Non-human animal modified by CRTAM gene

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) CRTAM protein 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) CRTAM protein and methods of using the same. Background Art

[0002] Traditional drug 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 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 human drug screening and evaluation 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 CRTAM protein. This animal model can express a human or chimeric CRTAM (e.g., humanized CRTAM) protein. It can be used for the study of the function of the CRTAM gene and also for the screening and evaluation of CRTAM signaling pathway modulators (e.g., therapeutic agents targeting CRTAM, including antibodies targeting CRTAM, nucleic acid drugs targeting CRTAM, 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 CRTAM target; this animal model can also be used to facilitate new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of the CRTAM protein and 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 comprising at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric cytotoxic and regulatory T cell molecule (CRTAM) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric CRTAM protein can be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the chimeric CRTAM protein is a humanized CRTAM protein. In some embodiments, the amino acid sequence of the chimeric CRTAM protein comprises an amino acid sequence that is identical to at least 50 to 393, such as at least 50, 100, 150, 200, 250, 270, 280, 287, 300, 350, 390 or 393 consecutive or non-consecutive amino acid sequences of the human CRTAM protein. In some embodiments, the chimeric CRTAM protein comprises a human or humanized extracellular region. In some embodiments, the chimeric CRTAM protein comprises a human or humanized extracellular region, an endogenous transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric CRTAM protein comprises a human extracellular region, an endogenous transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric CRTAM protein comprises or does not comprise a signal peptide, preferably, the signal peptide is an endogenous signal peptide of the non-human animal or a human or humanized signal peptide. In some embodiments, the chimeric CRTAM protein comprises a human or humanized signal peptide, a human or humanized extracellular region, an endogenous transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric CRTAM protein comprises a human signal peptide, a human extracellular region, an endogenous transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric CRTAM protein comprises all or part of the extracellular region of the human CRTAM protein, preferably further comprising all or part of the signal peptide of the human CRTAM protein. In some embodiments, the amino acid sequence of the chimeric CRTAM protein comprises an amino acid sequence that is identical to at least 50 to 270, such as at least 50, 100, 150, 200, 250 or 270 consecutive or non-consecutive amino acid sequences of the extracellular region of the human CRTAM protein. In some embodiments, the chimeric CRTAM protein comprises all or part of the extracellular region and the signal peptide of the human CRTAM protein. In some embodiments, the amino acid sequence of the chimeric CRTAM protein comprises an amino acid sequence that is identical to at least 50 to 287, such as at least 50, 100, 150, 200, 250, 270, 280 or 287 consecutive or non-consecutive amino acid sequences of the signal peptide and the extracellular region of the human CRTAM protein. In some embodiments, the nucleotide sequence encoding the human or chimeric CRTAM protein is operably linked to an endogenous regulatory element (e.g., an endogenous promoter, 5'UTR and / or 3'UTR) of the endogenous CRTAM locus of at least one chromosome.In some embodiments, the amino acid sequence of the chimeric CRTAM protein comprises amino acids 1-287 of SEQ ID NO: 2 or amino acids 18-287 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 1-287 of SEQ ID NO: 2 or amino acids 18-287 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the chimeric CRTAM protein comprises amino acids 290-393 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 290-393 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the chimeric CRTAM 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 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 CRTAM protein in the non-human animal is not expressed or has a reduced expression level compared to CRTAM in a wild-type animal. In some embodiments, one or more cells of the non-human animal express a human or chimeric CRTAM 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 a nucleotide sequence encoding a human or chimeric CRTAM replacing a nucleotide sequence encoding a corresponding region of endogenous CRTAM at the endogenous CRTAM locus. In some embodiments, the nucleotide sequence encoding a human or chimeric CRTAM is operably linked to an endogenous regulatory element of the endogenous CRTAM locus. In some embodiments, one or more cells of the non-human animal express a human or humanized CRTAM protein. In some embodiments, the endogenous CRTAM protein of the non-human animal is not expressed or has a reduced expression level compared to CRTAM in a wild-type animal. In some embodiments, the nucleotide sequence encoding the corresponding region of endogenous CRTAM comprises a nucleotide sequence encoding the extracellular region of the endogenous CRTAM of the non-human animal. In some embodiments, the nucleotide sequence encoding the corresponding region of endogenous CRTAM comprises a nucleotide sequence encoding the extracellular region and signal peptide of the endogenous CRTAM of the non-human animal. In some embodiments, the nucleotide sequence encoding the corresponding region of endogenous CRTAM comprises a nucleotide sequence encoding the endogenous extracellular region, endogenous transmembrane region, and / or endogenous cytoplasmic region of the non-human animal. In some embodiments, one or more cells of the non-human animal express a chimeric CRTAM, the chimeric CRTAM comprising an extracellular region, a transmembrane region, and a cytoplasmic region, the extracellular region comprising an amino acid sequence that is identical or has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the extracellular region of human CRTAM. In some embodiments, the extracellular region is identical to at least 50 to 270, such as at least 50, 100, 150, 200, 250, or 270 consecutive amino acids of the extracellular region of the human CRTAM protein. In some embodiments, the chimeric CRTAM comprises or does not comprise a signal peptide. In some embodiments, the chimeric CRTAM comprises a signal peptide, and the signal peptide is endogenous to the non-human animal or human or humanized. In some embodiments, the signal peptide of the chimeric CRTAM comprises an amino acid sequence that is identical or has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the signal peptide of human CRTAM. In some embodiments, the signal peptide of the chimeric CRTAM is identical to at least 1 to 17, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids of the signal peptide of the human CRTAM protein.In some embodiments, the nucleotide sequence encoding human or chimeric CRTAM comprises a portion of exon 1 of the human CRTAM gene, all of exons 2-7, and / or a portion of exon 8 (preferably also comprising intron 1 and / or intron 7). Preferably, the nucleotide sequence encoding human or chimeric CRTAM comprises a portion of exon 1 of the human CRTAM gene, all of exons 2-7, and / or a portion of exon 8 (preferably also comprising intron 1 and / or intron 7) with at least 200-1000, 2000-8000, 10000-15000, 20000-25000 bp or 26000-30000 nucleotides. In some embodiments, the nucleotide sequence encoding human or chimeric CRTAM 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: 5. In some embodiments, the nucleotide sequence encoding the corresponding region of endogenous CRTAM comprises a portion of exon 1 of the endogenous CRTAM gene of a non-human animal, all of exons 2-7, and / or a portion of exon 8 (preferably also comprising intron 1 and / or intron 7). In some embodiments, the modified CRTAM 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 CRTAM locus of the non-human animal, the nucleotide sequence corresponding to the endogenous CRTAM of the non-human animal is replaced with a nucleotide sequence comprising the nucleotide sequence of human CRTAM. In some embodiments, the nucleotide sequence of the human CRTAM comprises a nucleotide sequence encoding a human or chimeric CRTAM protein. In some embodiments, the nucleotide sequence of the human CRTAM comprises a nucleotide sequence encoding all or part of the extracellular region of the human CRTAM protein, preferably comprising a nucleotide sequence encoding all or part of the signal peptide of the human CRTAM protein. In some embodiments, the nucleotide sequence of the human CRTAM comprises a nucleotide sequence encoding all of the signal peptide and all of the extracellular region of the human CRTAM protein. In some embodiments, the nucleotide sequence of the human CRTAM comprises a nucleotide sequence encoding positions 1-287 of SEQ ID NO: 2 or positions 18-287 of SEQ ID NO: 2; or, a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence encoding positions 1-287 of SEQ ID NO: 2 or positions 18-287 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence of the human CRTAM may be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the nucleotide sequence of the human CRTAM comprises all or part of the human CRTAM gene. In some embodiments, the nucleotide sequence of the human CRTAM comprises at least 50 to 34144 bp, such as at least 50, 100, 500, 800, 810, 861, 1000, 25000, 28000, 28906, 30000, 34000, 34100 or 34144 bp of continuous or discontinuous nucleotide sequences that are identical to the human CRTAM gene. In some embodiments, the nucleotide sequence of the human CRTAM comprises a part of exon 1 to a part of exon 8 of the human CRTAM gene. In some embodiments, the nucleotide sequence of the human CRTAM comprises the start codon of the human CRTAM gene to a part of exon 8. In some embodiments, the part of exon 1 of the human CRTAM gene comprises at least 5-93 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 46, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 93 bp of continuous nucleotide sequences, preferably comprising the nucleotide sequence of the coding region.In some embodiments, a portion of exon 8 of the human CRTAM gene comprises at least 5-147 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 44, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 145 or 147 bp of contiguous nucleotide sequence, preferably a nucleotide sequence comprising a coding region. In some embodiments, the nucleotide sequence of the human CRTAM comprises SEQ ID NO: 5; 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: 5. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous CRTAM comprises a nucleotide sequence encoding the non-human animal endogenous CRTAM protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous CRTAM comprises a nucleotide sequence encoding all or part of the extracellular region of the non-human animal endogenous CRTAM protein, preferably a nucleotide sequence encoding all or part of the signal peptide of the non-human animal endogenous CRTAM protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous CRTAM comprises a nucleotide sequence encoding all of the signal peptide and all of the extracellular region of the non-human animal endogenous CRTAM protein. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous CRTAM encodes the nucleotide sequence of amino acids 1-289 of SEQ ID NO: 1 or amino acids 17-289 of SEQ ID NO: 1; 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 1-289 of SEQ ID NO: 1 or amino acids 17-289 of SEQ ID NO: 1. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous CRTAM comprises a portion of exon 1 to a portion of exon 8 of the non-human animal endogenous CRTAM gene. In some embodiments, the nucleotide sequence of the corresponding region of the non-human animal endogenous CRTAM comprises the start codon of the non-human animal endogenous CRTAM gene to a portion of exon 8. In some embodiments, a portion of exon 1 of the non-human animal endogenous CRTAM gene comprises at least 5-86 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 46, 50, 55, 60, 65, 70, 75, 80, 85 or 86 bp of contiguous nucleotide sequence, preferably a nucleotide sequence comprising a coding region.In some embodiments, a portion of exon 8 of the endogenous CRTAM gene in a non-human animal preferably comprises at least 5-147 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 44, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 145 or 147 bp of contiguous nucleotide sequence, preferably comprising a nucleotide sequence of the coding region. In some embodiments, the nucleotide sequence encoding a human or chimeric CRTAM protein or the nucleotide sequence of human CRTAM is operably linked to an endogenous regulatory element (such as a promoter, 5'UTR and / or 3'UTR) of the endogenous CRTAM locus. In some embodiments, the endogenous CRTAM protein in the non-human animal is not expressed or has a reduced expression level compared to CRTAM in a wild-type animal. In some embodiments, the modified CRTAM gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous locus being replaced. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent is a rat or a mouse. In some embodiments, the mRNA transcribed from the modified CRTAM gene in the genome of the non-human animal 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, the other human or chimeric protein comprising at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0008] 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 CRTAM protein. In some embodiments, the human or chimeric CRTAM protein comprises at least 50 to 393 contiguous amino acid sequences corresponding to the corresponding region of the human CRTAM protein, such as at least 50, 100, 150, 200, 250, 270, 280, 287, 300, 350, 390 or 393 contiguous amino acid sequences. In some embodiments, the non-human animal expresses a human or chimeric CRTAM protein. In some embodiments, the human or chimeric CRTAM protein comprises an amino acid sequence identical or having an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the amino acids at positions 1-287 of SEQ ID NO: 2 or the amino acids at positions 18-287 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human or chimeric CRTAM protein is operably linked to an endogenous CRTAM regulatory element, such as a promoter, 5'UTR and / or 3'UTR. In some embodiments, the nucleotide sequence encoding the human or chimeric CRTAM protein is integrated into the endogenous CRTAM locus of the non-human animal. In some embodiments, the chimeric CRTAM protein has at least one activity, such as endogenous CRTAM activity and / or human CRTAM activity of the non-human animal.

[0009] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. In some embodiments, in at least one cell of the non-human animal, at the endogenous CRTAM locus of the non-human animal, the nucleotide sequence encoding the endogenous CRTAM region of the non-human animal is replaced by the nucleotide sequence encoding the corresponding region of human CRTAM. In some embodiments, the endogenous CRTAM protein of the non-human animal is not expressed or has a reduced expression level compared to CRTAM in a wild-type animal. In some embodiments, the corresponding region of human CRTAM is the extracellular region of human CRTAM. In some embodiments, the corresponding region of human CRTAM is the extracellular region and signal peptide of human CRTAM. In some embodiments, the nucleotide sequence encoding the corresponding region of human CRTAM comprises a portion of exon 1 of the human CRTAM gene, all of exons 2-7, and / or a portion of exon 8 (preferably also comprising intron 1 and / or intron 7). Preferably, the nucleotide sequence encoding the corresponding region of human CRTAM comprises a portion of exon 1 of the human CRTAM gene, all of exons 2-7, and / or a portion of exon 8 (preferably also comprising intron 1 and / or intron 7) of at least 200-1000, 2000-8000, 10000-15000, 20000-25000, or 26000-30000 nucleotides. In some embodiments, the amino acid sequence of the corresponding region of human CRTAM 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 1-287 of SEQ ID NO: 2 or the amino acids at positions 18-287 of SEQ ID NO: 2. In some embodiments, the endogenous CRTAM region of the non-human animal is the endogenous extracellular region of CRTAM. In some embodiments, the endogenous CRTAM region of the non-human animal is the endogenous extracellular region and signal peptide of CRTAM. In some embodiments, the endogenous CRTAM region of the non-human animal is the endogenous extracellular region, endogenous transmembrane region, and / or endogenous cytoplasmic region of CRTAM. In some embodiments, the amino acid sequence of the corresponding region of human CRTAM 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: 2 or SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the corresponding region of human CRTAM 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: 5. In some embodiments, the nucleotide sequence encoding the endogenous CRTAM region of the non-human animal comprises a portion of exon 1 of the endogenous CRTAM gene of the non-human animal, all of exons 2-7, and / or a portion of exon 8 (preferably also comprising intron 1 and / or intron 7).In some embodiments, the nucleotide sequence encoding the corresponding region of human CRTAM is operably linked to a non-human animal endogenous CRTAM 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 CRTAM, the construction method comprising replacing, at the endogenous CRTAM locus of the non-human animal, the nucleotide sequence encoding the endogenous CRTAM region with the nucleotide sequence encoding the corresponding region of human CRTAM to generate a genetically modified non-human animal cell, and the non-human animal cell expressing human or chimeric CRTAM protein. In some embodiments, the chimeric CRTAM protein comprises a human or humanized extracellular region. In some embodiments, the chimeric CRTAM protein comprises a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the chimeric CRTAM protein comprises a human extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the chimeric CRTAM protein comprises or does not comprise a signal peptide. In some embodiments, the signal peptide is an endogenous signal peptide of the non-human animal or a human or humanized signal peptide. In some embodiments, the chimeric CRTAM protein comprises a human or humanized signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the chimeric CRTAM protein comprises a human signal peptide, a human extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the corresponding region of human CRTAM comprises a portion of exon 1 of the human CRTAM gene, all of exons 2-7, and / or a portion of exon 8 (preferably also comprising intron 1 and / or intron 7), preferably, the nucleotide sequence encoding the corresponding region of human CRTAM comprises a portion of exon 1 of the human CRTAM gene, all of exons 2-7, and / or a portion of exon 8 (preferably also comprising intron 1 and / or intron 7) of at least 200-1000, 2000-8000, 10000-15000, 20000-25000, or 26000-30000 nucleotides. In some embodiments, the amino acid sequence of the corresponding region of human CRTAM 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 1-287 of SEQ ID NO: 2 or the amino acid sequence of positions 18-287 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the corresponding region of human CRTAM 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: 2 or SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the corresponding region of human CRTAM 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: 5. In some embodiments, the endogenous CRTAM region comprises an endogenous extracellular region, preferably also comprising an endogenous signal peptide.In some embodiments, the endogenous CRTAM region comprises an endogenous CRTAM extracellular region, an endogenous transmembrane region, and / or an endogenous cytoplasmic region (preferably further comprising an endogenous signal peptide). In some embodiments, the nucleotide sequence encoding the endogenous CRTAM region comprises a portion of exon 1 of the non-human animal CRTAM gene, all of exons 2-7, and / or a portion of exon 8 (preferably further comprising intron 1 and / or intron 7). In some embodiments, the nucleotide sequence encoding the corresponding region of human CRTAM is operably linked to regulatory elements of endogenous CRTAM, 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 method for determining the efficacy 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 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 CRTAM, a nucleic acid drug targeting CRTAM, and / or a polypeptide drug. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent, which preferably comprises one or more antibodies specifically binding to 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 gastrointestinal pancreatic cancer, lymphocytic tumors, breast cancer, head and neck cancer, liver cancer, or lung cancer. In some embodiments, the immune disease comprises one or more of asthma, atopic dermatitis, psoriasis, rheumatoid arthritis, or multiple sclerosis. In some embodiments, the inflammation comprises inflammatory bowel disease (IBD).

[0012] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating cancer (tumor). In some embodiments, the method comprises: 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 cancer; 2) determining the inhibitory effect of the therapeutic agent on cancer. In some embodiments, the therapeutic agent comprises an antibody targeting CRTAM, a nucleic acid drug targeting CRTAM, 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 animal body. In some embodiments, the cancer includes solid tumors or hematological tumors. In some embodiments, the cancer includes one or more of gastrointestinal pancreatic cancer, lymphocyte tumors, breast 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.

[0013] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating immune diseases. In some embodiments, the method comprises: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the preparation method, wherein the non-human animal has an immune disease; 2) determining the therapeutic effect of the therapeutic agent on the immune disease. In some embodiments, the therapeutic agent comprises an antibody targeting CRTAM, a nucleic acid drug targeting CRTAM, and / or a polypeptide drug. In some embodiments, the immune diseases include one or more of asthma, atopic dermatitis, psoriasis, rheumatoid arthritis, or multiple sclerosis.

[0014] 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 comprises: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the preparation 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 comprises an antibody targeting CRTAM, a nucleic acid drug targeting CRTAM, and / or a polypeptide drug. In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0015] 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 preparation method; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the therapeutic agent includes an antibody targeting CRTAM, a nucleic acid drug targeting CRTAM, 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.

[0016] In one aspect, the present invention provides a humanized CRTAM protein, which comprises all or part of the human CRTAM protein. In some embodiments, the humanized CRTAM protein comprises all or part of the extracellular region of the human CRTAM protein, preferably all or part of the signal peptide of the human CRTAM protein. In some embodiments, the humanized CRTAM protein comprises all of the extracellular region and all of the signal peptide of the human CRTAM protein. In some embodiments, the humanized CRTAM protein comprises the extracellular region and signal peptide of the human CRTAM protein, as well as the cytoplasmic region and transmembrane region of a non-human animal. In some embodiments, the humanized CRTAM protein comprises amino acids 1-287 of SEQ ID NO: 2 or amino acids 18-287 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 1-287 of SEQ ID NO: 2 or amino acids 18-287 of SEQ ID NO: 2. In some embodiments, the humanized CRTAM 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 the amino acid sequence shown in SEQ ID NO: 11.

[0017] In one aspect, the present invention provides a humanized CRTAM gene, and the humanized CRTAM gene encodes the humanized CRTAM protein. In some embodiments, the humanized CRTAM gene comprises a portion from exon 1 to exon 8 of the human CRTAM gene. In some embodiments, the humanized CRTAM gene comprises a portion from the start codon of the human CRTAM gene to exon 8. In some embodiments, the portion of exon 1 of the human CRTAM gene preferably comprises at least 5 consecutive nucleotide sequences; the portion of exon 8 of the human CRTAM gene preferably comprises at least 5 consecutive nucleotide sequences. In some embodiments, the humanized CRTAM gene further comprises a portion of exon 1, a portion of exon 8, and / or all of exons 9-10 (preferably also including intron 8) of the endogenous CRTAM gene of a non-human animal, wherein the portion of exon 1 of the endogenous CRTAM gene of the non-human animal preferably comprises at least 5 consecutive nucleotide sequences, and the portion of exon 8 of the endogenous CRTAM gene of the non-human animal preferably comprises at least 5 consecutive nucleotide sequences. In some embodiments, the nucleotide sequence comprised by the humanized CRTAM gene comprises SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 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 in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10.

[0018] In one aspect, the present invention provides a cell, tissue or organ, and in some embodiments, the cell, tissue or organ expresses the humanized CRTAM protein, and / or, the genome of the cell, tissue or organ comprises the humanized CRTAM gene.

[0019] In one aspect, the present invention provides an animal model, and in some embodiments, the animal model expresses the humanized CRTAM protein, and / or, the genome of the animal model comprises the humanized CRTAM gene.

[0020] In one aspect, the present invention provides the use of the non-human animal, the non-human animal obtained by the construction method, the humanized CRTAM protein, the humanized CRTAM gene, the cell, tissue or organ or the animal model, and the use includes: A) use in the development of products related to the CRTAM-related immune process involving human cells; B) use as a model system related to CRTAM in pharmacological, immunological, microbiological and medical research; C) use involved in the production and utilization of animal experimental disease models for etiological research related to CRTAM 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 human CRTAM signaling pathway regulators; or, E) use in studying the function of the CRTAM gene, studying the drugs and drug effects targeting human CRTAM target sites, and studying therapeutic drugs for cancers, inflammations or immune diseases related to CRTAM.

[0021] 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.

[0022] The term "locus" in the present invention generally represents the position occupied by a gene on a chromosome, and specifically 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 "CRTAM locus" includes a DNA fragment arbitrarily selected from exons 1-10 of the CRTAM gene.

[0023] The term "part of exon XX" in the present invention means that several, dozens or hundreds of nucleotides are consecutive or spaced apart and are identical to the entire exon nucleotide sequence. For example, the part of exon 1 of the human CRTAM gene contains at least 5-93 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 46, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 93 bp of consecutive nucleotide sequences, preferably including the nucleotide sequence of the coding region.

[0024] 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 containing one exon to another exon and the introns therebetween. For example, exon 1-10 contains the whole of exon 1, 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, and the whole of exon 10 nucleotide sequence.

[0025] The term "part of exon XX to part of exon XXX" in the present invention refers to the part containing a part of one exon to a part of another exon and the introns therebetween. For example, part of exon 1 to part of exon 8 contains the part of exon 1, 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, and the part of exon 8 nucleotide sequence.

[0026] The term "comprising" or "including" in the present invention is an open-ended writing method, including the specified components or steps described, as well as other specified components or steps that will not be substantially affected. 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 has the same or similar activity as the original sequence.

[0027] The term "and / or" in the present invention 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".

[0028] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. This application describes methods and materials for this invention; other suitable methods and materials known in the art can be used. The materials, methods, and examples are illustrative only 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.

[0029] Those skilled in the art can easily insight into other aspects and advantages of this application from the following detailed description.

[0030] CRTAM

[0031] In the human genome, the CRTAM gene (NCBI Gene ID: 56253, UniProt ID: O95727, located at positions 122838500 to 122872643 of chromosome 11 NC_000011.10) contains 10 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, and exon 10. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_019604.4 and its encoded protein NP_062550.2 (SEQ ID NO: 2) are shown in Table 1.

[0032] Table 1

[0033]

[0034] In the mouse genome, the CRTAM gene (NCBI Gene ID: 54698, UniProt ID: Q149L7, located at positions 40880987 to 40915922 of chromosome 9 NC_000075.7) contains 10 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, and exon 10. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_001281954.1 and its encoded protein NP_001268883.1 (SEQ ID NO: 1) are shown in Table 2.

[0035] Table 2

[0036]

[0037]

[0038] CRTAM genes, proteins, and genetic 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), and the relevant information of these genes (such as intron sequences, exon sequences, and amino acid sequences) can all be found in NCBI, the entire content of which is incorporated herein by reference.

[0039] To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison purposes). 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, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced to achieve the optimal alignment of the two sequences. For example, the comparison of sequences and the determination of the percent 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.

[0040] The percentage of conserved residues with similar physicochemical properties (percent homology), such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues 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 (such as aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (such as 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.

[0041] vector

[0042] The present invention provides a targeting vector, comprising: a) a DNA fragment (5' arm or 5' homology arm) homologous to the 5' end of the conversion region to be altered, which is selected from the genomic DNA of the CRTAM gene and has a length of 100 - 10,000 nucleotides; b) a donor region; and c) a DNA fragment (3' arm or 3' homology arm) homologous to the 3' end of the conversion region to be altered, which is selected from the genomic DNA of the CRTAM gene and has a length of 100 - 10,000 nucleotides.

[0043] 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_000075.7; 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_000075.7.

[0044] 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 positions 40915883 to 40918964 of NCBI accession number NC_000075.7; c) the DNA fragment homologous to the 3' end of the conversion region to be altered is selected from the nucleotide sequence positions 40886561 to 40889506 of NCBI accession number NC_000075.7.

[0045] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed 0.8 kb, 1 kb,

[0046] 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.

[0047] In some embodiments, the conversion region to be altered is located on the endogenous CRTAM gene locus of a non-human animal, preferably on exons 1 to 10 of the endogenous CRTAM gene of the non-human animal, and more preferably on exons 1 to 8 of the endogenous CRTAM gene of the non-human animal.

[0048] In some embodiments, the 5' arm comprises SEQ ID NO: 3. In some embodiments, the 3' arm comprises SEQ ID NO: 4.

[0049] In some embodiments, the donor region in b) comprises a human sequence. In some embodiments, the human sequence comprises the nucleotide sequence from position 122838547 to 122867452 of NCBI accession number NC_000011.10 (SEQ ID NO: 5).

[0050] In some embodiments, the targeting vector comprises one or more marker genes (or resistance genes), for example, a resistance gene for positive clone screening or a coding gene for a negative selection marker. In some embodiments, the resistance gene for positive clone screening 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.

[0051] 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 CRTAM 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'.

[0052] In some embodiments, the present invention relates to a plasmid construct containing 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.

[0053] 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.

[0054] 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.

[0055] 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 CRTAM protein.

[0056] Genetically modified non-human animals

[0057] 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, there is provided a genetically modified non-human animal, the non-human animal comprising a modified endogenous CRTAM locus comprising an exogenous sequence (e.g., a human sequence), e.g., replacing one or more non-human sequences with one or more human sequences, or inserting one or more human and / or non-human sequences. The non-human animal is generally capable of transmitting the genetic modification to its offspring through germline transmission.

[0058] 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 a portion of the 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.

[0059] 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 a portion of the 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 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.

[0060] The "humanized (X) protein" or "humanized (X) polypeptide" as used in the present invention refers to a protein or polypeptide. In some embodiments, at least a part of the protein or polypeptide is derived from a human (X) protein or a human (X) polypeptide. In some embodiments, at least a part 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.

[0061] The "humanized (X) nucleic acid" or "humanized (X) gene" as used in the present invention refers to a nucleic acid or a gene. In some embodiments, at least a part of the gene or nucleic acid is derived from a human (X) nucleic acid or a human (X) gene. In some embodiments, at least a part of the gene or nucleic acid is derived from a non-human animal (X) nucleic acid or a non-human animal (X) gene. 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 (X) nucleic acid or humanized (X) gene refers to a humanized exon, which can be a human exon or a chimeric exon.

[0062] In some embodiments, the chimeric CRTAM gene or chimeric CRTAM nucleic acid is a humanized CRTAM gene or humanized CRTAM nucleic acid. In some embodiments, at least a part of the humanized CRTAM gene or humanized CRTAM nucleic acid is derived from a human CRTAM gene. In some embodiments, at least a part of the humanized CRTAM gene or humanized CRTAM nucleic acid is derived from a non-human animal CRTAM gene. In some embodiments, the humanized CRTAM gene or humanized CRTAM nucleic acid contains a sequence encoding a CRTAM protein. In some embodiments, the encoded CRTAM protein has at least one activity, such as the activity of a human CRTAM protein or a non-human animal CRTAM protein.

[0063] In some embodiments, the chimeric CRTAM protein or chimeric CRTAM polypeptide is a humanized CRTAM protein or humanized CRTAM polypeptide. In some embodiments, at least one or more parts of the humanized CRTAM protein or humanized CRTAM polypeptide are derived from a human CRTAM protein. In some embodiments, at least one or more parts of the amino acid sequence of the humanized CRTAM protein or humanized CRTAM polypeptide are derived from a non-human animal CRTAM protein. In some embodiments, the humanized CRTAM protein or humanized CRTAM polypeptide is functional or has at least one activity, such as the activity of a human CRTAM protein or a non-human animal CRTAM protein.

[0064] In some embodiments, a humanized CRTAM protein or a humanized CRTAM polypeptide comprises an extracellular region, a cytoplasmic region, and a transmembrane region. In some embodiments, the humanized CRTAM protein or the humanized CRTAM 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. In some embodiments, the humanized CRTAM protein or the humanized CRTAM polypeptide comprises a human extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the humanized CRTAM protein or the humanized CRTAM polypeptide comprises a human signal peptide, a human extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region.

[0065] Genetically modified non-human animals include modification of an endogenous non-human animal endogenous CRTAM gene locus (locus). In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature CRTAM 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 a nucleotide sequence encoding a mature CRTAM protein (preferably the extracellular region of the CRTAM protein)). Cells (e.g., ES cells, somatic cells) that may comprise such genetic modification are provided in the present invention.

[0066] Genetically modified non-human animals can express a human CRTAM protein and / or a chimeric (e.g., humanized) CRTAM protein at the endogenous non-human animal CRTAM gene locus. In some embodiments, the endogenous CRTAM gene in the non-human animal genome has been replaced or inserted with a gene of human CRTAM and / or a nucleotide sequence encoding a human CRTAM 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 CRTAM sequence. In some embodiments, the endogenous non-human animal CRTAM gene locus is modified with a nucleic acid sequence comprising all or a portion of the coding sequence of a human mature CRTAM protein.

[0067] In some embodiments, the genetically modified non-human animal can express human CRTAM and / or chimeric CRTAM (e.g., humanized CRTAM) 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 CRTAM or chimeric CRTAM (e.g., humanized CRTAM) in appropriate cells and in a manner that does not result in potential pathologies observed in some other transgenic mice known in the art. The human CRTAM or chimeric CRTAM (e.g., humanized CRTAM) expressed in the non-human animal can maintain one or more functions of the wild-type non-human animal or human CRTAM in the non-human animal. Additionally, in some embodiments, the non-human animal does not express endogenous CRTAM. In some embodiments, the level of endogenous CRTAM expression in the non-human animal is reduced compared to the level of CRTAM expression in a wild-type animal. As used herein, the term "endogenous CRTAM" refers to the CRTAM protein expressed from the endogenous CRTAM nucleotide sequence of a non-human animal (e.g., mouse) prior to any genetic modification.

[0068] In some embodiments, the genetically modified non-human animal can have one or more cells that express human or chimeric CRTAM (e.g., humanized CRTAM), and the humanized CRTAM has an extracellular domain, a transmembrane domain, and a cytoplasmic domain (preferably with or without a signal peptide). In some embodiments, the human CRTAM and the endogenous CRTAM of the non-human animal (e.g., mouse CRTAM) sequences are different, so antibodies that bind to human CRTAM do not necessarily have the same affinity or effect on the endogenous CRTAM of the non-human animal. Thus, a genetically modified non-human animal having a human or humanized extracellular domain (preferably also having a human or humanized signal peptide) can be used to better evaluate the effect of therapeutic agents targeting human CRTAM in an animal model.

[0069] In some embodiments, the humanized CRTAM gene contains the 5'UTR of the human CRTAM gene. In some embodiments, the humanized CRTAM locus contains the 5'UTR of the endogenous (e.g., mouse endogenous) CRTAM gene of the non-human animal. In some embodiments, the humanized CRTAM gene contains the 3'UTR of the human CRTAM gene. In some embodiments, the humanized CRTAM gene contains the 3'UTR of the endogenous (e.g., mouse endogenous) CRTAM gene of the non-human animal. In appropriate cases, it can be reasonably assumed that, based on the similarity of the non-human animal and human CRTAM gene sequences, they appear to be regulated similarly. As shown in the present application, in a non-human animal with an insertion or replacement of humanized CRTAM at the endogenous CRTAM locus, which retains the endogenous regulatory elements of the non-human animal but contains a humanized CRTAM coding sequence, no pathologies are exhibited. Both heterozygous and homozygous genetically modified mice with humanized CRTAM are normal.

[0070] The genetically modified non-human animals can be various non-human animals, such as mice, rats, rabbits, pigs, cows (e.g., cattle, bulls, buffaloes), 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 non-ES cell genome (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., the modified oocyte) in a non-human animal under appropriate conditions to form an embryo. The construction methods described above 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.

[0071] 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.

[0072] 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 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, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant content of the above documents 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 the 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 strain. 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.

[0073] The non-human animal can have one or more other genetic modifications and / or other modifications 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 non-human animal's immune system. Impairment, inactivation, or disruption of the non-human animal's immune system 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 / γcnull mice (Ito, M. et al., NOD / SCID / γcnull 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, there is provided a genetically modified mouse that can include humanization of at least a portion of the non-human animal's endogenous CRTAM locus and also includes a modification that impairs, inactivates, or partially disrupts the non-human animal's immune system (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 - / -

[0074] (NRG) mice, Rag2 - / - IL2rg - / -(RG) Mice and their combined modifications. These transgenic animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety. In some embodiments, the mouse may include replacing all or part of the endogenous mature CRTAM coding sequence of the mouse with all or part of the human mature CRTAM coding sequence.

[0075] The present invention further relates to the genomic DNA sequence of humanized mouse CRTAM, the DNA sequence obtained by reverse transcription of mRNA is identical or complementary to this DNA sequence; the construct expressing its amino acid sequence; the cell containing its construct; the tissue or organ including its cell.

[0076] The present invention further relates to non-human mammals produced by the above method. In some embodiments, its genome contains the human CRTAM gene.

[0077] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0078] In some embodiments, the non-human mammal expresses the protein encoded by the humanized CRTAM gene.

[0079] In addition, the present invention also provides a non-human mammalian model carrying a tumor, and the non-human mammalian model 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).

[0080] The present invention also provides a cell or cell line derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a primary cell culture, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, a tissue, an organ or its culture derived from a non-human mammal or its offspring. When carrying a tumor, it is derived from the tumor tissue of a non-human mammal or its offspring or a non-human mammal carrying a tumor.

[0081] The present invention provides a non-human mammal produced by any method described in the present application. In some embodiments, a non-human mammal, a genetically modified non-human animal is provided, and the genome of the genetically modified non-human animal contains DNA of human or humanized CRTAM.

[0082] In some embodiments, the non-human mammal includes the gene construct described in the present application. In some embodiments, a non-human mammal expressing human or humanized CRTAM protein is provided. In some embodiments, a cell, tissue or organ specifically expressing human or humanized CRTAM protein is provided.

[0083] In some embodiments, the expression of human or humanized CRTAM protein in a non-human animal is controllable, e.g., 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).

[0084] 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 includes rodents. In some embodiments, the non-human mammal is a mouse.

[0085] 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.

[0086] 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 CRTAM protein can be detected by various methods.

[0087] There are many analytical methods available for detecting exogenous 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 transcriptional 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 CRTAM protein.

[0088] In some embodiments, the genetically modified non-human animals described in this application (e.g., homozygous humanized CRTAM gene mice or heterozygous humanized CRTAM gene mice) can express human or humanized CRTAM protein in one or more cells.

[0089] Method for constructing genetically modified non-human animals

[0090] 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.

[0091] In some embodiments, the nucleotide sequence encoding the endogenous CRTAM 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 CRTAM. In some embodiments, the expression level of the endogenous CRTAM 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.

[0092] The present invention provides a targeting vector, which comprises a 5' homologous arm, a human or humanized CRTAM gene fragment and a 3' homologous arm. This process involves introducing the human or humanized CRTAM gene fragment into the endogenous locus of the non-human animal by 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 the human or humanized CRTAM gene fragment is replaced into the endogenous CRTAM locus of the non-human animal by using homologous recombination.

[0093] In some embodiments, the method for preparing a genetically modified humanized animal comprises replacing the nucleotide sequence encoding the endogenous CRTAM region in the genome of the non-human animal with the nucleotide sequence encoding the corresponding region of human CRTAM at the endogenous CRTAM locus (or site) of the non-human animal.

[0094] Preferably, the non-human animal further comprises other genetic modifications. More preferably, the other genes include at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0095] Preferably, the human or humanized CRTAM gene and / or other genes are homozygous for the endogenous modified (preferably replaced or inserted) locus.

[0096] Preferably, the human or humanized CRTAM gene and / or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) locus.

[0097] Preferably, the non-human animal can be selected from any non-human animal capable of gene editing for gene humanization, such as rodents, pigs, rabbits, monkeys, etc.

[0098] 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.

[0099] Therefore, the present invention provides a method for constructing a non-human animal with humanized CRTAM gene, wherein the construction method enables the expression of human or humanized CRTAM 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 CRTAM gene or a humanized CRTAM gene.

[0100] In some embodiments, the method of modifying the CRTAM locus of a non-human animal to express a human / non-human animal chimeric CRTAM polypeptide may include replacing the nucleotide sequence encoding all or part of the non-human animal CRTAM (preferably including the extracellular region of the non-human animal CRTAM, and more preferably further including the signal peptide of the non-human animal endogenous CRTAM) at the endogenous CRTAM locus of the non-human animal with a nucleotide sequence encoding all or part of the human CRTAM (preferably including the extracellular region of the human CRTAM, and more preferably further including the signal peptide of the human CRTAM), thereby generating a sequence encoding a human / non-human animal chimeric CRTAM. In some embodiments, the method may include inserting a nucleotide sequence encoding a human / non-human animal chimeric CRTAM at the endogenous CRTAM locus of the non-human animal, thereby generating a sequence encoding a chimeric CRTAM.

[0101] The present invention also provides a method for establishing a humanized animal model of CRTAM gene, comprising the following steps:

[0102] (a) Providing cells (such as fertilized egg cells) based on the method described in the present application;

[0103] (b) Cultivate the cells (preferably cultivate the cells in a liquid medium);

[0104] (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;

[0105] (d) Identify germline transmission in the offspring of the genetically modified humanized non-human mammals of the pregnant female in step (c).

[0106] In some embodiments, the non-human mammal in the above method is a mouse (such as a C57BL / 6 mouse).

[0107] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0108] 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.

[0109] 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.

[0110] In some embodiments, the method for preparing a genetically modified non-human animal includes modifying the coding frame of the CRTAM gene of the non-human animal. For example, under the control of the endogenous regulatory element of the CRTAM gene of the non-human animal, the nucleic acid sequence encoding the endogenous CRTAM region is replaced with a nucleotide sequence encoding the corresponding region of human CRTAM (such as genomic DNA, CDS sequence, or cDNA sequence). For example, one or more functional region sequences of the CRTAM gene of the non-human animal can be knocked out or inserted with sequences, so that the endogenous CRTAM protein of the non-human animal cannot be expressed or the expression level is reduced.

[0111] In some embodiments, a method of producing a genetically modified non-human animal comprises inserting a nucleotide sequence encoding a human or humanized CRTAM protein and / or a helper sequence after an endogenous regulatory element of the CRTAM gene in the non-human animal. In some embodiments, the helper sequence may be a stop codon such that the humanized CRTAM gene animal model can express a human or humanized CRTAM protein in vivo but not the CRTAM protein of the non-human animal. In some embodiments, the helper sequence includes WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0112] In some embodiments, a method for producing a genetically modified non-human animal comprises:

[0113] (1) providing a plasmid containing a human CRTAM 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 CRTAM of the non-human animal;

[0114] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous CRTAM gene of the non-human animal;

[0115] (3) modifying the genome of a cell (fertilized egg or embryonic stem cell) by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0116] (4) transplanting 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 a humanized CRTAM protein. Preferably, the method further comprises:

[0117] (5) mating the offspring mice obtained in step (4) to obtain homozygous mice.

[0118] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal target site and a 3'-terminal target site.

[0119] In some embodiments, the sequence encoding the humanized CRTAM protein or the human CRTAM gene fragment is operably linked to an endogenous regulatory element at the endogenous CRTAM locus of the non-human animal.

[0120] In some embodiments, the genetically modified non-human animal does not express the endogenous CRTAM protein.

[0121] In some embodiments, a method for producing a genetically modified non-human animal comprises:

[0122] (1) Provide a plasmid containing a human or chimeric CRTAM 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 non-human animal endogenous CRTAM;

[0123] (2) Provide one or more guide RNAs (sgRNAs) targeting the non-human animal endogenous CRTAM gene;

[0124] (3) Modify the genome of a cell (such as a fertilized egg or embryonic stem cell) by inserting the human or chimeric CRTAM gene fragment into the non-human animal genome.

[0125] In some embodiments, the nucleotide sequence encoding the endogenous CRTAM protein in the non-human animal genome is deleted. In some embodiments, the nucleotide sequence encoding the extracellular region of the endogenous CRTAM protein in the non-human animal genome is deleted. In some embodiments, the nucleotide sequence encoding the signal peptide of the endogenous CRTAM protein in the non-human animal genome is deleted. In some embodiments, the nucleotide sequences encoding the signal peptide and the extracellular region of the endogenous CRTAM protein in the non-human animal genome are deleted. In some embodiments, the nucleotide sequence encoding positions 1-289 of SEQ ID NO: 1 or positions 17-289 of SEQ ID NO: 1 in the non-human animal genome is deleted. In some embodiments, all or part of the endogenous CRTAM gene in the non-human animal genome is deleted. In some embodiments, a portion of exon 1 to a portion of exon 8 of the endogenous CRTAM gene in the non-human animal genome is deleted. In some embodiments, the start codon of the endogenous CRTAM gene to a portion of exon 8 in the non-human animal genome is deleted.

[0126] In some embodiments, the construction method includes replacing the nucleotide sequence encoding the endogenous CRTAM protein in the genome of a non-human animal with the nucleotide sequence encoding the human CRTAM protein (such as a genomic DNA sequence, a CDS sequence, or a cDNA sequence). 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 the nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the construction method includes replacing the nucleotide sequence encoding the extracellular region of the human CRTAM protein with the nucleotide sequence encoding the extracellular region of the endogenous CRTAM protein in the genome of a non-human animal. In some embodiments, the construction method includes replacing the nucleotide sequence at positions 18-287 of SEQ ID NO: 2 with the nucleotide sequence at positions 17-289 of SEQ ID NO: 1 in the genome of a non-human animal. In some embodiments, the construction method includes replacing the nucleotide sequence encoding the signal peptide and the extracellular region of the human CRTAM protein with the nucleotide sequence encoding the signal peptide and the extracellular region of the endogenous CRTAM protein in the genome of a non-human animal. In some embodiments, the construction method includes replacing the nucleotide sequence at positions 1-287 of SEQ ID NO: 2 with the nucleotide sequence at positions 1-289 of SEQ ID NO: 1 in the genome of a non-human animal. In some embodiments, the construction method includes replacing all or part of the human CRTAM gene (such as a genomic DNA sequence, a CDS sequence, or a cDNA sequence) with all or part of the endogenous CRTAM gene in the genome of a non-human animal. In some embodiments, the construction method includes replacing the part from exon 1 to exon 8 of the human CRTAM gene with the part from exon 1 to exon 8 of the endogenous CRTAM gene in the genome of a non-human animal. In some embodiments, the construction method includes replacing the part from the start codon to exon 8 of the human CRTAM gene with the part from the start codon to exon 8 of the endogenous CRTAM gene in the genome of a non-human animal. In some embodiments, the construction method includes replacing SEQ ID NO: 5 with the nucleotide sequence encoding SEQ ID NO: 1 at positions 1-289 in the genome of a non-human animal. In some embodiments, the construction method includes replacing SEQ ID NO: 10 with the nucleotide sequence encoding SEQ ID NO: 1 in the genome of a non-human animal.

[0127] Application of Genetically Modified Non-Human Animals

[0128] 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 an 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. In human transgenes, the upstream and / or downstream of the human sequence provides appropriate support for the expression and / or regulation of the transgene.

[0129] Genetically modified non-human animals that express human or humanized CRTAM proteins, for example, in a physiologically appropriate manner, have a variety of uses, including but not limited to the development of therapeutic methods for human diseases and disorders, and the evaluation of the toxicity and / or efficacy of these human therapeutic methods in animal models.

[0130] The present invention also provides an application of the above-mentioned CRTAM gene-modified non-human animal and the non-human animal obtained by any of the above construction methods.

[0131] In some embodiments, the application includes:

[0132] A) Application in the development of products related to CRTAM-related immune processes involving human cells;

[0133] B) Application as a model system related to CRTAM in pharmacological, immunological, microbiological, and medical research;

[0134] C) Application involving the production and utilization of animal experimental disease models for CRTAM-related etiological research and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0135] D) Application in the screening, pharmacodynamic detection, evaluation of efficacy, verification, or evaluation of human CRTAM signaling pathway modulators in vivo; or,

[0136] E) Application in studying the function of the CRTAM gene, studying the drugs and pharmacodynamics targeting human CRTAM target sites, and studying therapeutic drugs for cancers, inflammations, or immune diseases related to CRTAM.

[0137] The present invention provides a non-human animal expressing a human or humanized CRTAM protein, which can be used for screening human CRTAM-specific therapeutic agents. The therapeutic agents can reduce or block the interaction between CRTAM and the CRTAM receptor complex, test whether the therapeutic agents can increase or decrease the immune response, and / or determine whether the therapeutic agents are CRTAM agonists or antagonists. 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 a genetically modified human xenograft, including human solid tumors (e.g., breast cancer) or hematological tumors (e.g., lymphocytic tumors (including B or T cell tumors)).

[0138] In some embodiments, the genetically modified non-human animal can be used to determine the effectiveness of therapeutic agents (such as antibodies targeting CRTAM, nucleic acid drugs targeting CRTAM, and / or polypeptide drugs) in treating 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, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, multiple sclerosis, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, etc. In some embodiments, the immune diseases include asthma, atopic dermatitis, psoriasis, rheumatoid arthritis or multiple sclerosis.

[0139] In some embodiments, the genetically modified non-human animal can be used to determine the effectiveness of therapeutic agents (such as antibodies targeting CRTAM, nucleic acid drugs targeting CRTAM, and / or polypeptide drugs) in treating tumors. In some embodiments, the method involves administering a therapeutic agent to a non-human animal, wherein the non-human animal has cancer or a tumor; and determining 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 reduced risk of metastasis or a reduced risk of one or more additional metastases, an increased survival rate and an extended life expectancy, etc. 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 CRTAM.

[0140] In some embodiments, the tumor comprises one or more cancer cells (e.g., human or murine cancer cells) injected into an animal. In some embodiments, the therapeutic agent activates or inhibits the CRTAM signaling pathway. In some embodiments, the therapeutic agent does not activate or does not inhibit the CRTAM signaling pathway.

[0141] In some embodiments, genetically modified non-human animals can be used to determine whether a therapeutic agent (e.g., an antibody targeting CRTAM, a nucleic acid drug targeting CRTAM, and / or a polypeptide drug) is a CRTAM agonist or antagonist. In some embodiments, the methods are also designed to determine the effect of a therapeutic agent (e.g., an antibody targeting CRTAM, a nucleic acid drug targeting CRTAM, and / or a polypeptide drug) on CRTAM, e.g., whether the therapeutic agent can upregulate or downregulate the immune response, and / or whether the reagent 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, such as cancer (tumor), inflammation, or immune disease, in a subject.

[0142] The inhibitory effect on the tumor can also be determined by methods known in the art, e.g., measuring the tumor volume in a non-human animal, and / or determining the tumor (volume) inhibition rate (TGI TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%) = (1 – T Vt / T Vc ) × 100, where T Vt and T Vc are the average tumor volumes (or weights) of the treatment group and the control group.

[0143] In some embodiments, therapeutic agents (e.g., antibodies targeting CRTAM, nucleic acid drugs targeting CRTAM, and / or polypeptide drugs) are designed for treating various cancers. As used herein, the term "cancer" or "tumor" refers to cells with the ability of autonomous growth, i.e., an abnormal state or condition characterized by rapid proliferation of cells. This term is intended to include all types of cancerous growths or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or stage of invasiveness. The term "tumor" as used in this application refers to cancer cells, such as a mass of cancer cells. Cancers that can be treated or diagnosed using the methods described in this application include malignant tumors of various organ systems, such as malignant tumors affecting the lung, breast, thyroid, lymph, gastrointestinal, and genitourinary tracts, as well as adenocarcinomas, which include malignant tumors such as colon cancer, renal cell carcinoma, prostate cancer, and / or testicular tumors, lung cancer (e.g., non-small cell lung cancer). In some embodiments, the therapeutic agents described in this application are designed for treating or diagnosing cancer in a subject. The term "carcinoma" is well recognized and refers to a malignant tumor 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., including malignant tumors 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 malignant tumor of mesenchymal origin.

[0144] In some embodiments, the cancers described in the present 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 lymphoblastic (lymphocytic) 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 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 cancers include solid tumors or hematological tumors. In some embodiments, the cancer is one or more of gastrointestinal pancreatic cancer, lymphocyte tumor, breast cancer, head and neck cancer, liver cancer, or lung cancer.

[0145] In some embodiments, therapeutic agents (e.g., antibodies targeting CRTAM, nucleic acid drugs targeting CRTAM, and / or polypeptide drugs) are 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. Accordingly, the methods described in the present application can be used to determine the effectiveness of therapeutic agents (such as antibodies targeting CRTAM, nucleic acid drugs targeting CRTAM, and / or polypeptide drugs) in inhibiting immune responses. In some embodiments, the immune diseases described in the present application are graft-versus-host disease (GVHD), psoriasis, allergy, asthma, myocarditis, atopic dermatitis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological diseases, etc.

[0146] In some embodiments, therapeutic agents (e.g., antibodies targeting CRTAM, nucleic acid drugs targeting CRTAM, and / or polypeptide drugs) are designed to treat various inflammations, such as viral inflammations. In some embodiments, the inflammations described in the present application include acute inflammations and chronic inflammations. Specifically, the inflammations include, but are not limited to, degenerative inflammations, exudative inflammations (e.g., serous inflammation, fibrin inflammation, suppurative inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammations, specific inflammations (such as tuberculosis, syphilis, leprosy, or lymphogranuloma). In some embodiments, the inflammations described in the present application include infections, and an 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).

[0147] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (such as an antibody targeting CRTAM, a nucleic acid drug targeting CRTAM, and / or a polypeptide drug). The method includes administering the therapeutic agent to the aforementioned non-human animal 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 therapeutic agent can reduce the number of red blood cells (RBCs), hematocrit, or hemoglobin content 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 (e.g., the average weight of non-human animals not treated with the therapeutic agent).

[0148] 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 as a model system for pharmacological, immunological, microbiological, and medical research.

[0149] In some embodiments, there is provided an animal model generated by the method described in the present application for producing and utilizing an animal experimental disease model of the immune process of human cells, studying pathogens, or formulating new diagnostic strategies and / or treatment strategies.

[0150] The present invention also provides an animal model generated by the method described in the present application for screening, validating, evaluating, or studying the function of the CRTAM gene, human CRTAM antibodies, drugs for diseases related to the human CRTAM target (such as tumors, inflammations, or immune diseases), or effectiveness.

[0151] 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 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 CRTAM gene humanized non-human animals prepared by the method, the CRTAM 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 methods described in the present application, non-human animals expressing human or humanized CRTAM proteins, or the tumor-bearing or inflammatory animal models described in the present application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies can treat CRTAM-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 CRTAM-related diseases (e.g., tumors, inflammation, or immune diseases) described in the present application.

[0152] Non-human animal models of two or more human or chimeric genes

[0153] 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 comprise a human or chimeric CRTAM gene and sequences encoding additional human or chimeric proteins.

[0154] In some embodiments, the additional human or chimeric proteins include at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the non-human animal also expresses at least one of human or humanized ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 proteins.

[0155] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the construction method comprising:

[0156] (i) Providing a non-human animal obtained by the above construction method;

[0157] (ii) Mating the non-human animal provided in step (i) 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.

[0158] In some embodiments, the other genetically modified non-human animals include non-human animals humanized with one or a combination of two or more of the genes ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0159] In some embodiments, the humanization of the CRTAM gene is carried out directly on a non-human animal having at least one modification of the human or chimeric ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 gene.

[0160] Since these proteins may be involved in different mechanisms, combination therapies targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are underway and showing good results. Multigene-modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, for example, antibodies targeting CRTAM, nucleic acid drugs and / or polypeptide drugs targeting CRTAM, 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, where the non-human animal has a disease (e.g., a tumor, inflammation, or immune disease), and determining the effect of the combination therapy on the disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to 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 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 tumor includes one or more tumor cells expressing PD-L1 and / or PD-L2.

[0161] In some embodiments, the combination therapy is used to treat various cancers (tumors). In some embodiments, the combination therapy is designed to treat the immune diseases described in the present application, such as psoriasis. In some embodiments, the methods described in the present application can be used to evaluate combination therapies with some other methods. Methods for treating cancer that can be used alone or in combination with the methods 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, bleomycin, nitrosourea, dactinomycin, daunorubicin, bleomycin, plicamycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, carboplatin, 5-fluorouracil, vincristine, vinblastine, and / or methotrexate. Alternatively, in addition, the method can include performing surgery on the subject to remove at least a portion of the cancer, such as removing a part or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] Figure 1 : Schematic diagram (not to scale) of the comparison between the mouse CRTAM locus and the human CRTAM locus;

[0163] Figure 2 : Schematic diagram (not to scale) of the CRTAM gene targeting strategy and the design of the targeting vector;

[0164] Figure 3 : PCR identification results of the F1 generation of CRTAM gene humanized mice, where WT is the wild-type control, H2O is the water control, and M is the Marker;

[0165] Figure 4 : RT-PCR detection results, where + / + is the wild-type C57BL / 6 mouse, H / H is the CRTAM gene humanized homozygous mouse, H2O is the water control, and GAPDH is the internal reference. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0166] 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 as the description progresses. 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 modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0167] Materials and Methods

[0168] In each of the following embodiments, the equipment and materials were obtained from the several companies indicated below:

[0169] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center, Chinese National Institute for Food and Drug Control;

[0170] BioLegend PerCP anti-mouse CD45 Antibody was purchased from Biolegend, with the catalog number 103130;

[0171] Brilliant Violet 510 TM anti-mouse CD4 was purchased from Biolegend, with the catalog number 100559;

[0172] Brilliant Violet 711 TM anti-mouse CD8a was purchased from Biolegend, with the catalog number 100759;

[0173] APC anti-mouse CD355(CRTAM)Antibody was purchased from Biolegend, with the catalog number 142007;

[0174] PE anti-human CD355(CRTAM)Antibody was purchased from Biolegend, with the catalog number 339106;

[0175] Brilliant Violet 421 TM anti-mouse TCRβchain Antibody was purchased from Biolegend, with the catalog number 109229.

[0176] Example 1: Humanized mice of CRTAM gene

[0177] The comparison schematic diagram of mouse CRTAM gene (NCBI Gene ID: 54698, located at positions 40880987 to 40915922 of NC_000075.7 on chromosome 9, based on transcript NM_001281954.1 and its encoded protein NP_001268883.1 (SEQ ID NO: 1)) and human CRTAM gene (NCBI Gene ID: 56523, located at positions 122838500 to 122872643 of NC_000011.10 on chromosome 11, based on transcript NM_019604.4 and its encoded protein NP_062550.2 (SEQ ID NO: 2)) is as Figure 1 shown.

[0178] To achieve the object of the present invention, a nucleotide sequence encoding a human CRTAM protein may be introduced into the endogenous CRTAM gene locus of a mouse, such that the mouse expresses a human or humanized CRTAM protein. Specifically, using gene editing technology, under the control of the mouse CRTAM gene regulatory elements, a partial sequence of exon 1 to a partial sequence of exon 8 of the human CRTAM gene, approximately 28.9 kb, is used to replace a partial sequence of exon 1 to a partial sequence of exon 8 of the mouse, approximately 26.4 kb, to obtain a humanized CRTAM gene locus, thereby achieving the humanization of the mouse CRTAM gene.

[0179] To implement the present invention, a targeting vector ( Figure 2 ) was constructed. The targeting vector V1 contains homologous arm sequences upstream and downstream of the mouse CRTAM gene, and a fragment A containing a human CRTAM gene fragment. Among them, the upstream 5' homologous arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence from position 40915883 to 40918964 of NCBI accession number NC_000075.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence from position 40886561 to 40889506 of NCBI accession number NC_000075.7. The nucleotide sequence of the human CRTAM gene fragment (SEQ ID NO: 5) is identical to the nucleotide sequence from position 122838547 to 122867452 of NCBI accession number NC_000011.10; the connection design of the upstream of the human CRTAM fragment sequence to the mouse is as follows: Among them, the last "C" in the sequence " CCAGC " is the last nucleotide at the junction of the upstream of the mouse and human CRTAM fragment sequences, and the "A" in the sequence is the first nucleotide of the human CRTAM fragment sequence. The connection design of the downstream of the human CRTAM fragment sequence to the mouse is as follows: Among them, the "C" in the sequence " GTGGC " is the last nucleotide of the human CRTAM fragment sequence, and the "A" in the sequence is the first nucleotide at the junction of the downstream of the mouse and human CRTAM fragment sequences.

[0180] 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 CRTAM gene is as follows: Among them, the last "C" in the sequence " CTGGC " is the last nucleotide at the junction of the human CRTAM gene and the 5' end of the Neo cassette, and the first "A" in the sequence is the first nucleotide of the Neo cassette; the connection of the 3' end of the Neo cassette to the human CRTAM gene is designed as: Among them, the last "C" in the sequence " GAGCC " is the last nucleotide of the Neo cassette, and the "A" in the sequence is the first nucleotide at the junction of the human CRTAM gene and the 3' end of the Neo cassette. The mRNA sequence transcribed from the CRTAM gene in the modified humanized mouse is shown in SEQ ID NO: 10, and the protein sequence expressed is shown in SEQ ID NO: 11.

[0181] 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 selection marker gene to screen out the correct positive clone cells. The correct positive clone cells (black mice) screened out are introduced into the 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 interbred to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone selection marker gene, and then the CRTAM gene humanized homozygous mice can be obtained by interbreeding.

[0182] The genotype of F1 generation mouse somatic cells can be identified by PCR method. PCR detection is carried out using the primers described in Table 3, and the exemplary results are as Figure 3 shown. Combining the PCR and sequencing results, 4 mice numbered F1-1 to F1-4 are positive mice (the unlabeled lanes are irrelevant bands). This indicates that the humanized mice with CRTAM gene that can be stably passed on and have no random insertion can be constructed using this method.

[0183] Table 3 PCR primer sequences and target fragment sizes

[0184]

[0185] The expression of mRNA in the humanized CRTAM gene mice can be detected by RT-PCR. Specifically, one 7-week-old C57BL / 6 mouse (+ / +) and one 7-week-old male humanized homozygous CRTAM gene mouse (H / H) prepared in this example were selected. After sacrificing by cervical dislocation, liver tissues were taken and RT-PCR was performed using the primer sequences shown in Table 4. The detection results are as Figure 4 shown. It can be seen from Figure 4 that only mouse CRTAM mRNA was detected in wild-type C57BL / 6 mice, and human CRTAM mRNA was not detected; only humanized CRTAM mRNA was detected in humanized homozygous CRTAM gene mice.

[0186] Table 4 RT-PCR primer sequences and target fragment sizes

[0187]

[0188] The expression of humanized CRTAM protein in mice was confirmed by flow cytometry. Specifically, spleen cells of 6-week-old male wild-type C57BL / 6 mice and 6-week-old male humanized homozygous CRTAM gene mice were selected. After staining with leukocyte marker antibodies BioLegend PerCP anti-mouse CD45 Antibody (mCD45), BrilliantViolet510 TM anti-mouse CD4 (mCD4), Brilliant Violet 711 TM anti-mouse CD8a (mCD8a), Brilliant Violet 421 TM anti-mouse TCRβ chain Antibody (mTCRβ), anti-mouse CRTAM antibody APCanti-mouse CD355 (CRTAM) Antibody (mCRTAM), and anti-human CRTAM antibody PE anti-human CD355 (CRTAM) Antibody (hCRTAM), flow cytometry was performed.

[0189] The results showed that among CD8+ T cells in C57BL / 6 mice, 72.0% were mCRTAM-positive cells (characterized by mCD45+mTCRβ+mCD19-mCD4+mCD8+mCRTAM+), 3.15% were hCRTAM-positive cells (characterized by mCD45+mTCRβ+mCD19-mCD4+mCD8+hCRTAM+), among CD8+ T cells in homozygous humanized CRTAM gene mice, 2.12% were mCRTAM-positive cells (characterized by mCD45+mTCRβ+mCD19-mCD4+mCD8+mCRTAM+), and 20.7% were hCRTAM-positive cells (characterized by mCD45+mTCRβ+mCD19-mCD4+mCD8+hCRTAM+). It was shown that the humanized CRTAM gene mice prepared in this application could successfully express humanized CRTAM protein.

[0190] Furthermore, ELISA was used to detect the secretion of IFN-γ in wild-type mice and humanized CRTAM gene mice. Specifically, 2 six-week-old wild-type C57BL / 6 mice (+ / +) and 2 six-week-old homozygous humanized CRTAM gene mice (H / H) were selected. After euthanasia, the spleen tissues of the mice were taken and processed into single-cell suspensions. After sorting out CD8+ T cells, anti-mouse CD3 antibody (anti-mCD3ε, concentration 0.25 μg / mL) (BioXcell, catalog number: BE0001-1), anti-mouse CD28 antibody (anti-mCD28, concentration 0.5 μg / mL) (BioXcell, catalog number: BE0015-1), and human recombinant NECL2 protein (hNECL2, concentration 5 μg / mL) (ACROB, catalog number: CA1-H5225) were added. Among them, the control group was not treated with hNECL2. After culturing at 37°C for 72 hours, the cell culture supernatant was collected for ELISA experiment, and ELISA MAX TM Deluxe Set Mouse IFN-γ kit (Biolegend, catalog number: 430804) was used to detect the secretion of mIFN-γ. The results showed that after treatment with hNECL2, only the mIFN-γ in homozygous humanized CRTAM gene mice (H / H) increased. It was shown that the humanized CRTAM protein expressed by humanized CRTAM gene mice could recognize human NECL2 and mediate downstream IFN-γ secretion (data not shown).

[0191] Example 2 Pharmacodynamic model

[0192] The humanized mice of CRTAM gene prepared by using Example 1 of the present application can be used to evaluate the efficacy of regulators targeting human CRTAM (for example, antibodies targeting CRTAM, nucleic acid drugs targeting CRTAM, and / or polypeptide drugs) in diseases (such as tumors, inflammation, or immune diseases). For example, take homozygous humanized mice of CRTAM gene and subcutaneously inoculate them with MC38 cells. Wait until the tumor volume grows to about 100 mm 3 After that, divide them into a control group or a treatment group according to the tumor volume. For the treatment group, randomly select drugs targeting human CRTAM, and for the control group, inject an equal volume of normal saline. Regularly measure the tumor volume and weigh the mice. The in vivo safety and in vivo efficacy of the compound can be effectively evaluated by comparing the changes in mouse body weight and tumor size.

[0193] Example 3 Preparation of double-gene or multi-gene humanized mice

[0194] The humanized mice of CRTAM gene prepared by using Example 1 of the present application can also be used to prepare a multi-gene humanized mouse model. For example, in the aforementioned Example 1, the embryonic stem cells used for microinjection can be selected from mice containing at least one gene modification of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. Alternatively, on the basis of the humanized mice of CRTAM gene, by using the technique of isolating mouse ES embryonic stem cells and gene recombination and targeting, a double-gene humanized or multi-gene humanized mouse model can be obtained. The homozygous or heterozygous CRTAM mice obtained by this method 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 humanized mice with humanized CRTAM gene and other gene modifications. Then, mating the heterozygotes with each other can obtain homozygotes with double-gene or multi-gene modifications.

[0195] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0196] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable way. To avoid unnecessary repetition, the present invention will not separately describe various possible combination ways.

[0197] Furthermore, any combination can be made between various different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

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 cytotoxic and regulatory T cell molecule (CRTAM) protein.

2. The construction method according to claim 1, characterized in that: The chimeric CRTAM protein comprises all or part of the extracellular region of the human CRTAM protein, and preferably also comprises all or part of the signal peptide of the human CRTAM protein; Preferably, the amino acid sequence of the chimeric CRTAM protein comprises amino acids 1-287 of SEQ ID NO: 2 or amino acids 18-287 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 amino acids 1-287 of SEQ ID NO: 2 or amino acids 18-287 of SEQ ID NO: 2; Preferably, the chimeric CRTAM protein comprises a human or humanized extracellular region, an endogenous transmembrane region and an endogenous cytoplasmic region; further preferably, the chimeric CRTAM protein comprises a human or humanized 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 CRTAM 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 endogenous CRTAM locus of the non-human animal, the nucleotide sequence of the corresponding region of the endogenous CRTAM of the non-human animal is replaced with a nucleotide sequence comprising human CRTAM.

4. The construction method according to claim 3, characterized in that: The nucleotide sequence of human CRTAM comprises a nucleotide sequence encoding a human or chimeric CRTAM protein, preferably comprises a nucleotide sequence encoding all or part of the extracellular region of a human CRTAM protein, and further preferably comprises a nucleotide sequence encoding all or part of the signal peptide of a human CRTAM protein; Preferably, the nucleotide sequence of human CRTAM comprises a portion from exon 1 to exon 8 of the human CRTAM gene. Further preferably, the nucleotide sequence of human CRTAM comprises a portion from the start codon to exon 8 of the human CRTAM gene. Wherein, the portion of exon 1 of the human CRTAM gene preferably comprises at least 5 bp of continuous nucleotide sequence; the portion of exon 8 of the human CRTAM gene preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence of human CRTAM comprises SEQ ID NO: 5; 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:

5.

5. The construction method according to claim 3 or 4, characterized in that: The nucleotide sequence of the corresponding region of the non-human animal endogenous CRTAM comprises a nucleotide sequence encoding the non-human animal endogenous CRTAM protein, preferably comprises all or part of the nucleotide sequence encoding the extracellular region of the non-human animal endogenous CRTAM protein, and further preferably comprises all or part of the nucleotide sequence encoding the signal peptide of the non-human animal endogenous CRTAM protein; Preferably, the nucleotide sequence of the corresponding region of endogenous CRTAM of the non-human animal comprises a nucleotide sequence encoding amino acids 1-289 of SEQ ID NO: 1 or 17-289 of SEQ ID NO: 1; 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 1-289 of SEQ ID NO: 1 or 17-289 of SEQ ID NO: 1; Preferably, the nucleotide sequence of the corresponding region of the endogenous CRTAM of the non-human animal includes a portion from exon 1 to exon 8 of the endogenous CRTAM gene of the non-human animal. Further preferably, the nucleotide sequence of the corresponding region of the endogenous CRTAM of the non-human animal includes a portion from the start codon to exon 8 of the endogenous CRTAM gene of the non-human animal. Among them, the part of exon 1 of the endogenous CRTAM gene of non-human animals preferably contains at least 5 bp of continuous nucleotide sequence; the part of exon 8 of the endogenous CRTAM gene of non-human animals preferably contains at least 5 bp of continuous nucleotide sequence.

6. The construction method according to claims 1-5, characterized in that: The nucleotide sequence encoding the human or chimeric CRTAM protein or the nucleotide sequence of human CRTAM is operably linked to an endogenous regulatory element of an endogenous CRTAM locus; Preferably, the endogenous CRTAM protein of the non-human animal is not expressed or is expressed at a reduced level compared to CRTAM in wild-type animals; Preferably, the modified CRTAM 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 is a mammal, such as a monkey or a rodent; preferably, the rodent is a rat or a mouse; Preferably, the mRNA transcribed from the modified CRTAM 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, wherein the other human or chimeric proteins comprise at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

8. 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 according to any one of claims 1 to 7; 2) Determining the effects of therapeutic agents on non-human animals or diseases; Preferably, the therapeutic agent includes an antibody targeting CRTAM, a nucleic acid drug targeting CRTAM and / or a polypeptide drug; further preferably, the therapeutic agent also includes an additional therapeutic agent, and the additional therapeutic agent preferably 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 gastrointestinal and pancreatic cancer, lymphocytic tumors, breast cancer, head and neck cancer, liver cancer or lung cancer; Preferably, the immune disease includes one or more of asthma, atopic dermatitis, psoriasis, rheumatoid arthritis or multiple sclerosis; Preferably, the inflammation comprises inflammatory bowel disease (IBD).

9. A humanized CRTAM protein, characterized in that: The humanized CRTAM protein comprises all or part of the human CRTAM protein; preferably comprises all or part of the extracellular region of the human CRTAM protein, and further preferably comprises all or part of the signal peptide of the human CRTAM protein; Preferably, the humanized CRTAM protein comprises amino acids 1-287 of SEQ ID NO: 2 or amino acids 18-287 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 amino acids 1-287 of SEQ ID NO: 2 or amino acids 18-287 of SEQ ID NO: 2; Further preferably, the humanized CRTAM 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 the amino acid sequence shown in SEQ ID NO:

11.

10. A humanized CRTAM gene, characterized in that: The humanized CRTAM gene encodes the humanized CRTAM protein according to claim 9; Preferably, the humanized CRTAM gene comprises a portion from exon 1 to exon 8 of the human CRTAM gene. Further preferably, the humanized CRTAM gene comprises a portion from the start codon to exon 8 of the human CRTAM gene. Wherein, the portion of exon 1 of the human CRTAM gene preferably comprises at least 5 bp of continuous nucleotide sequence; the portion of exon 8 of the human CRTAM gene preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the humanized CRTAM gene comprises the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 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 in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10.

11. A cell, tissue or organ, characterized in that: The cell, tissue or organ expresses the humanized CRTAM protein according to claim 9; and / or the genome of the cell, tissue or organ contains the humanized CRTAM gene according to claim 10.

12. Use of the non-human animal obtained by the construction method according to any one of claims 1 to 7, the humanized CRTAM protein according to claim 9, the humanized CRTAM gene according to claim 10, and the cell, tissue or organ according to claim 11, characterized in that: The application includes: A) Application in product development involving CRTAM-related immune processes in human cells; B) Application as a model system related to CRTAM for pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for the study of CRTAM-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human CRTAM signaling pathway modulators; or E) Study the function of the CRTAM gene, study the drugs and efficacy targeting the human CRTAM target site, and study the application of therapeutic drugs for cancer, inflammation or immune diseases related to CRTAM.

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