KLRG1 gene modified non-human animal

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) KLRG1 protein and methods of use thereof.

Description

Technical Field

[0001] The present invention provides a non-human animal expressing a human or chimeric (eg, humanized) KLRG1 protein and methods of using the same. Background Art

[0002] Traditional drug development usually uses in vitro screening methods, but these screening methods cannot provide the body environment (such as tumor microenvironment, stromal cells, extracellular matrix components and immune cell interactions, etc.), resulting in a high failure rate of drug development. In addition, given the differences between humans and animals, the test results obtained from in vivo pharmacology tests using conventional experimental animals may not reflect the actual disease state and the interaction of the target site, resulting in significant differences between the results of many clinical trials and the results of animal experiments.

[0003] Therefore, developing a humanized animal model suitable for screening and evaluation of human antibodies will significantly improve the efficiency of new drug development and reduce the cost of drug development.

[0004] However, the construction of humanized animal models is very difficult. For example, the non-patent literature: Generation and utility of genetically humanized mouse models (Scheer N, Snaith M, Wolf CR, Seibler J., Drug Discovery Today; 18(23-24): 1200-11, 2013) discloses that the humanization of any gene carries certain risks, and even with carefully designed strategies, the expression and function of human genes in animals cannot be ensured. Summary of the invention

[0005] The present application provides an animal model with human or chimeric KLRG1 protein. The animal model can express human or chimeric KLRG1 (e.g., humanized KLRG1) protein. It can be used to study the function of the KLRG1 gene, and can also be used for the screening and evaluation of KLRG1 signaling pathway regulators (e.g., anti-human KLRG1 antibodies, oligonucleotide drugs and / or polypeptide drugs). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamics research, treatment of immune diseases and treatment of diseases at human KLRG1 target sites; the model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of the KLRG1 protein and provides a platform for screening related drugs.

[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 at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric killer cell lectin-like receptor G1 (KLRG1) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric KLRG1 protein is operably linked to a regulatory element of at least one chromosome (e.g., a human or non-human animal endogenous regulatory element, such as a promoter, an endogenous 5'UTR and / or a 3'UTR). In some embodiments, the chimeric KLRG1 protein is a humanized KLRG1 protein. In some embodiments, the chimeric KLRG1 protein comprises a human or humanized extracellular region, a human or humanized transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the chimeric KLRG1 protein is consistent with at least 50 to at least 189 consecutive amino acids of the human KLRG1 protein. In some embodiments, the amino acid sequence of the human or chimeric KLRG1 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189; or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189. In some embodiments, the amino acid sequence of the chimeric KLRG1 protein comprises SEQ ID NO: 7; or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 7. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a mouse or rat). In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal does not express endogenous KLRG1 protein or expresses reduced levels compared to KLRG1 in wild-type animals. In some embodiments, one or more cells of the non-human animal express human or chimeric KLRG1 protein.

[0007] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the endogenous KLRG1 gene of the non-human animal is replaced with a nucleotide sequence comprising human KLRG1 at the endogenous KLRG1 locus of the non-human animal. In some embodiments, the nucleotide sequence of human KLRG1 is operably linked to a regulatory element (e.g., an endogenous regulatory element of a human or non-human animal, such as a promoter, 5'UTR and / or 3'UTR). In some embodiments, the endogenous KLRG1 protein of the non-human animal is not expressed or the expression level is reduced compared to KLRG1 in wild-type animals. In some embodiments, the nucleotide sequence of human KLRG1 comprises a nucleotide sequence encoding a human KLRG1 protein. In some embodiments, the nucleotide sequence of human KLRG1 comprises a nucleotide sequence encoding an extracellular region of a human KLRG1 protein. In some embodiments, the nucleotide sequence of human KLRG1 comprises a nucleotide sequence encoding a transmembrane region of a human KLRG1 protein. In some embodiments, the nucleotide sequence of human KLRG1 comprises a nucleotide sequence encoding a cytoplasmic region of a human KLRG1 protein. In some embodiments, the nucleotide sequence of human KLRG1 comprises all or part of exon 1 to exon 5 of the human KLRG1 gene. In some embodiments, the nucleotide sequence of human KLRG1 comprises any one of the following groups: A) part of exon 2 to all of exon 5 of the human KLRG1 gene, preferably, the part of exon 2 of the human KLRG1 gene comprises at least 5bp of continuous nucleotides, preferably also including at least 50-1000bp downstream of 3'UTR. B) all of exon 1 to exon 5 of the human KLRG1 gene, preferably including the nucleotide sequence from the start codon to the stop codon, further preferably including 5'UTR and / or 3'UTR, more preferably also including at least 50-50000bp of continuous nucleotides upstream of 5'UTR and / or at least 50-5000bp of continuous nucleotides downstream of 3'UTR. In some embodiments, the nucleotide sequence of human or chimeric KLRG1 comprises the nucleotide sequence of SEQ ID NO:5 or NCBI accession number NC_000012.12 from 8944044 to 9012751; or comprises a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence of SEQ ID NO:5 or NCBI accession number NC_000012.12 from 8944044 to 9012751. In some embodiments, the replacement of the endogenous KLRG1 gene of the non-human animal is to replace any one or more of the extracellular region, endogenous transmembrane region or endogenous cytoplasmic region of the endogenous KLRG1 of the non-human animal.In some embodiments, one or more cells of the non-human animal express human or chimeric KLRG1, wherein the chimeric KLRG1 comprises an extracellular region, a transmembrane region and a cytoplasmic region, wherein the extracellular region is identical or identical to the extracellular region of human KLRG1 by at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5%. In some embodiments, the extracellular region is identical to the extracellular region of human KLRG1 by at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive amino acids. In some embodiments, the endogenous KLRG1 gene of the non-human animal comprises all or part of exon 1 to exon 5 of the endogenous KLRG1 gene of the non-human animal. In some embodiments, the replaced endogenous KLRG1 gene of a non-human animal comprises any one of the following groups: a) part of exon 2 to all of exon 5 of an endogenous KLRG1 gene of a non-human animal, preferably also comprising at least 50-1000bp of continuous nucleotides downstream of the 3'UTR; preferably, part of exon 2 of an endogenous KLRG1 gene of a non-human animal comprises at least 5bp of continuous nucleotides; b) all of exon 1 to exon 5 of an endogenous KLRG1 gene of a non-human animal, preferably also comprising at least 50-6000bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the modified KLRG1 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent comprises a mouse or a rat. In some embodiments, the mRNA transcribed from the modified KLRG1 gene in the genome of the non-human animal comprises SEQ ID NO: 6 or SEQ ID NO: 20; or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 6 or SEQ ID NO: 20. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins, preferably, the human or chimeric protein is selected from at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0008] In one aspect, the present invention provides a non-human animal or a method for constructing the same. In some embodiments, the non-human animal comprises at least one cell encoding a nucleotide sequence of a human or chimeric KLRG1 protein. In some embodiments, the amino acid sequence of the human or chimeric KLRG1 protein is identical to at least 50 to 189, such as at least 50, 100, 120, 130, 131, 140, 150, 160, 170, 180 or 189 consecutive amino acid sequences of a human KLRG1 protein. In some embodiments, the chimeric KLRG1 protein comprises at least 50 to 130, such as 50, 60, 70, 80, 90, 100, 110, 120 or 130 consecutive amino acids of the extracellular region of human KLRG1. In some embodiments, the chimeric KLRG1 protein comprises at least 1 to 21, such as 1, 5, 10, 15, 20 or 21 consecutive amino acids of the transmembrane region of human KLRG1. In some embodiments, the human or chimeric KLRG1 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189; or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189. In some embodiments, the nucleotide sequence encoding the human or chimeric KLRG1 protein is operably linked to a human or non-human animal endogenous KLRG1 regulatory element. In some embodiments, the nucleotide sequence encoding the human or chimeric KLRG1 protein can be integrated into a non-human animal endogenous KLRG1 locus. In some embodiments, the chimeric KLRG1 protein has at least one activity, such as a non-human animal KLRG1 activity and / or a human KLRG1 activity.

[0009] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, in some embodiments, the construction method includes replacing a nucleotide sequence encoding an endogenous KLRG1 region of a non-human animal with a nucleotide sequence encoding a corresponding region of human or chimeric KLRG1 at the endogenous KLRG1 locus of the non-human animal. In some embodiments, the endogenous KLRG1 protein of the non-human animal is not expressed or the expression level is reduced compared to KLRG1 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric KLRG1 comprises all or part of exon 1 to exon 5 of the human KLRG1 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric KLRG1 comprises any one of the following groups: A) part of exon 2 to all of exon 5 of the human KLRG1 gene, preferably, the part of exon 2 of the human KLRG1 gene comprises at least 5bp of continuous nucleotides, and preferably also includes at least 50bp downstream of the 3'UTR; wherein, the nucleotide sequence downstream of the 3'UTR of the human KLRG1 gene contained in the nucleotide sequence encoding the corresponding region of human or chimeric KLRG1 preferably comprises at least 50-1000bp, preferably 200-400bp, for example, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000bp of continuous nucleotide sequence. Wherein, the part of exon 2 of human KLRG1 gene comprises at least 5-105bp, such as 5, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 105bp continuous nucleotide sequence. B) All of exon 1 to exon 5 of human KLRG1 gene, preferably comprising the nucleotide sequence from the start codon to the stop codon, further preferably comprising 5'UTR and / or 3'UTR, more preferably further comprising at least 50bp continuous nucleotides upstream of 5'UTR and / or at least 50bp continuous nucleotides downstream of 3'UTR.The nucleotide sequence upstream of the 5'UTR of the human KLRG1 gene contained in the nucleotide sequence encoding the corresponding region of human or chimeric KLRG1 preferably comprises at least 50-50000 bp, preferably 30000-50000 bp, for example 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 450 00, 45502 or 50000 bp continuous nucleotide sequence; and / or, the 3'UTR downstream nucleotide sequence of the human KLRG1 gene contained in the nucleotide sequence encoding the corresponding region of human or chimeric KLRG1 preferably comprises at least 50-5000 bp, preferably 1000-3000 bp, for example 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000 bp continuous nucleotide sequence. In some embodiments, the amino acid sequence of the corresponding region of human or chimeric KLRG1 comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189; or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189. In some embodiments, the amino acid sequence of the corresponding region of chimeric KLRG1 comprises SEQ ID NO: 7; or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric KLRG1 comprises the nucleotide sequence of 8944044 to 9012751 of SEQ ID NO:5 or NCBI accession number NC_000012.12; or comprises a nucleotide sequence of 8944044 to 9012751 of SEQ ID NO:5 or NCBI accession number NC_000012.12 that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in positions 8944044 to 9012751. In some embodiments, the nucleotide sequence encoding the endogenous KLRG1 region of the non-human animal that is replaced is all or part of the nucleotide sequence encoding the extracellular region, endogenous transmembrane region or endogenous cytoplasmic region of the endogenous KLRG1. In some embodiments, the nucleotide sequence encoding the endogenous KLRG1 region of the non-human animal that is replaced comprises all or part of exon 1 to exon 5 of the KLRG1 gene of the non-human animal.In some embodiments, the replaced non-human animal endogenous KLRG1 gene comprises any one of the following groups: a) part of exon 2 to all of exon 5 of the non-human animal endogenous KLRG1 gene, preferably also comprising at least 50 bp of continuous nucleotides downstream of the 3'UTR; preferably, the part of exon 2 of the non-human animal endogenous KLRG1 gene comprises at least 5 bp of continuous nucleotides; wherein the replaced nucleotide sequence encoding the non-human animal endogenous KLRG1 region comprises a nucleotide sequence downstream of the 3'UTR of the non-human animal KLRG1 gene; Contains at least 50-1000bp, preferably 100-300bp, for example 50, 100, 170, 200, 300, 400, 500, 600, 700, 800, 900 or 1000bp of continuous nucleotide sequence; wherein the replaced portion of exon 2 of the endogenous KLRG1 gene of the non-human animal contains at least 5-105bp, for example 5, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 105bp of continuous nucleotide sequence. b) all of exon 1 to exon 5 of the endogenous KLRG1 gene of a non-human animal, preferably further comprising at least 50 bp of continuous nucleotides upstream of the 5'UTR, wherein the 5'UTR upstream nucleotide sequence of the non-human animal KLRG1 gene contained in the replaced nucleotide sequence encoding the endogenous KLRG1 region of the non-human animal comprises at least 50-6000 bp, preferably 5000-6000 bp, such as 50, 100, 170, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 5100, 5148, 5500 or 6000 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric KLRG1 is operably linked to a human or non-human animal endogenous KLRG1 regulatory element, such as a promoter, 5'UTR and / or 3'UTR. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (eg, a mouse or rat).

[0010] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric KLRG1, the construction method comprising replacing a nucleotide sequence encoding an endogenous KLRG1 region of a non-human animal with a nucleotide sequence encoding a corresponding region of human KLRG1 at an endogenous KLRG1 locus of a non-human animal, thereby producing a genetically modified non-human animal cell, wherein the non-human animal cell expresses a human or chimeric KLRG1 protein. In some embodiments, the chimeric KLRG1 protein comprises a human or humanized extracellular region, a human or humanized transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the corresponding region of human KLRG1 comprises all or part of exon 1 to exon 5 of the human KLRG1 gene. In some embodiments, the amino acid sequence of the human KLRG1 corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2 59-189 or SEQ ID NO: 2 60-189. In some embodiments, the nucleotide sequence encoding the endogenous KLRG1 region to be replaced comprises all or part of exon 1 to exon 5 of the endogenous KLRG1 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding the human KLRG1 corresponding region is operably linked to a regulatory element of endogenous KLRG1 of a human or non-human animal, such as a promoter, 5'UTR and / or 3'UTR. In some embodiments, the non-human animal is a mouse.

[0011] On the one hand, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating a disease. In some embodiments, the method comprises: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the above-mentioned construction method, wherein the non-human animal suffers from a disease; 2) determining the inhibitory effect of the therapeutic agent on the disease. In some embodiments, the therapeutic agent comprises an anti-KLRG1 antibody, and preferably further comprises an additional therapeutic agent (e.g., 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 cancer comprises one or more of melanoma, colon cancer, rectal cancer, pancreatic cancer, glioma, breast cancer, ovarian cancer, solid tumor, hematological tumor, head and neck cancer, liver cancer, or lung cancer. In some embodiments, the immune disease comprises one or more of systemic lupus erythematosus, asthma, rheumatoid arthritis, psoriasis, or multiple sclerosis. In some embodiments, the inflammation comprises inflammatory bowel disease. In some embodiments, the cancer is a tumor, and the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the non-human animal. In some embodiments, the cancer comprises injecting one or more cancer cells into the animal.

[0012] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent, and in some embodiments, the method comprises: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the above construction method; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the determining the effect of the therapeutic agent on the non-human animal comprises measuring the weight of the non-human animal or a blood test. In some embodiments, the blood test comprises one or more of red blood cell count, hematocrit, or hemoglobin content.

[0013] In one aspect, the present invention provides a humanized KLRG1 protein. In some embodiments, the humanized KLRG1 protein comprises all or part of a human KLRG1 protein. In some embodiments, the amino acid sequence of all or part of the human KLRG1 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189. In some embodiments, the humanized KLRG1 protein further comprises all or part of a non-human animal KLRG1 protein. In some embodiments, the humanized KLRG1 protein comprises SEQ ID NO:7; 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:7.

[0014] In one aspect, the present invention provides a humanized KLRG1 gene. In some embodiments, the humanized KLRG1 gene encodes the humanized KLRG1 protein. In some embodiments, the humanized KLRG1 gene comprises a portion of exon 2 to all of exon 5 of the human KLRG1 gene, and preferably further comprises at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the humanized KLRG1 gene comprises the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 16, 17, 18, 19, 20 or NCBI Accession No. NC_000012.12 from 8944044 to 9012751; or comprises SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 16, 17, 18, 19, 20 or NCBI Accession No. NC_000012.12 from 8944044 to 9012751, and the identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

[0015] In one aspect, the present invention provides a cell, tissue or organ. In some embodiments, the cell, tissue or organ expresses the humanized KLRG1 protein; or the genome of the cell, tissue or organ comprises the humanized KLRG1 gene.

[0016] In one aspect, the present invention provides an animal model. In some embodiments, the animal model expresses the humanized KLRG1 protein; or the genome of the animal model contains the humanized KLRG1 gene.

[0017] In one aspect, the present invention provides an application of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the non-human animal cell obtained by the above-mentioned construction method, the above-mentioned humanized KLRG1 protein, the above-mentioned humanized KLRG1 gene, the above-mentioned cell, tissue or organ or the above-mentioned animal model, the application comprising: A) application in the development of products involving immune processes related to KLRG1 in human cells; B) application as a model system related to KLRG1 in pharmacology, immunology, microbiology and medical research; C) application involving the production and use of animal experimental disease models for pathological research related to KLRG1 and / or for the development of diagnostic strategies and / or for the development of treatment strategies; D) application in the screening, efficacy detection, efficacy evaluation, verification or evaluation of human KLRG1 signaling pathway regulators in vivo; or, E) application in studying the function of the KLRG1 gene, studying drugs and efficacy targeting human KLRG1 target sites, and studying drugs for cancer, inflammation or immune diseases related to KLRG1.

[0018] The term "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense, and refers to a DNA fragment on a gene in a narrow sense, which can be a gene or a part of a gene. For example, the "KLRG1 locus" refers to a DNA fragment selected from exons 1 to 5 of the KLRG1 gene. In some embodiments, the replaced endogenous KLRG1 locus of a non-human animal can be a DNA fragment selected from exons 1 to 5 of the endogenous KLRG1 gene of a non-human animal.

[0019] The term "all of exon XX to exon XXX" or "exon XX to exon XXX" or "exon XX-XXX" of the present invention refers to the entire nucleotide sequence including exons and introns in between, for example, exon 1 to exon 5 includes exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4 and exon 5.

[0020] The term "part of exon XX to all of exon XXX" in the present invention refers to the nucleotide sequence of part of exon XX to all of exon XXX and introns in between, for example, part of exon 2 to all of exon 5 includes part of exon 2, all of intron 2, all of exon 3, all of intron 3, all of exon 4, all of introns and all of exon 5.

[0021] The term "intron xx" in the present invention refers to an intron between two exons, for example, intron 2 refers to an intron between exon 2 and exon 3.

[0022] The term "comprising" or "including" in the present invention is an open-ended writing method, which contains the specified components or steps described, as well as other specified components or steps that will not be substantially affected. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed 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.

[0023] The term "and / or" of the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the present application. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0024] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. This application describes methods and materials for use in the present invention; other suitable methods and materials known in the art may be used. The materials, methods, and examples are exemplary and non-limiting only. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0025] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description.

[0026] KLRG1

[0027] In the human genome, the KLRG1 gene (NCBI Gene ID: 10219, UniProt ID: Q96E93, located at positions 8950044 to 9215570 of chromosome 12 NC_000012.12) contains 5 exons, namely exon 1, exon 2, exon 3, exon 4 and exon 5. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_005810.4 and its encoded protein NP_005801.3 (SEQ ID NO: 2) is shown in Table 1.

[0028] Table 1

[0029]

[0030] In the mouse genome, the KLRG1 gene (NCBI Gene ID: 50928, UniProt ID: O88713, located at positions 122247555 to 122259792 of chromosome 6 NC_000072.7) contains 5 exons, namely exon 1, exon 2, exon 3, exon 4 and exon 5. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_016970.1 and its encoded protein NP_058666.1 (SEQ ID NO: 1) is shown in Table 2.

[0031] Table 2

[0032]

[0033] KLRG1 genes, proteins and gene loci of other species are also known in the art, for example, Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog) and Sus scrofa (pig), and the relevant information of these genes (e.g., intron sequences, exon sequences and amino acid sequences) can be found in NCBI, the entire contents of which are incorporated into this application by reference.

[0034] In order to determine the percent identity of two amino acid sequences or two nucleotide sequences, the sequences are compared for the purpose of optimal comparison (e.g., for optimal comparison, gaps can be introduced in one or both of the first and second amino acid or nucleotide sequences, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, which needs to be introduced to achieve the optimal comparison of the two sequences, taking into account the number of gaps and the length of each gap. For example, the comparison of the sequences and the determination of the percent identity between the two sequences can be completed 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.

[0035] The percentage (homology percentage) of conservative residues with similar physicochemical properties, such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage.

[0036] Carrier

[0037] The present invention provides a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of the switch region to be changed (5' arm or 5' homology arm), which is selected from the genomic DNA of the KLRG1 gene and has a length of 100 to 10,000 bp nucleotides; b) a donor region; and c) a DNA fragment homologous to the 3' end of the switch region to be changed (3' arm or 3' homology arm), which is selected from the genomic DNA of the KLRG1 gene and has a length of 100 to 10,000 bp nucleotides.

[0038] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000072.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000072.7.

[0039] In some embodiments, a) a DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 122256611 to 122259610 with NCBI Accession No. NC_000072.7; c) a DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 122244385 to 122247384 with NCBI Accession No. NC_000072.7.

[0040] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 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.

[0041] In some embodiments, the switch region to be altered is located at least 50 bp downstream of exon 1 to 3'UTR of the endogenous KLRG1 gene of a non-human animal, preferably at least 50 bp (eg, at least 170 bp) downstream of exon 2 to 3'UTR of the endogenous KLRG1 gene of a non-human animal.

[0042] In some embodiments, the switch region to be changed is located at least 50 bp upstream of the 5'UTR of the endogenous KLRG1 gene of the non-human animal to all or part of exon 5, preferably at least 50 bp (e.g., at least 5148 bp) upstream of the 5'UTR of the endogenous KLRG1 gene of the non-human animal to all of exon 5.

[0043] In some embodiments, the 5' homology arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 3 or 16; in some embodiments, the 3' homology arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 4 or 17.

[0044] In some embodiments, the donor sequence in the targeting vector comprises a human sequence (eg, nucleotide sequence 8992298 to 9011051 of NCBI accession number NC_000012.12, or nucleotide sequence 8944044 to 9012751 of NC_000012.12).

[0045] In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes), such as a positive screening marker gene or a negative screening marker gene. In some embodiments, the positive screening marker gene is a neomycin phosphotransferase coding sequence Neo. Preferably, the targeting vector further comprises two Frt recombination sites arranged in the same direction on both sides of the marker gene.

[0046] The targeting vector also includes a self-excision cassette, which includes the following elements from 5'-3': a recombination site loxp for a specific recombination system, a tissue-specific promoter, a Cre recombinase, a termination sequence (such as PloyA), a promoter, and a resistance gene (such as Neo) for positive clone screening, a termination sequence, and loxp. During germline transmission, Cre is expressed under the regulation of a specific promoter and is removed from the mouse cell genome together with Neo. 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 KLRG1 gene, and the sgRNA is unique on the target sequence (target site) of the gene to be changed, and satisfies the sequence arrangement rules of 5'-NNN(20)-NGG-3' or 5'-CCN-N(20)-3'. In some embodiments, the target site of the sgRNA in the KLRG1 gene of a non-human animal is located from exon 1 to exon 5. In some embodiments, the target site of the sgRNA in the KLRG1 gene of a non-human animal is located on the 5'UTR upstream nucleotide sequence and / or the 3'UTR downstream nucleotide sequence. In some embodiments, the target site of the sgRNA in the KLRG1 gene of a non-human animal is located on exon 2, and / or, on the 3'UTR downstream nucleotide sequence. In some embodiments, the target site of the sgRNA in the KLRG1 gene of a non-human animal is located on the 5'UTR upstream nucleotide sequence, and / or, on exon 5. In some embodiments, the target site of the sgRNA in the KLRG1 gene of a non-human animal is located on the 5'UTR upstream nucleotide sequence, and / or, on the 3'UTR downstream nucleotide sequence.

[0047] In some embodiments, the present invention also provides a plasmid construct (sgRNA vector, such as pT7-sgRNA) comprising an sgRNA sequence and / or a cell comprising the plasmid construct.

[0048] In some embodiments, the present invention also provides a cell comprising the above-mentioned targeting vector and / or sgRNA vector.

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

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

[0051] In some embodiments, the non-human mammal cell is a mouse cell. In some embodiments, the non-human mammal cell is a fertilized egg cell. In some embodiments, the non-human mammal cell is an embryonic stem cell. In some embodiments, the non-human mammal cell can be any cell capable of expressing KLRG1.

[0052] Genetically modified non-human animals

[0053] The "genetically modified non-human animal" described in the present invention refers to a non-human animal in which at least one chromosome in the genome of the non-human animal has exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal have exogenous DNA. Cells with exogenous DNA can be various cells, for example, somatic cells, immune cells (e.g., 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 KLRG1 locus, the endogenous KLRG1 locus comprising an exogenous sequence (e.g., a human sequence), for example, replacing one or more non-human sequences with one or more human sequences, or inserting one or more human sequences into one or more non-human sequences. Non-human animals are generally able to pass genetic modifications to offspring through germline transmission.

[0054] The "chimeric (x) gene" or "chimeric (x) nucleic acid" of the present invention refers to a gene or nucleic acid, wherein two or more parts of the gene or nucleic acid are from different species, or at least a portion of the sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, a chimeric (x) gene or chimeric (x) nucleic acid has at least a portion of the sequence having two or more different species sources, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, a chimeric (x) gene or chimeric (x) nucleic acid refers to a humanized (x) gene or a humanized (x) nucleic acid.

[0055] The "chimeric (x) protein" or "chimeric (x) polypeptide" of the present invention refers to a protein or polypeptide, wherein two or more parts of the polypeptide or protein are from different species, or at least a portion of the sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of a chimeric (x) protein or a chimeric (x) polypeptide has two or more different species sources, for example, the same (or homologous) proteins of different species. In some embodiments, a chimeric (x) protein or a chimeric (x) polypeptide refers to a humanized (x) protein or a humanized (x) polypeptide.

[0056] The "humanized (x) protein" or "humanized (x) polypeptide" of the present invention refers to a protein or polypeptide. In some embodiments, at least a portion of the protein or polypeptide is derived from a human (x) protein or a human (x) polypeptide. In some embodiments, at least a portion of the protein or polypeptide is derived from a non-human animal (x) protein or a non-human animal (x) polypeptide. In some embodiments, a humanized (x) protein or a humanized (x) polypeptide refers to a human (x) protein or a human (x) polypeptide.

[0057] The "humanized (x) nucleic acid" or "humanized (x) gene" of the present invention refers to a gene or a nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human (x) gene or a human (x) nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a non-human animal (x) gene or a non-human animal (x) nucleic acid. In some embodiments, all of the gene or nucleic acid is derived from a human (x) gene or a human (x) nucleic acid. In some embodiments, a humanized (x) nucleic acid or a humanized (x) gene refers to a humanized exon, which may be a human exon or a chimeric exon.

[0058] In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid is a humanized KLRG1 gene or a humanized KLRG1 nucleic acid. In some embodiments, at least a portion of the humanized KLRG1 gene or the humanized KLRG1 nucleic acid is derived from a human KLRG1 gene. In some embodiments, at least a portion of the humanized KLRG1 gene or the humanized KLRG1 nucleic acid is derived from a non-human animal KLRG1 gene. In some embodiments, the humanized KLRG1 gene or the humanized KLRG1 nucleic acid comprises a sequence encoding a KLRG1 protein. The encoded KLRG1 protein has at least one activity, such as the activity of a human KLRG1 protein and / or a non-human animal KLRG1 protein.

[0059] In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide is a humanized KLRG1 protein or a humanized KLRG1 polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the humanized KLRG1 protein or humanized KLRG1 polypeptide are from a human KLRG1 protein. In some embodiments, at least one or more portions of the amino acid sequence of the humanized KLRG1 protein or humanized KLRG1 polypeptide are from a non-human animal KLRG1 protein. The humanized KLRG1 protein or humanized KLRG1 polypeptide is functional, or has at least one activity, such as an activity of a human KLRG1 protein and / or a non-human animal KLRG1 protein.

[0060] In some embodiments, the humanized KLRG1 protein comprises an extracellular region, a transmembrane region and / or a cytoplasmic region. In some embodiments, the extracellular region is human or humanized. In some embodiments, the cytoplasmic region is endogenous to a human or non-human animal or humanized. In some embodiments, the transmembrane region is endogenous to a human or non-human animal or humanized. In some embodiments, the humanized KLRG1 protein comprises or does not comprise a signal peptide. In some embodiments, the signal peptide is human or humanized.

[0061] In some embodiments, the genetically modified non-human animal comprises a modification of an endogenous KLRG1 gene site in a non-human animal. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature KLRG1 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a mature KLRG1 protein amino acid sequence). Although cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein are provided in the present invention, in some embodiments, the genetically modified non-human animal comprises a modification of an endogenous KLRG1 gene site in a non-human animal.

[0062] The genetically modified non-human animal can express human KLRG1 and / or chimeric (e.g., humanized) KLRG1 at the endogenous locus of the non-human animal, wherein the endogenous KLRG1 gene of the non-human animal has been replaced or inserted with a nucleotide sequence of a human KLRG1 gene and / or a nucleotide sequence encoding a human KLRG1 sequence region or a nucleotide sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99% identical to the human KLRG1 sequence. In some embodiments, the endogenous KLRG1 locus of the non-human animal is modified by a nucleic acid sequence that comprises all or part of the nucleic acid sequence encoding the mature human KLRG1 protein.

[0063] In some embodiments, the genetically modified non-human animal can express human KLRG1 and / or chimeric KLRG1 (e.g., humanized KLRG1) under the control of regulatory elements (e.g., human or non-human animal endogenous regulatory elements, such as promoters, 5'UTRs, and / or 3'UTRs). Insertion or substitution at the endogenous KLRG1 locus of the non-human animal provides a non-human animal that expresses human KLRG1 or chimeric KLRG1 (e.g., humanized KLRG1) in suitable cells and in a manner that does not cause potential pathologies observed in some other transgenic non-human animals known in the art. Human KLRG1 or chimeric KLRG1 (e.g., humanized KLRG1) expressed in the non-human animal can maintain one or more functions of wild-type non-human animals or human KLRG1 in the non-human animal. In addition, in some embodiments, the non-human animal does not express endogenous KLRG1. In some embodiments, the endogenous KLRG1 expression level of the non-human animal is reduced compared to the KLRG1 expression level in the wild-type animal. As used herein, the term "endogenous KLRG1" refers to the KLRG1 protein expressed by the endogenous KLRG1 nucleotide sequence of a non-human animal (eg, mouse) before any genetic modification.

[0064] The genetically modified non-human animal may have one or more cells expressing human or chimeric KLRG1 (e.g., humanized KLRG1), wherein the KLRG1 has a cytoplasmic region, a transmembrane region, and an extracellular region. In some embodiments, the KLRG1 comprises or does not comprise a signal peptide. In many cases, the sequences of human KLRG1 and non-human animal KLRG1 (e.g., mouse KLRG1) are different, so antibodies that bind to human KLRG1 do not necessarily have the same affinity as non-human animal KLRG1 or have the same effect on non-human animal KLRG1. Therefore, genetically modified non-human animals having a human or humanized extracellular region (preferably also comprising a human or humanized transmembrane region) can be used to better evaluate the effects of anti-human KLRG1 antibodies in animal models.

[0065] In some embodiments, the entire transmembrane region and the entire cytoplasmic region of the humanized KLRG1 are derived from the endogenous KLRG1 sequence of a non-human animal. In some embodiments, the extracellular region of the humanized KLRG1 described in the present application is a humanized extracellular region, comprising a human KLRG1 sequence and an endogenous KLRG1 sequence of a non-human animal.

[0066] In some embodiments, the humanized KLRG1 gene comprises a human 5'UTR. In some embodiments, the humanized KLRG1 gene comprises a 5'UTR endogenous to a non-human animal (e.g., a mouse). In some embodiments, the humanized KLRG1 gene comprises a human 3'UTR. In some embodiments, the humanized KLRG1 gene comprises a 3'UTR endogenous to a non-human animal (e.g., a mouse). Where appropriate, it is reasonable to assume that, based on the similarity of the non-human animal and human KLRG1 gene sequences, they appear to be similarly regulated. As shown in the present application, a humanized KLRG1 comprising an insertion or replacement in the endogenous KLRG1 locus of a non-human animal, comprising a humanization of the KLRG1 coding sequence, does not exhibit pathological phenomena. Both genetically modified non-human animals that are heterozygous or homozygous for humanized KLRG1 are normal.

[0067] The non-human animal of genetic modification can be various animals, for example, mouse, rat, rabbit, pig, cattle (for example, cattle, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (for example, marmoset, rhesus monkey).For the non-human animal of suitable genetically modified embryonic stem cell (ES) that is not easy to obtain, adopt alternative methods to construct the non-human animal comprising genetic modification.Such method includes, for example, modifying non-ES cell genome (for example, fibroblast or induced pluripotent stem cell) and adopting nuclear transplantation to transfer the modified genome to suitable cell, for example oocyte, and incubating the modified cell (for example, modified oocyte) in non-human animal under appropriate conditions to form embryo.The above-mentioned construction method is known in the art, and is described in "A.Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition), " Cold Spring Harbor Laboratory Press, 2006 ", the entire contents of which are incorporated into the present application by reference.

[0068] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. Rodents can be selected from mice, rats and hamsters. In one embodiment, the rodent is selected from the family of Muridae. In one embodiment, the genetically modified non-human animal is from a family selected from the family of Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (mice and rats, gerbils, spiny mice, crested rats), Isla Muscicapidae (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Spiny Dormouse (e.g., spiny dormouse) and Muridae (e.g., mole rats, bamboo rats and zokors). In a specific 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 specific embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0069] In some embodiments, the non-human animal can be an immunodeficient non-human mammal. For example, an immunodeficient rodent, an immunodeficient rabbit, an immunodeficient pig, an immunodeficient monkey, etc. In some embodiments, the animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described in, for example, Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse is a hybrid of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid strain (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a BALB / c, BALB / cHeAn, BALB / cJ, BALB / cR1, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st or CBA / H strain.

[0070] In some embodiments, the non-human animal is a rat. The rat can be selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, 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.

[0071] The non-human animal may have one or more other genetic modifications and / or other modifications that are suitable for the specific purpose of preparing a humanized non-human animal. For example, a suitable mouse for maintaining a xenograft (e.g., a human cancer or tumor) may have one or more modifications that damage, inactivate, or destroy all or part of the immune system of the non-human animal. Damage, inactivation, or destruction of the immune system of a non-human animal may include, for example, chemical means (e.g., administration of toxins), physical means (e.g., irradiation of the animal), and / or genetic modification (e.g., knocking out one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γc null Mice (Ito, M. et al., NOD / SCID / γc null mouse:an excellent recipient mouse model for engraftment of human cells, Blood 100(9):3175-3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice can be optionally irradiated or otherwise treated to destroy one or more immune cell types. Therefore, in various embodiments, a genetically modified mouse is provided, which can include humanization of at least a portion of the endogenous KLRG1 locus of a non-human animal, and also includes damage, inactivation or partial destruction of the immune system (or one or more cell types of the immune system) of the non-human animal. In some embodiments, the mouse modification type 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 Mouse, NOD Rag 1 - / - IL - / - (NRG) mouse, Rag2 - / - IL - / -(RG) Modification of mice and combinations thereof In some embodiments, the mice may include replacing all or part of the mouse endogenous mature KLRG1 coding sequence with all or part of the human mature KLRG1 coding sequence, respectively.

[0072] The present invention further relates to a non-human mammal or its offspring produced by the above method. In some embodiments, the genome of the non-human mammal or its offspring comprises human genes.

[0073] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0074] In some embodiments, the non-human mammal expresses a protein encoded by a humanized KLRG1 gene.

[0075] In addition, the present invention also provides a non-human mammal model carrying a tumor, wherein the non-human mammal model is obtained by the method described in the present application. In some embodiments, the non-human mammal is a rodent (eg, mouse).

[0076] The present invention also provides a cell or cell line derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a primary cell culture, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a tissue, organ, or culture thereof derived from a non-human mammal or its offspring. When it carries 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.

[0077] The present invention provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal or a genetically modified non-human animal is provided, wherein the genome of the genetically modified non-human animal comprises DNA of human or humanized KLRG1.

[0078] In some embodiments, a non-human mammal comprises a gene construct described herein. In some embodiments, a non-human mammal expressing a human or humanized KLRG1 protein is provided. In some embodiments, a cell, tissue or organ specifically expressing a human or humanized KLRG1 protein is provided.

[0079] In some embodiments, the expression of the human or humanized KLRG1 protein expressed by the genetically modified non-human animal or the portion of the human KLRG1 gene or the humanized KLRG1 gene contained in the genome of the genetically modified 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).

[0080] The non-human mammal can be any non-human animal known in the art that can be used in the methods described herein. Preferred non-human mammals are mammals (eg, rodents). In some embodiments, the non-human mammal is a mouse.

[0081] The non-human mammals described above are subjected to genetic, molecular and behavioral analyses. The present invention provides offspring produced by mating with non-human mammals of the same genotype or other genotypes.

[0082] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its progeny. For example, a cell culture-based model can be prepared by the following method. The cell culture can be obtained by isolation from a non-human mammal, or cells can be obtained from a cell culture established using the same construct and standard cell transfection techniques. The integration of a genetic construct comprising a DNA sequence encoding a human KLRG1 protein can be detected by a variety of methods.

[0083] There are many analytical methods that can be used to detect exogenous DNA, including nucleic acid level methods (including the use of reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein level methods (including histochemical analysis, 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 achieve quantitative detection. For example, RT-PCR and hybridization methods can be used to detect transcription levels, including RNase protection assays, Southern Blots, and RNA dot hybridization assays (RNAdot). Immunohistochemical staining, flow cytometry, and Western blots can also be used to detect the presence of human or humanized KLRG1 protein.

[0084] In some embodiments, the genetically modified non-human animals described herein (eg, homozygous or heterozygous mice with humanized KLRG1 gene) can express human or humanized KLRG1 in one or more cells.

[0085] Methods for constructing genetically modified non-human animals

[0086] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting technology using embryonic stem cells, homologous recombination technology, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology technology. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR / Cas9 gene editing technology is used to produce genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399. " by Yin et al., which is incorporated into this application 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.

[0087] In some embodiments, the nucleotide sequence encoding the endogenous KLRG1 region of the non-human animal in the endogenous genome of at least one cell of the non-human animal is replaced by the nucleotide sequence encoding the corresponding region of human KLRG1 to form a humanized KLRG1 gene, and / or, the obtained non-human animal expresses human or humanized KLRG1 protein. In some embodiments, the expression level of the endogenous KLRG1 protein of the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0088] Preferably, the non-human animal further comprises other gene modifications, more preferably, the other genes include at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0089] Preferably, the human or humanized KLRG1 gene and / or other genes are homozygous for the endogenous modified (preferably replaced or inserted) locus.

[0090] Preferably, the human or humanized KLRG1 gene and / or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) locus.

[0091] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, pigs, rabbits, monkeys, etc.

[0092] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0093] Therefore, the present invention provides a method for constructing a non-human animal with a humanized KLRG1 gene, wherein the non-human animal expresses human or humanized KLRG1 protein in vivo, and / or the genome of the non-human animal contains a portion of the human KLRG1 gene or a humanized KLRG1 gene.

[0094] In some embodiments, the construction method allows the non-human animal to express human or humanized KLRG1 protein in vivo, and / or, the construction method allows the genome of the non-human animal to contain a portion of the human KLRG1 gene or a humanized KLRG1 gene.

[0095] In some embodiments, the method of modifying the KLRG1 locus of a non-human animal to express a chimeric human / non-human animal chimeric KLRG1 polypeptide may include replacing a nucleotide sequence encoding a non-human animal KLRG1 at an endogenous KLRG1 locus of the non-human animal with a nucleotide sequence encoding human KLRG1, thereby generating a chimeric human / non-human animal chimeric KLRG1 sequence. In some embodiments, the method may include inserting a nucleotide sequence encoding a chimeric human / non-human animal chimeric KLRG1 at an endogenous KLRG1 locus of the non-human animal, thereby generating a chimeric human / non-human animal chimeric KLRG1 sequence.

[0096] The present invention provides a targeting vector, which comprises a 5' homology arm, a human or humanized KLRG1 gene fragment and a 3' homology arm. The process involves using homologous recombination technology to replace the corresponding sequence of endogenous KLRG1 of non-human animals with the human or humanized KLRG1 gene fragment. In some embodiments, cutting upstream and downstream of the target site (for example, by zinc finger nuclease, TALEN or CRISPR) can cause DNA double-strand breaks, and homologous recombination is used to replace the corresponding sequence of endogenous KLRG1 of non-human animals with the human or humanized KLRG1 gene fragment.

[0097] Therefore, in some embodiments, the method for preparing a genetically modified humanized non-human animal comprises replacing a nucleotide sequence encoding a non-human animal endogenous KLRG1 region with a nucleotide sequence of a corresponding region of human KLRG1 at the non-human animal endogenous KLRG1 locus (or site).

[0098] The present invention also provides a method for establishing a KLRG1 gene humanized animal model, comprising the following steps:

[0099] (a) providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0100] (b) culturing the cells, preferably culturing the cells in a liquid culture medium;

[0101] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal;

[0102] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0103] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0104] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0105] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0106] The fertilized egg can be from any non-human animal, such as any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human animal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0107] In some embodiments, the method for preparing a genetically modified animal includes modifying the coding frame of the KLRG1 gene of a non-human animal, for example, by replacing the nucleotide sequence encoding the endogenous KLRG1 region of the non-human animal with a nucleotide sequence encoding the corresponding region of human KLRG1 (e.g., a genomic DNA sequence, a CDS sequence, or a cDNA sequence) under the control of an endogenous regulatory element of a human or non-human animal. For example, one or more functional region sequences of the KLRG1 gene of a non-human animal can be knocked out or a sequence can be inserted so that the endogenous KLRG1 protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the modified non-human animal KLRG1 gene can be all or part of the nucleotide sequence of exon 1 to exon 5 of the KLRG1 gene of a non-human animal, preferably part of exon 2 to all of exon 5, or, all of exon 1 to exon 5, or, the nucleotide sequence of the start codon to the stop codon.

[0108] In some embodiments, the method for preparing a genetically modified non-human animal comprises inserting a nucleotide sequence encoding a human or humanized KLRG1 protein and / or an auxiliary sequence after an endogenous regulatory element of a human or non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, so that the KLRG1 gene humanized animal model can express a human or humanized KLRG1 protein in vivo, but does not express the KLRG1 protein of a non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0109] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0110] (1) providing a plasmid comprising a human KLRG1 gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target endogenous KLRG1 of a non-human animal;

[0111] (2) providing one or more guide RNAs (sgRNAs) targeting endogenous KLRG1 genes of non-human animals;

[0112] (3) modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2) and Cas9;

[0113] (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express the humanized KLRG1 protein. Preferably, the method further comprises:

[0114] (5) The offspring mice obtained in step (4) are mated to obtain homozygous mice.

[0115] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal targeting site and a 3'-terminal targeting site.

[0116] In some embodiments, the sequence encoding the humanized KLRG1 protein is operably linked to human regulatory elements or non-human animal endogenous regulatory elements.

[0117] In some embodiments, the genetically modified non-human animal does not express endogenous KLRG1 protein.

[0118] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0119] (1) providing a plasmid comprising a human or chimeric KLRG1 gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target endogenous KLRG1 of a non-human animal;

[0120] (2) providing one or more guide RNAs (sgRNAs) targeting endogenous KLRG1 genes of non-human animals;

[0121] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric KLRG1 gene fragment into the genome.

[0122] In some embodiments, all or part of the nucleotide sequence encoding the endogenous KLRG1 protein of the non-human animal is deleted. In some embodiments, all or part of exon 1 to exon 5 of the endogenous KLRG1 gene of the non-human animal is deleted.

[0123] In some embodiments, a portion of exon 2 to all of exon 5 of the endogenous KLRG1 gene of a non-human animal is deleted. In some embodiments, the deleted portion further comprises at least 50 bp of nucleotide sequence downstream of the 3'UTR of the endogenous KLRG1 gene of the non-human animal. In some embodiments, a portion of exon 2 to at least 50 bp of nucleotide sequence downstream of the 3'UTR of the endogenous KLRG1 gene of the non-human animal is deleted (e.g., a 170 bp nucleotide sequence). In some embodiments, the deleted portion further comprises intron 2, intron 3, and / or intron 4 of the endogenous KLRG1 gene of the non-human animal.

[0124] In some embodiments, all of exon 1 to exon 5 of the endogenous KLRG1 gene of a non-human animal are deleted. In some embodiments, the deleted portion further comprises at least 50 bp of nucleotide sequence (e.g., 5148 bp of nucleotide sequence) upstream of the 5'UTR of the endogenous KLRG1 gene of a non-human animal. In some embodiments, all of the nucleotide sequence from at least 50 bp upstream of the 5'UTR of the endogenous KLRG1 gene of a non-human animal to exon 5 is deleted. In some embodiments, the deleted portion further comprises intron 1, intron 2, intron 3, and / or intron 4 of the endogenous KLRG1 gene of a non-human animal.

[0125] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the endogenous KLRG1 protein in the genome of a non-human animal with the nucleotide sequence encoding the human KLRG1 protein. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or SEQ ID NO: 1 at positions 59-188 in the genome of a non-human animal with the nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 2 at positions 59-189 or SEQ ID NO: 2 at positions 60-189. In some embodiments, the construction method comprises replacing the part of exon 2 to the whole of exon 5 of the endogenous KLRG1 gene of a non-human animal with the part of exon 2 to the whole of exon 5 of the human KLRG1 gene. In some embodiments, the construction method comprises replacing the part of exon 2 to the whole of exon 5 of the endogenous KLRG1 gene of a non-human animal with the part of exon 2 to the 3'UTR downstream of at least 50 bp of nucleotide sequence. In some embodiments, the construction method comprises replacing a portion of exon 2 of the human KLRG1 gene to a 300bp nucleotide sequence downstream of the 3'UTR of the non-human animal endogenous KLRG1 gene with a portion of exon 2 of the human KLRG1 gene to a 300bp nucleotide sequence downstream of the 3'UTR. In some embodiments, the construction method comprises replacing a nucleotide sequence encoding SEQ ID NO: 1 or SEQ ID NO: 1 at positions 59-188 in the genome of the non-human animal with SEQ ID NO: 5 or 6. In some embodiments, the construction method comprises replacing all of exon 1 to exon 5 of the non-human animal endogenous KLRG1 gene with all of exon 1 to exon 5 of the human KLRG1 gene. In some embodiments, the construction method comprises replacing a nucleotide sequence from the start codon to the stop codon of the non-human animal endogenous KLRG1 gene with a nucleotide sequence from the start codon to the stop codon of the human KLRG1 gene. In some embodiments, the construction method comprises replacing the entirety of the nucleotide sequence from at least 50 bp upstream of the 5'UTR to exon 5 of the endogenous KLRG1 gene of a non-human animal with the nucleotide sequence from at least 50 bp upstream of the 5'UTR to at least 50 bp downstream of the 3'UTR of the human KLRG1 gene. In some embodiments, the construction method comprises replacing the entirety of the nucleotide sequence from 5148 bp upstream of the 5'UTR to exon 5 of the endogenous KLRG1 gene of a non-human animal with the nucleotide sequence from 45502 bp upstream of the 5'UTR to 2000 bp downstream of the 3'UTR of the human KLRG1 gene.In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or SEQ ID NO: 1 at positions 59-188 in the genome of a non-human animal with the nucleotide sequence of positions 8944044 to 9012751 of SEQ ID NO: 5, 6, 20 or NCBI accession number NC_000012.12. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or SEQ ID NO: 1 at positions 59-188 in the genome of a non-human animal with the nucleotide sequence encoding human or humanized KLRG1 protein or the humanized KLRG1 gene.

[0126] Use of genetically modified non-human animals

[0127] Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence or inserting a homologous or orthologous human gene or human sequence into a non-human animal at a non-human animal endogenous locus and under the control of human or non-human animal endogenous regulatory elements (e.g., promoter, 5'UTR and / or 3'UTR) can produce a non-human animal with qualities and characteristics that may be significantly different from typical knockout plus transgenic animals. In typical knockout plus transgenic animals, the endogenous locus is removed or destroyed, and the full human transgene is inserted into the genome of the animal and may be randomly integrated into the genome. Typically, the location of the integrated transgene is unknown; the expression of human proteins is measured by transcription of human gene and / or protein assays and / or functional assays. In human transgenes, the upstream and / or downstream of the human sequence provide suitable support for the expression and / or regulation of the transgene.

[0128] Genetically modified non-human animals that express human or humanized KLRG1 protein, e.g., in a physiologically appropriate manner, provide a variety of uses, including but not limited to developing treatments for human diseases and disorders, and evaluating the toxicity and / or efficacy of these human treatments in animal models.

[0129] The present invention also provides an application of the above-mentioned KLRG1 gene-modified non-human animal or a non-human animal obtained by any of the above-mentioned construction methods. In some embodiments, the application comprises:

[0130] A) Application in product development involving KLRG1-related immune processes in human cells;

[0131] B) Application as a model system related to KLRG1 for pharmacology, immunology, microbiology and medical research;

[0132] C) Applications involving the production and use of animal experimental disease models for the study of KLRG1-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0133] D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human KLRG1 signaling pathway modulators; or

[0134] E) To study the function of KLRG1 gene, the drugs and efficacy targeting human KLRG1 target sites, and the application of KLRG1-related drugs in cancer, inflammation or immune diseases.

[0135] The present invention provides a non-human animal expressing human or humanized KLRG1 protein, which can be used for screening of human KLRG1-specific therapeutic agents. The therapeutic agent can reduce or block the interaction between KLRG1 and the KLRG1 receptor complex, test whether the therapeutic agent can increase or decrease the immune response, and / or determine whether the therapeutic agent is a KLRG1 agonist or antagonist. In some embodiments, the non-human animal is an animal model of human disease. For example, the disease is genetically induced (knock-in or knock-out). In different embodiments, the genetically modified non-human animal also comprises an impaired immune system, such as a genetically modified human-derived tissue xenograft, including a human solid tumor (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor).

[0136] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-KLRG1 antibodies) in treating various immune diseases. In some embodiments, the immune diseases include, but are not limited to, GVHD (graft versus host disease), psoriasis, allergies, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty liver disease), systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, 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 systemic lupus erythematosus, asthma, rheumatoid arthritis, psoriasis or multiple sclerosis.

[0137] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-KLRG1 antibodies; or drugs targeting the KLRG1 signaling pathway) in treating tumors. In some embodiments, the method involves administering a therapeutic agent to an animal as described herein, wherein the non-human animal suffers from cancer or a tumor; and determining the inhibitory effect of the therapeutic agent on the cancer or tumor. Inhibitory effects that can be determined include, for example, a reduction in tumor size or tumor volume, a reduction in tumor growth, a reduction in the rate of increase of tumor volume in a subject (e.g., compared to the rate of increase of tumor volume in the same subject before treatment or in another subject that has not received such treatment), a reduction in the risk of metastasis or the risk of one or more additional metastases, an increase in survival rate and an increase in life expectancy, etc. The tumor volume of a subject can be determined by various methods, such as by direct measurement, MRI, or CT.

[0138] In some embodiments, the tumor comprises one or more cancer cells (e.g., the cancer cells are derived from a human or non-human animal) injected into an animal. In some embodiments, the therapeutic agent activates or inhibits the KLRG1 signaling pathway. In some embodiments, the therapeutic agent does not activate or inhibit the KLRG1 signaling pathway.

[0139] In some embodiments, the genetically modified animals can be used to determine whether an antibody is a KLRG1 agonist or antagonist. In some embodiments, the methods described herein are also designed to determine the effect of a therapeutic agent (e.g., an antibody targeting KLRG1; or a drug targeting the KLRG1 signaling pathway) on KLRG1, for example, whether the therapeutic agent can upregulate an immune response or downregulate an immune response, and / or whether the agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent for treating a disease (e.g., cancer, inflammation, or immune disease) in a subject.

[0140] The inhibitory effect on tumors can also be determined by methods known in the art, for example, by measuring tumor volume in animals, and / or determining the tumor (volume) inhibition rate (TGI). TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%)=(1-TV t / TVc)×100, where TV t and TVc are the mean tumor volumes (or weights) of the treatment and control groups.

[0141] In some embodiments, therapeutic agents (e.g., antibodies targeting KLRG1; or drugs targeting the KLRG1 signaling pathway) are designed to treat various cancers. As used in this application, the term "cancer" refers to cells with autonomous growth capacity, that is, an abnormal state or state characterized by rapid proliferating cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasive stage. The term "tumor" used in this application refers to cancer cells, such as cancer cell clusters. Cancers that can be treated or diagnosed using the methods described in this application include malignant tumors of various organ systems, such as malignant tumors affecting the lungs, breasts, thyroid, lymph nodes, gastrointestinal and genital urinary tracts, and adenocarcinomas, including malignant tumors such as colon cancer, renal cell carcinoma, prostate cancer, testicular tumors, or non-small cell lung cancer. In some embodiments, the therapeutic agents described in this application are designed to treat or diagnose cancer in a subject. The term "cancer" is generally recognized and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, urogenital cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include cancers formed by cervical, lung, prostate, breast, head and neck, colon, and ovarian tissue. The term also includes carcinosarcoma, for example, including malignant tumors composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or tumor cells forming a recognizable glandular structure. The term "sarcoma" is generally recognized and refers to a malignant tumor of mesenchymal origin.

[0142] In some embodiments, the cancer described herein is lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, cancer, bladder cancer, glioma, cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome and sarcoma. In some embodiments, the leukemia is selected from acute lymphocytic (lymphocytic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia and chronic myelogenous leukemia. In certain embodiments, lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma and T cell lymphoma, and Waldenstrom macroglobulinemia. In certain embodiments, sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma and chondrosarcoma. In a specific embodiment, tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer or lung cancer. In certain embodiments, the cancer is melanoma, colon cancer, rectal cancer, pancreatic cancer, glioma, breast cancer, ovarian cancer, solid tumor, hematologic tumor, head and neck cancer, liver cancer or lung cancer.

[0143] In some embodiments, therapeutic agents (e.g., antibodies targeting KLRG1; or drugs targeting the KLRG1 signaling pathway) are designed to treat various immune diseases including rheumatoid arthritis, multiple sclerosis, Crohn's disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease (IBD), ulcerative colitis, or scleroderma. Therefore, the methods described herein can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting KLRG1; or drugs targeting the KLRG1 signaling pathway) in suppressing immune responses. In some embodiments, the immune diseases described herein are graft-versus-host disease (GVHD), psoriasis, allergies, multiple sclerosis, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological diseases, etc.

[0144] In some embodiments, therapeutic agents (e.g., antibodies targeting KLRG1; or drugs targeting the KLRG1 signaling pathway) are designed to treat various inflammations, such as viral inflammation. In some embodiments, the inflammation described herein includes acute inflammation and chronic inflammation. Specifically, inflammation includes, but is not limited to, degenerative inflammation, exudative inflammation (e.g., serous inflammation, fibrin inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (e.g., tuberculosis, syphilis, leprosy, or lymphogranuloma). In some embodiments, the inflammation described herein includes infection, and infection refers to local tissue and systemic inflammatory responses caused by bacteria, viruses, fungi, parasites, and / or other pathogens that invade the human body. In some embodiments, inflammation is inflammatory bowel disease (IBD).

[0145] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (e.g., an anti-KLRG1 antibody). The method comprises administering a therapeutic agent to the non-human animal described above or the non-human animal obtained by the above-mentioned construction method, and evaluating the weight change or blood test of the non-human animal. In some embodiments, the blood test includes one or more of red blood cell count, hematocrit, or hemoglobin content. In some embodiments, the therapeutic agent can reduce red blood cells (RBC), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the body weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% less than that of the control group (e.g., the average body weight of non-human animals not treated with antibodies).

[0146] The present invention also provides an animal model constructed by the method described in the present application for developing products related to human cellular immune processes, producing human antibodies, or a model system for pharmacology, immunology, microbiology and medical research.

[0147] In some embodiments, an animal model generated by the methods described herein is provided for producing and utilizing animal experimental disease models of immune processes of human cells, studying pathogens, or developing new diagnostic strategies and / or therapeutic strategies.

[0148] The present invention also provides an animal model generated by the method described in the present application to screen, verify, evaluate or study KLRG1 gene function, human KLRG1 antibodies, drugs or effectiveness of human KLRG1 target sites, drugs for immune diseases and anti-tumor drugs.

[0149] In some embodiments, the present application provides a method for verifying the in vivo efficacy of TCR-T, CAR-T and / or other immunotherapies (e.g., T cell adoptive transfer therapy). For example, the method includes transplanting human tumor cells into non-human animals described in the present application, and applying human CAR-T to non-human animals with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animal is selected from a KLRG1 gene humanized non-human animal prepared by the method described in the present application, a KLRG1 gene humanized non-human animal described in the present application, a double or multiple humanized non-human animal (or its offspring) produced by the method described in the present application, a non-human animal expressing a human or humanized KLRG1 protein, or a tumor-bearing or inflammatory animal model described in the present application. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat KLRG1-related diseases described in the present application. In some embodiments, TCA-T, CAR-T and / or other immunotherapies provide an evaluation method for treating KLRG1-related diseases described in the present application.

[0150] Non-human animal models with two or more human or chimeric genes

[0151] The present invention also provides a transgenic animal model or non-human animal having two or more human or chimeric genes. The non-human animal or animal model may contain a human or chimeric KLRG1 gene and a sequence encoding an additional human or chimeric protein.

[0152] In some embodiments, the additional human or chimeric protein comprises at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the non-human animal also expresses at least one of human or humanized ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 protein.

[0153] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising:

[0154] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0155] (ii) mating, in vitro fertilization, or direct gene editing of the non-human animal provided in step (i) with other genetically modified non-human animals, and screening to obtain two or more genetically modified non-human animals.

[0156] In some embodiments, the other genetically modified non-human animals include non-human animals humanized with one or a combination of two or more of the genes ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0157] In some embodiments, KLRG1 gene humanization is performed directly on a non-human animal with at least one genetic modification of human or chimeric ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0158] Since these proteins may involve different mechanisms, a combination therapy targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are underway and show 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, anti-KLRG1 antibodies, and additional therapeutic agents for treating cancer or immune diseases (e.g., asthma or atopic dermatitis). The method includes administering an anti-KLRG1 antibody and an additional therapeutic agent to an animal, wherein the animal has a tumor or immune disease, and determining the effect of the combined therapy on the immune tumor or immune disease. In some embodiments, the additional therapeutic agent includes an antibody that specifically binds to ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and / or CTLA4. In some embodiments, the additional therapeutic agent includes an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., nivolumab) or an anti-PD-L1 antibody. In some embodiments, the non-human animal described above further comprises a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the tumor described above comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0159] In some embodiments, the combination therapy is used to treat various cancers described herein, for example, breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer or lung cancer. In some embodiments, the combination therapy is designed to treat immune diseases described herein, such as psoriasis.

[0160] In some embodiments, the method described in the present application can be used to evaluate the combined treatment with some other methods. The method for treating cancer that can be used alone or in combination with the method described in the present application includes, for example, treating the subject with chemotherapy, for example, camphor alkali, doxorubicin, cisplatin, carboplatin, procarbazine, methylclorazepam, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chlorbutiazine, bithiofen, nitrosourea, dacrynic acid, daunorubicin, bleomycin, plimycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatin, 5-fluorouric acid, vincristine, vinblastine and / or methotrexate. Alternatively, in addition, the method can include surgery on the subject to remove at least a portion of the cancer, for example, removing a portion or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0161] Figure 1 : Schematic diagram comparing the mouse KLRG1 locus and the human KLRG1 locus (not to scale);

[0162] Figure 2 : Schematic diagram of KLRG1 gene targeting strategy and targeting vector design (not to scale);

[0163] Figure 3 : PCR identification results of the F1 generation of KLRG1 humanized mice constructed in Example 1, wherein WT is the wild-type control, M is the marker, and H2O is the water control;

[0164] Figure 4 : PCR identification results of the F1 generation of KLRG1 gene humanized mice constructed in Example 2, wherein WT is the wild-type control, M is the marker, and H2O is the water control. DETAILED DESCRIPTION

[0165] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0166] Materials and methods

[0167] In each of the following examples, equipment and materials were obtained from the following companies:

[0168] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center of the China Food and Drug Administration;

[0169] KLRG1 Monoclonal Antibody(13A2),PE,eBioscience TM Purchased from Thermo, product number: 12-9487-42.

[0170] Example 1 KLRG1 gene humanized mice (I)

[0171] A comparison diagram of the mouse KLRG1 gene (NCBI Gene ID: 50928, located at positions 122247555 to 122259792 of chromosome 6 NC_000072.7, based on transcript NM_016970.1 and its encoded protein NP_058666.1 (SEQ ID NO: 1)) and the human KLRG1 gene (NCBI Gene ID: 10219, located at positions 8950044 to 9215570 of chromosome 12 NC_000012.12, based on transcript NM_005810.4 and its encoded protein NP_005801.3 (SEQ ID NO: 2)) is shown in FIG. Figure 1 shown.

[0172] In order to achieve the purpose of the present invention, a nucleotide sequence encoding a human KLRG1 protein can be introduced into the mouse endogenous KLRG1 locus, so that the mouse genome contains a humanized KLRG1 gene, and / or the mouse expresses a human or humanized KLRG1 protein. Specifically, using gene editing technology, under the control of the mouse KLRG1 gene regulatory element, a portion of exon 2 of the mouse KLRG1 gene to a nucleotide sequence downstream of about 18.8 kb from 3'UTR is used to replace a portion of exon 2 of the mouse KLRG1 gene to a nucleotide sequence downstream of about 9.2 kb from 3'UTR, to obtain a humanized KLRG1 locus, thereby achieving humanization of the mouse KLRG1 gene.

[0173] In order to realize the targeting strategy of the present invention, a targeting vector was constructed. Figure 2) contains upstream and downstream homology arm sequences of the mouse KLRG1 gene, and an A fragment containing a human KLRG1 gene fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence of positions 122256611 to 122259610 of NCBI accession number NC_000072.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence of positions 122244385 to 122247384 of NCBI accession number NC_000072.7. The nucleotide sequence of the human KLRG1 gene fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 8992298 to 9011051 of NCBI accession number NC_000012.12; the connection design of the upstream of the human KLRG1 fragment sequence and the mouse is:

[0175] The sequence " AACGG The last "G" in the sequence is the last nucleotide in the mouse. The "A" in is the first nucleotide of the human sequence. The downstream connection of the human KLRG1 fragment sequence and the mouse sequence is designed as:

[0177] The sequence " TGTGG The last "G" in the sequence is the last nucleotide of the human sequence. The "A" in is the first nucleotide of the mouse sequence.

[0178] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the human KLRG1 gene is designed as follows:

[0180] The sequence " ATCAA The last "A" in " is the last nucleotide of the human KLRG1 gene, the sequence The "G" in is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the human KLRG1 gene is designed as:

[0182] The sequence " AGCCC The last "C" in the sequence is the last nucleotide of the Neo box. The first "G" in is the first nucleotide of the human KLRG1 gene. The mRNA sequence transcribed from the modified humanized KLRG1 gene is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 7.

[0183] The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified to be correct by sequencing is electroporated and transfected into the embryonic stem cells of C57BL / 6 mice, and the obtained cells are screened using the positive clone screening marker gene to screen the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to the technology known in the art, and the obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white) can be produced. F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. Positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene, and then mated with each other to obtain KLRG1 gene humanized homozygous mice.

[0184] The PCR method can be used to identify the somatic cell genotype of F1 generation mice, and the primers are shown in Table 3. The identification results of exemplary F1 generation mice are shown in Figure 3 A-3B, 7 mice numbered F1-01 to F1-07 were all positive.

[0185] Table 3 Primer sequences and recombinant fragment sizes for PCR detection of F1 genotypes

[0186]

[0187] The expression of humanized KLRG1 protein (chiKLRG1) in mice was confirmed by flow cytometry. Specifically, 6-week-old female wild-type C57BL / 6 mice and 6-week-old female KLRG1 gene heterozygous mice were selected, and spleen tissue was obtained after euthanasia by cervical dislocation. Specific anti-human antibody KLRG1 Monoclonal Antibody (13A2), PE, eBioscience was used. TM The results showed that humanized KLRG1 protein expression was not detected in the spleen of C57BL / 6 mice, and 18.2% chiKLRG1 positive cells (characterized by CD45+ KLRG1 + ), indicating that the humanized KLRG1 protein can be normally expressed in the transformed KLRG1 gene heterozygous mice.

[0188] Example 2 KLRG1 gene humanized mice (II)

[0189] In order to achieve the purpose of the present invention, a nucleotide sequence encoding a human KLRG1 protein can be introduced into the mouse endogenous KLRG1 locus, so that the mouse genome contains a humanized KLRG1 gene, and / or the mouse expresses a human or humanized KLRG1 protein. Specifically, using gene editing technology, under the control of the human KLRG1 gene regulatory element, the entire sequence from the mouse 5'UTR upstream nucleotide sequence to exon 5, about 17.4 kb, is replaced with a sequence containing about 68.7 kb of the 5'UTR upstream nucleotide sequence to the 3'UTR downstream nucleotide sequence of the human KLRG1 gene, to obtain a humanized KLRG1 locus, thereby achieving humanization of the mouse KLRG1 gene.

[0190] In order to realize the targeting strategy of the present invention, a targeting vector was constructed, which contains homology arm sequences upstream and downstream of the mouse KLRG1 gene, and an A1 fragment containing a human KLRG1 fragment. Among them, the upstream 5' homology arm sequence is SEQ ID NO: 16, the downstream 3' homology arm sequence is SEQ ID NO: 17, and the nucleotide sequence of the human KLRG1 gene fragment is the same as the nucleotide sequence from position 8944044 to position 9012751 of NCBI accession number NC_000012.12; the connection design of the upstream of the human KLRG1 fragment sequence and the mouse is:

[0192] The sequence The last "C" in the sequence is the last nucleotide in the mouse. GGTA The first "G" in " is the first nucleotide of the human sequence. The downstream connection of the human KLRG1 fragment sequence and the mouse sequence is designed as:

[0194] The sequence The last "C" in the human sequence is the last nucleotide in the sequence " TACT The first "T" in " is the first nucleotide of the mouse sequence. The mRNA sequence transcribed from the modified humanized KLRG1 is shown in SEQ ID NO: 20, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0195] The targeting vector also includes a self-excision cassette, which includes the following elements from 5' to 3': a recombination site loxp for a specific recombination system, a tissue-specific promoter (such as a testis-specific promoter, an ovary-specific promoter), a Cre recombinase, a termination sequence (such as PloyA), a promoter (such as a PGK promoter), and a resistance gene (such as Neo) for positive clone screening, a termination sequence, and loxp. During germline transmission, Cre is expressed under the regulation of a specific promoter and is removed from the mouse cell genome together with Neo.

[0196] The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified by sequencing is electroporated and transfected into the embryonic stem cells of C57BL / 6 mice, and the obtained cells are screened using positive clone screening marker genes to screen the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to the techniques known in the art, and the obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white) can be produced. F0 generation chimeric mice (male mice) are backcrossed with wild-type mice (female mice) to obtain F1 generation mice, and then the F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice.

[0197] The genotype of somatic cells of F1 mice can be identified by PCR, using the primers shown in Table 4 below for detection. The exemplary results are shown in Figure 4 As shown in A-4B, five mice numbered F1-1 to F1-5 were positive mice.

[0198] Table 4 Primer sequences and recombinant fragment sizes for PCR detection of F1 genotypes

[0199]

[0200] The expression of human KLRG1 protein in mice was confirmed by flow cytometry. Specifically, spleen tissues of 6-week-old female wild-type C57BL / 6 mice and 5-week-old female KLRG1 gene heterozygous mice were selected and leukocyte marker antibodies Invitrogen CD45 Monoclonal Antibody (30-F11), eFluor TM 506,eBioscience TM (mCD45), T cell marker antibody Alexa 700anti-mouse CD3 Antibody(mCD3), B cell marker antibody Brilliant Violet 650 TM anti-mouse CD19 Antibody(mCD19), CD4 positive T cell marker antibody Brilliant Violet 785 TM anti-mouse CD4 Antibody (mCD4), CD8 positive T cell marker antibody APCanti-mouse CD8a (mCD8), natural killer cell (NK cell) marker antibody Brilliant Violet711 TM anti-mouse NK-1.1Antibody(mNK1.1), natural killer cell receptor (NKp46) labeled antibodyBrilliantViolet421 TM anti-mouse CD335(NKp46)Antibody(mNKp46)Anti-human KLRG1 antibodyeBioscienceKLRG1 Monoclonal Antibody(13A2),PE(hKLRG1), flow cytometry detection after recognition staining.

[0201] The expression of human KLRG1 (hKLRG1) protein in the spleen of C57BL / 6 mice and humanized heterozygous mice of KLRG1 gene was analyzed. In humanized heterozygous mice, the positive proportion of hKLRG1 in T cells was 4.64% (characterized by mCD45+mCD19-mCD3+hKLRG1+); the positive proportion of hKLRG1 in NK cells was 14.0% (characterized by mCD45+mCD19-mCD3-mNK1.1+mNKp46+hKLRG1+). This indicates that the humanized heterozygous mice of KLRG1 gene can express human KLRG1 protein normally.

[0202] Example 3 Pharmacodynamic Model

[0203] The KLRG1 gene humanized mice prepared by the construction method described in Example 1 or 2 of the present application can be used to evaluate the efficacy of regulators targeting human KLRG1 in tumor diseases. For example, homozygous KLRG1 humanized mice are subcutaneously inoculated with MC38 cells, and the tumor volume grows to about 100 mm 3 The mice were then divided into a control group or a treatment group according to the tumor volume. The treatment group randomly selected a drug targeting human KLRG1, and the control group was injected with an equal volume of saline. The tumor volume was measured regularly and the mice were weighed. By comparing the weight changes of the mice and the tumor size, the in vivo safety and in vivo efficacy of the compound can be effectively evaluated.

[0204] Example 3 Preparation of double-gene or multi-gene humanized mice

[0205] The KLRG1 gene humanized mouse prepared by the construction method described in Example 1 or 2 of the present application can also prepare a double gene or multi-gene humanized mouse model. For example, in the aforementioned Example 1 or 2, the embryonic stem cells used for microinjection can be selected from mice containing at least one gene modification of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4, or, on the basis of the KLRG1 gene humanized mouse, the double gene humanized or multi-gene humanized mouse model can be obtained by separating mouse ES embryonic stem cells and gene recombination targeting technology. The homozygous or heterozygous humanized mouse of the KLRG1 gene obtained in the present application can also be mated with other gene-modified mice, and the offspring can be screened. According to the Mendel's law of inheritance, there is a certain probability of obtaining a multi-gene mouse modified with a humanized KLRG1 gene and other genes, and then the heterozygotes can be mated with each other to obtain a homozygous modified with double genes or multiple genes.

[0206] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0207] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0208] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A 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 killer cell lectin-like receptor G1 (KLRG1) protein.

2. The construction method according to claim 1, characterized in that: The chimeric KLRG1 protein comprises a human or humanized extracellular region, a human or humanized transmembrane region and an endogenous cytoplasmic region; Preferably, the amino acid sequence of the chimeric KLRG1 protein is identical to that of the human KLRG1 protein over at least 50 to 189 consecutive amino acids; Preferably, the amino acid sequence of the human or chimeric KLRG1 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189; or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189; Further preferably, the amino acid sequence of the chimeric KLRG1 protein comprises SEQ ID NO: 7; or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO:

7.

3. A method for constructing a genetically modified non-human animal, characterized in that: At the non-human animal endogenous KLRG1 gene locus, replacing the non-human animal endogenous KLRG1 gene with a nucleotide sequence comprising human KLRG1; Preferably, the nucleotide sequence of human KLRG1 comprises all or part of exon 1 to exon 5 of the human KLRG1 gene; Further preferably, the nucleotide sequence of human KLRG1 comprises any one of the following groups: A) part of exon 2 to all of exon 5 of the human KLRG1 gene, preferably including at least 50 bp of continuous nucleotides downstream of the 3'UTR; preferably, the part of exon 2 of the human KLRG1 gene comprises at least 5 bp of continuous nucleotides; B) the entirety of exon 1 to exon 5 of the human KLRG1 gene, preferably including the nucleotide sequence from the start codon to the stop codon, further preferably including the 5'UTR and / or the 3'UTR, and more preferably further including at least 50 bp of continuous nucleotides upstream of the 5'UTR and / or at least 50 bp of continuous nucleotides downstream of the 3'UTR; More preferably, the nucleotide sequence of human KLRG1 comprises the nucleotide sequence of SEQ ID NO: 5 or NCBI accession number NC_000012.12 from 8944044 to 9012751; or comprises a nucleotide sequence of SEQ ID NO: 5 or NCBI accession number NC_000012.12 from 8944044 to 9012751 that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical; Preferably, the non-human animal endogenous KLRG1 gene comprises all or part of exon 1 to exon 5 of the non-human animal endogenous KLRG1 gene; Further preferably, the non-human animal endogenous KLRG1 gene to be replaced comprises any one of the following groups: a) a portion of exon 2 to the entirety of exon 5 of an endogenous KLRG1 gene of a non-human animal, preferably further comprising at least 50 bp of continuous nucleotides downstream of the 3'UTR; preferably, the portion of exon 2 of an endogenous KLRG1 gene of a non-human animal comprises at least 5 bp of continuous nucleotides; b) the entire exon 1 to exon 5 of the endogenous KLRG1 gene of a non-human animal, preferably further comprising at least 50 bp of continuous nucleotides upstream of the 5'UTR.

4. The construction method according to any one of claims 1 to 3, characterized in that: The nucleotide sequence of human KLRG1 or the nucleotide sequence encoding human or chimeric KLRG1 protein is operably linked to a regulatory element, preferably, the regulatory element comprises a regulatory element of endogenous KLRG1 of a human or non-human animal, such as a promoter, 5'UTR and / or 3'UTR; Preferably, the endogenous KLRG1 protein of the non-human animal is not expressed or is expressed at a reduced level compared to KLRG1 in wild-type animals; Preferably, the modified KLRG1 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

5. The construction method according to any one of claims 1 to 4, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the rodent includes a mouse or a rat; Preferably, the mRNA transcribed from the modified KLRG1 gene in the genome of the non-human animal comprises SEQ ID NO: 6 or SEQ ID NO: 20; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 6 or SEQ ID NO:

20.

6. The construction method according to any one of claims 1 to 5, characterized in that: The non-human animal also includes a nucleotide sequence encoding other human or chimeric proteins; preferably, the human or chimeric protein includes at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

7. 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 6; 2) Determine the effects of therapeutic agents on non-human animals; Preferably, the non-human animal suffers from a disease; Preferably, the therapeutic agent includes an anti-KLRG1 antibody; further preferably, it also includes an additional therapeutic agent (such as one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody); Preferably, the disease includes one or more of cancer, immune disease or inflammation.

8. A humanized KLRG1 protein, characterized in that The humanized KLRG1 protein comprises all or part of the human KLRG1 protein; Preferably, the amino acid sequence of all or part of the human KLRG1 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189; or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity with SEQ ID NO: 2 or SEQ ID NO: 2 positions 59-189 or SEQ ID NO: 2 positions 60-189; Further preferably, the humanized KLRG1 protein comprises SEQ ID NO:7; or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO:

7.

9. A humanized KLRG1 gene, characterized in that: The humanized KLRG1 gene encodes the humanized KLRG1 protein according to claim 8; Preferably, the humanized KLRG1 gene comprises the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 16, 17, 18, 19, 20 or NCBI Accession No. NC_000012.12 from 8944044 to 9012751; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 16, 17, 18, 19, 20 or NCBI Accession No. NC_000012.12 from 8944044 to 9012751.

10. A cell, tissue or organ, characterized in that: The cell, tissue or organ expresses the humanized KLRG1 protein according to claim 8; or the genome of the cell, tissue or organ contains the humanized KLRG1 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 6, the humanized KLRG1 protein according to claim 8, the humanized KLRG1 gene according to claim 9, or the cell, tissue or organ according to claim 10, characterized in that: The application includes: A) Application in product development involving KLRG1-related immune processes in human cells; B) Application as a model system related to KLRG1 for pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for the study of KLRG1-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human KLRG1 signaling pathway modulators; or E) To study the function of KLRG1 gene, the drugs and efficacy targeting human KLRG1 target sites, and the application of KLRG1-related drugs in cancer, inflammation or immune diseases.

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