Non-human animal modified by MICB gene

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) MICB protein and methods of use thereof.

Description

Technical Field

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

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

[0003] Therefore, the development of humanized animal models suitable for the screening and evaluation of human drugs will significantly improve the efficiency of new drug development and reduce the cost of drug research and development. Summary of the Invention The present application provides an animal model having a human or chimeric MICB protein. This animal model can express a human or chimeric MICB (e.g., humanized MICB) protein. It can be used for the study of the function of the MICB gene and also for the screening and evaluation of MICB signaling pathway regulators (such as antibodies targeting MICB, nucleic acid drugs targeting MICB, and / or polypeptide drugs). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamic studies, and the treatment of MICB target-related diseases (such as tumors, inflammation, or immune diseases); this animal model can also be used to facilitate the development and design of new drugs, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of the MICB protein and a platform for screening therapeutic drugs for related diseases.

[0005] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. The genome of the non-human animal comprises at least one chromosome, and the chromosome comprises a nucleotide sequence encoding a human or chimeric major histocompatibility complex class I polypeptide-related sequence B (MICB) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric MICB protein can be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the amino acid sequence of the human or chimeric MICB protein comprises SEQ ID NO: 1; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the human or chimeric MICB protein is regulated by an endogenous regulatory element or an exogenous regulatory element. In some embodiments, the exogenous regulatory element is a human MICB promoter, 5'UTR and / or 3'UTR. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, one or more cells of the non-human animal express the human or chimeric MICB protein.

[0006] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises a nucleotide sequence encoding a human or chimeric MICB introduced into the endogenous MICB genome, preferably, the introduction is an insertion or replacement. In some embodiments, the introduction is an insertion into a safe site of the non-human animal, preferably, the introduction is an insertion into the ROSA26 site or Hipp11 site of the non-human animal. In some embodiments, the nucleotide sequence encoding a human or chimeric MICB is operably linked to an endogenous regulatory element or an exogenous regulatory element. In some embodiments, the nucleotide sequence encoding a human or chimeric MICB comprises all or part of exons 1 to 6 of the human MICB gene. In some embodiments, the nucleotide sequence encoding a human or chimeric MICB comprises all of exons 1 to 6 of the human MICB gene and at least 50 bp of continuous nucleotides downstream of the human MICB genome, preferably further comprising at least 50 bp of continuous nucleotides upstream of the human MICB genome. In some embodiments, the nucleotide sequence encoding a human or chimeric MICB comprises all of exons 1 to 6 of the human MICB gene and at least 23441 bp of continuous nucleotides upstream of the human MICB genome and at least 7218 bp of continuous nucleotides downstream of the human MICB genome. In some embodiments, the nucleotide sequence encoding a human or chimeric MICB comprises all of exons 1 to 6 of the human MICB gene and at least 23443 bp of continuous nucleotides upstream of the human MICB genome and at least 7213 bp of continuous nucleotides downstream of the human MICB genome. In some embodiments, the nucleotide sequence encoding a human or chimeric MICB comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequence shown in SEQ ID NO: 19. In some embodiments, the nucleotide sequence encoding a human or chimeric MICB comprises a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequence shown in SEQ ID NO: 4 and / or 20. In some embodiments, the nucleotide sequence encoding a human or chimeric MICB comprises, in order from the 5' end to the 3' end, a nucleotide sequence that is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequences shown in SEQ ID NO: 4, 19 and 20. In some embodiments, the modified MICB gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous modified (e.g., inserted or replaced) genome.

[0007] In one aspect, the present invention provides a non-human animal or a method for constructing the same, in which a nucleotide sequence containing human MICB is introduced into the endogenous genome of the non-human animal. In some embodiments, the nucleotide sequence of human MICB can be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the introduction is an insertion or a replacement. In some embodiments, the introduction is an insertion into a safe site of the endogenous genome of the non-human animal. In some embodiments, the safe site includes the ROSA26 site or the Hipp11 site. In some embodiments, the nucleotide sequence of human MICB contains a nucleotide sequence encoding a human or chimeric MICB protein. In some embodiments, the nucleotide sequence of human MICB contains at least 5-46868 bp of the genomic sequence of the human MICB gene, such as at least 5, 50, 100, 500, 1000, 5000, 10000, 12967, 15000, 16207, 20000, 25000, 30000, 35000, 40000, 43000,

[0008] A nucleotide sequence that is identical to a continuous or discontinuous nucleotide sequence of 43623, 43625, 43626, 45000, 46863 or 46866 bp. In some embodiments, the nucleotide sequence of human MICB comprises exons 1 to 6 of the human MICB gene. In some embodiments, the nucleotide sequence of human MICB comprises the nucleotide sequence from the start codon to the stop codon of the human MICB gene, preferably further comprising the 5'UTR and / or 3'UTR. In some embodiments, the nucleotide sequence of human MICB comprises SEQ ID NO: 19; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 19. In some embodiments, the nucleotide sequence of human MICB further comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR and / or at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human MICB comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR of the human MICB gene to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the nucleotide sequence upstream of the 5'UTR of the human MICB gene comprised in the nucleotide sequence of human MICB comprises at least 50 bp, preferably at least 50 bp to at least 30000 bp, such as at least 50, 100, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 20214, 20216, 23000, 23441, 23443, 25000 or 30000 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence downstream of the 3'UTR of the human MICB gene comprised in the nucleotide sequence of human MICB comprises at least 50 bp, preferably at least 50 bp to at least 10000 bp, such as at least 50, 100, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 7200, 7213, 7218, 8000, 9000 or 10000 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of human MICB comprises at least 23441 bp of continuous nucleotide sequence upstream of the 5'UTR of the human MICB gene to at least 7218 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human MICB comprises at least 23443 bp of continuous nucleotide sequence upstream of the 5'UTR of the human MICB gene to at least 7213 bp of continuous nucleotide sequence downstream of the 3'UTR.In some embodiments, the nucleotide sequence of the human MICB further comprises SEQ ID NO: 4 and / or 20; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 4 and / or 20. In some embodiments, the nucleotide sequence of the human MICB sequentially comprises SEQ ID NO: 4, 19 and 20 from the 5'-end to the 3'-end; or sequentially comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequences shown in SEQ ID NO: 4, 19 and 20. In some embodiments, the nucleotide sequence encoding the human or chimeric MICB protein or the nucleotide sequence of the human MICB is operably linked to an endogenous regulatory element or an exogenous regulatory element. In some embodiments, the exogenous regulatory element includes the human MICB promoter, 5'UTR and / or 3'UTR. In some embodiments, the modified MICB gene in the non-human animal genome is homozygous or heterozygous for the endogenously modified (e.g., inserted or replaced) genome. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the mRNA transcribed from the modified MICB gene in the non-human animal genome comprises the nucleotide sequence shown in NM_005931.5 or SEQ ID NO: 23; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in NM_005931.5 or SEQ ID NO: 23. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding another human or chimeric protein, and the other human or chimeric protein preferably comprises at least one of MICA, NKG2D, TFR1, NKP46, ICOS, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0009] In one aspect, the present invention provides a non-human animal or a method for constructing the same, the non-human animal comprising at least one cell encoding a nucleotide sequence of a human or humanized MICB protein. In some embodiments, the humanized MICB protein comprises an amino acid sequence that is identical to at least 50, 100, 150, 200, 250, 287, 300, 309 or 383 consecutive amino acid sequences of the human MICB protein. In some embodiments, the non-human animal expresses a human or humanized MICB protein. In some embodiments, the nucleotide sequence encoding the human or humanized MICB protein is regulated by an endogenous regulatory element or an exogenous regulatory element. Preferably, the exogenous regulatory element is a human MICB promoter, 5'UTR and / or 3'UTR. In some embodiments, the nucleotide sequence encoding the human or humanized MICB protein can be integrated into the endogenous genome of the non-human animal. In some embodiments, the nucleotide sequence encoding the human or humanized MICB protein can be inserted into a safe site of the endogenous genome of the non-human animal. Preferably, the safe site includes the ROSA26 site or the Hipp11 site.

[0010] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, in which a nucleotide sequence encoding a human or humanized MICB protein is introduced into the endogenous genome of the non-human animal in at least one cell of the non-human animal. In some embodiments, the introduction is an insertion or a replacement. In some embodiments, the nucleotide sequence encoding a human or humanized MICB protein may be the genomic DNA sequence, CDS sequence or cDNA sequence of human MICB. In some embodiments, the introduction is an insertion, and preferably, the introduction is an insertion into the ROSA26 locus or Hipp11 locus of the endogenous genome of the non-human animal. In some embodiments, the nucleotide sequence encoding a human or humanized MICB protein comprises all or part of exons 1 to 6 of the human MICB gene. In some embodiments, the nucleotide sequence encoding a human or humanized MICB protein comprises all of exons 1 to 6 of the human MICB gene and at least 50 consecutive nucleotides downstream of the human MICB genome, and more preferably further comprises at least 50 consecutive nucleotides upstream of the human MICB genome. In some embodiments, the nucleotide sequence encoding a human or humanized MICB protein comprises all of exons 1 to 6 of the human MICB gene and at least 23441 bp or 23443 bp of consecutive nucleotides upstream of the human MICB genome and at least 7213 bp or 7218 bp of consecutive nucleotides downstream of the human MICB genome. In some embodiments, the amino acid sequence of the human or humanized MICB protein is identical to the amino acid sequence shown in SEQ ID NO: 1 or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding a human or humanized MICB protein comprises a nucleotide sequence that is identical to the nucleotide sequence shown in SEQ ID NO: 19 or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding a human or humanized MICB protein comprises a nucleotide sequence that is identical to the nucleotide sequence shown in SEQ ID NO: 4 and / or 20 or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding a human or humanized MICB protein sequentially comprises, from the 5' end to the 3' end, a nucleotide sequence that is identical to the nucleotide sequences shown in SEQ ID NO: 4, 19 and 20 or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding a human or humanized MICB protein is operably linked to an endogenous regulatory element or an exogenous regulatory element. In some embodiments, the exogenous regulatory element is, for example, the human MICB promoter, 5'UTR and / or 3'UTR.In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat.

[0011] In one aspect, the present invention provides a method for constructing a non-human animal cell expressing a human or chimeric MICB gene modification, the construction method comprising inserting a nucleotide sequence encoding human MICB into a safe site of the endogenous genome of a non-human animal to generate a gene-modified non-human animal cell. In some embodiments, the safe site includes the ROSA26 site or the Hipp11 site. In some embodiments, the non-human animal cell expresses a human or chimeric MICB protein. In some embodiments, the nucleotide sequence encoding human MICB comprises all or part of exons 1 to 6 of the human MICB gene. In some embodiments, the nucleotide sequence encoding human MICB comprises all of exons 1 to 6 of the human MICB gene and at least 50 bp of continuous nucleotides downstream of the human MICB genome, preferably further comprising at least 50 bp of continuous nucleotides upstream of the human MICB genome. In some embodiments, the nucleotide sequence encoding human MICB comprises all of exons 1 to 6 of the human and at least 23441 bp or 23443 bp of continuous nucleotides upstream of the human MICB genome and at least 7213 bp or 7218 bp of continuous nucleotides downstream of the human MICB genome. In some embodiments, the amino acid sequence of the human MICB is identical to the amino acid sequence shown in SEQ ID NO: 1 or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding human MICB comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO: 19 or having an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding human MICB comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO: 4 and / or 20 or having an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding human MICB comprises, in order from the 5' end to the 3' end, nucleotide sequences identical to the nucleotide sequences shown in SEQ ID NO: 4, 19 and 20 or having an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding human MICB is operably linked to an endogenous regulatory element or an exogenous regulatory element. In some embodiments, the exogenous regulatory element is, for example, the human MICB promoter, 5'UTR and / or 3'UTR. In some embodiments, the non-human animal is a mouse.

[0012] In one aspect, the present invention provides a method for determining the efficacy or toxicity of a therapeutic agent in treating a disease, the method comprising: 1) administering the therapeutic agent to the non-human animal obtained by the construction method or the non-human animal; 2) determining the effect of the therapeutic agent on the non-human animal or the disease. In some embodiments, the therapeutic agent comprises an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises an antibody specifically binding to MICA, NKG2D, TFR1, NKP46, ICOS, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and / or CTLA4. In some embodiments, the additional therapeutic agent preferably comprises one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the disease comprises one or more of cancer, immune disease, or inflammation. In some embodiments, the disease is cancer. In some embodiments, the cancer comprises one or more of melanoma, skin cancer, colorectal cancer, breast cancer, endocrine cancer, lymphocyte tumor, head and neck cancer, liver cancer, or lung cancer. In some embodiments, the immune disease is one or more of asthma, psoriasis, atopic dermatitis, rheumatoid arthritis, or multiple sclerosis. In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0013] In one aspect, the present invention provides a method for determining the efficacy of a therapeutic agent in treating cancer (tumor), the method comprising: 1) administering the therapeutic agent to the non-human animal obtained by the construction method or the non-human animal, wherein the non-human animal has cancer (tumor); 2) determining the inhibitory effect of the therapeutic agent on the tumor. In some embodiments, the therapeutic agent comprises an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug. In some embodiments, the tumor comprises one or more tumor cells injected into the animal body. In some embodiments, determining the inhibitory effect of the therapeutic agent on the tumor involves measuring the tumor volume in the non-human animal. In some embodiments, the tumor comprises one or more tumor cells injected into the non-human animal body. In some embodiments, the tumor comprises a solid tumor or a hematological tumor. In some embodiments, the non-human animal has one or more of melanoma, skin cancer, colorectal cancer, breast cancer, endocrine cancer, lymphocyte tumor, head and neck cancer, liver cancer, or lung cancer. In some embodiments, the non-human animal further comprises a sequence encoding human or chimeric PD-1, human or chimeric PD-L1, and / or human or chimeric CTLA4. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0014] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating an immune disease, the method comprising: 1) administering the therapeutic agent to the non-human animal obtained by the construction method or the non-human animal, wherein the non-human animal suffers from an immune disease; 2) determining the therapeutic effect of the therapeutic agent on the immune disease. In some embodiments, the therapeutic agent comprises an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug. In some embodiments, the immune disease comprises one or more of asthma, psoriasis, atopic dermatitis, rheumatoid arthritis, or multiple sclerosis.

[0015] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating inflammation, the method comprising: 1) administering the therapeutic agent to the non-human animal obtained by the construction method or the non-human animal, wherein the non-human animal has inflammation; 2) determining the effectiveness of the therapeutic agent in treating inflammation. In some embodiments, the therapeutic agent comprises an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug. In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0016] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent, the method comprising: 1) administering the therapeutic agent to the non-human animal obtained by the construction method or the non-human animal; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, determining the effect of the therapeutic agent on the non-human animal involves measuring the body weight of the non-human animal or performing a blood test. In some embodiments, the blood test includes, but is not limited to, red blood cell count, hematocrit, and / or hemoglobin content.

[0017] In one aspect, the present invention provides a humanized MICB gene, the humanized MICB gene comprising any one of the following nucleotide sequences:

[0018] A) a nucleotide sequence encoding the protein of SEQ ID NO: 1;

[0019] B) the nucleotide sequence shown in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 19, 20, 21, 22, 23, or NM_005931.5;

[0020] C) a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 19, 20, 21, 22, 23, or NM_005931.5;

[0021] D) nucleotide sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequences shown in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 19, 20, 21, 22, 23 or NM_005931.5.

[0022] In one aspect, the present invention provides a cell, tissue or organ, wherein the genome of the cell, tissue or organ contains the humanized MICB gene, and / or the cell, tissue or organ expresses human MICB protein.

[0023] In one aspect, the present invention provides an animal model, wherein the genome of the animal model contains the humanized MICB gene, and / or the animal model expresses human MICB protein.

[0024] In one aspect, the present invention provides the use of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned humanized MICB gene, the above-mentioned cell, tissue or organ or the above-mentioned animal model, and the use includes: A) use in the development of products related to the MICB-related immune process involving human cells; B) use as a model system related to MICB in pharmacological, immunological, microbiological and medical research; C) use in the production and utilization of animal experimental disease models for MICB-related etiology research and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) use in the in vivo screening, efficacy detection, evaluation of efficacy, verification or evaluation of human MICB signaling pathway regulators; or, E) use in studying the function of the MICB gene, studying the drugs and drug effects targeting human MICB, and studying the therapeutic drugs for tumors, inflammation or immune diseases (preferably tumors) related to MICB.

[0025] The term "all or part" in the present invention, "all" refers to the whole, and "part" refers to the local part in the whole or the individual parts constituting the whole.

[0026] The term "exon XX to exon XXX" or "exon XX-XXX" or "the whole of exon XX-XXX" or "the whole of exon XX to the whole of exon XXX" in the present invention refers to the entire nucleotide sequence containing exon XX to exon XXX and the introns therebetween. For example, exon 1 to exon 6 contains 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 and exon 6.

[0027] The term "intron xx" in the present invention refers to the intron between two exons. For example, intron 1 is the intron between exon 1 and exon 2.

[0028] As used in the present invention, the term "comprising" or "including" is an open-ended expression, including the specified components or steps described, as well as other specified components or steps that do not substantially affect. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0029] As used in the present invention, the term "and / or" includes all combinations of the items connected by this term, and should be regarded as each combination having been separately listed in this application. For example, "A and / or B" includes "A", "B", and "A and B". Another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0030] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. This application describes the methods and materials for the present invention; other suitable methods and materials known in the art can be used. The materials, methods, and examples are merely exemplary and not restrictive. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated by reference in their entirety. In case of conflict, the present specification (including definitions) shall prevail.

[0031] Those skilled in the art can easily insight into other aspects and advantages of this application from the following detailed description.

[0032] MICB

[0033] In the human genome, the MICB gene (NCBI Gene ID: 4277, UniProt ID: Q29980, located at positions 31494918 to 31511124 on chromosome 6) contains 6 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, and exon 6. The corresponding positions of protein domains in the nucleotide sequence and amino acid sequence based on transcript NM_005931.5 (SEQ ID NO: 23) and its encoded protein NP_005922.2 (SEQ ID NO: 1) are shown in Table 1.

[0034] Table 1

[0035]

[0036]

[0037] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). Then the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced to achieve optimal alignment of the two sequences. For example, the comparison of sequences and the determination of the percent identity between two sequences can be accomplished using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0038] The percentage of conserved residues (percent homology) 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 (e.g., lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (such as threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percent homology is higher than the percent identity.

[0039] vector

[0040] The present invention provides a targeting vector, comprising: a) a DNA fragment (5'-arm or 5'-homologous arm) homologous to the 5'-end of the conversion region to be altered, which is selected from genomic DNA of a safe site (e.g., ROSA26 site or Hipp11 site) and has a length of 100 - 10000 nucleotides; b) a donor region; and c) a DNA fragment (3'-arm or 3'-homologous arm) homologous to the 3'-end of the conversion region to be altered, which is selected from genomic DNA of a safe site (e.g., ROSA26 site or Hipp11 site) and has a length of 100 - 10000 nucleotides.

[0041] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed 0.1 kb, 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, or 20 kb.

[0042] In some embodiments, the conversion region to be altered is located at a safe site in the endogenous genome of a non-human animal. In some embodiments, the safe site includes the ROSA26 locus or the Hipp11 locus.

[0043] In some embodiments, the conversion region to be altered is located in intron 1 of the ROSA26 locus of the endogenous genome of a non-human animal.

[0044] In some embodiments, the 5' arm comprises SEQ ID NO: 2. In some embodiments, the 5' arm comprises SEQ ID NO: 3.

[0045] In some embodiments, b) the donor region comprises a human sequence. In some embodiments, b) the donor region comprises SEQ ID NO: 19. In some embodiments, b) the donor region comprises SEQ ID NO: 4 and / or 20. In some embodiments, b) the donor region sequentially comprises SEQ ID NO: 4, 19, and 20 from the 5' end to the 3' end.

[0046] In some embodiments, the targeting vector comprises one or more marker genes (or resistance genes), for example, a resistance gene for positive clone screening or a coding gene for a negative selection marker. In some embodiments, the resistance gene for positive clone screening is the coding sequence Neo of neomycin phosphotransferase. Preferably, the targeting vector further comprises two directly repeated Frt recombination sites flanking the marker gene. In some embodiments, the coding gene for the negative selection marker is the coding gene (DTA) for the A subunit of diphtheria toxin.

[0047] 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 ROSA26 gene. In some embodiments, the target site of the sgRNA on the conversion region to be altered is unique and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'.

[0048] In some embodiments, the present invention relates to plasmid constructs comprising sgRNA (sgRNA vectors, such as pT7-sgRNA) and / or cells comprising such constructs. In some embodiments, the present invention also relates to cells comprising the targeting vector and / or sgRNA vector as described above.

[0049] In some embodiments, the present invention also provides a non-human mammalian cell having any one of the above vectors and one or more in vitro transcripts of the plasmid constructs described in the present application. In some embodiments, Cas9 mRNA or its in vitro transcript is included in the non-human mammalian cell.

[0050] In some embodiments, the gene in the non-human mammalian cell is heterozygous. In some embodiments, the gene in the non-human mammalian cell is homozygous.

[0051] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the non-human mammalian cell is a fertilized egg cell. In some embodiments, the non-human mammalian cell is an embryonic stem cell. In some embodiments, the non-human mammalian cell is any cell capable of expressing MICB.

[0052] Genetically modified non-human animal

[0053] As used herein, the term "genetically modified non-human animal" or "genetically engineered non-human animal" refers to a non-human animal in which at least one chromosome in the genome of the non-human animal has exogenous DNA. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal or genetically engineered non-human animal have exogenous DNA. The cells having exogenous DNA can be various cells, e.g., somatic cells, immune cells (such as 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, the non-human animal comprising a modified endogenous genome comprising an exogenous sequence (such as a human sequence), e.g., by inserting one or more human sequences into a safe site of the non-human animal (such safe sites include, for example, the ROSA26 site or the Hipp11 site). The non-human animal is generally capable of transmitting the genetic modification to its offspring through the germ line.

[0054] As used herein, the term "chimeric (X) gene" or "chimeric (X) nucleic acid" refers to a gene or nucleic acid. In some embodiments, two or more portions of the gene or nucleic acid are from different species, or at least one sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, the chimeric (X) gene or chimeric (X) nucleic acid has at least a portion of the sequence with two or more different origins, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species or sequences having both human and non-human animal sequences. In some embodiments, the chimeric (X) gene or chimeric (X) nucleic acid refers to a humanized (X) gene or humanized (X) nucleic acid.

[0055] As used herein, the term "chimeric (X) protein" or "chimeric (X) polypeptide" refers to a protein or polypeptide. In some embodiments, two or more portions of the polypeptide or protein are from different species, or at least one sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (X) protein or chimeric (X) polypeptide has two or more different origins, e.g., the same (or homologous) proteins of different species. In some embodiments, the chimeric (X) protein or chimeric (X) polypeptide refers to a humanized (X) protein or humanized (X) polypeptide. In some embodiments, the chimeric (X) protein or chimeric (X) polypeptide refers to a human (X) protein or human (X) polypeptide.

[0056] As used herein, the term "humanized (X) protein" or "humanized (X) polypeptide" refers to a protein or polypeptide, wherein at least a portion of the protein or polypeptide is from a human (X) protein or human (X) polypeptide. In some embodiments, the humanized (X) protein or humanized (X) polypeptide refers to a human (X) protein or human (X) polypeptide.

[0057] As used herein, the term "humanized (X) nucleic acid" or "humanized (X) gene" refers to a nucleic acid or gene, wherein at least a portion of the nucleic acid or gene is from a human (X) nucleic acid or human (X) gene. In some embodiments, the nucleic acid or gene in the humanized (X) nucleic acid or humanized (X) gene is entirely from humans. In some embodiments, the humanized (X) nucleic acid refers to a humanized exon, which may be a human exon or a chimeric exon.

[0058] In some embodiments, the chimeric MICB gene or chimeric MICB nucleic acid is a humanized MICB gene or humanized MICB nucleic acid. In some embodiments, at least a portion of the humanized MICB gene or humanized MICB nucleic acid is derived from the human MICB gene. In some embodiments, at least a portion of the humanized MICB gene or humanized MICB nucleic acid is derived from a non-human animal, such as a sequence of a non-human animal safe site. In some embodiments, the humanized MICB gene or humanized MICB nucleic acid comprises a sequence encoding the MICB protein. The encoded MICB protein has at least one activity, such as the activity of the human MICB protein.

[0059] In some embodiments, the chimeric MICB protein or chimeric MICB polypeptide is a humanized MICB protein or humanized MICB polypeptide. In some embodiments, at least one or more portions of the humanized MICB protein or humanized MICB polypeptide are from the human MICB protein. In some embodiments, the humanized MICB protein or humanized MICB polypeptide refers to the human MICB protein. In some embodiments, the humanized MICB protein or humanized MICB polypeptide is functional or has at least one activity, such as the activity of the human MICB protein.

[0060] The genetically modified non-human animals can be various non-human animals, such as mice, rats, rabbits, pigs, cows (e.g., cattle, bulls, water buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals for which it is not easy to obtain suitable genetically modifiable embryonic stem cells (ES), other methods are used to construct non-human animals containing genetic modifications. Such methods include, for example, modifying the non-ES cell genome (e.g., fibroblasts or induced pluripotent stem cells) and using nuclear transfer to transfer the modified genome into a suitable cell, such as an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under appropriate conditions to form an embryo. The above-described construction methods are known in the art and are described in "A. Nagy, et al., 'Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),' Cold Spring Harbor Laboratory Press, 2006", the entire content of which is incorporated herein by reference.

[0061] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. In some embodiments, the rodent can be selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the Muridae family. In one embodiment, the genetically modified non-human animal is from a family selected from Cricetidae (e.g., mouse-like hamsters), Muridae (e.g., hamsters, New World rats and mice, voles), Muroidea (mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (mountain mice, rock mice, tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats, bamboo rats, and zokors). In a particular embodiment, the genetically modified rodent is selected from mice or rats (Muroidea), gerbils, spiny mice, and crested rats. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one embodiment, the non-human animal is a rodent. In a particular embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0062] In some embodiments, the non-human animal can be an immunodeficient non-human mammal. For example, immunodeficient rodents, immunodeficient rabbits, immunodeficient pigs, immunodeficient monkeys, etc. In some embodiments, the non-human animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mouse is of the 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described, for example, in Festing et al., Revised nomenclature for strain129mice, Mammalian Genome 10:836(1999); Auerbach et al., Establishment andChimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem CellLines(2000), the relevant content of the above documents is incorporated herein by reference in its entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is of the BALB strain, such as the BALB / c strain. In some embodiments, the mouse is a hybrid of the BALB strain and another strain. In some embodiments, the mouse is from a hybrid strain (e.g., 50%

[0063] BALB / c - 50% 12954 / Sv; or 50% C57BL / 6 - 50% 129). In some embodiments, the non - human animal is a rodent. In some embodiments, the non - human animal is a mouse having a strain of BALB / c, BALB / cHeAn, BALB / cJ, BALB / cRl, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st or CBA / H. In some embodiments, the non - human animal is a rat. The rat can be selected from Wistar rats, LEA strain, Sprague - Dawley strain, Fischer strain, F344, F6 and Dark Agouti. In some embodiments, the rat strain is a hybrid species of two or more strains selected from Wistar, LEA, Sprague - Dawley, Fischer, F344, F6 and Dark Agouti. The non - human animal can have one or more other genetic modifications and / or other modifications that are suitable for a particular purpose of preparing a humanized animal. For example, a suitable mouse for maintaining xenografts (e.g., human cancers or tumors) can have one or more modifications that impair, inactivate or disrupt all or part of the immune system of the non - human animal. Impairment, inactivation or disruption of the immune system of the non - human animal can include, for example, by chemical means (e.g., administration of toxins), physical means (e.g., irradiation of the animal), and / or genetic modification (e.g., knockout of one or more genes). Non - limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γc null mice (Ito, M. et al., NOD / SCID / γc nullmouse: an excellent recipient mouse model for engraftment of human cells, Blood 100(9):3175-3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice can be optionally irradiated or otherwise treated to destroy one or more immune cell types. Thus, in various embodiments, a genetically modified mouse is provided that can include humanization of at least a portion of a safe site (e.g., the ROSA26 site or the Hipp11 site) and also includes a modification that impairs, inactivates, or partially destroys the immune system of the non-human animal (or one or more cell types of the immune system). In some embodiments, the type of mouse modification is selected from NOD mice, SCID mice, NOD / SCID mice, IL-2Rγ knockout mice, NOD / SCID / γc null mice, nude mice, Rag1 and / or Rag2 knockout mice, NOD Prkdc scid IL-2Rγ null mice, NOD Rag 1 - / - IL2rg - / - (NRG) mice, Rag2 - / - IL2rg - / - (RG) mice and combinations thereof. These transgenic non-human animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety. In some embodiments, the mouse can include insertion of all or part of the human mature MICB coding sequence into a safe site of the mouse endogenous genome, preferably into intron 1 of the ROSA26 site.

[0064] Genetically modified non-human animals include modifications to non-human animal endogenous gene loci. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of the mature MICB protein (e.g., a nucleotide sequence that is identical or has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence encoding the mature MICB protein). Although cells (e.g., ES cells, somatic cells) that can include such genetic modifications are provided in the present invention. In some embodiments, genetically modified non-human animals include modifications to the endogenous ROSA26 site or Hipp11 site in the non-human animal.

[0065] Genetically modified non-human animals can express human MICB protein in the endogenous genome of the non-human animals. In some embodiments, the endogenous ROSA26 locus or Hipp11 locus in the non-human animals has been replaced or inserted with the gene of human MICB and / or the nucleotide sequence encoding the human MICB sequence region or a nucleotide sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99% identity to the human MICB sequence. In various embodiments, the endogenous ROSA26 locus or Hipp11 locus in the non-human animals is modified with all or part of the nucleic acid sequence encoding the human mature MICB protein.

[0066] In some embodiments, the genetically modified non-human animals can express human MICB protein under the control of endogenous or exogenous regulatory elements. Insertion or replacement at a safe locus in the endogenous genome of the non-human animals provides non-human animals that express human MICB in appropriate cells and in a manner that does not result in the potential pathologies observed in some other transgenic non-human animals known in the art. The human MICB protein expressed in the non-human animals can maintain one or more functions of the human MICB protein. In some embodiments, the sequences of human MICB and non-human animal MICB (e.g., mouse MICB) are different, so antibodies that bind to human MICB do not necessarily have the same affinity or effect on non-human animal MICB. Therefore, genetically modified non-human animals having the human MICB coding sequence can be better used to evaluate the effects of therapeutic agents targeting human MICB in animal models.

[0067] In some embodiments, the humanized genome contains the 5’UTR of the human MICB gene. In some embodiments, the humanized genome contains the 3’UTR of the human MICB gene. As shown in this application, humanized mice with the MICB gene inserted or replaced at the endogenous ROSA26 locus or Hipp11 locus in mice contain humanization of the MICB coding sequence and use human regulatory elements and do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for the humanized MICB gene are normal.

[0068] The present invention further relates to the genomic DNA sequence of the humanized MICB gene mouse, the DNA sequence obtained by reverse transcription of mRNA is identical or complementary to this DNA sequence; the construct expressing its amino acid sequence; the cell containing its construct; the tissue or organ including its cell.

[0069] The present invention further relates to non-human mammals produced by the above method. In some embodiments, their genomes contain all or part of the human MICB gene.

[0070] In some embodiments, the non-human mammal is a rodent, preferably a mouse.

[0071] In some embodiments, the non-human mammal expresses a protein encoded by a human or humanized MICB gene.

[0072] In addition, the present invention also provides a non-human mammalian model carrying a tumor, which is obtained by the construction method described in the present application. In some embodiments, the non-human mammal is a rodent (such as a mouse).

[0073] The present invention also provides a cell or cell line, or a primary cell culture, derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, a tissue, organ or its culture derived from a non-human mammal or its offspring. When carrying a tumor, it is derived from a tumor tissue of a non-human mammal or its offspring or a non-human mammal carrying a tumor.

[0074] The present invention provides a non-human mammal produced by any of the methods described in the present application. In some embodiments, a non-human mammal, a genetically modified non-human animal is provided, the genome of which contains DNA of human or humanized MICB.

[0075] In some embodiments, the non-human mammal includes the gene construct described in the present application. In some embodiments, a non-human mammal expressing a human or humanized MICB protein is provided. In some embodiments, a cell, tissue or organ specifically expressing a human or humanized MICB protein is provided.

[0076] In some embodiments, the expression of the human or humanized MICB protein expressed in the non-human animal is controllable. Such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0077] The non-human mammal can be any non-human animal known in the art and can be used in the methods described in the present application. Preferably, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0078] Genetic, molecular and behavioral analyses are performed on the non-human mammals described above. The present invention provides an offspring produced by mating with non-human mammals of the same genotype or other genotypes.

[0079] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its offspring. For example, a cell culture-based model can be prepared by the following methods. The cell culture can be obtained by isolation from a non-human mammal, or cells can be obtained from a cell culture established using the same construct and standard cell transfection techniques. The integration of a genetic construct containing a DNA sequence encoding the human MICB protein can be detected by a variety of methods.

[0080] There are many analytical methods available for detecting foreign DNA, including methods at the nucleic acid level (including the use of reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and methods at the protein level (including histochemical analysis, immunoblot analysis, and in vitro binding studies)). In addition, the expression level of the target gene can be quantified by ELISA methods well-known to those skilled in the art. Many standard analytical methods can be used to complete quantitative detection. For example, RT-PCR and hybridization methods can be used to detect the transcriptional level, including ribonuclease protection assay, Southern Blot, RNA dot blot analysis (RNA dot). Immunohistochemical staining, flow cytometry, and Western blot can also be used to detect the presence of human or humanized MICB protein.

[0081] In some embodiments, the genetically modified non-human animals described in the present application (e.g., homozygous or heterozygous mice with humanized MICB gene) can express human or humanized MICB in one or more tissue cells.

[0082] Method for constructing genetically modified non-human animals

[0083] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting techniques using embryonic stem cells, homologous recombination techniques, CRISPR / Cas9 techniques, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases, or other molecular biology techniques. In some embodiments, homologous recombination techniques are preferably used. In some embodiments, CRISPR / Cas9 gene editing techniques can be used to construct genetically modified non-human animals. Many of these genome editing techniques are known in the art and are described in Yin et al., "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

[0084] In some embodiments, the endogenous genome of at least one cell of a non-human animal contains a nucleotide sequence encoding human MICB. In some embodiments, the nucleotide sequence encoding human MICB is inserted into a safe site of the endogenous genome of the non-human animal, preferably into the ROSA26 site or the Hipp11 site of the endogenous genome of the non-human animal. In some embodiments, the insertion occurs in cells such as germ cells, somatic cells, blastocysts or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0085] The present invention provides a targeting vector. The targeting vector is a vector composed of a 5' homologous arm, a human or humanized MICB gene sequence and a 3' homologous arm. This process involves introducing a human or humanized MICB gene sequence into the endogenous genome of a non-human animal by homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs or CRISPRs) can result in double-strand breaks in the DNA, and the human or humanized MICB gene sequence is inserted into the ROSA26 or Hipp11 site of a mouse by homologous recombination.

[0086] Thus, in some embodiments, a method for preparing a genetically modified humanized non-human animal comprises introducing a nucleotide sequence encoding human MICB into the endogenous ROSA26 or Hipp11 site.

[0087] The present invention also provides a method for establishing a humanized animal model of the MICB gene, comprising the following steps:

[0088] (a) Providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0089] (b) Culturing the cells (preferably culturing the cells in a liquid medium);

[0090] (c) Transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0091] (d) Identifying germline transmission in the offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0092] In some embodiments, the non-human mammal in the above method is a mouse (e.g., a C57BL / 6 mouse).

[0093] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0094] In some embodiments, the fertilized eggs used in the above method are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the method described in the present application include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, or DBA / 2 fertilized eggs.

[0095] The fertilized eggs can be from any non-human animal, such as any non-human animal described in the present application. In some embodiments, the fertilized egg cells are derived from rodents. The gene construct can introduce DNA into the fertilized eggs by microinjection. For example, by culturing the fertilized eggs after microinjection, the cultured fertilized eggs can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human animal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0096] In some embodiments, the method for preparing a genetically modified non-human animal includes modifying an endogenous safe site of the non-human animal. For example, by introducing a nucleotide sequence encoding human MICB (e.g., genomic DNA sequence, CDS sequence, or cDNA sequence) under the control of a human MICB gene regulatory element into an endogenous safe site of the non-human animal (e.g., the ROSA26 or Hipp11 site of the non-human animal), so that human MICB is expressed in the non-human animal.

[0097] In some embodiments, the method for preparing a genetically modified non-human animal includes inserting a nucleotide sequence encoding a human or humanized MICB protein and / or an auxiliary sequence at the ROSA26 or Hipp11 site of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon or an insulator, so that the humanized animal model of the MICB gene can express a human or humanized MICB protein in vivo. In some embodiments, the auxiliary sequence includes an insulator, WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0098] In some embodiments, the method for preparing a genetically modified non-human animal includes:

[0099] (1) Providing a plasmid containing a human MICB gene fragment, the plasmid flanked by a 5' homologous arm and a 3' homologous arm, wherein the 5' homologous arm and the 3' homologous arm target the endogenous ROSA26 or Hipp11 site of the non-human animal;

[0100] (2) Providing one or more guide RNAs (sgRNAs) targeting the endogenous ROSA26 or Hipp11 site of the non-human animal;

[0101] (3) Modifying the genome of a cell (e.g., a fertilized egg or an embryonic stem cell) by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0102] (4) Transfer the fertilized eggs obtained in step (3) into the oviduct of pseudopregnant female mice, or transfer the embryonic stem cells obtained in step (3) into blastocysts, and then transfer the blastocysts into the oviduct of pseudopregnant female mice to produce offspring mice that functionally express human or humanized MICB protein. Preferably, the method further comprises:

[0103] (5) Mate the offspring mice obtained in step (4) to obtain homozygous mice.

[0104] In some embodiments, the fertilized eggs are modified by CRISPR with sgRNAs targeting 5'-terminal target sites and 3'-terminal target sites.

[0105] In some embodiments, the sequence encoding human or humanized MICB protein is operably linked to a human regulatory element.

[0106] In some embodiments, the sequence encoding human or humanized MICB protein is operably linked to a non-human animal endogenous regulatory element.

[0107] In some embodiments, the method for preparing a genetically modified non-human animal comprises:

[0108] (1) Provide a plasmid containing a human MICB gene fragment, the plasmid flanked by a 5' homologous arm and a 3' homologous arm, wherein the 5' homologous arm and the 3' homologous arm target a non-human animal endogenous ROSA26 locus or Hipp11 locus;

[0109] (2) Provide one or more guide RNAs (sgRNAs) targeting a non-human animal endogenous ROSA26 locus or Hipp11 locus;

[0110] (3) Modify the genome of a cell (such as a fertilized egg or an embryonic stem cell) by inserting the human MICB gene fragment into the genome.

[0111] In some embodiments, the construction method includes inserting a nucleotide sequence encoding a human MICB protein into the endogenous genome of a non-human animal, preferably into a safe site of the endogenous genome of the non-human animal, and more preferably into the ROSA26 site or the Hipp11 site of the endogenous genome of the non-human animal. In some embodiments, the construction method includes inserting the nucleotide sequence of SEQ ID NO: 1 into intron 1 of the ROSA26 site of the endogenous genome of a non-human animal. In some embodiments, the construction method includes inserting all or part of the human MICB gene into intron 1 of the ROSA26 site of the endogenous genome of a non-human animal. In some embodiments, the construction method includes inserting all of exon 1 to all of exon 6 of the human MICB gene into intron 1 of the ROSA26 site of the endogenous genome of a non-human animal. In some embodiments, the construction method includes inserting the start codon to the stop codon of the human MICB gene into intron 1 of the ROSA26 site of the endogenous genome of a non-human animal. In some embodiments, the construction method includes inserting the nucleotide sequence from the 5' UTR to the 3' UTR of the human MICB gene into intron 1 of the ROSA26 site of the endogenous genome of a non-human animal. In some embodiments, the construction method includes inserting SEQ ID NO: 19 into intron 1 of the ROSA26 site of the endogenous genome of a non-human animal. In some embodiments, the construction method includes inserting a continuous nucleotide sequence of at least 50 bp upstream of the 5' UTR to a continuous nucleotide sequence of at least 50 bp downstream of the 3' UTR of the human MICB gene into intron 1 of the ROSA26 site of the endogenous genome of a non-human animal. In some embodiments, the construction method includes inserting a continuous nucleotide sequence of at least 23441 bp upstream of the 5' UTR to a continuous nucleotide sequence of at least 7218 bp downstream of the 3' UTR of the human MICB gene into intron 1 of the ROSA26 site of the endogenous genome of a non-human animal. In some embodiments, the construction method includes inserting a continuous nucleotide sequence of at least 23443 bp upstream of the 5' UTR to a continuous nucleotide sequence of at least 7213 bp downstream of the 3' UTR of the human MICB gene into intron 1 of the ROSA26 site of the endogenous genome of a non-human animal. In some embodiments, the construction method includes inserting a nucleotide sequence sequentially comprising SEQ ID NOs: 4, 19, and 20 into intron 1 of the ROSA26 site of the endogenous genome of a non-human animal.

[0112] Application of Genetically Modified Non-Human Animals

[0113] 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 within the endogenous genome of the non-human animal and under the control of a human regulatory element (e.g., a promoter, 5'UTR, and / or 3'UTR) can produce non-human animals having qualities and characteristics that may be significantly different from those of typical knockout plus transgenic animals. In typical knockout plus transgenic animals, the endogenous locus is removed or disrupted, and a fully human transgene is inserted into the genome of the non-human animal and may integrate randomly into the genome. Generally, the location of the integrated transgene is unknown; the expression of the human protein is measured by transcription of the human gene and / or protein assay and / or functional assay.

[0114] Genetically modified non-human animals that express a human or humanized MICB protein, for example, in a physiologically appropriate manner, provide a variety of uses, including but not limited to the development of therapeutic methods for human diseases and disorders, and the evaluation of the toxicity and / or efficacy of these human therapeutic methods in animal models.

[0115] The present invention also provides an application of the above-mentioned MICB gene-modified non-human animal and the non-human animal obtained by any of the above construction methods.

[0116] In some embodiments, the application includes:

[0117] A) Application in the development of products related to MICB-related immune processes involving human cells;

[0118] B) Application as a model system related to MICB in pharmacological, immunological, microbiological, and medical research;

[0119] C) Application involving the production and utilization of animal experimental disease models for MICB-related etiological research and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0120] D) Application in the screening, pharmacodynamic detection, evaluation of efficacy, verification, or assessment of human MICB signaling pathway regulators in vivo; or,

[0121] E) Application in studying the function of the MICB gene, studying the drugs and pharmacodynamics targeting the human MICB target, and studying the therapeutic drugs for tumors, inflammation, or immune diseases (preferably tumors) related to MICB.

[0122] The present invention provides a non-human animal expressing human or humanized MICB protein, which can be used for screening human MICB-specific regulators. In some embodiments, the non-human animal is a human disease animal model. For example, the disease is genetically induced (knock-in or knock-out). In different embodiments, the genetically modified non-human animal also comprises a damaged immune system, such as genetically modified human xenotransplantation, including human solid tumors (e.g., breast cancer) or hematological tumors (e.g., lymphocytic tumors (e.g., B or T cell tumors)).

[0123] In some embodiments, a therapeutic agent (e.g., an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug) blocks or inhibits or enhances or activates the MICB-mediated signaling pathway. In some embodiments, the therapeutic agents described in the present application (e.g., an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug) can block or enhance the interaction between MICB complexes, thereby inhibiting or activating the MICB signaling pathway.

[0124] In some embodiments, the genetically modified non-human animal can be used to determine the effectiveness of a therapeutic agent (e.g., an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug) in treating various immune diseases. In some embodiments, the immune diseases include but are not limited to GVHD (graft-versus-host disease), psoriasis, allergy, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty liver disease), systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, atopic dermatitis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders, etc. In some embodiments, the immune disease is asthma, psoriasis, atopic dermatitis, rheumatoid arthritis, or multiple sclerosis.

[0125] In some embodiments, the genetically modified non-human animal can be used to determine the effectiveness of a therapeutic agent (e.g., an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug) in treating various inflammations (infections). In some embodiments, the inflammations include acute inflammations and also chronic inflammations. Specifically, they include but are not limited to degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.). In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0126] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting MICB, nucleic acid drugs targeting MICB, and / or polypeptide drugs) in treating cancer (tumors). In some embodiments, a therapeutic agent is administered to a non-human animal that 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 methods include measurement with a vernier caliper, flow cytometry, and / or in vivo imaging of the animal. In some embodiments, the detection includes assessing an individual's body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, which include changes in food consumption or water consumption.

[0127] In some embodiments, the tumor cells include one or more cancer cells (e.g., cancer cells derived from a human or non-human animal) that are injected into the animal. In some embodiments, a therapeutic agent (e.g., an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug) inhibits the MICB signaling pathway. In some embodiments, a therapeutic agent (e.g., an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug) does not inhibit the MICB signaling pathway.

[0128] In some embodiments, genetically modified non-human animals can be used to detect whether a therapeutic agent (an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug) is an agonist or antagonist. In some embodiments, the methods described in this application can be used to detect the function of a therapeutic agent (e.g., an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug), for example, whether the therapeutic agent can upregulate or downregulate the immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cell cytotoxicity (ADCC). In some embodiments, genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent for treating a subject's disease (e.g., a tumor, an immune disease, or an inflammation). The inhibitory effect on the tumor can also be determined by methods known in the art, for example, measuring the tumor volume in the non-human animal, and / or determining the tumor growth inhibition rate (TGI TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%) = (1 – T Vt / T Vc ) x 100, where T Vt and T Vc are the average tumor volumes (or weights) of the treatment group and the control group.

[0129] In some embodiments, therapeutic agents (e.g., antibodies targeting MICB, nucleic acid drugs targeting MICB, and / or polypeptide drugs) can be used to treat various cancers (tumors). As used herein, "cancer" refers to cells with the ability of autonomous growth, i.e., an abnormal state or disorder characterized by rapid proliferation of cell growth. This term is intended to include all types of cancerous growths or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. As used herein, "tumor" includes, but is not limited to, lymphoma, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, glioblastoma, lung cancer (e.g., non-small cell 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 multiforme, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Among them, the leukemia is selected from acute lymphoblastic (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 lymphoma and non-Hodgkin lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In a specific embodiment of the present invention, the tumor is breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor, mesothelial tissue tumor, oropharyngeal tumor, female reproductive system cancer, or meningioma. In some embodiments, the tumor includes solid tumors or hematological tumors. In some embodiments, the tumor includes melanoma, skin cancer, colorectal cancer, breast cancer, endocrine cancer, lymphocyte tumor, head and neck cancer, liver cancer, or lung cancer.

[0130] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (such as an antibody targeting MICB, a nucleic acid drug targeting MICB, and / or a polypeptide drug). The method includes administering the therapeutic agent to the non-human animal obtained by the construction method or the non-human animal, and evaluating the weight change of the non-human animal or performing a blood test. In some embodiments, the blood test includes, but is not limited to, red blood cell count, hematocrit, and / or hemoglobin content. In some embodiments, the therapeutic agent can reduce the red blood cells (RBC), hematocrit, or hemoglobin content by 20%, 30%, 40%, or more than 50%. In some embodiments, the weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% less than that of the control group (such as the average weight of non-human animals not treated with the therapeutic agent).

[0131] The present invention also provides an animal model constructed by the method described in the present application for developing products related to the human cellular immune process, manufacturing human antibodies, or for use in model systems for pharmacological, immunological, microbiological, and medical research.

[0132] In some embodiments, there is provided an animal model generated by the method described in the present application for producing and utilizing an animal experimental disease model of the immune process of human cells, studying pathogens, or formulating new diagnostic strategies and / or treatment strategies.

[0133] The present invention also provides an animal model generated by the method described in the present application for screening, validating, evaluating, or studying the function of the MICB gene, human MICB antibodies, drugs for MICB target-related diseases (such as tumors, inflammation, or immune diseases, preferably tumors), or effectiveness.

[0134] In some embodiments, the present disclosure provides a method for verifying the in vivo efficacy of TCR-T, CAR-T, and / or other immunotherapies (such as adoptive T cell transfer therapy). For example, the method includes transplanting human tumor cells into the non-human animals described in the present application, and applying human CAR-T to the non-human animals with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animals are selected from MICB gene humanized non-human animals prepared by the method described in the present application, double-gene or multi-gene humanized non-human animals (or their offspring) generated by the method described in the present application, non-human animals expressing human or humanized MICB protein, or the tumor or inflammatory animal models described in the present application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies can treat the MICB-related diseases described herein (such as tumors, inflammation, or immune diseases, preferably tumors). In some embodiments, TCA-T, CAR-T, and / or other immunotherapies provide an evaluation method for treating the MICB-related diseases described in the present application (such as tumors, inflammation, or immune diseases, preferably tumors).

[0135] Non-human animal models of two or more human or chimeric genes

[0136] The present invention also provides a method for an animal model or non-human animal having two or more human or chimeric genes. The non-human animal or animal model may comprise a human or chimeric MICB gene and a sequence encoding an additional human or chimeric protein.

[0137] In some embodiments, the additional human or chimeric protein comprises at least one of MICA, NKG2D, TFR1, NKP46, ICOS, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the non-human animal also expresses at least one of human or humanized MICA, NKG2D, TFR1, NKP46, ICOS, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4 proteins.

[0138] The present invention also provides a method for constructing a non-human animal of two or more human or chimeric genes, the construction method comprising:

[0139] (1) Providing a non-human animal obtained by the above construction method;

[0140] (2) Mating the non-human animal provided in step (1) with other genetically modified non-human animals, performing in vitro fertilization or directly performing gene editing, and screening to obtain a multi-gene modified non-human animal.

[0141] 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 MICA, NKG2D, TFR1, NKP46, ICOS, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0142] In some embodiments, humanization of the MICB gene is directly performed on a non-human animal having a genetic modification of human or chimeric MICA, NKG2D, TFR1, NKP46, ICOS, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4 genes.

[0143] Since these proteins may be involved in different mechanisms, combination therapies targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are underway and showing good results. Multigene-modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins. For example, therapeutic agents (including antibodies targeting MICB, nucleic acid drugs targeting MICB, and / or polypeptide drugs), and additional therapeutic agents for treating diseases (such as tumors, inflammatory or immune diseases, preferably tumors). The method includes administering a therapeutic agent and an additional therapeutic agent to a non-human animal, where the non-human animal has a disease, and determining the effect of the combination treatment on the disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to MICA, NKG2D, TFR1, NKP46, ICOS, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), or an anti-PD-L1 antibody. In some embodiments, the above-mentioned non-human animal further includes a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the above-mentioned tumor includes one or more tumor cells expressing PD-L1 and / or PD-L2.

[0144] In some embodiments, the combination treatment is used to treat various cancers described in the present application. In some embodiments, the combination treatment is designed to treat immune diseases described in the present application, such as psoriasis. In some embodiments, the methods described in the present application can be used to evaluate combination treatments with some other methods. Methods for treating cancer that can be used alone or in combination with the methods described in the present application include, for example, treating a subject with chemotherapy, such as camptothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, bleomycin, nitrosourea, dactinomycin, daunorubicin, bleomycin, plicamycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, carboplatin, 5-fluorouracil, vincristine, vinblastine, and / or methotrexate. Alternatively, in addition to this, the method can include performing surgery on the subject to remove at least a part of the cancer, such as removing a part or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1 : Schematic diagram of the MICB gene targeting strategy and the design of the targeting vector V1 (not to scale);

[0146] Figure 2 : PCR identification results of F1 generation of humanized mice with MICB gene. Among them, WT is the wild-type control, M is the Marker, and H2O is the water control;

[0147] Figure 3 : RT-PCR identification results of F1 generation of humanized mice with MICB gene. Among them, + / + is the wild-type mouse, H / H is the homozygous humanized mouse with MICB gene, H2O is the water control, and GAPDH is the internal reference. Specific implementation manners

[0148] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

[0149] In each of the following embodiments, the equipment and materials are obtained from the following several companies:

[0150] C57BL / 6 mice and Flp transgenic mice are purchased from the National Rodent Laboratory Animal Seed Center, National Institutes for Food and Drug Control.

[0151] Example 1 Humanized mice with MICB gene

[0152] To achieve the purpose of the present invention, the nucleotide sequence of the human MICB gene can be introduced into the endogenous ROSA26 locus of mice. Specifically, using gene editing technology, under the control of the regulatory elements of the human MICB gene, the nucleotide sequence of 43.6 kb in total, including all of exon 1 to all of exon 6 of the human MICB gene and the upstream and downstream sequences, is inserted into the ROSA26 locus of mice.

[0153] Design the targeting strategy as Figure 1 shown, Figure 1 The targeting vector V1 shown contains the homologous arm sequences upstream and downstream of the mouse endogenous ROSA26 gene, the 2xInsulator sequence, and the A fragment containing the human MICB gene. Among them, the upstream 5' homologous arm sequence is as shown in SEQ ID NO: 2, and the downstream 3' homologous arm sequence is as shown in SEQ ID NO: 3. The nucleotide sequence of the human MICB gene fragment contains SEQ ID NO: 4, SEQ ID NO: 19, and SEQ ID NO: 20 in sequence from the 5' end to the 3' end. Among them, the connection design of SEQ ID NO: 4 and SEQ ID NO: 19 is: wherein the "C" in the sequence " GTTTC " is the last nucleotide of SEQ ID NO: 4, and the "A" in the sequence is the first nucleotide of SEQ ID NO: 19; the connection of SEQ ID NO: 19 and SEQ ID NO: 20 is designed as: wherein the last "A" in the sequence " TAGA " is the last nucleotide of SEQ ID NO: 19, and the "C" in the sequence is the first nucleotide of SEQ ID NO: 20. Figure 1 The connection of the 2xInsulator sequence (5'-end) upstream and the mouse sequence is designed as: wherein the "G" in the sequence " CTCCG " is the last nucleotide at the junction of the mouse sequence and the 2xInsulator sequence (5'-end), and the first "G" in the sequence is the first nucleotide of the 2xInsulator sequence (5'-end); the connection of the 2xInsulator sequence (5'-end) downstream and the upstream of the human MICB gene fragment sequence is designed as: wherein the last "C" in the sequence " ATCGC " is the last nucleotide of the 2xInsulator sequence (5'-end), and the first "G" in the sequence is the first nucleotide of the human MICB gene fragment sequence. The connection of the 2xInsulator sequence (3'-end) and the mouse sequence is designed as: wherein the last "C" in the sequence " ATCG C" is the last nucleotide of the 2xInsulator sequence (3'-end), and the first "G" in the sequence is the first nucleotide at the junction of the mouse sequence and the 2xInsulator sequence (3'-end).

[0154] The targeting vector also includes a resistance gene for positive clone screening, i.e., the neomycin phosphotransferase coding sequence Neo, and two directly repeated site-specific recombination systems Frt recombination sites are installed on both sides of the resistance gene to form a Neo cassette. The connection of the 5'-end of the Neo cassette and the human MICB gene is designed as:

[0155] Among them, the last "T" in the sequence " AATCT " is the last nucleotide of the human MICB gene, and the "G" in the sequence is the first nucleotide of the Neo cassette; the connection design of the 3'-end of the Neo cassette and the 2xInsulator sequence (3'-end) is as follows: Among them, the last "C" in the sequence " GTACC " is the last nucleotide of the Neo cassette, and the first "G" in the sequence is the first nucleotide of the 2xInsulator sequence (3'-end). The mRNA transcribed from the MICB gene in the humanized mouse of the modified MICB gene is NM_005931.5 (SEQ ID NO: 23), and the expressed protein is as shown in SEQ ID NO: 1.

[0156] The construction of the targeting vector can be carried out by conventional methods, such as restriction enzyme digestion and ligation. After the constructed targeting vector is preliminarily verified by restriction enzyme digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into the embryonic stem cells of C57BL / 6 mice by electroporation, and the obtained cells are screened using the positive clone selection marker gene to screen out the correct positive clone cells. The correct positive clone cells (black mice) screened out are introduced into the isolated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient female mouse (white mouse) to produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are interbred to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone selection marker gene, and then the MICB gene humanized homozygous mice can be obtained by interbreeding.

[0157] The somatic cell genotype of F1 generation mice can be identified by PCR method, and the primers are shown in Table 2. The identification results of exemplary F1 generation mice are shown in Figure 2 (A) and Figure 2 (B) ( Figure 2 The unlabeled numbers in it are irrelevant results), and 7 mice numbered F1-01 to F1-07 are all positive.

[0158] Table 2 Primer sequences and recombinant fragment sizes for PCR detection of F1 generation genotypes

[0159]

[0160] The expression of mRNA in humanized mice of the MICB gene can be detected by RT-PCR. Specifically, one C57BL / 6 mouse (+ / +) and one homozygous humanized MICB gene mouse (H / H) prepared in this example were selected respectively. After sacrificing the mice by cervical dislocation, spleen tissues were taken and RT-PCR was performed using the primer sequences shown in Table 3. The detection results are as Figure 3 shown. It can be seen from Figure 3 that human MICB mRNA was only detected in the homozygous humanized mice of the MICB gene.

[0161] Table 3 RT-PCR primer sequences and target fragment sizes

[0162]

[0163] Example 2 Pharmacodynamic model

[0164] The humanized mice of the MICB gene prepared in Example 1 of the present application can be used to evaluate the pharmacodynamic effects of therapeutic agents targeting human MICB (such as antibodies targeting MICB, nucleic acid drugs targeting MICB, and / or polypeptide drugs) in tumor diseases. For example, homozygous humanized mice of the MICB gene were subcutaneously inoculated with MC38 cells. When the tumor volume grew to about 100 mm 3 and then divided into a control group or a treatment group according to the tumor volume. The treatment group was randomly selected with a therapeutic agent targeting human MICB, and the control group was injected with an equal volume of normal saline. The tumor volume was measured regularly and the body weight of the mice was weighed. The in vivo safety and in vivo pharmacodynamic effects of the compound can be effectively evaluated by comparing the changes in the body weight of the mice and the tumor size.

[0165] Example 3 Preparation of double-gene or multi-gene humanized mice

[0166] The humanized mice of the MICB gene prepared in Example 1 of the present application can also be used to prepare a multi-gene humanized mouse model. For example, in the aforementioned Example 1, the embryonic stem cells used for microinjection can be selected from mice containing at least one gene modification of MICA, NKG2D, TFR1, NKP46, ICOS, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. Alternatively, on the basis of humanized MICB mice, double-gene humanized or multi-gene humanized mouse models can be obtained by using the techniques of isolating mouse ES embryonic stem cells and gene recombination and targeting. The homozygous or heterozygous humanized mice of the MICB gene obtained by this method can also be mated with other gene-modified mice, and their offspring can be screened. According to Mendelian inheritance, there is a certain probability of obtaining multi-gene mice with humanized MICB genes and other gene modifications. Then, the heterozygotes can be mated with each other to obtain homozygotes with double-gene or multi-gene modifications.

[0167] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0168] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

[0169] Furthermore, any combination can be made among various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric MHC class I polypeptide-related sequence B (MICB) protein.

2. The construction method according to claim 1, characterized in that: The amino acid sequence of the human or chimeric MICB protein comprises SEQ ID NO: 1; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

1.

3. A method for constructing a genetically modified non-human animal, characterized in that: A nucleotide sequence comprising human MICB is introduced into the endogenous genome of a non-human animal. Preferably, the introduction is by insertion or replacement.

4. The construction method according to claim 3, characterized in that: The introduction is inserted into a safe site of the endogenous genome of a non-human animal. Preferably, the safe site includes a ROSA26 site or a Hipp11 site.

5. The construction method according to any one of claims 3-4, characterized in that: The nucleotide sequence of human MICB comprises a nucleotide sequence encoding a human or chimeric MICB protein; Preferably, the nucleotide sequence of human MICB comprises exon 1 to exon 6 of the human MICB gene; Preferably, the nucleotide sequence of human MICB comprises a nucleotide sequence from the start codon to the stop codon of the human MICB gene, further preferably further comprises a 5'UTR and / or a 3'UTR, more preferably comprises SEQ ID NO: 19; or comprises a nucleotide sequence having an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the nucleotide sequence shown in SEQ ID NO: 19; Preferably, the nucleotide sequence of human MICB further comprises at least 50 bp of continuous nucleotide sequence upstream of 5'UTR and / or at least 50 bp of continuous nucleotide sequence downstream of 3'UTR; further preferably comprises SEQ ID NO: 4 and / or 20; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity with the nucleotide sequence shown in SEQ ID NO: 4 and / or 20; Preferably, the nucleotide sequence of human MICB comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR of the human MICB gene; Preferably, the nucleotide sequence of human MICB comprises SEQ ID NOs: 4, 19 and 20 from the 5' end to the 3' end; or comprises nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity with the nucleotide sequences shown in SEQ ID NOs: 4, 19 and 20.

6. The construction method according to any one of claims 3 to 5, characterized in that: The nucleotide sequence encoding the human or chimeric MICB protein or the nucleotide sequence of human MICB is operably linked to an endogenous regulatory element or an exogenous regulatory element; Preferably, the modified MICB gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous modified (eg, inserted or replaced) genome.

7. The construction method according to any one of claims 3 to 6, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent, preferably, the rodent includes a mouse or a rat; Preferably, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins, and the other human or chimeric proteins preferably comprise at least one of MICA, NKG2D, TFR1, NKP46, ICOS, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

8. A method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, characterized in that: The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 7; 2) Determine the effect of therapeutic agents on non-human animals or diseases; Preferably, the therapeutic agent comprises an antibody targeting MICB, a nucleic acid drug targeting MICB and / or a polypeptide drug; Preferably, the therapeutic agent further comprises an additional therapeutic agent, and the additional therapeutic agent preferably comprises one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; Preferably, the disease is cancer; Preferably, the cancer includes one or more of melanoma, skin cancer, colorectal cancer, breast cancer, endocrine cancer, lymphocytic tumor, head and neck cancer, liver cancer or lung cancer.

9. A humanized MICB gene, characterized in that: The humanized MICB gene comprises any of the following nucleotide sequences: A) a nucleotide sequence encoding the protein of SEQ ID NO: 1; B) the nucleotide sequence shown in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 19, 20, 21, 22, 23 or NM_005931.5; C) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 19, 20, 21, 22, 23 or NM_005931.5; D) a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 19, 20, 21, 22, 23 or NM_005931.

5.

10. A cell, tissue or organ, characterized in that: The genome of the cell, tissue or organ comprises the humanized MICB gene according to claim 9.

11. Use of the non-human animal obtained by the construction method according to any one of claims 1 to 7, the humanized MICB gene according to claim 9, or the cell, tissue or organ according to claim 10, characterized in that: The application includes: A) Applications in product development for MICB-related immune processes involving human cells; B) Application as a model system relevant to MICB for pharmacological, immunological, microbiological and medical research; C) Applications involving the production and use of animal experimental disease models for the study of the etiology of MICB 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 modulators of the human MICB signaling pathway; or, E) Study the function of MICB gene, study the drugs and efficacy targeting human MICB targets, and study the application of therapeutic drugs for MICB-related tumors.

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