NKG2d gene-modified non-human animal

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g. humanized) NKG2D protein and a method of using same.

Description

A non-human animal modified with NKG2D gene

[0001] Cross-references to related applications

[0002] This application claims the benefit of priority of Chinese invention patent application No. 202310952970.0 filed on July 31, 2023, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention provides a non-human animal expressing a human or chimeric (eg, humanized) NKG2D protein and methods of use thereof. Background Art

[0004] Traditional drug development typically uses in vitro screening methods. However, these screening methods cannot provide the body's environment (such as the tumor microenvironment, stromal cells, extracellular matrix components, and immune cell interactions), resulting in a high rate of drug development failure. In addition, given the differences between humans and animals, the results of in vivo pharmacology trials using conventional experimental animals may not reflect the actual disease state and the interaction of target sites, resulting in significant differences between the results of many clinical trials and those of animal experiments.

[0005] Therefore, developing humanized animal models suitable for screening and evaluation of human antibodies will significantly improve the efficiency of new drug development and reduce drug development costs.

[0006] Summary of the Invention

[0007] The present application provides an animal model with a human or chimeric NKG2D protein. The animal model can express a human or chimeric NKG2D (e.g., humanized NKG2D) protein and / or contain a human or chimeric NKG2D (e.g., humanized NKG2D) gene. It can be used to study the function of the NKG2D gene, and can also be used for screening and evaluation of NKG2D / NKG2DL signaling pathway regulators (e.g., anti-human NKG2D antibodies). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamic studies, treatment of immune-related diseases, and cancer treatment of human NKG2D target sites; the model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of the NKG2D protein and provides a platform for screening anti-cancer drugs.

[0008] On the one hand, the present invention provides a genetically modified non-human animal, the genome of the non-human animal comprising at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric killer cell lectin-like receptor K1 (NKG2D) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric NKG2D protein is operably linked to an endogenous regulatory element or a human regulatory element (e.g., 5'UTR and / or 3'UTR) of the endogenous NKG2D locus of at least one chromosome. In some embodiments, the chimeric NKG2D protein is a humanized NKG2D protein, and the humanized NKG2D protein comprises all or part of the extracellular region of a human NKG2D protein. In some embodiments, the humanized NKG2D protein comprises at least 50 to 144 consecutive amino acids identical to the extracellular region of the human NKG2D protein. In some embodiments, the humanized NKG2D protein further comprises all or part of the transmembrane region and / or cytoplasmic region of a human or non-human animal NKG2D protein. In some embodiments, the humanized NKG2D protein comprises a continuous amino acid sequence that is identical to a human NKG2D protein for at least 50 to 216 amino acids. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a mouse or rat). In some embodiments, the non-human animal is a mouse. In some embodiments, the human or chimeric NKG2D protein comprises SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9; or, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to the amino acid sequence as set forth in SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9; or, comprises an amino acid sequence that differs from SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid, or comprises an amino acid sequence that is a substitution, deletion and / or insertion of one or more amino acids as set forth in SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the human or chimeric NKG2D protein comprises SEQ ID NO: 7 or 48, or is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 7 or 48. In some embodiments, the non-human animal does not express endogenous NKG2D protein or expresses it at a reduced level compared to NKG2D in wild-type animals. In some embodiments, one or more cells of the non-human animal express the human or chimeric NKG2D protein. In some embodiments, the human or chimeric NKG2D protein can bind to an endogenous NKG2DL receptor, inducing activation of downstream signaling pathways.In some embodiments, the human or chimeric NKG2D protein can bind to the human NKG2DL receptor and induce activation of downstream signaling pathways.

[0009] On the one hand, the present invention provides a kind of genetically modified non-human animal, the genome of the non-human animal is included in the endogenous NKG2D locus, and the endogenous NKG2D gene is replaced with the nucleotide sequence comprising human NKG2D. In some embodiments, the nucleotide sequence of the human NKG2D is operably linked to the endogenous regulatory element or human regulatory element (such as, 5'UTR and / or 3'UTR) of the endogenous NKG2D locus, and preferably, one or more cells of the non-human animal express human or chimeric NKG2D protein. In some embodiments, the endogenous NKG2D protein of the non-human animal is not expressed or the protein expression level is reduced compared with NKG2D in wild-type animals. In some embodiments, the nucleotide sequence of the human NKG2D comprises all or part of exon 4, all of exons 5-7 and / or all or part of exon 8 of the human NKG2D gene. In some embodiments, it also comprises all of exons 1-3 of the human NKG2D gene. In some embodiments, the nucleotide sequence of the human NKG2D comprises the nucleotide sequence of the coding region of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D comprises the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D further comprises the 5'UTR and / or 3'UTR of the human NKG2D gene, and may further comprise a nucleotide sequence of at least 50bp of continuous nucleotides upstream of the 5'UTR of the human NKG2D gene and / or a nucleotide sequence of at least 50bp of continuous nucleotides downstream of the 3'UTR of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D comprises the 5'UTR of the human NKG2D gene and at least 50bp of continuous nucleotides upstream of the 5'UTR of the human NKG2D gene, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene, and the 3'UTR of the human NKG2D gene and at least 50bp of continuous nucleotides downstream of the 3'UTR of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D comprises part of exon 4 to part of exon 8 of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D comprises SEQ ID NO: 7 or 48; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to the nucleotide sequence of SEQ ID NO: 7 or 48; or comprises a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 7 or 48 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or comprises a nucleotide sequence that comprises a substitution, deletion, and / or insertion of one or more nucleotides from the nucleotide sequence of SEQ ID NO: 7 or 48. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a rat or mouse).In some embodiments, the mRNA transcribed from the modified nucleotide sequence of endogenous NKG2D in the genome of the non-human animal comprises SEQ ID NO: 8 or 38; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to the nucleotide sequence shown in SEQ ID NO: 8 or 38; or, comprises a nucleotide sequence that differs from SEQ ID NO: 8 or 38 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 bp of nucleotides; or, comprises a nucleotide sequence that is substituted, deleted and / or inserted with one or more nucleotides shown in SEQ ID NO: 8 or 38. In some embodiments, all or part of exon 4, all of exons 5-7, and / or all or part of exon 8 of an endogenous NKG2D gene are replaced. Preferably, the replaced endogenous NKG2D gene also includes all of exons 1-3, and more preferably, also includes at least 50 bp of contiguous nucleotides upstream of the 5' UTR and / or at least 50 bp of nucleotides downstream of the 3' UTR of the endogenous NKG2D gene. In some embodiments, a portion of exon 4 to a portion of exon 8 of an endogenous NKG2D gene is replaced. In some embodiments, a portion of at least 50 bp of contiguous nucleotides upstream of the 5' UTR to at least 50 bp of nucleotides downstream of the 3' UTR of the endogenous NKG2D gene is replaced. In some embodiments, the modified NKG2D gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the human or chimeric NKG2D protein expressed by the non-human animal has at least one NKG2D activity, e.g., a non-human animal NKG2D activity and / or a human NKG2D activity. In some embodiments, the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins are selected from at least one of NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40 and B7-H3.

[0010] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or chimeric NKG2D protein, wherein the human or chimeric NKG2D protein comprises at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 150, 160, 170, 180, 190, 200, 210, or 216 consecutive amino acids identical to a human NKG2D protein. In some embodiments, the human or chimeric NKG2D protein comprises all or part of the extracellular region of a human NKG2D protein, the transmembrane region of a human or non-human animal NKG2D protein, and / or the cytoplasmic region of a human or non-human animal NKG2D protein. In some embodiments, the human or chimeric NKG2D protein comprises SEQ ID NO: 2, or is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the human or chimeric NKG2D protein comprises all or part of the extracellular region of a human NKG2D protein. In some embodiments, the amino acid sequence of the extracellular region of the human or chimeric NKG2D protein comprises the amino acid sequence of positions 78-216 of SEQ ID NO: 2, or is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of positions 78-216 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the human or chimeric NKG2D protein comprises SEQ ID NO: 9, or is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of positions 78-216 of SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the human or chimeric NKG2D protein is operably linked to an endogenous regulatory element or a human regulatory element (e.g., 5'UTR and / or 3'UTR) of an endogenous NKG2D locus in at least one chromosome. In some embodiments, the nucleotide sequence encoding the human or chimeric NKG2D protein can be integrated into the endogenous NKG2D locus of the non-human animal. In some embodiments, the chimeric (e.g., humanized) NKG2D protein has at least one activity of a mouse NKG2D and / or a human NKG2D.

[0011] In one aspect, the present invention provides a non-human animal genome comprising at least one chromosome comprising a nucleotide sequence encoding a human or chimeric killer cell lectin-like receptor K1 (NKG2D) protein. In some embodiments, the endogenous NKG2D gene is silenced or destroyed (e.g., deleted). In some embodiments, the endogenous NKG2D gene is deleted, and the deleted region is replaced by a nucleotide sequence encoding a human or chimeric NKG2D protein. In some embodiments, the endogenous NKG2D gene is deleted, and the deleted region is replaced by a nucleotide sequence of a human NKG2D. In some embodiments, the chimeric NKG2D protein is a humanized NKG2D protein, comprising a portion of a human NKG2D protein and a portion of a non-human animal NKG2D protein. In some embodiments, the humanized NKG2D protein comprises a continuous amino acid sequence that is at least 50 to 216 amino acids identical to a human NKG2D protein. In some embodiments, the human or chimeric NKG2D protein comprises SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9; or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to the amino acid sequence set forth in SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9; or an amino acid sequence that differs from SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid, or an amino acid sequence that comprises a substitution, deletion, and / or insertion of one or more amino acids from SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9. In some embodiments, the endogenous NKG2D gene is replaced at the endogenous NKG2D locus of a non-human animal with a nucleotide sequence comprising a human NKG2D. In some embodiments, the nucleotide sequence of human NKG2D comprises all or part of exon 4, all of exons 5-7, and / or all or part of exon 8 of the human NKG2D gene, and preferably also comprises all of exons 1-3 of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D comprises the nucleotide sequence of the coding region of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D comprises the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D comprises part of exon 4 to part of exon 8 of the human NKG2D gene.In some embodiments, the nucleotide sequence of human NKG2D comprises a nucleotide sequence of at least 50 bp of consecutive nucleotides upstream of the 5'UTR of the human NKG2D gene to a nucleotide sequence of at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D comprises a nucleotide sequence of human NKG2D comprising SEQ ID NO: 7 or 48; or, a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology to the nucleotide sequence of SEQ ID NO: 7 or 48; or, a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 7 or 48 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, a nucleotide sequence comprising a substitution, deletion and / or insertion of one or more nucleotides from the nucleotide sequence of SEQ ID NO: 7 or 48. In some embodiments, all or part of exon 4, all of exons 5-7, and / or all or part of exon 8 of the endogenous NKG2D gene are replaced. Preferably, the replaced endogenous NKG2D gene also includes all of exons 1-3, and more preferably, also includes at least 50 bp of consecutive nucleotides upstream of the 5'UTR and / or at least 50 bp of nucleotides downstream of the 3'UTR of the endogenous NKG2D gene. In some embodiments, a portion of exon 4 to a portion of exon 8 of the endogenous NKG2D gene is replaced. In some embodiments, a portion of at least 50 bp of consecutive nucleotides upstream of the 5'UTR to at least 50 bp of nucleotides downstream of the 3'UTR of the endogenous NKG2D gene is replaced. In some embodiments, the deleted endogenous NKG2D gene includes a nucleotide sequence encoding an endogenous NKG2D protein, such as a sequence encoding SEQ ID NO: 1 or nucleotides 90-232 thereof. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a rat or mouse). In some embodiments, the mRNA transcribed from the modified nucleotide sequence of endogenous NKG2D in the genome of the non-human animal comprises SEQ ID NO: 8 or 38; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to the nucleotide sequence shown in SEQ ID NO: 8 or 38; or, comprises a nucleotide sequence that differs from SEQ ID NO: 8 or 38 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 bp of nucleotides; or, comprises a nucleotide sequence that is substituted, deleted and / or inserted with one or more nucleotides shown in SEQ ID NO: 8 or 38.

[0012] In one aspect, the present invention provides a cell comprising the non-human animal genome described above.

[0013] In one aspect, the present invention provides a non-human animal comprising the above-mentioned non-human animal genome or the above-mentioned cell.

[0014] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal. In some embodiments, the non-human animal expresses a human or chimeric NKG2D protein. In some embodiments, in at least one cell of the non-human animal, the endogenous NKG2D gene is replaced with a nucleotide sequence comprising human NKG2D at the non-human animal's endogenous NKG2D locus. In some embodiments, the non-human animal's endogenous NKG2D protein is not expressed or the protein expression level is reduced compared to NKG2D in wild-type animals. In some embodiments, the nucleotide sequence of the human NKG2D comprises a nucleotide sequence encoding all or part of the extracellular region of the human NKG2D protein, and preferably further comprises a nucleotide sequence encoding all or part of the transmembrane region and / or cytoplasmic region of the human or non-human animal NKG2D protein. In some embodiments, the nucleotide sequence of human NKG2D comprises all or part of exon 4, all of exons 5-7, and / or all or part of exon 8 of the human NKG2D gene, preferably further comprises all of exons 1-3 of the human NKG2D gene, further preferably comprises the nucleotide sequence of the coding region of the human NKG2D gene, and more preferably comprises the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D further comprises the 5'UTR and / or 3'UTR of the human NKG2D gene, further preferably further comprises a nucleotide sequence of at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human NKG2D gene and / or a nucleotide sequence of at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D comprises the 5'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides upstream of the 5'UTR and its outer side, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene, and the 3'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR and its outer side. In some embodiments, the nucleotide sequence of human NKG2D comprises part of exon 4 to part of exon 8 of the human NKG2D gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence of human NKG2D comprises SEQ ID NO: 2 or any one of positions 78-216 thereof, or SEQ ID NO: 9, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to the amino acid sequence of SEQ ID NO: 2 or any one of positions 78-216 thereof, or SEQ ID NO: 9.In some embodiments, the nucleotide sequence of human NKG2D comprises SEQ ID NO: 7 or 48; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to the nucleotide sequence of SEQ ID NO: 7 or 48; or, comprises a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 7 or 48 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotides; or, comprises a nucleotide sequence that is substituted, deleted and / or inserted with one or more nucleotides as shown in the nucleotide sequence of SEQ ID NO: 7 or 48. In some embodiments, all or part of exon 4 of the endogenous NKG2D gene, all of exons 5-7, and / or all or part of exon 8 are replaced. Preferably, the replaced endogenous NKG2D gene also includes all of exons 1-3, and further preferably also includes at least 50bp of continuous nucleotides upstream of the 5'UTR of the endogenous NKG2D gene and / or at least 50bp of nucleotides downstream of the 3'UTR. In some embodiments, part of exon 4 to part of exon 8 of the endogenous NKG2D gene are replaced. In some embodiments, at least 50bp of continuous nucleotides upstream of the 5'UTR of the endogenous NKG2D gene to at least 50bp of nucleotides downstream of the 3'UTR are replaced. In some embodiments, the nucleotide sequence of the human NKG2D is operably linked to an NKG2D regulatory element of the NKG2D locus of at least one chromosome, such as an endogenous regulatory element or a human NKG2D regulatory element, such as a promoter. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by other genes, wherein the human or chimeric protein is selected from at least one of NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40, and B7-H3. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a mouse or rat). In some embodiments, the non-human animal is a mouse.

[0015] In one aspect, the present invention provides a method for constructing cells of a genetically modified non-human animal that expresses a human or chimeric NKG2D protein. In some embodiments, the construction method comprises replacing the endogenous NKG2D gene at the non-human animal's endogenous NKG2D locus with a nucleotide sequence comprising human NKG2D. In some embodiments, the amino acid sequence encoded by the nucleotide sequence of the human NKG2D comprises all or part of the extracellular region of the human NKG2D protein, and preferably also comprises all or part of the transmembrane region and / or cytoplasmic region of the human or non-human animal NKG2D protein. In some embodiments, the nucleotide sequence of the human NKG2D comprises all or part of exon 4, all of exons 5-7, and / or all or part of exon 8 of the human NKG2D gene, and preferably also comprises all of exons 1-3 of the human NKG2D gene. In some embodiments, the nucleotide sequence of the human NKG2D comprises the nucleotide sequence of the coding region of the human NKG2D gene. In some embodiments, the nucleotide sequence of the human NKG2D comprises the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D further comprises the 5'UTR and / or 3'UTR of the human NKG2D gene, and more preferably further comprises a nucleotide sequence of at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human NKG2D gene and / or a nucleotide sequence of at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D includes the 5'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides upstream of the 5'UTR, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene, and the 3'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human NKG2D comprises a portion of exon 4 to a portion of exon 8 of the human NKG2D gene. In some embodiments, the nucleotide sequence of human NKG2D encodes an amino acid sequence comprising SEQ ID NO: 2, or any one of positions 78 to 216 thereof, or SEQ ID NO: 9, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2, or any one of positions 78 to 216 thereof, or SEQ ID NO: 9. In some embodiments, the nucleotide sequence of human NKG2D comprises SEQ ID NO: 7 or 48, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 7 or 48.In some embodiments, all or part of exon 4 of the endogenous NKG2D gene, all of exons 5-7, and / or all or part of exon 8 are replaced. Preferably, the replaced endogenous NKG2D gene also includes all of exons 1-3, and further preferably also includes at least 50bp of continuous nucleotides upstream of the 5'UTR of the endogenous NKG2D gene and / or at least 50bp of nucleotides downstream of the 3'UTR. In some embodiments, part of exon 4 of the endogenous NKG2D gene to part of exon 8 is replaced. In some embodiments, at least 50bp of continuous nucleotides upstream of the 5'UTR of the endogenous NKG2D gene to at least 50bp of nucleotides downstream of the 3'UTR are replaced. In some embodiments, the expression of the nucleotide sequence of the human NKG2D is regulated by a regulatory element, such as an endogenous regulatory element or a human regulatory element, and the regulatory element can be a promoter. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a mouse or rat). In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins are selected from at least one of NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40 and B7-H3.

[0016] In one aspect, the present invention provides a humanized NKG2D protein comprising all or part of a human NKG2D protein. In some embodiments, the humanized NKG2D protein comprises all or part of the extracellular domain of a human NKG2D protein, and preferably further comprises all or part of the transmembrane and / or cytoplasmic domains of a human or non-human animal NKG2D protein. In some embodiments, the humanized NKG2D protein amino acid sequence comprises SEQ ID NO: 2 or any one of positions 78-216 thereof or SEQ ID NO: 9; or, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to SEQ ID NO: 2 or any one of positions 78-216 thereof or SEQ ID NO: 9; or, comprises an amino acid sequence that differs from SEQ ID NO: 2 or any one of positions 78-216 thereof or SEQ ID NO: 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid, or comprises an amino acid sequence that is substituted, deleted and / or inserted with one or more amino acids as shown in SEQ ID NO: 2 or any one of positions 78-216 thereof or SEQ ID NO: 9.

[0017] In one aspect, the present invention provides a humanized NKG2D gene, which encodes the above-mentioned humanized NKG2D protein. In some embodiments, the humanized NKG2D gene comprises all or part of exon 4 of a human NKG2D gene, all of exons 5-7, and / or all or part of exon 8, and preferably further comprises all of exons 1-3 of a human NKG2D gene. In some embodiments, the humanized NKG2D gene comprises the nucleotide sequence of the coding region of a human NKG2D gene. In some embodiments, the humanized NKG2D gene comprises the nucleotide sequence from the start codon to the stop codon of a human NKG2D gene. In some embodiments, the humanized NKG2D gene further comprises the 5'UTR and / or 3'UTR of a human NKG2D gene, and further preferably further comprises a nucleotide sequence of at least 50bp of continuous nucleotides upstream of the 5'UTR of a human NKG2D gene and / or a nucleotide sequence of at least 50bp of continuous nucleotides downstream of the 3'UTR of a human NKG2D gene. In some embodiments, the humanized NKG2D gene comprises the 5'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides upstream of the 5'UTR, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene, and the 3'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the humanized NKG2D gene comprises a portion of exon 4 to a portion of exon 8 of the human NKG2D gene. In some embodiments, the humanized NKG2D gene comprises the nucleotide sequence of any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 10, 11, 35, 36, 38, 39, 40, and 48; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to the nucleotide sequence of any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 10, 11, 35, 36, 38, 39, 40, and 48.

[0018] In one aspect, a targeting vector is provided, comprising a 5' arm, a donor region, and a 3' arm, wherein the 5' arm is homologous to the 5' end of the switch region to be altered, the 3' arm is homologous to the 3' end of the switch region to be altered, and the donor region comprises a nucleotide sequence encoding a human or chimeric NKG2D protein. In some embodiments, the switch region to be altered is located at the endogenous NKG2D locus of a non-human animal, preferably the switch region to be altered comprises at least one exon or at least one intron of an endogenous NKG2D gene of the non-human animal, for example, from a portion of exon 4 to a portion of exon 8 of an endogenous NKG2D gene, or, at least 50 bp upstream of the outer side of the 5'UTR of the endogenous NKG2D gene to at least 50 bp downstream of the outer side of the 3'UTR of the endogenous NKG2D gene. In some embodiments, the 5' end of the switch region to be altered is located at exon 4 of an endogenous NKG2D gene of the non-human animal, and / or the 3' end of the switch region to be altered is located at exon 8 of an endogenous NKG2D gene of the non-human animal. In some embodiments, the 5' end of the switch region to be altered is located at least 50 bp upstream of the 5'UTR of the endogenous NKG2D gene of the non-human animal, and / or the 3' end of the switch region to be altered is located at least 50 bp downstream of the 3'UTR of the endogenous NKG2D gene of the non-human animal. In some embodiments, the 5' arm sequence is set forth in SEQ ID NO:3, and the 3' arm sequence is set forth in SEQ ID NO:4. In some embodiments, the 5' arm sequence is set forth in SEQ ID NO:5, and the 3' arm sequence is set forth in SEQ ID NO:6. In some embodiments, the 5' arm sequence is set forth in SEQ ID NO:35, and the 3' arm sequence is set forth in SEQ ID NO:36. In some embodiments, the chimeric NKG2D protein is a humanized NKG2D protein, which comprises portions of a human NKG2D protein and a non-human NKG2D protein. In some embodiments, the humanized NKG2D protein comprises a contiguous amino acid sequence of at least 50 to 216 amino acids identical to a human NKG2D protein. In some embodiments, the human or chimeric NKG2D protein comprises SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9; or, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to the amino acid sequence as set forth in SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9; or, comprises an amino acid sequence that differs from SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid, or comprises an amino acid sequence that is a substitution, deletion and / or insertion of one or more amino acids as set forth in SEQ ID NO: 2, or any one of positions 78-216 thereof, or SEQ ID NO: 9.In some embodiments, the donor region comprises all or part of exon 4, all of exons 5-7, and / or all or part of exon 8 of the human NKG2D gene, and preferably also comprises all of exons 1-3 of the human NKG2D gene. In some embodiments, the donor region comprises the nucleotide sequence of the coding region of the human NKG2D gene. In some embodiments, the donor region comprises the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene. In some embodiments, the donor region further comprises the 5'UTR and / or 3'UTR of the human NKG2D gene, and further preferably further comprises the nucleotide sequence of at least 50 bp of consecutive nucleotides upstream of the 5'UTR of the human NKG2D gene and / or the nucleotide sequence of at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human NKG2D gene. In some embodiments, the donor region includes the 5'UTR of the human NKG2D gene and at least 50 bp of consecutive nucleotides upstream of the 5'UTR, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene, and the 3'UTR of the human NKG2D gene and at least 50 bp of consecutive nucleotides downstream of the 5'UTR. In some embodiments, in some embodiments, the donor region includes a portion of exon 4 to a portion of exon 8 of the human NKG2D gene. In some embodiments, the donor region comprises SEQ ID NO: 7 or 48; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to the nucleotide sequence of SEQ ID NO: 7 or 48; or, comprises a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 7 or 48 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence that is substituted, deleted and / or inserted with one or more nucleotides as set forth in the nucleotide sequence of SEQ ID NO: 7 or 48.

[0019] In one aspect, the present invention provides a cell, tissue or organ, which expresses the above-mentioned humanized NKG2D protein, or comprises the above-mentioned humanized NKG2D gene.

[0020] In one aspect, the present invention provides an animal model, characterized in that the animal model expresses the above-mentioned humanized NKG2D protein, or comprises the above-mentioned humanized NKG2D gene, or comprises the above-mentioned cells, tissues or organs.

[0021] In one aspect, the present invention provides a method for determining the effectiveness of an NKG2D therapeutic agent in treating a disease, the method comprising: 1) administering an anti-NKG2D therapeutic agent to a non-human animal described herein, or a non-human animal obtained by the construction method, or an animal model described herein, wherein the non-human animal or animal model suffers from a disease; and 2) determining the inhibitory effect of the anti-NKG2D therapeutic agent on the disease. In some embodiments, the disease comprises a tumor, a viral infection-related disease, graft-versus-host disease, or an autoimmune disease. In some embodiments, the tumor comprises one or more tumor cells, wherein the tumor cells are injected into the non-human animal or animal model. In some embodiments, determining the inhibitory effect of the anti-NKG2D therapeutic agent on the disease comprises measuring tumor volume in the non-human animal or animal model. In some embodiments, the tumor comprises one or more of a solid tumor, gastrointestinal cancer, acute myeloid leukemia, colorectal cancer, gastrointestinal pancreatic cancer, breast cancer, multiple myeloma, genitourinary cancer, prostate cancer, glioblastoma, liver cancer, lung cancer, melanoma, lymphoma, bile duct cancer, ovarian cancer, nasopharyngeal tumor, nasopharyngeal carcinoma, or medulloblastoma. In some embodiments, the autoimmune disease includes one or more of rheumatoid arthritis, type I diabetes, alopecia areata, Crohn's disease, or atherosclerosis. In some embodiments, the viral infection-related disease includes one or more of the following viruses: cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis virus, Kaposi's sarcoma-associated herpes virus (KSHV), human papillomavirus (HPV), molluscum contagiosum virus (MCV), human T-cell leukemia virus 1 (HTLV-1), or HIV (human immunodeficiency virus). One or more of the following.

[0022] In one aspect, the present invention provides a method for determining the effectiveness of anti-NKG2D therapeutic agents and other therapeutic agents in treating a disease, the method comprising: 1) administering an anti-NKG2D therapeutic agent and other therapeutic agents to the non-human animal described herein or the non-human animal obtained by the construction method or the animal model, wherein the non-human animal or animal model suffers from a disease; 2) determining the inhibitory effect of the anti-NKG2D therapeutic agent and other therapeutic agents on the disease. In some embodiments, the disease comprises a tumor or an autoimmune disease. In some embodiments, the other therapeutic agents comprise anti-PD-1 antibodies, anti-PD-L1 antibodies, and / or anti-B7-H3 antibodies. In some embodiments, the tumor comprises one or more tumor cells, wherein the tumor cells are injected into the non-human animal or animal model. In some embodiments, determining the inhibitory effect of the anti-NKG2D therapeutic agent on the disease comprises measuring the tumor volume in the non-human animal or animal model. In some embodiments, the tumor includes one or more of solid tumors, gastrointestinal cancer, acute myeloid leukemia, colorectal cancer, gastrointestinal pancreatic cancer, breast cancer, multiple myeloma, genitourinary cancer, prostate cancer, glioblastoma, liver cancer, lung cancer, melanoma, lymphoma, bile duct cancer, ovarian cancer, nasopharyngeal tumor, nasopharyngeal carcinoma or medulloblastoma. In some embodiments, the autoimmune disease includes one or more of rheumatoid arthritis, type I diabetes, alopecia areata, Crohn's disease or atherosclerosis. In some embodiments, the viral infection-related disease includes a disease caused by one or more of the following viruses: cytomegalovirus (CMV), Epstein-Barr virus (Epstein-Barr Virus; EBV), hepatitis virus, Kaposi's sarcoma-associated herpes virus (KSHV), human papilloma virus (HPV), molluscum contagiosum virus (MCV), human T-cell leukemia virus 1 (HTLV-1) or HIV (human immunodeficiency virus). One or more of.

[0023] In one aspect, the present invention provides a method for determining the toxicity of an anti-NKG2D therapeutic agent, comprising: 1) administering the anti-NKG2D therapeutic agent to a non-human animal described herein, or a non-human animal obtained by the construction method, or an animal model described herein; and 2) determining the effect of the anti-NKG2D therapeutic agent on the non-human animal or animal model. In some embodiments, determining the effect of the anti-NKG2D therapeutic agent on the animal involves measuring the weight of the non-human animal or animal model or performing a blood test. Preferably, the blood test includes one or more of red blood cell count, hematocrit, or hemoglobin.

[0024] In one aspect, provided is an application of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal genome, the above-mentioned cell obtained by the above-mentioned construction method, the above-mentioned chimeric NKG2D protein, the above-mentioned humanized NKG2D gene, the above-mentioned cell, tissue or organ, the above-mentioned animal model or the above-mentioned targeting vector, wherein the application includes: A) application in the development of products involving immune processes related to NKG2D in human cells; B) application as a model system related to NKG2D in pharmacological, immunological, microbiological and medical research; C) application involving the production and use of animal experimental disease models for NKG2D-related pathological research and / or for developing diagnostic strategies and / or for developing therapeutic strategies; D) application in in vivo screening, efficacy testing, efficacy assessment, verification or evaluation of human NKG2D signaling pathway regulators; or, E) application in studying NKG2D gene function, studying drugs and drug efficacy targeting human NKG2D target sites, studying drugs for immune-related diseases or anti-tumor drugs related to NKG2D.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. This application describes methods and materials for use with the present invention; other suitable methods and materials known in the art may be used. The materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of conflict, the present specification, including definitions, will control.

[0026] The term "all or part" in the present invention, "all" refers to the whole, and "part" refers to a part of the whole, or an individual part that makes up the whole.

[0027] The term "humanized NKG2D protein" herein includes portions derived from human NKG2D proteins, and preferably also includes portions of non-human animal NKG2D proteins. For example, the "human NKG2D protein" is identical to the entire human NKG2D protein, i.e., the amino acid sequence is identical to the full-length amino acid sequence of the human NKG2D protein. The "portion of the human NKG2D protein" is 5-216 consecutive or intermittent amino acids, preferably 10-216 or 50-216 consecutive or intermittent amino acids, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 139, 140, 144, 150, 160, 170, 180, 190, 200, 210, or 216 amino acids, that are identical to the sequence of the human NKG2D protein.

[0028] The term "humanized NKG2D gene" herein encompasses portions derived from a human NKG2D gene. It preferably also encompasses portions of a non-human animal NKG2D gene. For example, the "human NKG2D gene" encompasses the entire human NKG2D gene, i.e., the nucleotide sequence is identical to the full-length nucleotide sequence of a human NKG2D gene. The "part of the human NKG2D gene" is a continuous or intermittent 20-1576bp, or 20-420, or 20-17689bp nucleotide sequence that is consistent with the nucleotide sequence of the human NKG2D gene, for example, a 20, 50, 100, 200, 300, 400, 420, 432, 450, 500, 1000, 1200, 1500, 1550, 1576, 2000, 2500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 17000, 17600 or 17689bp nucleotide sequence that is consistent with the nucleotide sequence of the human NKG2D gene.

[0029] The term "locus" in the present invention broadly refers to the location of a gene on a chromosome, and narrowly refers to a DNA segment on a gene, which can be either a gene or a portion of a gene. For example, the "NKG2D locus" described herein refers to a DNA segment selected from exons 1-8 of the NKG2D gene. In some embodiments, the endogenous NKG2D locus of a non-human animal to be replaced can be a DNA segment selected from exons 1-8 of the endogenous NKG2D gene of the non-human animal.

[0030] The term "portion of an exon" in the present invention means that continuous or intermittent several, dozens or hundreds of nucleotide sequences are consistent with the entire exon nucleotide sequence, for example, a portion of exon 4 of the human NKG2D gene, including continuous or intermittent 5-93bp, for example, 5, 6, 7, 8, 9, 10, 11, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 93bp nucleotide sequences are consistent with the nucleotide sequence of exon 1 of the human NKG2D gene.

[0031] The term "exon XX to exon XXX" or "exons XX-XXX" or "the entirety of exon XX to exon XXX" of the present invention refers to exons and introns therebetween. For example, exon 1 to exon 8 includes the entire nucleotide sequence of exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, and exon 8. For another example, exons 5-7 include the entire nucleotide sequence of exon 5, intron 5, exon 6, intron 6, and exon 7. For another example, part of exon 4 to part of exon 8 includes part of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, and a portion of the nucleotide sequence of exon 8.

[0032] The term "intron xx" in the present invention refers to an intron between two exons, for example, intron 1 is an intron between exon 1 and exon 2.

[0033] The terms "comprising" or "including" in the present invention are open-ended and encompass the specified components or steps being described, as well as other specified components or steps that do not substantially affect them. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still having the same or similar activity as the original sequence.

[0034] The term "and / or" in this invention includes all combinations of the items connected by the term, and each combination should be deemed to have been listed separately in this application. For example, "A and / or B" includes "A", "A and B", and "B". For 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".

[0035] Those skilled in the art will readily appreciate other aspects and advantages of the present application from the following detailed description.

[0036] Detailed description

[0037] NKG2D

[0038] NKG2D (killer cell lectin like receptor K1, member D), also known as KLRK1, was discovered on the surface of human NK cells in 1991. NKG2D belongs to the NKG2 family and is a type II transmembrane protein that is mainly expressed on cytotoxic immune cells. NKG2D is abundantly present in all NK cells, NKT cells, and γδT cell subsets. +It is also expressed on T cells, but mouse CD8 + T cells only upregulate expression after activation. + Even after activation, T cells do not express NKG2D, but its expression can be induced under specific pathological conditions in humans. As an activating receptor, NKG2D plays an important role in innate immunity, participating in the recognition of virus-infected cells and the killing of tumor cells by NK cells.

[0039] In the field of anti-tumor, NK cells have recently been at the forefront of many immunotherapy strategies, and new approaches are being developed to fully exploit the anti-tumor potential of NK cells. One of the most relevant NK cell activating receptors is NKG2D, and the human NKG2D ligand is NKG2DL. Immunotherapy targeting NKG2D / NKG2DL is under investigation, which can reshape the tumor microenvironment, release NK cells and cytotoxic CD8 + Anti-tumor effects of T cells.

[0040] In terms of graft-versus-host disease (GVHD), NKG2D is involved in the immune rejection reaction of mouse bone marrow transplantation. Blocking the NKG2D / NKG2DL pathway using NKG2D monoclonal antibodies can alleviate GVHD in BALB / c mouse bone marrow transplantation.

[0041] In the area of ​​autoimmune diseases, studies have reported that NKG2D ligands can be detected in tissues from patients with autoimmune diseases, including rheumatoid arthritis, type 1 diabetes, Crohn's disease, and atherosclerosis. Studies have shown that anti-NKG2D antibodies can significantly reduce the severity of collagen-induced arthritis.

[0042] In the human genome, the NKG2D gene (Gene ID: 22914) contains eight exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 (Figure 1). The nucleotide sequence of human NKG2D mRNA is NM_007360.4, and the amino acid sequence of human NKG2D is NP_031386.2 (SEQ ID NO: 2). Based on the nucleotide and amino acid sequences of transcript NM_007360.4 and its encoded protein NP_031386.2, the corresponding positions of each exon are as follows:

[0043] Table 1

[0044] The human NKG2D gene (NCBI Gene ID: 22914) is located at positions 10372353 to 10390041 of NC_000012.12 on chromosome 12 (GRCh38.p13 (GCF_000001405.39)). The specific positions of each exon based on transcript NM_007360.4 are as follows: the 5'UTR is located at positions 10389943 to 10390041 and 10388811 to 10388875 of NC_000012.12, exon 1 is located at positions 10390041 to 10389943 of NC_000012.12, intron 1 is located at positions 10389942 to 10388876 of NC_000012.12, and exon 2 is located at positions 10389943 to 10390041 of NC_000012.12. 012.12 at positions 10388875 to 10388771, intron 2 is located at positions 10388770 to 10387011 of NC_000012.12, exon 3 is located at positions 10387010 to 10386903 of NC_000012.12, intron 3 is located at positions 10386902 to 10379793 of NC_000012.12, and exon 4 is located at positions 10379792 to 10379797 of NC_000012.12. 00, intron 4 is located at positions 10379699 to 10379483 of NC_000012.12, exon 5 is located at positions 10379482 to 10379447 of NC_000012.12, intron 5 is located at positions 10379446 to 10378706 of NC_000012.12, exon 6 is located at positions 10378705 to 10378554 of NC_000012.12. 2 is located at positions 10378553 to 10378236, exon 7 is located at positions 10378235 to 10378132 of NC_000012.12, intron 7 is located at positions 10378131 to 10373232 of NC_000012.12, exon 8 is located at positions 10373231 to 10372353 of NC_000012.12, and the 3' UTR is located at positions 10372353 to 10373113 of NC_000012.12. All relevant information about the human NKG2D locus can be retrieved on the NCBI website (Gene ID: 22914). The entire content of this application is incorporated by reference.

[0045] In the mouse genome, the NKG2D gene (Gene ID: 27007) contains eight exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 (Figure 1). The nucleotide sequence of mouse NKG2D mRNA is NM_033078.4, and the amino acid sequence of mouse NKG2D is NP_149069.1 (SEQ ID NO: 1). Based on the nucleotide and amino acid sequences of transcript NM_033078.4 and its encoded protein NP_149069.1, the corresponding positions of each exon are as follows:

[0046] Table 2

[0047] The mouse NKG2D gene (NCBI Gene ID: 27007) is located at positions 129587286 to 129600863 of NC_000072.7 on chromosome 6 (GRCm39 (GCF_000001635.27)). The specific positions of each exon based on transcript NM_033078.4 are as follows: the 5'UTR is located at positions 129599537 to 129599735 of NC_000072.7, exon 1 is located at positions 129599735 to 129599500 of NC_000072.7, intron 1 is located at positions 129599499 to 129598206 of NC_000072.7, and exon 2 is located at positions 129599537 to 129599735 of NC_000072.7. to 129598098, intron 2 is located at NC_000072.7 positions 129598097 to 129594485, exon 3 is located at NC_000072.7 positions 129594484 to 129594446, intron 3 is located at NC_000072.7 positions 129594445 to 129593737, exon 4 is located at NC_000072.7 positions 129593736 to 129593632, intron 4 is located at The exon 5 is located at positions 129593631 to 129593296 of NC_000072.7, the intron 5 is located at positions 129593295 to 129593260 of NC_000072.7, the intron 5 is located at positions 129593259 to 129592424 of NC_000072.7, the exon 6 is located at positions 129592423 to 129592272 of NC_000072.7 The NKG2D locus is located at positions 92271 to 129591670, exon 7 is located at positions 129591669 to 129591566 of NC_000072.7, intron 7 is located at positions 129591565 to 129589867 of NC_000072.7, exon 8 is located at positions 129589866 to 129589377 of NC_000072.7, and the 3' UTR is located at positions 129589377 to 129589748 of NC_000072.7. All relevant information about the mouse NKG2D locus can be retrieved on the NCBI website (Gene ID: 27007). The entire content of this application is incorporated by reference.

[0048] Figure 15 shows an alignment of the human NKG2D amino acid sequence (NP_031386.2; SEQ ID NO: 2) and the mouse NKG2D amino acid sequence (NP_149069.1; SEQ ID NO: 1). Therefore, the corresponding amino acid residues or regions between human and mouse NKG2D can be found in Figure 15 and Table 3.

[0049] Table 3

[0050] NKG2D genes, proteins, and gene loci from other species are also known in the art. For example, Rattus norvegicus (rat) NKG2D has Gene ID: 24934, Macaca mulatta (rhesus monkey) NKG2D has Gene ID: 574240, and Sus scrofa (pig) NKG2D has Gene ID: 396737. Relevant information (e.g., intron sequences, exon sequences, and amino acid sequences) for these genes can be found in NCBI, the entire contents of which are incorporated into this application by reference.

[0051] Figure 16 shows the amino acid sequence of human NKG2D (NP_031386.2; SEQ ID NO: 2) and rat NKG2D (NP_598196.1; SEQ ID NO: 44). Therefore, the corresponding amino acid residues or regions between human and rat NKG2D can be retrieved in Figure 16 and Table 4.

[0052] Table 4

[0053] The present invention provides a human or chimeric (e.g., humanized) NKG2D protein, or a human or chimeric (e.g., humanized) NKG2D gene. In some embodiments, the humanized NKG2D protein comprises a portion of a human NKG2D protein, and preferably also comprises a portion of a non-human animal NKG2D protein. In some embodiments, the humanized NKG2D protein comprises a sequence of at least 50 to 144 consecutive amino acids identical to the extracellular region of a human NKG2D protein. In some embodiments, the humanized NKG2D protein further comprises all or part of the transmembrane region and / or cytoplasmic region of a human or non-human animal NKG2D protein. In some embodiments, the humanized NKG2D protein comprises at least 50 to 216 (e.g., 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 139, 140, 144, 150, 160, 170, 180, 190, 200, 210, or 216) consecutive amino acid sequences identical to those of a human NKG2D protein. In some embodiments, the humanized NKG2D protein comprises a human NKG2D extracellular region and a non-human animal transmembrane region and a non-human animal cytoplasmic region. In some embodiments, the humanized NKG2D protein comprises SEQ ID NO: 2 or any one of positions 78-216 thereof, or SEQ ID NO: 9; or, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to the amino acid sequence as set forth in SEQ ID NO: 2 or any one of positions 78-216 thereof, or SEQ ID NO: 9; or, comprises an amino acid sequence that differs from SEQ ID NO: 2 or any one of positions 78-216 thereof, or SEQ ID NO: 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid, or comprises an amino acid sequence that is substituted, deleted and / or inserted with one or more amino acids as set forth in SEQ ID NO: 2 or any one of positions 78-216 thereof, or SEQ ID NO: 9.

[0054] In some embodiments, the humanized NKG2D gene encodes the above-mentioned humanized NKG2D protein. Preferably, the humanized NKG2D gene comprises a portion of a human NKG2D gene and a portion of an endogenous NKG2D gene of a non-human animal. In some embodiments, the portion of the human NKG2D gene can be a genomic sequence, a CDS, or a cDNA sequence. In some embodiments, the portion of the human NKG2D gene comprises at least one exon and / or at least one intron of the human NKG2D gene. In some embodiments, the portion of the human NKG2D gene comprises all or part of exon 4, all of exons 5-7, and / or all or part of exon 8 of the human NKG2D gene. In some embodiments, it also comprises all of exons 1-3 of the human NKG2D gene. In some embodiments, the portion of the human NKG2D gene comprises the nucleotide sequence of the coding region of the human NKG2D gene. In some embodiments, the portion of the human NKG2D gene comprises the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene. In some embodiments, the portion of the human NKG2D gene further comprises the 5'UTR and / or 3'UTR of the human NKG2D gene, and may further comprise a nucleotide sequence of at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human NKG2D gene and / or a nucleotide sequence of at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human NKG2D gene. In some embodiments, the portion of the human NKG2D gene includes the 5'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides upstream of the 5'UTR, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene, and the 3'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the portion of the human NKG2D gene includes a portion of exon 4 to a portion of exon 8 of the human NKG2D gene. In some embodiments, the portion of the human NKG2D gene comprises SEQ ID NO: 7 or 48; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to the nucleotide sequence of SEQ ID NO: 7 or 48; or comprises a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 7 or 48 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or comprises a nucleotide sequence that is substituted, deleted and / or inserted with one or more nucleotides as set forth in the nucleotide sequence of SEQ ID NO: 7 or 48. In some embodiments, all or part of the nucleotide sequence from the upstream nucleotide sequence outside the 5'UTR to the downstream nucleotide sequence outside the 3'UTR or the encoded amino acid sequence of the endogenous NKG2D genome of the non-human animal is replaced with the corresponding nucleotide sequence or encoded amino acid sequence of the human NKG2D gene.In some embodiments, the 5'UTR-outside upstream nucleotide sequence to the 3'UTR-outside downstream nucleotide sequence of the non-human animal endogenous NKG2D genome comprises the 5'UTR-outside upstream nucleotide sequence, the 5'UTR, the endogenous NKG2D gene coding region sequence (including all or part of endogenous NKG2D exons 1-8), the 3'UTR, and / or the 3'UTR-outside downstream nucleotide sequence. In some embodiments, a portion of exon 4 to a portion of exon 8, or a nucleotide sequence encoding the extracellular region, of the non-human animal endogenous NKG2D is replaced with the corresponding nucleotide sequence of the human NKG2D gene or a nucleotide sequence encoding the extracellular region.

[0055] In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 200, 300, 500, 800, 1400, 2200, 3000, 4000, 5000, 6000, 6100, 6200, 6220, 6240, 6260, 6264, 7000, 7481 or 8000 bp of nucleotides. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises 7481 bp. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises a nucleotide sequence of at least 100-500 bp, 1000-3000 bp or 5000-8000 bp. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 120, 140, 180, 200, 220, 240, 280, 300, 350, 400, 450 or 500 bp nucleotides. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises 200-400 bp. In some embodiments, the "part" of the non-human animal endogenous NKG2D genome 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence or the encoded amino acid sequence comprises at least 50, 60, 70, 80, 90, 100, 110, 120、130、140、150、160、170、180、190、200、250、300、350、400、420、430、431、432、500、600、800、1400、1800、2200、2600、3000、3100、3200、3220、3240、3260、3270、3271、3272、3300、3600、3700、3900、3901、3902、3903、3904、3905、4000、5000、6000、6100、6200、6220、6240、6260、6264、 141, 142, 143, 150, 160, 170, 180, 190, 200, 210, 220, 230, 231, or 232 consecutive amino acid sequences.In some embodiments, the "portion" is identical or has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99% homology to the mouse NKG2D genome 5'UTR outer upstream nucleotide sequence to 3'UTR outer downstream nucleotide sequence or the encoded amino acid sequence. In some embodiments, the 5'UTR outer upstream nucleotide sequence to 3'UTR outer downstream nucleotide sequence includes 5'UTR outer upstream nucleotide sequence, 5'UTR, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, 3'UTR and / or 3'UTR outer downstream sequence (preferably further includes at least one of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7).

[0056] In some embodiments, "part" or "all" of the 5'UTR outer upstream nucleotide sequence, 5'UTR, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, 3'UTR, and / or the 3'UTR outer downstream nucleotide sequence of the mouse NKG2D genome (e.g., the entire 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence or a part of exon 4, all of exons 5-7 and exon 8) The present invention relates to a method for replacing a human NKG2D gene with a “partial” or “complete” nucleotide sequence of the human NKG2D gene (e.g., the entire nucleotide sequence from the upstream nucleotide sequence outside the 5'UTR of the exon to the downstream nucleotide sequence outside the 3'UTR, or a part of exon 4, all of exons 5-7, and a part of exon 8) by a nucleotide sequence of the human NKG2D gene.

[0057] In some embodiments, "partial" deletion of the upstream nucleotide sequence outside the 5'UTR, 5'UTR, exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, 3'UTR and / or the downstream nucleotide sequence outside the 3'UTR of the endogenous NKG2D gene of the non-human animal.

[0058] In some embodiments, all or part of exons 1-8 of the endogenous NKG2D gene of the non-human animal is replaced, preferably part of exon 4, all of exons 5-7, and part of exon 8 is replaced. In some embodiments, part of exon 4 to part of exon 8 of the endogenous NKG2D gene of the non-human animal is replaced. In some embodiments, all or part of the nucleotide sequence encoding the extracellular region in the NKG2D gene of the non-human animal is replaced. In some embodiments, part of exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, and part of exon 8 of the endogenous NKG2D gene of the non-human animal is deleted. In some embodiments, the portion of exon 4 of the endogenous NKG2D gene of a non-human animal includes at least 5-105 bp, for example, 5, 10, 15, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 83, 85, 90, 95, 100 or 105 bp, preferably a nucleotide sequence comprising the coding region. In some embodiments, the portion of exon 8 of the endogenous NKG2D gene includes at least 50-2581 bp, for example, 50, 100, 118, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2460, 2461, 2462, 2463, 2464, 2465, 2500, 2550, 2580 or 2581 bp, preferably including the nucleotide sequence of the coding region.

[0059] In some embodiments, the humanized NKG2D gene comprises, from 5' to 3' end, all of exons 1-3 of the mouse NKG2D gene, a portion of exon 4 of the mouse NKG2D gene, a portion of exon 4 of the human NKG2D gene, all of exons 5-7 of the human NKG2D gene, a portion of exon 8 of the human NKG2D gene, and a portion of exon 8 of the mouse NKG2D gene. In some embodiments, the humanized NKG2D gene comprises, from 5' to 3' end: A) SEQ ID NOs: 3, 7, 4; B) SEQ ID NOs: 3, 48, 4; C) SEQ ID NOs: 5, 7, 6; D) SEQ ID NOs: 5, 48, 6; E) SEQ ID NOs: 35, 7, 36; F) SEQ ID NOs: 35, 48, 36. In some embodiments, the humanized NKG2D gene comprises one or more of SEQ ID NOs: 10, 11, 39, or 40. In some embodiments, the mRNA transcribed from the humanized NKG2D gene comprises SEQ ID NO: 8 or 38; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to the nucleotide sequence of SEQ ID NO: 8 or 38; or comprises a nucleotide sequence that differs from SEQ ID NO: 8 or 38 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide sequence; or comprises a nucleotide sequence that is substituted, deleted, and / or inserted with one or more nucleotides from SEQ ID NO: 8 or 38. In some embodiments, the humanized NKG2D gene further comprises a resistance gene. In some embodiments, the humanized NKG2D gene further comprises two Frt recombination sites arranged in the same direction on either side of the resistance gene. In some embodiments, the resistance gene is the neomycin phosphotransferase encoding sequence Neo. In some embodiments, the humanized NKG2D gene further comprises a specific inducer or repressor, and the specific inducer or repressor is preferably a conventional substance that can be induced or repressed. In some embodiments, the specific inducer includes but is not limited to the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System). In some embodiments, the humanized NKG2D gene is regulated in a non-human animal by a regulatory element. In some embodiments, the regulatory element is an endogenous regulatory element or an exogenous regulatory element (e.g., a human regulatory element). In some embodiments, the regulatory element is a promoter.

[0060] In some embodiments, the present invention provides a genetically modified non-human animal, wherein the genome of the non-human animal comprises a human or chimeric (e.g., humanized) NKG2D gene. In some embodiments, the nucleotide sequence of the human or chimeric (e.g., humanized) NKG2D gene encodes a protein comprising SEQ ID NO: 2 or 78 to 216 thereof, or SEQ ID NO: 9, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 78 to 216 thereof, or SEQ ID NO: 9. In some embodiments, the non-human animal genome comprises a nucleotide sequence comprising SEQ ID NO: 3, 4, 5, 6, 7, 8, 10, 11, 35, 36, 38, 39, 40 and 48, or a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95% or 99% homologous to SEQ ID NO: 3, 4, 5, 6, 7, 8, 10, 11, 35, 36, 38, 39, 40 and 48.

[0061] In some embodiments, the non-human animal of the present invention comprises a human or humanized NKG2D gene. In some embodiments, the humanized NKG2D gene comprises 8 exons. In some embodiments, the humanized NKG2D gene comprises human exon 1, human exon 2, human exon 3, human exon 4, human exon 5, human exon 6, human exon 7, and / or human exon 8. In some embodiments, the humanized NKG2D gene comprises human intron 1, human intron 2, human intron 3, human intron 4, human intron 5, human intron 6, and / or human intron 7. In some embodiments, the humanized NKG2D gene comprises non-human animal endogenous exon 1, non-human animal endogenous exon 2, non-human animal endogenous exon 3, humanized exon 4, human exon 5, human exon 6, human exon 7, and / or humanized exon 8. In some embodiments, the humanized NKG2D gene comprises a human or humanized 5'UTR. In some embodiments, the humanized NKG2D gene comprises a human or humanized 3'UTR. In some embodiments, the humanized NKG2D gene comprises an endogenous 5'UTR. In some embodiments, the humanized NKG2D gene comprises an endogenous 3'UTR. In some embodiments, the humanized NKG2D gene comprises a human 5'UTR. In some embodiments, the humanized NKG2D gene comprises a human 3'UTR.

[0062] In some embodiments, the genetically modified non-human animal can express a human NKG2D and / or chimeric (e.g., humanized) NKG2D protein, wherein the endogenous NKG2D gene sequence of the non-human animal is replaced by a human NKG2D gene and / or nucleotide sequence. Furthermore, the amino acid sequence encoded by the human NKG2D gene and / or nucleotide sequence is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the human NKG2D protein set forth in SEQ ID NO: 2 or positions 78-216 thereof. In some embodiments, the endogenous NKG2D gene is replaced in whole or in part by a nucleotide sequence encoding a mature human NKG2D protein.

[0063] In some embodiments, the human or humanized NKG2D protein comprises all or part of the extracellular region, transmembrane region and / or cytoplasmic region of a human NKG2D protein. In some embodiments, the human or humanized NKG2D protein comprises all or part of the extracellular region of a human NKG2D protein. In some embodiments, the humanized NKG2D protein comprises at least 50 consecutive amino acids in the extracellular region of a human NKG2D protein, for example, at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 or 144 consecutive amino acids. In some embodiments, the humanized NKG2D protein comprises all or part of the transmembrane region and / or cytoplasmic region of a human or non-human animal NKG2D protein. In some embodiments, the humanized NKG2D protein comprises at least 50 consecutive amino acids of a human NKG2D protein, such as at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 150, 160, 170, 180, 190, 200, 210, or 216 consecutive amino acids. In some embodiments, the humanized NKG2D protein comprises an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in positions 78-216 of SEQ ID NO: 2 or SEQ ID NO: 9.

[0064] In some embodiments, the humanized NKG2D protein comprises all or part of the extracellular region, transmembrane region and / or cytoplasmic region of a non-human NKG2D protein. In some embodiments, the humanized NKG2D protein comprises all or part of the transmembrane region and cytoplasmic region of a non-human NKG2D protein. In some embodiments, the humanized NKG2D protein comprises all or part of the transmembrane region of a non-human NKG2D protein. In some embodiments, the humanized NKG2D protein comprises at least one consecutive amino acid in the transmembrane region of a non-human NKG2D protein, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 21, 22, or 23 consecutive amino acids. In some embodiments, the transmembrane region of the humanized NKG2D protein comprises an amino acid sequence that is identical or has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of positions 67-89 of SEQ ID NO: 1. In some embodiments, the humanized NKG2D protein comprises all or part of the cytoplasmic region of a non-human NKG2D protein. In some embodiments, the humanized NKG2D protein comprises at least 10 consecutive amino acids of the cytoplasmic region of a non-human NKG2D protein, for example, at least 10, 12, 15, 17, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65 or 66 consecutive amino acids.

[0065] In some embodiments, the cytoplasmic region of the humanized NKG2D protein comprises amino acids that are identical or have at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence shown in positions 1-66 of SEQ ID NO: 1.

[0066] In some embodiments, the human or humanized NKG2D protein comprises an amino acid sequence that is identical or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 2 positions 78-216, SEQ ID NO: 9, or SEQ ID NO: 1 positions 1-89.

[0067] In some embodiments, the genetically modified non-human animal expresses human NKG2D and / or chimeric NKG2D proteins (e.g., humanized NKG2D proteins) under endogenous or human regulatory elements (e.g., promoters). Replacement of the endogenous locus provides a non-human animal that expresses human or chimeric NKG2D proteins (e.g., humanized NKG2D proteins) in the same cell type. The genetically modified mice do not exhibit potential diseases observed in certain other transgenic mice known in the art. The human NKG2D or chimeric NKG2D proteins expressed in non-human animals can maintain the functions of one or more wild-type or human NKG2D proteins, for example, the expressed NKG2D proteins can bind to human or non-human NKG2DL proteins. In some embodiments, the genetically modified non-human animal does not express endogenous NKG2D proteins. In some embodiments, the expression of the genetically modified non-human animal endogenous NKG2D protein is reduced compared to NKG2D in wild-type animals. The "endogenous NKG2D protein" mentioned in the present application refers to the NKG2D protein encoded by the endogenous NKG2D nucleotide sequence of a non-human animal (eg, mouse) before genetic modification.

[0068] The genome of the non-human animal comprises a nucleotide sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to or homologous to the amino acid sequence of human NKG2D protein (NP_031386.2; SEQ ID NO: 2 or SEQ ID NO: 2, positions 78-216). In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to or homologous to the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 48, and SEQ ID NO: 38.

[0069] In some embodiments, the nucleotide sequence encoding the endogenous NKG2D region in the non-human animal genome is replaced by the nucleotide sequence encoding the corresponding region of human NKG2D. In some embodiments, the nucleotide sequence encoding the endogenous NKG2D region comprises all or part of the 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence of the non-human animal NKG2D gene. In some embodiments, the 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence comprises the 5'UTR outer upstream nucleotide sequence, 5'UTR, the endogenous NKG2D gene coding region sequence, 3'UTR and / or the 3'UTR outer downstream nucleotide sequence. In some embodiments, the endogenous NKG2D gene coding region sequence comprises all or part of endogenous NKG2D exons 1-8, wherein the portion comprises a portion of exon 4, all of exons 5-7, and a portion of exon 8. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 200, 300, 500, 800, 1400, 2200, 3000, 4000, 5000, 6000, 6100, 6200, 6220, 6240, 6260, 6264, 7000, 7481 or 8000 bp of nucleotides. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises 7481 bp. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises a nucleotide sequence of at least 100-500 bp, 1000-3000 bp or 5000-8000 bp. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 120, 140, 180, 200, 220, 240, 280, 300, 350, 400, 450 or 500 bp of nucleotides. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises 200-400 bp.

[0070] In some embodiments, the replaced nucleotide sequence encoding the endogenous NKG2D region comprises a portion of exon 4, all of exons 5-7, and a portion of exon 8 of an endogenous NKG2D gene of a non-human animal, or comprises a portion of exon 4 to exon 8 of an endogenous NKG2D gene of a non-human animal. In some embodiments, the portion of exon 4 of an endogenous NKG2D gene of a non-human animal comprises at least 5-105 bp, for example, 5, 10, 15, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 83, 85, 90, 95, 100, or 105 bp, preferably comprises a nucleotide sequence of the coding region. In some embodiments, the portion of exon 8 of an endogenous NKG2D gene comprises at least 50-258 bp, for example, 50, 100, 118, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2460, 2461, 2462, 2463, 2464, 2465, 2500, 2550, 2580 or 2581 bp, preferably comprising a nucleotide sequence of the coding region.

[0071] In some embodiments, the nucleotide sequence encoding the endogenous NKG2D region is located within the endogenous NKG2D regulatory region. In some embodiments, the nucleotide sequence encoding the endogenous NKG2D region is located within the region from the upstream nucleotide sequence outside the 5'UTR to the downstream nucleotide sequence outside the 3'UTR of the endogenous NKG2D gene of the non-human animal. In some embodiments, the nucleotide sequence encoding the endogenous NKG2D region is located from a portion of exon 4 to a portion of exon 8 of the endogenous NKG2D gene of the non-human animal.

[0072] In some embodiments, one or more cells of the genetically modified non-human animal express a human or humanized NKG2D protein. In some embodiments, the human or humanized NKG2D protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 144, 150, 160, 170, 180, 190, 200, 210, or 216 consecutive amino acids identical to the amino acid sequence of SEQ ID NO: 2.

[0073] In some embodiments, the genome of the genetically modified non-human animal comprises all or part of the nucleotide sequence from the upstream nucleotide sequence outside the 5'UTR of the human NKG2D gene to the downstream nucleotide sequence outside the 3'UTR. In some embodiments, the nucleotide sequence from the upstream nucleotide sequence outside the 5'UTR of the human NKG2D gene to the downstream nucleotide sequence outside the 3'UTR comprises the upstream nucleotide sequence outside the 5'UTR, the 5'UTR, the NKG2D gene coding region sequence, the 3'UTR and / or the downstream nucleotide sequence outside the 3'UTR. In some embodiments, the NKG2D gene coding region sequence comprises all or part of human NKG2D exons 1-8, wherein the portion comprises a portion of exon 4, all of exons 5-7 and a portion of exon 8, or comprises a portion of exon 4 to a portion of exon 8. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 450, 600, 1000, 1500, 2000, 2500, 3000, 3500, 5000, 7000, 9000, 12000, 12100, 12300, 12600, 12800, 12900, 12910, 12920, 12930, 12932, 12934, 12936, 12937, 12938, 12939, 14000 or 20000 bp of nucleotides. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises 12939 bp. In some embodiments, the 5'UTR outer upstream nucleotide sequence includes a nucleotide sequence of at least 100-800bp, 2000-6000bp or 8000-13000bp. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 450, 600, 800, 900, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1200, 1400, 1800 or 2000bp of continuous nucleotides. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises 800-1500bp. In some embodiments, the portion of exon 4 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, or 93 bp of continuous nucleotide sequence.In some embodiments, the portion of exon 8 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 112, 114, 116, 117, 118, 150, 200, 250, 350, 450, 600, 700, 800, 820, 840, 860, 870, 871, 873, 875, 876, 877, 878, or 879 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 8 comprises at least 118 bp of continuous nucleotide sequence.

[0074] In some embodiments, the portion of the human NKG2D gene 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence comprises at least 5, 10, 50, 60, 70, 80, 90, 91, 92, 93, 100, 110, 112, 114, 116, 118, 120, 130, 140, 150, 160, 170, 180, 1 90, 200, 400, 500, 600, 800, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1800, 2200, 2600, 3000, 3600, 4000, 5000, 6000, 6200, 6400, 6500, 6540, 6560 、6590、6591、6593、6594、6596、7000、9000、12000、12100、12300、12600、12800、12900、12910、12920、12930、12932、12934、12936、12937、12938、12939、14000、1600 0, 17000, 17200, 17400, 17600, 17640, 17660, 17680, 17682, 17686, 17687, 17689, 18696, 20000, 25000, 30000, 30628, 31000, 31200, 31400, 31600 or 31635 bp of continuous nucleotide sequence.

[0075] In some embodiments, the genetically modified non-human animal genome comprises a portion of a human NKG2D gene. In some embodiments, the portion of the human NKG2D gene comprises all or part of exons 1-8. In some embodiments, the portion of the human NKG2D gene comprises a portion of exon 4, all of exons 5-7, and a portion of exon 8, or comprises a portion of exon 4 to a portion of exon 8. In some embodiments, the portion of exon 4 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, or 93 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 4 comprises at least 10 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 8 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 112, 114, 116, 117, 118, 150, 200, 250, 350, 450, 600, 700, 800, 820, 840, 860, 870, 871, 873, 875, 876, 877, 878, or 879 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 8 comprises at least 118 bp of continuous nucleotide sequence.

[0076] In some embodiments, the non-human animal genome comprises a nucleotide sequence encoding all or part of the amino acid sequence of a human NKG2D protein. In some embodiments, the non-human animal genome comprises all or part of the nucleotide sequence of SEQ ID NO: 48 or SEQ ID NO: 7.

[0077] In some embodiments, the genetically modified non-human animal genome comprises all of exons 1-3, a portion of exon 4, and a portion of exon 8 of an endogenous NKG2D gene of the non-human animal (e.g., mouse). In some embodiments, the portion of exon 4 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 81, 82, 83, 90, 100, 101, 102, 103, 104, or 105 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 4 comprises 83 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 8 includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22, 23, 28, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 350, 450, 600, 700, 800, 1000, 1400, 1800, 2000, 2200, 2400, 2420, 2430, 2440, 2450, 2460, 2461, 2462, 2463, 2464, 2500, 2540, 2560, 2570, 2572, 2574, 2576, 2577, 2578, 2579, 2580, or 2581 bp of contiguous nucleotide sequence. In some embodiments, the portion of exon 8 comprises at least 2463 bp of contiguous nucleotide sequence.

[0078] In some embodiments, the modified gene in the modified animal genome is homozygous or heterozygous for the endogenous modified (preferably replaced) locus. In some embodiments, the modified NKG2D gene in the genome is heterozygous or homozygous for the endogenous modified (preferably replaced) locus.

[0079] In some embodiments, the humanized NKG2D gene comprises the 5'UTR of a human NKG2D gene. In some embodiments, the humanized NKG2D genome comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanized NKG2D genome comprises an endogenous (e.g., mouse) 3'UTR. In some embodiments, the humanized NKG2D genome comprises the 3'UTR of a human NKG2D gene. Where appropriate, based on sequence similarity, it is reasonable to infer that mouse and human NKG2D genes are subject to similar regulation. As described herein, humanized NKG2D mice comprise replacements of endogenous mouse loci that retain or do not retain mouse endogenous regulatory elements but comprise human NKG2D coding sequences. Expression of NKG2D in genetically modified heterozygous or homozygous mice is completely normal.

[0080] On the other hand, the present invention provides a genetically modified non-human animal, the non-human animal genome comprising a deletion of an endogenous NKG2D gene. In some embodiments, the deletion of the endogenous NKG2D gene of the non-human animal comprises all of the 5'UTR outer upstream nucleotide sequence, the 5'UTR, the NKG2D gene coding region sequence, the 3'UTR and / or the 3'UTR outer downstream nucleotide sequence, or a partial deletion thereof. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 200, 300, 500, 800, 1400, 2200, 3000, 4000, 5000, 6000, 6100, 6200, 6220, 6240, 6260, 6264, 7000, 7481 or 8000 bp of nucleotides. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises 7481 bp of nucleotides. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises a nucleotide sequence of at least 100-500bp, 1000-3000bp, or 5000-8000bp. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 120, 140, 180, 200, 220, 240, 280, 300, 350, 400, 450, or 500bp of nucleotides. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises 200-400bp.

[0081] In some embodiments, all or part of exons 1-8 of the endogenous NKG2D of the non-human animal, preferably part of exon 4, all of exons 5-7 and part of exon 8, is deleted, or part of exon 4 to part of exon 8 is deleted. In some embodiments, the portion of exon 4 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22, 25, 30, 40, 50, 60, 70, 80, 83, 90, 100, 101, 102, 103, 104 or 105 bp of continuous nucleotide sequence or more. In some embodiments, the portion of exon 4 comprises at least 22 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 8 comprises 50, 60, 70, 80, 90, 100, 112, 113, 114, 115, 116, 117, 118, 119, 120, 150, 200, 250, 350, 450, 600, 700, 800, 1000, 1400, 1800, 2000, 2200, 2400, 2420, 2430, 2440, 2450, 2460, 2461, 2462, 2463, 2500, 2540, 2560, 2570, 2572, 2574, 2576, 2577, 2578, 2579, 2580, or 2581 bp of contiguous nucleotide sequence, or more. In some embodiments, the portion of exon 8 comprises at least 118 bp of contiguous nucleotide sequence.

[0082] In some embodiments, the deletion of the endogenous NKG2D gene further comprises one or more introns selected from intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7.

[0083] In some embodiments, the deletion comprises at least 1 bp to 21359 bp of the endogenous NKG2D gene, and in some embodiments, the deletion comprises at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 420, 430, 431, 432, 500, 600, 800, 1400, 1800, 2200, 2600, 3000, 1350, 13520, 13576, 13578, 16000, 20000, 20142 or 21359 bp of continuous nucleotide sequence or more.

[0084] In some embodiments, the deletion of the endogenous NKG2D gene comprises at least 50 bp to at least 3272 bp of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7 and / or exon 8, such as 50, 100, 200, 300, 400, 430, 432, 435, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3200, 3200 or 3272 bp of contiguous nucleotide sequence or more, (e.g., all of exons 1-8, or, at least 10 bp of contiguous nucleotide sequence of exon 4 to at least 10 bp of contiguous nucleotide sequence of exon 8).

[0085] The present invention provides a humanized mouse NKG2D genomic DNA sequence; provides a construct expressing a humanized NKG2D protein amino acid sequence; a cell comprising the construct; and a tissue comprising the cell. Therefore, in some embodiments, the present invention provides a chimeric (e.g., humanized) NKG2D nucleotide sequence and / or amino acid sequence, wherein in some embodiments, the chimeric (e.g., humanized) NKG2D nucleotide sequence and / or amino acid sequence is homologous to mouse endogenous NKG2D mRNA (e.g., NM_033078.4), mouse NKG2D amino acid sequence (e.g., NP_149069.1, SEQ ID NO: 1) or a portion thereof (e.g., all of exons 1-3, a portion of exon 4, and a portion of exon 8) is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or identical to the sequence shown in SEQ ID NO: 1) or a portion thereof (e.g., all of exons 1-3, a portion of exon 4, and a portion of exon 8). In some embodiments, the humanized nucleotide sequence is identical to or identical to the human NKG2D mRNA sequence (e.g., NM_007360.4), NKG2D amino acid sequence (e.g., NP_031386.2, SEQ ID NO: 2) or a portion thereof (e.g., a portion of exon 4 to a portion of exon 8, preferably including a portion of exon 4, all of exons 5-7 and a portion of exon 8), or the 5'UTR of human NKG2D gene and at least 50 bp of continuous nucleotides upstream thereof, the nucleotide sequence from the start codon to the stop codon of human NKG2D gene and the 3'UTR of human NKG2D gene and at least 50 bp of continuous nucleotides downstream thereof are identical or have an identity of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0086] In some embodiments, the nucleotide sequence encoding mouse NKG2D (SEQ ID NO: 1) or amino acids 90-232 thereof is replaced with a nucleotide sequence encoding human NKG2D (SEQ ID NO: 2) or amino acids 78-216 thereof.

[0087] In some embodiments, the extracellular region encoding mouse NKG2D is replaced with all or part of the nucleotide sequence encoding the extracellular region of human NKG2D.

[0088] In some embodiments, the human NKG2D nucleotide sequence described above is operably linked to a regulatory element, for example, a human or mouse NKG2D promoter, an inducible promoter, an enhancer, and / or a human or mouse regulatory element.

[0089] In some embodiments, at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) of the chimeric nucleotide sequence of the present invention is different from all or part of the mouse NKG2D nucleotide sequence (e.g., all of exons 1-8 or part of exon 4, all of exons 5-7, and part of exon 8 of the mouse NKG2D gene transcript NM_033078.4).

[0090] In some embodiments, at least a portion of the chimeric nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) is identical to all or a portion of a mouse NKG2D nucleotide sequence (e.g., all of exons 1-3, a portion of exon 4, and a portion of exon 8 of mouse NKG2D gene transcript NM_033078.4).

[0091] In some embodiments, at least a portion of the chimeric nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) is different from all or a portion of a human NKG2D nucleotide sequence (e.g., all of exons 1-3, a portion of exon 4, and a portion of exon 8 of the human NKG2D gene transcript NM_007360.4).

[0092] In some embodiments, at least a portion of the chimeric nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) is identical to all or part of a human NKG2D nucleotide sequence (e.g., all of exons 1-8 or part of exon 4, all of exons 5-7, and part of exon 8 of the human NKG2D gene transcript NM_007360.4).

[0093] In some embodiments, at least a portion of the amino acids encoded by the chimeric nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, such as, consecutive or non-consecutive amino acid residues) is different from all or part of the mouse NKG2D protein amino acid sequence (e.g., amino acids 1-232 (SEQ ID NO: 1) or amino acids 90-232 of mouse NKG2D protein sequence NP_149069.1).

[0094] In some embodiments, at least a portion of the amino acids encoded by the chimeric nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, such as continuous or non-contiguous amino acid residues) are identical to all or part of the amino acid sequence of mouse NKG2D protein (e.g., amino acids 1-89 of mouse NKG2D protein sequence NP_149069.1 (SEQ ID NO: 1)).

[0095] In some embodiments, at least a portion of the amino acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) is different from all or part of the amino acid sequence of human NKG2D protein (e.g., amino acids 1-77 of human NKG2D protein sequence NP_031386.2 (SEQ ID NO: 2)).

[0096] In some embodiments, at least a portion of the amino acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) is identical to all or part of the amino acid sequence of human NKG2D protein (e.g., amino acids 1-216 (SEQ ID NO: 2) or 78-216 of human NKG2D protein sequence NP_031386.2).

[0097] The present invention also provides a humanized NKG2D protein, in some embodiments, whose amino acid sequence comprises any one of the following groups:

[0098] A) amino acid sequence shown in SEQ ID NO: 2 or 78-216, 9 thereof;

[0099] B) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence of SEQ ID NO: 2 or 78-216, 9;

[0100] C) differs from the amino acid sequence of SEQ ID NO: 2 or 78-216, 9 thereof by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0101] D) an amino acid sequence as shown in SEQ ID NO: 2 or 78-216, 9 thereof, comprising substitution, deletion and / or insertion of one or more amino acid residues.

[0102] In some embodiments, the amino acid sequence comprises any one of the following groups:

[0103] A) the amino acid sequence shown in SEQ ID NO: 1, positions 1-89;

[0104] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in positions 1-89 of SEQ ID NO: 1;

[0105] C) differs from the amino acid sequence of SEQ ID NO: 1 at positions 1-89 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0106] D) an amino acid sequence comprising substitutions, deletions and / or insertions of one or more amino acid residues as shown in positions 1 to 89 of SEQ ID NO: 1.

[0107] The present invention also provides a humanized NKG2D nucleotide (eg, DNA or RNA) sequence, wherein the nucleotide sequence comprises any one of the following groups:

[0108] A) a nucleic acid sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 10, 11, 35, 36, 38, 39, 40 or 48, or a nucleic acid sequence encoding a humanized mouse NKG2D homologous amino acid sequence;

[0109] B) a nucleic acid sequence capable of hybridizing to the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 10, 11, 35, 36, 38, 39, 40 or 48 under low stringency conditions or stringent conditions;

[0110] C) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 10, 11, 35, 36, 38, 39, 40 or 48;

[0111] D) the amino acid sequence encoded by it is identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:2 positions 78-216 or SEQ ID NO:1 positions 1-89 or SEQ ID NO:9;

[0112] E) the encoded amino acid sequence differs from the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 2 positions 78-216 or SEQ ID NO: 1 positions 1-89 or SEQ ID NO: 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0113] F) The amino acid sequence encoded by the amino acid sequence is the same as that shown in SEQ ID NO: 2 or SEQ ID NO: 2 positions 78-216 or SEQ ID NO: 1 positions 1-89 or SEQ ID NO: 9, including substitutions, deletions and / or insertions of one or more amino acid residues.

[0114] The present invention further provides a humanized mouse NKG2D genomic DNA sequence, which is obtained by reverse transcription of the mRNA transcribed from the NKG2D, and is identical to or complementary to a DNA sequence homologous to the sequence shown in SEQ ID NO: 7, 8, 38 or 48.

[0115] In order to determine the percent identity of two amino acid sequences or two nucleotide sequences, for the best comparison purpose, the sequence is compared (for example, for best comparison, a gap can be introduced in one or both of the first and second amino acid or nucleotide sequences, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides on the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the amino acid residue or nucleotide identical to the corresponding position in the second sequence, the molecule is identical at that position. The percent identity between the two sequences is a function of the quantity of the same positions shared by the sequences, which, considering the number of gaps and the length of each gap, needs to be introduced to realize the best comparison of the two sequences. For example, the comparison of the sequence and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix of gap penalty 12, gap extension penalty 4 and frameshift gap penalty 5.

[0116] The percentage ratio (homology percentage) of conserved residues with similar physicochemical properties, 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 (such as lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (such as threonine, valine and isoleucine) and aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage ratio is higher than the identity percentage ratio.

[0117] In some embodiments, the "homology" refers to the use of amino acid sequences or nucleotide sequences. Under the premise of ensuring similar structure or function to known sequences, those skilled in the art can adjust the sequence according to actual work needs, so that the used sequence has (including but not limited to) at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 112%, 113 6%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity or consistency.

[0118] The present invention also provides cells, tissues and animals (eg, mice) comprising the nucleotide sequences of the present invention, as well as cells, tissues and animals (eg, mice) expressing human or chimeric (eg, humanized) NKG2D at an endogenous non-human NKG2D locus.

[0119] Genetically modified non-human animals

[0120] The "genetically modified non-human animal" or "genetically modified non-human animal" described in the present invention refers to a non-human animal in which at least one chromosome in the genome of the animal has an exogenous NKG2D. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50% of the cells in the genetically modified non-human animal have exogenous DNA. The cells with exogenous DNA can be various cells, for example, somatic cells, immune cells (T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, a genetically modified non-human animal is provided, which comprises an endogenous NKG2D locus and an exogenous NKG2D locus (e.g., a human sequence), for example, replacing one or more non-human sequences with one or more human sequences, or inserting one or more human and / or non-human sequences. Animals are generally able to pass genetic modifications to offspring through germline transmission.

[0121] The "chimeric (x) gene" or "chimeric (x) nucleic acid" of the present invention refers to a gene or nucleic acid, wherein two or more parts of the gene or nucleic acid are from different species, or at least one sequence of the gene or nucleic acid is different from the wild-type nucleic acid in an animal. In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid has at least a portion of the sequence derived from two or more different species, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins 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.

[0122] The "chimeric (X) protein" or "chimeric (X) polypeptide" of the present invention refers to a protein or polypeptide, wherein two or more portions of the polypeptide or protein are derived from different species, or at least one sequence of the protein or polypeptide differs from the wild-type amino acid sequence in an animal. In some embodiments, at least a portion of the sequence of the chimeric (X) protein or chimeric (X) polypeptide is derived from two or more different species, for example, the same (or homologous) proteins from different species. In some embodiments, the chimeric (X) protein or chimeric (X) polypeptide is a humanized (X) protein or humanized (X) polypeptide.

[0123] As used herein, a "humanized (X) protein" or "humanized (X) polypeptide" refers to a protein or polypeptide, at least a portion of which is derived from a human protein or polypeptide. In some embodiments, a humanized (X) protein or humanized (X) polypeptide refers to a human protein or polypeptide.

[0124] As used herein, "humanized (x) nucleic acid" or "humanized (x) gene" refers to a nucleic acid, at least a portion of which is derived from human nucleic acid. In some embodiments, all nucleic acids in the humanized (x) nucleic acid or humanized (x) gene are derived from humans. In some embodiments, the humanized (x) nucleic acid or humanized (x) gene refers to humanized exons, which may be human exons or chimeric exons. In some embodiments, the humanized (x) nucleic acid or humanized (x) gene refers to humanized exons and humanized introns, which may be human introns or chimeric introns.

[0125] In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid is a humanized NKG2D gene or humanized NKG2D nucleic acid. In some embodiments, at least a portion of the humanized NKG2D gene or humanized NKG2D nucleic acid is derived from a human NKG2D gene, or at least a portion of the gene or nucleic acid is derived from a non-human animal NKG2D gene. In some embodiments, the humanized NKG2D gene or humanized NKG2D nucleic acid comprises a sequence encoding an NKG2D protein. In some embodiments, the encoded NKG2D protein has at least one activity of a human NKG2D protein or a non-human animal NKG2D protein.

[0126] In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide is a humanized NKG2D protein or humanized NKG2D polypeptide. In some embodiments, at least one or more portions of the humanized NKG2D protein or humanized NKG2D polypeptide are derived from a human NKG2D protein, or at least one or more portions of the humanized NKG2D protein or humanized NKG2D polypeptide are derived from a non-human animal NKG2D protein. The humanized NKG2D protein or humanized NKG2D polypeptide is functional or has at least one activity of a human NKG2D protein or a non-human animal NKG2D protein.

[0127] The non-human animal of genetic modification can be various animals, for example, mouse, rat, rabbit, pig, cattle (for example, cattle, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (for example, marmoset, rhesus monkey).For the non-human animal of suitable genetically modified embryonic stem cell (ES) that is not easy to obtain, adopt alternative methods to construct the non-human animal comprising genetic modification.Such method includes, for example, modifying non-ES cell genome (for example, fibroblast or induced pluripotent stem cell) and adopting nuclear transplantation to transfer the modified genome to suitable cell, for example oocyte, and incubating the modified cell (for example, modified oocyte) to form embryo in non-human animal under appropriate conditions.Above-mentioned construction method is known in the art, and is described in " A.Nagy, et al., " Manipulating the Mouse Embryo:A Laboratory Manual (Third Edition), " Cold Spring Harbor Laboratory Press, 2003 ", the entire contents of which are incorporated herein by reference.

[0128] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the family Muridae. In one embodiment, the genetically modified animal is selected from the families Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny mice, crested rats), Malinomys (climbing mice, rock mice, tailed rats, Madagascar rats, and mice), Spiny Mouse (e.g., spiny dormouse), and Murid (e.g., mole rats, bamboo rats, and zokors). In a specific embodiment, the genetically modified rodent is selected from the families true mice or rats (Muroidea), gerbils, spiny mice, and crested rats. In one embodiment, the genetically modified mouse is from a member of the family Muridae. In one embodiment, the non-human animal is a rodent. In a specific embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0129] In some embodiments, the non-human animal is a mouse of the C57BL strain, wherein the C57BL strain is selected from the group consisting of C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / min, C57BL6J, C57B1 / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL10SnSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2. These mice are described, 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 contents of which are incorporated herein by reference in their 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 BL / 6 strain. In some embodiments, the mouse is a 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 system (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a BALB / c, a, a / He, a / J, a / WySN, AKR, AKR / a, AKR / J, AKR / N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA / 2, KM, NIH, ICR, CFW, FACA, C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL6, C57L / 6J, C57BL / 6ByJ, or C5C57BL / 6NJ.Mice of C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st or CBA / H strains and NOD, NOD / SCID, NOD-Prkdc. scid IL-2rg null Background of mice.

[0130] The genetically modified non-human animal comprises a modification of an endogenous non-human NKG2D gene site (or locus). In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature NKG2D protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to a mature NKG2D protein amino acid sequence). Although the present invention provides cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein, in some embodiments, the genetically modified non-human animal comprises a modification of an endogenous NKG2D gene site in the non-human animal.

[0131] The genetically modified animal can express human NKG2D and / or chimeric (e.g., humanized) NKG2D at an endogenous mouse locus, wherein the non-human animal (e.g., mouse) endogenous NKG2D gene has been replaced or inserted with a human NKG2D gene and / or a nucleotide sequence encoding a region of a human NKG2D sequence, or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% homologous to a human NKG2D sequence. In various embodiments, the non-human animal's endogenous NKG2D locus is modified by comprising all or part of a nucleic acid sequence encoding a mature human NKG2D protein.

[0132] In some embodiments, genetically modified non-human animals (e.g., mice) can express human NKG2D and / or chimeric NKG2D (e.g., humanized NKG2D) under the control of endogenous or human regulatory elements (including 5'UTR, 3'UTR, enhancer, or promoter) in non-human animals. In some embodiments, genetically modified non-human animals can express human NKG2D and / or chimeric NKG2D (e.g., humanized NKG2D) under the control of human promoters and / or regulatory elements. Insertion or substitution at the endogenous locus of a non-human animal provides a non-human animal that expresses human NKG2D or chimeric NKG2D (e.g., humanized NKG2D) in suitable cells and does not result in potential pathological changes observed in some other transgenic non-human animals known in the art. Human NKG2D or chimeric NKG2D (e.g., humanized NKG2D) expressed in a non-human animal can maintain one or more functions of wild-type animals or human NKG2D in the non-human animal. In some embodiments, the non-human animal does not express endogenous NKG2D. In some embodiments, the non-human animal's endogenous NKG2D expression level is reduced compared to the NKG2D expression level in a wild-type animal. As used herein, the term "endogenous NKG2D" refers to the NKG2D protein expressed by the endogenous NKG2D nucleotide sequence of a non-human animal (e.g., mouse) before any genetic modification.

[0133] In some embodiments, the genome of the genetically modified non-human animal comprises a nucleotide sequence that is identical or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to the amino acid sequence of human NKG2D (NP_031386.2; SEQ ID NO: 2). In some embodiments, the genome comprises a nucleotide sequence that is identical or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to SEQ ID NO: 7, 8, 38, or 48. In some embodiments, the genome comprises a nucleotide sequence that is identical or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to positions 396-815 or 1-1576 of NM_007360.4.

[0134] In some embodiments, the genome of a non-human animal comprises: all or part of the nucleotide sequence from the upstream nucleotide sequence outside the 5'UTR of a human NKG2D gene to the downstream nucleotide sequence outside the 3'UTR. In some embodiments, the nucleotide sequence from the upstream nucleotide sequence outside the 5'UTR of the human NKG2D genome to the downstream nucleotide sequence outside the 3'UTR comprises all or part of the nucleotide sequence outside the 5'UTR, the 5'UTR, the NKG2D gene coding region sequence, the 3'UTR, and / or the downstream nucleotide sequence outside the 3'UTR. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 450, 600, 1000, 1500, 2000, 2500, 3000, 3500, 5000, 7000, 9000, 12000, 12100, 12300, 12600, 12800, 12900, 12910, 12920, 12930, 12932, 12934, 12936, 12937, 12938, 12939, 14000 or 20000 bp of nucleotides. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises 12939 bp. In some embodiments, the 5'UTR outer upstream nucleotide sequence includes a nucleotide sequence of at least 100-800bp, 2000-6000bp or 8000-13000bp. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 450, 600, 800, 900, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1200, 1400, 1800 or 2000bp of continuous nucleotides. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises 800-1500bp.

[0135] In some embodiments, the genome of a non-human animal comprises: all or part of exons 1-8 of the human NKG2D gene, preferably part of exon 4, all of exons 5-7, and part of exon 8. In some embodiments, the portion of exon 4 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, or 93 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 4 comprises 10 bp of continuous nucleotide sequence. The portion of exon 8 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 112, 114, 116, 117, 118, 150, 200, 250, 350, 450, 600, 700, 800, 820, 840, 860, 870, 871, 873, 875, 876, 877, 878 or 879 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 8 comprises at least 118 bp of continuous nucleotide sequence.

[0136] In some embodiments, the non-human animal genome comprises a nucleotide sequence encoding all or part of the amino acid sequence of human NKG2D. In some embodiments, the non-human animal genome comprises all or part of the nucleotide sequence of SEQ ID NO: 48 or SEQ ID NO: 7.

[0137] In some embodiments, the genetically modified animal genome comprises all of exons 1-3, a portion of exon 4, and a portion of exon 8 of an endogenous NKG2D gene (e.g., mouse NKG2D). In some embodiments, the portion of exon 4 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22, 23, 28, 30, 40, 50, 60, 70, 80, 81, 82, 83, 84, 85, 90, 100, or 105 bp of contiguous nucleotide sequence. In some embodiments, the portion of exon 4 comprises 83 bp of contiguous nucleotide sequence. 70, 2572, 2574, 2576, 2577, 2578, 2579, 2580, or 2581 bp of contiguous nucleotide sequence. In some embodiments, the portion of exon 8 comprises at least 2463 bp of contiguous nucleotide sequence.

[0138] In some embodiments, a non-human animal harbors a nucleotide sequence encoding a chimeric human / non-human NKG2D polypeptide at an endogenous NKG2D locus, and the non-human animal expresses functional NKG2D on the surface of its cells. In some embodiments, the human portion of the chimeric human / non-human NKG2D polypeptide may comprise an amino acid sequence encoded by a portion of exon 4, exons 5-7, and / or a portion of exon 8 of a human NKG2D gene. In some embodiments, the human portion of the chimeric human / non-human NKG2D polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 2, or positions 78-216 thereof.

[0139] In some embodiments, the modified gene in the genome of the modified animal is homozygous or heterozygous for the endogenous modified (preferably replaced) locus. In some embodiments, the modified NKG2D gene in the genome is homozygous or heterozygous for the endogenous modified (preferably replaced) locus.

[0140] In some embodiments, the humanized NKG2D gene comprises the 5'UTR of a human NKG2D gene. In some embodiments, the humanized NKG2D gene comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the chimeric NKG2D gene comprises an endogenous (e.g., mouse) 3'UTR. In some embodiments, the humanized NKG2D gene comprises the 3'UTR of a human NKG2D gene. Where appropriate, it can be reasonably inferred that, based on the similarity of mouse and human NKG2D sequences, mouse and human NKG2D genes are subject to similar regulation. As described herein, a humanized NKG2D mouse comprising an insertion or replacement in a non-human animal endogenous NKG2D locus retains mouse endogenous regulatory elements but comprises a human NKG2D coding sequence and does not exhibit case phenomena. The expression of NKG2D in genetically modified heterozygous or homozygous mice is completely normal.

[0141] On the other hand, the present invention provides a genetically modified non-human animal, the non-human animal genome comprising a deletion of an endogenous NKG2D gene. In some embodiments, the deletion of the endogenous NKG2D gene comprises all or part of the 5'UTR outer upstream nucleotide sequence, the 5'UTR, the NKG2D gene coding region sequence, the 3'UTR and / or the 3'UTR outer downstream nucleotide sequence. In some embodiments, the deleted 5'UTR outer upstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 200, 300, 500, 800, 1400, 2200, 3000, 4000, 5000, 6000, 6100, 6200, 6220, 6240, 6260, 6264, 7000, 7481 or 8000 bp of nucleotides. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises 7481 bp of nucleotides. In some embodiments, the deleted 5'UTR outer upstream nucleotide sequence includes at least 100-500bp, 1000-3000bp or 5000-8000bp of nucleotide sequence. In some embodiments, the deleted 3'UTR outer downstream nucleotide sequence contains at least 50, 60, 70, 80, 90, 100, 120, 140, 180, 200, 220, 240, 280, 300, 350, 400, 450 or 500bp of nucleotides. In some embodiments, the 3'UTR outer downstream nucleotide sequence contains 300bp.

[0142] In some embodiments, the deletion of the endogenous NKG2D gene comprises a portion of endogenous NKG2D exon 4, all of exons 5-7, and a portion of exon 8. In some embodiments, the deleted portion of exon 4 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22, 25, 30, 40, 50, 60, 70, 80, 83, 90, 100, 101, 102, 103, 104, or 105 bp of contiguous nucleotide sequence or more. In some embodiments, the deleted portion of exon 4 comprises at least 22 bp of contiguous nucleotide sequence. In some embodiments, the portion of endogenous exon 8 that is deleted comprises 50, 60, 70, 80, 90, 100, 112, 113, 114, 115, 116, 117, 118, 119, 120, 150, 200, 250, 350, 450, 600, 700, 800, 1000, 1400, 1800, 2000, 2200, 2400, 2420, 2430, 2440, 2450, 2460, 2461, 2462, 2463, 2500, 2540, 2560, 2570, 2572, 2574, 2576, 2577, 2578, 2579, 2580, or 2581 bp of contiguous nucleotide sequence, or more. In some embodiments, the deleted portion of endogenous exon 8 comprises at least 118 bp of contiguous nucleotide sequence.

[0143] In some embodiments, the deletion of the endogenous NKG2D gene further comprises one or more introns selected from intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7.

[0144] In some embodiments, the deletion comprises at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 420, 430, 431, 432, 500, 600, 800, 1400, 1800, 2200, 2600, 3000, 3100, 3200, 3220 , 3240, 3260, 3270, 32716100, 6200, 6220, 6240, 6260, 6264, 7000, 7481, 8000, 10000, 12000, 13000, 13200, 13400, 13500, 13520, 13576, 13578, 16000, 20000, 20142 or 21359 bp of continuous nucleotide sequence or more.

[0145] The present invention further relates to the NKG2D genomic DNA sequence of humanized mice, a DNA sequence obtained by reverse transcription of mRNA that is identical to or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell comprising the construct thereof; and a tissue comprising the cells thereof.

[0146] The present invention further provides a non-human mammal constructed using the above method. In some embodiments, the non-human mammal comprises the above-described non-human animal genome. In some embodiments, the non-human mammal is a rodent. In some embodiments, the rodent is a mouse. In some embodiments, the non-human mammal expresses a protein encoded by a humanized NKG2D gene.

[0147] In addition, the present invention also provides a non-human mammal model carrying a tumor, wherein the non-human mammal model is obtained by the method described in the present application. In some embodiments, the non-human mammal is a rodent (eg, a mouse).

[0148] The present invention also provides a cell or cell line, or primary cell culture derived from a non-human mammal or its progeny, or a non-human mammal carrying a tumor, wherein the cell or cell line is derived from a non-human mammal or its progeny, or a non-human mammal carrying a tumor, or a tissue, organ, or culture thereof derived from a non-human mammal or its progeny. In the case of a tumor, the cell or cell line is derived from a tumor tissue of a non-human mammal or its progeny, or a non-human mammal carrying a tumor.

[0149] The present invention provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal or a genetically modified non-human animal is provided, wherein the genome of the genetically modified non-human animal comprises human or humanized NKG2D DNA.

[0150] In some embodiments, a non-human mammal comprises a genetic construct described herein (e.g., a genetic construct as shown in Figures 2 and 12). In some embodiments, a non-human mammal expressing a human or humanized NKG2D protein is provided. In some embodiments, a tissue specifically expressing a human or humanized NKG2D protein is provided.

[0151] In some embodiments, the expression of non-human animal human or humanized NKG2D protein is controllable, such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0152] The non-human mammal can be any non-human animal known in the art that can be used in the methods described herein. Preferred non-human mammals are mammals (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

[0153] The non-human mammal described above is subjected to genetic, molecular and behavioral analysis. The present invention provides offspring produced by mating with a non-human mammal of the same genotype or another genotype.

[0154] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its progeny. For example, a cell culture-based model can be prepared by the following methods. The cell culture can be obtained by isolating cells from the non-human mammal, or by obtaining cells from a cell culture established using the same construct and cell transfection techniques. Integration of a genetic construct containing a DNA sequence encoding a human NKG2D protein can be detected by various methods.

[0155] There are many analytical methods that can be used to detect exogenous DNA, including nucleic acid level methods (including the use of reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein level methods (including histochemical analysis, immunoblotting analysis and in vitro binding studies). In addition, the expression level of the target gene can be quantified by the ELSA method 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 transcription levels, including RNAse protection assays, Southern Blots, and RNA dot hybridization analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blots can also be used to detect the presence of human or humanized NKG2D proteins.

[0156] In some embodiments, the genetically modified non-human animals described herein (eg, mice homozygous for humanized NKG2D genes) can express human or humanized NKG2D in one or more B cells.

[0157] carrier

[0158] The present invention provides a targeting vector targeting the NKG2D gene, comprising: a) a DNA fragment (5' arm) homologous to the 5' end of the conversion region to be changed, which is selected from the genomic DNA of the NKG2D gene of a non-human animal and has a length of 100-10,000 nucleotides; b) a DNA sequence encoding a donor region; c) a DNA fragment (3' arm) homologous to the 3' end of the conversion region to be changed, which is selected from the genomic DNA of the NKG2D gene of a non-human animal and has a length of 100-10,000 nucleotides.

[0159] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000072.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000072.7;

[0160] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from nucleotides 129593654 to 129597258 of NCBI Accession No. NC_000072.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from nucleotides 129586050 to 129589748 of NCBI Accession No. NC_000072.7;

[0161] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from nucleotides 129593654 to 129594939 of NCBI Accession No. NC_000072.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from nucleotides 129588308 to 129589748 of NCBI Accession No. NC_000072.7;

[0162] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from nucleotides 129607128 to 129612145 of NCBI Accession No. NC_000072.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from nucleotides 129582548 to 129586985 of NCBI Accession No. NC_000072.7;

[0163] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 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, or 10 kb.

[0164] In some embodiments, the switch region to be altered is located in exons 1 to 8 of the NKG2D gene of a non-human animal. In some embodiments, the switch region to be altered is located in exons 4 and 8 of the NKG2D gene of a non-human animal (e.g., positions 378-809 of NM_033078.4).

[0165] In some embodiments, the switch region to be changed is located on the 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence of the non-human animal NKG2D gene. In some embodiments, the switch region to be changed is located on the 5'UTR outer upstream nucleotide sequence and the 3'UTR outer downstream nucleotide sequence of the non-human animal NKG2D gene (for example, located at 7481 bp upstream of the 5'UTR outer side of the mouse NKG2D genome and 300 bp downstream of the 3'UTR outer side of the mouse NKG2D genome).

[0166] In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes). For example, a positive screening marker gene or a negative screening marker gene. In some embodiments, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo. Preferably, the targeting vector further comprises two Frt recombination sites arranged in the same direction on both sides of the marker gene. In some embodiments, the coding gene for the negative screening marker is a coding gene for the diphtheria toxin A subunit (DTA).

[0167] In some embodiments, the 5' arm sequence is the nucleotide sequence set forth in SEQ ID NO: 3; and the 3' arm sequence is the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, the 5' arm sequence is the nucleotide sequence set forth in SEQ ID NO: 5; and the 3' arm sequence is the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, the 5' arm sequence is the nucleotide sequence set forth in SEQ ID NO: 35; and the 3' arm sequence is the nucleotide sequence set forth in SEQ ID NO: 36.

[0168] In some embodiments, the 5' arm comprises a nucleotide sequence that is at least 90% homologous to NCBI Accession No. NC_000072.7, and more preferably, the 5' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO: 3, 5, or 35. In some embodiments, the 3' arm comprises a nucleotide sequence that is at least 90% homologous to NCBI Accession No. NC_000072.7, and more preferably, the 3' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO: 4, 6, or 36.

[0169] In some embodiments, the targeting vector comprises a human sequence (e.g., positions 10373114-10379709 of NC_000012.12 or positions 10371346-10402980 of NC_000012.12). For example, the targeting region in the targeting vector includes: all or part of the nucleotide sequence of the human NKG2D gene. In some embodiments, the human NKG2D sequence comprises the upstream nucleotide sequence outside the 5'UTR of the human NKG2D genome to the downstream nucleotide sequence outside the 3'UTR. In some embodiments, the human NKG2D sequence comprises part of exon 4, all of exons 5-7, and part of exon 8 of the human NKG2D gene. In some embodiments, the nucleotide sequence of the humanized NKG2D gene encodes all or part of the nucleotide sequence of the human NKG2D protein (e.g., positions 78-216 of the human NKG2D protein), and the NCBI protein number is NP_031386.2 (SEQ ID NO: 2).

[0170] In some embodiments, the targeting vector comprises, from the 5' end to the 3' end, all of exons 1-3 of the mouse NKG2D gene, part of exon 4 of the mouse NKG2D gene, part of exon 4 of the human NKG2D gene, all of exons 5-7 of the human NKG2D gene, part of exon 8 of the human NKG2D gene, and part of exon 8 of the mouse NKG2D gene. In some embodiments, the targeting vector comprises, from the 5' end to the 3' end, a 5' arm, the 5'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides upstream of the 5'UTR, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene, the 3'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides downstream of the 5'UTR, and the 3' arm.

[0171] In some embodiments, the targeting vector comprises, from the 5' end to the 3' end:

[0172] A) SEQ ID NO: 3, 7, 4;

[0173] B) SEQ ID NO: 3, 48, 4;

[0174] C) SEQ ID NO: 5, 7, 6;

[0175] D) SEQ ID NO: 5, 48, 6;

[0176] E) SEQ ID NO: 35, 7, 36;

[0177] F) SEQ ID NO: 35, 48, 36.

[0178] In some embodiments, the targeting vector further comprises a Neo cassette. The Neo cassette can be inserted into the 5' homology arm, the 3' homology arm, or the donor region.

[0179] In some embodiments, the targeting vector further comprises one or more of SEQ ID NO: 10, 11, 39 or 40.

[0180] 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 NKG2D gene, and the sgRNA is unique on the target sequence of the gene to be changed and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'; and in some embodiments, the targeting site of the sgRNA in the mouse NKG2D gene is located at exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, the upstream nucleotide sequence of exon 1 of the mouse NKG2D gene and / or the downstream nucleotide sequence of exon 8.

[0181] In some embodiments, the targeting sequence is shown as SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23. Therefore, the present invention provides sgRNA sequences for constructing genetically modified animal models. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 16 and 19. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 18 and 20. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 17 and 22. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 21 and 23.

[0182] In some embodiments, the present invention relates to a plasmid construct comprising an sgRNA sequence (e.g., pT7-sgRNA) and / or a cell comprising the construct.

[0183] The present invention also relates to a cell comprising the targeting vector or sgRNA as described above.

[0184] In addition, the present invention also provides a non-human mammalian cell having any one of the above-mentioned targeting vectors and one or more in vitro transcripts of the constructs described herein. In some embodiments, the cell comprises Cas9 mRNA or its in vitro transcript.

[0185] In some embodiments, the cell is heterozygous for the gene. In some embodiments, the cell is homozygous for the gene.

[0186] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell.

[0187] The present invention also relates to the use of the above-mentioned targeting vector or sgRNA or sgRNA vector in NKG2D gene modification.

[0188] Method for constructing genetically modified non-human animals

[0189] Genetically modified non-human animals can be prepared by several gene editing technologies known in the art, including homologous recombination technology using embryonic stem cells, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR / Cas9 genome editing is used to produce genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in “Delivery technologies for genome editing,” Nature Reviews Drug Discovery 16.6 (2017): 387-399 by Yin et al., the entire contents of which are 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.

[0190] In some embodiments, the nucleotide sequence encoding the endogenous NKG2D region in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding the corresponding region of human NKG2D. In some embodiments, the non-human animal's endogenous NKG2D protein is expressed at a reduced level or is absent compared to wild-type. In some embodiments, the replacement occurs in a germ cell, somatic cell, blastocyst, or fibroblast. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0191] Figures 3, 4, 5, 13 and 14 show a humanized targeting strategy for targeting the mouse NKG2D site. The targeting vector comprises a vector consisting of a 5' homology arm, a human or humanized NKG2D gene fragment and a 3' homology arm. The process involves using homologous recombination to replace the endogenous corresponding NKG2D nucleotide sequence with a human or humanized nucleotide sequence. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs or CRISPR) can result in a double-strand break of the DNA, and homologous recombination is used to replace the mouse endogenous NKG2D sequence with the human or humanized NKG2D sequence to form a chimeric NKG2D gene (e.g., a humanized NKG2D gene).

[0192] In some embodiments, the nucleotide sequence encoding the corresponding region of human NKG2D comprises all or part of the nucleotide sequence encoding the extracellular region, transmembrane region and / or cytoplasmic region of the human NKG2D protein.

[0193] In some embodiments, the nucleotide sequence encoding the corresponding region of human NKG2D comprises a nucleotide sequence encoding at least 50 to 216, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 144, 150, 160, 170, 180, 190, 200, 210 or 216 consecutive amino acids of human NKG2D protein; further preferably comprises a nucleotide sequence encoding SEQ ID NO: 2 positions 78 to 216 or the amino acid sequence of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 positions 78 to 216 or the amino acid sequence of SEQ ID NO: NO:2; or, comprising a nucleotide sequence encoding at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:2; or, comprising a nucleotide sequence encoding a nucleotide sequence that differs from SEQ ID NO:2 at positions 78-216 or the amino acid sequence set forth in SEQ ID NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprising a nucleotide sequence encoding one or more amino acids as set forth in positions 78-216 of SEQ ID NO:2 or the amino acid sequence set forth in SEQ ID NO:2, including substitutions, deletions and / or insertions.

[0194] In some embodiments, the nucleotide sequence encoding the corresponding region of human NKG2D comprises part of human NKG2D gene exon 4 to part of exon 8, or comprises all or part of the nucleotide sequence from the upstream nucleotide sequence outside the 5'UTR of the human NKG2D gene to the downstream nucleotide sequence outside the 3'UTR. In some embodiments, the nucleotide sequence from the upstream nucleotide sequence outside the 5'UTR of the human NKG2D genome to the downstream nucleotide sequence outside the 3'UTR comprises the upstream nucleotide sequence outside the 5'UTR, the 5'UTR, the NKG2D gene coding region sequence, the 3'UTR and / or the downstream nucleotide sequence outside the 3'UTR. In some embodiments, the NKG2D gene coding region sequence comprises all or part of human NKG2D exons 1-8, wherein the portion comprises part of exon 4, all of exons 5-7 and part of exon 8. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 450, 600, 1000, 1500, 2000, 2500, 3000, 3500, 5000, 7000, 9000, 12000, 12100, 12300, 12600, 12800, 12900, 12910, 12920, 12930, 12932, 12934, 12936, 12937, 12938, 12939, 14000 or 20000 bp of nucleotides. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises 12939 bp. In some embodiments, the 5'UTR outer upstream nucleotide sequence includes a nucleotide sequence of at least 100-800bp, 2000-6000bp or 8000-13000bp. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 450, 600, 800, 900, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1200, 1400, 1800 or 2000bp of continuous nucleotides. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises 800-1500bp.

[0195] In some embodiments, the portion of human NKG2D gene exon 4 comprises human NKG2D gene exon 4 including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92 or 93 bp of continuous nucleotide sequence. In some embodiments, the portion of human NKG2D gene exon 8 includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 112, 114, 116, 117, 118, 150, 200, 250, 350, 450, 600, 700, 800, 820, 840, 860, 870, 871, 873, 875, 876, 877, 878, or 879 bp of contiguous nucleotide sequence of human NKG2D gene exon 8. In some embodiments, the portion of exon 8 comprises at least 118 bp of contiguous nucleotide sequence.

[0196] In some embodiments, the nucleotide sequence encoding the corresponding region of human NKG2D comprises the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 48; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 48; or, comprises a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 48 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotides; or, comprises a nucleotide sequence having the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 48, including substitutions, deletions and / or insertions of one or more nucleotides.

[0197] In some embodiments, the nucleotide sequence encoding the corresponding region of human NKG2D is operably linked to an endogenous regulatory element of an endogenous NKG2D gene on at least one chromosome.

[0198] In some embodiments, the nucleotide sequence encoding the corresponding region of human NKG2D is operably linked to human regulatory elements of the endogenous NKG2D gene of at least one chromosome.

[0199] In some embodiments, the replaced endogenous NKG2D nucleotide sequence in the non-human animal includes a portion of exon 4 to a portion of exon 8 of the mouse NKG2D gene, or includes all or part of the 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence of the mouse NKG2D gene. In some embodiments, the mouse NKG2D genome 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence includes the 5'UTR outer upstream nucleotide sequence, 5'UTR, NKG2D gene coding region sequence, 3'UTR and / or 3'UTR outer downstream nucleotide sequence. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 200, 300, 500, 800, 1400, 2200, 3000, 4000, 5000, 6000, 6100, 6200, 6220, 6240, 6260, 6264, 7000, 7481 or 8000 bp of nucleotides. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises 7481 bp of nucleotides. In some embodiments, the 5'UTR outer upstream nucleotide sequence comprises at least 100-500 bp, 1000-3000 bp or 5000-6000 bp of nucleotide sequence. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises at least 50, 60, 70, 80, 90, 100, 120, 140, 180, 200, 220, 240, 280, 300, 350, 400, 450 or 500 bp of nucleotides. In some embodiments, the 3'UTR outer downstream nucleotide sequence comprises 200-400 bp of nucleotides.

[0200] In some embodiments, the replaced endogenous NKG2D nucleotide sequence comprises part of endogenous NKG2D exon 4, all of exons 5-7, and part of exon 8. In some embodiments, the replaced endogenous NKG2D nucleotide sequence encodes the amino acid sequence shown in positions 90-232 of SEQ ID NO: 1.

[0201] In some embodiments, the human or humanized NKG2D gene is regulated in a non-human animal by a regulatory element. For example, the regulatory element can be endogenous or exogenous. In a specific embodiment of the present invention, the endogenous regulatory element is derived from a non-human animal NKG2D gene, and the exogenous regulatory element is derived from a human NKG2D gene.

[0202] In some embodiments, the construction method includes replacing all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7 and / or exon 8 of a non-human animal NKG2D gene with all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7 and / or exon 8 of a human NKG2D gene, preferably replacing part of exon 4, all of 5-7 and part of exon 8 (preferably part of exon 4 to part of exon 8) of a non-human animal NKG2D gene with part of exon 4, all of 5-7 and part of exon 8 (preferably part of exon 4 to part of exon 8) of a human NKG2D gene.

[0203] In some embodiments, the construction method comprises replacing the 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence of the non-human animal NKG2D gene with the 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence of the human NKG2D gene.

[0204] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 in the genome of a non-human animal with a nucleotide sequence encoding a human or humanized NKG2D protein, or a nucleotide sequence of a human or humanized NKG2D gene.

[0205] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 in the genome of a non-human animal with a genomic DNA sequence, cDNA sequence or CDS sequence comprising human NKG2D.

[0206] In some embodiments, the construction method comprises replacing a nucleotide sequence encoding the extracellular region of non-human animal NKG2D with a nucleotide sequence encoding the extracellular region of human NKG2D.

[0207] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 in the genome of a non-human animal with a nucleotide sequence encoding SEQ ID NO: 2 or the amino acid sequence shown at positions 78-216 thereof.

[0208] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or the amino acid sequence shown at positions 90-232 thereof in the genome of a non-human animal with a nucleotide sequence encoding a human or humanized NKG2D protein, or a nucleotide sequence of a human or humanized NKG2D gene.

[0209] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or the amino acid sequence shown at positions 90-232 thereof in the genome of a non-human animal with a genomic DNA sequence, cDNA sequence or CDS sequence comprising a human NKG2D gene.

[0210] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or positions 90-232 thereof in the genome of a non-human animal with a nucleotide sequence encoding SEQ ID NO: 2 or the amino acid sequence shown in positions 78-216 thereof.

[0211] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the amino acid sequence shown at positions 90-232 of SEQ ID NO: 1 in the genome of a non-human animal with the nucleotide sequence shown in SEQ ID NO: 7.

[0212] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 in the genome of a non-human animal with the nucleotide sequence shown in SEQ ID NO: 48.

[0213] In some embodiments, the construction method of a non-human animal comprises the construction of a genetically modified non-human animal NKG2D gene coding frame, wherein the genetically modified non-human animal NKG2D gene coding frame construction can be achieved by knocking out a functional region of the non-human animal NKG2D gene or by inserting a sequence so that the non-human animal NKG2D protein is not expressed or the expression is reduced or the expressed protein is non-functional. In some embodiments, the genetically modified non-human animal NKG2D gene coding frame construction comprises knocking out all or part of the nucleotide sequence of exons 1-8 of the non-human animal NKG2D gene. In some embodiments, the modified non-human animal NKG2D gene coding frame construction can knock out part of exon 4, all of 5-7, and part of exon 8 of the non-human animal NKG2D gene, or knock out part of exon 4 to part of exon 8 of the non-human animal NKG2D gene.

[0214] In some embodiments, the method for constructing a genetically modified non-human animal comprises replacing the sequence following the endogenous regulatory elements of the non-human animal's NKG2D gene with a nucleotide sequence encoding a human or humanized NKG2D protein. In some embodiments, the replaced sequence further comprises an auxiliary sequence. In some embodiments, the auxiliary sequence can be a stop codon, such that the NKG2D genetically modified non-human animal expresses a human or humanized NKG2D protein but does not express the non-human animal's NKG2D protein.

[0215] In some embodiments, the method for constructing a genetically modified non-human animal comprises replacing the upstream nucleotide sequence outside the 5'UTR to the downstream nucleotide sequence outside the 3'UTR of the non-human animal NKG2D gene, or replacing a portion of exon 4 to a portion of exon 8 of the non-human animal NKG2D gene, with a nucleotide sequence encoding a human NKG2D protein. In some embodiments, the replaced sequence further comprises an auxiliary sequence. In some embodiments, the nucleotide sequence encoding the human NKG2D protein comprises a human regulatory element. In some embodiments, the auxiliary sequence can be a stop codon, such that the NKG2D gene humanized animal model expresses a human or humanized NKG2D protein in vivo without expressing the non-human animal NKG2D protein.

[0216] In some embodiments, in order to improve the recombination efficiency, a sgRNA targeting the NKG2D gene can also be used together with the targeting vector of the NKG2D gene to construct a non-human animal. The sgRNA targets the NKG2D gene of a non-human animal, and the sequence of the sgRNA is on the target sequence on the NKG2D gene to be changed. In some embodiments, the sgRNA target site is located on the sequence from exon 1 to exon 8 of the NKG2D gene. In some embodiments, the sgRNA target site is located on the sequence of exon 4 and exon 8 of the NKG2D gene. In some embodiments, the target sequence of the sgRNA on the NKG2D gene is shown in SEQ ID NO: 14 or SEQ ID NO: 15.

[0217] In some embodiments, the construction method includes introducing the above-mentioned NKG2D gene targeting vector, sgRNA targeting the NKG2D gene and Cas9 into non-human animal cells, culturing the cells (preferably fertilized eggs), and then transplanting the cultured cells into the fallopian tubes of female non-human animals (such as non-human mammals), allowing them to develop, and identifying and screening to obtain NKG2D gene-modified non-human animals.

[0218] In some embodiments, the construction method includes introducing the above-mentioned targeting vector into embryonic stem cells of a non-human animal (e.g., a non-human mammal), and after a brief culture, introducing it into a previously isolated blastocyst. The resulting chimeric blastocyst is transplanted into the oviduct of a recipient mother mouse, allowed to develop, and non-human animals with humanized NKG2D genes are obtained by identification and screening.

[0219] In some embodiments, the construction method further comprises: mating a non-human animal with a humanized NKG2D gene with other genetically modified non-human animals, fertilizing in vitro, or directly performing gene editing, and screening to obtain a multi-gene modified non-human animal. In some embodiments of the present invention, the other genes are at least one of NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40, and B7-H3. In some embodiments, the non-human animal also expresses at least one of human or chimeric NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40, and B7-H3.

[0220] In some embodiments, some of the non-human animals have been described in patents PCT / CN2019 / 127084, PCT / CN2017 / 106024, PCT / CN2018 / 110069, PCT / CN2017 / 099574, PCT / CN2017 / 117984, PCT / CN2018 / 091846, PCT / CN2018 / 081628, PCT / CN2017 / 120388, PCT / CN2018 / 091845, PCT / CN2017 / 099576, PCT / CN2017 / 110494, PCT / CN2017 / 099575, and PCT / CN2021 / 095273, the entire contents of which are incorporated herein by reference.

[0221] In some embodiments, each of the multiple genes modified in the genome of the multi-gene modified non-human animal is homozygous or heterozygous for the endogenous modified locus.

[0222] In some embodiments, the non-human animal can be selected from rodents, pigs, rabbits, monkeys, and any other non-human animals that can be gene-edited to produce humanized genes.

[0223] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0224] Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit, or an immunodeficient monkey. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Still further preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - Mouse, Rag 2 - / - -IL2rg - / - mice, NOD / SCID mice, or nude mice.

[0225] Use of genetically modified non-human animals

[0226] 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 non-human animal locus and under the control of an endogenous promoter and / or regulatory elements can produce a non-human animal with qualities and characteristics that may be significantly different from those of a typical knockout plus transgenic animal. In a typical knockout plus transgenic animal, the endogenous locus is removed or destroyed and a fully human transgene is inserted into the genome of the animal and may integrate randomly into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of the human gene and / or protein assays and / or functional assays. In a human transgene, the human sequences upstream and / or downstream provide suitable support for expression and / or regulation of the transgene.

[0227] Genetically modified animals that express human or humanized NKG2D proteins, e.g., in a physiologically appropriate manner, offer a variety of uses, including but not limited to developing treatments for human diseases and disorders, and evaluating the toxicity and / or efficacy of these human treatments in animal models.

[0228] The present invention also provides a use of the above-mentioned NKG2D gene-modified non-human animal or a non-human animal obtained by any of the above-mentioned construction methods.

[0229] In some embodiments, the application comprises:

[0230] A) Applications in product development involving NKG2D-related immune processes in human cells;

[0231] B) Application as a model system for NKG2D-related research in pharmacology, immunology, microbiology, and medicine;

[0232] C) Applications involving the production and use of animal experimental disease models for the study of NKG2D-related etiologies and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0233] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human NKG2D signaling pathway modulators; or

[0234] E) Study the function of the NKG2D gene, study the drugs and efficacy targeting the human NKG2D target site, and study the application of NKG2D-related immune disease drugs or anti-tumor drugs.

[0235] The present invention provides a non-human animal expressing a human or humanized NKG2D protein, which can be used to screen for human NKG2D-specific modulators. In some embodiments, the non-human animal is an animal model for human disease. For example, the disease is genetically induced (knock-in or knock-out). In various embodiments, the genetically modified non-human animal further comprises an impaired immune system, such as a genetically modified human tissue xenograft, including human solid tumors (e.g., prostate cancer) or blood cell tumors (e.g., lymphocyte tumors, B or T cell tumors), etc.

[0236] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-NKG2D antibodies) in treating diseases associated with NKG2D expression or abnormal expression (e.g., overexpression). In some embodiments, the diseases include diseases for which targeting NKG2D or downregulating NKG2D expression is beneficial. In some embodiments, the diseases include autoimmune diseases or cancer. In some embodiments, the autoimmune diseases include, but are not limited to, rheumatoid arthritis, type I diabetes, alopecia areata, Crohn's disease, and atherosclerosis.

[0237] In some embodiments, a therapeutic agent (e.g., an anti-NKG2D antibody) is administered to a non-human animal, wherein the non-human animal has cancer or a tumor, and the inhibitory effect of the therapeutic agent on the cancer or tumor is detected. In some embodiments, the detection includes determining the size and / or proliferation rate of tumor cells. In some embodiments, the detection method includes vernier caliper measurement, flow cytometry, and / or animal in vivo imaging detection. In some embodiments, the detection includes assessing individual body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, including changes in food consumption or water consumption.

[0238] In some embodiments, the tumor cells include one or more cancer cells injected into an animal (e.g., cancer cells derived from a human or non-human animal). In some embodiments, the therapeutic agent inhibits the NKG2D / NKG2DL-mediated signaling pathway. In some embodiments, the therapeutic agent does not inhibit the NKG2D / NKG2DL-mediated signaling pathway.

[0239] In some embodiments, genetically modified non-human animals can be used to detect whether an anti-NKG2D antibody is an agonist or an antagonist. In some embodiments, the methods described herein can be used to detect the function of a therapeutic agent (e.g., an anti-NKG2D antibody), for example, whether the therapeutic agent can upregulate an immune response or downregulate an immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent for treating a disease in a subject (e.g., an immune disease). The inhibitory effect on tumors can also be determined by methods known in the art, for example, measuring the tumor volume in an 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-TVt / TVc) x 100, where TVt and TVc are the mean tumor volumes (or weights) of the treatment and control groups.

[0240] In some embodiments, therapeutic agents (such as, anti-NKG2D antibodies) can be used to treat various cancers. "Cancer" described in the present invention refers to cells with autonomous growth ability, i.e., an abnormal state or disease characterized by rapid proliferation of cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of the histopathological type or invasive stage. "Tumor" described in the present invention includes but is not limited to lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome and sarcoma. wherein the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In a specific embodiment of the present invention, the tumor is a solid tumor, gastrointestinal cancer, acute myeloid leukemia, colorectal cancer, gastrointestinal pancreatic cancer, breast cancer, multiple myeloma, genitourinary system cancer, prostate cancer, glioblastoma, liver cancer, lung cancer, melanoma, lymphoma, bile duct cancer, ovarian cancer, nasopharyngeal tumor, nasopharyngeal carcinoma, medulloblastoma.

[0241] The present invention also provides a method for determining the toxicity of a therapeutic agent (e.g., an anti-NKG2D antibody). The method comprises administering an anti-NKG2D antibody to a non-human animal as described above and assessing the animal's weight change, red blood cell count, hematocrit, and / or hemoglobin. In some embodiments, the antibody can reduce red blood cells (RBCs), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the animal's weight is at least 5%, 10%, 20%, 30%, or 40% less than a control group (e.g., the average weight of an animal not treated with the antibody).

[0242] The present invention also provides an animal model constructed by the method described in the present application for developing products related to human cellular immune processes, producing human antibodies, or a model system for pharmacology, immunology, microbiology and medical research.

[0243] In some embodiments, an animal model generated by the methods described herein is provided for producing and utilizing animal experimental disease models of immune processes in human cells, studying pathogens, or developing new diagnostic and / or therapeutic strategies.

[0244] The present invention also provides an animal model generated by the method described herein to screen, verify, evaluate or study NKG2D gene function, human NKG2D antibodies, drugs or effectiveness of human NKG2D target sites, drugs for immune-related diseases and anti-tumor drugs.

[0245] Non-human animal models with two or more human or chimeric (x) genes

[0246] The present invention also provides a non-human animal or animal model having two or more human or chimeric (x) genes. In some embodiments, the non-human animal or animal model comprises a human or chimeric NKG2D gene and a nucleic acid sequence encoding other human or chimeric (x) proteins. In some embodiments, the chimeric (x) protein comprises at least one of NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40, and B7-H3. In some embodiments, the non-human animal or animal model further expresses at least one of human or humanized NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40, and B7-H3.

[0247] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric (x) genes, the method comprising:

[0248] (1) Providing the above-mentioned construction method to obtain non-human animals or animal models;

[0249] (2) mating, in vitro fertilization, or direct gene editing of the non-human animal or animal model provided in step (1) with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0250] 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 NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40 and B7-H3.

[0251] In some embodiments, NKG2D humanization is performed directly on non-human animals with human or chimeric NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40, and B7-H3 gene modifications.

[0252] Since these proteins may be involved in different mechanisms, combination therapy targeting two or more of them may be a more effective treatment method. In fact, many related clinical trials are underway and have shown good results. Multigene modified non-human animal models can be used to determine the effectiveness of combination therapy targeting two or more proteins, for example, anti-NKG2D antibodies, and additional therapeutic agents for treating cancer or metabolic diseases (e.g., obesity or cardiovascular disease). The method comprises administering an anti-NKG2D antibody and an additional therapeutic agent to an animal, wherein the non-human animal has a tumor or immune disease, and determining the effect of the combination therapy on the immune tumor or immune disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40 or B7-H3. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., nivolumab) or an anti-PD-L1 antibody. In some embodiments, the non-human animal further comprises a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized B7-H3. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PD-L1 antibody, or an anti-B7-H3 antibody. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1 and / or B7-H3.

[0253] In some embodiments, the combination therapy can also be used to treat various cancers described herein, such as solid tumors, gastrointestinal cancer, acute myeloid leukemia, colorectal cancer, gastrointestinal pancreatic cancer, breast cancer, multiple myeloma, genitourinary system cancer, prostate cancer, glioblastoma, liver cancer, lung cancer, melanoma, lymphoma, bile duct cancer, ovarian cancer, nasopharyngeal tumor, nasopharyngeal carcinoma or one or more of medulloblastoma.

[0254] In some embodiments, the above-described methods of treatment can be used in combination with conventional cancer chemotherapy drugs. In some embodiments, the methods of treating cancer can be used alone or in combination with the methods described herein, including treating the subject with chemotherapy, such as camphor, doxorubicin, cisplatin, carboplatin, procarbazine, methylchloroethylamine, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chlorambucil, endosulfan, nitrosulfamethoxazole, dactinomycin, daunorubicin, bleomycin, prinomycin, mitomycin, etoposide, verapir, podophyllotoxin, tamoxifen, paclitaxel, transplatin, 5-fluorouracil, vincristine, vinblastine and / or methotrexate. The methods may include performing surgery on the subject to remove at least a portion of the cancer, such as removing part or all of a tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0255] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0256] Figure 1: Schematic comparison of the mouse NKG2D gene and the human NKG2D locus (not to scale);

[0257] Figure 2: Schematic diagram of the humanized mouse NKG2D gene (not to scale);

[0258] Figure 3: Schematic diagram of NKG2D gene targeting strategy and targeting vector design (not to scale);

[0259] Figure 4: Schematic diagram of the FRT recombination process for humanizing the NKG2D gene in mice (not to scale);

[0260] Figure 5: Schematic diagram 2 of NKG2D gene targeting strategy and targeting vector design (not to scale);

[0261] Figure 6: PCR identification results of the tail of F1 generation humanized mice carrying the NKG2D gene, where PC is the positive control, WT is the wild-type control, H2O is the water control, and M is a marker;

[0262] Figure 7: Schematic diagram of the results of Southern Blot detection of positive clones, WT is the wild type;

[0263] Figure 8: NKG2D mRNA detection results in the spleens of C57BL / 6 wild-type mice (+ / +) and NKG2D gene homozygous mice (H / H), where H2O is a water control;

[0264] Figure 9: Percentages of leukocyte subtypes (A) and T cell subtypes (B) in the spleens of wild-type C57BL / 6 mice (+ / +) and NKG2D homozygous mice (H / H);

[0265] Figure 10: Percentages of leukocyte subtypes (A) and T cell subtypes (B) in lymph nodes of wild-type C57BL / 6 mice (+ / +) and NKG2D humanized homozygous mice (H / H);

[0266] Figure 11: Percentages of leukocyte subtypes (A) and T cell subtypes (B) in the blood of wild-type C57BL / 6 mice (+ / +) and NKG2D humanized homozygous mice (H / H);

[0267] FIG12 : Schematic diagram of the second humanized modification of the mouse NKG2D gene (not to scale);

[0268] Figure 13: Schematic diagram of NKG2D gene targeting strategy and targeting vector design (not to scale);

[0269] Figure 14: Schematic diagram of the FRT recombination process of NKG2D gene humanized mice (not to scale);

[0270] Figure 15: Human NKG2D amino acid sequence (NP_031386.2; SEQ ID NO: 2) and mouse NKG2D amino acid sequence (NP_149069.1; SEQ ID NO: 1);

[0271] Figure 16: Human NKG2D amino acid sequence (NP_031386.2; SEQ ID NO: 2) and rat NKG2D amino acid sequence (NP_598196.1; SEQ ID NO: 44). DETAILED DESCRIPTION

[0272] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0273] In each of the following examples, equipment and materials were obtained from the following companies:

[0274] BspHI, NcoI, and AvrII enzymes were purchased from NEB with catalog numbers R0517S, R3193S, and R0174S, respectively;

[0275] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Center of the China Food and Drug Administration;

[0276] Brilliant Violet 510 TMAnti-mouse CD45 Antibody was purchased from Biolegend, catalog number 103138;

[0277] APC Hamster Anti-Mouse TCRβChain was purchased from BD Pharmingen, catalog number 553174;

[0278] PE / Cy TM 7. Mouse anti-mouse NK1.1 was purchased from BD Pharmingen, catalog number 552878;

[0279] FITC anti-mouse CD314 (NKG2D) Antibody was purchased from Biolegend, catalog number 115711;

[0280] CD314 (NKG2D) Monoclonal Antibody (1D11), PerCP-eFluor 710, eBioscience™ was purchased from ThermoFisher, catalog number 46-5878-42;

[0281] Zombie NIR TM Fixable Viability Kit was purchased from Biolegend, catalog number 423106;

[0282] Purified anti-mouse CD16 / 32 Antibody was purchased from Biolegend, catalog number 101302;

[0283] Trizol kit was purchased from TakaRa, catalog number 6110A;

[0284] PerCP / Cyanine5.5 anti-mouse TCRβchain Antibody was purchased from Biolegend, catalog number 109228;

[0285] Brilliant Violet 421 TM Anti-mouse CD4 Antibody was purchased from Biolegend, catalog number 100438;

[0286] PE anti-mouse CD8a Antibody was purchased from Biolegend, catalog number 100708;

[0287] Brilliant Violet 605 TMAnti-mouse CD19 Antibody was purchased from Biolegend, catalog number 115540;

[0288] PerCP anti-mouse Ly-6G / Ly-6C (Gr-1) Antibody was purchased from Biolegend, catalog number 108426;

[0289] Brilliant Violet 605 TM Anti-mouse CD11c Antibody was purchased from Biolegend, catalog number 117334;

[0290] PE anti-mouse / human CD11b Antibody was purchased from Biolegend, catalog number 101208;

[0291] FITC anti-mouse F4 / 80 Antibody was purchased from Biolegend, catalog number 123108;

[0292] APC anti-mouse / rat Foxp3 Antibody was purchased from eBioscience, catalog number 17-5773-82;

[0293] V450 Rat Anti-mouse CD3 Molecular Complex was purchased from BD Pharmingen, catalog number 561389;

[0294] Brilliant Violet 785 TM Anti-mouse CD4 Antibody was purchased from Biolegend, catalog number 100453;

[0295] Brilliant Violet 711 TM Anti-mouse CD8a was purchased from Biolegend, catalog number 100759;

[0296] Brilliant Violet 650 TM Anti-mouse CD19 Antibody was purchased from Biolegend, catalog number 115541.

[0297] Example 1 Method 1 for constructing NKG2D gene humanized mice

[0298] A schematic diagram of the comparison of the mouse NKG2D gene (NCBI Gene ID: 27007, Primary source: MGI: 1196250, UniProt ID: O54709, located at positions 129587286 to 129600863 of chromosome 6 NC_000072.7, based on transcript NM_033078.4 and its encoded protein NP_149069.1 (SEQ ID NO: 1)) and the human NKG2D gene (NCBI Gene ID: 22914, Primary source: HGNC: 18788, UniProt ID: P26718, located at positions 10372353 to 10390041 of chromosome 12 NC_000012.12, based on transcript NM_007360.4 and its encoded protein NP_031386.2 (SEQ ID NO: 2)) is shown in Figure 1.

[0299] To achieve the objectives of the present invention, a nucleotide sequence encoding a human NKG2D protein can be introduced into the endogenous NKG2D locus of a mouse, such that the mouse expresses a human or humanized NKG2D protein. Specifically, using gene editing technology, under the control of the mouse NKG2D gene regulatory elements, a partial sequence from exon 4 to exon 8 of the mouse, approximately 3.9 kb, is replaced with a partial sequence from exon 4 to exon 8 of the human NKG2D gene, comprising approximately 6.6 kb. A schematic diagram of the resulting humanized NKG2D locus is shown in Figure 2, thereby achieving humanization of the mouse NKG2D gene.

[0300] A targeting strategy was designed as shown in Figure 3, which shows the targeting vector containing upstream and downstream homology arm sequences for the mouse NKG2D gene and Segment A containing the human NKG2D fragment. The upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to nucleotides 129593654 to 129597258 of NCBI Accession No. NC_000072.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to nucleotides 129586050 to 129589748 of NCBI Accession No. NC_000072.7. The nucleotide sequence of the human NKG2D fragment in Segment A (SEQ ID NO: 7) is identical to nucleotides 10373114 to 10379709 of NCBI Accession No. NC_000012.12.

[0301] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system FRT recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The 5' end of the Neo cassette was designed to link to the human gene as follows: 5'-TGGCACATGCCTGTAATCC CAGTCGACGGTATCGATAAGCTTGATATCGAATTCCGAAGTTCCTATTCTCTA-3' (SEQ ID NO: 10), where the "A" in the "CCCA" sequence is the last nucleotide of the human gene, and the first G in the "GTCG" sequence is the first nucleotide of the Neo cassette. The 3' end of the Neo cassette was designed to link to the human gene as follows: 5'-CTCTAGAAAGTATAGGAACTTCATCAGTCAGGTACATAATGGTGGATCCGCTACTTACGAAACTGACACAGGAGAATCACT-3' (SEQ ID NO: 11), where the last "C" in the "ATCC" sequence is the last nucleotide of the Neo cassette, and the "G" in the "GCTA" sequence is the first nucleotide of the human gene. In addition, a gene encoding a negative selection marker (the gene encoding the diphtheria toxin A subunit (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the modified humanized mouse NKG2D is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 9.

[0302] Given that human NKG2D has multiple isoforms or transcripts, the methods described herein can be applied to other isoforms or transcripts.

[0303] The targeting vector construction can be carried out by conventional methods, such as enzyme digestion and connection. After the constructed targeting vector is preliminarily verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector electroporation transfection correct for sequencing verification is introduced into the embryonic stem cells of C57BL / 6 mice, and the cells obtained are screened using positive clone screening marker genes, and PCR and Southern Blot techniques are utilized to detect the integration of the foreign gene, and correct positive clone cells are screened. After PCR identification (primers are shown in Table 5), it is confirmed that there is no random insertion of the clone, and then the correct clone is further sequenced and verified to be carried out the next step of the experiment.

[0304] Table 5 PCR primer names and specific sequences

[0305] The correctly selected positive clone cells (black mice) are introduced into isolated blastocysts (white mice) according to techniques known in the art. The resulting chimeric blastocysts are transferred to culture medium for a short period of time and then transplanted into the oviducts of recipient female mice (white mice), producing F0 generation chimeric mice (black and white). F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice. F1 generation heterozygous mice are then mated with each other to obtain F2 generation homozygous mice. Positive mice can also be mated with Flp tool mice to remove the positive clone selection marker gene (see Figure 4 for a schematic diagram of this process). Mice homozygous for the humanized NKG2D gene can then be obtained by mating with each other.

[0306] In addition, the CRISPR / Cas system can be introduced for gene editing, and a targeting strategy can be designed as shown in Figure 5, which shows that the targeting vector contains upstream and downstream homology arm sequences of the mouse NKG2D gene, as well as the human NKG2D fragment sequence. The upstream homology arm sequence (5' homology arm, SEQ ID NO: 5) is identical to the nucleotide sequence from positions 129593654 to 129594939 of NCBI Accession No. NC_000072.7, the downstream homology arm sequence (3' homology arm, SEQ ID NO: 6) is identical to the nucleotide sequence from positions 129588308 to 129589748 of NCBI Accession No. NC_000072.7, and the nucleotide sequence of the human NKG2D fragment is identical to the nucleotide sequence from positions 10373114 to 10379709 of NCBI Accession No. NC_000012.12 (SEQ ID NO: 7). The mRNA sequence of the modified humanized mouse NKG2D is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 9.

[0307] Targeting vector construction can be performed using conventional methods, such as enzyme digestion, ligation, and direct synthesis. After initial verification of the constructed targeting vector through enzyme digestion, it is sent to a sequencing company for sequencing verification. Targeting vectors that have been verified to be correct by sequencing are used in subsequent experiments.

[0308] The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are prerequisites for constructing sgRNA expression vectors. sgRNA sequences that recognize the target site were designed and synthesized. The target sequences of each sgRNA on the NKG2D gene are as follows:

[0309] sgRNA1 target site (SEQ ID NO: 14): 5'-TATGAGGGTACCCATGTACCAGG-3';

[0310] sgRNA2 target site (SEQ ID NO: 15): 5'-CTGCTGCAGGTTAACTCTGGTGG-3';

[0311] The activity of sgRNA was detected using a UCA kit. After confirming that it could mediate efficient cleavage, enzyme cleavage sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences (see Table 6). After annealing, the annealed products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain expression vectors pT7-NKG2D-1 and pT7-NKG2D-2.

[0312] Table 6 Sequence list of sgRNA1 and sgRNA2

[0313] The pT7-sgRNA vector is synthesized by a plasmid synthesis company. The fragment DNA containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 24) is then ligated to the backbone vector (source: Takara, Cat. No. 3299) by enzyme digestion (EcoRI and BamHI). Sequencing verification by a professional sequencing company indicates that the target plasmid has been obtained. Pronuclear fertilized eggs of mice, such as C57BL / 6 mice, are taken. In vitro transcription products of the pT7-NKG2D-1 and pT7-NKG2D-2 plasmids (transcribed using the Ambion in vitro transcription kit according to the instructions), the targeting vector, and Cas9 mRNA are premixed and injected into the cytoplasm or nucleus of the mouse fertilized eggs using a microinjector. Microinjection of fertilized eggs was performed according to the method described in the "Mouse Embryo Handbook (3rd Edition)" (Andras Nagy, Chemical Industry Press, 2006). The injected fertilized eggs were transferred to culture medium for a short period of incubation and then transplanted into the oviduct of a recipient female mouse for development. The resulting mice (F0 generation) were then hybridized and selfed to expand the population and establish a stable NKG2D gene humanized mouse strain. The genotype of somatic cells in the F0 generation mice was identified by PCR (primers are shown in Table 5). Mice that tested positive by PCR were then subjected to Southern Blot analysis. Mice that tested positive by Southern Blot analysis were further sequenced to confirm the presence of random insertions.

[0314] The success of the mice prepared by the above method can be determined by conventional methods. For example, the genotype of the somatic cells of the F1 generation mice can be determined by PCR (primers are shown in Table 5). The results of the identification of exemplary F1 generation mice are shown in Figure 6, wherein the mice numbered F1-01, F1-02, and F1-03 are positive heterozygous mice.

[0315] Southern blot analysis was performed on F1 generation mice that tested positive by PCR to confirm the presence of random insertions. Genomic DNA was extracted from the tails and digested with either BspHI or NcoI, transferred to a membrane, and hybridized. Specific probe and target fragment lengths are shown in Table 7. The test results, shown in Figure 7, show that F1-01, F1-02, and F1-03 were all heterozygous positive mice. This demonstrates that this method successfully generated humanized NKG2D mice that can be stably passaged and are free of random insertions.

[0316] Table 7 Specific probe and target fragment lengths

[0317] The probe synthesis primers are as follows:

[0318] 3'Probe-F (SEQ ID NO: 27): 5'-TGCCTAGGGGAAAGGCAACTTAACAC-3',

[0319] 3'Probe-R (SEQ ID NO: 28): 5'-TCTACGTTCTCCTCCATCCCCAAAC-3';

[0320] LR Probe (5')-F (SEQ ID NO: 29): 5'-GTCACTAGAAAGCATAACATCTGTGC-3',

[0321] LR Probe (5')-R (SEQ ID NO: 30): 5'-TGTTTTAACAACACACATTTTCAGGT-3';

[0322] The expression of human NKG2D mRNA in NKG2D gene homozygous humanized mice can be confirmed by conventional detection methods, such as RT-PCR. Specifically, one female C57BL / 6 wild-type mouse aged 4-6 weeks and one female NKG2D gene homozygous mouse prepared in this example were euthanized by cervical dislocation, and the mouse spleens were removed. Cellular RNA was extracted according to the Trizol kit instructions, reverse transcribed into cDNA, and then subjected to RT-PCR (primers are shown in Table 8). The test results are shown in Figure 8: only mouse NKG2D mRNA was detected in C57BL / 6 wild-type mice, and no human NKG2D mRNA was detected; human NKG2D mRNA was detected only in NKG2D gene homozygous mice.

[0323] Table 8 RT-PCR primer names and specific sequences

[0324] Flow cytometry was used to further detect the expression of humanized NKG2D protein in NKG2D gene homozygous mice. Specifically, 6-week-old female C57BL / 6 wild-type mice and NKG2D gene homozygous mice were euthanized by cervical dislocation, spleen tissues were obtained, and anti-mouse CD45 antibodies Brilliant Violet 510 and Brilliant Violet 511 were used to detect the expression of humanized NKG2D protein in NKG2D gene homozygous mice. TM anti-mouse CD45 Antibody (mCD4), anti-mouse TCRβ antibody APC Hamster Anti-Mouse TCRβChain (mTCRβ), anti-mouse NK1.1 antibody PE / Cy TM 7Mouse anti-mouse NK1.1 (mNK1.1), anti-mouse NKG2D antibody FITC anti-mouse CD314 (NKG2D) Antibody (mNKG2D), anti-human NKG2D antibody CD314 (NKG2D) Monoclonal Antibody (1D11) (hNKG2D), and purified anti-mouse CD16 / 32 antibody were used for identification and staining, and then flow cytometry detection was performed.

[0325] The data showed that spleen NK cells (characterized by mCD45 + mTCRβ - mNK1.1 + ) in mNKG2D-positive cells (characterized by mCD45 + mTCRβ - mNK1.1 + mNKG2D + ) accounted for 40.4%, hNKG2D positive cells (characterized by mCD45 + mTCRβ - mNK1.1 + hNKG2D + ) ratio was 0.56%; in the spleen NK cells of mice homozygous for the humanized NKG2D gene, the proportion of hNKG2D-positive cells was 29.8%, and the proportion of mNKG2D-positive cells was 0.73%. These results indicate that humanized NKG2D protein expression was successfully detected in NKG2D-humanized mice.

[0326] Furthermore, flow cytometry was used to detect the immunophenotyping of NKG2D gene humanized homozygous mice. Specifically, 3 female C57BL / 6 wild-type mice (+ / +) aged 8 weeks and 3 female NKG2D gene humanized homozygous mice (H / H) aged 9 weeks were selected, and spleen, lymph nodes and blood tissues were collected after euthanasia by cervical dislocation. Brilliant Violet 510 was used to detect the immunophenotyping of NKG2D gene humanized homozygous mice. TM anti-mouse CD45 Antibody, PerCP / Cyanine5.5 anti-mouse TCRβchain Antibody, Brilliant Violet 421 TM anti-mouse CD4 Antibody, PE anti-mouse CD8aAntibody, Brilliant Violet 605 TM anti-mouse CD19 Antibody, PerCP anti-mouse Ly-6G / Ly-6C(Gr-1)Antibody, Brilliant Violet 605 TM anti-mouse CD11c Antibody, PE anti-mouse / human CD11b Antibody, FITC anti-mouse F4 / 80Antibody, PE / Cy TM 7. Mouse anti-mouse NK1.1 and APC anti-mouse / rat Foxp3 Antibody were used for recognition and staining, and then flow cytometry was performed.

[0327] The results of the detection of leukocyte subtypes and T cell subtypes in the spleen and blood are shown in Figures 9 and 11, respectively. As can be seen from the figures, the percentages of leukocyte subtypes such as T cells, B cells, NK cells, granulocytes, dendritic cells, macrophages, and monocytes in the spleen and blood tissues of NKG2D gene humanized mice are basically the same as those of C57BL / 6 wild-type mice (Figures 9A and 11A). + T cells, CD8 +The percentages of T cell subtypes, such as T cells and Treg cells, were basically the same as those of C57BL / 6 wild-type mice (Figures 9B and 11B). The results of the detection of leukocyte subtypes and T cell subtypes in the lymph nodes are shown in Figures 10A and 10B, respectively. As can be seen from the figures, the leukocyte subtypes, such as B cells, T cells, and NK cells, in the lymph nodes of NKG2D gene humanized mice were basically the same as those of C57BL / 6 wild-type mice (Figure 10A). + T cells, CD8 + The percentages of T cell subtypes, including T cells and Treg cells, were essentially the same as those in C57BL / 6 wild-type mice (Figure 10B). These results indicate that humanization of the NKG2D gene did not affect the overall development, differentiation, or distribution of leukocyte and T cell subtypes in mice.

[0328] Example 2 Method 2 for constructing NKG2D gene humanized mice

[0329] To achieve the purpose of the present invention, a nucleotide sequence encoding a human NKG2D protein can be introduced into the mouse endogenous NKG2D locus, so that the mouse expresses a fully human NKG2D protein. Specifically, using gene editing technology, under the control of human NKG2D gene regulatory elements, the mouse 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence (about 20kb) is replaced with a nucleotide sequence comprising the 5'UTR outer upstream nucleotide sequence to the 3'UTR outer downstream nucleotide sequence (about 32kb) of the human NKG2D gene, to obtain a humanized NKG2D locus schematic diagram as shown in Figure 12, thereby achieving humanization of the mouse NKG2D gene.

[0330] A targeting strategy was designed as shown in Figure 13 , which shows the targeting vector containing upstream and downstream homology arm sequences for the mouse NKG2D gene and the A3 segment containing the human NKG2D gene. The upstream 5' homology arm sequence (SEQ ID NO: 35) is identical to nucleotides 129607128 to 129612145 of NCBI Accession No. NC_000072.7, and the downstream 3' homology arm sequence (SEQ ID NO: 36) is identical to nucleotides 129582548 to 129586985 of NCBI Accession No. NC_000072.7. The nucleotide sequence of the human NKG2D segment in the A3 segment (SEQ ID NO: 48) is identical to nucleotides 10371346 to 10402980 of NCBI Accession No. NC_000012.12.

[0331] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system FRT recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The 5' end of the Neo cassette was designed to link to the human gene as follows: 5'-TATCCTTTCATTAAATATACTTAACAATCATCATCTGGCGAATCGGACCCACAAGAGCACTGAGGTCGGAAGTTCCTATTCTCTAGAA-3' (SEQ ID NO: 39), where the "C" in the "AATC" sequence is the last nucleotide of the human nucleotide, and the first "A" in the "ATCA" sequence is the first nucleotide of the Neo cassette. The 3' end of the Neo cassette was designed to link to the human gene as follows: 5'-TCTCTAGAAAGTATAGGAACTTCATCAGTCCAGGATACATAGATTACCACAACTCCGAGCC CTGTATACAGGTGTTATTTTAACCTAATTCTAA-3' (SEQ ID NO: 40), where the last "C" in the "AGCC" sequence is the last nucleotide of the Neo cassette, and the "C" in the "CTGT" sequence is the first nucleotide of the human nucleotide. In addition, a gene encoding a negative selection marker (the gene encoding the diphtheria toxin A subunit (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the modified humanized mouse NKG2D is shown in SEQ ID NO: 38, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0332] Given that human NKG2D has multiple isoforms or transcripts, the methods described herein can be applied to other isoforms or transcripts.

[0333] The targeting vector construction can adopt ordinary method to carry out, as enzyme digestion and connection etc.The targeting vector built is carried out preliminary verification by enzyme digestion, then sends sequencing company to carry out sequencing verification.The correct targeting vector electroporation transfection of sequencing verification is entered in the embryonic stem cell of C57BL / 6 mice, utilizes positive clone screening marker gene to screen the cell that obtains, and utilizes PCR (primer such as Table 9) and Southern Blot technology (concrete probe and purpose fragment length are shown in Table 10) to detect the integration situation of confirmed foreign gene, screen out correct positive clone cell.Identified as positive clone through PCR, then carry out Southern Blot detection, further sequencing verification, result display ES-01, ES-02, ES-03, ES-04, ES-05, ES-06 and ES-09 clone all do not have random insertion, and correct clone is carried out next step experiment.

[0334] Table 9 PCR primer names and specific sequences

[0335] Table 10 Specific probes and target fragment lengths

[0336] The probe synthesis primers are as follows:

[0337] A1 Probe:

[0338] A1 Probe-F (SEQ ID NO: 45): 5'-TTCTATGGGGAGCAAGTGCTGG-3';

[0339] A1 Probe-R (SEQ ID NO: 46): 5'-CCAAGATGCTAAGCAAACTAATGAC-3';

[0340] A2 Probe:

[0341] A2 Probe-F (SEQ ID NO: 47): 5'-GCTGCTGGAGAAAGTGGGATTG-3';

[0342] A2 Probe-R (SEQ ID NO: 37): 5'-ACAGGCTGCAGCAGCACTGC-3';

[0343] The correct positive clones (black mice) screened are introduced into isolated blastocysts (white mice) according to techniques known in the art. The resulting chimeric blastocysts are transferred to culture medium for a short period of time and then transplanted into the oviducts of recipient female mice (white mice), producing F0 generation chimeric mice (black and white). F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice. F1 generation heterozygous mice are then mated with each other to obtain F2 generation homozygous mice. Positive mice can also be mated with Flp tool mice to remove the positive clone selection marker gene (see Figure 14 for a schematic diagram of this process). Mice homozygous for the humanized NKG2D gene can then be obtained by mating with each other.

[0344] The expression of humanized NKG2D protein in positive mice was detected by flow cytometry. Specifically, 7-week-old female C57BL / 6 wild-type mice and NKG2D gene humanized heterozygous mice were euthanized by cervical dislocation and spleen tissue was obtained. The spleen tissue was detected by anti-mouse CD45 antibody Brilliant Violet 510 and anti-mouse CD45 antibody Brilliant Violet 510. TManti-mouse CD45 Antibody (mCD4), anti-mouse CD3 antibody V450 Rat Anti-mouse CD3 Molecular Complex (mCD3), anti-mouse CD4 antibody Brilliant Violet 785 TM anti-mouse CD4 Antibody (mCD4), anti-mouse CD8a antibody Brilliant Violet 711 TM anti-mouse CD8a (mCD8), anti-mouse CD19 antibody Brilliant Violet 650 TM anti-mouse CD19 Antibody (mCD19), anti-mouse NK1.1 antibody PE / Cy TM 7 Mouse anti-mouse NK1.1 (mNK1.1), anti-mouse NKG2D Flow cytometry detection was performed after identification and staining with antibodies such as FITC anti-mouse CD314(NKG2D)Antibody(mNKG2D), anti-human NKG2D antibody APC anti-human CD314(NKG2D)Antibody(hNKG2D), and Purified anti-mouse CD16 / 32.

[0345] NK cell characteristics: mCD45 + mCD3 - mNK1.1 + Among them, the positive NK cell characteristics of mouse NKG2D are: mCD45 + mCD3 - mNK1.1 + mNKG2D + ; The positive NK cell characteristics of human NKG2D are: mCD45 + mCD3 - mNK1.1 + hNKG2D + ;

[0346] CD8 + T cell characteristics: mCD45 + mCD3 + mCD19 - mCD4 - mCD8 + Among them, the positive CD8 + T cell characteristics: mCD45 + mCD3 +mCD19 - mCD4 - mCD8 + mNKG2D + ; Human NKG2D positive CD4 + T cell characteristics: mCD45 + mCD3 + mCD19 - mCD4 - mCD8 + hNKG2D + ;

[0347] Table 11 Flow cytometry results of NKG2D in humanized NKG2D heterozygous mice

[0348] The results in Table 11 show that the expression of mouse NKG2D protein was detected only in the NK cells of wild-type C57BL / 6 mice, and the expression of NK cells and CD8 + Only the expression of human NKG2D protein was detected in T cells. In summary, humanized heterozygous mice of the NKG2D gene were successfully constructed.

[0349] Example 3: Efficacy Verification

[0350] The NKG2D gene humanized mice prepared by this method can be used to evaluate the efficacy of antibodies targeting human NKG2D. For example, NKG2D gene humanized homozygous mice were subcutaneously inoculated with colon cancer cells MC38, and the tumor volume grew to about 100 mm. 3 Afterwards, mice were divided into a control group and a treatment group based on tumor volume. The treatment group was injected with an antibody targeting human NKG2D, while the control group was injected with an equal volume of saline. Tumor volume was measured and mice were weighed regularly. Comparing changes in mouse weight and tumor volume effectively assessed the safety and efficacy of the antibody in humanized NKG2D mice.

[0351] Example 4 Preparation of Double Humanized or Multiply Double Humanized Mice

[0352] The NKG2D mice prepared by this method can also be used to prepare double-humanized or multi-humanized mouse models. For example, in Examples 1 or 2 above, the embryonic stem cells used for blastocyst microinjection can be derived from mice modified with other genes such as NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40, and B7-H3. Alternatively, based on humanized NKG2D mice, mouse ES embryonic stem cells can be isolated and gene-targeted to obtain double- or multi-gene modified mouse models with NKG2D and other genes. The homozygous or heterozygous NKG2D mice obtained by this method can also be mated with homozygous or heterozygous mice with other genetic modifications, and their offspring can be screened. According to Mendel's law of inheritance, there is a certain probability of obtaining heterozygous mice with humanized NKG2D and other genetic modifications, double-gene or multi-gene modifications. The heterozygotes can then be mated with each other to obtain homozygous mice with double-gene or multi-gene modifications. These double-gene or multi-gene modified mice can be used to conduct in vivo efficacy verification of drugs targeting human NKG2D and other gene regulators.

[0353] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0354] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0355] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. 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 killer cell lectin-like receptor K1 (NKG2D) protein.

2. The non-human animal according to claim 1, characterized in that The nucleotide sequence encoding the human or chimeric NKG2D protein is operably linked to endogenous regulatory elements or human regulatory elements (eg, 5'UTR and / or 3'UTR) of the endogenous NKG2D locus of at least one chromosome.

3. The non-human animal according to claim 1 or 2, characterized in that: The chimeric NKG2D protein is a humanized NKG2D protein, and the humanized NKG2D protein comprises all or part of the extracellular region of the human NKG2D protein.

4. The non-human animal according to claim 3, characterized in that The humanized NKG2D protein comprises a sequence of at least 50 to 144 consecutive amino acids identical to those of the extracellular region of the human NKG2D protein.

5. The non-human animal according to claim 4, characterized in that The humanized NKG2D protein also comprises all or part of the transmembrane region and / or cytoplasmic region of the human or non-human animal NKG2D protein.

6. The non-human animal according to any one of claims 3 to 5, characterized in that: The humanized NKG2D protein comprises a continuous amino acid sequence of at least 50 to 216 amino acids that is identical to a human NKG2D protein.

7. The non-human animal 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 (eg, a rat or a mouse).

8. The non-human animal according to any one of claims 1 to 7, characterized in that: The human or chimeric NKG2D protein comprises SEQ ID NO: 2 or any one of its 78-216 or SEQ ID NO: 9; or, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to the amino acid sequence shown in SEQ ID NO: 2 or any one of its 78-216 or SEQ ID NO: 9; or, comprises an amino acid sequence that differs from SEQ ID NO: 2 or any one of its 78-216 or SEQ ID NO: 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid, or comprises an amino acid sequence that substitutes, deletes and / or inserts one or more amino acids shown in SEQ ID NO: 2 or any one of its 78-216 or SEQ ID NO:

9.

9. The non-human animal according to any one of claims 1 to 8, characterized in that: The non-human animal does not express endogenous NKG2D protein or the expression level is reduced compared with NKG2D in wild-type animals.

10. A genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises a replacement of the endogenous NKG2D gene at the endogenous NKG2D locus with a nucleotide sequence comprising human NKG2D.

11. The non-human animal according to claim 10, characterized in that The nucleotide sequence of human NKG2D is operably linked to an endogenous regulatory element or a human regulatory element (eg, 5'UTR and / or 3'UTR) of an endogenous NKG2D locus, Preferably, one or more cells of the non-human animal express human or chimeric NKG2D protein.

12. The non-human animal according to claim 10 or 11, characterized in that: The endogenous NKG2D protein of the non-human animal is not expressed or the protein expression level is reduced compared with NKG2D in wild-type animals.

13. The non-human animal according to any one of claims 10 to 12, characterized in that: The nucleotide sequence of human NKG2D includes all or part of exon 4, all of exons 5-7 and / or all or part of exon 8 of the human NKG2D gene, preferably also includes all of exons 1-3 of the human NKG2D gene, further preferably includes the nucleotide sequence of the coding region of the human NKG2D gene, and more preferably includes the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene.

14. The non-human animal according to claim 13, characterized in that The nucleotide sequence of human NKG2D also includes the 5'UTR and / or 3'UTR of the human NKG2D gene, and further preferably includes a nucleotide sequence of at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human NKG2D gene and / or a nucleotide sequence of at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human NKG2D gene.

15. The non-human animal according to claim 14, characterized in that The nucleotide sequence of human NKG2D includes the 5'UTR of the human NKG2D gene and at least 50bp of continuous nucleotides upstream of it, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene and the 3'UTR of the human NKG2D gene and at least 50bp of continuous nucleotides downstream of it, or, includes part of exon 4 to part of exon 8 of the human NKG2D gene.

16. The non-human animal according to any one of claims 10 to 15, characterized in that: The nucleotide sequence of human NKG2D comprises SEQ ID NO: 7 or 48, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO: 7 or 48; or, a nucleotide sequence differing from the nucleotide sequence shown in SEQ ID NO: 7 or 48 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotides; or, a nucleotide sequence comprising substitution, deletion and / or insertion of one or more nucleotides shown in the nucleotide sequence shown in SEQ ID NO: 7 or 48.

17. The non-human animal according to any one of claims 10 to 16, characterized in that: All or part of exon 4, all of exons 5-7 and / or all or part of exon 8 of the endogenous NKG2D gene are replaced. Preferably, the replaced endogenous NKG2D gene also includes all of exons 1-3, and further preferably also includes at least 50bp of continuous nucleotides upstream of the 5'UTR outside and / or at least 50bp of nucleotides downstream of the 3'UTR outside of the endogenous NKG2D gene.

18. The non-human animal according to any one of claims 10 to 17, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent (eg, a rat or a mouse).

19. The non-human animal according to any one of claims 10 to 18, characterized in that: The mRNA transcribed from the modified nucleotide sequence of endogenous NKG2D in the genome of the non-human animal comprises SEQ ID NO: 8 or 38; or, comprises a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology to the nucleotide sequence shown in SEQ ID NO: 8 or 38; or, comprises a nucleotide sequence that differs from SEQ ID NO: 8 or 38 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 bp nucleotides; or, comprises a nucleotide sequence that replaces, deletes and / or inserts one or more nucleotides shown in SEQ ID NO: 8 or 38.

20. The non-human animal according to any one of claims 1 to 19, characterized in that: The modified NKG2D gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

21. The non-human animal according to any one of claims 1 to 20, characterized in that: The human or chimeric NKG2D protein expressed by the non-human animal has at least one NKG2D activity, such as a non-human animal NKG2D activity and / or a human NKG2D activity.

22. The non-human animal according to any one of claims 1 to 21, characterized in that: The non-human animal comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins are selected from at least one of NKG2DL, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, OX40 and B7-H3.

23. A non-human animal genome, characterized in that The non-human animal genome comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric killer cell lectin-like receptor K1 (NKG2D) protein.

24. The non-human animal genome according to claim 23, characterized in that At the endogenous NKG2D locus of the non-human animal, the endogenous NKG2D gene is replaced with a nucleotide sequence comprising human NKG2D.

25. The non-human animal genome according to any one of claims 23-24, characterized in that: The nucleotide sequence of human NKG2D comprises all or part of exon 4, all of exons 5-7 and / or all or part of exon 8 of the human NKG2D gene, preferably comprises part of exon 4 to part of exon 8 of the human NKG2D gene; Preferably, it also comprises all of exons 1-3 of the human NKG2D gene, further preferably comprises the nucleotide sequence of the coding region of the human NKG2D gene, and more preferably comprises the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene.

26. The non-human animal genome according to claim 25, characterized in that The nucleotide sequence of human NKG2D also includes the 5'UTR and / or 3'UTR of the human NKG2D gene, and further preferably includes a nucleotide sequence of at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human NKG2D gene and / or a nucleotide sequence of at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human NKG2D gene.

27. The non-human animal genome according to any one of claims 24-26, characterized in that: All or part of exon 4, all of exons 5-7 and / or all or part of exon 8 of the endogenous NKG2D gene are replaced. Preferably, the replaced endogenous NKG2D gene also includes all of exons 1-3, and further preferably also includes at least 50bp of continuous nucleotides upstream of the 5'UTR outside and / or at least 50bp of nucleotides downstream of the 3'UTR outside of the endogenous NKG2D gene.

28. A method for constructing a genetically modified non-human animal, characterized in that: In at least one cell of the non-human animal, an endogenous NKG2D gene is replaced with a nucleotide sequence comprising human NKG2D at the endogenous NKG2D locus of the non-human animal.

29. The construction method according to claim 28, characterized in that: The nucleotide sequence of human NKG2D includes all or part of exon 4, all of exons 5-7 and / or all or part of exon 8 of the human NKG2D gene, preferably also includes all of exons 1-3 of the human NKG2D gene, further preferably includes the nucleotide sequence of the coding region of the human NKG2D gene, and more preferably includes the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene.

30. The construction method according to claim 29, characterized in that: The nucleotide sequence of human NKG2D also includes the 5'UTR and / or 3'UTR of the human NKG2D gene, and further preferably includes a nucleotide sequence of at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human NKG2D gene and / or a nucleotide sequence of at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human NKG2D gene.

31. The construction method according to any one of claims 28 to 30, characterized in that: The nucleotide sequence of human NKG2D includes the 5'UTR of human NKG2D gene and at least 50bp continuous nucleotides upstream of its outer side, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene and the 3'UTR of human NKG2D gene and at least 50bp continuous nucleotides downstream of its outer side, or, includes part of exon 4 to part of exon 8 of the human NKG2D gene.

32. A method for constructing a cell of a genetically modified non-human animal expressing a human or chimeric NKG2D protein, the construction method comprising replacing the endogenous NKG2D gene at the endogenous NKG2D locus of the non-human animal with a nucleotide sequence comprising human NKG2D.

33. The construction method according to claim 32, characterized in that: The nucleotide sequence of human NKG2D includes all or part of exon 4, all of exons 5-7 and / or all or part of exon 8 of the human NKG2D gene, preferably also includes all of exons 1-3 of the human NKG2D gene, further preferably includes the nucleotide sequence of the coding region of the human NKG2D gene, and more preferably includes the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene.

34. The construction method according to claim 33, characterized in that: The nucleotide sequence of human NKG2D also includes the 5'UTR and / or 3'UTR of the human NKG2D gene, and further preferably includes a nucleotide sequence of at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human NKG2D gene and / or a nucleotide sequence of at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human NKG2D gene.

35. The construction method according to any one of claims 32 to 34, characterized in that: The nucleotide sequence of human NKG2D includes the 5'UTR of the human NKG2D gene and at least 50bp of continuous nucleotides upstream of it, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene and the 3'UTR of the human NKG2D gene and at least 50bp of continuous nucleotides downstream of it, or, includes part of exon 4 to part of exon 8 of the human NKG2D gene.

36. The construction method according to any one of claims 32-25, characterized in that: The expression of the nucleotide sequence of human NKG2D is regulated by a regulatory element, and the regulatory element is an endogenous regulatory element or a human regulatory element.

37. A humanized NKG2D protein, characterized in that The humanized NKG2D protein comprises all or part of the human NKG2D protein, preferably comprises all or part of the extracellular region of the human NKG2D protein, and further preferably comprises all or part of the transmembrane region and / or cytoplasmic region of the human or non-human animal NKG2D protein.

38. The humanized NKG2D protein according to claim 37, characterized in that The humanized NKG2D protein amino acid sequence comprises SEQ ID NO: 2 or any one of its 78-216 or SEQ ID NO: 9; or, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to SEQ ID NO: 2 or any one of its 78-216 or SEQ ID NO: 9; or, comprises an amino acid sequence that differs from SEQ ID NO: 2 or any one of its 78-216 or SEQ ID NO: 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid, or comprises an amino acid sequence that substitutes, deletes and / or inserts one or more amino acids as shown in SEQ ID NO: 2 or any one of its 78-216 or SEQ ID NO:

9.

39. A humanized NKG2D gene, characterized in that: The humanized NKG2D gene encodes the humanized NKG2D protein according to any one of claims 37-38; Preferably, the humanized NKG2D gene comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 10, 11, 35, 36, 38, 39, 40 and 48; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homologous to a nucleotide sequence as shown in any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 10, 11, 35, 36, 38, 39, 40 and 48.

40. A cell, tissue or organ, characterized in that: The cells, tissues or organs express the humanized NKG2D protein according to any one of claims 37-38, or contain the humanized NKG2D gene according to claim 39.

41. An animal model, characterized in that The animal model expresses the humanized NKG2D protein described in any one of claims 37-38, or comprises the humanized NKG2D gene described in claim 39, or comprises the cell, tissue or organ described in claim 40.

42. A method for determining the effectiveness of an anti-NKG2D therapeutic agent in treating a disease, characterized in that: The method comprises: 1) administering an anti-NKG2D therapeutic agent to the non-human animal according to any one of claims 1 to 22, the non-human animal obtained by the construction method according to any one of claims 28 to 31, or the animal model according to claim 41, wherein the non-human animal or animal model suffers from a disease; 2) Determine the inhibitory effect of anti-NKG2D therapeutic agents on the disease; Preferably, the disease includes tumors, viral infection-related diseases, graft-versus-host disease or autoimmune diseases.

43. The method according to claim 42, characterized in that The determining of the inhibitory effect of the anti-NKG2D therapeutic agent on the disease comprises measuring the tumor volume in the non-human animal or animal model.

44. A method for determining the effectiveness of anti-NKG2D therapeutic agents and other therapeutic agents in treating diseases, characterized in that: The method comprises: 1) administering an anti-NKG2D therapeutic agent and other therapeutic agents to the non-human animal according to any one of claims 1 to 22, the non-human animal obtained by the construction method according to any one of claims 28 to 31, or the animal model according to claim 41, wherein the non-human animal or animal model suffers from a disease; 2) Determine the inhibitory effects of anti-NKG2D therapeutics and other therapeutic agents on the disease; Preferably, the disease includes tumors, viral infection-related diseases, graft-versus-host disease or autoimmune diseases.

45. The method according to claim 44, characterized in that The other therapeutic agents include anti-PD-1 antibodies, anti-PD-L1 antibodies and / or anti-B7-H3 antibodies.

46. ​​The method according to any one of claims 44-45, characterized in that: The determination of the inhibitory effect of the anti-NKG2D therapeutic agent and other therapeutic agents on the disease comprises measuring the tumor volume in the non-human animal or animal model.

47. The method according to any one of claims 42 to 46, characterized in that: The tumor includes one or more of solid tumors, gastrointestinal cancer, acute myeloid leukemia, colorectal cancer, gastrointestinal pancreatic cancer, breast cancer, multiple myeloma, genitourinary system cancer, prostate cancer, glioblastoma, liver cancer, lung cancer, melanoma, lymphoma, bile duct cancer, ovarian cancer, nasopharyngeal tumor, nasopharyngeal carcinoma or medulloblastoma.

48. The method according to any one of claims 42 to 47, characterized in that: The autoimmune disease includes one or more of rheumatoid arthritis, type I diabetes, alopecia areata, Crohn's disease or atherosclerosis.

49. The method according to any one of claims 42 to 48, characterized in that: The viral infection-related diseases include diseases caused by one or more of the following viruses: cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis virus, Kaposi's sarcoma-associated herpes virus (KSHV), human papilloma virus (HPV), molluscum contagiosum virus (MCV), human T-cell leukemia virus 1 (HTLV-1) or HIV (human immunodeficiency virus). One or more of the following.

50. A method for determining the toxicity of an anti-NKG2D therapeutic agent, characterized in that: The method comprises: 1) administering an anti-NKG2D therapeutic agent to the non-human animal according to any one of claims 1 to 22, the non-human animal obtained by the construction method according to any one of claims 28 to 31, or the animal model according to claim 41; 2) Determine the effects of anti-NKG2D therapeutics on non-human animals or animal models.

51. The method according to claim 50, characterized in that Determining the effect of the anti-NKG2D therapeutic agent on the non-human animal or animal model involves measuring the body weight or blood test of the non-human animal or animal model; preferably, the blood test includes one or more of red blood cell count, hematocrit or hemoglobin.

52. A targeting vector, characterized in that The targeting vector comprises a 5' arm, a donor region and a 3' arm, wherein the 5' arm is homologous to the 5' end of the switch region to be changed, the 3' arm is homologous to the 3' end of the switch region to be changed, and the donor region comprises a nucleotide sequence encoding a human or chimeric NKG2D protein.

53. The targeting vector according to claim 52, characterized in that The conversion region to be changed is located at the endogenous NKG2D locus of the non-human animal. Preferably, the conversion region to be changed includes at least one exon or at least one intron of the endogenous NKG2D gene of the non-human animal, for example, part of exon 4 to part of exon 8 of the endogenous NKG2D gene, or at least 50bp upstream of the outer side of the 5'UTR to at least 50bp downstream of the outer side of the 3'UTR of the endogenous NKG2D gene.

54. The targeting vector according to any one of claims 52-53, characterized in that The donor region includes all or part of exon 4, all of exons 5-7 and / or all or part of exon 8 of the human NKG2D gene, preferably also includes all of exons 1-3 of the human NKG2D gene, further preferably includes the nucleotide sequence of the coding region of the human NKG2D gene, and more preferably includes the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene.

55. The targeting vector according to claim 54, characterized in that The donor region further comprises the 5'UTR and / or 3'UTR of the human NKG2D gene, and further preferably comprises a nucleotide sequence of at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human NKG2D gene and / or a nucleotide sequence of at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human NKG2D gene; Preferably, the donor region comprises the 5'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides upstream of it, the nucleotide sequence from the start codon to the stop codon of the human NKG2D gene, and the 3'UTR of the human NKG2D gene and at least 50 bp of continuous nucleotides downstream of it, or, the donor region comprises part of exon 4 to part of exon 8 of the human NKG2D gene.

56. A use of the non-human animal according to any one of claims 1-22, the non-human animal genome according to any one of claims 23-27, the non-human animal obtained by the construction method according to any one of claims 28-31, the cell obtained by the construction method according to any one of claims 32-36, the humanized NKG2D protein according to any one of claims 37-38, the humanized NKG2D gene according to claim 39, the cell, tissue or organ according to claim 40, the animal model according to claim 41, and the targeting vector according to any one of claims 52-55, characterized in that: The applications described include: A) Application in the development of products involving NKG2D-related immune processes in human cells; B) Application as a model system related to NKG2D for pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for NKG2D-related etiology studies and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human NKG2D signaling pathway modulators; or, E) Study the function of NKG2D gene, study the drugs and efficacy targeting human NKG2D target sites, and study the application of NKG2D-related immune-related disease drugs or anti-tumor drugs.

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