2 protein comprises amino acids 24-622 of SEQ ID NO: 13, amino acids 24-643 of SEQ ID NO: 13, amino acids 24-623 of SEQ ID NO: 13, amino acids 1-640 of SEQ ID NO: 13, or amino acids 1-643 of SEQ ID NO: 13; or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to amino acids 24-622 of SEQ ID NO: 13, amino acids 24-643 of SEQ ID NO: 13, amino acids 24-623 of SEQ ID NO: 13, amino acids 1-640 of SEQ ID NO: 13, or amino acids 1-643 of SEQ ID NO: 13. In some embodiments, the chimeric IL12RB2 protein comprises all or part of the non-human animal endogenous IL12RB2 protein. In some embodiments, the chimeric IL12RB2 protein comprises all or part of the transmembrane region of the non-human animal endogenous IL12RB2 protein, and preferably also comprises all or part of the cytoplasmic region of the non-human animal endogenous IL12RB2 protein. In some embodiments, the chimeric IL12RB2 protein comprises amino acids 638-874 of SEQ ID NO: 12 or amino acids 639-874 of SEQ ID NO: 12; or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to amino acids 638-874 of SEQ ID NO: 12 or amino acids 639-874 of SEQ ID NO: 12. In some embodiments, the amino acid sequence of the chimeric IL12RB2 protein comprises SEQ ID NO: 21 or 56; or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 21 or 56. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal endogenous IL12RB2 protein is not expressed or is expressed at a reduced level compared to IL12RB2 in a wild-type animal. In some embodiments, one or more cells of the non-human animal express a human or chimeric IL12RB2 protein.

[0012] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises replacing the nucleotide sequence encoding the corresponding region of endogenous IL12RB2 with a nucleotide sequence encoding a human or chimeric IL12RB2 region at the endogenous IL12RB2 locus, or introducing a nucleotide sequence encoding a human or chimeric IL12RB2 region into the endogenous IL12RB2 locus of the non-human animal (the introduction preferably includes insertion). In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB2 region is operably linked to an endogenous regulatory element (such as a promoter, 5'UTR and / or 3'UTR) of the endogenous IL12RB2 locus. In some embodiments, the endogenous IL12RB2 protein of the non-human animal is not expressed or has a reduced expression level compared to IL12RB2 in a wild-type animal. In some embodiments, the corresponding region of the endogenous IL12RB2 is the endogenous extracellular region and / or the endogenous transmembrane region (preferably further comprising an endogenous signal peptide). In some embodiments, one or more cells of the non-human animal express a chimeric IL12RB2, which comprises an extracellular region, a transmembrane region, and a cytoplasmic region (preferably with or without a signal peptide). In some embodiments, the signal peptide comprises an amino acid sequence that is identical or has an identity of at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% to the signal peptide of the human IL12RB2 protein. In some embodiments, the signal peptide comprises an amino acid sequence that is identical or has an identity of at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% to the signal peptide of the endogenous IL12RB2 protein of the non-human animal. In some embodiments, the extracellular region comprises an amino acid sequence that is identical or has an identity of at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% to the extracellular region of the human IL12RB2 protein. In some embodiments, the transmembrane region comprises an amino acid sequence that is identical or has an identity of at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% to the transmembrane region of the human IL12RB2 protein. In some embodiments, the signal peptide comprises an amino acid sequence that is identical to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive amino acids of the signal peptide of the human IL12RB2 protein (such as positions 1-23 of SEQ ID NO: 13). In some embodiments, the signal peptide comprises an amino acid sequence that is identical to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive amino acids of the signal peptide of the endogenous IL12RB2 protein of the non-human animal (such as positions 1-23 of SEQ ID NO: 12).In some embodiments, the extracellular region comprises an amino acid sequence that is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 550, 590, or 599 consecutive amino acids identical to the extracellular region of the human IL12RB2 protein (e.g., positions 24-622 of SEQ ID NO: 13). In some embodiments, the transmembrane region comprises an amino acid sequence that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 consecutive amino acids identical to the transmembrane region of the human IL12RB2 protein (e.g., positions 623-643 of SEQ ID NO: 13). In some embodiments, the transmembrane region further comprises an amino acid sequence that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 consecutive amino acids identical to the transmembrane region of the endogenous IL12RB2 of a non-human animal (e.g., positions 639-658 or positions 638-658 of SEQ ID NO: 12). In some embodiments, the cytoplasmic region comprises an amino acid sequence that is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, or 216 consecutive amino acids identical to the cytoplasmic region of the endogenous IL12RB2 of a non-human animal (e.g., positions 659-874 of SEQ ID NO: 12). In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB2 region comprises a portion of exon 2, all of exons 3-13, and a portion of exon 14 of the human IL12RB2 gene (preferably further comprising intron 2 and / or intron 13). In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB2 region comprises the CDS sequence of a portion of exon 2, all of exons 3-13, and a portion of exon 14 of the human IL12RB2 gene. In some embodiments, the nucleotide sequence encoding the chimeric IL12RB2 region comprises a portion of exon 14, all of exon 15, and all or a portion of exon 16 of the endogenous IL12RB2 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding the chimeric IL12RB2 region comprises the CDS sequence of a portion of exon 14, all of exon 15, and all or a portion of exon 16 of the endogenous IL12RB2 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding the chimeric IL12RB2 region comprises a portion of exon 14, all of exon 15, all of exon 16, and 300 bp of consecutive nucleotide sequences downstream of the 3' UTR of the endogenous IL12RB2 gene of a non-human animal.In some embodiments, the nucleotide sequence encoding the chimeric IL12RB2 region comprises a partial sequence of exon 14 of the non-human animal endogenous IL12RB2 gene, the entire exon 15, and the CDS sequence, 3'UTR sequence, and 300 bp of consecutive nucleotide sequence downstream of the 3'UTR of exon 16. In some embodiments, the nucleotide sequence encoding the chimeric IL12RB2 region comprises the human IL12RB2 nucleotide sequence and the non-human animal IL12RB2 nucleotide sequence. In some embodiments, the nucleotide sequence encoding the chimeric IL12RB2 region comprises the human IL12RB2 nucleotide sequence, the non-human animal IL12RB2 nucleotide sequence, and an auxiliary sequence. In some embodiments, the auxiliary sequence is a STOP sequence. In some embodiments, the human IL12RB2 nucleotide sequence can be a CDS, cDNA, or genomic DNA sequence. In some embodiments, the non-human animal IL12RB2 nucleotide sequence can be a CDS, cDNA, or genomic DNA sequence. In some embodiments, the human IL12RB2 nucleotide sequence in the nucleotide sequence encoding the chimeric IL12RB2 region comprises a nucleotide sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% to the nucleotide sequence shown at positions 67320369 to 67386643 of SEQ ID NO: 24 or NCBI accession number NC_000001.11. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence encoding the chimeric IL12RB2 region comprises a nucleotide sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% to the nucleotide sequence shown in SEQ ID NO: 67. In some embodiments, the STOP sequence in the nucleotide sequence encoding the chimeric IL12RB2 region comprises a nucleotide sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% to the nucleotide sequence shown in SEQ ID NO: 68. In some embodiments, the introduction of the non-human animal endogenous IL12RB2 locus is an insertion into exon 2 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is after the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein in the non-human animal genome. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is between the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein and the nucleotide sequence encoding the extracellular region of the endogenous IL12RB2 protein in the non-human animal genome.In some embodiments, the insertion of the non-human animal endogenous IL12RB2 locus is between the nucleotide sequence at the 23rd position encoding SEQ ID NO: 12 and the nucleotide sequence at the 24th position encoding SEQ ID NO: 12 in the non-human animal genome. In some embodiments, the insertion of the non-human animal endogenous IL12RB2 locus is between the 257th and 258th positions of the non-human animal genome with the NCBI accession number NM_008354.4.

[0013] In some embodiments, the modified IL12RB2 gene in the non-human animal genome is homozygous or heterozygous for the locus of the endogenous gene that is modified (e.g., inserted or replaced).

[0014] In one aspect, the present invention provides a non-human animal or a method for constructing the same, and the construction method includes introducing a nucleotide sequence containing a human or chimeric IL12RB2 into the endogenous IL12RB2 locus of the non-human animal. In some embodiments, the nucleotide sequence of the human or chimeric IL12RB2 is operably linked to an endogenous regulatory element of the endogenous IL12RB2 locus. In some embodiments, the endogenous regulatory element includes a promoter, 5'UTR, and / or 3'UTR. In some embodiments, the endogenous IL12RB2 protein of the non-human animal is not expressed or has a reduced expression level compared to IL12RB2 in a wild-type animal. In some embodiments, the modified IL12RB2 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous modified (e.g., inserted or replaced) locus. In some embodiments, the nucleotide sequence of the human or chimeric IL12RB2 can be one or a combination of two or more of a CDS, cDNA, or genomic DNA sequence. In some embodiments, the nucleotide sequence of the human or chimeric IL12RB2 encodes a human or chimeric IL12RB2 protein. In some embodiments, the nucleotide sequence of the human or chimeric IL12RB2 contains a nucleotide sequence encoding the extracellular region of the human IL12RB2 protein, preferably further containing all or part of the nucleotide sequence encoding the signal peptide and / or all or part of the transmembrane region. In some embodiments, the nucleotide sequence of the human or chimeric IL12RB2 contains a nucleotide sequence encoding positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, positions 24-643 of SEQ ID NO: 13, positions 1-640 of SEQ ID NO: 13, or positions 1-643 of SEQ ID NO: 13; or contains a nucleotide sequence encoding at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, positions 24-643 of SEQ ID NO: 13, positions 1-640 of SEQ ID NO: 13, or positions 1-643 of SEQ ID NO: 13. In some embodiments, the nucleotide sequence of the human or chimeric IL12RB2 contains a portion of exon 2 to a portion of exon 14 of the human IL12RB2 gene. In some embodiments, the nucleotide sequence of the human or chimeric IL12RB2 contains the start codon of human IL12RB2 to a portion of exon 14. In some embodiments, the nucleotide sequence of the human or chimeric IL12RB2 contains a CDS sequence of a portion of exon 2 to a portion of exon 14 of the human IL12RB2 gene. In some embodiments, the introduction is an insertion or replacement.In some embodiments, the nucleotide sequence of the human or chimeric IL12RB2 comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequence shown in SEQ ID NO: 24 or positions 67320369 to 67386643 of NCBI accession number NC_000001.11. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the mRNA transcribed from the modified IL12RB2 gene in the genome of the non-human animal comprises SEQ ID NO: 20 or 55; or comprises a nucleotide sequence that has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with SEQ ID NO: 20 or 55. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding another human or chimeric protein, and the other human or chimeric protein preferably comprises at least one of IL12A, IL12B, IL12RB1, IL23R, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the other human or chimeric protein comprises IL12RB1, IL23R and / or PD-1. In some embodiments, the other human or chimeric protein is a humanized IL12RB1 protein, and the amino acid sequence of the humanized IL12RB1 protein preferably comprises SEQ ID NO: 11; or comprises an amino acid sequence that has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with SEQ ID NO: 11. In some embodiments, the other human or chimeric protein is a humanized IL23R protein, and the amino acid sequence of the humanized IL23R protein preferably comprises SEQ ID NO: 78; or comprises an amino acid sequence that has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with SEQ ID NO: 78. In some embodiments, the other human or chimeric protein is a humanized PD-1 protein.

[0015] In some embodiments, the construction method includes inserting a nucleotide sequence of chimeric IL12RB2 into the endogenous IL12RB2 locus of a non-human animal. In some embodiments, the nucleotide sequence of chimeric IL12RB2 comprises a human IL12RB2 nucleotide sequence and a non-human animal IL12RB2 nucleotide sequence. In some embodiments, the nucleotide sequence of chimeric IL12RB2 comprises a human IL12RB2 nucleotide sequence, a non-human animal IL12RB2 nucleotide sequence, and an auxiliary sequence. In some embodiments, the auxiliary sequence is a STOP sequence. In some embodiments, the human IL12RB2 nucleotide sequence can be a CDS, cDNA or genomic DNA sequence. In some embodiments, the non-human animal IL12RB2 nucleotide sequence can be a CDS, cDNA or genomic DNA sequence. In some embodiments, the human IL12RB2 nucleotide sequence in the nucleotide sequence of chimeric IL12RB2 comprises a nucleotide sequence encoding a human or chimeric IL12RB2 protein. In some embodiments, the human IL12RB2 nucleotide sequence in the nucleotide sequence of chimeric IL12RB2 comprises a nucleotide sequence encoding all or part of the extracellular region of the human IL12RB2 protein, preferably comprising a nucleotide sequence encoding all or part of the transmembrane region of the human IL12RB2 protein. In some embodiments, the human IL12RB2 nucleotide sequence in the nucleotide sequence of chimeric IL12RB2 comprises a nucleotide sequence encoding positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, or positions 24-643 of SEQ ID NO: 13; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence encoding positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, or positions 24-643 of SEQ ID NO: 13. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of chimeric IL12RB2 comprises a nucleotide sequence encoding the endogenous IL12RB2 protein of the non-human animal. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of chimeric IL12RB2 comprises a nucleotide sequence encoding all or part of the transmembrane region of the endogenous IL12RB2 protein of the non-human animal, preferably comprising a nucleotide sequence encoding all or part of the cytoplasmic region of the endogenous IL12RB2 protein of the non-human animal.In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the chimeric IL12RB2 nucleotide sequence comprises a nucleotide sequence encoding positions 638-874 of SEQ ID NO: 12 or positions 639-874 of SEQ ID NO: 12; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence encoding positions 638-874 of SEQ ID NO: 12 or positions 639-874 of SEQ ID NO: 12. In some embodiments, the human IL12RB2 nucleotide sequence in the chimeric IL12RB2 nucleotide sequence includes the portion from exon 2 to exon 14 of human IL12RB2. In some embodiments, the human IL12RB2 nucleotide sequence in the chimeric IL12RB2 nucleotide sequence comprises the CDS sequence of the portion from exon 2 to exon 14 of the human IL12RB2 gene. In some embodiments, the portion of exon 2 of the human IL12RB2 gene preferably comprises at least 1-112 bp, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 112 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region. The portion of exon 14 of the human IL12RB2 gene preferably comprises at least 5-91 bp, such as at least 5, 10, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 91 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the chimeric IL12RB2 nucleotide sequence comprises all or part of exon 16 from the portion of exon 14 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the chimeric IL12RB2 nucleotide sequence comprises the CDS sequence of all or part of exon 16 from the portion of exon 14 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the portion of exon 14 of the non-human animal endogenous IL12RB2 gene preferably comprises at least 5-91 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 76, 77, 78, 79, 80, 85, 90 or 91 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region.In some embodiments, a portion of exon 16 of the non-human animal endogenous IL12RB2 gene preferably comprises at least 5 - 719 bp, such as at least 5, 50, 100, 200, 300, 400, 500, 530, 531, 600, 700, or 719 bp of contiguous nucleotide sequence, and preferably comprises a nucleotide sequence of the coding region. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of the chimeric IL12RB2 comprises the nucleotide sequence from a portion of exon 14 of the non-human animal endogenous IL12RB2 gene to the stop codon, preferably further comprises the 3'UTR, and further preferably comprises at least 50 bp of contiguous nucleotide sequence downstream of the 3'UTR. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of the chimeric IL12RB2 comprises the CDS sequence of the nucleotide sequence from a portion of exon 14 of the non-human animal endogenous IL12RB2 gene to the stop codon, preferably further comprises the 3'UTR, and further preferably comprises at least 50 bp of contiguous nucleotide sequence downstream of the 3'UTR, preferably comprises at least 50 bp to 500 bp of contiguous nucleotide sequence downstream of the 3'UTR, such as at least 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 bp of contiguous nucleotide sequence. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of the chimeric IL12RB2 comprises the nucleotide sequence from a portion of exon 14 of the non-human animal endogenous IL12RB2 gene to at least 50 bp of contiguous nucleotide sequence downstream of the 3'UTR. In some embodiments, the human IL12RB2 nucleotide sequence in the nucleotide sequence of the chimeric IL12RB2 comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% with the nucleotide sequence shown in SEQ ID NO: 24. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of the chimeric IL12RB2 comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% with the nucleotide sequence shown in SEQ ID NO: 67. In some embodiments, the STOP sequence in the nucleotide sequence of the chimeric IL12RB2 comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% with the nucleotide sequence shown in SEQ ID NO: 68. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is an insertion into exon 2 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is an insertion after the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein in the non-human animal genome.In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is between the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein and the nucleotide sequence encoding the extracellular region of the endogenous IL12RB2 protein in the non-human animal genome. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is between the nucleotide sequence encoding the 23rd position of SEQ ID NO: 12 and the nucleotide sequence encoding the 24th position of SEQ ID NO: 12 in the non-human animal genome. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is between the 257th and 258th positions of the NCBI accession number NM_008354.4 in the non-human animal genome.

[0016] In some embodiments, the construction method includes replacing the nucleotide sequence of the corresponding region of the endogenous IL12RB2 in a non-human animal with a nucleotide sequence comprising the human IL12RB2 at the endogenous IL12RB2 locus of the non-human animal. In some embodiments, the nucleotide sequence of the human IL12RB2 may be a CDS, cDNA or genomic DNA sequence. In some embodiments, the nucleotide sequence of the human IL12RB2 comprises a nucleotide sequence encoding a human or chimeric IL12RB2 protein. In some embodiments, the nucleotide sequence of the human IL12RB2 comprises a nucleotide sequence encoding all or part of the extracellular region of the human IL12RB2 protein, preferably comprising a nucleotide sequence encoding all or part of the transmembrane region of the human IL12RB2 protein. In some embodiments, the nucleotide sequence of the human IL12RB2 comprises a nucleotide sequence encoding all or part of the signal peptide of the human IL12RB2 protein. In some embodiments, the nucleotide sequence of the IL12RB2 comprises a nucleotide sequence encoding all of the signal peptide, all of the extracellular region, and all or part of the transmembrane region of the human IL12RB2 protein. In some embodiments, the nucleotide sequence of the human IL12RB2 comprises a nucleotide sequence encoding positions 1-640 of SEQ ID NO: 13 or positions 1-643 of SEQ ID NO: 13; or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence encoding positions 1-640 of SEQ ID NO: 13 or positions 1-643 of SEQ ID NO: 13. In some embodiments, the nucleotide sequence of the human IL12RB2 comprises all or part of the human IL12RB2 gene. In some embodiments, the nucleotide sequence of the human IL12RB2 comprises a portion of exon 2 to a portion of exon 14 of the human IL12RB2 gene. In some embodiments, the nucleotide sequence of the human IL12RB2 comprises the start codon of the human IL12RB2 gene to a portion of exon 14. In some embodiments, the portion of exon 2 of the human IL12RB2 gene preferably comprises at least 5-112 bp, such as at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 75, 76, 80, 90, 100, 110 or 112 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region, and the portion of exon 14 of the human IL12RB2 gene preferably comprises at least 5-91 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90 or 91 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region.In some embodiments, the nucleotide sequence of human IL12RB2 comprises the nucleotide sequence from position 67320369 to 67386643 of NCBI accession number NC_000001.11; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence from position 67320369 to 67386643 of NCBI accession number NC_000001.11. In some embodiments, the nucleotide sequence of the corresponding region of endogenous IL12RB2 in the non-human animal comprises a nucleotide sequence encoding the endogenous IL12RB2 protein of the non-human animal. In some embodiments, the nucleotide sequence of the corresponding region of endogenous IL12RB2 in the non-human animal comprises a nucleotide sequence encoding all or part of the extracellular region of the endogenous IL12RB2 protein of the non-human animal, preferably comprises a nucleotide sequence encoding all or part of the transmembrane region of the endogenous IL12RB2 protein of the non-human animal. In some embodiments, the nucleotide sequence of the corresponding region of endogenous IL12RB2 in the non-human animal comprises a nucleotide sequence encoding all or part of the signal peptide of the endogenous IL12RB2 protein of the non-human animal. In some embodiments, the nucleotide sequence of the corresponding region of endogenous IL12RB2 in the non-human animal comprises a nucleotide sequence encoding positions 1-655 of SEQ ID NO: 12 or positions 1-658 of SEQ ID NO: 12; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence encoding positions 1-655 of SEQ ID NO: 12 or positions 1-658 of SEQ ID NO: 12. In some embodiments, the nucleotide sequence of the corresponding region of endogenous IL12RB2 in the non-human animal comprises all or part of the endogenous IL12RB2 gene of the non-human animal. In some embodiments, the nucleotide sequence of the corresponding region of endogenous IL12RB2 in the non-human animal comprises a part of exon 2 to a part of exon 14 of the endogenous IL12RB2 gene of the non-human animal. In some embodiments, the nucleotide sequence of the corresponding region of endogenous IL12RB2 in the non-human animal comprises a part from the start codon of the endogenous IL12RB2 gene of the non-human animal to exon 14.In some embodiments, a portion of exon 2 of the non-human animal endogenous IL12RB2 gene preferably comprises at least 5-110 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 76, 80, 85, 90, 95, 100, 105 or 110 bp of continuous nucleotide sequence, preferably comprising a nucleotide sequence of the coding region, and a portion of exon 14 of the non-human animal endogenous IL12RB2 gene preferably comprises at least 5-91 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 91 bp of continuous nucleotide sequence, preferably comprising a nucleotide sequence of the coding region.

[0017] In one aspect, the present invention provides a non-human animal or a method for constructing the same, the non-human animal comprising at least one cell encoding a nucleotide sequence of a human or chimeric IL12RB2 protein. In some embodiments, the human or chimeric IL12RB2 protein comprises an amino acid sequence that is identical to at least 50, 100, 200, 300, 400, 500, 590, 599, 600, 620, 640, 643, 700, 800, 850 or 862 consecutive amino acid sequences of the human IL12RB2 protein. In some embodiments, the non-human animal expresses the human or chimeric IL12RB2 protein. In some embodiments, the human or chimeric IL12RB2 protein comprises an amino acid sequence that is identical to at least 50 consecutive amino acids of the extracellular region of human IL12RB2. In some embodiments, the chimeric IL12RB2 protein comprises an amino acid sequence that is identical to at least 1 consecutive amino acid of the transmembrane region of human IL12RB2. In some embodiments, the chimeric IL12RB2 protein comprises an amino acid sequence that is identical to at least 1 consecutive amino acid of the signal peptide of human IL12RB2. In some embodiments, the human or chimeric IL12RB2 protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acids at positions 24-622, positions 24-643, positions 24-623, positions 1-640 or positions 1-643 of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB2 protein is operably linked to an endogenous IL12RB2 regulatory element (such as a promoter, 5'UTR and / or 3'UTR). In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB2 protein is integrated into the endogenous IL12RB2 locus of the non-human animal. In some embodiments, the chimeric IL12RB2 protein has at least one activity, such as endogenous IL12RB2 activity of the non-human animal and / or human IL12RB2 activity.

[0018] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, in which in at least one cell of the non-human animal, a nucleotide sequence encoding a human or chimeric IL12RB2 is introduced into the endogenous IL12RB2 locus of the non-human animal. In some embodiments, the introduction includes replacement or insertion. In some embodiments, the endogenous IL12RB2 protein of the non-human animal is not expressed or has a reduced expression level compared to IL12RB2 in a wild-type animal. In some embodiments, the nucleotide sequence encoding the chimeric IL12RB2 contains the nucleotide sequence of the human IL12RB2 gene and the nucleotide sequence of the endogenous IL12RB2 gene of the non-human animal. In some embodiments, the nucleotide sequence encoding the chimeric IL12RB2 contains the nucleotide sequence of the human IL12RB2 gene, the nucleotide sequence of the endogenous IL12RB2 gene of the non-human animal, and an auxiliary sequence (such as a STOP sequence). In some embodiments, the nucleotide sequence of the human IL12RB2 gene in the nucleotide sequence encoding the chimeric IL12RB2 contains the portion from exon 2 to exon 14 of the human IL12RB2 gene. In some embodiments, the nucleotide sequence of the human IL12RB2 gene in the nucleotide sequence encoding the chimeric IL12RB2 contains the portion from the start codon of the human IL12RB2 gene to exon 14. In some embodiments, the nucleotide sequence of the human IL12RB2 gene in the nucleotide sequence encoding the chimeric IL12RB2 contains the portion of exon 2 of the human IL12RB2 gene, all of exons 3-13, and the portion of exon 14 of the human IL12RB2 gene (preferably including intron 2 and / or intron 13). In some embodiments, the nucleotide sequence encoding the chimeric IL12RB2 contains the portion of exon 2 of the human IL12RB2 gene, all of exon 3, all of exon 4, all of exon 5, all of exon 6, all of exon 7, all of exon 8, all of exon 9, all of exon 10, all of exon 11, all of exon 12, all of exon 13, and the portion of exon 14 of the human IL12RB2 gene. In some embodiments, the amino acid sequence of the chimeric IL12RB2 contains an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% to the amino acid sequence at positions 24-623 of SEQ ID NO: 13, positions 24-643 of SEQ ID NO: 13, positions 24-622 of SEQ ID NO: 13, positions 1-640 of SEQ ID NO: 13, or positions 1-643 of SEQ ID NO: 13.In some embodiments, the amino acid sequence of the chimeric IL12RB2 comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the amino acids shown in SEQ ID NO: 21 or 56. In some embodiments, the human IL12RB2 nucleotide sequence in the nucleotide sequence encoding the chimeric IL12RB2 comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequence shown at positions 67320369 to 67386643 of SEQ ID NO: 24 or NCBI accession number NC_000001.11. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence encoding the chimeric IL12RB2 comprises a part of exon 14, all of exon 15 and all or part of exon 16 (preferably also including intron 14 and / or intron 16) of the non-human animal endogenous IL12RB2 gene. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence encoding the chimeric IL12RB2 comprises the CDS sequence of a part of exon 14, all of exon 15 and all or part of exon 16 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of the chimeric IL12RB2 comprises the CDS sequence of a part of exon 14, all of exon 15 and all or part of exon 16 of the non-human animal endogenous IL12RB2 gene, preferably also including the 3'UTR, and more preferably also including at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of the chimeric IL12RB2 comprises a part of exon 14 of the non-human animal endogenous IL12RB2 gene to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of the chimeric IL12RB2 comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequence shown in SEQ ID NO: 67. In some embodiments, the STOP sequence in the nucleotide sequence of the chimeric IL12RB2 comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequence shown in SEQ ID NO: 68. In some embodiments, the introduction of the non-human animal endogenous IL12RB2 locus is an insertion into exon 2 of the non-human animal endogenous IL12RB2 gene.In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is after the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein in the non-human animal genome. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is between the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein and the nucleotide sequence encoding the extracellular region of the endogenous IL12RB2 protein in the non-human animal genome. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is between the nucleotide sequence encoding the 23rd position of SEQ ID NO: 12 and the nucleotide sequence encoding the 24th position of SEQ ID NO: 12 in the non-human animal genome. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is between the 257th and 258th positions of the NCBI accession number NM_008354.4 in the non-human animal genome. In some embodiments, the introduction into the non-human animal endogenous IL12RB2 locus is to replace the corresponding region of the non-human animal endogenous IL12RB2. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 contains the nucleotide sequence encoding the non-human animal endogenous IL12RB2 protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 contains all or part of the nucleotide sequence encoding the extracellular region of the non-human animal endogenous IL12RB2 protein, preferably contains all or part of the nucleotide sequence encoding the transmembrane region of the non-human animal endogenous IL12RB2 protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 contains all or part of the nucleotide sequence encoding the signal peptide of the non-human animal endogenous IL12RB2 protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 contains the nucleotide sequence encoding positions 1-655 or positions 1-658 of SEQ ID NO: 12; or contains a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence encoding positions 1-655 or positions 1-658 of SEQ ID NO: 12. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 contains a part of exon 2 to a part of exon 14 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 contains the start codon of the non-human animal endogenous IL12RB2 gene to a part of exon 14. In some embodiments, the nucleotide sequence encoding the human or chimeric IL12RB2 is operably linked to the regulatory elements of the non-human animal endogenous IL12RB2, such as, a promoter, 5'UTR and / or 3'UTR.In some embodiments, the non-human animal is a mammal, such as a monkey, a rodent, or a rodent (mouse or rat).

[0019] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric IL12RB2. The construction method includes introducing a nucleotide sequence encoding human or chimeric IL12RB2 into the endogenous IL12RB2 locus of a non-human animal at the endogenous IL12RB2 locus of the non-human animal to generate a genetically modified non-human animal cell. In some embodiments, the introduction is an insertion or replacement. In some embodiments, the non-human animal cell expresses human or chimeric IL12RB2 protein. In some embodiments, the nucleotide sequence encoding chimeric IL12RB2 includes the nucleotide sequence of human IL12RB2 and the nucleotide sequence of the endogenous IL12RB2 of the non-human animal. In some embodiments, the nucleotide sequence encoding chimeric IL12RB2 includes the nucleotide sequence of human IL12RB2, the nucleotide sequence of the endogenous IL12RB2 of the non-human animal, and an auxiliary sequence (such as a STOP sequence). In some embodiments, the nucleotide sequence of the human IL12RB2 gene in the nucleotide sequence encoding chimeric IL12RB2 includes the part from exon 2 to exon 14 of the human IL12RB2 gene. In some embodiments, the nucleotide sequence of the human IL12RB2 gene in the nucleotide sequence encoding chimeric IL12RB2 includes the part from the start codon of the human IL12RB2 gene to exon 14. In some embodiments, the nucleotide sequence of human IL12RB2 in the nucleotide sequence encoding chimeric IL12RB2 includes the part of exon 2 of the human IL12RB2 gene, all of exons 3-13, and the part of exon 14 (preferably including intron 2 and / or intron 13). In some embodiments, the nucleotide sequence encoding chimeric IL12RB2 includes the part of exon 2 of the human IL12RB2 gene, all of exon 3, all of exon 4, all of exon 5, all of exon 6, all of exon 7, all of exon 8, all of exon 9, all of exon 10, all of exon 11, all of exon 12, all of exon 13, and the part of exon 14 of the human IL12RB2 gene. In some embodiments, the amino acid sequence of the chimeric IL12RB2 includes an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% to the amino acid sequence shown in SEQ ID NO: 21 or 56. In some embodiments, the amino acid sequence of the chimeric IL12RB2 includes an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% to the amino acid sequence at positions 24-622 of SEQ ID NO: 2, positions 24-643 of SEQ ID NO: 2, positions 24-623 of SEQ ID NO: 2, positions 1-640 of SEQ ID NO: 2, or positions 1-643 of SEQ ID NO: 2.In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence encoding chimeric IL12RB2 comprises a part of exon 14, the whole of exon 15, and all or part of exon 16 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence encoding chimeric IL12RB2 comprises a part of exon 14, the whole of exon 15, and the CDS sequence of all or part of exon 16 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of chimeric IL12RB2 comprises a part of exon 14, the whole of exon 15, and the CDS sequence of all or part of exon 16 of the non-human animal endogenous IL12RB2 gene, preferably further comprises a 3'UTR, and more preferably further comprises at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of chimeric IL12RB2 comprises a part of exon 14 of the non-human animal endogenous IL12RB2 gene to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the non-human animal IL12RB2 nucleotide sequence in the nucleotide sequence of chimeric IL12RB2 comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequence shown in SEQ ID NO: 67. In some embodiments, the STOP sequence in the nucleotide sequence of chimeric IL12RB2 comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequence shown in SEQ ID NO: 68. In some embodiments, the introduction of the non-human animal endogenous IL12RB2 locus is an insertion into exon 2 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is after the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein in the non-human animal genome. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is between the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein and the nucleotide sequence encoding the extracellular region of the endogenous IL12RB2 protein in the non-human animal genome. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is between the nucleotide sequence encoding the 23rd position of SEQ ID NO: 12 and the nucleotide sequence encoding the 24th position of SEQ ID NO: 12. In some embodiments, the insertion into the non-human animal endogenous IL12RB2 locus is an insertion into the non-human animal genome, between positions 257-258 of NCBI accession number NM_008354.4.In some embodiments, the introduction of the non-human animal endogenous IL12RB2 locus is to replace the corresponding region of the non-human animal endogenous IL12RB2. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 comprises the nucleotide sequence encoding the non-human animal endogenous IL12RB2 protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 comprises the nucleotide sequence encoding all or part of the extracellular region of the non-human animal endogenous IL12RB2 protein, preferably comprises the nucleotide sequence encoding all or part of the transmembrane region of the non-human animal endogenous IL12RB2 protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 comprises the nucleotide sequence encoding all or part of the signal peptide of the non-human animal endogenous IL12RB2 protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 comprises the nucleotide sequence encoding positions 1-655 of SEQ ID NO: 12 or positions 1-658 of SEQ ID NO: 12; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence encoding positions 1-655 of SEQ ID NO: 12 or positions 1-658 of SEQ ID NO: 12. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 comprises the part of exon 2 to the part of exon 14 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 comprises the part from the start codon of the non-human animal endogenous IL12RB2 gene to exon 14. In some embodiments, the nucleotide sequence encoding human or chimeric IL12RB2 is operably linked to the regulatory elements of the non-human animal endogenous IL12RB2, such as, a promoter, 5'UTR and / or 3'UTR. In some embodiments, the non-human animal is a mouse.

[0020] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. The genome of the non-human animal comprises at least one chromosome, and the chromosome comprises a nucleotide sequence encoding a human or chimeric interleukin-23 receptor (IL23R) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric IL23R protein can be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the nucleotide sequence encoding the human or chimeric IL23R protein is operably linked to an endogenous regulatory element (such as a promoter, 5'UTR and / or 3'UTR) of the endogenous IL23R locus of at least one chromosome. In some embodiments, the chimeric IL23R protein is a humanized IL23R protein. In some embodiments, the chimeric IL23R protein contains or does not contain a signal peptide. In some embodiments, the chimeric IL23R protein contains a signal peptide, and the signal peptide is endogenous to the non-human animal. In some embodiments, the chimeric IL23R protein contains a human or humanized extracellular region. In some embodiments, the chimeric IL23R protein contains an endogenous signal peptide, a human or humanized extracellular region, an endogenous transmembrane region and / or an endogenous cytoplasmic region. In some embodiments, the chimeric IL23R protein contains all or part of the human IL23R protein. In some embodiments, the chimeric IL23R protein contains at least 50 to 629, such as 50, 100, 150, 200, 250, 300, 320, 327, 330, 332, 350, 353, 355, 400, 450, 500, 550, 600, 620 or 629 consecutive amino acids identical to the human IL23R protein. In some embodiments, the chimeric IL23R protein contains all or part of the extracellular region of the human IL23R protein. In some embodiments, the amino acid sequence of the chimeric IL23R protein comprises amino acids at positions 24-355 of SEQ ID NO: 59 or amino acids at positions 27-353 of SEQ ID NO: 59; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acids at positions 24-355 of SEQ ID NO: 59 or the amino acids at positions 27-353 of SEQ ID NO: 59. In some embodiments, the chimeric IL23R protein contains all or part of the endogenous IL23R protein of the non-human animal. In some embodiments, the chimeric IL23R protein contains all or part of the signal peptide, transmembrane region and / or cytoplasmic region of the endogenous IL23R protein of the non-human animal.In some embodiments, the amino acid sequence of the chimeric IL23R protein comprises positions 1-26 and 373-644 of SEQ ID NO: 27, or positions 1-23 and 375-644 of SEQ ID NO: 27; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to positions 1-26 and 373-644 of SEQ ID NO: 27, or positions 1-23 and 375-644 of SEQ ID NO: 27. In some embodiments, the amino acid sequence of the chimeric IL23R protein comprises SEQ ID NO: 78; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 78. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous IL23R protein of the non-human animal is not expressed or has a reduced expression level compared to IL23R in wild-type animals. In some embodiments, one or more cells of the non-human animal express a human or chimeric IL23R protein.

[0021] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises a nucleotide sequence encoding all or part of the endogenous IL23R protein at the endogenous IL23R locus replaced by a nucleotide sequence encoding a human or chimeric IL23R protein. In some embodiments, the nucleotide sequence encoding the human or chimeric IL23R protein is operably linked to an endogenous regulatory element (such as a promoter, 5'UTR, and / or 3'UTR) of the non-human endogenous IL23R locus. In some embodiments, one or more cells of the non-human animal express a human or humanized IL23R protein. In some embodiments, the endogenous IL23R protein of the non-human animal is not expressed or has a reduced expression level compared to IL23R in a wild-type animal. In some embodiments, the humanized IL23R protein comprises or does not comprise a signal peptide. In some embodiments, the humanized IL23R protein comprises a human or humanized extracellular region. In some embodiments, the humanized IL23R protein comprises an endogenous signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the humanized extracellular region comprises an amino acid sequence that is identical or has an identity of at least 50%, 60%, 70%, 80%, 90%, 95%, or 99.5% to the extracellular region of the human IL23R protein. In some embodiments, the humanized extracellular region comprises an amino acid sequence that is identical to at least 10 to 332, such as at least 10, 50, 100, 150, 200, 250, 300, 320, 327, 330, or 332 consecutive amino acids of the extracellular region of the human IL23R. In some embodiments, the nucleotide sequence encoding the human or chimeric IL23R protein comprises all or part of exon 3, all of exons 4-8, and part of exon 9 of the human IL23R gene (preferably comprising intron 3 and / or intron 8). In some embodiments, the nucleotide sequence encoding the human or chimeric IL23R protein comprises the CDS sequence of all or part of exon 3, all of exons 4-8, and part of exon 9 of the human IL23R gene. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises part of exon 2 to part of exon 3, and part of exon 9 to all or part of exon 11 of the endogenous IL23R gene of the non-human animal. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the CDS sequence of part of exon 2 to part of exon 3, and part of exon 9 to all or part of exon 11 of the endogenous IL23R gene of the non-human animal. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the 3'UTR of the endogenous IL23R gene of the non-human animal, preferably further comprising at least 50 bp of consecutive nucleotide sequence downstream of the 3'UTR.In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises a partial CDS sequence from exon 2 to exon 3, and a partial CDS sequence from exon 9 to all or part of exon 11 of the endogenous IL23R gene of a non-human animal. Preferably, it further comprises a 3'UTR, and more preferably, it further comprises at least 300 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises: A) an encoded amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acid sequence of positions 1-23 of SEQ ID NO: 27 or positions 1-26 of SEQ ID NO: 27; B) an encoded amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acid sequence of positions 24-355 of SEQ ID NO: 59 or positions 27-353 of SEQ ID NO: 59; and 3) an encoded amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acid sequence of positions 373-644 of SEQ ID NO: 27 or positions 375-644 of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises a nucleotide sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the nucleotide sequence shown in SEQ ID NO: 69 or SEQ ID NO: 71. In some embodiments, the amino acid sequence of the chimeric IL23R protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acid sequence shown in SEQ ID NO: 78. In some embodiments, the modified IL23R gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous locus being replaced.

[0022] In one aspect, the present invention provides a non-human animal or a method for constructing the same, wherein at the endogenous IL23R locus of the non-human animal, the nucleotide sequence of the endogenous IL23R of the non-human animal is replaced with a nucleotide sequence comprising a nucleotide sequence encoding a chimeric IL23R protein. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein may be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the nucleotide sequence of human IL23R and the nucleotide sequence of the endogenous IL23R of the non-human animal. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises all or part of exon 3 to part of exon 9 of the human IL23R gene. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the CDS sequence of all or part of exon 3 to part of exon 9 of the human IL23R gene. In some embodiments, the part of exon 3 of the human IL23R gene preferably comprises at least 5-297 bp, such as at least 5, 20, 50, 100, 150, 200, 250, 280, 289, 290 or 297 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region, and the part of exon 9 of the human IL23R gene preferably comprises at least 5-103 bp, such as at least 5, 10, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 103 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the part of exon 2 to part of exon 3 of the endogenous IL23R gene of the non-human animal and the part of exon 9 to all or part of exon 11 of the endogenous IL23R gene of the non-human animal. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the CDS sequence of the part of exon 2 to part of exon 3 of the endogenous IL23R gene of the non-human animal and the part of exon 9 to all or part of exon 11 of the endogenous IL23R gene of the non-human animal, preferably further comprising a 3'UTR sequence, more preferably further comprising at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR, preferably comprising at least 50 bp to 500 bp of continuous nucleotide sequence downstream of the 3'UTR, such as at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the part of exon 2 to part of exon 3 of the endogenous IL23R gene of the non-human animal and the nucleotide sequence from part of exon 9 of the endogenous IL23R gene of the non-human animal to the stop codon, preferably further comprising a 3'UTR, more preferably further comprising at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR.In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises a portion from exon 2 to exon 3 of the endogenous IL23R gene of a non-human animal and a portion from exon 9 of the endogenous IL23R gene of the non-human animal to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the portion of exon 2 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5-103 bp, such as at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 103 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region; the portion of exon 3 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5-297 bp, such as at least 5, 8, 10, 20, 50, 100, 150, 200, 250, 290 or 297 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region; the portion of exon 9 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5-103 bp, such as at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 89, 90, 100 or 103 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region; the portion of exon 11 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5-1082 bp, such as at least 5, 100, 200, 300, 400, 500, 600, 636, 700, 800, 900, 1000 or 1082 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises SEQ ID NO: 69; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 69. In some embodiments, the nucleotide sequence of the endogenous IL23R of the non-human animal to be replaced comprises the nucleotide sequence encoding the endogenous IL23R protein of the non-human animal. In some embodiments, the nucleotide sequence of the endogenous IL23R of the non-human animal to be replaced comprises the nucleotide sequence encoding all or part of the extracellular region of the endogenous IL23R protein of the non-human animal. In some embodiments, the nucleotide sequence of the endogenous IL23R of the non-human animal to be replaced comprises the nucleotide sequence encoding amino acids 24-122 of SEQ ID NO: 27 or amino acids 33-122 of SEQ ID NO: 27; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence encoding amino acids 24-122 of SEQ ID NO: 27 or amino acids 33-122 of SEQ ID NO: 27. In some embodiments, the nucleotide sequence of the endogenous IL23R of the non-human animal to be replaced comprises all or part of the endogenous IL23R gene of the non-human animal.In some embodiments, the nucleotide sequence of the non-human animal endogenous IL23R to be replaced comprises a portion of exon 3 of the non-human animal endogenous IL23R gene, and the portion of exon 3 of the non-human animal endogenous IL23R gene to be replaced preferably comprises at least 5-297 bp, such as at least 5, 10, 20, 50, 100, 150, 200, 250, 270, 271, 290 or 297 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of the non-human animal endogenous IL23R to be replaced further comprises all or part of intron 3 of the non-human animal endogenous IL23R gene, and the portion of intron 3 of the non-human animal endogenous IL23R gene preferably comprises at least 5-7295 bp, such as at least 5, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,...

[0023] A continuous nucleotide sequence of 5251, 5500, 6000, 6500, 7000 or 7295 bp. In some embodiments, the nucleotide sequence of the non-human animal endogenous IL23R to be replaced comprises at least 5251 bp of continuous nucleotide sequence from a part of exon 3 to intron 3 of the non-human animal endogenous IL23R gene. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein is operably linked to endogenous regulatory elements (such as, a promoter, 5'UTR and / or 3'UTR) of the endogenous IL23R locus. In some embodiments, the endogenous IL23R protein of the non-human animal is not expressed or has a reduced expression level compared to IL23R in a wild-type animal. In some embodiments, the modified IL23R gene in the non-human animal genome is homozygous or heterozygous for the endogenous locus to be replaced. In some embodiments, the non-human animal is a mammal, such as, a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the mRNA transcribed from the modified IL23R gene in the non-human animal genome comprises SEQ ID NO: 71; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 71. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding another human or chimeric protein, and the other human or chimeric protein preferably comprises at least one of IL12A, IL12B, IL12RB1, IL12RB2, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the other human or chimeric protein comprises IL12RB1, IL12RB2 and / or PD-1. In some embodiments, the other human or chimeric protein is a humanized IL12RB1 protein, and the amino acid sequence of the humanized IL12RB1 protein preferably comprises SEQ ID NO: 11; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 11. In some embodiments, the other human or chimeric protein is a humanized IL12RB2 protein, and the amino acid sequence of the humanized IL12RB2 protein preferably comprises SEQ ID NO: 21 or 56; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 21 or 56. In some embodiments, the other human or chimeric protein is a humanized PD-1 protein.

[0024] In one aspect, the present invention provides a non-human animal or a method for constructing the same, wherein the non-human animal comprises at least one cell encoding a nucleotide sequence of a human or humanized IL23R protein, and the humanized IL23R protein comprises an amino acid sequence that is identical to at least 50 to 629, such as at least 50, 100, 150, 200, 250, 300, 320, 327, 330, 332, 350, 353, 355, 400, 450, 500, 550, 600, 620 or 629 consecutive amino acid sequences of the human IL23R protein. In some embodiments, the non-human animal expresses a human or humanized IL23R protein. In some embodiments, the human or humanized IL23R protein comprises an amino acid sequence that is identical to at least 10 to 332, such as at least 10, 50, 100, 150, 200, 250, 300, 320, 327, 330 or 332 consecutive amino acids of the extracellular region of the human IL23R protein. In some embodiments, the humanized IL23R protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acid sequence at positions 24-355 of SEQ ID NO: 59 or positions 27-353 of SEQ ID NO: 59. In some embodiments, the humanized IL23R protein comprises an amino acid sequence that is identical to at least 1 to 23, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive amino acids of the signal peptide of the endogenous IL23R protein of the non-human animal. In some embodiments, the humanized IL23R protein comprises an amino acid sequence that is identical to at least 1 to 351, such as at least 1, 2, 3, 4, 5, 10, 50, 100, 150, 200, 250, 300, 350 or 351 consecutive amino acids of the extracellular region of the endogenous IL23R protein of the non-human animal. In some embodiments, the humanized IL23R protein comprises an amino acid sequence that is identical to at least 1 to 21, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive amino acids of the transmembrane region of the endogenous IL23R protein of the non-human animal. In some embodiments, the humanized IL23R protein comprises an amino acid sequence that is identical to at least 50 to 249, such as at least 50, 100, 150, 200, 210, 220, 230, 240 or 249 consecutive amino acids of the cytoplasmic region of the endogenous IL23R protein of the non-human animal.In some embodiments, the humanized IL23R protein comprises an amino acid sequence that is identical or has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to amino acids 1-23 of SEQ ID NO: 27, amino acids 1-26 of SEQ ID NO: 27, amino acids 373-644 of SEQ ID NO: 27, or amino acids 375-644 of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the human or humanized IL23R protein is operably linked to an endogenous IL23R regulatory element (e.g., a promoter, 5'UTR, and / or 3'UTR). In some embodiments, the nucleotide sequence encoding the human or humanized IL23R protein is integrated into the endogenous IL23R locus of the non-human animal, preferably by insertion or replacement. In some embodiments, the humanized IL23R protein has at least one activity, such as endogenous IL23R activity of the non-human animal and / or human IL23R activity.

[0025] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. In at least one cell of the non-human animal, at the endogenous IL23R locus of the non-human animal, the nucleotide sequence encoding the endogenous IL23R protein is replaced by a nucleotide sequence encoding a chimeric IL23R protein. In some embodiments, the endogenous IL23R protein of the non-human animal is not expressed or has a reduced expression level compared to IL23R in a wild-type animal. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein is a human-non-human animal chimeric genomic DNA sequence, a chimeric CDS sequence, or a chimeric cDNA sequence. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein is a human-non-human animal chimeric CDS sequence. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein is a chimeric sequence of a human-non-human animal chimeric CDS sequence and the genomic DNA sequence of the endogenous IL23R of the non-human animal. In some embodiments, the chimeric IL23R protein comprises a human or humanized extracellular region. In some embodiments, the chimeric IL23R protein comprises an endogenous signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises all or part of exon 3 of the human IL23R gene, all of exons 4-8, and part of exon 9. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the CDS sequence of all or part of exon 3 of the human IL23R gene, all of exons 4-8, and part of exon 9. In some embodiments, the amino acid sequence of the chimeric IL23R protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% to the amino acid sequence at positions 24-355 of SEQ ID NO: 59 or positions 27-353 of SEQ ID NO: 59. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises part of exon 2 to part of exon 3, and part of exon 9 to all or part of exon 11 of the non-human animal IL23R gene. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the CDS sequence of part of exon 2 to part of exon 3, and part of exon 9 to all or part of exon 11 of the non-human animal IL23R gene. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein further comprises the 3'UTR of the endogenous IL23R gene of the non-human animal, preferably further comprising at least 50 bp of continuous nucleotides downstream of the 3'UTR.In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the CDS sequence of the partial exon 2 to partial exon 3, partial exon 9 to all or part of exon 11 of the non-human animal IL23R gene, preferably further comprises the 3'UTR sequence, and more preferably further comprises at least 300 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the amino acid sequence of the chimeric IL23R protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to positions 1-23 of SEQ ID NO: 27, positions 1-26 of SEQ ID NO: 27, positions 373-644 of SEQ ID NO: 27 or positions 375-644 of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises a nucleotide sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the nucleotide sequence shown in SEQ ID NO: 69 or SEQ ID NO: 71. In some embodiments, the amino acid sequence of the chimeric IL23R protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acid sequence shown in SEQ ID NO: 78. In some embodiments, the nucleotide sequence encoding the endogenous IL23R protein comprises part or all of exon 3 of the non-human animal IL23R gene. In some embodiments, the nucleotide sequence encoding the endogenous IL23R protein comprises part of exon 3 of the non-human animal IL23R gene. In some embodiments, the nucleotide sequence encoding the endogenous IL23R protein comprises all or part of intron 3 of the non-human animal IL23R gene. In some embodiments, the nucleotide sequence encoding the endogenous IL23R protein comprises part of exon 3 of the non-human animal IL23R gene and at least 50 bp of continuous nucleotides of intron 3. In some embodiments, the nucleotide sequence encoding the endogenous IL23R protein comprises part of exon 3 of the non-human animal IL23R gene and at least 5251 bp of continuous nucleotides of intron 3. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the endogenous IL23R protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the nucleotide sequence of positions 24-122 of SEQ ID NO: 27 or positions 33-122 of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein is operably linked to a non-human animal endogenous IL23R regulatory element (such as, a promoter, 5'UTR and / or 3'UTR).In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (mouse or rat).

[0026] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing a human or chimeric IL23R protein. The construction method includes replacing the nucleotide sequence encoding the endogenous IL23R protein with a nucleotide sequence encoding a chimeric IL23R protein at the endogenous IL23R locus of the non-human animal to generate a genetically modified non-human animal cell that expresses a human or chimeric IL23R protein. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein is a human and non-human animal chimeric genomic DNA sequence, a chimeric CDS sequence, or a chimeric cDNA sequence. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein is a human and non-human animal chimeric CDS sequence. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein is a chimeric sequence of a human and non-human animal chimeric CDS sequence and the genomic DNA sequence of the non-human animal endogenous IL23R. In some embodiments, the chimeric IL23R protein comprises a human or humanized extracellular region. In some embodiments, the chimeric IL23R protein comprises an endogenous signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises all or part of exon 3 of the human IL23R gene, all of exons 4-8, and part of exon 9. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the CDS sequence of all or part of exon 3 of the human IL23R gene, all of exons 4-8, and part of exon 9. In some embodiments, the amino acid sequence of the chimeric IL23R protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% to the amino acid sequence at positions 24-355 of SEQ ID NO: 59 or positions 27-353 of SEQ ID NO: 59. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises part of exon 2 to part of exon 3 of the non-human animal IL23R gene, part of exon 9 to all or part of exon 11. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises the CDS sequence of part of exon 2 to part of exon 3 of the non-human animal IL23R gene, part of exon 9 to all or part of exon 11. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein further comprises the 3'UTR of the non-human animal endogenous IL23R gene, preferably further comprising at least 50 bp of continuous nucleotides downstream of the 3'UTR.In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises a CDS sequence from a part of exon 2 to a part of exon 3, a part of exon 9 to all or part of exon 11 of the non-human animal IL23R gene, preferably further comprises a 3'UTR sequence, and more preferably further comprises at least 300 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the amino acid sequence of the chimeric IL23R protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to positions 1-23 of SEQ ID NO: 27, positions 1-26 of SEQ ID NO: 27, positions 373-644 of SEQ ID NO: 27 or positions 375-644 of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein comprises a nucleotide sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the nucleotide sequence shown in SEQ ID NO: 69 or SEQ ID NO: 71. In some embodiments, the amino acid sequence of the chimeric IL23R protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acid sequence shown in SEQ ID NO: 78. In some embodiments, the nucleotide sequence encoding the endogenous IL23R protein comprises a part or all of exon 3 of the non-human animal IL23R gene. In some embodiments, the nucleotide sequence encoding the endogenous IL23R protein comprises a part or all of intron 3 of the non-human animal IL23R gene. In some embodiments, the nucleotide sequence encoding the endogenous IL23R protein comprises a part of exon 3 of the non-human animal IL23R gene and at least 50 bp of continuous nucleotides of intron 3. In some embodiments, the nucleotide sequence encoding the endogenous IL23R protein comprises a part of exon 3 of the non-human animal IL23R gene and at least 5251 bp of continuous nucleotides of intron 3. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the endogenous IL23R protein comprises an amino acid sequence that is identical or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acid sequence of positions 24-122 of SEQ ID NO: 27 or positions 33-122 of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the chimeric IL23R protein is operably linked to an endogenous IL23R regulatory element (such as a promoter, 5'UTR and / or 3'UTR). In some embodiments, the non-human animal is a mouse.

[0027] In one aspect, the present invention provides a method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, the method comprising: 1) administering the therapeutic agent to the non-human animal or the non-human animal obtained by the above construction method; 2) determining the effect of the therapeutic agent on the non-human animal or the disease. In some embodiments, the therapeutic agent comprises an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug. In some embodiments, the therapeutic agent comprises an additional therapeutic agent, and the additional therapeutic agent comprises an antibody specifically binding to IL12A, IL12B, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the additional therapeutic agent comprises one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the disease comprises one or more of cancer, immune diseases, or inflammation. In some embodiments, the cancer comprises one or more of colorectal cancer, breast cancer, hepatobiliary cancer, lymphocyte tumors, head and neck cancer, liver cancer, or lung cancer. In some embodiments, the immune diseases comprise one or more of psoriasis, atopic dermatitis, asthma, rheumatoid arthritis, or multiple sclerosis. In some embodiments, the inflammation comprises inflammatory bowel disease (IBD).

[0028] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating cancer. In some embodiments, the method comprises: 1) administering the therapeutic agent to the non-human animal or the non-human animal obtained by the above construction method, wherein the non-human animal has cancer; 2) determining the inhibitory effect of the therapeutic agent on cancer. In some embodiments, the therapeutic agent comprises an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug. In some embodiments, the cancer is a tumor, and the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the non-human animal. In some embodiments, the cancer comprises injecting one or more cancer cells into the non-human animal. In some embodiments, it is a solid tumor or a hematological tumor. In some embodiments, the cancer is one or more of colorectal cancer, breast cancer, hepatobiliary cancer, head and neck cancer, liver cancer, or lung cancer. In some embodiments, the non-human animal further comprises a sequence encoding human or chimeric PD-1, human or chimeric PD-L1, and / or human or chimeric CTLA4. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0029] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating an immune disease. In some embodiments, the method includes: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal suffers from an immune disease; 2) determining the therapeutic effect of the therapeutic agent on the immune disease. In some embodiments, the therapeutic agent includes an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug. In some embodiments, the immune disease includes one or more of psoriasis, atopic dermatitis, asthma, rheumatoid arthritis, or multiple sclerosis.

[0030] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating inflammation. In some embodiments, the method includes: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has inflammation; 2) determining the effectiveness of the therapeutic agent in treating inflammation. In some embodiments, the therapeutic agent includes an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug. In some embodiments, the inflammation includes inflammatory bowel disease (IBD).

[0031] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent. In some embodiments, the method includes: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the therapeutic agent includes an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug. In some embodiments, determining the effect of the therapeutic agent on the non-human animal involves measuring the body weight of the non-human animal or performing a blood test. In some embodiments, the blood test includes, but is not limited to, red blood cell count, hematocrit, and / or hemoglobin content.

[0032] In one aspect, the present invention provides a humanized IL12RB1 protein, which comprises all or part of the human IL12RB1 protein. In some embodiments, the humanized IL12RB1 protein comprises all or part of the extracellular region of the human IL12RB1 protein, preferably all or part of the transmembrane region of the human IL12RB1 protein. In some embodiments, the humanized IL12RB1 protein comprises all or part of the signal peptide of the human IL12RB1 protein. In some embodiments, the humanized IL12RB1 protein comprises all or part of the signal peptide, all or part of the extracellular region, and all or part of the transmembrane region of the human IL12RB1 protein. In some embodiments, the humanized IL12RB1 protein comprises all of the signal peptide, all of the extracellular region, and part of the transmembrane region of the human IL12RB1 protein, and part of the transmembrane region and all of the cytoplasmic region of the endogenous IL12RB1 protein of a non-human animal. In some embodiments, the humanized IL12RB1 protein comprises positions 1-549 of SEQ ID NO: 2, positions 1-570 of SEQ ID NO: 2, positions 24-549 of SEQ ID NO: 2, positions 24-570 of SEQ ID NO: 2, positions 1-545 of SEQ ID NO: 2, or positions 24-545 of SEQ ID NO: 2; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to positions 1-549 of SEQ ID NO: 2, positions 1-570 of SEQ ID NO: 2, positions 24-549 of SEQ ID NO: 2, positions 24-570 of SEQ ID NO: 2, positions 1-545 of SEQ ID NO: 2, or positions 24-545 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the humanized IL12RB1 protein comprises SEQ ID NO: 11; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 11.

[0033] In one aspect, the present invention provides a humanized IL12RB1 gene, and the humanized IL12RB1 gene encodes the humanized IL12RB1 protein. In some embodiments, the humanized IL12RB1 gene comprises a portion of exon 1 to a portion of exon 14 of the human IL12RB1 gene. In some embodiments, the humanized IL12RB1 gene comprises a portion from the start codon of the human IL12RB1 gene to exon 14. In some embodiments, the portion of exon 1 of the human IL12RB1 gene preferably comprises at least 5 consecutive nucleotide sequences, and the portion of exon 14 of the human IL12RB1 gene preferably comprises at least 5 consecutive nucleotide sequences. In some embodiments, the humanized IL12RB1 gene further comprises a portion of exon 1, a portion of exon 14 to all or part of exon 16 of the non-human animal IL12RB1 gene. In some embodiments, the humanized IL12RB1 gene comprises the nucleotide sequence shown at positions 18062249 to 18086823 of SEQ ID NO: 3, 4, 6, 7, 8, 9, 10 or NCBI accession number NC_000019.10; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown at positions 18062249 to 18086823 of SEQ ID NO: 3, 4, 6, 7, 8, 9, 10 or NCBI accession number NC_000019.10.

[0034] In one aspect, the present invention provides a humanized IL12RB2 protein, which comprises all or part of the human IL12RB2 protein. In some embodiments, the humanized IL12RB2 protein comprises all or part of the extracellular region of the human IL12RB2 protein, preferably further comprises all or part of the transmembrane region of the human IL12RB2 protein. In some embodiments, the humanized IL12RB2 protein comprises all or part of the signal peptide of the human IL12RB2 protein. In some embodiments, the humanized IL12RB2 protein comprises positions 24-622 of SEQ ID NO: 13, positions 24-643 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, positions 1-640 of SEQ ID NO: 13, or positions 1-643 of SEQ ID NO: 13; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, positions 24-643 of SEQ ID NO: 13, positions 1-640 of SEQ ID NO: 13, or positions 1-643 of SEQ ID NO: 13. In some embodiments, the humanized IL12RB2 protein further comprises all or part of the endogenous IL12RB2 protein of a non-human animal. In some embodiments, the humanized IL12RB2 protein further comprises all or part of the transmembrane region of the endogenous IL12RB2 protein of a non-human animal, preferably further comprises all or part of the cytoplasmic region of the endogenous IL12RB2 protein of a non-human animal. In some embodiments, the humanized IL12RB2 protein comprises positions 638-874 of SEQ ID NO: 12, positions 656-874 of SEQ ID NO: 12, or positions 639-874 of SEQ ID NO: 12; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to positions 638-874 of SEQ ID NO: 12, positions 656-874 of SEQ ID NO: 12, or positions 639-874 of SEQ ID NO: 12. In some embodiments, the amino acid sequence of the humanized IL12RB2 protein comprises SEQ ID NO: 21 or 56; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 21 or 56.

[0035] In one aspect, the present invention provides a humanized IL12RB2 gene, and the humanized IL12RB2 gene encodes the humanized IL12RB2 protein. In some embodiments, the humanized IL12RB1 gene comprises a portion from exon 2 to exon 14 of the human IL12RB2 gene. In some embodiments, the humanized IL12RB1 gene comprises the CDS sequence of a portion from exon 2 to exon 14 of the human IL12RB2 gene. In some embodiments, the portion of exon 2 of the human IL12RB2 gene preferably comprises at least 1 bp of continuous nucleotide sequence, and the portion of exon 14 of the human IL12RB2 gene preferably comprises at least 5 bp of continuous nucleotide sequence. In some embodiments, the humanized IL12RB2 gene further comprises all or part of the portion from exon 14 to exon 16 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the humanized IL12RB2 gene further comprises the CDS sequence of all or part of the portion from exon 14 to exon 16 of the non-human animal endogenous IL12RB2 gene. In some embodiments, the portion of exon 14 of the non-human animal endogenous IL12RB2 gene preferably comprises at least 5 bp of continuous nucleotide sequence, and the portion of exon 16 of the non-human animal endogenous IL12RB2 gene preferably comprises at least 5 bp of continuous nucleotide sequence. In some embodiments, the humanized IL12RB2 gene further comprises the nucleotide sequence from the portion of exon 14 of the non-human animal endogenous IL12RB2 gene to the stop codon, preferably further comprises the 3'UTR, and more preferably further comprises at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the humanized IL12RB2 gene further comprises the CDS sequence from the portion of exon 14 of the non-human animal endogenous IL12RB2 gene to the stop codon, preferably further comprises the 3'UTR, and more preferably further comprises at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the humanized IL12RB2 gene further comprises the portion from exon 14 of the non-human animal endogenous IL12RB2 gene to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR.In some embodiments, the humanized IL12RB2 gene comprises the nucleotide sequence shown at positions 67320369 to 67386643 of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 48, 49, 51, 52, 53, 54, 55, 67, 68 or NCBI accession number NC_000001.11; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown at positions 67320369 to 67386643 of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 48, 49, 51, 52, 53, 54, 55, 67, 68 or NCBI accession number NC_000001.11.

[0036] In one aspect, the present invention provides a humanized IL23R protein, which comprises all or part of the human IL23R protein. In some embodiments, the humanized IL23R protein comprises all or part of the extracellular region of the human IL23R protein. In some embodiments, the amino acid sequence of the humanized IL23R protein comprises amino acids at positions 24 - 355 of SEQ ID NO: 59 or amino acids at positions 27 - 353 of SEQ ID NO: 59; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acids at positions 24 - 355 of SEQ ID NO: 59 or the amino acids at positions 27 - 353 of SEQ ID NO: 59. In some embodiments, the humanized IL23R protein comprises all or part of the endogenous IL23R protein of a non-human animal. In some embodiments, the humanized IL23R protein comprises all or part of the signal peptide, transmembrane region and / or cytoplasmic region of the endogenous IL23R protein of a non-human animal. In some embodiments, the amino acid sequence of the humanized IL23R protein comprises amino acids at positions 1 - 26 and 373 - 644 of SEQ ID NO: 27, or amino acids at positions 1 - 23 and 375 - 644 of SEQ ID NO: 27; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acids at positions 1 - 26 and 373 - 644 of SEQ ID NO: 27, or the amino acids at positions 1 - 23 and 375 - 644 of SEQ ID NO: 27. In some embodiments, the amino acid sequence of the humanized IL23R protein comprises SEQ ID NO: 78; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 78.

[0037] In one aspect, the present invention provides a humanized IL23R gene. In some embodiments, the humanized IL23R gene encodes the humanized IL23R protein. In some embodiments, the humanized IL23R gene comprises all or part of the human IL23R gene. In some embodiments, the humanized IL23R gene comprises all or part of exon 3 to part of exon 9 of the human IL23R gene. In some embodiments, the humanized IL23R gene comprises the CDS sequence of all or part of exon 3 to part of exon 9 of the human IL23R gene. In some embodiments, the part of exon 3 of the human IL23R gene preferably comprises at least 5 consecutive nucleotide sequences, and the part of exon 9 of the human IL23R gene preferably comprises at least 5 consecutive nucleotide sequences. In some embodiments, the humanized IL23R gene comprises all or part of the endogenous IL23R gene of a non-human animal. In some embodiments, the humanized IL23R gene comprises part of exon 2 to part of exon 3 of the endogenous IL23R gene of a non-human animal and all or part of exon 9 to exon 11 of the endogenous IL23R gene of a non-human animal. In some embodiments, the humanized IL23R gene comprises the CDS sequence of part of exon 2 to part of exon 3 of the endogenous IL23R gene of a non-human animal and all or part of exon 9 to exon 11 of the endogenous IL23R gene of a non-human animal. In some embodiments, the humanized IL23R gene comprises the nucleotide sequence of part of exon 2 to part of exon 3 of the endogenous IL23R gene of a non-human animal and part of exon 9 to the stop codon of the endogenous IL23R gene of a non-human animal, preferably further comprising the 3'UTR, and more preferably further comprising at least 50 consecutive nucleotide sequences downstream of the 3'UTR. In some embodiments, the humanized IL23R gene comprises the CDS sequence of part of exon 2 to part of exon 3 of the endogenous IL23R gene of a non-human animal and part of exon 9 to the stop codon of the endogenous IL23R gene of a non-human animal, preferably further comprising the 3'UTR, and more preferably further comprising at least 50 consecutive nucleotide sequences downstream of the 3'UTR. In some embodiments, the humanized IL23R gene comprises part of exon 2 to part of exon 3 of the endogenous IL23R gene of a non-human animal and part of exon 9 to at least 50 consecutive nucleotide sequences downstream of the 3'UTR. In some embodiments, the part of exon 2 of the endogenous IL23R gene of a non-human animal preferably comprises at least 5 consecutive nucleotide sequences, the part of exon 3 of the endogenous IL23R gene of a non-human animal preferably comprises at least 5 consecutive nucleotide sequences, the part of exon 9 of the endogenous IL23R gene of a non-human animal preferably comprises at least 5 consecutive nucleotide sequences, and the part of exon 11 of the endogenous IL23R gene of a non-human animal preferably comprises at least 5 consecutive nucleotide sequences.In some embodiments, the humanized IL23R gene comprises the nucleotide sequence shown in SEQ ID NO: 65, 66, 69, 71, 72, 74, 75, 76, 77, 79 or 80; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 65, 66, 69, 71, 72, 74, 75, 76, 77, 79 or 80.

[0038] In one aspect, the present invention provides a cell, tissue or organ that expresses the above-mentioned humanized IL12RB1 protein, the above-mentioned humanized IL12RB2 protein and / or the above-mentioned humanized IL23R protein, and / or, the genome of the cell, tissue or organ comprises the above-mentioned humanized IL12RB1 gene, the above-mentioned humanized IL12RB2 gene and / or the above-mentioned humanized IL23R gene.

[0039] In one aspect, the present invention provides an animal model that expresses the above-mentioned humanized IL12RB1 protein, the above-mentioned humanized IL12RB2 protein and / or the above-mentioned humanized IL23R protein, and / or, the genome of the animal model comprises the above-mentioned humanized IL12RB1 gene, the above-mentioned humanized IL12RB2 gene and / or the above-mentioned humanized IL23R gene.

[0040] In one aspect, the present invention provides the use of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned humanized IL12RB1 protein, the above-mentioned humanized IL12RB2 protein, the above-mentioned humanized IL23R protein, the above-mentioned humanized IL12RB1 gene, the above-mentioned humanized IL12RB2 gene, the above-mentioned humanized IL23R gene, the above-mentioned cell, tissue or organ or the above-mentioned animal model, and the uses include: A) use in the development of products related to the immune processes involving human cells and related to IL12RB1, IL12RB2 and / or IL23R; B) use as a model system related to IL12RB1, IL12RB2 and / or IL23R in pharmacological, immunological, microbiological and medical research; C) use related to the production and utilization of animal experimental disease models for etiological research related to IL12RB1, IL12RB2 and / or IL23R and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) use in the in vivo screening, efficacy detection, evaluation of efficacy, verification or evaluation of regulators of the human IL12RB1, IL12RB2 and / or IL23R signaling pathways; or, E) use in studying the functions of the IL12RB1, IL12RB2 and / or IL23R genes, studying the drugs and drug effects targeting the human IL12RB1, IL12RB2 and / or IL23R target sites, and studying the therapeutic drugs for tumors, inflammation or immune diseases related to IL12RB1, IL12RB2 and / or IL23R.

[0041] The term "all or part" in the present invention, "all" refers to the whole, and "part" refers to the local part in the whole, or the individual parts constituting the whole.

[0042] The term "locus" in the present invention broadly represents the position occupied by a gene on a chromosome, and narrowly represents a DNA fragment on a certain gene, which can be either a gene or a part of a gene or a regulatory region of a gene, etc. For example, the so-called "non-human animal endogenous IL12RB1 locus" includes a DNA fragment of any segment selected from exons 1-16 of the non-human animal IL12RB1 gene (such as the mouse IL12RB1 gene).

[0043] The part of the term "exon XX" in the present invention means that several, dozens or hundreds of nucleotides are consecutive or spaced and are identical to the nucleotide sequence of the entire exon. For example, the part of exon 1 of the human IL12RB1 gene preferably contains at least 5-175 bp, such as at least 5, 20, 50, 60, 64, 65, 70, 100, 110, 120, 130, 140, 150, 160, 170 or 175 bp of consecutive nucleotide sequences, and preferably contains the nucleotide sequence of the coding region.

[0044] The term "exon XX to exon XXX" or "exon XX-XXX" or "the whole of exon XX-XXX" or "the whole of exon XX to the whole of exon XXX" in the present invention refers to the entire nucleotide sequence including one exon to another exon and the introns therebetween. For example, exon 1-16 includes the entire nucleotide sequences of exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15 and exon 16.

[0045] The term "part of exon XX to part of exon XXX" or "the whole of exon XX to part of exon XXX" or "part of exon XX to all or part of exon XXX" in the present invention refers to all or part of one exon to all or part of another exon, preferably also including the introns therebetween. For example, the part of exon 1 to part of exon 14 of the human IL12RB1 gene includes the part of exon 1 of the human IL12RB1 gene, the whole of intron 1, the whole of exon 2, the whole of intron 2, the whole of exon 3, the whole of intron 3, the whole of exon 4, the whole of intron 4, the whole of exon 5, the whole of intron 5, the whole of exon 6, the whole of intron 6, the whole of exon 7, the whole of intron 7, the whole of exon 8, the whole of intron 8, the whole of exon 9, the whole of intron 9, the whole of exon 10, the whole of intron 10, the whole of exon 11, the whole of intron 11, the whole of exon 12, the whole of intron 12, the whole of exon 13, the whole of intron 13 and the part of nucleotide sequence of exon 14. When the sequence is a CDS sequence, the intron sequence is not included. For example, the CDS sequence of the part of exon 2 to part of exon 14 of the human IL12RB2 gene includes the part of exon 2 of the human IL12RB2 gene, the whole of exon 3, the whole of exon 4, the whole of exon 5, the whole of exon 6, the whole of exon 7, the whole of exon 8, the whole of exon 9, the whole of exon 10, the whole of exon 11, the whole of exon 12, the whole of exon 13 and the part of nucleotide sequence of exon 14.

[0046] The term "intron" in the present invention refers to the intron between two exons. For example, intron 2 is the intron between exon 2 and exon 3.

[0047] As used in this invention, the term "comprising" or "including" is an open - ended expression, which includes the specified components or steps described, as well as other specified components or steps that do not substantially affect. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the said sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0048] As used in this invention, the term "and / or" includes all combinations of the items connected by this term, and should be regarded as each combination having been separately listed in this application. For example, "A and / or B" includes "A", "B", and "A and B". Another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0049] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. This application describes the methods and materials for this invention; other suitable methods and materials known in the art can be used. The materials, methods, and examples are merely exemplary and not restrictive. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated by reference in their entirety. In case of conflict, the present specification (including definitions) shall prevail.

[0050] Those skilled in the art can easily perceive other aspects and advantages of this application from the following detailed description.

[0051] IL12RB1

[0052] In the human genome, the IL12RB1 gene (NCBI Gene ID: 3594, UniProt ID: P42701, located at positions 18058994 to 18099027 of chromosome 19, NC_000019.10) contains 17 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and exon 17. The corresponding positions of protein domains in the nucleotide sequence and amino acid sequence based on transcript NM_005535.3 and its encoded protein NP_005526.1 (SEQ ID NO: 2) are shown in Table 1.

[0053] Table 1

[0054]

[0055]

[0056] In the genome of mice, the IL12RB1 gene (NCBI Gene ID: 16161, UniProt ID: Q60837, located at positions 71261005 to 71276186 on chromosome 8, NC_000074.7) contains 16 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and exon 16. The corresponding positions of protein domains in the nucleotide sequence of transcript NM_008353.2 and its encoded protein NP_032379.2 (SEQ ID NO: 1) and the amino acid sequence are shown in Table 2.

[0057] Table 2

[0058]

[0059] IL12RB1 genes, proteins, and gene loci of other species in the art are also known. For example, Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog), and Sus scrofa (pig). The relevant information of these genes (such as intron sequences, exon sequences, and amino acid sequences) can all be found in NCBI, and the entire content is incorporated herein by reference.

[0060] IL12RB2

[0061] In the genome of humans, the IL12RB2 gene (NCBI Gene ID: 3595, UniProt ID: Q99665, located at positions 67307351 to 67398724 on chromosome 1, NC_000001.11) contains 16 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and exon 16. The corresponding positions of protein domains in the nucleotide sequence of transcript NM_001559.3 and its encoded protein NP_001550.1 (SEQ ID NO: 13) and the amino acid sequence are shown in Table 3.

[0062] Table 3

[0063]

[0064]

[0065] In the genome of mice, the IL12RB2 gene (NCBI Gene ID: 16162, UniProt ID: P97378, located at positions 67263914 to 67353277 of NC_000072.7 on chromosome 6) contains 16 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and exon 16. The corresponding positions of protein domains in the nucleotide sequence and amino acid sequence based on transcript NM_008354.4 and its encoded protein NP_032380.1 (SEQ ID NO: 12) are shown in Table 4.

[0066] Table 4

[0067]

[0068] IL12RB2 genes, proteins, and gene loci of other species in the art are also known. For example, in Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog), and Sus scrofa (pig), relevant information about these genes (such as intron sequences, exon sequences, and amino acid sequences) can all be found in NCBI, and the entire content is incorporated herein by reference.

[0069] IL23R

[0070] In the genome of humans, the IL23R gene (NCBI Gene ID: 149233, UniProt ID: Q5VWK5, located at positions 67138637 to 67265903 of NC_000001.11 on chromosome 1) contains 11 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and exon 11. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_144701.3 and its encoded protein NP_653302.2 (SEQ ID NO: 59) are shown in Table 5.

[0071] Table 5

[0072]

[0073] In the genome of mice, the IL23R gene (NCBI Gene ID: 209590, UniProt ID: Q8K4B4, located at positions 67399906 to 67468838 of NC_000072.7 on chromosome 6) contains 11 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and exon 11. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_144548.2 and its encoded protein NP_653131.3 (SEQ ID NO: 27) are shown in Table 6.

[0074] Table 6

[0075]

[0076] IL23R genes, proteins, and gene loci of other species in the art are also known. For example, Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog), and Sus scrofa (pig). The relevant information of these genes (such as intron sequences, exon sequences, and amino acid sequences) can all be found in NCBI, and the entire content is incorporated herein by reference.

[0077] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (e.g., for optimal alignment, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non - homologous sequences may be ignored for the purpose of comparison). Then the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between two sequences is a function of the number of positions shared by the sequences, taking into account the number of gaps and the length of each gap, which are introduced to achieve the optimal alignment of the two sequences. For example, the comparison of sequences and the determination of the percentage identity between two sequences can be done using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0078] The percentage of conserved residues (percent homology) with similar physicochemical properties, such as leucine and isoleucine, can also be used to measure sequence similarity. Amino acid residue families with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percent homology is higher than the percent identity.

[0079] Vector targeting the endogenous IL12RB1 gene in non-human animals

[0080] The present invention provides a targeting vector, comprising: a) a DNA fragment (5'-arm or 5'-homologous arm) homologous to the 5'-end of the conversion region to be altered, which is selected from the genomic DNA of the IL12RB1 gene and has a length of 100-10,000 nucleotides; b) a donor region; and c) a DNA fragment (3'-arm or 3'-homologous arm) homologous to the 3'-end of the conversion region to be altered, which is selected from the genomic DNA of the IL12RB1 gene and has a length of 100-10,000 nucleotides.

[0081] In some embodiments, a) the DNA fragment homologous to the 5'-end of the conversion region to be altered is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000074.7; c) the DNA fragment homologous to the 3'-end of the conversion region to be altered is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000074.7.

[0082] In some embodiments, a) the DNA fragment homologous to the 5'-end of the conversion region to be altered is selected from the nucleotide sequence at positions 71257469 to 71261195 of NCBI accession number NC_000074.7; c) the DNA fragment homologous to the 3'-end of the conversion region to be altered is selected from the nucleotide sequence at positions 71271998 to 71274804 of NCBI accession number NC_000074.7.

[0083] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, or 20 kb.

[0084] In some embodiments, the conversion region to be altered is located at the endogenous IL12RB1 locus of a non-human animal. In some embodiments, the conversion region to be altered is located on exons 1-16 of the endogenous IL12RB1 gene of a non-human animal. In some embodiments, the conversion region to be altered is located on exons 1-14 of the endogenous IL12RB1 gene of a non-human animal.

[0085] In some embodiments, the 5' arm comprises SEQ ID NO: 3. In some embodiments, the 3' arm comprises SEQ ID NO: 4.

[0086] In some embodiments, the donor region of b) comprises a human sequence. In some embodiments, the human sequence comprises the nucleotide sequence from positions 18,062,249 to 18,086,823 of NCBI accession number NC_000019.10.

[0087] In some embodiments, the targeting vector comprises one or more marker genes (or resistance genes), for example, a resistance gene for positive clone selection or a coding gene for a negative selection marker. In some embodiments, the resistance gene for positive clone selection is the coding sequence Neo of neomycin phosphotransferase. Preferably, the targeting vector further comprises two directly repeated Frt recombination sites flanking the marker gene. In some embodiments, the coding gene for the negative selection marker is the coding gene (DTA) for the A subunit of diphtheria toxin.

[0088] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the IL12RB1 gene. In some embodiments, the target site of the sgRNA on the conversion region to be altered is unique and satisfies the sequence arrangement rules of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'.

[0089] In some embodiments, the present invention relates to a plasmid construct (sgRNA vector, such as pT7-sgRNA) comprising an sgRNA and / or a cell comprising the construct. In some embodiments, the present invention also relates to a cell comprising the targeting vector and / or the sgRNA vector as described above.

[0090] In some embodiments, the present invention also provides a non-human mammalian cell having any one of the above vectors and one or more in vitro transcripts of the plasmid constructs described in the present application. In some embodiments, the non-human mammalian cell contains Cas9 mRNA or its in vitro transcript.

[0091] In some embodiments, the gene in the non-human mammalian cell is heterozygous. In some embodiments, the gene in the non-human mammalian cell is homozygous.

[0092] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the non-human mammalian cell is a fertilized egg cell. In some embodiments, the non-human mammalian cell is an embryonic stem cell. In some embodiments, the non-human mammalian cell is any cell capable of expressing the IL12RB1 protein.

[0093] Vector targeting the endogenous IL12RB2 gene of non-human animals

[0094] The present invention provides a targeting vector, comprising: a) a DNA fragment (5' arm or 5' homologous arm) homologous to the 5' end of the conversion region to be altered, which is selected from the genomic DNA of the IL12RB2 gene and has a length of 100-10,000 nucleotides; b) a donor region; and c) a DNA fragment (3' arm or 3' homologous arm) homologous to the 3' end of the conversion region to be altered, which is selected from the genomic DNA of the IL12RB2 gene and has a length of 100-10,000 nucleotides.

[0095] In some embodiments, a) the DNA fragment homologous to the 5' end of the conversion region to be altered is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000072.7; c) the DNA fragment homologous to the 3' end of the conversion region to be altered is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000072.7.

[0096] In some embodiments, a) the DNA fragment homologous to the 5' end of the conversion region to be altered is selected from the nucleotide sequence at positions 67338866 to 67343072 of NCBI accession number NC_000072.7; c) the DNA fragment homologous to the 3' end of the conversion region to be altered is selected from the nucleotide sequence at positions 67334870 to 67338865 of NCBI accession number NC_000072.7, wherein the base G at position 67338729 is mutated to base C.

[0097] In some embodiments, a) the DNA fragment homologous to the 5'-end of the conversion region to be altered is selected from the nucleotide sequence at positions 67338866 to 67339936 of NCBI accession number NC_000072.7; c) the DNA fragment homologous to the 3'-end of the conversion region to be altered is selected from the nucleotide sequence at positions 67337484 to 67338865 of NCBI accession number NC_000072.7.

[0098] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb or 20 kb.

[0099] In some embodiments, the conversion region to be altered is located at the IL12RB2 locus of a non-human animal. In some embodiments, the conversion region to be altered is located on exon 2 of the IL12RB2 gene (preferably after the coding sequence of the signal peptide is inserted, more preferably between the coding sequence of the signal peptide and the coding sequence of the extracellular region, still more preferably between the nucleotide sequences encoding positions 23 and 24 of SEQ ID NO: 12, and even more preferably between positions 257-258 of NCBI accession number NM_008354.4); alternatively, it is located from exon 2 to exon 14.

[0100] In some embodiments, the 5'-arm comprises SEQ ID NO: 14, 22 or 48. In some embodiments, the 3'-arm comprises SEQ ID NO: 15, 23 or 49.

[0101] In some embodiments, the b) donor sequence comprises a human sequence. In some embodiments, the nucleotide sequence of the b) donor sequence comprising the human IL12RB2 gene fragment is from positions 67320369 to 67386643 of NCBI accession number NC_000001.11.

[0102] In some embodiments, the b) donor sequence comprises a chimeric sequence of human and non-human animals.

[0103] In some embodiments, the b) donor sequence sequentially comprises a human IL12RB2 fragment and a non-human animal endogenous IL12RB2 fragment from the 5'-end to the 3'-end.

[0104] In some embodiments, the b) donor sequence comprises SEQ ID NO: 24 and SEQ ID NO: 67.

[0105] In some embodiments, the donor sequence of b) contains an auxiliary sequence.

[0106] In some embodiments, the donor sequence of b) sequentially contains a human IL12RB2 fragment, a non-human animal endogenous IL12RB2 fragment, and an auxiliary sequence from the 5'-end to the 3'-end.

[0107] In some embodiments, the auxiliary sequence is, for example, a STOP sequence, and the STOP sequence contains SEQ ID NO: 68.

[0108] In some embodiments, the donor sequence of b) contains SEQ ID NO: 24, SEQ ID NO: 67, and SEQ ID NO: 68.

[0109] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector contains an sgRNA sequence, wherein the sgRNA sequence targets the IL12RB2 gene. In some embodiments, the target site of the sgRNA on the conversion region to be altered is unique and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'.

[0110] In some embodiments, the target site of the sgRNA in the non-human animal endogenous IL12RB2 gene is located on exon 2 of the non-human animal endogenous IL12RB2 gene; or, located on exons 2 to 14.

[0111] In some embodiments, the target site is shown as SEQ ID NO: 73. Therefore, the present invention provides an sgRNA for constructing a gene-modified animal model. In some embodiments, the oligonucleotide sequence of the sgRNA sequence is listed in SEQ ID NO: 35 and 37. In some embodiments, the oligonucleotide sequence of the sgRNA sequence is listed in SEQ ID NO: 36 and 38.

[0112] In some embodiments, the present invention relates to a plasmid construct containing an sgRNA (sgRNA vector, such as pT7-sgRNA) and / or a cell containing the construct. In some embodiments, the present invention also relates to a cell containing the targeting vector and / or sgRNA vector as described above.

[0113] In some embodiments, the present invention also provides a non-human mammalian cell having any one of the above vectors and one or more in vitro transcripts of the plasmid constructs described in the present application. In some embodiments, the non-human mammalian cell contains Cas9 mRNA or its in vitro transcript.

[0114] In some embodiments, the gene in the non-human mammalian cell is heterozygous. In some embodiments, the gene in the non-human mammalian cell is homozygous.

[0115] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the non-human mammalian cell is a fertilized egg cell. In some embodiments, the non-human mammalian cell is an embryonic stem cell. In some embodiments, the non-human mammalian cell is any cell capable of expressing the IL12RB2 protein.

[0116] Vector targeting the endogenous IL23R gene of a non-human animal

[0117] The present invention provides a targeting vector, comprising: a) a DNA fragment (5'-arm or 5'-homologous arm) homologous to the 5'-end of the conversion region to be altered, which is selected from the genomic DNA of the IL23R gene and has a length of 100-10,000 nucleotides; b) a donor region; and c) a DNA fragment (3'-arm or 3'-homologous arm) homologous to the 3'-end of the conversion region to be altered, which is selected from the genomic DNA of the IL23R gene and has a length of 100-10,000 nucleotides.

[0118] In some embodiments, a) the DNA fragment homologous to the 5'-end of the conversion region to be altered is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000072.7; c) the DNA fragment homologous to the 3'-end of the conversion region to be altered is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000072.7.

[0119] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb or 20 kb.

[0120] In some embodiments, the conversion region to be altered is located at the endogenous IL23R gene locus of a non-human animal, preferably on exons 1 to 11 of the endogenous IL23R gene of a non-human animal, and more preferably on exon 3 to intron 3 of the endogenous IL23R gene of a non-human animal.

[0121] In some embodiments, the 5'-arm comprises SEQ ID NO: 65 or 79. In some embodiments, the 3'-arm comprises SEQ ID NO: 66 or 80.

[0122] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the IL23R gene. In some embodiments, the target site of the sgRNA on the conversion region to be altered is unique and satisfies the sequence arrangement rules of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'.

[0123] In some embodiments, the target site of the sgRNA in the endogenous IL23R gene of the non-human animal is located on exon 3 to intron 3 of the endogenous IL23R gene of the non-human animal.

[0124] In some embodiments, the target sites are shown as SEQ ID NO: 81 and / or 82. Accordingly, the present invention provides an sgRNA for constructing a gene-modified animal model. In some embodiments, the oligonucleotide sequence of the sgRNA sequence is listed in SEQ ID NO: 83 and 85. In some embodiments, the oligonucleotide sequence of the sgRNA sequence is listed in SEQ ID NO: 84 and 86. In some embodiments, the oligonucleotide sequence of the sgRNA sequence is listed in SEQ ID NO: 87 and 89. In some embodiments, the oligonucleotide sequence of the sgRNA sequence is listed in SEQ ID NO: 88 and 90.

[0125] In some embodiments, the present invention relates to a plasmid construct containing an sgRNA (sgRNA vector, such as pT7-sgRNA) and / or a cell containing the construct. In some embodiments, the present invention also relates to a cell containing the targeting vector and / or sgRNA vector as described above.

[0126] In some embodiments, the present invention also provides a non-human mammalian cell having any one of the above vectors and one or more in vitro transcripts of the plasmid constructs described in the present application. In some embodiments, the non-human mammalian cell contains Cas9 mRNA or its in vitro transcript.

[0127] In some embodiments, the gene in the non-human mammalian cell is heterozygous. In some embodiments, the gene in the non-human mammalian cell is homozygous.

[0128] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the non-human mammalian cell is a fertilized egg cell. In some embodiments, the non-human mammalian cell is an embryonic stem cell. In some embodiments, the non-human mammalian cell is any cell capable of expressing the IL23R protein.

[0129] Genetically modified non-human animals

[0130] As used herein, the term "genetically modified non-human animal" or "genetically engineered non-human animal" refers to a non-human animal in which at least one chromosome in the genome of the non-human animal has exogenous DNA. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in a genetically modified non-human animal or genetically engineered non-human animal, have exogenous DNA. The cells having exogenous DNA can be various cells, e.g., somatic cells, immune cells (including T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, provided is a genetically modified non-human animal, wherein the non-human animal comprises a modified endogenous IL12RB1, IL12RB2 and / or IL23R locus, and comprises an exogenous sequence (e.g., a human sequence), e.g., replacing or inserting one or more non-human sequences with one or more human sequences, or a chimeric sequence of human and non-human animal. The non-human animal is generally capable of transmitting the genetic modification to offspring through germline transmission.

[0131] As used herein, the term "chimeric (X) gene" or "chimeric (X) nucleic acid" refers to a gene or nucleic acid. In some embodiments, two or more portions of the gene or nucleic acid are from different species, or at least one sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (X) gene or chimeric (X) nucleic acid has two or more different sources, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) protein of two or more different species. In some embodiments, the chimeric (X) gene or chimeric (X) nucleic acid refers to a humanized (X) gene or humanized (X) nucleic acid.

[0132] As used herein, the term "chimeric (X) protein" or "chimeric (X) polypeptide" refers to a protein or polypeptide. In some embodiments, two or more portions of the polypeptide or protein are from different species, or at least one sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (X) protein or chimeric (X) polypeptide has two or more different species sources, e.g., the same (or homologous) protein of different species. In some embodiments, the chimeric (X) protein or chimeric (X) polypeptide refers to a humanized (X) protein or humanized (X) polypeptide.

[0133] As used herein, the term "humanized (X) protein" or "humanized (X) polypeptide" refers to a protein or polypeptide. In some embodiments, at least a portion of the protein or polypeptide is derived from a human (X) protein or a human (X) polypeptide. In some embodiments, at least a portion of the protein or polypeptide is derived from a non-human animal (X) protein or a non-human animal (X) polypeptide. In some embodiments, the humanized (X) protein or humanized (X) polypeptide refers to a human protein or polypeptide.

[0134] As used herein, the term "humanized (X) nucleic acid" or "humanized (X) gene" refers to a nucleic acid or a gene. In some embodiments, at least a portion of the nucleic acid or gene is derived from a human (X) gene or a human (X) nucleic acid. In some embodiments, at least a portion of the nucleic acid or gene is derived from a non-human animal (X) gene or a non-human animal (X) nucleic acid. In some embodiments, the nucleic acid or gene in the humanized (X) nucleic acid or humanized (X) gene is entirely derived from a human (X) nucleic acid or a human (X) gene. In some embodiments, the humanized nucleic acid refers to a humanized exon, which can be a human exon or a chimeric exon.

[0135] A non-human animal having a humanized IL12RB1 locus

[0136] In some embodiments, a chimeric IL12RB1 gene or a chimeric IL12RB1 nucleic acid is a humanized IL12RB1 gene or a humanized IL12RB1 nucleic acid. In some embodiments, at least a portion of the humanized IL12RB1 gene or humanized IL12RB1 nucleic acid is derived from the human IL12RB1 gene. In some embodiments, at least a portion of the humanized IL12RB1 gene or humanized IL12RB1 nucleic acid is derived from a non-human animal IL12RB1 gene. In some embodiments, the humanized IL12RB1 gene or humanized IL12RB1 nucleic acid contains a sequence encoding the IL12RB1 protein. The encoded IL12RB1 protein has at least one activity, such as the activity of the human IL12RB1 protein and / or the non-human animal IL12RB1 protein.

[0137] In some embodiments, the chimeric IL12RB1 protein or chimeric IL12RB1 polypeptide is a humanized IL12RB1 protein or humanized IL12RB1 polypeptide. In some embodiments, at least one or more portions of the humanized IL12RB1 protein or humanized IL12RB1 polypeptide are from the human IL12RB1 protein. In some embodiments, at least one or more portions of the humanized IL12RB1 protein or humanized IL12RB1 polypeptide are from a non-human animal IL12RB1 protein. In some embodiments, the humanized IL12RB1 protein or humanized IL12RB1 polypeptide is functional or has at least one activity, such as the activity of the human IL12RB1 protein and / or the non-human animal IL12RB1 protein.

[0138] In some embodiments, the humanized IL12RB1 protein or humanized IL12RB1 polypeptide comprises an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized IL12RB1 protein or humanized IL12RB1 polypeptide comprises or does not comprise a signal peptide. In some embodiments, the extracellular region is human or humanized. In some embodiments, the signal peptide is human or humanized. In some embodiments, the cytoplasmic region is endogenous to a non-human animal or human or humanized. In some embodiments, the transmembrane region is endogenous to a non-human animal or human or humanized.

[0139] Genetically modified non-human animals include modification of the endogenous non-human animal endogenous IL12RB1 gene locus (locus). In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of the mature IL12RB1 protein (e.g., comprising a nucleotide sequence that is identical or has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence encoding the mature IL12RB1 protein). Cells (e.g., ES cells, somatic cells) that may comprise the genetic modification described herein are provided in the present invention.

[0140] Genetically modified non-human animals can express the human IL12RB1 protein and / or a chimeric (e.g., humanized) IL12RB1 protein at the endogenous IL12RB1 gene locus of the non-human animal. In some embodiments, the endogenous IL12RB1 gene in the non-human animal genome has been replaced or inserted with the gene of human IL12RB1 and / or a nucleotide sequence encoding a human IL12RB1 sequence region or a nucleotide sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99.5% identity to the human IL12RB1 sequence. In some embodiments, the endogenous IL12RB1 gene locus of the non-human animal is modified with a nucleic acid sequence comprising all or part of the sequence encoding the human mature IL12RB1 protein.

[0141] In some embodiments, a genetically modified non-human animal can express a human IL12RB1 protein and / or a chimeric IL12RB1 (e.g., humanized IL12RB1) protein under the control of an endogenous regulatory element of the non-human animal. Insertion or replacement at the endogenous IL12RB1 locus of the non-human animal provides a non-human animal that expresses human IL12RB1 or chimeric IL12RB1 (e.g., humanized IL12RB1) in appropriate cells and in a manner that does not result in potential pathologies observed in some other transgenic non-human animals known in the art. The human IL12RB1 or chimeric IL12RB1 (e.g., humanized IL12RB1) expressed in the non-human animal can maintain one or more functions of a wild-type animal or human IL12RB1 in the non-human animal. In addition, in some embodiments, the non-human animal does not express endogenous IL12RB1. In some embodiments, the expression level of endogenous IL12RB1 in the non-human animal is reduced compared to the expression level of IL12RB1 in a wild-type animal. As used herein, the term "endogenous IL12RB1" refers to the IL12RB1 protein expressed from the endogenous IL12RB1 nucleotide sequence of a non-human animal (e.g., a mouse) prior to any genetic modification.

[0142] The genetically modified non-human animal has one or more cells that express a human or chimeric IL12RB1 (e.g., humanized IL12RB1) protein, and the chimeric IL12RB1 protein has an extracellular region, a transmembrane region, and a cytoplasmic region (preferably also including a signal peptide). In many cases, the human IL12RB1 and non-human animal IL12RB1 (e.g., mouse IL12RB1) sequences are different, so an antibody that binds to human IL12RB1 may not have the same affinity or effect on non-human animal IL12RB1. Thus, a genetically modified non-human animal having a human or humanized extracellular region (preferably also including a human or humanized signal peptide and / or a human or humanized transmembrane region) can be used to better evaluate the effect of a therapeutic agent targeting human IL12RB1 in an animal model.

[0143] In some embodiments, the humanized IL12RB1 locus comprises the 5'UTR of the human IL12RB1 gene. In some embodiments, the humanized IL12RB1 locus comprises the 3'UTR of the human IL12RB1 gene. In some embodiments, the humanized IL12RB1 locus comprises the 5'UTR of the non-human animal endogenous (e.g., mouse endogenous) IL12RB1 gene. In some embodiments, the humanized IL12RB1 locus comprises the 3'UTR of the non-human animal endogenous (e.g., mouse endogenous) IL12RB1 gene. In appropriate cases, it can be reasonably assumed that based on the similarity of the non-human animal and human IL12RB1 gene sequences, they seem to be regulated similarly. As shown in the present application, in a humanized mouse in which the endogenous IL12RB1 locus in the mouse contains an inserted or replaced IL12RB1 gene, which retains the mouse regulatory elements but contains the humanization of the IL12RB1 coding sequence, no pathological phenomena are exhibited. Both the heterozygous and homozygous gene-modified mice with humanized IL12RB1 are normal.

[0144] Non-human animals having a humanized IL12RB2 locus

[0145] In some embodiments, the chimeric IL12RB2 gene or chimeric IL12RB2 nucleic acid is a humanized IL12RB2 gene or humanized IL12RB2 nucleic acid. In some embodiments, at least a portion of the humanized IL12RB2 gene or humanized IL12RB2 nucleic acid is derived from the human IL12RB2 gene. In some embodiments, at least a portion of the humanized IL12RB2 gene or humanized IL12RB2 nucleic acid is derived from the non-human animal IL12RB2 gene. In some embodiments, the humanized IL12RB2 gene or humanized IL12RB2 nucleic acid comprises a sequence encoding an IL12RB2 protein. In some embodiments, the encoded IL12RB2 protein has at least one activity, such as the activity of the human IL12RB2 protein and / or the non-human animal IL12RB2 protein.

[0146] In some embodiments, the chimeric IL12RB2 protein or chimeric IL12RB2 polypeptide is a humanized IL12RB2 protein or humanized IL12RB2 polypeptide. In some embodiments, at least one or more portions of the humanized IL12RB2 protein or humanized IL12RB2 polypeptide are from the human IL12RB2 protein. In some embodiments, at least one or more portions of the humanized IL12RB2 protein or humanized IL12RB2 polypeptide are from the non-human animal IL12RB2 protein. In some embodiments, the humanized IL12RB2 protein or humanized IL12RB2 polypeptide is functional or has at least one activity, such as the activity of the human IL12RB2 protein and / or the non-human animal IL12RB2 protein.

[0147] In some embodiments, a humanized IL12RB2 protein or a humanized IL12RB2 polypeptide comprises an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized IL12RB2 protein or the humanized IL12RB2 polypeptide comprises or does not comprise a signal peptide. In some embodiments, the extracellular region is human or humanized. In some embodiments, the cytoplasmic region is endogenous to a non-human animal or human or humanized. In some embodiments, the transmembrane region is endogenous to a non-human animal or human or humanized. In some embodiments, the signal peptide is endogenous to a non-human animal or human or humanized.

[0148] Genetically modified non-human animals include modification of an endogenous non-human animal endogenous IL12RB2 gene locus (locus). In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of the mature IL12RB2 protein (e.g., comprising a nucleotide sequence that is identical or has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence encoding all or part of the mature IL12RB2 protein). Cells (e.g., ES cells, somatic cells) that may comprise such genetic modification are provided in the present invention.

[0149] Genetically modified non-human animals can express a human IL12RB2 protein and / or a chimeric (e.g., humanized) IL12RB2 protein at the endogenous IL12RB2 gene locus of the non-human animal. In some embodiments, the endogenous IL12RB2 gene in the genome of the non-human animal has been replaced or inserted with all or part of the gene of human IL12RB2 and / or the nucleotide sequence encoding the human IL12RB2 sequence region or a nucleotide sequence that is identical or has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 99.5% identity to the human IL12RB2 sequence. In some embodiments, the endogenous IL12RB2 gene locus of the non-human animal is modified with all or part of the nucleic acid sequence encoding the human mature IL12RB2 protein.

[0150] In some embodiments, the genetically modified non-human animal can express human IL12RB2 and / or chimeric IL12RB2 (e.g., humanized IL12RB2) under the control of non-human animal endogenous regulatory elements. Insertion or replacement at the non-human animal endogenous locus provides a non-human animal that expresses human IL12RB2 or chimeric IL12RB2 (e.g., humanized IL12RB2) in appropriate cells and in a manner that does not result in potential pathologies observed in some other transgenic non-human animals known in the art. The human IL12RB2 or chimeric IL12RB2 (e.g., humanized IL12RB2) expressed in the non-human animal can maintain one or more functions of wild-type animal or human IL12RB2 in the non-human animal. Additionally, in some embodiments, the non-human animal does not express endogenous IL12RB2. In some embodiments, the non-human animal endogenous IL12RB2 expression level is reduced compared to the IL12RB2 expression level in wild-type animals. As used herein, the term "endogenous IL12RB2" refers to the IL12RB2 protein expressed from the endogenous IL12RB2 nucleotide sequence of a non-human animal (e.g., mouse) prior to any genetic modification.

[0151] In some embodiments, the genetically modified non-human animal can have one or more cells that express human or chimeric IL12RB2 (e.g., humanized IL12RB2), the chimeric IL12RB2 having an extracellular domain, a transmembrane domain, and a cytoplasmic domain (preferably with or without a signal peptide). In some embodiments, the human IL12RB2 and non-human animal IL12RB2 (e.g., mouse IL12RB2) sequences are different, so antibodies that bind to human IL12RB2 do not necessarily have the same affinity or effect on non-human animal IL12RB2. Thus, a genetically modified non-human animal having a human or humanized extracellular domain (preferably also including a human or humanized signal peptide and / or transmembrane domain) can be used to better evaluate the effects of therapeutic agents targeting human IL12RB2 in an animal model.

[0152] In some embodiments, the humanized IL12RB2 locus comprises the 5'UTR of the human IL12RB2 gene. In some embodiments, the humanized IL12RB2 locus comprises the 3'UTR of the human IL12RB2 gene. In some embodiments, the humanized IL12RB2 locus comprises the 5'UTR of the non-human animal endogenous (e.g., mouse endogenous) IL12RB2 gene. In some embodiments, the humanized IL12RB2 locus comprises the 3'UTR of the non-human animal endogenous (e.g., mouse) IL12RB2 gene. In appropriate cases, it can be reasonably assumed that, based on the similarity of the non-human animal and human IL12RB2 gene sequences, they appear to be regulated similarly. As shown in this application, in humanized mice in which the endogenous mouse IL12RB2 locus contains an inserted or replaced IL12RB2 gene, which retain mouse regulatory elements but contain humanization of the IL12RB2 coding sequence, do not exhibit pathological phenomena. Both gene-modified mice that are heterozygous or homozygous for humanized IL12RB2 are normal.

[0153] Non-human animals having a humanized IL23R locus

[0154] In some embodiments, the chimeric IL23R gene or chimeric IL23R nucleic acid is a humanized IL23R gene or humanized IL23R nucleic acid. In some embodiments, at least a portion of the humanized IL23R gene or humanized IL23R nucleic acid is derived from the human IL23R gene. In some embodiments, at least a portion of the humanized IL23R gene or humanized IL23R nucleic acid is derived from the non-human animal IL23R gene. In some embodiments, the humanized IL23R gene or humanized IL23R nucleic acid comprises a sequence encoding an IL23R protein. In some embodiments, the encoded IL23R protein has at least one activity, such as the activity of the human IL23R protein and / or the non-human animal IL23R protein.

[0155] In some embodiments, the chimeric IL23R protein or chimeric IL23R polypeptide is a humanized IL23R protein or humanized IL23R polypeptide. In some embodiments, at least one or more portions of the humanized IL23R protein or humanized IL23R polypeptide are from the human IL23R protein. In some embodiments, at least one or more portions of the humanized IL23R protein or humanized IL23R polypeptide are from the non-human animal IL23R protein. In some embodiments, the humanized IL23R protein or humanized IL23R polypeptide is functional or has at least one activity, such as the activity of the human IL23R protein and / or the non-human animal IL23R protein.

[0156] In some embodiments, a humanized IL23R protein or a humanized IL23R polypeptide comprises an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, a humanized IL23R protein or a humanized IL23R polypeptide comprises or does not comprise a signal peptide. In some embodiments, the extracellular region is human or humanized. In some embodiments, the cytoplasmic region is non-human animal endogenous or human or humanized. In some embodiments, the transmembrane region is non-human animal endogenous or human or humanized. In some embodiments, the signal peptide is non-human animal endogenous or human or humanized.

[0157] In some embodiments, the genetically modified non-human animal comprises a modification of the endogenous non-human animal endogenous IL23R gene locus (locus).

[0158] In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature IL23R protein (e.g., comprising a nucleotide sequence that is identical or has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence encoding all or part of the mature IL23R protein). Cells (e.g., ES cells, somatic cells) that may comprise the genetic modification are provided in the present invention.

[0159] The genetically modified non-human animal can express a human IL23R protein and / or a chimeric (e.g., humanized) IL23R protein at the endogenous non-human animal IL23R gene locus. In some embodiments, the endogenous IL23R gene in the genome of the non-human animal has been replaced or inserted with the gene of human IL23R and / or the nucleotide sequence encoding the human IL23R sequence region or a nucleotide sequence that is identical or has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 99.5% identity to the human IL23R sequence in whole or in part. In some embodiments, the endogenous non-human animal IL23R gene locus is modified with a nucleic acid sequence comprising all or part of the nucleotide sequence encoding the human mature IL23R protein.

[0160] In some embodiments, a genetically modified non-human animal can express human IL23R and / or a chimeric IL23R (e.g., humanized IL23R) under the control of an endogenous regulatory element of the non-human animal. Insertion or replacement at an endogenous locus of the non-human animal provides a non-human animal that expresses human IL23R or a chimeric IL23R (e.g., humanized IL23R) in appropriate cells and in a manner that does not result in potential pathologies observed in some other transgenic non-human animals known in the art. The human IL23R or chimeric IL23R (e.g., humanized IL23R) expressed in the non-human animal can maintain one or more functions of a wild-type animal or human IL23R in the non-human animal. In addition, in some embodiments, the non-human animal does not express endogenous IL23R. In some embodiments, the endogenous IL23R expression level in the non-human animal is reduced compared to the IL23R expression level in a wild-type animal. As used herein, the term "endogenous IL23R" refers to the IL23R protein expressed from the endogenous IL23R nucleotide sequence of a non-human animal (e.g., mouse) prior to any genetic modification.

[0161] In some embodiments, the genetically modified non-human animal can have one or more cells that express a human or chimeric IL23R (e.g., humanized IL23R), the chimeric IL23R having an extracellular domain, a transmembrane domain, and a cytoplasmic domain (optionally including or excluding a signal peptide). In some embodiments, the human IL23R and non-human animal IL23R (e.g., mouse IL23R) sequences are different, so an antibody that binds to human IL23R may not have the same affinity or effect on non-human animal IL23R. Thus, a genetically modified non-human animal having a human or humanized extracellular domain can be used to better evaluate the effect of a therapeutic agent targeting human IL23R in an animal model.

[0162] In some embodiments, the humanized IL23R locus contains the 5'UTR of the human IL23R gene. In some embodiments, the humanized IL23R locus contains the 3'UTR of the human IL23R gene. In some embodiments, the humanized IL23R locus contains the 5'UTR of the endogenous (e.g., mouse endogenous) IL23R gene of the non-human animal. In some embodiments, the humanized IL23R locus contains the 3'UTR of the endogenous (e.g., mouse endogenous) IL23R gene of the non-human animal. In appropriate cases, it can be reasonably assumed that, based on the similarity of the non-human and human IL23R gene sequences, they appear to be regulated similarly. As shown in the present application, humanized mice having an inserted or replaced IL23R gene at the mouse endogenous IL23R locus, which retain mouse regulatory elements but contain a humanized IL23R coding sequence, do not exhibit pathologies. Both heterozygous and homozygous gene-modified mice with humanized IL23R are normal.

[0163] The genetically modified non-human animals can be various non-human animals, such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, water buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals for which it is not easy to obtain suitable genetically modifiable embryonic stem cells (ES), other methods are used to construct non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of non-ES cells (e.g., fibroblasts or induced pluripotent stem cells) and using nuclear transfer to transfer the modified genome to a suitable cell, such as an oocyte, and gestating the modified cell (e.g., modified oocyte) in a non-human animal under appropriate conditions to form an embryo. The above-described construction methods are known in the art and are described in “A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2006”, the entire content of which is incorporated herein by reference.

[0164] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. In some embodiments, the rodent can be selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the Muridae family. In one embodiment, the genetically modified non-human animal is selected from the families Cricetidae (e.g., mouse-like hamsters), Muridae (e.g., hamsters, New World rats and mice, voles), Muroidea (mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (mountain mice, rock mice, tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats, bamboo rats, and zokors). In a particular embodiment, the genetically modified rodent is selected from mice or rats (Muroidea), gerbils, spiny mice, and crested rats. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one embodiment, the non-human animal is a rodent. In a particular embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0165] In some embodiments, the non-human animal can be an immunodeficient non-human mammal. For example, immunodeficient rodents, immunodeficient rabbits, immunodeficient pigs, immunodeficient monkeys, etc. In some embodiments, the non-human animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mouse is of the 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described in, for example, Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant content of which is incorporated herein by reference in its entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is of the BALB strain, such as the BALB / c strain. In some embodiments, the mouse is a hybrid of the BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50% BALB / c - 50% 12954 / Sv; or 50% C57BL / 6 - 50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a strain of BALB / c, BALB / cHeAn, BALB / cJ, BALB / cRl, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, or CBA / H. In some embodiments, the non-human animal is a rat. The rat can be selected from Wistar rats, LEA strain, Sprague-Dawley strain, Fischer strain, F344, F6, and Dark Agouti.In some embodiments, the rat strain is a hybrid species of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti. The non-human animal can have one or more other genetic modifications and / or other modifications that are suitable for a particular purpose of preparing a humanized animal. For example, a suitable mouse for maintaining xenografts (such as human cancers or tumors) can have one or more modifications that impair, inactivate, or disrupt all or part of the immune system of the non-human animal. Impairment, inactivation, or disruption of the immune system of the non-human animal can include, for example, by chemical means (such as administration of toxins), physical means (such as irradiating the animal), and / or genetic modification (such as knocking out one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γc. null mouse (Ito, M. et al., NOD / SCID / γc null mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100(9):3175 - 3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice can optionally be irradiated or otherwise treated to destroy one or more immune cell types. Thus, in various embodiments, a genetically modified mouse is provided that can include humanization of at least a portion of the non-human animal endogenous IL12RB1, IL12RB2, and / or IL23R loci and also includes a modification that impairs, inactivates, or partially disrupts the immune system of the non-human animal (or one or more cell types of the immune system). In some embodiments, the type of mouse modification is selected from NOD mice, SCID mice, NOD / SCID mice, IL-2Rγ knockout mice, NOD / SCID / γc null mice, nude mice, Rag1 and / or Rag2 knockout mice, NOD Prkdc scid IL-2Rγ null mice, NOD Rag 1 - / - IL2rg - / - (NRG) mice, Rag2 - / - IL2rg - / - (RG) mice and combinations thereof. These transgenic non-human animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety.

[0166] The present invention further relates to non-human mammals produced by the above method. In some embodiments, their genomes comprise the human IL12RB1 gene, the human IL12RB2 gene, and / or the human IL23R gene.

[0167] In some embodiments, the non-human mammal is a rodent, preferably a mouse.

[0168] In some embodiments, the non-human mammal expresses a protein encoded by a humanized IL12RB1, humanized IL12RB2, and / or humanized IL23R gene.

[0169] In addition, the present invention also provides a non-human mammal model carrying a tumor, which is obtained by the construction method described in the present application. In some embodiments, the non-human mammal is a rodent (such as a mouse).

[0170] The present invention also provides a cell or cell line, or a primary cell culture, derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, a tissue, an organ, or a culture thereof derived from a non-human mammal or its offspring. When carrying a tumor, it is derived from a tumor tissue of a non-human mammal or its offspring or a non-human mammal carrying a tumor.

[0171] The present invention provides a non-human mammal produced by any of the methods described in the present application. In some embodiments, a non-human mammal, a genetically modified non-human animal is provided, the genome of which comprises DNA of human or humanized IL12RB1, human or humanized IL12RB2, and / or human or humanized IL23R.

[0172] In some embodiments, the non-human mammal comprises the gene construct described in the present application. In some embodiments, a non-human mammal expressing human or humanized IL12RB1, human or humanized IL12RB2, and / or human or humanized IL23R protein is provided. In some embodiments, a cell, tissue, or organ specifically expressing human or humanized IL12RB1, human or humanized IL12RB2, and / or human or humanized IL23R protein is provided.

[0173] In some embodiments, the expression of human or humanized IL12RB1 protein, human or humanized IL12RB2 protein, and / or human or humanized IL23R protein expressed in a non-human animal is controllable, such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0174] The non-human mammal can be any non-human animal known in the art and can be used in the methods described in this application. Preferably, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0175] Genetic, molecular, and behavioral analyses are performed on the non-human mammals described above. The present invention provides an offspring produced by mating with non-human mammals of the same genotype or other genotypes.

[0176] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its offspring. For example, a cell culture-based model can be prepared by the following method. The cell culture can be obtained by isolation from a non-human mammal, or cells can be obtained from a cell culture established using the same construct and standard cell transfection techniques. The integration of a genetic construct containing a DNA sequence encoding a human or humanized IL12RB1 protein, a human or humanized IL12RB2 protein, and / or a human or humanized IL23R protein can be detected by various methods.

[0177] There are many analytical methods available for detecting foreign DNA, including methods at the nucleic acid level (including using reverse transcription-polymerase chain reaction (RT-PCR), Southern Blot, and in situ hybridization) and methods at the protein level (including histochemical analysis, immunoblot analysis, and in vitro binding studies). In addition, the expression level of the target gene can be quantified by ELISA methods well-known to those skilled in the art. Many standard analytical methods can be used to complete quantitative detection. For example, RT-PCR and hybridization methods can be used to detect the transcription level, including ribonuclease protection assay, Southern Blot, and RNA dot blot analysis (RNA dot). Immunohistochemical staining, flow cytometry, and Western blot can also be used to detect the presence of human or humanized IL12RB1 protein, human or humanized IL12RB2 protein, and / or human or humanized IL23R protein.

[0178] In some embodiments, the genetically modified non-human animal (homozygous or heterozygous) described in the present application can express human or humanized IL12RB1 protein, human or humanized IL12RB2 protein, and / or human or humanized IL23R protein in one or more cells.

[0179] Method for constructing a genetically modified non-human animal

[0180] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting techniques using embryonic stem cells, homologous recombination techniques, CRISPR / Cas9 techniques, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases, or other molecular biology techniques. In some embodiments, homologous recombination techniques are preferably used. In some embodiments, CRISPR / Cas9 gene editing techniques can be used to construct genetically modified non-human animals. Many of these genome editing techniques are known in the art and are described in Yin et al., "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

[0181] In some embodiments, the nucleotide sequence encoding the endogenous IL12RB1 region in the endogenous genome of at least one cell of the non-human animal is replaced with the nucleotide sequence encoding the corresponding region of human IL12RB1. In some embodiments, the expression level of the endogenous IL12RB1 protein in the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts, or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0182] The present invention provides a targeting vector, which comprises a vector composed of a 5' homologous arm, a human or humanized IL12RB1 gene fragment, and a 3' homologous arm. This process involves using homologous recombination to introduce the human or humanized IL12RB1 sequence into the endogenous IL12RB1 locus of a non-human animal. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nuclease, TALEN, or CRISPR) can result in double-strand breaks in the DNA, and the human or humanized IL12RB1 sequence is replaced into the endogenous IL12RB1 locus of the non-human animal using homologous recombination.

[0183] Preferably, the non-human animal further comprises other gene modifications. More preferably, the other genes include at least one of IL12RB2, IL12A, IL12B, IL23R, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0184] Preferably, the human or humanized IL12RB1 gene and / or other genes are homozygous for the endogenous modified (preferably replaced or inserted) locus.

[0185] Preferably, the human or humanized IL12RB1 gene and / or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) locus.

[0186] Preferably, the non-human animal can be selected from any non-human animal that can be genetically edited to prepare a gene humanized animal, such as a rodent, a pig, a rabbit, a monkey, etc.

[0187] Preferably, the non-human animal is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0188] Therefore, the present invention provides a method for constructing a non-human animal with a humanized IL12RB1 gene, wherein the construction method enables the expression of a human or humanized IL12RB1 protein in the non-human animal, and / or, the construction method enables the genome of the non-human animal to contain a part of the human IL12RB1 gene or a humanized IL12RB1 gene.

[0189] In some embodiments, the method of modifying the IL12RB1 locus of a non-human animal to express a chimeric human / mouse IL12RB1 polypeptide may include replacing the nucleotide sequence encoding the endogenous IL12RB1 of the non-human animal at the endogenous IL12RB1 locus with a nucleotide sequence encoding all or part of the human IL12RB1 protein, thereby generating a nucleotide sequence encoding a chimeric human / non-human animal chimeric IL12RB1 sequence. In some embodiments, the method may include inserting a nucleotide sequence encoding a chimeric human / non-human animal chimeric IL12RB1 at the endogenous IL12RB1 locus of the non-human animal, thereby generating a nucleotide sequence encoding a chimeric human / non-human animal chimeric IL12RB1 sequence.

[0190] The present invention also provides a method for establishing a humanized animal model of the IL12RB1 gene, comprising the following steps:

[0191] (a) Providing cells (such as fertilized egg cells) based on the method described in the present application;

[0192] (b) Cultivate the cells (preferably cultivate the cells in a liquid culture medium);

[0193] (c) Transplant the cultivated cells into the oviduct or uterus of a recipient female non - human mammal, and allow the cells to develop in the uterus of the female non - human mammal;

[0194] (d) Identify germline transmission in the offspring of the genetically modified humanized non - human mammal of the pregnant female in step (c).

[0195] In some embodiments, the non - human mammal in the above - mentioned method is a mouse (such as a C57BL / 6 mouse).

[0196] In some embodiments, the non - human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0197] In some embodiments, the fertilized egg used in the above - mentioned method is a C57BL / 6 fertilized egg. Other fertilized eggs that can also be used in the method described in this application include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0198] The fertilized egg can be from any non - human animal, such as any non - human animal described in this application. In some embodiments, the fertilized egg cells are derived from rodents. The gene construct can introduce DNA into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non - human animal, and then the pseudopregnant non - human animal gives birth to a non - human mammal, thereby producing the non - human mammal mentioned in the above - mentioned method.

[0199] In some embodiments, the method for preparing a genetically modified non - human animal includes modifying the coding frame of the IL12RB1 gene of the non - human animal. For example, under the control of the endogenous regulatory elements of the non - human animal IL12RB1 gene, the nucleic acid sequence encoding the endogenous IL12RB1 region is replaced with a nucleotide sequence encoding the corresponding region of human IL12RB1 (such as a genomic DNA sequence, a CDS sequence, or a cDNA sequence). For example, one or more functional region sequences of the IL12RB1 gene of the non - human animal can be knocked out or inserted with sequences, such that the endogenous IL12RB1 protein of the non - human animal cannot be expressed or the expression level is reduced.

[0200] In some embodiments, a method of producing a genetically modified non-human animal includes inserting a nucleotide sequence encoding a human or humanized IL12RB1 protein and / or a helper sequence after the endogenous regulatory elements of the IL12RB1 gene in the non-human animal. In some embodiments, the helper sequence may be a stop codon such that the IL12RB1 gene humanized animal model can express the human or humanized IL12RB1 protein in vivo but not the IL12RB1 protein of the non-human animal. In some embodiments, the helper sequence includes WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0201] In some embodiments, a method for producing a genetically modified non-human animal includes:

[0202] (1) Providing a plasmid comprising a human IL12RB1 gene fragment flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target the endogenous IL12RB1 in the non-human animal;

[0203] (2) Providing one or more guide RNAs (sgRNAs) targeting the endogenous IL12RB1 gene in the non-human animal;

[0204] (3) Modifying the genome of a cell (e.g., a fertilized egg or an embryonic stem cell) by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0205] (4) Transferring the fertilized eggs obtained in step (3) into the oviduct of a pseudopregnant female mouse, or transferring the embryonic stem cells obtained in step (3) into a blastocyst, and then transferring the blastocyst into the oviduct of a pseudopregnant female mouse to produce offspring mice that functionally express the humanized IL12RB1 protein. Preferably, the method further includes:

[0206] (5) Mating the offspring mice obtained in step (4) to obtain homozygous mice.

[0207] In some embodiments, the fertilized eggs are modified by CRISPR with sgRNAs targeting the 5'-terminal target site and the 3'-terminal target site.

[0208] In some embodiments, the sequence encoding the humanized IL12RB1 protein is operably linked to the endogenous regulatory elements at the endogenous IL12RB1 locus in the non-human animal.

[0209] In some embodiments, the genetically modified non-human animal does not express the endogenous IL12RB1 protein.

[0210] In some embodiments, a method for producing a genetically modified non-human animal includes:

[0211] (1) Provide a plasmid containing a human or chimeric IL12RB1 gene fragment, the plasmid flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target the endogenous IL12RB1 of a non-human animal;

[0212] (2) Provide one or more guide RNAs (sgRNAs) targeting the endogenous IL12RB1 gene of a non-human animal;

[0213] (3) Modify the genome of a cell (e.g., a fertilized egg or an embryonic stem cell) by inserting the human or chimeric IL12RB1 gene fragment into the genome of the non-human animal.

[0214] In some embodiments, the nucleotide sequence encoding the endogenous IL12RB1 protein in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 1 in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding all or part of the extracellular region of the endogenous IL12RB1 protein in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding positions 20-565 of SEQ ID NO: 1 in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding all or part of the transmembrane region of the endogenous IL12RB1 protein in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding positions 566-570 of SEQ ID NO: 1 in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding all or part of the signal peptide of the endogenous IL12RB1 protein in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding positions 1-19 of SEQ ID NO: 1 in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding positions 1-570 of SEQ ID NO: 1 in the genome of the non-human animal is deleted. In some embodiments, all or part of exons 1-16 of the endogenous IL12RB1 gene in the genome of the non-human animal is deleted. In some embodiments, a partial deletion from exon 1 to exon 14 of the endogenous IL12RB1 gene in the genome of the non-human animal is deleted. In some embodiments, a partial deletion from the start codon of the endogenous IL12RB1 gene to exon 14 in the genome of the non-human animal is deleted.

[0215] In some embodiments, the construction method includes replacing the nucleotide sequence encoding the human IL12RB1 protein with the nucleotide sequence encoding the endogenous IL12RB1 protein in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 2 or 11 with the nucleotide sequence encoding SEQ ID NO: 1 in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence encoding the extracellular region of the human IL12RB1 protein with the nucleotide sequence encoding the extracellular region of the endogenous IL12RB1 protein in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence at positions 24-545 of SEQ ID NO: 2 with the nucleotide sequence at positions 20-565 of SEQ ID NO: 1 in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence encoding the signal peptide and extracellular region of the human IL12RB1 protein with the nucleotide sequence encoding the signal peptide and extracellular region of the endogenous IL12RB1 protein in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence at positions 1-545 of SEQ ID NO: 2 with the nucleotide sequence at positions 1-565 of SEQ ID NO: 1 in the non-human animal genome. In some embodiments, the construction method includes replacing all or part of the nucleotide sequence encoding the signal peptide, extracellular region, and transmembrane region of the human IL12RB1 protein with all or part of the nucleotide sequence encoding the signal peptide, extracellular region, and transmembrane region of the endogenous IL12RB1 protein in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence at positions 1-549 of SEQ ID NO: 2 with the nucleotide sequence at positions 1-570 of SEQ ID NO: 1 in the non-human animal genome. In some embodiments, the construction method includes replacing all or part of the human IL12RB1 gene with all or part of the endogenous IL12RB1 gene in the non-human animal genome. In some embodiments, the construction method includes replacing the part of exon 1 to the part of exon 14 of the human IL12RB1 gene with the part of exon 1 to the part of exon 14 of the endogenous IL12RB1 gene in the non-human animal genome. In some embodiments, the construction method includes replacing the start codon to the part of exon 14 of the human IL12RB1 gene with the start codon to the part of exon 14 of the endogenous IL12RB1 gene in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence shown at positions 18062249 to 18086823 of NCBI accession number NC_000019.10 with the nucleotide sequence encoding positions 1-570 of SEQ ID NO: 1 in the non-human animal genome.In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 1 in the genome of a non-human animal with SEQ ID NO: 10.

[0216] In some embodiments, the nucleotide sequence encoding endogenous IL12RB2 in the endogenous genome of at least one cell of a non-human animal is inserted or replaced with a nucleotide sequence encoding human or chimeric IL12RB2. In some embodiments, the expression level of the endogenous IL12RB2 protein in the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts, or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0217] Preferably, the non-human animal further comprises other gene modifications. More preferably, the other genes include at least one of IL12RB1, IL12A, IL12B, IL23R, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0218] The present invention provides a targeting vector. The targeting vector comprises a vector consisting of a 5' homologous arm, a human or humanized IL12RB2 sequence, and a 3' homologous arm. This process involves introducing a human or humanized IL12RB2 sequence into the endogenous IL12RB2 locus of a non-human animal using homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs, or CRISPR) can result in double-strand breaks in the DNA, and a human or humanized IL12RB2 sequence is introduced into the endogenous IL12RB2 sequence of the non-human animal using homologous recombination. In some embodiments, the introduction includes insertion or replacement.

[0219] The present invention also provides a method for establishing a humanized animal model of the IL12RB2 gene, comprising the following steps:

[0220] (a) Providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0221] (b) Culturing the cells (preferably culturing the cells in a liquid medium);

[0222] (c) Transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0223] (d) Identifying germline transmission in the offspring of the genetically modified humanized non-human mammals of the pregnant female in step (c).

[0224] In some embodiments, the non-human mammal in the above method is a mouse (such as a C57BL / 6 mouse).

[0225] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0226] In some embodiments, the fertilized egg used in the above method is a C57BL / 6 fertilized egg. Other fertilized eggs that can also be used in the method described in this application include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0227] The fertilized egg can be from any non-human animal, such as any non-human animal described in this application. In some embodiments, the fertilized egg cells are derived from rodents. The gene construct can introduce DNA into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human animal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0228] In some embodiments, the method for preparing a genetically modified non-human animal includes modifying the coding frame of the IL12RB2 gene of the non-human animal. For example, under the control of the endogenous regulatory elements of the non-human animal IL12RB2 gene, a nucleotide sequence encoding a human or chimeric IL12RB2 (such as a genomic DNA sequence, CDS sequence, or cDNA sequence) is introduced into the endogenous IL12RB2 gene locus of the non-human animal. For example, one or more functional region sequences of the IL12RB2 gene of the non-human animal can be knocked out or inserted with sequences, such that the endogenous IL12RB2 protein of the non-human animal cannot be expressed or the expression level is reduced.

[0229] In some embodiments, the method for preparing a genetically modified non-human animal includes inserting a nucleotide sequence encoding a human or humanized IL12RB2 protein and / or auxiliary sequences after the endogenous regulatory elements of the IL12RB2 gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, such that the IL12RB2 gene humanized animal model can express a human or humanized IL12RB2 protein in vivo, but does not express the IL12RB2 protein of the non-human animal. In some embodiments, the auxiliary sequences include WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0230] In some embodiments, the method for preparing a gene-modified non-human animal includes:

[0231] (1) Provide a plasmid containing a human or chimeric IL12RB2 gene fragment, the plasmid flanked by a 5'-homologous arm and a 3'-homologous arm, wherein the 5'-homologous arm and the 3'-homologous arm target the endogenous IL12RB2 of a non-human animal;

[0232] (2) Provide one or more guide RNAs (sgRNAs) targeting the endogenous IL12RB2 gene of a non-human animal;

[0233] (3) Modify the genome of a cell (such as a fertilized egg or embryonic stem cell) by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0234] (4) Transfer the fertilized eggs obtained in step (3) into the oviduct of a pseudopregnant female mouse, or transfer the embryonic stem cells obtained in step (3) into a blastocyst, and then transfer the blastocyst into the oviduct of a pseudopregnant female mouse to produce offspring mice that functionally express a humanized IL12RB2 protein. Preferably, the method further includes:

[0235] (5) Mate the offspring mice obtained in step (4) to obtain homozygous mice.

[0236] In some embodiments, the fertilized eggs are modified by CRISPR with sgRNAs targeting a 5'-terminal target site and a 3'-terminal target site.

[0237] In some embodiments, the sequence encoding the humanized IL12RB2 protein or the human or chimeric IL12RB2 gene fragment is operably linked to a non-human animal endogenous regulatory element.

[0238] In some embodiments, the genetically modified non-human animal does not express the endogenous IL12RB2 protein.

[0239] In some embodiments, the method for preparing a genetically modified non-human animal includes:

[0240] (1) Provide a plasmid containing a human or chimeric IL12RB2 gene fragment, the plasmid flanked by a 5'-homologous arm and a 3'-homologous arm, wherein the 5'-homologous arm and the 3'-homologous arm target the endogenous IL12RB2 of a non-human animal;

[0241] (2) Provide one or more guide RNAs (sgRNAs) targeting the endogenous IL12RB2 gene of a non-human animal;

[0242] (3) Modify the genome of a cell (such as a fertilized egg or embryonic stem cell) by inserting the human or chimeric IL12RB2 gene fragment into the genome of a non-human animal.

[0243] In some embodiments, the nucleotide sequence encoding the endogenous IL12RB2 protein in the non-human animal genome is deleted. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 12 in the non-human animal genome is deleted. In some embodiments, the nucleotide sequence encoding all or part of the extracellular region of the endogenous IL12RB2 protein in the non-human animal genome is deleted. In some embodiments, the nucleotide sequence encoding positions 24-637 of SEQ ID NO: 12 in the non-human animal genome is deleted. In some embodiments, the nucleotide sequence encoding all or part of the transmembrane region of the endogenous IL12RB2 protein in the non-human animal genome is deleted. In some embodiments, the nucleotide sequence encoding positions 638-655 or positions 638-658 of SEQ ID NO: 12 in the non-human animal genome is deleted. In some embodiments, the nucleotide sequence encoding all or part of the signal peptide of the endogenous IL12RB2 protein in the non-human animal genome is deleted. In some embodiments, the nucleotide sequence encoding positions 1-23 of SEQ ID NO: 12 in the non-human animal genome is deleted. In some embodiments, the nucleotide sequence encoding positions 1-655 of SEQ ID NO: 12 in the non-human animal genome is deleted. In some embodiments, all or part of exons 1-16 of the endogenous IL12RB2 gene in the non-human animal genome is deleted. In some embodiments, part of exon 2 to part of exon 14 of the endogenous IL12RB2 gene in the non-human animal genome is deleted. In some embodiments, the start codon of the endogenous IL12RB2 gene to part of exon 14 in the non-human animal genome is deleted.

[0244] In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding a human or chimeric IL12RB2 protein into or with the nucleotide sequence encoding the endogenous IL12RB2 protein in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding SEQ ID NO: 13, 21, or 56 into or with the nucleotide sequence encoding SEQ ID NO: 12 in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding the extracellular region of the human IL12RB2 protein into or with the nucleotide sequence encoding the extracellular region of the endogenous IL12RB2 protein in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding positions 24-622 of SEQ ID NO: 13 into or with the nucleotide sequence encoding positions 24-637 of SEQ ID NO: 12 in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding the signal peptide and extracellular region of the human IL12RB2 protein into or with the nucleotide sequence encoding the signal peptide and extracellular region of the endogenous IL12RB2 protein in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding positions 1-622 of SEQ ID NO: 13 into or with the nucleotide sequence encoding positions 1-637 of SEQ ID NO: 12 in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding all or part of the signal peptide, extracellular region, and transmembrane region of the human IL12RB2 protein into or with the nucleotide sequence encoding all or part of the signal peptide, extracellular region, and transmembrane region of the endogenous IL12RB2 protein in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding positions 1-640 of SEQ ID NO: 13 into or with the nucleotide sequence encoding positions 1-655 of SEQ ID NO: 12 in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing all or part of the human IL12RB2 gene into or with all or part of the endogenous IL12RB2 gene in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing the part of exon 2 to the part of exon 14 of the human IL12RB2 gene into or with the part of exon 2 to the part of exon 14 of the endogenous IL12RB2 gene in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing the start codon to the part of exon 14 of the human IL12RB2 gene into or with the start codon to the part of exon 14 of the endogenous IL12RB2 gene in the genome of a non-human animal.In some embodiments, the construction method includes inserting or replacing the nucleotide sequence shown at positions 67320369 to 67386643 of NCBI accession number NC_000001.11 with the nucleotide sequence encoding amino acids 1 to 655 of SEQ ID NO: 12 in the genome of a non-human animal. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence of SEQ ID NO: 20 or 55 with the nucleotide sequence encoding SEQ ID NO: 12 in the genome of a non-human animal. In some embodiments, the construction method includes inserting the chimeric CDS sequence of human and non-human animal IL12RB2 into the endogenous IL12RB2 locus of the non-human animal. In some embodiments, the construction method includes inserting the chimeric CDS sequence of human and non-human animal IL12RB2 into exons 1-16 of the endogenous IL12RB2 gene in the genome of a non-human animal, preferably into exon 2. In some embodiments, the construction method includes inserting the nucleotide sequence of chimeric IL12RB2 into exons 1-16 of the endogenous IL12RB2 gene in the genome of a non-human animal, preferably into exon 2. In some embodiments, the construction method includes inserting the nucleotide sequence encoding the chimeric IL12RB2 protein into exon 2 of the endogenous IL12RB2 gene in the genome of a non-human animal. In some embodiments, the construction method includes inserting the nucleotide sequence encoding amino acids 24 to 623 of SEQ ID NO: 13 and amino acids 639 to 874 of SEQ ID NO: 12 into exon 2 of the endogenous IL12RB2 gene in the genome of a non-human animal. In some embodiments, the construction method includes inserting the nucleotide sequence encoding amino acids 24 to 622 of SEQ ID NO: 13 and amino acids 638 to 874 of SEQ ID NO: 12 into exon 2 of the endogenous IL12RB2 gene in the genome of a non-human animal. In some embodiments, the construction method includes inserting the chimeric CDS sequence of human and non-human animal IL12RB2 and the 3'UTR and / or the nucleotide sequence downstream of the 3'UTR of the non-human animal IL12RB2 gene into exon 2 of the endogenous IL12RB2 gene in the genome of a non-human animal. In some embodiments, the construction method includes inserting the nucleotide sequence encoding amino acids 244 to 622 of SEQ ID NO: 13 and amino acids 638 to 874 of SEQ ID NO: 12 and the 3'UTR and the nucleotide sequence downstream of the 3'UTR of the non-human animal endogenous IL12RB2 gene into exon 2 of the endogenous IL12RB2 gene in the genome of a non-human animal.In some embodiments, the construction method includes inserting SEQ ID NO: 24, SEQ ID NO: 67, and SEQ ID NO: 68 into exon 2 of the endogenous IL12RB2 gene in the non-human animal genome, preferably after the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein in the non-human animal genome, more preferably between the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein and the nucleotide sequence encoding the extracellular region of the endogenous IL12RB2 protein in the non-human animal genome, even more preferably between the nucleotide sequences encoding positions 23 and 24 of SEQ ID NO: 12 in the non-human animal genome, and still more preferably between positions 257 - 258 of the sequence with NCBI accession number NM_008354.4 in the non-human animal genome.

[0245] In some embodiments, the nucleotide sequence encoding the endogenous IL23R region in the endogenous genome of at least one cell of the non-human animal is replaced with the nucleotide sequence encoding the human IL23R region. In some embodiments, the expression level of the endogenous IL23R protein in the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts, or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0246] The present invention provides a targeting vector. The targeting vector comprises a vector composed of a 5' homologous arm, a human or humanized IL23R gene fragment, and a 3' homologous arm. This process involves replacing the endogenous IL23R sequence with a human or humanized IL23R sequence using homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs, or CRISPRs) can result in double-strand breaks in the DNA, and the human or humanized IL23R sequence is replaced at the endogenous IL23R locus of the non-human animal using homologous recombination.

[0247] Therefore, in some embodiments, the method for preparing a gene-modified humanized animal includes replacing the nucleic acid sequence encoding the endogenous IL23R with the nucleotide sequence encoding the human or chimeric IL23R region at the endogenous IL23R locus (or site).

[0248] Preferably, the non-human animal further comprises other gene modifications. More preferably, the other genes include at least one of IL12RB1, IL12RB2, IL12A, IL12B, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0249] The present invention also provides a method for establishing a humanized animal model of the IL23R gene, comprising the following steps:

[0250] (a) Providing cells (such as fertilized egg cells) based on the method described in the present application;

[0251] (b) Culturing the cells (preferably culturing the cells in a liquid medium);

[0252] (c) Transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0253] (d) Identifying germline transmission in the offspring of the genetically modified humanized non-human mammals of the pregnant female in step (c).

[0254] In some embodiments, the non-human mammal in the above method is a mouse (such as a C57BL / 6 mouse).

[0255] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0256] In some embodiments, the fertilized egg used in the above method is a C57BL / 6 fertilized egg. Other fertilized eggs that can also be used in the method described in the present application include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0257] The fertilized egg can be from any non-human animal, such as any non-human animal described in the present application. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can introduce DNA into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human animal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0258] In some embodiments, the method for preparing a genetically modified non-human animal includes modifying the coding frame of the IL23R gene of the non-human animal. For example, under the control of the endogenous regulatory elements of the non-human animal IL23R gene, the nucleotide sequence encoding the endogenous IL23R is replaced with a nucleotide sequence encoding a human or chimeric IL23R (such as a genomic DNA sequence, a CDS sequence, or a cDNA sequence). For example, one or more functional region sequences of the IL23R gene of the non-human animal can be knocked out or inserted with sequences, such that the endogenous IL23R protein of the non-human animal cannot be expressed or the expression level is reduced.

[0259] In some embodiments, a method of producing a genetically modified non-human animal includes inserting a nucleotide sequence encoding a human or chimeric humanized IL23R protein and / or a helper sequence after the endogenous regulatory elements of the IL23R gene in the non-human animal. In some embodiments, the helper sequence can be a stop codon such that the IL23R gene humanized animal model can express a human or chimeric IL23R protein in vivo but not the IL23R protein of the non-human animal. In some embodiments, the helper sequence includes GSG, P2A, WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0260] In some embodiments, a method for producing a genetically modified non-human animal includes:

[0261] (1) Providing a plasmid containing a human or chimeric IL23R gene fragment, the plasmid flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target the endogenous IL23R of the non-human animal;

[0262] (2) Providing one or more guide RNAs (sgRNAs) targeting the endogenous IL23R gene of the non-human animal;

[0263] (3) Modifying the genome of a cell (e.g., a fertilized egg or an embryonic stem cell) by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0264] (4) Transferring the fertilized egg obtained in step (3) into the oviduct of a pseudopregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudopregnant female mouse to produce offspring mice that functionally express the humanized IL23R protein. Preferably, the method further includes:

[0265] (5) Mating the offspring mice obtained in step (4) to obtain homozygous mice.

[0266] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal target site and a 3'-target site.

[0267] In some embodiments, the sequence encoding the humanized IL23R protein is operably linked to the endogenous regulatory elements at the endogenous IL23R locus of the non-human animal.

[0268] In some embodiments, the genetically modified non-human animal does not express the endogenous IL23R protein.

[0269] In some embodiments, a method for producing a genetically modified non-human animal includes:

[0270] (1) Provide a plasmid containing a human or chimeric IL23R gene fragment, the plasmid flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target endogenous IL23R;

[0271] (2) Provide one or more guide RNAs (sgRNAs) targeting the endogenous IL23R gene of a non-human animal;

[0272] (3) Modify the genome of a cell (e.g., a fertilized egg or an embryonic stem cell) by inserting the human or chimeric IL23R gene fragment into the genome of the non-human animal.

[0273] In some embodiments, the nucleotide sequence encoding the endogenous IL23R protein in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 27 in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding all or part of the extracellular region of the endogenous IL23R protein in the genome of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding positions 24-122 of SEQ ID NO: 27 or positions 33-122 of SEQ ID NO: 27 in the genome of the non-human animal is deleted. In some embodiments, all or part of exons 1-11 of the endogenous IL23R gene in the genome of the non-human animal is deleted. In some embodiments, all or part of exon 3 of the endogenous IL23R gene in the genome of the non-human animal is deleted. In some embodiments, part of exon 3 to all or part of intron 3 of the endogenous IL23R gene in the genome of the non-human animal is deleted.

[0274] In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding the human IL23R protein with the nucleotide sequence encoding the endogenous IL23R protein in the non-human animal genome. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding SEQ ID NO: 59 or 78 with the nucleotide sequence encoding SEQ ID NO: 27 in the non-human animal genome. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding all or part of the extracellular region of the endogenous IL23R in the non-human animal genome with the chimeric CDS sequence of human and non-human animal IL23R. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding all or part of the extracellular region of the endogenous IL23R in the non-human animal genome with the chimeric CDS sequence of human and non-human animal IL23R and the 3'UTR and / or the nucleotide sequence downstream of the 3'UTR of the non-human animal IL23R. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence encoding positions 1-26 of SEQ ID NO: 27, positions 27-353 of SEQ ID NO: 59, and positions 373-644 of SEQ ID NO: 27 with the nucleotide sequence encoding positions 33-122 or positions 24-122 of SEQ ID NO: 27 in the non-human animal genome. In some embodiments, the construction method includes inserting or replacing the nucleotide sequence of SEQ ID NO: 69 or SEQ ID NO: 71 with the nucleotide sequence encoding positions 33-122 or positions 24-122 of SEQ ID NO: 27 in the non-human animal genome.

[0275] Application of Genetically Modified Non-Human Animals

[0276] Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence or inserting a homologous or orthologous human gene or human sequence into a non-human animal at the endogenous locus of the non-human animal and under the control of endogenous regulatory elements of the non-human animal (such as promoters, 5'UTRs, and / or 3'UTRs) can produce non-human animals with qualities and characteristics that may be significantly different from those of typical knockout plus transgenic animals. In typical knockout plus transgenic animals, the endogenous locus is removed or disrupted, and a fully human transgene is inserted into the genome of the non-human animal and may integrate randomly into the genome. Generally, the location of the integrated transgene is unknown; the expression of human proteins is measured by transcription of human genes and / or protein assays and / or functional assays.

[0277] Genetically modified non-human animals that express human or humanized IL12RB1, IL12RB2, and / or IL23R proteins, for example, in a physiologically appropriate manner, have a variety of uses, including but not limited to developing therapeutic methods for human diseases and disorders and evaluating the toxicity and / or efficacy of these human therapeutic methods in animal models.

[0278] The present invention also provides the use of the above-mentioned non-human animals genetically modified with IL12RB1, IL12RB2, and / or IL23R genes and the non-human animals obtained by any of the above construction methods.

[0279] In some embodiments, the use includes:

[0280] A) Use in the development of products related to immune processes associated with IL12RB1, IL12RB2, and / or IL23R involving human cells;

[0281] B) Use as a model system related to IL12RB1, IL12RB2, and / or IL23R in pharmacological, immunological, microbiological, and medical research;

[0282] C) Use related to the production and utilization of animal experimental disease models for etiological research related to IL12RB1, IL12RB2, and / or IL23R and / or for developing diagnostic strategies and / or for developing therapeutic strategies;

[0283] D) Use in the in vivo screening, pharmacodynamic detection, efficacy evaluation, verification, or assessment of regulators of the human IL12RB1, IL12RB2, and / or IL23R signaling pathway; or,

[0284] E) Use in studying the gene functions of IL12RB1, IL12RB2, and / or IL23R, studying the drugs and pharmacodynamics targeting human IL12RB1, IL12RB2, and / or IL23R target sites, and studying therapeutic drugs for tumors, inflammations, or immune diseases related to IL12RB1, IL12RB2, and / or IL23R.

[0285] The present invention provides a non-human animal that expresses human or humanized IL12RB1, IL12RB2, and / or IL23R proteins, which can be used for screening human IL12RB1, IL12RB2, and / or IL23R-specific therapeutic agents. The therapeutic agents can reduce or block the interaction between IL12RB1, IL12RB2, and / or IL23R receptor complexes. In some embodiments, the non-human animal can be used to test whether a therapeutic agent can increase or decrease the immune response and / or determine whether the therapeutic agent is an agonist or antagonist of IL12RB1, IL12RB2, and / or IL23R. In some embodiments, the non-human animal is a human disease animal model. For example, the disease is genetically induced (knock-in or knock-out). In different embodiments, the genetically modified non-human animal also comprises a damaged immune system, such as human-derived tissue xenotransplantation that has been genetically modified, including human solid tumors (e.g., breast cancer) or hematological tumors (e.g., lymphocytic tumors (including B or T cell tumors)).

[0286] In some embodiments, the genetically modified non-human animal can be used to determine the effectiveness of therapeutic agents (such as antibodies targeting IL12RB1, IL12RB2, and / or IL23R, nucleic acid drugs targeting IL12RB1, IL12RB2, and / or IL23R, and / or polypeptide drugs) in the treatment of various immune diseases. In some embodiments, the immune diseases include but are not limited to GVHD (graft-versus-host disease), psoriasis, allergy, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic steatohepatitis), systemic lupus erythematosus, rheumatoid arthritis, atopic dermatitis, multiple sclerosis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders, etc. In some embodiments, the immune disease is psoriasis, atopic dermatitis, asthma, rheumatoid arthritis, or multiple sclerosis.

[0287] In some embodiments, the genetically modified non-human animal can be used to determine the effectiveness of therapeutic agents (such as antibodies targeting IL12RB1, IL12RB2, and / or IL23R, nucleic acid drugs targeting IL12RB1, IL12RB2, and / or IL23R, and / or polypeptide drugs) in the treatment of tumors.

[0288] In some embodiments, the method administers a therapeutic agent to a non-human animal, wherein the non-human animal has cancer or a tumor; and determines the inhibitory effect of the therapeutic agent on the cancer or tumor. The inhibitory effects that can be determined include, for example, a reduction in tumor size or tumor volume, a reduction in tumor growth, a decrease in the rate of increase in tumor volume in a subject (e.g., compared to the rate of increase in tumor volume in the same subject before treatment or in another subject not treated with such treatment), a reduction in the risk of metastasis or the risk of developing one or more additional metastases, an increase in survival rate, and an extension of life expectancy. The tumor volume of the subject can be determined by various methods, such as by direct measurement, MRI, or CT. In some embodiments, the therapeutic agent can directly target cells expressing IL12RB1, IL12RB2, and / or IL23R.

[0289] In some embodiments, the tumor comprises one or more cancer cells (e.g., human or murine cancer cells) injected into the non-human animal. In some embodiments, the therapeutic agent activates or inhibits the IL12RB1, IL12RB2, and / or IL23R signaling pathway. In some embodiments, the therapeutic agent does not activate or does not inhibit the IL12RB1, IL12RB2, and / or IL23R signaling pathway.

[0290] In some embodiments, genetically modified non-human animals can be used to determine whether a therapeutic agent (e.g., an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug) is an agonist or antagonist of IL12RB1, IL12RB2, and / or IL23R. In some embodiments, the methods described in the present application are also designed to determine the effect of a therapeutic agent (e.g., an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug) on IL12RB1, IL12RB2, and / or IL23R, e.g., whether the therapeutic agent can upregulate or downregulate the immune response, and / or whether the agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cell cytotoxicity (ADCC). In some embodiments, genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent to treat a disease in a subject, such as cancer, inflammation, or an immune disease.

[0291] In some embodiments, the inhibitory effect on the tumor can also be determined by methods known in the art, such as measuring the tumor volume in a non-human animal and / or determining the tumor (volume) growth inhibition rate (TGI TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%) = (1 – T Vt / T Vc)×100 is calculated, where T Vt and T Vc are the average tumor volumes (or weights) of the treatment group and the control group.

[0292] In some embodiments, therapeutic agents (e.g., antibodies targeting IL12RB1, IL12RB2, and / or IL23R, nucleic acid drugs targeting IL12RB1, IL12RB2, and / or IL23R, and / or polypeptide drugs) are designed for the treatment of various cancers (tumors). As used herein, the terms “cancer” or “tumor” refer to cells having the ability to grow autonomously, i.e., an abnormal state or condition characterized by the growth of rapidly proliferating cells. The term is intended to include all types of cancerous growths or carcinogenic processes, metastatic tissues, or malignant transformed cells, tissues, or organs, without regard to histopathologic type or stage of invasiveness. The term “tumor” as used in this application refers to cancer cells, e.g., a mass of cancer cells. Cancers that can be treated or diagnosed using the methods described in this application include malignancies of various organ systems, such as malignancies affecting the lung, breast, thyroid, lymph, gastrointestinal, and genitourinary tracts, as well as adenocarcinomas, which include malignancies such as colon cancer, renal cell carcinoma, prostate cancer, and / or testicular tumors, lung cancer (e.g., non-small cell lung cancer), or cancers. In some embodiments, the therapeutic agents described in this application are designed for the treatment or diagnosis of cancer in a subject. The term “carcinoma” is well recognized and refers to a malignancy of epithelial or endocrine tissues, including carcinomas of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary carcinomas include carcinomas formed from cervical, lung, prostate, breast, head and neck, colon, and ovarian tissues. The term also includes carcinosarcomas, e.g., malignancies composed of carcinoma tissue and sarcoma tissue. “Adenocarcinoma” refers to a cancer that originates from glandular tissue or in which tumor cells form recognizable glandular structures. The term “sarcoma” is well recognized and refers to a malignancy of mesenchymal origin. In some embodiments, the cancers described in this application are lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, cancer, bladder cancer, glioma, cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. In some embodiments, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia. In some embodiments, the lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, and T-cell lymphoma, as well as Waldenstrom macroglobulinemia.In some embodiments, the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In a specific embodiment, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer, or cancer. In some embodiments, the tumor includes solid tumors or hematological tumors. In some embodiments, the cancer includes breast cancer, lymphocyte tumors, colorectal cancer, hepatobiliary cancer, head and neck cancer, liver cancer, or lung cancer.

[0293] In some embodiments, the therapeutic agent (e.g., an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug) is designed to treat various immune diseases, including rheumatoid arthritis, Crohn's disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease (IBD), ulcerative colitis, or scleroderma. Thus, the method described in the present application can be used to determine the effectiveness of a therapeutic agent (e.g., an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug) in inhibiting an immune response. In some embodiments, the immune diseases described in the present application are graft-versus-host disease (GVHD), psoriasis, allergy, asthma, myocarditis, nephritis, hepatitis, atopic dermatitis, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological diseases, etc. In some embodiments, the therapeutic agent (e.g., an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug) is designed to treat various inflammations, such as viral inflammations. In some embodiments, the inflammations described in the present application include acute inflammation and chronic inflammation. Specifically, the inflammation includes, but is not limited to, degenerative inflammation, exudative inflammation (e.g., serous inflammation, fibrin inflammation, suppurative inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (such as tuberculosis, syphilis, leprosy, or lymphogranuloma). In some embodiments, the inflammations described in the present application include infections, and the infection refers to a local tissue and systemic inflammatory response caused by bacteria, viruses, fungi, parasites, and / or other pathogens invading the human body. In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0294] The present invention also provides a method for detecting the toxicity of a therapeutic agent (such as an antibody targeting IL12RB1, IL12RB2, and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2, and / or IL23R, and / or a polypeptide drug). The method includes administering the therapeutic agent to the non-human animal described above and evaluating the weight change of the non-human animal or performing a blood test. In some embodiments, the blood test includes, but is not limited to, red blood cell count, hematocrit, and / or hemoglobin content. In some embodiments, the antibody can reduce red blood cells (RBCs), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% less than that of the control group (such as the average weight of non-human animals not treated with the therapeutic agent).

[0295] The present invention also provides an animal model constructed by the method described in the present application for developing products related to the human cellular immune process, manufacturing human antibodies, or for use in model systems for pharmacological, immunological, microbiological, and medical research.

[0296] In some embodiments, there is provided an animal model generated by the method described in the present application for use in animal experimental disease models of the immune process of human cells, studying pathogens, or formulating new diagnostic strategies and / or treatment strategies.

[0297] The present invention also provides an animal model generated by the method described in the present application for screening, validating, evaluating, or studying the gene functions of IL12RB1, IL12RB2, and / or IL23R, human antibodies against IL12RB1, IL12RB2, and / or IL23R, or therapeutic drugs or efficacy for diseases related to the target sites of human IL12RB1, IL12RB2, and / or IL23R (such as tumors, inflammation, or immune diseases).

[0298] In some embodiments, the present application provides a method for verifying the in vivo efficacy of TCR-T, CAR-T, and / or other immunotherapies (e.g., adoptive T cell transfer therapy). For example, the method includes transplanting human tumor cells into the non-human animals described in the present application, and applying human CAR-T to the non-human animals with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animals are selected from IL12RB1, IL12RB2, and / or IL23R gene humanized non-human animals prepared by the construction method, IL12RB1, IL12RB2, and / or IL23R gene humanized non-human animals described in the present application, double-gene or multi-gene humanized non-human animals (or their offspring) generated by the method described in the present application, non-human animals expressing human or humanized IL12RB1, IL12RB2, and / or IL23R proteins, or tumor- or inflammation-bearing animal models described in the present application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies can treat IL12RB1, IL12RB2, and / or IL23R-related diseases (e.g., tumors, inflammation, or immune diseases) described in the present application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies provide an evaluation method for treating IL12RB1, IL12RB2, and / or IL23R-related diseases (e.g., tumors, inflammation, or immune diseases) described in the present application.

[0299] Non-human animal models of two or more human or chimeric genes

[0300] The present invention also provides an animal model or non-human animal having two or more human or chimeric genes. The non-human animal or animal model may include a human or chimeric IL12RB1 gene, a human or chimeric IL12RB2 gene, and / or a human or chimeric IL23R gene and a sequence encoding an additional human or chimeric protein.

[0301] In some embodiments, the additional human or chimeric protein includes at least one of IL12A, IL12B, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0302] In some embodiments, the non-human animal also expresses at least one of human or humanized IL12A, IL12B, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 proteins.

[0303] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, and the construction method includes:

[0304] (1) Providing a non-human animal obtained by the above construction method;

[0305] (2) Mating the non-human animal provided in step (1) with other genetically modified non-human animals, performing in vitro fertilization, or directly performing gene editing, and screening to obtain a multi-gene modified non-human animal.

[0306] In some embodiments, the other genetically modified non-human animals include non-human animals in which one or a combination of two or more of the genes IL12A, IL12B, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4 are humanized.

[0307] In some embodiments, gene humanization is directly performed on a non-human animal having at least one modification of the human or chimeric IL12A, IL12B, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4 gene.

[0308] Since these proteins may be involved in different mechanisms, combination therapies targeting two or more of these proteins may be a more effective treatment. In fact, many related clinical trials are underway and showing promising results. Multigene-modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, such as antibodies targeting IL12RB1, IL12RB2, and / or IL23R, nucleic acid drugs and / or polypeptide drugs targeting IL12RB1, IL12RB2, and / or IL23R, and additional therapeutic agents for treating diseases (e.g., tumors, inflammation, or immune diseases). The method includes administering a therapeutic agent and an additional therapeutic agent to a non-human animal, wherein the non-human animal has a disease (e.g., a tumor, inflammation, or immune disease), and determining the effect of the combination treatment on the disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to IL12A, IL12B, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4, or a nucleic acid drug and / or polypeptide drug targeting the above targets. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), or an anti-PD-L1 antibody. In some embodiments, the above-mentioned non-human animal further includes a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the above-mentioned tumor includes one or more tumor cells expressing PD-L1 and / or PD-L2.

[0309] In some embodiments, the combination treatment method is used to treat various cancers (tumors). In some embodiments, the combination treatment is designed to treat the immune diseases described in the present application, such as psoriasis. In some embodiments, the method described in the present application can be used to evaluate combination treatments with some other methods. Methods of treating cancer that can be used alone or in combination with the method described in the present application include, for example, treating a subject with chemotherapy, such as camptothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, bisantrene, nitrosourea, dactinomycin, daunorubicin, bleomycin, plicamycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, carboplatin, 5-fluorouracil, vincristine, vinblastine, and / or methotrexate. Alternatively, in addition, the method may include performing surgery on the subject to remove at least a part of the cancer, such as removing a part or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0310] Figure 1 : Schematic diagram of the comparison between the murine IL12RB1 locus and the human IL12RB1 locus (not to scale);

[0311] Figure 2 : Schematic diagram of the IL12RB1 gene targeting strategy and the design of targeting vector V1 (not to scale);

[0312] Figure 3 : PCR identification results of the F1 generation of humanized IL12RB1 gene mice. Among them, WT is the wild-type control, H2O is the water control, and M is the Marker;

[0313] Figure 4 : RT-PCR detection results. Among them, + / + is the wild-type C57BL / 6 mouse, H / + is the humanized heterozygous IL12RB1 gene mouse, H2O is the water control, GAPDH is the internal reference, and M is the Marker;

[0314] Figure 5 : Schematic diagram of the comparison between the murine IL12RB2 locus and the human IL12RB2 locus (not to scale);

[0315] Figure 6 : Schematic diagram of the IL12RB2 gene targeting strategy and the design of targeting vector V2 (not to scale);

[0316] Figure 7 : Schematic diagram of the IL12RB2 gene targeting strategy and the design of targeting vector V3 (not to scale);

[0317] Figure 8 : Southern blot identification results of the F1 generation of humanized IL12RB2 gene mice (V1). Among them, WT is the wild-type control;

[0318] Figure 9 : RT-PCR detection results. Among them, + / + is the wild-type C57BL / 6 mouse, H / + is the humanized heterozygous IL12RB2 gene mouse, H2O is the water control, and GAPDH is the internal reference;

[0319] Figure 10 : Schematic diagram of the IL12RB2 gene targeting strategy and the design of targeting vector V4 (not to scale);

[0320] Figure 11 : Southern blot identification results of the F1 generation of humanized IL12RB2 gene mice (V2). Among them, WT is the wild-type control;

[0321] Figure 12: RT-PCR test results, where + / + are wild-type C57BL / 6 mice, H / + are IL12RB2 gene humanized heterozygous mice, H2O is the water control, GAPDH is the internal reference, and M is the Marker;

[0322] Figure 13 : Schematic diagram of the comparison between the mouse IL23R locus and the human IL23R locus (not to scale);

[0323] Figure 14 : Schematic diagram of the IL23R gene targeting strategy and the design of the targeting vector V5 (not to scale);

[0324] Figure 15 : Schematic diagram of the IL23R gene targeting strategy and the design of the targeting vector V6 (not to scale);

[0325] Figure 16 : PCR identification results of the F1 generation of IL23R gene humanized mice. Among them, PC is the positive heterozygous control, WT is the wild-type control, H2O is the water control, and M is the Marker;

[0326] Figure 17 : ELISA test results, where + / + are wild-type C57BL / 6 mice, H / +; H / + are IL12RB1 / IL12RB2 double-gene humanized heterozygous mice;

[0327] Figure 18 : ELISA test results, where + / + are wild-type C57BL / 6 mice, IL12RB1 / IL12RB2 are IL12RB1 / IL12RB2 double-gene humanized homozygous mice, and IL12RB1 / IL12RB2 plus are IL12RB1 / IL12RB2plus double-gene humanized homozygous mice;

[0328] Figure 19 : ELISA test results, where + / + are wild-type C57BL / 6 mice, and H / H are PD-1 / IL12RB1 / IL12RB2 multi-gene humanized homozygous mice;

[0329] Figure 20 : RT-PCR test results, where Figure 20 A is the test result of IL12RB1, Figure 20 B is the test result of IL23R. + / + are wild-type C57BL / 6 mice, H / H are homozygous IL23R / IL12RB1 double-gene humanized mice, H2O is the water control, and GAPDH is the internal reference;

[0330] Figure 21: Mouse colon cancer cells MC38 were implanted into homozygous mice of IL12RB1 / IL12RB2 double - gene humanized mice, and anti - tumor efficacy tests were carried out using recombinant human IL12 (hIL12). Among them Figure 21 A is the tumor volume of the experimental animals, Figure 21 B is the body weight of the experimental animals;

[0331] Figure 22 : Changes in mouse body weight and the ratios of liver and spleen to body weight;

[0332] Figure 23 : Results of mouse serum tests. Among them Figure 23 (A) is the concentration of mIFNγ in the serum; Figure 23 (B) and Figure 23 (C) are the levels of ALT and AST in mouse serum respectively;

[0333] Figure 24 : Changes in body weight and the ratios of liver and spleen to body weight of IL12RB1 / IL12RB2 mice and IL12RB1 / IL12RB2 plus double - gene humanized mice. Among them Figure 24 (A) is the change in body weight (%); Figure 24 (B) and Figure 24 (C) are the liver / body weight (%) and spleen / body weight (%) respectively;

[0334] Figure 25 : Results of serum tests of IL12RB1 / IL12RB2 mice and IL12RB1 / IL12RB2 plus double - gene humanized mice. Among them Figure 25 (A) is the concentration of mIFNγ in the serum; Figure 25 (B) and Figure 25 (C) are the levels of ALT and AST in mouse serum respectively.

[0335] Figure 26 : Detection results of the concentration of mIFNγ in the serum of IL12RB1 / IL12RB2 mice;

[0336] Figure 27 : Changes in the body weight of IL12RB1 / IL12RB2 mice;

[0337] Figure 28 : Ratios of liver and spleen to body weight and liver function results of IL12RB1 / IL12RB2 mice. Among them Figure 28 A is the ratio of liver weight to body weight, Figure 28 B is the ratio of spleen weight to body weight, Figure 28 C is the content of AST in the serum, Figure 28 D is the content of ALT in the serum. AST is alanine aminotransferase, and ALT is aspartate aminotransferase;

[0338] Figure 29 : IL17A ELISA test results;

[0339] In each of the figures, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Detailed implementation manners

[0340] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

[0341] Materials and methods

[0342] In each of the following embodiments, the equipment and materials were obtained from the several companies indicated below:

[0343] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center of the National Institutes for Food and Drug Control, China;

[0344] PD-1 humanized mice were from Beijing Biocytogen Co., Ltd.;

[0345] PshAI enzyme was purchased from NEB, catalog number R0593S;

[0346] Eco53KI enzyme was purchased from NEB, catalog number R0116S;

[0347] In VivoMAb anti-mouse CD3 was purchased from BioCell, catalog number BP0001-1;

[0348] In VivoMAb anti-mouse CD28 was purchased from BioCell, catalog number BE0015-1;

[0349] ELISA MAX TM Deluxe Set Mouse IFN-γ was purchased from Biolegend, catalog number 430804;

[0350] Mouse IL-12, research grade was purchased from Miltenyi Biotec, catalog number 130-096-708;

[0351] Human IL-12, premium grade was purchased from Miltenyi Biotec, with the product number 130-096-705.

[0352] Example 1 Construction of Humanized Mice with IL12RB1 Gene

[0353] The comparison schematic diagram of the mouse IL12RB1 gene (NCBI Gene ID: 16161, located at positions 71261005 to 71276186 of NC_000074.7 on chromosome 8, based on transcript NM_008353.2 and its encoded protein NP_032379.2 (SEQ ID NO: 1)) and the human IL12RB1 gene (NCBI Gene ID: 3594, located at positions 18058994 to 18099027 of NC_000019.10 on chromosome 19, based on transcript NM_005535.3 and its encoded protein NP_005526.1 (SEQ ID NO: 2)) is as Figure 1 shown.

[0354] To achieve the object of the present invention, a nucleotide sequence encoding the human IL12RB1 protein can be introduced into the mouse endogenous IL12RB1 gene locus, so that the mouse expresses the human or humanized IL12RB1 protein. Specifically, using gene editing technology, under the control of the mouse IL12RB1 gene regulatory element, about 24.6 kb of the partial sequence from exon 1 to exon 14 of the human IL12RB1 gene is used to replace about 10.8 kb of the partial sequence from exon 1 to exon 14 of the mouse exon 1 to obtain a humanized IL12RB1 gene locus, realizing the humanization transformation of the mouse IL12RB1 gene.

[0355] To implement the present invention, a targeting vector V1 ( Figure 2 ) was constructed. The targeting vector V1 contains homologous arm sequences upstream and downstream of the mouse IL12RB1 gene, and fragment A containing the human IL12RB1 gene fragment. Among them, the upstream 5' homologous arm sequence (SEQ ID NO: 3) is the same as the nucleotide sequence at positions 71257469 to 71261195 of NCBI accession number NC_000074.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 4) is the same as the nucleotide sequence at positions 71271998 to 71274804 of NCBI accession number NC_000074.7. The nucleotide sequence of the human IL12RB1 gene fragment is the same as the nucleotide sequence at positions 18062249 to 18086823 of NCBI accession number NC_000019.10; the connection design of the upstream of the human IL12RB1 fragment sequence and the mouse sequence is: wherein the last "T" in the sequence " CTCCT " is the last nucleotide at the upstream junction of the murine sequence and the human IL12RB1 fragment sequence, and the "C" in the sequence is the first nucleotide of the human IL12RB1 fragment sequence. The connection of the downstream of the human IL12RB1 fragment sequence with the murine sequence is designed as: wherein the last "C" in the sequence " TCTTC " is the last nucleotide of the human IL12RB1 fragment sequence, and the first "T" in the sequence is the first nucleotide at the downstream junction of the murine sequence and the human IL12RB1 fragment sequence.

[0358] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection of the 5'-end of the Neo cassette with the human IL12RB1 gene is designed as: wherein the "G" in the sequence " CACAG " is the last nucleotide at the junction of the human IL12RB1 gene and the 5'-end of the Neo cassette, and the "G" in the sequence is the first nucleotide of the Neo cassette; the connection of the 3'-end of the Neo cassette with the human IL12RB1 gene is designed as: wherein the last "C" in the sequence " CCACC " is the last nucleotide of the Neo cassette, and the first "C" in the sequence is the first nucleotide at the junction of the human IL12RB1 gene and the 3'-end of the Neo cassette. The mRNA sequence transcribed from the IL12RB1 gene in the engineered humanized mouse is as shown in SEQ ID NO: 10, and the protein sequence expressed is as shown in SEQ ID NO: 11.

[0361] The construction of the targeting vector can be carried out by conventional methods, such as digestion and ligation. After the constructed targeting vector is preliminarily verified by digestion, it is sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into the embryonic stem cells of C57BL / 6 mice by electroporation, and the obtained cells are screened using the positive clone screening marker gene to screen out the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the isolated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient female mouse (white mouse) to produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are interbred to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene, and then the IL12RB1 gene humanized homozygous mice can be obtained by interbreeding.

[0362] The genotypes of the somatic cells of F1 generation mice can be identified by PCR method, and the primers in Table 7 are used for detection. The exemplary results are as Figure 3 shown. Combining the PCR and sequencing results, 12 mice numbered from F1-1 to F1-12 are positive mice. This indicates that the IL12RB1 gene humanized mice constructed by this method can be stably passaged and have no random insertion.

[0363] Table 7 PCR primer sequences and target fragment sizes

[0364]

[0365] The expression of mRNA in the IL12RB1 gene humanized mice can be detected by RT-PCR. Specifically, one 7-week-old C57BL / 6 mouse (+ / +) and one 7-week-old male IL12RB1 gene humanized heterozygous mouse (H / +) prepared in this example are selected respectively. After euthanasia by cervical dislocation, thymus and spleen tissues are taken, and RT-PCR detection is carried out using the primer sequences shown in Table 8. The detection results are as Figure 4 shown. As can be seen from Figure 4 , only mouse IL12RB1 mRNA is detected in the wild-type C57BL / 6 mice, and no humanized IL12RB1 mRNA is detected; in the IL12RB1 gene humanized heterozygous mice, not only mouse IL12RB1 mRNA but also humanized IL12RB1 mRNA can be detected.

[0366] Table 8 RT-PCR primer sequences and target fragment sizes

[0367]

[0368] Example 2 Construction of Humanized IL12RB2 Gene Mice (V1)

[0369] The comparison schematic diagram of mouse IL12RB2 gene (NCBI Gene ID: 16162, located at positions 67263914 to 67353277 of NC_000072.7 on chromosome 6, based on transcript NM_008354.4 and its encoded protein NP_032380.1 (SEQ ID NO: 12)) and human IL12RB2 gene (NCBI Gene ID: 3595, located at positions 67307351 to 67398724 of NC_000001.11 on chromosome 1, based on transcript NM_001559.3 and its encoded protein NP_001550.1 (SEQ ID NO: 13)) is as Figure 5 shown.

[0370] To achieve the purpose of the present invention, a nucleotide sequence encoding a human or chimeric IL12RB2 protein can be introduced into the mouse endogenous IL12RB2 gene locus, so that the mouse expresses a human or humanized IL12RB2 protein. Specifically, using gene editing technology, under the control of the mouse IL12RB2 gene regulatory element, a human-mouse chimeric CDS sequence is inserted into exon 2 of the mouse IL12RB2 gene. In the human-mouse chimeric CDS sequence, the human part contains the partial sequence from exon 2 to exon 14 of the IL12RB2 gene, and the mouse part contains the partial sequence of exon 14 and the entire sequences of exons 15-16 and the partial nucleotide sequence downstream of the 3'UTR, to obtain a humanized IL12RB2 gene locus and achieve the humanization transformation of the mouse IL12RB2 gene.

[0371] To implement the present invention, a targeting vector V2 was constructed ( Figure 6) The targeting vector V2 contains homologous arm sequences upstream and downstream of the mouse IL12RB2 gene, as well as fragment A1 containing a human IL12RB2 gene fragment. Among them, the upstream 5' homologous arm sequence (SEQ ID NO: 14) is identical to the nucleotide sequence from position 67338866 to 67343072 of NCBI accession number NC_000072.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 15) has a 99% identity with the nucleotide sequence from position 67334870 to 67338865 of NCBI accession number NC_000072.7, where the base G at position 67338729 is mutated to base C. Fragment A1 sequentially contains a human IL12RB2 fragment, a mouse IL12RB2 fragment, and a STOP sequence from the 5' end to the 3' end; among them, the nucleotide sequence of the human IL12RB2 fragment (SEQ ID NO: 24) is identical to the nucleotide sequence from position 201 to 2000 of NM_001559.3; the connection design of the upstream of the human IL12RB2 fragment sequence and the mouse sequence is as follows: Among them, the last "A" in the sequence " AAGCA " is the last nucleotide at the junction of the mouse sequence and the upstream of the human IL12RB2 fragment sequence, and the first "A" in the sequence is the first nucleotide of the human IL12RB2 fragment sequence. The connection design of the downstream of the human IL12RB2 fragment sequence and the mouse sequence is as follows: Among them, the last "G" in the sequence " ATTGG " is the last nucleotide of the human IL12RB2 fragment sequence, and the first "A" in the sequence is the first nucleotide at the junction of the mouse sequence and the downstream of the human IL12RB2 fragment sequence. The nucleotide sequence of the mouse IL12RB2 fragment in fragment A1 is SEQ ID NO: 67, and the STOP sequence is SEQ ID NO: 68.

[0374] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. Among them, the connection design of the 5' end of the Neo cassette and STOP is as follows: Among them, the last "T" in the sequence " TTAAT " is the last nucleotide of the STOP sequence, and the sequence The first "G" in it is the first nucleotide of the Neo cassette; the connection of the 3' end of the Neo cassette to the mouse IL12RB2 gene is designed as: wherein the last "C" in the sequence " ACTTC " is the last nucleotide of the Neo cassette, and the first "A" in the sequence is the first nucleotide at the junction of the mouse IL12RB2 gene and the 3' end of the Neo cassette. The mRNA sequence transcribed from the IL12RB2 gene in the modified humanized mouse is shown in SEQ ID NO: 20, and the expressed protein sequence is shown in SEQ ID NO: 21.

[0377] The targeting vector can be constructed by conventional methods, such as restriction enzyme ligation, etc. After the constructed targeting vector is preliminarily verified by restriction enzyme digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into the embryonic stem cells of C57BL / 6 mice by electroporation, and the obtained cells are screened using a positive clone selection marker gene to screen out the correct positive clone cells. The correctly screened positive clone cells (black mice) are introduced into the pre-separated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient female mouse (white mouse) to produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clones.

[0378] In addition, the CRISPR / Cas9 technology can also be used for gene editing, and the targeting strategy and targeting vector V3 ( Figure 7 ) are further designed. Figure 7It shows that the targeting vector V3 contains homologous arm sequences upstream and downstream of the mouse IL12RB2 gene, as well as the A2 fragment containing human IL12RB2. Among them, the upstream 5' homologous arm sequence (SEQ ID NO: 22) is identical to the nucleotide sequence from position 67338866 to 67339936 of NCBI accession number NC_000072.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 23) is identical to the nucleotide sequence from position 67337484 to 67338865 of NCBI accession number NC_000072.7. The A2 fragment sequentially contains a human IL12RB2 fragment (SEQ ID NO: 24), a mouse IL12RB2 fragment (SEQ ID NO: 67), and a STOP sequence (SEQ ID NO: 68) from the 5' end to the 3' end. The mRNA sequence transcribed from the IL12RB2 gene in the modified humanized mouse is as shown in SEQ ID NO: 20, and the expressed protein sequence is as shown in SEQ ID NO: 21.

[0379] The construction of the targeting vector can be carried out by conventional methods such as digestion and ligation, direct synthesis, etc. After the constructed targeting vector is preliminarily verified by digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is used for subsequent experiments.

[0380] The target sequence (target site) determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, the selection and design of highly efficient and specific target sequences are the premise for constructing the sgRNA expression vector. Design and synthesize the sgRNA sequence that recognizes the target site. The target sequences (target sites) of the exemplary sgRNA on the IL12RB2 gene are as follows:

[0381] sgRNA1 target site (SEQ ID NO: 73): 5'-TAAATAAGCTAATACTATAGAGG-3';

[0382] After detecting the activity of the sgRNA using the UCA kit and determining that it can mediate high cleavage efficiency, restriction enzyme sites are added to the 5' end and its complementary strand respectively to obtain the forward and reverse oligonucleotide sequences as shown in Table 9. After annealing, the annealed product is ligated to the pT7-sgRNA plasmid (the plasmid is first linearized with BbsI) to obtain the expression vector pT7-IL12RB2-1. After screening the marker gene, the IL12RB2 gene humanized homozygous mice can be obtained by intercrossing.

[0383] Table 9 sgRNA1 sequence list

[0384]

[0385] The genotypes of somatic cells of F1 generation mice can be identified by PCR method. For the mice with positive PCR identification in F1 generation, Southern blot detection is carried out to confirm whether there is random insertion. Cut the mouse tail to extract genomic DNA, digest the genomic DNA with PshAI enzyme or Eco53KI enzyme respectively, transfer the membrane, and hybridize. The 3’ probe (3’ Probe) and LR probe (LR Probe) are located downstream of the 3’ homologous arm and on the 5’ homologous arm respectively. The specific probes and the lengths of the target fragments are shown in Table 10), and the exemplary results are as Figure 8 shown. Combining the PCR and sequencing results, 4 mice numbered F1-01, F1-02, F1-04 and F1-06 to are positive mice. This indicates that the method can be used to construct IL12RB2 gene humanized mice that can be stably passed on and have no random insertion.

[0386] Table 10 Specific probes and the lengths of the target fragments

[0387] Restriction endonuclease Probe Wild-type fragment size Recombinant sequence fragment size PshAI 3’Probe 9.4kb 4.4kb Eco53KI LR Probe 6.9kb 3.5kb

[0388] 3’Probe-F (SEQ ID NO: 39): 5’-TCTTCCAACTCGTCCAACACAGGC-3’,

[0389] 3’Probe-R (SEQ ID NO: 40): 5’-TGATGTTGTCACCTGGTGATTCCATTG-3’;

[0390] LR Probe-F (SEQ ID NO: 41): 5’-GGACTCTCATTCTGCCATTTTGTG-3’,

[0391] LR Probe-R (SEQ ID NO: 42): 5’-TGCTTTAATCAGCAGCCACATGAACAA-3’;

[0392] The expression of mRNA in the IL12RB2 gene humanized mice can also be detected by RT-PCR. Specifically, 1 10-week-old C57BL / 6 mouse (+ / +) and 1 10-week-old male IL12RB2 gene humanized heterozygote (H / +) prepared in this example are selected respectively. After being stimulated with anti-CD3e Anti-mCD3e antibody (7.5 μg / 200 μL) for 24 h, the spleen tissue is taken after decapitation and euthanasia, and RT-PCR detection is carried out using the primer sequences shown in Table 11. The detection results are as Figure 9 shown. From Figure 9As can be seen, only murine IL12RB2 mRNA was detected in wild-type C57BL / 6 mice, while humanized IL12RB2 mRNA was not detected; in heterozygous mice with humanized IL12RB2 gene, both murine IL12RB2 mRNA and humanized IL12RB2 mRNA were detected.

[0393] Table 11 RT-PCR primer sequences and target fragment sizes

[0394]

[0395]

[0396] Example 3 Construction of humanized IL12RB2 gene mice (V2)

[0397] To achieve the object of the present invention, the nucleotide sequence encoding human IL12RB2 protein can also be introduced into the murine endogenous IL12RB2 locus in other ways, so that the mouse expresses human or humanized IL12RB2 protein. Specifically, using gene editing technology, under the control of the murine IL12RB2 gene regulatory element, about 66.3 kb of the partial sequence from exon 2 to exon 14 of the human IL12RB2 gene is used to replace about 63.8 kb of the partial sequence from exon 2 to exon 14 of the murine exon, to obtain a humanized IL12RB2 locus, and realize the humanization of the murine IL12RB2 gene.

[0398] To implement the present invention, a targeting vector V4 ( Figure 10 ) was constructed. The targeting vector V4 contains homologous arm sequences upstream and downstream of the murine IL12RB2 gene, and an A3 fragment containing the human IL12RB2 gene fragment. Among them, the upstream 5' homologous arm sequence is SEQ ID NO: 48, the downstream 3' homologous arm sequence is SEQ ID NO: 49, and the nucleotide sequence of the human IL12RB2 gene fragment is the same as the nucleotide sequence at positions 67320369 to 67386643 of NCBI accession number NC_000001.11; the connection design of the upstream of the human IL12RB2 fragment sequence and the murine sequence is: Among them, the last "C" in the sequence " GACTC " is the last nucleotide at the junction of the murine sequence and the upstream of the human IL12RB2 fragment sequence, and the "A" in the sequence is the first nucleotide of the human IL12RB2 fragment sequence. The connection design of the downstream of the human IL12RB1 fragment sequence and the murine sequence is: Among them, the sequence " GCATTThe last "T" in "" is the last nucleotide of the human IL12RB2 fragment sequence, and the sequence The first "T" in the sequence is the first nucleotide at the downstream junction of the mouse sequence and the human IL12RB1 fragment sequence.

[0399] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection design of the 5' end of the Neo cassette to the human IL12RB2 sequence is as follows:

[0400] Among them, the sequence " ACATC " The last "C" in is the last nucleotide at the junction of the human IL12RB2 sequence and the 5' end of the Neo cassette, and the sequence The first "T" in is the first nucleotide of the Neo cassette; the connection design of the 3' end of the Neo cassette to the human IL12RB2 sequence is as follows: Among them, the sequence " AGCCC " The last "C" in is the last nucleotide of the Neo cassette, and the sequence The first "A" in is the first nucleotide at the junction of the human IL12RB2 sequence and the 3' end of the Neo cassette. The mRNA sequence transcribed from the IL12RB2 gene in the engineered humanized mouse is shown in SEQ ID NO: 55, and the expressed protein sequence is shown in SEQ ID NO: 56.

[0401] The construction of the targeting vector can be carried out by conventional methods, such as restriction enzyme ligation, etc. After the constructed targeting vector is preliminarily verified by restriction enzyme digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into the embryonic stem cells of C57BL / 6 mice by electroporation, and the obtained cells are screened using the positive clone screening marker gene to screen out the correct positive clone cells. The correct positive clone cells (black mice) screened out are introduced into the pre-separated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient female mouse (white mouse), and F0 generation chimeric mice (black and white) can be produced. The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clones.

[0402] The genotype of somatic cells of F1 generation mice can be identified by PCR method, detected using the primers in Table 12, and exemplary results are as Figure 11As shown in the figure, combining the PCR and sequencing results, one mouse numbered F1-1 was a positive mouse. This indicates that the method can construct humanized mice with the IL12RB2 gene that can be stably passaged and have no random insertion.

[0403] Table 12 PCR primer sequences and target fragment sizes

[0404]

[0405] The expression of mRNA in the humanized mice with the IL12RB2 gene can also be detected by RT-PCR. Specifically, one 12-week-old C57BL / 6 mouse (+ / +) and one 12-week-old male humanized heterozygous IL12RB2 gene mouse (H / +) prepared in this example were selected respectively. After cervical dislocation euthanasia, spleen tissues were taken and RT-PCR was performed using the primer sequences shown in Table 13. The detection results are as Figure 12 shown. As can be seen from Figure 12 , only mouse IL12RB2 mRNA was detected in wild-type C57BL / 6 mice, and humanized IL12RB2 mRNA was not detected; in humanized heterozygous IL12RB2 gene mice, not only mouse IL12RB2 mRNA but also humanized IL12RB2 mRNA was detected.

[0406] Table 13 RT-PCR primer sequences and target fragment sizes

[0407]

[0408]

[0409] Example 4 Humanized mice with the IL23R gene

[0410] The comparison schematic diagram of the mouse IL23R gene (NCBI Gene ID: 209590, located at positions 67399906 to 67468838 of NC_000072.7 on chromosome 6, based on transcript NM_144548.2 and its encoded protein NP_653131.3 (SEQ ID NO: 27)) and the human IL23R gene (NCBI Gene ID: 149233, located at positions 67138637 to 67265903 of NC_000001.11 on chromosome 1, based on transcript NM_144701.3 and its encoded protein NP_653302.2 (SEQ ID NO: 59)) is as Figure 13 shown.

[0411] To achieve the object of the present invention, a nucleotide sequence encoding a humanized IL23R protein can be introduced into the endogenous IL23R gene locus of a mouse, such that the mouse expresses the humanized IL23R protein. Specifically, using gene editing technology, a chimeric CDS containing the extracellular region of the human IL23R protein and the signal peptide, transmembrane region, and cytoplasmic region of the mouse IL23R protein, and the 3'UTR and downstream nucleotide sequence of the mouse IL23R gene are used to replace a partial sequence of approximately 5.52 kb from exon 3 to intron 3 of the mouse to obtain a humanized IL23R gene locus, thereby achieving humanization of the mouse IL23R gene.

[0412] To implement the targeting strategy of the present invention, a targeting vector V5 ( Figure 14 ) was constructed. The targeting vector V5 contains upstream and downstream homologous arm sequences, as well as an A4 fragment. Among them, the A4 fragment contains a P2A linker peptide sequence, a human-mouse chimeric IL23R CDS, the 3'UTR and downstream sequence of the mouse IL23R (SEQ ID NO: 69), and a 3*SV40 PloyA (PloyA) sequence. Among them, the upstream 5' homologous arm sequence is SEQ ID NO: 65, and the downstream 3' homologous arm sequence is SEQ ID NO: 66; the linker sequence between the mouse sequence and the upstream of the human-mouse chimeric IL23R CDS is: Among them, the last "T" in the sequence is the last nucleotide at the junction of the mouse sequence and the upstream of the human-mouse chimeric IL23R CDS, and the first "A" in the sequence " ATGA " is the first nucleotide of the human-mouse chimeric IL23R CDS. The underlined sequence with a curve is the P2A sequence. The linker sequence between the 3'UTR and downstream nucleotide sequence of the mouse IL23R and the upstream of the PloyA sequence is: Among them, the last "T" in the sequence is the last nucleotide of the 3'UTR and downstream nucleotide sequence of the mouse IL23R, and the "A" in the sequence " AGCG " is the first nucleotide of the PloyA sequence. The linker sequence between the downstream of the PloyA sequence and the mouse IL23R sequence is: Among them, the last "C" in the sequence is the last nucleotide of the PloyA sequence, and the first "C" in the sequence " CACT " is the first nucleotide at the junction of the mouse IL23R sequence and the downstream of the PloyA sequence. The obtained F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are interbred to obtain F2 generation homozygous mice.

[0417] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two directly repeated site-specific recombination systems, Frt recombination sites, are installed on both sides of the resistance gene to form a Neo cassette. The connection at the 5' end of the Neo cassette and downstream of the PloyA sequence is designed as: Among them, the last "C" in the sequence is the last nucleotide of the PloyA sequence, and the first "G" in the sequence " GAAG " is the first nucleotide of the Neo cassette; the connection at the 3' end of the Neo cassette and the murine IL23R gene is designed as: Among them, the last "C" in the sequence is the last nucleotide of the Neo cassette, and the first "C" in the sequence " CACT " is the first nucleotide at the junction of the murine IL23R gene and the 3' end of the Neo cassette. The mRNA sequence transcribed from the IL23R gene in the modified humanized mouse is as shown in SEQ ID NO: 71, and the expressed protein sequence is as shown in SEQ ID NO: 78.

[0421] The construction of the targeting vector can be carried out by conventional methods, such as digestion and ligation. After the constructed targeting vector is preliminarily verified by digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into the embryonic stem cells of C57BL / 6 mice by electroporation, and the obtained cells are screened using the positive clone screening marker gene to screen out the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the isolated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient female mouse (white mouse) to produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are interbred to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene, and then the IL23R gene humanized homozygous mice can be obtained by interbreeding.

[0422] In addition, the CRISPR / Cas9 technology can also be used for gene editing. Design the targeting vector V6 ( Figure 15) The targeting vector V6 contains homologous arm sequences upstream and downstream of the mouse IL23R gene, as well as the A5 fragment. Among them, the A5 fragment contains a P2A linker peptide sequence, a human-mouse chimeric IL23R CDS, the 3’UTR of the mouse IL23R and its downstream sequence (SEQ ID NO: 69), and a PloyA sequence. Among them, the upstream 5’ homologous arm sequence is SEQ ID NO: 79, and the downstream 3’ homologous arm sequence is SEQ ID NO: 80. The mRNA sequence transcribed from the IL23R gene in the modified humanized mouse is as shown in SEQ ID NO: 71, and the expressed protein sequence is as shown in SEQ ID NO: 78.

[0423] The construction of the targeting vector can be carried out by conventional methods, such as restriction enzyme ligation, direct synthesis, etc. After the constructed targeting vector is preliminarily verified by restriction enzyme digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is used for subsequent experiments.

[0424] The target sequence (target site) determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, the selection and design of highly efficient and specific target sequences are the premise for constructing the sgRNA expression vector. Design and synthesize the sgRNA sequence that recognizes the target site. The target sequences (target sites) of the exemplary sgRNA on the IL23R gene are as follows:

[0425] sgRNA2 target site (SEQ ID NO: 81): 5’-TCTATAGTACTTACGTCCAGAGG-3’;

[0426] sgRNA3 target site (SEQ ID NO: 82): 5’-AGTTACTGACATCCTTGCACTGG-3’;

[0427] After detecting the activity of the sgRNA using the UCA kit and determining that it can mediate high cleavage efficiency, restriction enzyme sites are added to the 5’ end and its complementary strand respectively to obtain the forward and reverse oligonucleotide sequences as shown in Table 14. After annealing, the annealed product is ligated to the pT7-sgRNA plasmid (the plasmid is first linearized with BbsI) to obtain the expression vectors pT7-IL23R-2 and pT7-IL23R-3.

[0428] Table 14 Sequence list of sgRNA2 and sgRNA3

[0429]

[0430] The pT7-sgRNA vector was synthesized by a plasmid synthesis company. A DNA fragment (SEQ ID NO: 47) containing the T7 promoter and the sgRNA scaffold was ligated to the backbone vector (source: Takara, catalog number 3299) through enzymatic digestion (EcoRI and BamHI) in sequence. After verification by a professional sequencing company, the results showed that the target plasmid was obtained. Pronuclear stage fertilized eggs of mice, such as C57BL / 6 mice, were taken. The in vitro transcription products of pT7-IL23R-1 and pT7-IL23R-2 plasmids (using the Ambion in vitro transcription kit and transcribing according to the instructions), the targeting vector and Cas9 mRNA were premixed and then injected into the cytoplasm or nucleus of the mouse fertilized eggs. Microinjection of the fertilized eggs was carried out according to the method in "Mouse Embryo Manipulation Experimental Manual (Third Edition)" (Andras Nagy, Chemical Industry Press, 2006). The injected fertilized eggs were transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient female mouse for development. The obtained mice (F0 generation) were hybridized and self-crossed to expand the population size and establish a stable humanized mouse strain of the IL23R gene.

[0431] The somatic cell genotypes of F1 generation mice can be identified by PCR method. The primers shown in Table 15 were used for detection. Exemplary results are as Figure 16 shown. Four mice numbered F1-1 to F1-4 are positive mice.

[0432] Table 15 Primer sequences for PCR detection of F1 generation genotypes and sizes of recombinant fragments

[0433]

[0434] Example 5 Construction of multi-gene humanized mice

[0435] The humanized mice of IL12RB1, IL12RB2 and / or IL23R genes prepared using Examples 1-4 can also be used to prepare multi-gene humanized mouse models. For example, in Example 1 above, the embryonic stem cells used for microinjection can be selected from mice modified with at least one gene among IL12RB2, IL12A, IL12B, IL23A, IL23R, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4. Alternatively, based on the humanized IL12RB1, IL12RB2 and / or IL23R mice, a double-gene humanized or multi-gene humanized mouse model can be obtained by separating mouse ES embryonic stem cells and using gene recombination and targeting technology. The homozygous or heterozygous IL12RB1, IL12RB2 and / or IL23R mice prepared above can also be mated with other gene-modified mice, and their offspring can be screened. According to Mendelian inheritance, there is a certain probability of obtaining multi-gene mice with humanized IL12RB1, IL12RB2 and / or IL23R genes and other gene modifications. Then, mating the heterozygotes with each other can obtain homozygotes with double-gene or multi-gene modifications.

[0436] Taking the IL12RB1 / IL12RB2 double-gene humanized mouse as an example, the IL12RB1 gene humanized mouse prepared using Example 1 and the IL12RB2 gene humanized homozygous mouse prepared using Example 2 were mated. After screening the offspring, the IL12RB1 / IL12RB2 double-gene humanized mouse was obtained.

[0437] The expression of mRNA in the IL12RB1 / IL12RB2 double-gene humanized mouse can be detected by RT-PCR. The results showed (data not shown) that only mouse IL12RB1 mRNA and mouse IL12RB2 mRNA were detected in wild-type C57BL / 6 mice, and no humanized IL12RB1 mRNA and humanized IL12RB2 mRNA were detected; humanized IL12RB1 mRNA and humanized IL12RB2 mRNA were detected in the IL12RB1 / IL12RB2 double-gene humanized mouse.

[0438] Furthermore, ELISA was used to detect the secretion of IFN-γ in wild-type mice and IL12RB1 / IL12RB2 double-gene humanized heterozygous mice. Specifically, three wild-type C57BL / 6 mice and three IL12RB1 / IL12RB2 double-gene humanized heterozygous mice were selected. After euthanasia, the spleen tissues of the mice were taken and processed into single-cell suspensions. After sorting out CD4+ T cells, anti-mouse CD3ε antibody (anti-mCD3ε, concentration 0.4 μg / mL), anti-mouse CD28 antibody (anti-mCD28, concentration 0.8 μg / mL), and mouse IL12 recombinant protein (mIL12, concentration 0.008 μg / mL, 0.04 μg / mL, 0.2 μg / mL, 1 μg / mL or 4 μg / mL) or human IL12 recombinant protein (hIL12, concentration 0.008 μg / mL, 0.04 μg / mL, 0.2 μg / mL, 1 μg / mL or 4 μg / mL) at different concentrations were added, and cultured at 37 °C for 48 h; the cell culture supernatant was collected for ELISA experiment to detect the secretion of mIFN-γ, and the results were as Figure 17 shown. The results showed that an increase in the content of mIFN-γ was detected in both wild-type mice and IL12RB1 / IL12RB2 double-gene humanized heterozygous mice under the stimulation of mIL12, while an increase in the content of mIFN-γ was only detected in IL12RB1 / IL12RB2 double-gene humanized heterozygous mice under the stimulation of hIL12.

[0439] In addition, the IL12RB1 gene humanized mice prepared in Example 1 and the IL12RB2 gene humanized homozygous mice prepared in Example 3 were mated, and after screening the offspring, IL12RB1 / IL12RB2 plus double-gene humanized mice were obtained. Similar to the above method, the expression of humanized IL12RB1 mRNA and humanized IL12RB2 mRNA was detected in IL12RB1 / IL12RB2 plus double-gene humanized mice by RT-PCR method (data not shown).

[0440] Furthermore, flow cytometry was further used to perform immunophenotyping detection on the spleen, lymph nodes and blood tissues of C57BL / 6 wild-type mice, homozygous IL12RB1 / IL12RB2 double-gene humanized mice, and homozygous IL12RB1 / IL12RB2 plus double-gene humanized mice. Specifically, three 6-week-old female C57BL / 6 wild-type mice, three 9-week-old IL12RB1 / IL12RB2 plus double-gene humanized mice, and three 6-week-old homozygous IL12RB1 / IL12RB2 double-gene humanized mice were respectively selected. After cervical dislocation euthanasia, the spleen, lymph nodes and blood tissues were taken, and immunophenotyping detection was performed using antibodies. The results showed that the leukocyte subtypes such as T, B, and NK cells in IL12RB1 / IL12RB2 plus double-gene humanized mice and IL12RB1 / IL12RB2 double-gene humanized mice were similar to those of the wild type, and the percentages of T cell subtypes such as CD4+ T cells, CD8+ T cells, and Tregs cells were similar to those of C57BL / 6 wild-type mice (data not shown). It was shown that the humanization of IL12RB1 / IL12RB2 plus double-gene humanized mice and IL12RB1 / IL12RB2 double-gene humanized mice did not affect the differentiation, development and distribution of leukocytes and T cells in the spleen, lymph nodes and blood in mice.

[0441] In another experiment, ELISA was used to detect the secretion of IFN-γ in wild-type mice (+ / +), IL12RB1 / IL12RB2 double-gene humanized mice, and IL12RB1 / IL12RB2 plus double-gene humanized mice. Similar to the previous experimental protocol, three wild-type C57BL / 6 mice, three homozygous IL12RB1 / IL12RB2 double-gene humanized mice, and three homozygous IL12RB1 / IL12RB2 plus double-gene humanized mice were selected. After euthanasia, the spleen tissues of the mice were taken and processed into single-cell suspensions. After sorting out CD4+ T cells, anti-mouse CD3ε antibody (anti-mCD3ε, concentration 0.4 μg / mL), anti-mouse CD28 antibody (anti-mCD28, concentration 0.8 μg / mL), and different concentrations of mouse IL12 recombinant protein (mIL12, concentrations of 0, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL or 1000 ng / mL) or human IL12 recombinant protein (hIL12, concentrations of 0, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL or 1000 ng / mL) were added, and cultured at 37 °C for 48 h; the cell culture supernatants were collected for ELISA experiments to detect the secretion of mIFN-γ, and the results were as Figure 18As shown. The results showed that different concentrations of mIL12 could induce mIFN-γ production in two types of humanized mice and wild-...

Claims

1. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric interleukin 12 receptor subunit β1 (IL12RB1) protein.

2. The construction method according to claim 1, characterized in that: The chimeric IL12RB1 protein comprises all or part of the extracellular region of the human IL12RB1 protein, and preferably also comprises all or part of the transmembrane region of the human IL12RB1 protein; Further preferably, the chimeric IL12RB1 protein comprises a human or humanized extracellular region, a human or humanized transmembrane region and an endogenous cytoplasmic region; Preferably, the amino acid sequence of the human or chimeric IL12RB1 protein comprises positions 1-549 of SEQ ID NO: 2, positions 1-570 of SEQ ID NO: 2, positions 24-549 of SEQ ID NO: 2, positions 24-570 of SEQ ID NO: 2, positions 1-545 of SEQ ID NO: 2, or positions 24-545 of SEQ ID NO: 2; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to positions 1-549 of SEQ ID NO: 2, positions 1-570 of SEQ ID NO: 2, positions 24-549 of SEQ ID NO: 2, positions 24-570 of SEQ ID NO: 2, positions 1-545 of SEQ ID NO: 2, or positions 24-545 of SEQ ID NO: 2; Preferably, the amino acid sequence of the chimeric IL12RB1 protein comprises SEQ ID NO: 11; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

11.

3. A method for constructing a genetically modified non-human animal, characterized in that: At the non-human animal endogenous IL12RB1 gene locus, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB1 is replaced with a nucleotide sequence comprising human IL12RB1.

4. The construction method according to claim 3, characterized in that: The nucleotide sequence of human IL12RB1 comprises a nucleotide sequence encoding a human or chimeric IL12RB1 protein, preferably comprises a nucleotide sequence encoding all or part of the extracellular region of the human IL12RB1 protein, and further preferably further comprises a nucleotide sequence encoding all or part of the transmembrane region of the human IL12RB1 protein; Preferably, the nucleotide sequence of human IL12RB1 comprises a portion from exon 1 to exon 14 of the human IL12RB1 gene; further preferably, the nucleotide sequence of human IL12RB1 comprises a portion from the start codon to exon 14 of the human IL12RB1 gene; Wherein, the portion of exon 1 of the human IL12RB1 gene preferably comprises at least 5 bp of continuous nucleotide sequence, and the portion of exon 14 of the human IL12RB1 gene preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence of human IL12RB1 comprises the nucleotide sequence shown at positions 18062249 to 18086823 of NCBI Accession No. NC_000019.10; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown at positions 18062249 to 18086823 of NCBI Accession No. NC_000019.

10.

5. The construction method according to any one of claims 3-4, characterized in that: The nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB1 comprises a nucleotide sequence encoding the non-human animal endogenous IL12RB1 protein, preferably comprises a nucleotide sequence encoding all or part of the extracellular region of the non-human animal endogenous IL12RB1 protein, and further preferably further comprises a nucleotide sequence encoding all or part of the transmembrane region of the non-human animal endogenous IL12RB1 protein; Preferably, the nucleotide sequence of the corresponding region of the endogenous IL12RB1 of the non-human animal comprises a nucleotide sequence encoding positions 1-570 of SEQ ID NO: 1, positions 1-591 of SEQ ID NO: 1, positions 20-565 of SEQ ID NO: 1, positions 20-570 of SEQ ID NO: 1, positions 20-591 of SEQ ID NO: 1, or positions 1-565 of SEQ ID NO: 1; Preferably, the nucleotide sequence of the corresponding region of the endogenous IL12RB1 of the non-human animal comprises a portion from exon 1 to exon 14 of the endogenous IL12RB1 gene of the non-human animal. Further preferably, the nucleotide sequence of the corresponding region of the endogenous IL12RB1 of the non-human animal comprises a portion from the start codon to exon 14 of the endogenous IL12RB1 gene of the non-human animal. Among them, the part of exon 1 of the endogenous IL12RB1 gene of the non-human animal preferably contains at least 5 bp of continuous nucleotide sequence, and the part of exon 14 of the endogenous IL12RB1 gene of the non-human animal preferably contains at least 5 bp of continuous nucleotide sequence.

6. The construction method according to any one of claims 1 to 5, characterized in that: The nucleotide sequence encoding the human or chimeric IL12RB1 protein or the nucleotide sequence of human IL12RB1 is operably linked to an endogenous regulatory element of an endogenous IL12RB1 locus; Preferably, the endogenous IL12RB1 protein of the non-human animal is not expressed or is expressed at a reduced level compared to IL12RB1 in wild-type animals; Preferably, the modified IL12RB1 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

7. The construction method according to any one of claims 1 to 6, characterized in that: The non-human animal includes a mammal, such as a monkey or a rodent, preferably, the rodent includes a mouse or a rat; Preferably, the mRNA transcribed from the modified IL12RB1 gene in the genome of the non-human animal comprises SEQ ID NO: 10; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 10; Preferably, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins, and the other human or chimeric proteins preferably comprise at least one of IL12A, IL12B, IL12RB2, IL23R, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4; Further preferably, the other human or chimeric protein is a humanized IL12RB2 protein, and the amino acid sequence of the humanized IL12RB2 protein preferably comprises SEQ ID NO: 21 or 56; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 21 or 56; Further preferably, the other human or chimeric protein is a humanized IL23R protein, and the amino acid sequence of the humanized IL23R protein preferably comprises SEQ ID NO: 78; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

78.

8. A humanized IL12RB1 protein, characterized in that The humanized IL12RB1 protein comprises all or part of the extracellular region of the human IL12RB1 protein, and preferably comprises all or part of the transmembrane region of the human IL12RB1 protein; Preferably, the humanized IL12RB1 protein comprises positions 1-549 of SEQ ID NO: 2, positions 1-570 of SEQ ID NO: 2, positions 24-549 of SEQ ID NO: 2, positions 24-570 of SEQ ID NO: 2, positions 1-545 of SEQ ID NO: 2, or positions 24-545 of SEQ ID NO: 2; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to positions 1-549 of SEQ ID NO: 2, positions 1-570 of SEQ ID NO: 2, positions 24-549 of SEQ ID NO: 2, positions 24-570 of SEQ ID NO: 2, positions 1-545 of SEQ ID NO: 2, or positions 24-545 of SEQ ID NO: 2; Preferably, the amino acid sequence of the humanized IL12RB1 protein comprises SEQ ID NO: 11; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

11.

9. A humanized IL12RB1 gene, characterized in that: The humanized IL12RB1 gene encodes the humanized IL12RB1 protein according to claim 8; Preferably, the humanized IL12RB1 gene comprises a portion from exon 1 to exon 14 of the human IL12RB1 gene. Further preferably, the humanized IL12RB1 gene comprises a portion from the start codon to exon 14 of the human IL12RB1 gene. Wherein, the portion of exon 1 of the human IL12RB1 gene preferably comprises at least 5 bp of continuous nucleotide sequence, and the portion of exon 14 of the human IL12RB1 gene preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the humanized IL12RB1 gene comprises SEQ ID NO: 3, 4, 6, 7, 8, 9, 10 or NCBI accession number The nucleotide sequence shown at positions 18062249 to 18086823 of NC_000019.10; or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown at positions 18062249 to 18086823 of NC_000019.

10.

10. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric interleukin 12 receptor subunit β2 (IL12RB2) protein.

11. The construction method according to claim 10, characterized in that: The chimeric IL12RB2 protein comprises all or part of the extracellular region of the human IL12RB2 protein, preferably comprises all or part of the transmembrane region of the human IL12RB2 protein; Preferably, the chimeric IL12RB2 protein comprises positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, positions 24-643 of SEQ ID NO: 13, positions 1-640 of SEQ ID NO: 13, or positions 1-643 of SEQ ID NO: 13; or comprises positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, positions 24-643 of SEQ ID NO: 13, positions 1-640 of SEQ ID NO: 13, or positions 1-643 of SEQ ID NO: 13 that are at least 70%, 75%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700 85%, 90%, 95%, 99% or 99.5% of the amino acid sequence; Preferably, the chimeric IL12RB2 protein comprises all or part of the transmembrane region of the endogenous IL12RB2 protein of a non-human animal, and further preferably comprises all or part of the cytoplasmic region of the endogenous IL12RB2 protein of a non-human animal; Preferably, the chimeric IL12RB2 protein comprises positions 638-874 of SEQ ID NO: 12 or positions 639-874 of SEQ ID NO: 12; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to positions 638-874 of SEQ ID NO: 12 or positions 639-874 of SEQ ID NO: 12; Further preferably, the chimeric IL12RB2 protein comprises a human or humanized extracellular region, a human or humanized transmembrane region and an endogenous cytoplasmic region; Preferably, the amino acid sequence of the chimeric IL12RB2 protein comprises SEQ ID NO: 21 or 56; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 21 or 56.

12. A method for constructing a genetically modified non-human animal, characterized in that: The construction method comprises the steps of: The nucleotide sequence of IL12RB2 is introduced into the endogenous IL12RB2 locus of a non-human animal. Preferably, the introduction is by insertion or replacement.

13. The construction method according to claim 12, characterized in that: The construction method comprises inserting the nucleotide sequence of the chimeric IL12RB2 into the endogenous IL12RB2 locus of a non-human animal; Preferably, the nucleotide sequence of the chimeric IL12RB2 comprises a nucleotide sequence encoding a human or chimeric IL12RB2 protein, preferably comprises a nucleotide sequence encoding all or part of the extracellular region of the human IL12RB2 protein, and further preferably comprises a nucleotide sequence encoding all or part of the transmembrane region of the human IL12RB2 protein; Preferably, the nucleotide sequence of the chimeric IL12RB2 comprises a nucleotide sequence encoding positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, or positions 24-643 of SEQ ID NO: 13; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence encoding positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, or positions 24-643 of SEQ ID NO: 13; Preferably, the nucleotide sequence of the chimeric IL12RB2 comprises a nucleotide sequence encoding an endogenous IL12RB2 protein of a non-human animal, preferably comprises a nucleotide sequence encoding all or part of a transmembrane region of an endogenous IL12RB2 protein of a non-human animal, and further preferably further comprises a nucleotide sequence encoding all or part of a cytoplasmic region of an endogenous IL12RB2 protein of a non-human animal; Preferably, the nucleotide sequence of the chimeric IL12RB2 comprises the nucleotide sequence encoding positions 638-874 of SEQ ID NO: 12 or positions 639-874 of SEQ ID NO: 12; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence encoding positions 638-874 of SEQ ID NO: 12 or positions 639-874 of SEQ ID NO: 12; Preferably, the nucleotide sequence of the chimeric IL12RB2 comprises a portion of exon 2 to a portion of exon 14 of the human IL12RB2 gene, wherein the portion of exon 2 of the human IL12RB2 gene preferably comprises at least 1 bp of continuous nucleotide sequence, and the portion of exon 14 of the human IL12RB2 gene preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence of the chimeric IL12RB2 comprises SEQ ID NO: 24; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 24; Preferably, the nucleotide sequence of the chimeric IL12RB2 comprises all or part of exon 14 to exon 16 of the endogenous IL12RB2 gene of a non-human animal, wherein the part of exon 14 of the endogenous IL12RB2 gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence, and the part of exon 16 of the endogenous IL12RB2 gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence of the chimeric IL12RB2 comprises a nucleotide sequence from part of exon 14 to the stop codon of the endogenous IL12RB2 gene of a non-human animal, preferably further comprises a 3'UTR, and further preferably further comprises at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; Preferably, the nucleotide sequence of the chimeric IL12RB2 comprises a portion of exon 14 of the endogenous IL12RB2 gene of a non-human animal to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; Preferably, the nucleotide sequence of the chimeric IL12RB2 comprises SEQ ID NO: 67; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 67; Preferably, the sequence inserted into the endogenous IL12RB2 locus of the non-human animal further comprises an auxiliary sequence, such as a STOP sequence. Preferably, the STOP sequence comprises SEQ ID NO: 68; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 68; Preferably, the insertion into the non-human animal endogenous IL12RB2 gene locus is insertion into exon 2 of the non-human animal endogenous IL12RB2 gene; Further preferably, the insertion into the non-human animal endogenous IL12RB2 locus is inserted into the genome of the non-human animal after the nucleotide sequence encoding the endogenous IL12RB2 protein signal peptide; More preferably, the insertion into the non-human animal endogenous IL12RB2 locus is inserted into the genome of the non-human animal between the nucleotide sequence encoding the signal peptide of the endogenous IL12RB2 protein and the nucleotide sequence encoding the extracellular region of the endogenous IL12RB2 protein; More preferably, the insertion into the endogenous IL12RB2 locus of the non-human animal is inserted between the nucleotide sequence encoding the 23rd position of SEQ ID NO: 12 and the nucleotide sequence encoding the 24th position of SEQ ID NO: 12 in the genome of the non-human animal.

14. The construction method according to claim 12, characterized in that: The construction method comprises replacing the nucleotide sequence of the corresponding region of the endogenous IL12RB2 of the non-human animal with a nucleotide sequence containing human IL12RB2 at the endogenous IL12RB2 locus of the non-human animal.

15. The construction method according to claim 14, characterized in that: The nucleotide sequence of human IL12RB2 comprises a nucleotide sequence encoding a human or chimeric IL12RB2 protein, preferably comprises a nucleotide sequence encoding all or part of the extracellular region of the human IL12RB2 protein, and further preferably further comprises a nucleotide sequence encoding all or part of the transmembrane region of the human IL12RB2 protein; Preferably, the nucleotide sequence of human IL12RB2 comprises a nucleotide sequence encoding positions 1-640 of SEQ ID NO: 13 or positions 1-643 of SEQ ID NO: 13; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence encoding positions 1-640 of SEQ ID NO: 13 or positions 1-643 of SEQ ID NO: 13; Preferably, the nucleotide sequence of human IL12RB2 comprises a portion from exon 2 to exon 14 of the human IL12RB2 gene. Further preferably, the nucleotide sequence of human IL12RB2 comprises a portion from the start codon to exon 14 of the human IL12RB2 gene. Wherein, the portion of exon 2 of the human IL12RB2 gene preferably comprises at least 5 bp of continuous nucleotide sequence, and the portion of exon 14 of the human IL12RB2 gene preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence of human IL12RB2 comprises the nucleotide sequence of positions 67320369 to 67386643 of NCBI Accession No. NC_000001.11; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence of positions 67320369 to 67386643 of NCBI Accession No. NC_000001.

11.

16. The construction method according to claim 14 or 15, characterized in that: The nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 comprises a nucleotide sequence encoding the non-human animal endogenous IL12RB2 protein, preferably comprises a nucleotide sequence encoding all or part of the extracellular region of the non-human animal endogenous IL12RB2 protein, and further preferably further comprises a nucleotide sequence encoding all or part of the transmembrane region of the non-human animal endogenous IL12RB2 protein; Preferably, the nucleotide sequence of the corresponding region of endogenous IL12RB2 of a non-human animal comprises a nucleotide sequence encoding positions 1-655 of SEQ ID NO: 12 or positions 1-658 of SEQ ID NO: 12; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence encoding positions 1-655 of SEQ ID NO: 12 or positions 1-658 of SEQ ID NO: 12; Preferably, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 includes a portion from exon 2 to exon 14 of the non-human animal endogenous IL12RB2 gene. Further preferably, the nucleotide sequence of the corresponding region of the non-human animal endogenous IL12RB2 includes a portion from the start codon to exon 14 of the non-human animal endogenous IL12RB2 gene. Among them, the portion of exon 2 of the endogenous IL12RB2 gene of the non-human animal preferably contains at least 5 bp of continuous nucleotide sequence, and the portion of exon 14 of the endogenous IL12RB2 gene of the non-human animal preferably contains at least 5 bp of continuous nucleotide sequence.

17. The construction method according to any one of claims 10 to 16, characterized in that: The nucleotide sequence encoding the human or chimeric IL12RB2 protein, the nucleotide sequence of the chimeric IL12RB2, or the nucleotide sequence of the human IL12RB2 is operably linked to an endogenous regulatory element of the endogenous IL12RB2 locus; Preferably, the endogenous IL12RB2 protein of the non-human animal is not expressed or is expressed at a reduced level compared to IL12RB2 in wild-type animals; Preferably, the modified IL12RB2 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous modified (eg, inserted or replaced) locus.

18. The construction method according to any one of claims 10 to 17, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the rodent includes a mouse or a rat; Preferably, the mRNA transcribed from the modified IL12RB2 gene in the genome of the non-human animal comprises SEQ ID NO: 20 or 55; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 20 or 55; Preferably, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins, and the other human or chimeric proteins preferably include IL12A, IL12B, IL12RB1, IL23R, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, at least one of TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4; Preferably, the other human or chimeric protein is a humanized IL12RB1 protein, preferably a humanized protein as described in claim 8. IL12RB1 protein; Preferably, the other human or chimeric protein is a humanized IL23R protein, and the amino acid sequence of the humanized IL23R protein preferably comprises SEQ ID NO: 78; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 80 ... 95%, 99% or 99.5% of the amino acid sequence.

19. A humanized IL12RB2 protein, characterized in that The humanized IL12RB2 protein comprises all or part of the extracellular region of the human IL12RB2 protein, and preferably also comprises all or part of the transmembrane region of the human IL12RB2 protein; Preferably, the humanized IL12RB2 protein comprises positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, positions 24-643 of SEQ ID NO: 13, positions 1-640 of SEQ ID NO: 13, or positions 1-643 of SEQ ID NO: 13; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to positions 24-622 of SEQ ID NO: 13, positions 24-623 of SEQ ID NO: 13, positions 24-643 of SEQ ID NO: 13, positions 1-640 of SEQ ID NO: 13, or positions 1-643 of SEQ ID NO: 13; Preferably, the humanized IL12RB2 protein further comprises all or part of the transmembrane region of the endogenous IL12RB2 protein of a non-human animal, and further preferably further comprises all or part of the cytoplasmic region of the endogenous IL12RB2 protein of a non-human animal; Preferably, the humanized IL12RB2 protein comprises positions 638-874 of SEQ ID NO: 12, positions 656-874 of SEQ ID NO: 12, or positions 639-874 of SEQ ID NO: 12; or comprises positions 638-874 of SEQ ID NO: 12, positions 656-874 of SEQ ID NO: 12, or positions 639-874 of SEQ ID NO: 12 that are at least 70%, 75%, 80%, 85%, 90%, 100%, 110%, 120%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 139%, 139%, 131%, 137%, 138%, 139 ... 95%, 99% or 99.5% of the amino acid sequence; Preferably, the amino acid sequence of the humanized IL12RB2 protein comprises SEQ ID NO: 21 or 56; or comprises the amino acid sequence of SEQ ID NO: An amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to NO:21 or 56.

20. A humanized IL12RB2 gene, characterized in that: The humanized IL12RB2 gene encodes the humanized IL12RB2 protein according to claim 19; Preferably, the humanized IL12RB2 gene comprises a portion from exon 2 to exon 14 of the human IL12RB2 gene, wherein the portion of exon 2 of the human IL12RB2 gene preferably comprises at least 1 bp of continuous nucleotide sequence, and the portion of exon 14 of the human IL12RB2 gene preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the humanized IL12RB2 gene further comprises part of exon 14 to all or part of exon 16 of the endogenous IL12RB2 gene of a non-human animal, wherein the part of exon 14 of the endogenous IL12RB2 gene of a non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence, and the part of exon 16 of the endogenous IL12RB2 gene of a non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the humanized IL12RB2 gene further comprises a nucleotide sequence from part of exon 14 to the stop codon of the endogenous IL12RB2 gene of a non-human animal, preferably further comprises a 3'UTR, and further preferably further comprises at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; Preferably, the humanized IL12RB2 gene further comprises a continuous nucleotide sequence of at least 50 bp from a portion of exon 14 of an endogenous IL12RB2 gene of a non-human animal to the downstream of the 3'UTR; Preferably, the humanized IL12RB2 gene comprises the nucleotide sequence shown in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 48, 49, 51, 52, 53, 54, 55, 67, 68 or NCBI accession number NC_000001.11 from position 67320369 to position 67386643; or comprises the nucleotide sequence shown in SEQ ID NO: NO:14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 48, 49, 51, 52, 53, 54, 55, 67, 68 or a nucleotide sequence having a nucleotide sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequence shown in positions 67320369 to 67386643 of NCBI Accession No. NC_000001.

11.

21. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric interleukin 23 receptor (IL23R) protein.

22. The construction method according to claim 21, characterized in that: The chimeric IL23R protein comprises all or part of the extracellular region of the human IL23R protein; Preferably, the amino acid sequence of the chimeric IL23R protein comprises amino acids 24-355 of SEQ ID NO:59 or amino acids 27-353 of SEQ ID NO:59; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 24-355 of SEQ ID NO:59 or amino acids 27-353 of SEQ ID NO:59; Preferably, the chimeric IL23R protein comprises all or part of the signal peptide, transmembrane region and / or cytoplasmic region of the endogenous IL23R protein of a non-human animal; Preferably, the amino acid sequence of the chimeric IL23R protein comprises amino acids 1-26 and 373-644 of SEQ ID NO: 27, or amino acids 1-23 and 375-644 of SEQ ID NO: 27; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 1-26 and 373-644 of SEQ ID NO: 27, or amino acids 1-23 and 375-644 of SEQ ID NO: 27; Preferably, the chimeric IL23R protein comprises an endogenous signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region; Preferably, the amino acid sequence of the chimeric IL23R protein comprises SEQ ID NO:78; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

78.

23. A method for constructing a genetically modified non-human animal, characterized in that: At the non-human animal endogenous IL23R gene locus, the nucleotide sequence of the non-human animal endogenous IL23R is replaced with a nucleotide sequence encoding a chimeric IL23R protein.

24. The construction method according to claim 23, characterized in that: The nucleotide sequence encoding the chimeric IL23R protein comprises all or part of exon 3 to part of exon 9 of the human IL23R gene; Wherein, the portion of exon 3 of the human IL23R gene preferably comprises at least 5 bp of continuous nucleotide sequence, and the portion of exon 9 of the human IL23R gene preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence encoding the chimeric IL23R protein comprises a portion of exon 2 to a portion of exon 3 of an endogenous IL23R gene of a non-human animal and all or part of a portion of exon 9 to exon 11 of an endogenous IL23R gene of a non-human animal; Preferably, the nucleotide sequence encoding the chimeric IL23R protein comprises a nucleotide sequence from a portion of exon 2 to a portion of exon 3 of an endogenous IL23R gene of a non-human animal and a portion of exon 9 to a stop codon, preferably further comprises a 3'UTR, and more preferably further comprises at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; Preferably, the nucleotide sequence encoding the chimeric IL23R protein comprises a portion of exon 2 to a portion of exon 3 of an endogenous IL23R gene of a non-human animal and a portion of exon 9 to at least 50 bp of a continuous nucleotide sequence downstream of the 3'UTR of an endogenous IL23R gene of a non-human animal; Among them, the portion of exon 2 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence, the portion of exon 3 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence, the portion of exon 9 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence, and the portion of exon 11 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence encoding the chimeric IL23R protein comprises SEQ ID NO: 69; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

69.

25. The construction method according to claim 23 or 24, characterized in that: The replaced nucleotide sequence of the endogenous IL23R of a non-human animal comprises a nucleotide sequence encoding an endogenous IL23R protein of a non-human animal, preferably comprises a nucleotide sequence encoding all or part of the extracellular region of an endogenous IL23R protein of a non-human animal; Preferably, the nucleotide sequence of the non-human animal endogenous IL23R to be replaced comprises a nucleotide sequence encoding amino acids 24-122 of SEQ ID NO: 27 or 33-122 of SEQ ID NO: 27; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence encoding amino acids 24-122 of SEQ ID NO: 27 or 33-122 of SEQ ID NO: 27; Preferably, the replaced nucleotide sequence of the non-human animal endogenous IL23R comprises a portion of exon 3 of the non-human animal endogenous IL23R gene, and the replaced portion of exon 3 of the non-human animal endogenous IL23R gene preferably comprises at least 5 bp of continuous nucleotide sequence; Further preferably, the replaced nucleotide sequence of the endogenous IL23R of the non-human animal also comprises all or part of intron 3 of the endogenous IL23R gene of the non-human animal, and the part of intron 3 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence.

26. The construction method according to any one of claims 21 to 25, characterized in that: The nucleotide sequence encoding the human or chimeric IL23R protein is operably linked to endogenous regulatory elements (eg, promoter, 5'UTR and / or 3'UTR) of the endogenous IL23R locus; Preferably, the endogenous IL23R protein of the non-human animal is not expressed or is expressed at a reduced level compared to IL23R in wild-type animals; Preferably, the modified IL23R gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

27. The construction method according to any one of claims 21 to 26, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent, preferably, the rodent includes a mouse or a rat; Preferably, the mRNA transcribed from the modified IL23R gene in the genome of the non-human animal comprises SEQ ID NO: 71; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 71; Preferably, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins, and the other human or chimeric proteins preferably comprise at least one of IL12A, IL12B, IL12RB1, IL12RB2, IL23A, ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4; Preferably, the other human or chimeric protein is a humanized IL12RB1 protein, preferably the humanized IL12RB1 protein according to claim 8; Preferably, the other human or chimeric protein is a humanized IL12RB2 protein, preferably the humanized IL12RB2 protein according to claim 19.

28. A humanized IL23R protein, characterized in that The humanized IL23R protein comprises all or part of the extracellular region of the human IL23R protein; Preferably, the amino acid sequence of the humanized IL23R protein comprises amino acids 24-355 of SEQ ID NO: 59 or amino acids 27-353 of SEQ ID NO: 59; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 24-355 of SEQ ID NO: 59 or amino acids 27-353 of SEQ ID NO: 59; Preferably, the humanized IL23R protein comprises all or part of the signal peptide, transmembrane region and / or cytoplasmic region of the endogenous IL23R protein of a non-human animal; Preferably, the amino acid sequence of the humanized IL23R protein comprises amino acids 1-26 and 373-644 of SEQ ID NO: 27, or amino acids 1-23 and 375-644 of SEQ ID NO: 27; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 1-26 and 373-644 of SEQ ID NO: 27, or amino acids 1-23 and 375-644 of SEQ ID NO: 27; Preferably, the amino acid sequence of the humanized IL23R protein comprises SEQ ID NO: 78; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

78.

29. A humanized IL23R gene, characterized in that: The humanized IL23R gene encodes the humanized IL23R protein according to claim 28; Preferably, the humanized IL23R gene comprises all or part of exon 3 to part of exon 9 of the human IL23R gene; Wherein, the portion of exon 3 of the human IL23R gene preferably comprises at least 5 bp of continuous nucleotide sequence, and the portion of exon 9 of the human IL23R gene preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the humanized IL23R gene comprises a portion of exon 2 to a portion of exon 3 of an endogenous IL23R gene of a non-human animal and all or a portion of exon 9 to exon 11 of an endogenous IL23R gene of a non-human animal; Preferably, the humanized IL23R gene comprises a nucleotide sequence from a portion of exon 2 to a portion of exon 3 of an endogenous IL23R gene of a non-human animal and a portion of exon 9 to a stop codon, preferably further comprises a 3'UTR, and more preferably further comprises at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; Preferably, the humanized IL23R gene comprises a portion of exon 2 to a portion of exon 3 of an endogenous IL23R gene of a non-human animal and a portion of exon 9 to at least 50 bp of a continuous nucleotide sequence downstream of the 3'UTR of an endogenous IL23R gene of a non-human animal; Among them, the portion of exon 2 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence, the portion of exon 3 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence, the portion of exon 9 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence, and the portion of exon 11 of the endogenous IL23R gene of the non-human animal preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the humanized IL23R gene comprises the nucleotide sequence shown in SEQ ID NO: 65, 66, 69, 71, 72, 74, 75, 76, 77, 79 or 80; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 65, 66, 69, 71, 72, 74, 75, 76, 77, 79 or 80.

30. A method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, characterized in that: The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method of any one of claims 1-7, 10-18, 21-27; 2) Determining the effects of therapeutic agents on non-human animals or diseases; Preferably, the therapeutic agent comprises an antibody targeting IL12RB1, IL12RB2 and / or IL23R, a nucleic acid drug targeting IL12RB1, IL12RB2 and / or IL23R and / or a polypeptide drug; Preferably, the therapeutic agent includes an additional therapeutic agent, and the additional therapeutic agent includes one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody; Preferably, the disease includes one or more of cancer, immune disease or inflammation; Preferably, the cancer includes one or more of colorectal cancer, hepatobiliary cancer, lymphocytic tumors, breast cancer, head and neck cancer, liver cancer or lung cancer; Preferably, the immune disease includes one or more of psoriasis, atopic dermatitis, asthma, rheumatoid arthritis or multiple sclerosis; Preferably, the inflammation comprises inflammatory bowel disease (IBD).

31. A cell, tissue or organ, characterized in that: The cell, tissue or organ expresses the humanized IL12RB1 protein of claim 8, the humanized IL12RB2 protein of claim 19 and / or the humanized IL23R protein of claim 28, and / or the genome of the cell, tissue or organ contains the humanized IL12RB1 gene of claim 9, the humanized IL12RB2 gene of claim 20 and / or the humanized IL23R gene of claim 29.

32. A use of a non-human animal obtained by the construction method of any one of claims 1-7, 10-18, 21-27, the humanized IL12RB1 protein of claim 8, the humanized IL12RB2 protein of claim 19, the humanized IL23R protein of claim 28, the humanized IL12RB1 gene of claim 9, the humanized IL12RB2 gene of claim 20, the humanized IL23R gene of claim 29, or the cell, tissue or organ of claim 31, characterized in that: The applications described include: A) Applications in the development of products involving immune processes associated with IL12RB1, IL12RB2 and / or IL23R in human cells; B) Use as a model system for pharmacological, immunological, microbiological and medical research related to IL12RB1, IL12RB2 and / or IL23R; C) Applications involving the production and use of animal experimental disease models for the study of etiology associated with IL12RB1, IL12RB2 and / or IL23R and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human IL12RB1, IL12RB2 and / or IL23R signaling pathway modulators; or, E) Study the gene functions of IL12RB1, IL12RB2 and / or IL23R, study the drugs and drug efficacy targeting human IL12RB1, IL12RB2 and / or IL23R target sites, and study the application of therapeutic drugs for tumors, inflammation or immune diseases related to IL12RB1, IL12RB2 and / or IL23R.

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