Non-human animal modified by CLDN1 gene

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) CLDN1 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) CLDN1 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 humanized animal models suitable for screening and evaluation of human drugs will significantly improve the efficiency of new drug development and reduce drug development costs. Summary of the invention

[0004] The present application provides an animal model with human or chimeric claudin 1 (CLDN1) protein. The animal model can express human or chimeric CLDN1 (e.g., humanized CLDN1) protein. It can be used for the study of CLDN1 gene function, and can also be used for the screening and evaluation of CLDN1 signaling pathway regulators (e.g., therapeutic agents targeting human CLDN1, such as anti-CLDN1 antibodies, nucleic acid 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, and the treatment of cancer, inflammation or immune diseases and diseases at human CLDN1 target sites; the animal 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 CLDN1 protein and provides a platform for screening related drugs.

[0005] 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 human or chimeric claudin 1 (CLDN1). In some embodiments, the chimeric CLDN1 protein is a humanized CLDN1 protein. In some embodiments, the nucleotide sequence encoding the human or chimeric CLDN1 protein may be a CDS, cDNA, or genomic DNA. In some embodiments, the nucleotide sequence encoding the human or chimeric CLDN1 protein is operably linked to an endogenous regulatory element (e.g., an endogenous promoter and / or UTR, preferably a 5'UTR and / or a 3'UTR) of an endogenous CLDN1 locus of at least one chromosome. In some embodiments, the amino acid sequence of the chimeric CLDN1 protein comprises an amino acid sequence that is identical to at least 50 to 211 consecutive amino acids of a human CLDN1 protein, such as at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 170, 175, 179, 180, 181, 182, 183, 184, 185, 190, 200, 210, or 211 consecutive amino acids. In some embodiments, the amino acid sequence of the human or chimeric CLDN1 protein comprises SEQ ID NO: 2, SEQ ID NO: 11, 22-211 of SEQ ID NO: 2, 29-211 of SEQ ID NO: 2, or 31-211 of SEQ ID NO: 2, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to SEQ ID NO: 2, SEQ ID NO: 11, 22-211 of SEQ ID NO: 2, 29-211 of SEQ ID NO: 2, or 31-211 of SEQ ID NO: 2. In some embodiments, the chimeric CLDN1 protein comprises a portion of a human CLDN1 protein and a portion of a non-human animal CLDN1 protein. In some embodiments, the portion of the human CLDN1 protein comprises SEQ ID NO: 2, SEQ ID NO: 11, 22-211 of SEQ ID NO: 2, 29-211 of SEQ ID NO: 2, or 31-211 of SEQ ID NO: 2. In some embodiments, the portion of the non-human animal CLDN1 protein comprises SEQ ID NO: 1, 1-21 of SEQ ID NO: 1, 1-28 of SEQ ID NO: 1, or 1-30 of SEQ ID NO: 1. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat. In some embodiments, the non-human animal is a mouse.In some embodiments, the non-human animal does not express endogenous CLDN1 protein or expresses it at a reduced level compared to CLDN1 in wild-type animals. In some embodiments, one or more cells of the non-human animal express human or chimeric CLDN1 protein.

[0006] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein the genome of the non-human animal comprises a nucleotide sequence of human CLDN1 replacing the nucleotide sequence of the corresponding region of endogenous CLDN1 at the endogenous CLDN1 locus. In some embodiments, the nucleotide sequence of human CLDN1 can be CDS, cDNA or genomic DNA. In some embodiments, the nucleotide sequence of human CLDN1 comprises at least 5 bp to 16740 bp of continuous nucleotides of the human CLDN1 gene, for example, at least 5, 50, 100, 200, 300, 400, 500, 540, 545, 546, 547, 548, 550, 600, 700, 800, 870, 873, 874, 875, 876, 877, 900, 10 00, 1100, 1500, 2000, 2500, 2730, 2733, 2734, 2735, 2500, 3000, 3446, 4000, 5000, 10000, 11000, 12000, 13000, 13800, 13839, 13840, 13850, 14000, 15000, 16000 or 16740 bp of consecutive nucleotides. In some embodiments, the expression of the nucleotide sequence encoding the human or chimeric CLDN1 protein or the nucleotide sequence of human CLDN1 is regulated by endogenous regulatory elements (such as promoters and / or UTRs) of non-human animals. In some embodiments, the nucleotide sequence encoding the human or chimeric CLDN1 protein or the nucleotide sequence of human CLDN1 is operably linked to endogenous regulatory elements (such as promoters and / or UTRs) of the endogenous CLDN1 locus. In some embodiments, the endogenous CLDN1 protein of the non-human animal is not expressed or is expressed at a reduced level compared to CLDN1 in wild-type animals. In some embodiments, the modified CLDN1 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the nucleotide sequence of human CLDN1 encodes a human or chimeric CLDN1 protein. In some embodiments, the nucleotide sequence of human CLDN1 encodes a portion of a human CLDN1 protein. In some embodiments, the nucleotide sequence of human CLDN1 encodes SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 2, 22-211, SEQ ID NO: 2, 29-211, or SEQ ID NO: 2. 31-211. In some embodiments, the nucleotide sequence of human CLDN1 includes a portion of exon 1 to a portion of exon 4 of the human CLDN1 gene.In some embodiments, the portion of exon 1 of the human CLDN1 gene includes at least 5 bp to 463 bp of continuous nucleotide sequence of exon 1, for example, at least 5, 10, 15, 50, 100, 110, 120, 130, 131, 132, 133, 134, 135, 140, 150, 200, 300, 400, 450 or 463 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 1 of the human CLDN1 gene includes a portion of the coding region. In some embodiments, the portion of exon 4 of the human CLDN1 gene includes at least 5 bp to 2733 bp of continuous nucleotide sequence of exon 4, for example, at least 5, 10, 15, 50, 100, 110, 120, 130, 140, 150, 160, 161, 162, 163, 164, 165, 170, 180, 190, 200, 300, 400, 500, 800, 1000, 1500, 2000, 2100, 2500 or 2733 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 4 of the human CLDN1 gene includes a portion of the coding region. In some embodiments, the nucleotide sequence of human CLDN1 comprises SEQ ID NO: 5, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequence of the corresponding region of endogenous CLDN1 encodes a portion of a non-human animal CLDN1 protein. In some embodiments, the nucleotide sequence of the corresponding region of endogenous CLDN1 encodes an amino acid sequence of SEQ ID NO: 1, 22-211 of SEQ ID NO: 1, 29-211 of SEQ ID NO: 1, or 31-211 of SEQ ID NO: 1. In some embodiments, the nucleotide sequence of the corresponding region of endogenous CLDN1 comprises a portion of exon 1 to a portion of exon 4 of a non-human animal CLDN1 gene. In some embodiments, the portion of exon 1 of the non-human animal CLDN1 gene includes at least 5 bp to 436 bp of continuous nucleotide sequence of exon 1, for example, at least 5, 10, 15, 50, 100, 110, 120, 130, 131, 132, 133, 134, 135, 140, 150, 200, 300, 400, 430 or 436 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 1 of the non-human animal CLDN1 gene includes a portion of the coding region.In some embodiments, the portion of exon 4 of the non-human animal CLDN1 gene includes a continuous nucleotide sequence of at least 5 bp to 2556 bp of exon 4, for example, at least 5, 10, 15, 50, 100, 110, 120, 130, 140, 150, 160, 161, 162, 163, 164, 165, 170, 180, 190, 200, 300, 400, 500, 800, 1000, 1500, 2000, 2550 or 2556 bp of exon 4. In some embodiments, the portion of exon 4 of the non-human animal CLDN1 gene includes a portion of the coding region. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat. In some embodiments, the mRNA transcribed from the modified gene in the genome of the non-human animal comprises SEQ ID NO: 10, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 10. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by other genes, the human or chimeric protein being selected from at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0007] In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises a nucleotide sequence encoding an endogenous CLDN1 region at an endogenous CLDN1 locus replaced by a nucleotide sequence encoding a corresponding region of human CLDN1. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 may be a CDS, cDNA, or genomic DNA. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 is operably linked to an endogenous regulatory element (such as a promoter and / or UTR) of the endogenous CLDN1 locus, and one or more cells of the animal express a human or humanized CLDN1 protein. In some embodiments, the endogenous CLDN1 protein of the non-human animal is not expressed or the expression level is reduced compared to CLDN1 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 comprises a portion of exon 1, all of exons 2-3, and a portion of exon 4 of the human CLDN1 gene, preferably also comprising intron 1 and / or intron 3. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 comprises a portion of exon 1 to a portion of exon 4 of the human CLDN1 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous CLDN1 region comprises part of exon 1, all of exons 2-3 and part of exon 4 of the CLDN1 gene of a non-human animal (such as a mouse), and preferably also comprises intron 1 and / or intron 3. In some embodiments, the nucleotide sequence encoding the endogenous CLDN1 region comprises part of exon 1 to part of exon 4 of the CLDN1 gene of a non-human animal (such as a mouse). In some embodiments, the modified CLDN1 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenously replaced locus.

[0008] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the human or chimeric CLDN1 protein with the nucleotide sequence encoding the endogenous CLDN1 protein of the non-human animal. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 2 or 11 with the nucleotide sequence encoding the endogenous CLDN1 protein of the non-human animal. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 2 or 11 with the nucleotide sequence encoding SEQ ID NO: 1 endogenously in the non-human animal. In some embodiments, the construction method comprises replacing the corresponding region of the endogenous CLDN1 of the non-human animal with the nucleotide sequence encoding SEQ ID NO: 2 at positions 22-211, SEQ ID NO: 2 at positions 29-211, or SEQ ID NO: 2 at positions 31-211. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 2 at positions 31-211 with the nucleotide sequence encoding SEQ ID NO: 1 endogenously in the non-human animal at positions 31-211. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding 29-211 of SEQ ID NO: 2 in the non-human animal endogenous nucleotide sequence encoding 29-211 of SEQ ID NO: 1. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding 22-211 of SEQ ID NO: 2 in the non-human animal endogenous nucleotide sequence encoding 22-211 of SEQ ID NO: 1. In some embodiments, the construction method comprises replacing the nucleotide sequence of the endogenous CLDN1 gene of the non-human animal with the nucleotide sequence of human CLDN1. In some embodiments, the construction method comprises replacing the nucleotide sequence of the endogenous CLDN1 gene of the non-human animal endogenous nucleotide sequence encoding SEQ ID NO: 1, SEQ ID NO: 1 22-211, SEQ ID NO: 1 29-211 or SEQ ID NO: 1 31-211 with a portion of exon 1 to exon 4 of the human CLDN1 gene. In some embodiments, the construction method comprises replacing a nucleotide sequence of a part of exon 1 to a part of exon 4 of an endogenous CLDN1 gene of a non-human animal with a part of exon 1 to a part of exon 4. In some embodiments, the construction method comprises replacing a nucleotide sequence of positions 31-211 of an endogenous non-human animal encoding SEQ ID NO: 1 with SEQ ID NO: 5. In some embodiments, the construction method comprises replacing a nucleotide sequence of an endogenous non-human animal encoding SEQ ID NO: 1 with SEQ ID NO: 10. In some embodiments, the construction method comprises replacing a nucleotide sequence of a part of exon 1 to a part of exon 4 of an endogenous non-human animal with SEQ ID NO: 5.In some embodiments, the construction method includes replacing the nucleotide sequence of SEQ ID NO: 10 with a portion of exon 1 to a portion of exon 4 of an endogenous non-human animal.

[0009] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or humanized CLDN1 protein, wherein the humanized CLDN1 protein comprises at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 170, 175, 179, 180, 181, 182, 183, 184, 185, 190, 200, 210 or 211 consecutive amino acid sequences that are identical to the continuous amino acid sequence of the corresponding region of a human, and the non-human animal expresses the human or humanized CLDN1 protein. In some embodiments, the nucleotide sequence encoding the human or humanized CLDN1 protein is operably linked to an endogenous CLDN1 regulatory element (such as a promoter and / or UTR). In some embodiments, the nucleotide sequence encoding the human or humanized CLDN1 protein can be integrated into the endogenous CLDN1 locus of the non-human animal. In some embodiments, the human or humanized CLDN1 protein has at least one non-human animal (eg, mouse) CLDN1 activity and / or human CLDN1 activity.

[0010] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein in at least one cell of the non-human animal, at the endogenous CLDN1 locus of the non-human animal, the nucleotide sequence encoding the endogenous CLDN1 region is replaced by the nucleotide sequence encoding the corresponding region of human CLDN1. In some embodiments, the endogenous CLDN1 protein of the non-human animal is not expressed or the expression level is reduced compared with CLDN1 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 comprises part of exon 1, all of exons 2-3, and part of exon 4 of the human CLDN1 gene, preferably further comprising intron 1 and / or intron 3. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 comprises part of exon 1 to part of exon 4 of the human CLDN1 gene. In some embodiments, the amino acid sequence of the corresponding region of human CLDN1 comprises SEQ ID NO: 11, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous CLDN1 region comprises a portion of exon 1, all of exons 2-3 and a portion of exon 4 of a CLDN1 gene of a non-human animal (such as a mouse), and preferably further comprises intron 1 and / or intron 3. In some embodiments, the nucleotide sequence encoding the endogenous CLDN1 region comprises a portion of exon 1 to exon 4 of a CLDN1 gene of a non-human animal (such as a mouse). In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 is operably linked to an endogenous CLDN1 regulatory element, such as a promoter and / or UTR. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat.

[0011] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric CLDN1, the construction method comprising replacing a nucleotide sequence encoding an endogenous CLDN1 region with a nucleotide sequence encoding a corresponding region of human CLDN1 at an endogenous CLDN1 locus of a non-human animal (such as a mouse), producing a genetically modified non-human animal cell, the non-human animal cell expressing a human or chimeric CLDN1 protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 comprises a portion of exon 1, all of exons 2-3, and a portion of exon 4 of the human CLDN1 gene, preferably further comprising intron 1 and / or intron 3. In some embodiments, the amino acid sequence of the corresponding region of human CLDN1 comprises SEQ ID NO: 11, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous CLDN1 region comprises a portion of exon 1, all of exons 2-3 and a portion of exon 4 of the CLDN1 gene of a non-human animal (such as a mouse), and preferably further comprises intron 1 and / or intron 3. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLDN1 is operably linked to a regulatory element of endogenous CLDN1, such as a promoter and / or UTR. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins include but are not limited to at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0012] In one aspect, the present invention provides a non-human animal obtained by the construction method or an application of the non-human animal, the application comprising: A) application in the development of products involving immune processes related to CLDN1 in human cells; B) application as a model system related to CLDN1 in pharmacology, immunology, microbiology and medical research; C) application involving the production and use of animal experimental disease models for CLDN1-related pathology research and / or for the development of diagnostic strategies and / or for the development of treatment strategies; D) application in in vivo research on the screening, efficacy testing, efficacy assessment, verification or evaluation of human CLDN1 signaling pathway regulators; or, E) application in studying the function of the CLDN1 gene, studying drugs and efficacy targeting human CLDN1 target sites, and studying cancer, immune diseases or inflammatory drugs related to CLDN1.

[0013] In one aspect, the present invention provides a method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, the method comprising: 1) administering a therapeutic agent to a non-human animal or the non-human animal obtained by the construction method; 2) determining the effect of the therapeutic agent on the disease or the non-human animal. In some embodiments, the therapeutic agent is a therapeutic agent targeting CLDN1, such as an anti-CLDN1 antibody, a nucleic acid drug, or a polypeptide drug. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the disease comprises cancer, inflammation, or an immune disease. In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the cancer is breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, head and neck cancer, liver cancer, lymphoma, or lung cancer. In some embodiments, the immune disease includes but is not limited to GVHD (graft versus host disease), psoriasis, atopic dermatitis, multiple sclerosis, allergy, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty hepatitis), systemic lupus erythematosus, 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 asthma, psoriasis, atopic dermatitis, rheumatoid arthritis or multiple sclerosis. In some embodiments, the inflammation includes acute inflammation and also includes chronic inflammation. Specifically, it includes but is not limited to degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.). In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0014] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating cancer, the method comprising:

[0015] 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has a tumor; 2) determining the inhibitory effect of the therapeutic agent on the tumor. In some embodiments, the therapeutic agent is a therapeutic agent targeting CLDN1, such as an anti-CLDN1 antibody, a nucleic acid drug, or a polypeptide drug. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the tumor comprises one or more tumor cells injected into a non-human animal. In some embodiments, determining the inhibitory effect of the therapeutic agent on the tumor involves measuring the tumor volume in a non-human animal. In some embodiments, the non-human animal further comprises a sequence encoding human or chimeric PD-1, human or chimeric PD-L1, and / or human or chimeric CTLA4. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1. In some embodiments, the tumor comprises one or more tumor cells injected into an animal.

[0016] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating an immune disease, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal suffers from an immune disease; 2) determining the therapeutic effect of the therapeutic agent on the immune disease.

[0017] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating inflammation, the method comprising:

[0018] 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has inflammation; 2) determining the effectiveness of the therapeutic agent in treating inflammation.

[0019] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the therapeutic agent is a therapeutic agent targeting CLDN1, such as an anti-CLDN1 antibody, a nucleic acid drug, or a polypeptide drug. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the determination of the effect of the therapeutic agent on the non-human animal involves measuring the weight change or blood test of the animal. In some embodiments, the blood test includes but is not limited to red blood cell count, hematocrit, and / or hemoglobin content.

[0020] In one aspect, the present invention provides a humanized CLDN1 protein, comprising a portion of a human CLDN1 protein and a portion of a non-human animal CLDN1 protein. In some embodiments, the portion of the human CLDN1 protein comprises the amino acid sequence of positions 22-211 of SEQ ID NO: 2, positions 29-211 of SEQ ID NO: 2, or positions 31-211 of SEQ ID NO: 2. In some embodiments, the portion of the non-human animal CLDN1 protein comprises the amino acid sequence of positions 1-21 of SEQ ID NO: 1, positions 1-28 of SEQ ID NO: 1, or positions 1-30 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the humanized CLDN1 protein comprises SEQ ID NO: 11.

[0021] In one aspect, the present invention provides a humanized CLDN1 gene, wherein the humanized CLDN1 gene encodes the humanized CLDN1 protein. In some embodiments, the humanized CLDN1 gene comprises any of the following nucleotide sequences: A) a nucleotide sequence encoding amino acids 22-211 of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 2 ...

[0022] In one aspect, the present invention provides a cell, tissue or organ, wherein the cell, tissue or organ expresses the humanized CLDN1 protein or comprises the humanized CLDN1 gene.

[0023] In one aspect, the present invention provides a non-human animal genome, the non-human animal genome comprising at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric CLDN1 protein. In some embodiments, the chromosome comprises a human or chimeric CLDN1 gene. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human CLDN1 protein. In some embodiments, the chromosome comprises a portion of exon 1 to a portion of exon 4 of the human CLDN1 gene. In some embodiments, the CLDN1 of the endogenous genome of the non-human animal is replaced. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 1, 22-211 of SEQ ID NO: 1, 29-211 of SEQ ID NO: 1, or 31-211 of SEQ ID NO: 1 in the endogenous genome of the non-human animal is replaced. In some embodiments, a portion of exon 1 to a portion of exon 4 of the endogenous CLDN1 gene of the non-human animal is replaced. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human or chimeric CLDN1 protein or a nucleotide sequence of human CLDN1 replaces the corresponding portion of the endogenous chromosome of the non-human animal. In some embodiments, the nucleotide sequence encoding nucleotides 31-211 of SEQ ID NO: 2 replaces the nucleotide sequence of nucleotides 31-211 of the non-human animal endogenous encoding SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding nucleotides 29-211 of SEQ ID NO: 2 replaces the nucleotide sequence of nucleotides 29-211 of the non-human animal endogenous encoding SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding nucleotides 22-211 of SEQ ID NO: 2 replaces the nucleotide sequence of nucleotides 22-211 of the non-human animal endogenous encoding SEQ ID NO: 1.

[0024] In one aspect, the present invention provides a cell, tissue or organ comprising the above-mentioned non-human animal genome.

[0025] In one aspect, the present invention provides an animal model, wherein the animal model comprises the above-mentioned humanized CLDN1 gene.

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

[0027] The term "locus" in 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, a part of a gene, or a regulatory region of a gene, etc. For example, the "CLDN1 locus" includes exons 1-4 of the CLDN1 gene and any DNA fragment in its regulatory region.

[0028] The term "part of exon XX" of the present invention means that a continuous or intermittent sequence of several, dozens or hundreds of nucleotides is consistent with the entire exon nucleotide sequence.

[0029] The term "exon XX to exon XXX" or "exon XX-XXX" or "exon XX to all of exon XXX" or "all of exon XX to all of exon XXX" of the present invention refers to exons and introns in between.

[0030] The term "a portion of exon x to a portion of exon xx" or "a portion of exon x-a portion of exon xx" of the present invention includes all or part of an exon and introns therebetween. For example, "a portion of exon 1 to a portion of exon 4" includes a portion of exon 1, all of intron 1, all of exon 2, all of intron 2, all of exon 3, all of intron 3, and a portion of exon 4.

[0031] The term "intron xx" in this application refers to an intron between two exons, for example, intron 3 is the intron between exon 3 and exon 4.

[0032] 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.

[0033] 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".

[0034] 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.

[0035] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description.

[0036] CLDN1

[0037] In the human genome, the CLDN1 gene (NCBI Gene ID: 9076, UniProt ID: O95832, located at positions 190305707 to 190322446 of chromosome 3 NC_000003.12) contains 4 exons, namely exon 1, exon 2, exon 3 and / or exon 4. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_021101.5 and its encoded protein NP_066924.1 (SEQ ID NO: 2) is shown in Table 1.

[0038] Table 1

[0039]

[0040] In the mouse genome, the CLDN1 gene (NCBI Gene ID: 12737, UniProt ID: O88551, located at positions 26175395 to 26190589 of chromosome 16 NC_000082.7) contains 4 exons, namely exon 1, exon 2, exon 3 and / or exon 4. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_016674.4 and its encoded protein NP_057883.1 (SEQ ID NO: 1) is shown in Table 2.

[0041] Table 2

[0042]

[0043]

[0044] CLDN1 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). The relevant information of these genes (such as intron sequence, exon sequence and amino acid sequence) can be found in NCBI, the entire contents of which are incorporated into this application by reference.

[0045] In order to determine the percent identity of two amino acid sequences or two nucleic acid 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 nucleic acid sequences, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides on the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the 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.

[0046] 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.

[0047] Carrier

[0048] The present invention provides a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of the region to be changed (5' arm or 5' homology arm), which is selected from the genomic DNA of the CLDN1 gene and has a length of 100 to 10,000 nucleotides; b) a donor region; and c) a DNA fragment homologous to the 3' end of the region to be changed (3' arm or 3' homology arm), which is selected from the genomic DNA of the CLDN1 gene and has a length of 100 to 10,000 nucleotides.

[0049] In some embodiments, a) the DNA fragment homologous to the 5' end of the region to be changed is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000082.7; c) the DNA fragment homologous to the 3' end of the region to be changed is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000082.7.

[0050] In some embodiments, the length of the genomic nucleotide sequence selected by the targeting vector can exceed about 0.8 kb, 1 kb,

[0051] 1.5kb, 2kb, 2.5kb, 3kb, 3.5kb, 4kb, 4.5kb, 6.5kb, 7kb, 7.5kb, 8kb, 8.5kb, 9kb, 9.5kb, 10kb, 15kb, 16kb, 18kb, 19kb or 20kb.

[0052] In some embodiments, the region to be changed is located on a non-human animal CLDN1 gene. In some embodiments, the region to be changed is located on exon 1 to exon 4 of a non-human animal CLDN1 gene.

[0053] In some embodiments, the 5' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO: 3. In some embodiments, the 3' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO:4.

[0054] In some embodiments, the targeting vector comprises a human sequence (eg, positions 190305707 to 190322446 of NC_000003.12). For example, preferably, a portion of exon 1 to a portion of exon 4 of the human CLDN1 gene. In some embodiments, the donor region in the targeting vector comprises SEQ ID NO: 5.

[0055] In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes). For example, a positive screening marker gene or a negative screening marker gene. In some embodiments, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo or a hygromycin resistance gene sequence HygR. Preferably, the targeting vector further comprises two Frt recombination sites arranged in the same direction on both sides of the marker gene. In some embodiments, the coding gene of the negative screening marker is a coding gene (DTA) of the diphtheria toxin A subunit.

[0056] 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 CLDN1 gene, and the sgRNA is unique on the target sequence of the gene to be changed, and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'. In some embodiments, the targeting site of the sgRNA in the CLDN1 gene of a non-human animal (e.g., mouse) is located at exon 1 to exon 4.

[0057] In some embodiments, the present application relates to a plasmid construct (e.g., pT7-sgRNA) comprising an sgRNA sequence and / or a cell comprising the construct.

[0058] The present invention also relates to a cell comprising a targeting vector or an sgRNA vector as described above.

[0059] In addition, the present invention also provides a non-human mammalian cell having any one of the above-mentioned targeting vectors and one or more in vitro transcripts of the construct described in the present application. In some embodiments, the cell comprises Cas9mRNA or its in vitro transcript.

[0060] In some embodiments, the cell is heterozygous for the gene. In some embodiments, the cell is homozygous for the gene.

[0061] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell.

[0062] Genetically modified non-human animals

[0063] The "genetically modified non-human animal" or "genetically modified non-human animal" described in the present invention refers to a non-human animal in which at least one chromosome in the genome of the 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. The cells with exogenous DNA can be various cells, for example, somatic cells, immune cells (such as T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, a genetically modified non-human animal is provided, wherein the non-human animal comprises a modified endogenous CLDN1 locus, comprising an exogenous sequence (such as a human sequence), for example, one or more non-human sequences are replaced with one or more human sequences, or one or more human and / or non-human sequences are inserted. Non-human animals are generally able to pass genetic modifications to offspring through germline transmission.

[0064] The "chimeric (x) gene" or "chimeric (x) nucleic acid" of the present invention refers to a gene or nucleic acid, wherein two or more parts of the gene or nucleic acid are from different species, or at least one sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, a chimeric (x) gene or a chimeric (x) nucleic acid has at least a portion of a 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 a chimeric (x) nucleic acid refers to a humanized (x) gene or a humanized (x) nucleic acid.

[0065] 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 one sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (x) protein or chimeric (x) polypeptide has two or more different species sources, for example, the same (or homologous) proteins of different species. In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide refers to a humanized (x) protein or humanized (x) polypeptide.

[0066] The "humanized (x) protein" or "humanized (x) polypeptide" of the present invention refers to a protein or polypeptide, wherein at least a portion of the protein or polypeptide is derived from a human protein or polypeptide. In some embodiments, the humanized (x) protein or humanized (x) polypeptide refers to a human protein or polypeptide.

[0067] The "humanized (x) nucleic acid" of the present invention refers to a nucleic acid, wherein at least a portion of the nucleic acid is derived from a human. In some embodiments, the nucleic acids in the humanized (x) nucleic acid are all derived from a human. In some embodiments, the humanized (x) nucleic acid refers to a humanized exon, and the humanized exon can be a human exon or a chimeric exon.

[0068] In some embodiments, the chimeric CLDN1 gene or chimeric CLDN1 nucleic acid is a humanized CLDN1 gene or humanized CLDN1 nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human CLDN1 gene, and at least a portion of the gene or nucleic acid is derived from a non-human CLDN1 gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding a CLDN1 protein. The encoded CLDN1 protein has at least one activity of a human CLDN1 protein or a non-human animal CLDN1 protein.

[0069] In some embodiments, the chimeric CLDN1 protein or chimeric CLDN1 polypeptide is a humanized CLDN1 protein or humanized CLDN1 polypeptide. In some embodiments, at least one or more parts of the amino acid sequence of the protein or polypeptide are from human CLDN1 protein, and at least one or more parts of the amino acid sequence of the protein or polypeptide are from non-human animal CLDN1 protein. The humanized CLDN1 protein or humanized CLDN1 polypeptide is functional, or has at least one activity of a human CLDN1 protein or a non-human animal CLDN1 protein.

[0070] Genetically modified non-human animals can be various animals, for example, mice, rats, rabbits, pigs, cattle (e.g., cattle, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals that are not easy to obtain suitable genetically modified embryonic stem cells (ES), other methods are used to construct non-human animals containing genetic modifications. Such methods include, for example, modifying the non-ES cell genome (e.g., fibroblasts or induced pluripotent stem cells) and using nuclear transplantation to transfer the modified genome to suitable cells, such as oocytes, and incubating the modified cells (e.g., modified oocytes) in non-human animals under appropriate conditions to form embryos. The above-mentioned construction methods are known in the art and are described in "A. Nagy, et al.,

[0071] “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2006”, the entire contents of which are incorporated herein by reference.

[0072] 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 some embodiments, the rodent is selected from the family Muridae. In some embodiments, the genetically modified animal is 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), Malboridae (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 some embodiments, the genetically modified rodent is selected from mice or rats (Muroidea), gerbils, spiny mice and crested rats. In some embodiments, the genetically modified mouse is from a member of the Muridae family. In some embodiments, the animal is a rodent. In some embodiments, the rodent is selected from mice and rats. In some embodiments, the non-human animal is a mouse.

[0073] 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 non-human animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the mouse is 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 certain embodiments, non-human animals are rodents. In certain embodiments, non-human animals are mice with 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 strains. In certain embodiments, non-human animals are rats. Rats 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 of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

[0074] The genetically modified non-human animal includes modification of the endogenous non-human animal CLDN1 gene site. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature CLDN1 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to a mature CLDN1 protein amino acid sequence). Although cells (e.g., ES cells, somatic cells) that may include the genetic modifications described herein are provided in the present invention, in many embodiments, the genetically modified non-human animal includes modification of the endogenous CLDN1 gene site in the non-human animal.

[0075] The genetically modified non-human animal can express human CLDN1 and / or chimeric (e.g., humanized) CLDN1 at an endogenous locus of a non-human animal (e.g., mouse), wherein the endogenous CLDN1 gene of the non-human animal (e.g., mouse) has been replaced or inserted with a gene of human CLDN1 and / or a nucleotide sequence encoding a region of a human CLDN1 sequence or a nucleotide sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to a human CLDN1 sequence. In some embodiments, the endogenous non-human animal CLDN1 locus is modified by a human nucleic acid sequence that comprises all or part of a mature CLDN1 protein.

[0076] In some embodiments, the genetically modified mouse can express human CLDN1 and / or chimeric CLDN1 (e.g., humanized CLDN1) under the control of mouse regulatory elements (promoters and / or UTRs). Insertion or replacement at the mouse endogenous locus provides a non-human animal that expresses human CLDN1 or chimeric CLDN1 (e.g., humanized CLDN1) in suitable cells and in a manner that does not cause potential pathology observed in some other transgenic mice known in the art. Human CLDN1 or chimeric CLDN1 (e.g., humanized CLDN1) expressed in a non-human animal can maintain one or more functions of wild-type mice or human CLDN1 in the non-human animal. In addition, in some embodiments, the non-human animal does not express endogenous CLDN1. In some embodiments, the expression level of endogenous CLDN1 in the non-human animal is reduced compared to the expression level of CLDN1 in wild-type animals. As used herein, the term "endogenous CLDN1" refers to a CLDN1 protein expressed by an endogenous CLDN1 nucleotide sequence of a non-human animal (e.g., a mouse) before any genetic modification.

[0077] In some embodiments, the humanized CLDN1 locus comprises a human 5'UTR. In some embodiments, the humanized CLDN1 locus comprises an endogenous 5'UTR of a non-human animal (such as a mouse). In some embodiments, the humanized CLDN1 locus comprises a human 3'UTR. In some embodiments, humanization comprises an endogenous 3'UTR of a non-human animal (such as a mouse). Where appropriate, it is reasonable to assume that, based on the similarity of the 5' flanking sequences of the mouse and human CLDN1 genes, they appear to be similarly regulated. As shown in the present application, humanized CLDN1 mice comprising insertions or substitutions in the endogenous mouse CLDN1 locus retain mouse regulatory elements but comprise humanization of the CLDN1 coding sequence and do not exhibit pathological phenomena. Both genetically modified mice that are heterozygous or homozygous for humanized CLDN1 are normal.

[0078] The present invention further relates to the CLDN1 genomic DNA sequence of humanized mice, a DNA sequence obtained by reverse transcription of mRNA that is identical to or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell comprising the construct thereof; and a tissue comprising the cell thereof.

[0079] The present invention further relates to a non-human mammal or its offspring produced by the above method. In some embodiments, its genome comprises human genes.

[0080] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0081] In some embodiments, the non-human mammal expresses a protein encoded by a humanized CLDN1 gene.

[0082] 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).

[0083] 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.

[0084] 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 CLDN1.

[0085] In some embodiments, a non-human mammal comprises a gene construct as described herein. In some embodiments, a non-human mammal expressing a human or humanized CLDN1 protein is provided. In some embodiments, a tissue specifically expressing a human or humanized CLDN1 protein is provided.

[0086] In some embodiments, the expression of non-human animal human or humanized CLDN1 protein is controllable, such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0087] The non-human mammal can be any non-human animal known in the art that can be used in the methods described herein. Preferably, the non-human animal is a mammal (eg, a rodent). In some embodiments, the non-human mammal is a mouse.

[0088] 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.

[0089] 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 CLDN1 protein can be detected by a variety of methods.

[0090] 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 perform quantitative detection. For example, RT-PCR and hybridization methods can be used to detect transcription levels, including RNase protection analysis, Southern Blot, RNA dot hybridization analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blot can also be used to detect the presence of human or humanized CLDN1 protein.

[0091] In some embodiments, the genetically modified non-human animal described herein (eg, a CLDN1 gene homozygous mouse) can express human or humanized CLDN1 in one or more liver tissue cells.

[0092] Methods for constructing genetically modified non-human animals

[0093] Genetically modified non-human animals can be prepared by several techniques known in the art, including the use of homologous recombination technology, gene targeting technology of embryonic stem cells, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR-Cas9 gene editing technology is used to produce genetically modified non-human animals. Many of these gene editing technologies are known in the art and are described in "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017) by Yin et al. : 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting genetically modified cells into enucleated oocytes and fusing enucleated oocytes with another genetically modified cell.

[0094] In some embodiments, the nucleotide sequence encoding the endogenous CLDN1 region 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 CLDN1. In some embodiments, the expression level of the endogenous CLDN1 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.

[0095] In order to achieve the humanization targeting strategy of the mouse CLDN1 locus. The targeting vector comprises a vector consisting of a 5' homology arm, a human or humanized CLDN1 gene fragment and a 3' homology arm. The process involves replacing the endogenous corresponding CLDN1 sequence with the human or humanized CLDN1 sequence by homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nuclease, TALEN or CRISPR) can result in DNA double-strand breaks, and homologous recombination is used to replace the mouse endogenous CLDN1 sequence with the human or humanized CLDN1 sequence.

[0096] Therefore, in some embodiments, the method of preparing a genetically modified humanized animal comprises replacing a nucleic acid sequence encoding an endogenous CLDN1 region with a nucleotide sequence encoding a corresponding region of human CLDN1 at the endogenous CLDN1 locus (or site).

[0097] The present invention also provides a method for establishing a CLDN1 gene humanized animal model, comprising the following steps:

[0098] (a) providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0099] (b) culturing the cells, preferably culturing the cells in a liquid culture medium;

[0100] (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;

[0101] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0102] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0103] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0104] 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.

[0105] 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.

[0106] In some embodiments, the method for preparing a genetically modified non-human animal comprises modifying the coding framework of the CLDN1 gene of the non-human animal, for example, by replacing the nucleic acid sequence encoding the endogenous CLDN1 region with a nucleotide sequence encoding the corresponding region of human CLDN1 (e.g., CDS, genomic DNA or cDNA sequence) under the control of the endogenous regulatory elements (e.g., promoter and / or UTR) of the CLDN1 gene of the non-human animal. For example, one or more functional region sequences of the CLDN1 gene of the non-human animal can be knocked out or inserted into a sequence, so that the endogenous CLDN1 protein of the non-human animal cannot be expressed or the expression level is reduced.

[0107] In some embodiments, the method for preparing a genetically modified non-human animal comprises inserting a nucleotide sequence and / or an auxiliary sequence encoding a human or humanized CLDN1 protein after the endogenous regulatory element of the CLDN1 gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, so that the CLDN1 gene humanized animal model can express a human or humanized CLDN1 protein in vivo, but does not express the CLDN1 protein of the non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0108] In some embodiments, the construction method includes obtaining using the above-mentioned vector. In some embodiments, the vector is a targeting vector and / or an sgRNA vector. In some embodiments, the construction method includes introducing the targeting vector into a non-human animal or its cells. In some embodiments, the construction method includes introducing the targeting vector and the sgRNA vector into a non-human animal or its cells.

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

[0110] (1) providing a plasmid (such as the above-mentioned vector) comprising a human CLDN1 gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target endogenous CLDN1;

[0111] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous CLDN1 gene;

[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 (or 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 humanized CLDN1 protein;

[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 CLDN1 protein is operably linked to endogenous regulatory elements (such as a promoter and / or UTR) at the endogenous CLDN1 locus.

[0117] In some embodiments, the genetically modified non-human animal does not express endogenous CLDN1 protein.

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

[0119] (1) providing a plasmid (e.g., the above-mentioned vector) comprising a human or chimeric CLDN1 gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target endogenous CLDN1;

[0120] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous CLDN1 gene;

[0121] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric CLDN1 gene fragment into the genome.

[0122] In some embodiments, the non-human animal endogenous nucleotide sequence encoding CLDN1 protein is deleted. In some embodiments, the non-human animal endogenous CLDN1 gene has a portion of exon 1 to a portion of exon 4 deleted.

[0123] Use of genetically modified non-human animals

[0124] Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence or inserting a homologous or orthologous human gene or human sequence into a non-human animal at an endogenous non-human animal locus and under the control of endogenous regulatory elements (promoters and / or UTRs) 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.

[0125] Genetically modified animals that express human or humanized CLDN1 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.

[0126] The present invention also provides a use of the above-mentioned CLDN1 gene-modified non-human animal and a non-human animal obtained by any of the above-mentioned construction methods.

[0127] In some embodiments, the application comprises:

[0128] A) Application in the development of products involving CLDN1-related immune processes in human cells;

[0129] B) Application as a model system related to CLDN1 for pharmacological, immunological, microbiological and medical research;

[0130] C) Applications involving the production and use of animal experimental disease models for the study of CLDN1-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0131] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human CLDN1 signaling pathway modulators; or,

[0132] E) Study the function of CLDN1 gene, study the drugs and efficacy targeting human CLDN1 target sites, and study the application of CLDN1-related drugs in cancer (tumor), inflammation, and immune diseases.

[0133] The present invention provides a non-human animal expressing human or humanized CLDN1 protein, which can be used for screening of human CLDN1 specific modulators. In some embodiments, the non-human animal is a human disease animal model. For example, the disease is genetically induced (knock-in or knock-out). In different embodiments, the genetically modified non-human animal also comprises an impaired immune system, such as a genetically modified human-derived tissue xenograft, including human solid tumors (e.g., breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, head and neck cancer, liver cancer or lung cancer) or hematological tumors (e.g., lymphoma, preferably B or T cell tumors).

[0134] In some embodiments, the therapeutic agent (therapeutic agent targeting CLDN1, such as anti-CLDN1 antibody, nucleic acid drug and / or polypeptide drug) blocks or inhibits the CLDN1-mediated signaling pathway. In some embodiments, the therapeutic agent described in the present application (therapeutic agent targeting CLDN1, such as anti-CLDN1 antibody, nucleic acid drug and / or polypeptide drug) can block the interaction between CLDN1 complexes, thereby inhibiting the CLDN1 signaling pathway.

[0135] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (therapeutics targeting CLDN1, such as antibodies, nucleic acid drugs and / or polypeptide drugs) in treating various immune diseases. In some embodiments, the immune diseases include, but are not limited to, GVHD (graft versus host disease), psoriasis, atopic dermatitis, multiple sclerosis, allergies, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty hepatitis), systemic lupus erythematosus, 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 asthma, psoriasis, atopic dermatitis, rheumatoid arthritis or multiple sclerosis.

[0136] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (therapeutic agents targeting CLDN1, such as anti-CLDN1 antibodies, nucleic acid drugs and / or polypeptide drugs) in treating various inflammations. In some embodiments, the inflammation is inflammation caused by infection. In some embodiments, the inflammation includes acute inflammation and chronic inflammation. Specifically, it includes but is not limited to degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.). In some embodiments, inflammation is inflammatory bowel disease (IBD).

[0137] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (therapeutic agents targeting CLDN1, such as anti-CLDN1 antibodies, nucleic acid drugs and / or polypeptide drugs) for treating cancer. In some embodiments, a therapeutic agent (a therapeutic agent targeting CLDN1, such as an anti-CLDN1 antibody, a nucleic acid drug and / or a polypeptide drug) is administered to a non-human animal, wherein the non-human animal has cancer or a tumor, and the inhibitory effect of the therapeutic agent on the cancer or tumor is detected. In some embodiments, the detection includes measuring the size and / or proliferation rate of tumor cells. In some embodiments, the detection method includes vernier caliper measurement, flow cytometry and / or animal in vivo imaging detection. In some embodiments, the detection includes assessing individual body weight, fat mass, activation pathways, neuroprotective activity or metabolic changes, and the metabolic changes include changes in food consumption or water consumption.

[0138] In some embodiments, the tumor cells include one or more cancer cells injected into an animal (e.g., cancer cells derived from a human or non-human animal). In some embodiments, the therapeutic agent inhibits the CLDN1 signaling pathway. In some embodiments, the therapeutic agent does not inhibit the CLDN1 signaling pathway.

[0139] In some embodiments, the genetically modified non-human animals can be used to detect whether a therapeutic agent (a therapeutic agent targeting CLDN1, such as an anti-CLDN1 antibody, a nucleic acid drug, and / or a polypeptide drug) is an agonist or an antagonist. In some embodiments, the methods described herein can be used to detect the function of a therapeutic agent (a therapeutic agent targeting CLDN1, such as an anti-CLDN1 antibody, a nucleic acid drug, and / or a polypeptide drug), for example, whether the therapeutic agent can upregulate an immune response or downregulate an immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent for treating a disease in a subject (e.g., an immune disease, inflammation, or cancer). The inhibitory effect on tumors can also be determined by methods known in the art, for example, by measuring the tumor volume in a non-human animal, and / or determining the tumor (volume) inhibition rate (TGI). TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%)=(1–T Vt / T Vc ) x 100, where T Vt and T Vc is the average tumor volume (or weight) of the treatment group and the control group.

[0140] In some embodiments, therapeutic agents (therapeutic agents targeting CLDN1, such as anti-CLDN1 antibodies, nucleic acid drugs and / or polypeptide drugs) can be used to treat various cancers. The "cancer" of the present invention refers to cells with autonomous growth ability, that is, abnormal states or conditions characterized by rapid cell growth and proliferation. 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 "tumor" of the present invention includes, but is not limited to, lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, brain glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In some embodiments, the tumor is breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor, mesothelial tumor, oropharyngeal tumor, female reproductive system cancer or meningioma. In some embodiments, the tumor is a solid tumor or a hematological tumor. In some embodiments, the cancer is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, liver cancer, head and neck cancer, lymphoma or lung cancer.

[0141] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (a therapeutic agent targeting CLDN1, such as an anti-CLDN1 antibody, a nucleic acid drug and / or a polypeptide drug). The method comprises administering a therapeutic agent to the non-human animal and evaluating the weight change or blood test of the non-human animal. In some embodiments, the blood test includes but is not limited to red blood cell count, hematocrit and / or hemoglobin content. In some embodiments, the therapeutic agent can reduce 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 a control group (e.g., the average body weight of an animal not treated with a therapeutic agent).

[0142] 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.

[0143] 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.

[0144] The present invention also provides an animal model generated by the method described in the present application to screen, verify, evaluate or study the function of the CLDN1 gene, drugs (such as anti-CLDN1 antibodies, nucleic acid drugs and / or polypeptide drugs) or effectiveness of human CLDN1 targeting sites, drugs for immune diseases, cancer or inflammation, and anti-tumor drugs.

[0145] 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 animals described in the present application, and applying human CAR-T to animals with human tumor cells. The effectiveness of CAR-T therapy can be determined and evaluated. In some embodiments, the non-human animal is selected from a CLDN1 gene humanized non-human animal prepared by the method described in the present application, a double or multiple gene 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 CLDN1 protein, or a tumor-bearing, immune disease or inflammatory animal model described in the present application. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat CLDN1-related diseases described in the present application. In some embodiments, TCR-T, CAR-T and / or other immunotherapies provide an evaluation method for treating CLDN1-related diseases described in the present application.

[0146] Non-human animal models with two or more human or chimeric genes

[0147] The present invention also provides a method for generating a genetically modified non-human animal model having two or more human or chimeric genes. The non-human animal may comprise a human or chimeric CLDN1 gene and a sequence encoding an additional human or chimeric protein.

[0148] In some embodiments, the additional human or chimeric proteins include, but are not limited to, at least one genetically modified non-human animal of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the non-human animal described above also expresses at least one of human or humanized LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 proteins.

[0149] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising:

[0150] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0151] (ii) mating, in vitro fertilization or direct gene editing of the non-human animals provided in step (i) with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0152] In some embodiments, the other genetically modified non-human animals include but are not limited to non-human animals humanized with one or a combination of two or more of the genes LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0153] In some embodiments, CLDN1 humanization is performed directly on a non-human animal with at least one genetic modification of human or chimeric LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0154] 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, therapeutic agents targeting CLDN1, and additional therapeutic agents for treating cancer or immune diseases or inflammation. The method includes administering a therapeutic agent targeting CLDN1 and an additional therapeutic agent to a non-human animal, wherein the non-human animal has a tumor or immune disease or inflammation, and determining the effect of the combined therapy on inflammation or tumor or immune disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab) or an anti-PD-L1 antibody. In some embodiments, the non-human animal further comprises a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0155] In some embodiments, the combination therapy is used to treat various cancers described herein. In some embodiments, the cancer is a solid tumor or a hematologic tumor. In some embodiments, the solid tumor includes breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, head and neck cancer, lung cancer, or liver cancer. In some embodiments, the hematologic tumor includes but is not limited to lymphocyte tumors, such as B or T cell tumors. In some embodiments, the combination therapy is designed to treat immune diseases described herein, such as psoriasis, asthma, atopic dermatitis, rheumatoid arthritis, or multiple sclerosis. In some embodiments, the method described herein can be used to evaluate the combination therapy with some other methods. Methods for treating cancer that can be used alone or in combination with the methods described herein include, for example, treating the subject with chemotherapy, such as camphor, doxorubicin, cisplatin, carboplatin, procarbazine, methylclorazepam, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chloramphenicol, pyrimidine, nitrosourea, dacrynic acid, bleomycin, primycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatin, 5-fluorouric acid, vincristine, vinblastine and / or methotrexate. Alternatively, in addition, the method can include performing surgery on the subject to remove at least a portion of the cancer, for example, removing a portion or all of a tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0156] Figure 1 : Schematic diagram comparing mouse CLDN1 locus and human CLDN1 locus (not to scale);

[0157] Figure 2 : Schematic diagram of CLDN1 gene targeting strategy and targeting vector design (not to scale);

[0158] Figure 3 : PCR identification results of the F1 generation of humanized mice with CLDN1 gene, where WT is the wild-type control, M is the marker, H is the 2 O is water control;

[0159] Figure 4 :RT-PCR test results of liver tissue, where + / + is wild-type C57BL / 6 mice, H / H is CLDN1 gene homozygous mice, H 2 O is water control;

[0160] Figure 5 : RT-PCR test results of liver, skin, spleen and small intestine tissues, where + / + is wild-type C57BL / 6 mice, H / H is CLDN1 gene homozygous mice, H 2 O is water control;

[0161] Figure 6 :Western Blot test results, where + / + is wild-type C57BL / 6 mice, H / H is CLDN1 gene humanized homozygous mice, and GADPH is the internal reference. DETAILED DESCRIPTION

[0162] 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.

[0163] In each of the following examples, equipment and materials were obtained from the following companies:

[0164] C57BL / 6 mice and Flp transgenic mice were purchased from the National Rodent Laboratory Animal Seed Center of the China Food and Drug Inspection Institutes.

[0165] Example 1 CLDN1 gene humanized mice

[0166] A schematic diagram of the comparison between the mouse CLDN1 gene (Gene ID: 12737, located at positions 26175395 to 26190589 on chromosome 16 NC_000082.7, based on transcript NM_016674.4 and its encoded protein NP_057883.1 (SEQ ID NO: 1)) and the human CLDN1 gene (Gene ID: 9076, located at positions 190305707 to 190322446 on chromosome 3 NC_000003.12, based on transcript NM_021101.5 and its encoded protein NP_066924.1 (SEQ ID NO: 2)) is shown in FIG. Figure 1 shown.

[0167] In order to achieve the purpose of the present invention, a nucleotide sequence encoding a human CLDN1 protein can be introduced into the mouse endogenous CLDN1 locus, so that the mouse expresses a human or humanized CLDN1 protein. Specifically, using gene editing technology, under the control of the mouse CLDN1 gene regulatory element, a partial sequence of exon 1 to a partial sequence coding region of about 13.9 kb of human CLDN1 gene exon 1 to exon 4 is used to replace a partial sequence of about 12.5 kb of mouse exon 4, thereby obtaining a humanized CLDN1 locus and realizing the humanization of the mouse CLDN1 gene.

[0168] In order to realize the targeting strategy of the present invention, a targeting vector ( Figure 2 ), the targeting vector contains homology arm sequences upstream and downstream of the mouse CLDN1 gene, and an A fragment containing a human CLDN1 fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence of positions 26190287 to 26193238 of NCBI accession number NC_000082.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence of positions 26174590 to 26177787 of NCBI accession number NC_000082.7. The nucleotide sequence of the human CLDN1 gene fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 190308277 to 190322116 of NCBI accession number NC_000003.12; the connection design of the upstream of the human CLDN1 fragment sequence and the mouse is: The sequence " AGTGG The last "G" in " is the last nucleotide upstream of the mouse sequence and the human CLDN1 fragment sequence. The first "A" in is the first nucleotide of the human sequence. The downstream connection of the human CLDN1 fragment sequence and the mouse sequence is designed as: The sequence " TGTGA "A" is the last nucleotide in the human sequence. The "C" in is the first nucleotide downstream of the connection between the mouse sequence and the human CLDN1 fragment sequence.

[0169] 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 CLDN1 gene is designed as follows: The sequence " GTAAC The "C" in " is the last nucleotide of the human CLDN1 gene connected to the 5' end of the Neo box. The first "G" in is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the human CLDN1 gene is designed as: The sequence " CAACT "T" in the sequence is the last nucleotide of the Neo box. The first "A" in is the first nucleotide connecting the human CLDN1 gene to the 3' end of the Neo box. The mRNA sequence transcribed from the modified humanized mouse CLDN1 gene is shown in SEQ ID NO: 10, and the expressed protein sequence is shown in SEQ ID NO: 11.

[0170] 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 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 CLDN1 gene humanized homozygous mice.

[0171] The genotype of the somatic cells of the F1 generation mice can be identified by PCR, and the primers described in Table 3 are used for PCR identification. The exemplary results are as follows: Figure 3 As shown, combined with the PCR and sequencing results, two mice numbered F1-1 and F1-2 were positive mice. This shows that the present method can be used to construct a CLDN1 gene humanized mouse that can be stably propagated and has no random insertion.

[0172] Table 3 PCR primer sequences and target fragment sizes

[0173]

[0174] The expression of mRNA in humanized CLDN1 gene mice can be detected by RT-PCR. Specifically, 1 6-week-old C57BL / 6 mouse (+ / +) and 1 6-week-old male homozygous CLDN1 gene humanized mouse (H / H) prepared in this example were selected, and liver tissues were obtained after euthanasia by cervical dislocation. RT-PCR detection was performed using the primer sequences shown in Table 4. The detection results are shown in FIG. Figure 4 As shown. Figure 4 As can be seen, only mouse CLDN1 mRNA was detected in wild-type C57BL / 6 mice, and no humanized CLDN1 mRNA was detected; only humanized CLDN1 mRNA was detected in mice homozygous for the humanized CLDN1 gene.

[0175] In another similar experiment, 6-week-old C57BL / 6 mice (+ / +) and 6-week-old male CLDN1 gene humanized homozygous (H / H) prepared in this example were selected, and liver, skin, spleen and small intestine tissues were obtained after euthanasia by cervical dislocation. RT-PCR detection was performed using the primer sequences shown in Table 4. The detection results are shown in FIG. Figure 5 As shown. Figure 5 It can be seen that only mouse CLDN1 mRNA was detected in the liver, skin, spleen and small intestine tissues of wild-type C57BL / 6 mice, and no humanized CLDN1 mRNA was detected; only humanized CLDN1 mRNA was detected in the liver, skin, spleen and small intestine tissues of CLDN1 gene homozygous mice.

[0176] Table 4 RT-PCR primer sequences and target fragment sizes

[0177]

[0178] In addition, the expression of humanized CLDN1 protein in CLDN1 humanized mice can be detected by conventional methods such as flow cytometry. Specifically, 6-week-old male C57BL / 6 mice (+ / +) and 6-week-old male CLDN1 homozygous humanized gene (H / H) prepared in this example were selected, liver tissues were taken, and anti-mouse CD45 antibody Brilliant Violet510 was used to detect the expression of humanized CLDN1 protein in CLDN1 humanized mice. TM After staining with anti-mouse CD45 (mCD45), anti-human CLDN1 antibody Anti-CLDN1-analogs-1 (hCLDN1-1) and anti-human CLDN1 antibody Anti-CLDN1-analogs-2 (hCLDN1-2), flow cytometry was performed to detect the expression of humanized CLDN1 protein.

[0179] The results showed that 1.25% of the positive cells (characterized by mCD45-hCLDN1-1+) were found in the liver tissue of wild-type C57BL / 6 mice, and 20.9% of the positive cells (characterized by mCD45-hCLDN1-1+) were found in the liver tissue of CLDN1 gene homozygous mice; 1.90% of the positive cells (characterized by mCD45-hCLDN1-2+) were found in the liver tissue of wild-type C57BL / 6 mice, and 16.5% of the positive cells (characterized by mCD45-hCLDN1-2+) were found in the liver tissue of CLDN1 gene homozygous mice.

[0180] The expression of humanized CLDN1 protein in CLDN1 humanized mice can also be detected by conventional methods such as Western Blot. Specifically, 6-week-old male C57BL / 6 mice (+ / +) and 6-week-old male CLDN1 humanized homozygotes (H / H) prepared in this example were selected, and liver, skin and spleen tissues were taken. Human-mouse cross antibody Claudin1 Monoclonal Antibody (purchased from Invitrogen, catalog number 37-4900) and Proteintech HRP-conjugated GAPDH Monoclonal antibody (purchased from Proteintech, catalog number HRP-60004) were used for detection. The test results are as follows: Figure 6 shown.

[0181] from Figure 6 As can be seen, CLDN1 protein expression can be detected in the liver and skin tissues of wild-type C57BL / 6 mice and CLDN1 humanized homozygous mice, indicating that humanized CLDN1 protein can be successfully expressed in CLDN1 gene homozygous mice.

[0182] Example 2 Pharmacodynamic Model

[0183] The CLDN1 humanized mice prepared by this method can be used to evaluate the efficacy of regulators targeting human CLDN1 in cancer. Taking the tumor model as an example, CLDN1 humanized mice homozygous for MC38 cells were subcutaneously inoculated, and the tumor volume grew 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 drugs targeting human CLDN1, 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.

[0184] Example 3 Preparation of double-gene or multi-gene humanized mice

[0185] The humanized CLDN1 gene mice prepared by the present method can also be used to prepare a multi-gene humanized mouse model. For example, in the above-mentioned Example 1, the embryonic stem cells used for microinjection can be selected from mice modified with at least one gene containing LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4, or, on the basis of humanized CLDN1 mice, mouse ES embryonic stem cells can be separated and gene recombination targeting technology can be used to obtain a double gene humanized or multi-gene humanized mouse model. The homozygous or heterozygous CLDN1 mouse obtained by the present method can also be mated with other gene-modified mice, and the offspring can be screened. According to Mendel's genetic law, there is a certain probability that a multi-gene modified mouse modified with a humanized CLDN1 gene and other genes can be obtained, and then the heterozygotes can be mated with each other to obtain a homozygous double gene or multi-gene modified mouse.

[0186] 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.

[0187] 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.

[0188] 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 human or chimeric claudin 1 (CLDN1).

2. The construction method according to claim 1, characterized in that: The amino acid sequence of the human or chimeric CLDN1 protein comprises SEQ ID NO: 2, SEQ ID NO: 11, positions 22-211 of SEQ ID NO: 2, positions 29-211 of SEQ ID NO: 2, or positions 31-211 of SEQ ID NO: 2, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 2, SEQ ID NO: 11, positions 22-211 of SEQ ID NO: 2, positions 29-211 of SEQ ID NO: 2, or positions 31-211 of SEQ ID NO:

2.

3. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises a nucleotide sequence of human CLDN1 replacing a nucleotide sequence of a corresponding region of endogenous CLDN1 at an endogenous CLDN1 locus.

4. The construction method according to any one of claims 1 to 3, characterized in that: The nucleotide sequence encoding the human or chimeric CLDN1 protein or the nucleotide sequence of human CLDN1 is operably linked to an endogenous regulatory element of an endogenous CLDN1 locus; Preferably, the endogenous CLDN1 protein of the non-human animal is not expressed or is expressed at a reduced level compared to CLDN1 in wild-type animals; Preferably, the modified CLDN1 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

5. The construction method according to claim 3 or 4, characterized in that: The nucleotide sequence of human CLDN1 encodes human or chimeric CLDN1 protein; Preferably, the nucleotide sequence of human CLDN1 encodes the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 2 positions 22-211, SEQ ID NO: 2 positions 29-211, or SEQ ID NO: 2 positions 31-211; Preferably, the nucleotide sequence of human CLDN1 comprises a portion of exon 1 to a portion of exon 4 of the human CLDN1 gene; More preferably, the nucleotide sequence of human CLDN1 comprises SEQ ID NO: 5, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

5.

6. The construction method according to any one of claims 3 to 5, characterized in that: The nucleotide sequence of the corresponding region of endogenous CLDN1 encodes the amino acid sequence of SEQ ID NO: 1, positions 22 to 211 of SEQ ID NO: 1, positions 29 to 211 of SEQ ID NO: 1, or positions 31 to 211 of SEQ ID NO: 1; Preferably, the nucleotide sequence of the corresponding region of endogenous CLDN1 includes a portion of exon 1 to a portion of exon 4 of the CLDN1 gene of a non-human animal.

7. The construction method according to any one of claims 1 to 6, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the non-human animal is a mouse or a rat; Preferably, the mRNA transcribed from the modified gene in the genome of the non-human animal comprises SEQ ID NO: 10, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

10.

8. The construction method according to any one of claims 1 to 7, characterized in that: The non-human animal also includes nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins are selected from at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

9. An application of a non-human animal obtained by the construction method according to any one of claims 1 to 8, characterized in that: The application includes: A) Application in the development of products involving CLDN1-related immune processes in human cells; B) Application as a model system related to CLDN1 for pharmacological, immunological, microbiological and medical research; C) Applications involving the production and use of animal experimental disease models for the study of CLDN1-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human CLDN1 signaling pathway modulators; or, E) Study the function of CLDN1 gene, study the drugs and efficacy targeting human CLDN1 target sites, and study the application of CLDN1-related cancer drugs.

10. 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 8; 2) Determine the effect of a therapeutic agent on a disease or non-human animal; Preferably, the therapeutic agent is a therapeutic agent targeting CLDN1, such as an anti-CLDN1 antibody, a nucleic acid drug or a polypeptide drug; further preferably, the therapeutic agent further comprises an additional therapeutic agent, such as an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; Preferably, the disease comprises cancer; Further preferably, the cancer is a solid tumor or a blood tumor; for example, breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, head and neck cancer, liver cancer, lymphoma or lung cancer.

11. A humanized CLDN1 protein, characterized in that: The humanized CLDN1 protein includes a portion of a human CLDN1 protein and a portion of a non-human animal CLDN1 protein; Preferably, the portion of the human CLDN1 protein includes the amino acid sequence of SEQ ID NO: 2, positions 22-211, SEQ ID NO: 2, positions 29-211, or SEQ ID NO: 2, positions 31-211; Further preferably, the amino acid sequence of the humanized CLDN1 protein comprises SEQ ID NO:

11.

12. A humanized CLDN1 gene, characterized in that: The humanized CLDN1 gene encodes the humanized CLDN1 protein according to claim 11; Preferably, the humanized CLDN1 gene comprises any of the following nucleotide sequences: A) a nucleotide sequence encoding amino acids 22-211 of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 2, SEQ ID NO: 2, SEQ ID NO: 2, SEQ ID NO: 2, or SEQ ID NO: 2; B) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10; C) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10; D) a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10.

13. A cell, tissue or organ, characterized in that: The cell, tissue or organ expresses the humanized CLDN1 protein according to claim 11, or contains the humanized CLDN1 gene according to claim 12.

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