Construction method and application of CD70 gene humanized non-human animals

Abstract

The present invention provides a method for constructing a non-human animal with a humanized CD70 gene, wherein a nucleotide sequence encoding a human CD70 protein is introduced into the non-human animal genome by homologous recombination, and the animal can normally express human or humanized CD70 protein in vivo, and can be used as an animal model for studying the human CD70 signaling mechanism, screening drugs for tumors and immune-related diseases, and has important application value for the development of new drugs for immune targets. The present invention also provides a humanized CD70 protein, a humanized CD70 gene, a targeting vector for the CD70 gene, and the application of the above-constructed non-human animal in the field of biomedicine.

Description

Technical Field

[0001] The present invention belongs to the field of animal genetic engineering and genetic modification, and specifically relates to a method for constructing a CD70 gene-modified non-human animal model and its application in the field of biomedicine. Background Art

[0002] CD70 is a member of the tumor necrosis factor (TNF) superfamily and a type II transmembrane protein that is mainly expressed in activated T cells, B cells, natural killer (NK) cells, and dendritic cells. The interaction between CD70 and its receptor CD27 can promote the activation, proliferation, and differentiation of T cells, B cells, and NK cells, and is a necessary condition for the generation of immune responses. In cellular immunity, the main biological function of CD70 / CD27 is to promote the activation of T cells and their proliferation, making them effector T cells and memory T cells; in humoral immunity, the main function of CD70 / CD27 is to promote B cell differentiation and plasma cell proliferation; for NK cells, the direct interaction between CD27 and CD70 on the surface of NK cells can increase the killing activity of NK cells, promote NK cell differentiation, and release IFN-γ.

[0003] Under physiological conditions, CD70 is highly precisely regulated by antibodies, antigens, cytokines, etc., so it is expressed on the surface of activated T cells and B cells for a short time. In addition to being expressed on normal lymphocytes, CD70 is also highly expressed on the surface of a variety of tumor cells, especially hematogenous tumors, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and Waldenstroms macroglobulinemia, which are potential targets for targeted and immunotherapy of some tumors; in non-hematogenous tumors, high expression of CD70 was first found in undifferentiated nasopharyngeal carcinoma and germ cell carcinoma. Subsequent studies have found that CD70 is also highly expressed in tumors such as thymoma, nephroblastoma, glioblastoma, astrocytoma, and ovarian cancer, especially renal cancer. It is currently considered a new specific tumor marker for renal cancer. In addition, studies have found that the costimulatory signal provided by the CD27 / CD70 interaction plays a promoting role in the occurrence and development of a variety of autoimmune diseases, and selective intervention in this costimulatory pathway can alleviate or reduce the development of certain autoimmune diseases. For example, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) may become a specific therapeutic target for autoimmune diseases.

[0004] Currently, monoclonal antibodies targeting CD70 have entered clinical trials in humans and have shown good safety and therapeutic effects. For example, the humanized monoclonal antibody Cusatuzumab, which Janssen acquired with an upfront payment of US$300 million and an equity investment of US$200 million, not only showed good safety and tolerability in a Phase I / II clinical trial of CD70-positive advanced cutaneous T-cell lymphoma, but also demonstrated a certain anti-cancer effect, demonstrating its potential in treating the disease; in another Phase I / II clinical trial of acute myeloid leukemia, the combination of Cusatuzumab and Azacitidine had a higher overall remission rate compared to historical data of Azacitidine alone.

[0005] Experimental animal disease models are indispensable research tools for studying the causes and pathogenesis of human diseases, developing prevention and treatment technologies, and developing drugs. However, due to the differences in the physiological structure and metabolic system between animals and humans, traditional animal models cannot well reflect the real condition of the human body. It is an urgent need for the biopharmaceutical industry to establish disease models in animals that are closer to human physiological characteristics. Therefore, due to the differences in physiology and pathology between animals and humans, coupled with the complexity of genes, how to construct an "effective" humanized animal model for new drug development is still the biggest challenge.

[0006] In view of the huge application potential of the CD27 / CD70 signaling pathway in the treatment of tumors and autoimmune diseases, in order to further explore its related biological characteristics, improve the effectiveness of preclinical efficacy tests, improve the success rate of research and development, make preclinical tests more effective and minimize research and development failures, the field urgently needs to develop non-human animal models of the CD27 / CD70 signaling pathway. In addition, the non-human animals obtained by this method can also be mated with other genetically humanized non-human animals to obtain a multi-gene humanized animal model, which is used to screen and evaluate the efficacy of human drugs and combination drugs targeting this signaling pathway. The present invention has broad application prospects in academic and clinical research. Summary of the invention

[0007] This application replaces the homologous gene of the animal genome with a normal or mutant gene of human origin, and establishes a normal or mutant gene animal model that is closer to human physiological or disease characteristics. It improves and enhances the humanized animal model of cell or tissue transplantation. More importantly, due to the insertion of human gene fragments, the animal can express or partially express human proteins, which can be used as targets for drugs that can only recognize human protein sequences, making it possible to screen anti-human antibodies and other drugs at the animal level.

[0008] According to a first aspect of the present invention, a humanized CD70 protein is provided, wherein the humanized CD70 protein comprises all or part of the human CD70 protein.

[0009] Preferably, the humanized CD70 protein comprises the extracellular region, transmembrane region and / or cytoplasmic region of the human CD70 protein.

[0010] Preferably, the humanized CD70 protein comprises all or part of the extracellular region of the human CD70 protein. It is further preferred that it comprises at least 50 to at least 154, such as 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154 consecutive amino acids of the extracellular region of human CD70. It is further preferred that the extracellular region comprises 0-20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids removed from the C-terminus and / or N-terminus of the extracellular region of human CD70.

[0011] Preferably, the humanized CD70 protein also comprises a portion of a non-human animal CD70 protein, and further preferably, the portion of the non-human animal CD70 protein includes the transmembrane region and / or cytoplasmic region of the non-human animal CD70 protein; even more preferably, it also comprises a portion of the extracellular region of the non-human animal CD70 protein, and even more preferably comprises a continuous 0-20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acid sequence at the C-terminus and / or N-terminus of the non-human animal extracellular region.

[0012] Preferably, the portion of the human CD70 protein and the portion of the non-human animal CD70 protein are directly connected or indirectly connected (eg, a peptide linker).

[0013] In a specific embodiment of the present invention, the humanized CD70 protein comprises the extracellular region of human CD70 protein, the transmembrane region and the cytoplasmic region of a non-human animal, and preferably also includes part of the extracellular region of a non-human animal.

[0014] Preferably, the portion of the human CD70 protein comprises the amino acid sequence as shown in positions 48-193 or 39-193 of SEQ ID NO:2; or, comprises at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence as shown in positions 48-193 or 39-193 of SEQ ID NO:2; or, comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference from the amino acid sequence as shown in positions 48-193 or 39-193 of SEQ ID NO:2; or, comprises the amino acid sequence as shown in positions 48-193 or 39-193 of SEQ ID NO:2, including substitution, deletion and / or insertion of one or more amino acid residues.

[0015] Preferably, the portion of the non-human animal CD70 protein comprises the amino acid sequence shown in positions 1-44 or 1-49 of SEQ ID NO: 1; or, comprises at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 1-44 or 1-49 of SEQ ID NO: 1; or, comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference from the amino acid sequence shown in positions 1-44 or 1-49 of SEQ ID NO: 1; or, comprises the amino acid sequence shown in positions 1-44 or 1-49 of SEQ ID NO: 1, including substitution, deletion and / or insertion of one or more amino acid residues.

[0016] In a specific embodiment of the present invention, the humanized CD70 protein comprises the amino acid sequence shown at positions 48-193 or 39-193 of SEQ ID NO:2 and the amino acid sequence shown at positions 1-44 or 1-49 of SEQ ID NO:1.

[0017] Preferably, the portion of human CD70 protein comprises all or part of the amino acid sequence encoded by the human CD70 gene.

[0018] Preferably, the portion of the human CD70 protein comprises all or part of the amino acid sequence encoded by exon 1, exon 2 and / or exon 3 of the human CD70 gene; preferably, the humanized CD70 protein comprises all or part of the amino acid sequence encoded by exons 1 to 3 of the human CD70 gene. Further preferably, the humanized CD70 protein comprises all or part of the amino acid sequence encoded by one, two, three or a combination of two consecutive exons from exons 1 to 3 of the human CD70 gene. Even further preferably, the humanized CD70 protein comprises part of exon 1 and amino acid sequences encoded by exons 2 to 3 of the human CD70 gene, wherein the portion of exon 1 is preferably a nucleotide sequence encoding a transmembrane region and / or an extracellular region.

[0019] Preferably, the portion of the non-human animal CD70 protein comprises a partial amino acid sequence encoded by a non-human animal CD70 gene, preferably a partial amino acid sequence encoded by exon 1 of a non-human animal CD70 gene.

[0020] In a specific embodiment of the present invention, the humanized CD70 protein comprises a portion of exon 1 of a non-human animal CD70 gene and the entire amino acid sequence encoded by exons 2 to 3 of the human CD70 gene, and preferably also comprises a partial amino acid sequence encoded by exon 1 of the human CD70 gene.

[0021] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0022] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0023] Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice or nude mice.

[0024] Preferably, the amino acid sequence derived from human CD70 protein in the humanized CD70 protein is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 2.

[0025] Preferably, the amino acid sequence of the non-human animal CD70 protein in the humanized CD70 protein is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0026] Preferably, the humanized CD70 protein contains at least 50 amino acid sequences that are identical to the corresponding amino acid sequences of human CD70. Furthermore, the 50 amino acid sequences are the amino acid sequences starting from the first amino acid at the C-terminus.

[0027] In a specific embodiment of the present invention, the amino acid sequence of the humanized CD70 protein comprises one of the following groups:

[0028] a) is all or part of the amino acid sequence of SEQ ID NO: 11;

[0029] b) at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 11;

[0030] c) differs from the amino acid sequence of SEQ ID NO: 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

[0031] d) an amino acid sequence as shown in SEQ ID NO: 11, comprising a substitution, deletion and / or insertion of one or more amino acid residues.

[0032] The second aspect of the present invention provides a nucleic acid encoding the humanized CD70 protein.

[0033] The third aspect of the present invention provides a humanized CD70 gene, wherein the humanized CD70 gene comprises a portion of the human CD70 gene.

[0034] Preferably, the humanized CD70 gene encodes the humanized CD70 protein.

[0035] Preferably, the humanized CD70 gene comprises all or part of exons 1 to 3 of the human CD70 gene, further preferably comprises any one, two or three or two consecutive exons of exons 1 to 3 of the human CD70 gene, and further preferably comprises part of exon 1, all of exon 2 and part of exon 3 of the human CD70 gene, wherein the part of exon 1 comprises at least 10 bp to at least 310 bp of exon 1, for example, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100, 150, 200, 250, 300, 310 bp of continuous nucleotide sequence, the portion of exon 3 at least includes at least 100 bp to at least 567 bp of exon 3, for example, 100, 200, 300, 350, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 500, 550, 567 bp of continuous nucleotide sequence, preferably also includes introns 1-2 and / or introns 2-3.

[0036] Preferably, the humanized CD70 gene comprises all or part of the genomic DNA sequence, cDNA sequence or CDS sequence of the human CD70 gene.

[0037] In a specific embodiment of the present invention, the partial nucleotide sequence of the human CD70 gene contained in the humanized CD70 gene comprises one of the following groups:

[0038] (A) all or part of the nucleotide sequence shown in SEQ ID NO: 5;

[0039] (B) is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:5;

[0040] (C) differs from the nucleotide sequence of SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or

[0041] (D) A nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0042] Preferably, the humanized CD70 gene also comprises a portion of a non-human animal CD70 gene, and further preferably comprises all or part of exon 1 and / or all or part of exon 3 of a non-human animal CD70 gene.

[0043] In a specific embodiment of the present invention, the humanized CD70 gene comprises part of exon 1 of non-human animal CD70, part of exon 1 of human CD70 gene, all of exon 2, part of exon 3, and part of exon 3 of non-human animal CD70 gene. Preferably, it also comprises introns 1-2 and / or introns 2-3 of human CD70 gene.

[0044] Preferably, the humanized CD70 gene comprises all or part of the nucleotide sequence encoding the transmembrane region, cytoplasmic region and / or extracellular region of the human CD70 protein. Further preferably, the humanized CD70 gene comprises all or part of the nucleotide sequence encoding the extracellular region of the human CD70 protein.

[0045] Preferably, the humanized CD70 gene comprises a nucleotide sequence encoding positions 39-193 or 48-193 of SEQ ID NO: 2; or, comprises a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to a nucleotide sequence encoding positions 39-193 or 48-193 of SEQ ID NO: 2; or, comprises a nucleotide sequence that differs from a nucleotide sequence encoding positions 39-193 or 48-193 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence that has a nucleotide sequence encoding positions 39-193 or 48-193 of SEQ ID NO: 2, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0046] Preferably, the humanized CD70 gene further comprises a partial nucleotide sequence encoding a non-human animal CD70 protein. Further preferably, it comprises all or part of a nucleotide sequence encoding a cytoplasmic region and / or a transmembrane region of a non-human animal CD70 protein, and even more preferably, it further comprises a partial nucleotide sequence encoding an extracellular region of a non-human animal CD70 protein.

[0047] In a specific embodiment of the present invention, the humanized CD70 gene comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence encoding an amino acid sequence at positions 1-44 or 1-49 of SEQ ID NO: 1 or is consistent with a nucleotide sequence encoding an amino acid sequence at positions 1-44 or 1-49 of SEQ ID NO: 1.

[0048] Preferably, the humanized CD70 gene further includes the 5'UTR and / or 3'UTR of the non-human animal CD70 gene.

[0049] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0050] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0051] Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice or nude mice.

[0052] Preferably, the nucleotide sequence of the humanized CD70 gene comprises a nucleotide sequence that is at least 90% or at least 95% identical to SEQ ID NO: 6 and / or SEQ ID NO: 7, or comprises the nucleotide sequence shown in SEQ ID NO: 6 and / or SEQ ID NO: 7.

[0053] Preferably, the nucleotide sequence of the humanized CD70 gene comprises a nucleotide sequence that is at least 90% or at least 95% identical to SEQ ID NO: 8 and / or SEQ ID NO: 9, or comprises the nucleotide sequence shown in SEQ ID NO: 8 and / or SEQ ID NO: 9.

[0054] In a specific embodiment of the present invention, the mRNA transcribed from the humanized CD70 gene comprises any one of the following groups:

[0055] (a) all or part of the nucleotide sequence shown in SEQ ID NO: 10;

[0056] (b) is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:10;

[0057] (c) differs from the nucleotide sequence of SEQ ID NO: 10 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or

[0058] (d) A nucleotide sequence comprising a substitution, deletion and / or insertion of one or more nucleotides as shown in the nucleotide sequence shown in SEQ ID NO: 10.

[0059] Preferably, the humanized CD70 gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a conventional substance that can be induced or repressed.

[0060] In a specific embodiment of the present invention, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0061] In a fourth aspect of the present invention, a targeting vector is provided, wherein the targeting vector comprises a donor nucleotide sequence, and the donor nucleotide sequence comprises one of the following groups:

[0062] A) all or part of a nucleotide sequence encoding a human or humanized CD70 protein;

[0063] B) all or part of a nucleotide sequence encoding the extracellular region, transmembrane region and / or cytoplasmic region of human CD70 protein, preferably all or part of a nucleotide sequence encoding the extracellular region of human CD70 protein, further preferably comprising a nucleotide sequence encoding at least 50 to at least 154, such as 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154 consecutive amino acids of the extracellular region of human CD70, further preferably comprising a nucleotide sequence encoding the extracellular region of the extracellular region of human CD70 with 0-20 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids removed from the C-terminus and / or N-terminus, more preferably encoding a nucleotide sequence comprising the amino acids shown in positions 39-193 or 48-193 of SEQ ID NO: 2; or, encoding a nucleotide sequence comprising the amino acids shown in positions 39-193 or 48-193 of SEQ ID NO: 2; a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence set forth in positions 39-193 or 48-193 of SEQ ID NO:2; or, comprises a nucleotide sequence that differs from the nucleotide sequence encoding positions 39-193 or 48-193 of SEQ ID NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence that has a nucleotide sequence encoding positions 39-193 or 48-193 of SEQ ID NO:2, including a substitution, deletion and / or insertion of one or more nucleotides;

[0064] C) all or part of the human or humanized CD70 gene; or,

[0065] D) all or part of exons 1 to 3 of the human CD70 gene, preferably any one, two or three or two consecutive exons of exons 1 to 3 of the human CD70 gene, further preferably part of exon 1, all of exon 2 and part of exon 3 of the human CD70 gene, wherein the part of exon 1 at least includes at least 10 bp to at least 310 bp of exon 1, for example 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100 , 150, 200, 250, 300, 310 bp of continuous nucleotide sequence, the portion of exon 3 at least includes at least 100 bp to at least 567 bp of exon 3, such as 100, 200, 300, 350, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 500, 550, 567 bp of continuous nucleotide sequence, preferably also includes introns 1-2 and / or introns 2-3; further preferably includes SEQ ID NO:5; or, comprising a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:5; or, comprising a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprising a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:5, including substitution, deletion and / or insertion of one or more nucleotides.

[0066] Preferably, the targeting vector further comprises a 5' arm and / or a 3' arm.

[0067] The 5' arm (or 5' homology arm) is a DNA fragment homologous to the 5' end of the switch region to be changed, which is selected from a nucleotide of 100-10000 length of the genomic DNA of the CD70 gene of a non-human animal. Preferably, the 5' arm has at least 90% homology with the nucleotide with NCBI accession number NC_000083.7. Further preferably, the 5' arm sequence has 90% homology with SEQ ID NO: 3, or is as shown in SEQ ID NO: 3.

[0068] The 3' arm (or 3' homology arm) is a DNA fragment homologous to the 3' end of the switch region to be changed, which is selected from a nucleotide of 100-10000 length of the genomic DNA of the CD70 gene of a non-human animal. Preferably, the 3' arm has at least 90% homology with the nucleotide with NCBI accession number NC_000083.7. Further preferably, the 3' arm sequence has 90% homology with SEQ ID NO: 4, or is as shown in SEQ ID NO: 4.

[0069] Preferably, the targeting vector further comprises a non-human animal 3'UTR.

[0070] Preferably, the targeting vector further comprises a non-human animal 5'UTR.

[0071] Preferably, the switch region to be changed is located at the non-human animal CD70 locus. Further preferably, the switch region to be changed is located at exons 1 to 3 of the non-human animal CD70 gene. Further preferably, it is located at exons 1 and / or 3 of the non-human animal CD70 gene.

[0072] Preferably, the targeting vector comprises a nucleotide sequence that is at least 90% or at least 95% identical to SEQ ID NO: 6 and / or SEQ ID NO: 7, or comprises a nucleotide sequence as shown in SEQ ID NO: 6 and / or SEQ ID NO: 7.

[0073] Preferably, the targeting vector comprises a nucleotide sequence that is at least 90% or at least 95% identical to SEQ ID NO: 8 and / or SEQ ID NO: 9, or comprises a nucleotide sequence as shown in SEQ ID NO: 8 and / or SEQ ID NO: 9.

[0074] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0075] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0076] Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is a NOD-Prkdc scidIL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice or nude mice.

[0077] Preferably, the targeting vector further comprises a marker gene. Further preferably, the marker gene is a gene encoding a negative selection marker. Even further preferably, the gene encoding the negative selection marker is a gene encoding a diphtheria toxin A subunit (DTA).

[0078] In a specific embodiment of the present invention, the targeting vector further comprises a resistance gene for positive clone screening. Further preferably, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo.

[0079] In a specific embodiment of the present invention, the targeting vector further comprises a specific recombination system. Further preferably, the specific recombination system is an Frt recombination site (a conventional LoxP recombination system may also be selected). The specific recombination system has two Frt recombination sites, which are respectively connected to both sides of the resistance gene.

[0080] The fifth aspect of the present invention provides a cell comprising the above-mentioned targeting vector.

[0081] In a sixth aspect of the present invention, the use of the above-mentioned targeting vector and cells containing the above-mentioned targeting vector in CD70 gene modification is provided. Preferably, the application includes but is not limited to knockout, insertion or replacement.

[0082] The seventh aspect of the present invention provides a method for constructing a non-human animal with a humanized CD70 gene, wherein the non-human animal expresses human or humanized CD70 protein in vivo, and / or the genome of the non-human animal contains a human or humanized CD70 gene.

[0083] Preferably, the humanized CD70 protein is the above-mentioned humanized CD70 protein.

[0084] Preferably, the humanized CD70 gene is the above-mentioned humanized CD70 gene.

[0085] Preferably, the expression of endogenous CD70 protein in the non-human animal is reduced or absent.

[0086] Preferably, the genome of the non-human animal comprises a portion of the human CD70 gene, further preferably comprises all or part of exons 1 to 3 of the human CD70 gene, further preferably comprises any one, two, three or two consecutive exons of exons 1 to 3 of the human CD70 gene, and further preferably comprises a portion of exon 1, all of exon 2 and a portion of exon 3 of the human CD70 gene, wherein the portion of exon 1 comprises at least 10 bp to at least 310 bp of exon 1, for example, 10, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 50, 100, 150, 200, 250, 300, 310 bp of continuous nucleotide sequence, the portion of exon 3 at least includes at least 100 bp to at least 567 bp of exon 3, such as 100, 200, 300, 350, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 500, 550, 567 bp of continuous nucleotide sequence, preferably also includes introns 1-2 and / or introns 2-3; further preferably includes SEQ ID NO:5; or, comprising a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:5; or, comprising a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprising a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:5, including substitution, deletion and / or insertion of one or more nucleotides.

[0087] Preferably, the genome of the non-human animal comprises all or part of a nucleotide sequence encoding a human CD70 protein, more preferably comprises all or part of a nucleotide sequence encoding an extracellular region, a cytoplasmic region and / or a transmembrane region of a human CD70 protein, and even more preferably comprises all or part of a nucleotide sequence encoding an extracellular region of a human CD70 protein, wherein the portion of the extracellular region of a human CD70 protein comprises at least 50 to at least 154, such as 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154 consecutive amino acids of the extracellular region of human CD70. It is further preferred that the extracellular region comprises 0-20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids removed from the C-terminus and / or N-terminus of the extracellular region of human CD70.

[0088] In a specific embodiment of the present invention, the non-human animal genome contains a nucleotide sequence encoding the amino acids shown in positions 39-193 or 48-193 of SEQ ID NO: 2; or, contains a nucleotide sequence encoding the amino acid sequence shown in positions 39-193 or 48-193 of SEQ ID NO: 2 that is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in positions 39-193 or 48-193 of SEQ ID NO: 2; or, contains a nucleotide sequence that differs from the nucleotide sequence encoding positions 39-193 or 48-193 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, contains a nucleotide sequence encoding positions 39-193 or 48-193 of SEQ ID NO: 2, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0089] Preferably, the construction method comprises introducing any of the following nucleotide sequences into the CD70 locus of a non-human animal:

[0090] A) all or part of a nucleotide sequence encoding a human or humanized CD70 protein;

[0091] B) all or part of a nucleotide sequence encoding the extracellular region, transmembrane region and / or cytoplasmic region of human CD70 protein, preferably all or part of a nucleotide sequence encoding the extracellular region of human CD70 protein, further preferably comprising a nucleotide sequence encoding at least 50 to at least 154, such as 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154 consecutive amino acids of the extracellular region of human CD70, further preferably comprising a nucleotide sequence encoding the extracellular region of the extracellular region of human CD70 with 0-20 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids removed from the C-terminus and / or N-terminus, more preferably encoding a nucleotide sequence comprising the amino acids shown in positions 39-193 or 48-193 of SEQ ID NO: 2; or, encoding a nucleotide sequence comprising the amino acids shown in positions 39-193 or 48-193 of SEQ ID NO: 2; a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence set forth in positions 39-193 or 48-193 of SEQ ID NO:2; or, comprises a nucleotide sequence that differs from the nucleotide sequence encoding positions 39-193 or 48-193 of SEQ ID NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence that has a nucleotide sequence encoding positions 39-193 or 48-193 of SEQ ID NO:2, including a substitution, deletion and / or insertion of one or more nucleotides;

[0092] C) all or part of the human or humanized CD70 gene; or,

[0093] D) all or part of exons 1 to 3 of the human CD70 gene, preferably any one, two or three or two consecutive exons of exons 1 to 3 of the human CD70 gene, further preferably part of exon 1, all of exon 2 and part of exon 3 of the human CD70 gene, wherein the part of exon 1 at least includes at least 10 bp to at least 310 bp of exon 1, for example 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100 , 150, 200, 250, 300, 310 bp of continuous nucleotide sequence, the portion of exon 3 at least includes at least 100 bp to at least 567 bp of exon 3, such as 100, 200, 300, 350, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 500, 550, 567 bp of continuous nucleotide sequence, preferably also includes introns 1-2 and / or introns 2-3; further preferably includes SEQ ID NO:5; or, comprising a nucleotide sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:5; or, comprising a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprising a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:5, including substitution, deletion and / or insertion of one or more nucleotides.

[0094] Preferably, the human or humanized CD70 gene, or the nucleotide sequence encoding the human or humanized CD70 protein is regulated in a non-human animal by a regulatory element, and the regulatory element is endogenous or exogenous.

[0095] Preferably, the regulatory element comprises an endogenous promoter.

[0096] Optionally, the endogenous regulatory element is a non-human animal CD70 gene regulatory element, and the exogenous regulatory element is a human CD70 gene regulatory element.

[0097] Preferably, the introduction is insertion or replacement.

[0098] Preferably, the introduction of the non-human animal CD70 locus is to replace the corresponding region of the non-human animal, preferably to replace all or part of the nucleotide sequence encoding the human CD70 protein in the non-human animal genome, further preferably to replace the nucleotide sequence encoding part of the extracellular region of the endogenous CD70 protein in the non-human animal genome, and even more preferably to replace all or part of exons 1 to 3 of the CD70 gene in the non-human animal genome.

[0099] In a specific embodiment of the present invention, the introduction of the CD70 locus into a non-human animal is to replace the nucleotide sequence encoding amino acids 50 to 195 of SEQ ID NO: 1 in the non-human animal.

[0100] In a specific embodiment of the present invention, part of exon 1, all of exon 2 and part of exon 3 of the CD70 gene of a non-human animal are replaced; further preferably, introns 1-2 and / or introns 2-3 of the non-human animal are also replaced.

[0101] Preferably, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the non-human animal CD70 protein at the non-human animal CD70 locus with a cDNA sequence encoding the human CD70 protein or all or part of the nucleotide sequence encoding the human or humanized CD70 protein.

[0102] Preferably, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the transmembrane region, cytoplasmic region and / or extracellular region of the non-human animal CD70 protein on the non-human animal CD70 locus with all or part of the nucleotide sequence encoding the transmembrane region, cytoplasmic region and / or extracellular region of the non-human animal CD70 protein. Further preferably, all or part of the nucleotide sequence encoding the extracellular region of the human CD70 protein is inserted or replaced with all or part of the nucleotide sequence encoding the extracellular region of the non-human animal CD70 protein on the non-human animal CD70 locus. In a specific embodiment, the nucleotide sequence encoding the 48th to 193rd amino acids of SEQ ID NO: 2 is inserted or replaced with the nucleotide sequence encoding the 50th to 195th amino acids of SEQ ID NO: 1 on the non-human animal CD70 locus. In a specific embodiment, the nucleotide sequence encoding the 39th to 193rd amino acids of SEQ ID NO: 2 is inserted or replaced with the nucleotide sequence encoding the 45th to 195th amino acids of SEQ ID NO: 1 on the non-human animal CD70 locus.

[0103] Preferably, the construction method comprises inserting or replacing a nucleotide sequence comprising the humanized CD70 gene into a non-human animal CD70 locus. Preferably, the construction method comprises inserting or replacing a portion of an endogenous CD70 gene on a non-human animal CD70 locus with a portion comprising the human CD70 gene. Preferably, the construction method comprises inserting or replacing all or part of exons 1 to 3 of a non-human animal CD70 gene with all or part of exons 1 to 3 of a human CD70 gene. Preferably, the construction method comprises inserting or replacing a portion of exon 1, all of exon 2, and a portion of exon 3 of a non-human animal CD70 gene with a portion of exon 1, all of exon 2, and a portion of exon 3 of a human CD70 gene, preferably introns 1-2 and / or introns 2-3 are replaced.

[0104] Preferably, the construction method includes modifying the coding frame of the non-human animal CD70 gene, inserting any nucleotide sequence as described above into the endogenous regulatory element of the non-human animal CD70 gene, wherein the coding frame of the modified non-human animal CD70 gene can be knocked out of the functional region of the non-human animal CD70 gene or inserted into a sequence, so that the non-human animal CD70 protein is not expressed or the expression is reduced or the expressed protein is non-functional. Further preferably, the coding frame of the modified non-human animal CD70 gene can be the whole or part of the nucleotide sequence of exon 1 to exon 3 of the non-human animal CD70 gene. Preferably, the insertion is to first destroy the coding frame of the endogenous CD70 gene of the non-human animal, and then perform the insertion operation. Alternatively, the insertion step can cause a frameshift mutation to the endogenous CD70 gene and realize the step of inserting the human sequence.

[0105] Preferably, at least one chromosome in the genome of the non-human animal contains a humanized CD70 gene.

[0106] Preferably, at least one cell in the non-human animal expresses human or humanized CD70 protein.

[0107] Preferably, gene editing technology is used to construct non-human animals, and the gene editing technology includes gene targeting technology using embryonic stem cells, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology technology.

[0108] Preferably, the targeting vector of the present invention is used for constructing non-human animals.

[0109] Preferably, the construction method comprises introducing the targeting vector into non-human animal cells, culturing the cells, and then transplanting the cultured cells into the fallopian tubes of female non-human animals, allowing them to develop, and identifying and screening to obtain non-human animals with humanized CD70 genes.

[0110] Preferably, the construction method further comprises mating, in vitro fertilization or direct gene editing of the CD70 gene humanized non-human animal with other gene-modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0111] Preferably, the other gene is selected from at least one of PD-1, PD-L1, CD27, CD40, OX40, 4-1BB, and LAG3. Preferably, the human or humanized CD70 gene or other gene is homozygous for the endogenously modified locus; preferably, the human or humanized CD70 gene or other gene is heterozygous for the endogenously modified locus.

[0112] The eighth aspect of the present invention provides a humanized non-human animal, wherein the non-human animal expresses human or humanized CD70 protein in its body, and / or the genome of the non-human animal contains human or humanized CD70 gene.

[0113] Preferably, the humanized CD70 protein is the above-mentioned humanized CD70 protein.

[0114] Preferably, the humanized CD70 gene is the above-mentioned humanized CD70 gene.

[0115] Preferably, the nucleotide sequence encoding the human or humanized CD70 protein or the nucleotide sequence encoding the human or humanized CD70 gene is operably linked to an endogenous regulatory element at the endogenous CD70 locus in at least one chromosome.

[0116] Preferably, the expression of endogenous CD70 protein in the non-human animal is reduced or absent.

[0117] Preferably, the non-human animal further comprises other gene modifications, and the other genes are selected from at least one of PD-1, PD-L1, CD27, CD40, OX40, 4-1BB, and LAG3.

[0118] Preferably, the human or humanized CD70 gene and / or said other gene is homozygous for the endogenous modified locus.

[0119] Preferably, the human or humanized CD70 gene and / or said other gene is heterozygous for the endogenous modified locus.

[0120] The ninth aspect of the present invention provides a non-human animal lacking the CD70 gene.

[0121] Preferably, the non-human animal lacks all or part of the nucleotide sequence of the CD70 gene.

[0122] Preferably, the non-human animal lacks all or part of exons 1 to 3 of the CD70 gene. Further preferably, the nucleotide sequence of one, two, three or a combination of two consecutive exons from exons 1 to 3 is completely or partially deleted. Even more preferably, the nucleotide sequence of part of exon 1, all of exon 2 and part of exon 3 is deleted. Most preferably, the nucleotide sequence of part of exon 1, introns 1-2, all of exon 2, introns 2-3 and part of exon 3 is deleted.

[0123] Preferably, the portion of exon 1 of the CD70 gene deleted in the non-human animal does not at least include the non-coding region, and further preferably, does not include the portion encoding the cytoplasmic region and / or the transmembrane region. Even more preferably, the non-human animal also lacks the nucleotide sequence encoding part of the extracellular region on exon 1 of the CD70 gene.

[0124] In a specific embodiment of the present invention, the partial nucleotide sequence of exon 3 of the endogenous CD70 gene deleted in the non-human animal does not at least include the non-coding region.

[0125] Preferably, the non-human animal lacks all or part of the nucleotide sequence encoding the cytoplasmic region, the transmembrane region and / or the extracellular region. Further preferably, the non-human animal lacks all or part of the nucleotide sequence encoding the extracellular region and / or the transmembrane region. In a specific embodiment of the present invention, the non-human animal lacks all or part of the nucleotide sequence encoding the extracellular region.

[0126] The tenth aspect of the present invention provides a method for constructing the above-mentioned CD70 gene-deficient non-human animal, the construction method comprising using the above-mentioned targeting vector to construct the non-human animal.

[0127] The eleventh aspect of the present invention provides a method for constructing a multi-gene modified non-human animal, comprising the following steps:

[0128] I) providing the above-mentioned non-human animal, or the non-human animal obtained by the above-mentioned construction method;

[0129] II) mating, in vitro fertilization or direct gene editing of the non-human animal provided by step I) with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0130] Preferably, the other genetically modified non-human animals include non-human animals modified with genes PD-1, PD-L1, CD27, CD40, OX40, 4-1BB and / or LAG3.

[0131] Preferably, the multi-gene modified non-human animal is a two-gene humanized non-human animal, a three-gene humanized non-human animal, a four-gene humanized non-human animal, a five-gene humanized non-human animal, a six-gene humanized non-human animal, a seven-gene humanized non-human animal, an eight-gene humanized non-human animal or a nine-gene modified non-human animal.

[0132] Preferably, each of the multiple genes modified in the genome of the multi-gene modified non-human animal can be homozygous for the endogenous modified locus.

[0133] Preferably, each of the multiple genes modified in the genome of the multi-gene modified non-human animal can be heterozygous for the endogenous modified locus.

[0134] The twelfth aspect of the present invention provides a non-human animal or its offspring obtained by the above-mentioned construction method.

[0135] The thirteenth aspect of the present invention provides an animal model, wherein the animal model is derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, or the above-mentioned non-human animal or its offspring. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

[0136] In a fourteenth aspect of the present invention, a method for preparing an animal model is provided, the method comprising using the above-constructed CD70 gene humanized non-human animal, CD70 gene-deficient non-human animal or multi-gene modified non-human animal or its progeny. Preferably, the method further comprises the step of implanting tumor cells.

[0137] The fifteenth aspect of the present invention provides the use of the above-mentioned CD70 gene humanized non-human animals, CD70 gene-deficient non-human animals, multi-gene modified non-human animals or their progeny, or the CD70 gene humanized non-human animals, CD70 gene-deficient non-human animals, multi-gene modified non-human animals or their progeny obtained by the above-mentioned construction method in preparing animal models.

[0138] The sixteenth aspect of the present invention provides a cell, tissue or organ, which expresses the humanized CD70 protein, or the genome of the cell, tissue or organ contains the humanized CD70 gene, or the cell, tissue or organ is derived from the non-human animal, or is derived from the non-human animal obtained by the above-mentioned construction method.

[0139] The seventeenth aspect of the present invention provides a tumor tissue after tumor bearing, wherein the tumor tissue expresses the above-mentioned humanized CD70 protein, or the genome of the tumor tissue contains the above-mentioned humanized CD70 gene, or the tumor tissue is derived from the above-mentioned non-human animal or its offspring, the non-human animal obtained by the above-mentioned construction method, or the above-mentioned animal model.

[0140] The eighteenth aspect of the present invention provides a CD70 gene humanized cell, wherein the cell expresses human or humanized CD70 protein, or the genome of the cell contains a humanized CD70 gene.

[0141] Preferably, the humanized CD70 protein is the humanized CD70 protein described in the first aspect of the present invention.

[0142] Preferably, the expression of endogenous CD70 protein in the cells is reduced or absent.

[0143] Preferably, the genome of the cell contains all or part of the human CD70 gene. Further preferably, the cell contains the humanized CD70 gene.

[0144] In a nineteenth aspect, the present invention provides uses of the humanized CD70 protein, the humanized CD70 gene, the non-human animal or its progeny, the non-human animal obtained by the construction method, the animal model, the cell, tissue or organ, and the tumor tissue after tumor bearing, the uses comprising:

[0145] (A) Application in product development involving CD70-related immune processes in human cells;

[0146] (B) Application as a model system related to CD70 for pharmacology, immunology, microbiology and medical research;

[0147] (C) Applications involving the production and use of animal experimental disease models for the study of CD70-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0148] (D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human CD70 signaling pathway modulators; or

[0149] (E) To study the function of CD70 gene, the drugs targeting human CD70 target site and their efficacy, and the application of CD70-related immune-related disease drugs and anti-tumor drugs;

[0150] (F) Study the application of CD27 / CD70 signaling pathway in the treatment of tumors and immune-related diseases.

[0151] Preferably, the use may be for therapeutic purposes.

[0152] Preferably, the application may be for non-therapeutic purposes.

[0153] The twentieth aspect of the present invention provides a method for screening human CD70-specific regulators, the screening method comprising applying a regulator to an individual implanted with tumor cells and detecting tumor inhibitory activity; wherein the individual is selected from the above-mentioned non-human animal or its offspring, the non-human animal obtained by the above-mentioned construction method, or the above-mentioned animal model.

[0154] Preferably, the regulator is selected from CAR-T and drugs. Further preferably, the drug is an antibody.

[0155] Preferably, the regulator is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

[0156] Preferably, said detecting comprises measuring the size and / or proliferation rate of tumor cells.

[0157] Preferably, the detection method includes vernier caliper measurement, flow cytometry detection and / or in vivo animal imaging detection.

[0158] Preferably, the testing comprises assessing an individual's body weight, fat mass, activated pathways, neuroprotective activity or metabolic changes, including changes in food consumption or water consumption.

[0159] Preferably, the tumor cells are derived from humans or non-human animals.

[0160] Preferably, the method for screening human CD70-specific regulators can be for therapeutic purposes.

[0161] Preferably, the screening method for human CD70-specific modulators can be for non-therapeutic purposes. The screening method is used to screen or evaluate drugs, detect and compare the efficacy of candidate drugs to determine which candidate drugs can be used as drugs and which cannot be used as drugs, or compare the sensitivity of the efficacy of different drugs, that is, the therapeutic effect is not inevitable, but only a possibility.

[0162] The twenty-first aspect of the present invention provides an intervention regimen evaluation method, the evaluation method comprising implanting tumor cells into an individual, applying the intervention regimen to the individual implanted with tumor cells, and detecting and evaluating the tumor inhibition effect on the individual after the application of the intervention regimen; wherein the individual is selected from the above-mentioned non-human animals, the non-human animals obtained by the above-mentioned construction method, the above-mentioned non-human animals or their offspring, or the above-mentioned animal models.

[0163] Preferably, the intervention regimen is selected from CAR-T and drug therapy. Further preferably, the drug is an antigen binding protein. The antigen binding protein is an antibody.

[0164] Preferably, the tumor cells are derived from humans or non-human animals.

[0165] Preferably, the intervention regimen evaluation method may be for therapeutic purposes.

[0166] Preferably, the evaluation method of the intervention scheme may be for non-therapeutic purposes. The evaluation method detects and evaluates the effect of the intervention scheme to determine whether the intervention scheme has a therapeutic effect, that is, the therapeutic effect is not inevitable, but only a possibility.

[0167] The twenty-second aspect of the present invention provides a use of the non-human animal or its offspring derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, or the above-mentioned animal model in the preparation of a human CD70-specific regulator.

[0168] The twenty-third aspect of the present invention provides a use of the non-human animal or its offspring derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, or the above-mentioned animal model in the preparation of drugs for treating tumors, inflammation or immune-related diseases.

[0169] The "immune-related diseases" described in the present invention include, but are not limited to, allergies, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, etc.

[0170] The "inflammation" described in the present invention includes both 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, and specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).

[0171] The "tumor" described in the present invention includes, but is not limited to, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, 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.

[0172] The non-human animal with humanized CD70 gene of the present invention can normally express human or humanized CD70 protein in its body, and can be used for drug screening, drug efficacy evaluation, immune disease and tumor treatment for human CD70 target site, which can accelerate the new drug development process, save time and cost. It provides effective guarantee for studying CD70 protein function and drug screening for related diseases.

[0173] The "cell" described in the present invention can be a fertilized egg cell, an embryonic stem cell, an immune cell (such as a T cell, a B cell, a natural killer (NK) cell, and a dendritic cell), a tumor cell, etc. The tumor cell can be a hematogenous tumor, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and Waldenstrom's macroglobulinemia, and can also be a thymoma, a Wilms' tumor, a glioblastoma, an astrocytoma, and ovarian cancer, etc. Therefore, depending on the different sources of the cells, some of the cells described in the present application can develop into animal individuals, while some cannot develop into animal individuals.

[0174] The term "comprising" or "including" described in the present invention is an open description, which contains the specified components or steps described, as well as other specified components or steps that will not be substantially affected. However, 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 activity described in the present invention.

[0175] The “all or part” mentioned in the present invention, “all” refers to the whole, and “part” refers to a part of the whole, or an individual that makes up the whole.

[0176] The "humanized CD70 protein" described in the present invention includes a portion derived from a human CD70 protein and a portion of a non-human CD70 protein. Among them, the "human CD70 protein" is the same as the entire human CD70 protein, that is, its amino acid sequence is consistent with the full-length amino acid sequence of the human CD70 protein. The "portion of the human CD70 protein" is a continuous or intermittent 5-193 amino acid sequence that is consistent with the amino acid sequence of the human CD70 protein. Preferably, it is continuous or intermittent 10-146, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 146, 150, 160, 170, 180, 190, 193 amino acid sequences that are consistent with the amino acid sequence of the human CD70 protein.

[0177] The “entire transmembrane region of the human CD70 protein”, “entire cytoplasmic region of the human CD70 protein” or “entire extracellular region of the human CD70 protein” mentioned in the present invention means that its amino acid sequence is consistent with the full-length amino acid sequence of the transmembrane region, cytoplasmic region or extracellular region of the human CD70 protein, respectively.

[0178] The “part of the extracellular region of the human CD70 protein” described in the present invention is a continuous or intermittent 5-155 (preferably 10-146) amino acid sequence that is consistent with the amino acid sequence of the extracellular region of the human CD70 protein, for example, a continuous 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 146, 150, 155 amino acid sequence that is consistent with the amino acid sequence of the extracellular region of the human CD70 protein.

[0179] The "part of a non-human animal CD70 protein" described in the present invention is a continuous or intermittent 5-195 (preferably 10-50) amino acid sequence that is consistent with the amino acid sequence of the non-human animal CD70 protein, for example, a continuous 5, 10, 20, 30, 40, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 195 amino acid sequence that is consistent with the amino acid sequence of the non-human animal CD70 protein.

[0180] The "humanized CD70 gene" of the present invention includes a portion derived from the human CD70 gene and a portion derived from the non-human CD70 gene. The "human CD70 gene" is the same as the entire human CD70 gene, that is, its nucleotide sequence is consistent with the full-length nucleotide sequence of the human CD70 gene. The “portion of the human CD70 gene” is a continuous or intermittent 20-9503bp (preferably 20-4842bp or 20-911 or 20-441) nucleotide sequence consistent with the nucleotide sequence of the human CD70 gene, for example, 20, 50, 100, 200, 300, 400, 441, 500, 600, 700, 800, 900, 911, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 4000, 4842, 5000, 6000, 7000, 8000, 9000 or 9503bp nucleotide sequence consistent with the nucleotide sequence of the human CD70 gene.

[0181] The "part of an exon" of the present invention means that a continuous or intermittent nucleotide sequence of several, dozens or hundreds of nucleotides is consistent with the nucleotide sequence of all exons. For example, a part of exon 1 of the human CD70 gene includes a continuous or intermittent 5-310bp (preferably 10-21bp) nucleotide sequence that is consistent with the nucleotide sequence of exon 1 of the human CD70 gene. For example, a part of exon 3 of the human CD70 gene includes a continuous or intermittent 5-567bp (preferably 10-386bp) nucleotide sequence that is consistent with the nucleotide sequence of exon 3 of the human CD70 gene. In a specific embodiment of the present invention, the "part of exon 1" contained in the "human CD70 gene" includes at least the nucleotide sequence encoding the 48th amino acid of the human extracellular region to the last nucleotide sequence of exon 1. In a specific embodiment of the present invention, the "part of exon 3" contained in the "humanized CD70 gene" includes at least the first nucleotide sequence of exon 3 to the stop codon.

[0182] The "exons xx to xxx" or "all of exons xx to xxx" mentioned in the present invention include the nucleotide sequences of exons and introns therebetween, for example, the "exons 1 to 3" mentioned above include the entire nucleotide sequences of exon 1, introns 1-2, exon 2, introns 2-3 and exon 3.

[0183] The "intron x-xx" of the present invention refers to the intron between exon x and exon xx. For example, "intron 1-2" refers to the intron between exon 1 and exon 2.

[0184] The "part of the non-human animal CD70 gene" of the present invention is a continuous or intermittent 20-3781bp (20-764bp or 20-441bp) nucleotide sequence that is consistent with the nucleotide sequence of the non-human animal CD70 gene, for example, 20, 50, 100, 200, 300, 400, 441, 500, 600, 700, 764, 800, 900, 1000, 1100, 1200, 1300, The nucleotide sequences of 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, and 3781 are consistent with the nucleotide sequence of the CD70 gene of non-human animals.

[0185] The "gene locus" described 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 or a part of a gene. For example, the "CD70 gene locus" refers to an arbitrary DNA fragment on exons 1 to 3 of the CD70 gene.

[0186] The "nucleotide sequence" of the present invention includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences, preferably DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, and tRNA.

[0187] The term "treatment" as used herein means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic interventions that improve the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

[0188] The "homology" mentioned in the present invention means that in terms of the use of amino acid sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs, while ensuring that the structures or functions are similar to those of known sequences, so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36% compared with the sequences obtained by the prior art. , 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity.

[0189] One skilled in the art is able to determine and compare sequence elements or degrees of identity to distinguish between additional mouse and human sequences.

[0190] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology. These techniques are explained in detail in the following literature. For example: Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (DNGlovered., 1985); Oligonucleotide Synthesis (MJGaited., 1984); Mullisetal. US Pat. No. 4, 683, 195; Nucleic Acid Hybridization (BDHames&S.J.Higginseds.1984); Transcription And Translation (BDHames&S.J.Higginseds.1984); Culture Of Animal Cells (RIFreshney, AlanR.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds.inchief, Academic Press, Inc., New York), specifically, Vols.154and 155 (Wuetal.eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and M.P. Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods InCell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes V (DMWeir and CCBlackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0191] In one aspect, the non-human animal is a mammal. Preferably, the non-human animal is a small mammal, such as the family of Jerboas. In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is selected from mice, rats and hamsters. In one embodiment, the rodent is selected from the family of Muridae. In one embodiment, the genetically modified animal is from a family selected from the family of Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny mice, crested rats), Malvinas (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Dormouseidae (e.g., spiny dormouse) and Muridae (e.g., mole rats, bamboo rats and zokors). In a specific embodiment, the genetically modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny mice and crested rats. In one embodiment, the genetically modified mouse is from a member of the family of Muridae. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from the group consisting of a mouse and a rat. In one embodiment, the non-human animal is a mouse.

[0192] In a specific embodiment, the non-human animal is a rodent selected from the group consisting of BALB / c, A, A / He, A / J, A / WySN, AKR, AKR / A, AKR / J, AKR / N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA / 2, KM, NIH, ICR, CFW, FACA, C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr and C57BL / Ola of C57BL, C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, CBA / H strains and NOD, NOD / SCID, NOD-Prkdc scidIL-2rg null Background of mice.

[0193] The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

[0194] All patents and publications mentioned in this specification are incorporated herein by reference as a whole. Those skilled in the art will recognize that certain changes may be made to the present invention without departing from the concept or scope of the present invention.

[0195] The following examples further illustrate the present invention and are not to be construed as limiting the scope of the present invention or the specific methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0196] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0197] Figure 1 : Schematic diagram comparing the mouse CD70 gene and the human CD70 locus (not to scale);

[0198] Figure 2 : Schematic diagram of the humanization of mouse CD70 gene (not to scale);

[0199] Figure 3 : Schematic diagram of CD70 gene targeting strategy and targeting vector design (not to scale);

[0200] Figure 4 :Southern Blot detection results, where WT is the wild-type control;

[0201] Figure 5 : Schematic diagram of the FRT recombination process of CD70 gene humanized mice (not to scale);

[0202] Figure 6 : Genotype identification results of F1 generation mice, where M is Marker, WT is wild type control, PC is positive control, and H2O is water control;

[0203] Figure 7 : RT-PCR test results of mouse thymus tissue, where M is Marker, + / + is wild-type C57BL / 6 mouse, H / H is CD70 gene humanized homozygous mouse, and H2O is water control. DETAILED DESCRIPTION

[0204] 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.

[0205] In each of the following examples, equipment and materials were obtained from the following companies:

[0206] BglII, DraIII, and AseI enzymes were purchased from NEB with catalog numbers R0144M, R0510L, and R0526M, respectively;

[0207] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center of the China Food and Drug Administration;

[0208] Brilliant Violet 510 TM Anti-mouse CD45 Antibody was purchased from Biolegend, catalog number 103138;

[0209] PerCP / Cy5.5 anti-mouse TCRβchain was purchased from Biolegend, catalog number 109228;

[0210] Brilliant Violet 605 TM anti-mouse CD11c Antibody was purchased from Biolegend, catalog number 117334;

[0211] APC anti-mouse CD70 Antibody was purchased from Biolegend, catalog number 2204066;

[0212] PE anti-human CD70 Antibody was purchased from Biolegend, catalog number 355103;

[0213] Zombie NIR TM Fixable Viability Kit was purchased from Biolegend, catalog number 423106;

[0214] Purified anti-mouse CD16 / 32 was purchased from Biolegend, catalog number 101302.

[0215] Example 1 Preparation of CD70 gene humanized mice

[0216] A comparison diagram of the mouse CD70 gene (NCBI Gene ID: 21948, Primary source: MGI: 1195273, UniProt: O55237, located at positions 57452997 to 57456777 on chromosome 17 NC_000083.7, based on transcript NM_011617.2 and its encoded protein NP_035747.1 (SEQ ID NO: 1)) and the human CD70 gene (NCBI Gene ID: 970, Primary source: HGNC: 11937, UniProt ID: P32970, located at positions 6581648 to 6591150 on chromosome 19 NC_000019.10, based on transcript NM_001252.5 and its encoded protein NP_001243.1 (SEQ ID NO: 2)) is shown in FIG. Figure 1 shown.

[0217] In order to achieve the purpose of the present invention, a nucleotide sequence encoding a human CD70 protein can be introduced into the mouse endogenous CD70 locus, so that the mouse expresses a human or humanized CD70 protein. Specifically, using gene editing technology, under the control of the mouse CD70 gene regulatory element, a nucleotide sequence encoding a human CD70 protein is used to replace the corresponding mouse sequence, and a humanized CD70 locus schematic diagram is obtained as shown in FIG. Figure 2 As shown, the humanized transformation of the mouse CD70 gene was achieved.

[0218] Further design such as Figure 3 The schematic diagram of the targeting strategy shown in the figure shows the upstream and downstream homology arm sequences of the mouse CD70 gene on the targeting vector, and the A fragment containing the nucleotide sequence encoding the human CD70 protein. Among them, the upstream homology arm sequence (5' homology arm, SEQ ID NO: 3) is the same as the nucleotide sequence of positions 57456455 to 57460839 of NCBI accession number NC_000083.7, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) is the same as the nucleotide sequence of positions 57448179 to 57452246 of NCBI accession number NC_000083.7; the human CD70 gene sequence (SEQ ID NO: 5) contained in the A fragment is the same as the nucleotide sequence of positions 6586020 to 6590861 of NCBI accession number NC_000019.10; the connection design between the upstream of the human CD70 sequence and the mouse in the A fragment is The sequence " aacag The "g" in the sequence is the last nucleotide in the mouse. The first "c" in is the first nucleotide of human; the downstream connection of human CD70 sequence and mouse is designed as The sequence " cctv "a" in the sequence is the last nucleotide in humans. The first "c" in is the first nucleotide in mouse.

[0219] 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 mouse gene is designed as follows: The sequence " cgcag The last "g" in the sequence is the last nucleotide in the mouse. The first "C" in the sequence is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the mouse gene is designed as The sequence " GCCGC The last "C" in the sequence is the last nucleotide of the Neo box. The first "T" in is the first nucleotide of the mouse. In addition, a coding gene with a negative selection marker (the coding gene of the diphtheria toxin A subunit (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the modified humanized mouse CD70 is shown in SEQ ID NO: 10, and the expressed protein sequence is shown in SEQ ID NO: 11.

[0220] 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, and the integration of the exogenous gene is confirmed by PCR and Southern Blot technology. The correct positive clone cells are screened, and the clones identified as positive by PCR are then subjected to Southern Blot (cell DNA is digested with BglII or DraIII or AseI and hybridized with 3 probes, and the probes and target fragment lengths are shown in Table 1). The exemplary results are shown in Table 1. Figure 4 As shown, further verification by sequencing revealed that four clones numbered 1-A11, 1-G02, 1-G12 and 3-E06 were positive clones without random insertions.

[0221] Table 1: Specific probes and target fragment lengths

[0222] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size BglII 5'Probe 8.4kb 5.8kb DraIII 3'Probe 20.7kb 12.1kb AseI Neo Probe - 10.4kb

[0223] Among them, the PCR assay includes the following primers:

[0224] PCR-F1: 5'-CACTGCCACGGAATTCTTGAGGTGA-3' (SEQ ID NO: 12),

[0225] PCR-R1: 5'-TCATTCTGTCTTTTCGGTCACGCG-3' (SEQ ID NO: 13);

[0226] PCR-F2: 5'-GCTCGACTAGAGCTTGCGGA-3' (SEQ ID NO: 14),

[0227] PCR-R2: 5'-ACAGTCTGGACCTCCGAGGCA-3' (SEQ ID NO: 15);

[0228] Southern Blot assay includes the following probe primers:

[0229] 5'Probe:

[0230] 5'Probe-F: 5'-AGAAGCAACTTGCAGAAGGGACAGG-3' (SEQ ID NO: 16),

[0231] 5'Probe-R: 5'-TTTAGAGGCCCTCGGAGATTGGTCT-3' (SEQ ID NO: 17);

[0232] 3'Probe:

[0233] 3'Probe-F: 5'-TCTCCTCTCTCTTTGTTGGCTCTGG-3' (SEQ ID NO: 18),

[0234] 3'Probe-R: 5'-AAGGCCTCTAGCACAGGATCTCCA-3' (SEQ ID NO: 19);

[0235] Neo Probe:

[0236] Neo Probe-F: 5'-GGATCGGCATTGAACAAGAT-3' (SEQ ID NO: 20),

[0237] Neo Probe-R: 5'-CAGAAGAACTCGTCAAGAAGGC-3' (SEQ ID NO: 21).

[0238] The correct positive clone cells (black mice) screened out are introduced into the separated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium for a short culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), which can produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and the F1 generation heterozygous mice are then mated with each other to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene (see the schematic diagram of this process). Figure 5 ) and then mating with each other to obtain CD70 gene humanized homozygous mice. The genotype of the somatic cells of the offspring mice can be identified by PCR (primers are shown in Table 2). The identification results of the exemplary F1 generation mice (Neo marker gene removed) are shown in Figure 6 Among them, 8 mice numbered F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, F1-07, and F1-08 were all positive heterozygous mice. This shows that the present method can be used to construct CD70 gene humanized mice that can be stably propagated and have no random insertion.

[0239] Table 2: Primer names and specific sequences

[0240]

[0241] The expression of humanized CD70 mRNA in positive mice can be confirmed by conventional detection methods, such as RT-PCR. Specifically, 1 9-week-old wild-type C57BL / 6 mouse and 10-week-old CD70 gene humanized homozygous mouse prepared by this method were selected, and the thymus tissue was obtained after euthanasia by cervical dislocation. The primer sequences shown in Table 3 were designed to detect the mRNA expression in the thymus tissue of C57BL / 6 mice and CD70 gene humanized homozygous mice. The results showed that only the expression of mouse CD70 mRNA was detected in the thymus tissue of C57BL / 6 mice ( Figure 7 A); only humanized CD70 mRNA expression was detected in the thymus tissue of CD70 homozygous mice ( Figure 7 B), no expression of mouse CD70 mRNA was detected ( Figure 7 A).

[0242] Table 3: RT-PCR primer sequences

[0243]

[0244] The expression of humanized CD70 protein in positive mice can be confirmed by flow cytometry. Specifically, 8-week-old wild-type C57BL / 6 female mice and 9-week-old CD70 homozygous mice prepared in this example were selected, euthanized by cervical dislocation, and bone marrow cells were obtained. After stimulation with 1ug / ml lipopolysaccharide (LPS) for 24h, the anti-mouse CD45 antibody Brilliant Violet 510 was used to detect the expression of humanized CD70 protein in positive mice. TM anti-mouse CD45 Antibody(mCD45), mouse specific T cell surface antibody PerCP / Cy5.5 anti-mouse TCRβchain(mTCRβ), anti-mouse CD11c antibody Brilliant Violet605 TM Flow cytometry was performed after identification and staining with anti-mouse CD11c Antibody (mCD11c), anti-mouse CD70 antibody APC anti-mouse CD70Antibody (mCD70) or anti-human CD70 antibody PE anti-human CD70 Antibody (hCD70). The results showed that among the dendritic cells (DC) (characterized by mCD45+mTCRβ-mCD11c+) in the bone marrow of wild-type C57BL / 6 mice, the proportion of mCD70-labeled cells (characterized by mCD45+mTCRβ-mCD11c+) was 14.1%, and the proportion of hCD70 (characterized by mCD45+mTCRβ-mCD11c+hCD70+)-labeled cells was 0.95%; while among the bone marrow DC cells of CD70 gene humanized mice, the proportion of hCD70-labeled cells was 20.1%, and the proportion of mCD70-labeled cells was 0.94%. The above results indicate that humanized CD70 protein can be successfully expressed in the bone marrow cells of CD70 gene humanized mice.

[0245] Example 2 Preparation of double humanized or multiply double humanized mice

[0246] The CD70 gene humanized mouse prepared by the present method can also be used to prepare a double humanized or multi-humanized mouse model. For example, in the above-mentioned Example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice modified with other genes such as PD-1, PD-L1, CD27, CD40, OX40, 4-1BB, LAG3, etc. Alternatively, on the basis of humanized CD70 mice, mouse ES embryonic stem cells can be separated and gene recombination targeting technology can be used to obtain a double or multi-gene modified mouse model of CD70 and other genes. The homozygous or heterozygous CD70 mice obtained by this method can also be mated with homozygous or heterozygous mice modified with other genes, and their offspring can be screened. According to Mendel's law of inheritance, there is a certain probability of obtaining heterozygous mice with humanized CD70 and double or multiple genes modified with other genes. The heterozygotes can then be mated with each other to obtain homozygous mice modified with double or multiple genes. These double or multiple genes modified mice can be used to perform in vivo efficacy verification of targeted human CD70 and other gene regulators.

[0247] Example 3: Efficacy Verification

[0248] The CD70 gene humanized mice or multi-gene modified mice prepared by this method can be used to evaluate the efficacy of antibodies targeting human CD70. For example, a tumor animal model is constructed with CD70 gene humanized homozygous mice, and the mice are divided into a control group or a treatment group. The treatment group is injected with an antibody drug targeting human CD70, and the control group is injected with an equal volume of normal saline. The body weight, tumor volume and tumor-related indicators of each group of mice are then monitored, which can effectively evaluate the safety and in vivo efficacy of antibody drugs in humanized CD70 mice.

[0249] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present invention. 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. In addition, the various different embodiments of the present invention can also be arbitrarily combined, 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. Sequence Listing <110> Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd. <120> Construction method and application of CD70 gene humanized non-human animals <130> BHCD70.002CN1-P0102021030231Z <150> 2021106044751 <151> 2021-05-31 <160> 35 <170> SIPOSequenceListing 1.0 <210> 1 <211> 195 <212> PRT <213> Mus musculus <400> 1 Met Pro Glu Glu Gly Arg Pro Cys Pro Trp Val Arg Trp Ser Gly Thr 1 5 10 15 Ala Phe Gln Arg Gln Trp Pro Trp Leu Leu Leu Val Val Phe Ile Thr 20 25 30 Val Phe Cys Cys Trp Phe His Cys Ser Gly Leu Leu Ser Lys Gln Gln 35 40 45 Gln Arg Leu Leu Glu His Pro Glu Pro His Thr Ala Glu Leu Gln Leu 50 55 60 Asn Leu Thr Val Pro Arg Lys Asp Pro Thr Leu Arg Trp Gly Ala Gly 65 70 75 80 Pro Ala Leu Gly Arg Ser Phe Thr His Gly Pro Glu Leu Glu Glu Gly 85 90 95 His Leu Arg Ile His Gln Asp Gly Leu Tyr Arg Leu His Ile Gln Val 100 105 110 Thr Leu Ala Asn Cys Ser Ser Pro Gly Ser Thr Leu Gln His Arg Ala 115 120 125 Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ala His Gly Ile Ser Leu 130 135 140 Leu Arg Gly Arg Phe Gly Gln Asp Cys Thr Val Ala Leu Gln Arg Leu 145 150 155 160 Thr Tyr Leu Val His Gly Asp Val Leu Cys Thr Asn Leu Thr Leu Pro 165 170 175 Leu Leu Pro Ser Arg Asn Ala Asp Glu Thr Phe Phe Gly Val Gln Trp 180 185 190 Ile Cys Pro 195 <210> 2 <211> 193 <212> PRT <213> Homo sapiens <400> 2 Met Pro Glu Glu Gly Ser Gly Cys Ser Val Arg Arg Arg Pro Tyr Gly 1 5 10 15 Cys Val Leu Arg Ala Ala Leu Val Pro Leu Val Ala Gly Leu Val Ile 20 25 30 Cys Leu Val Val Cys Ile Gln Arg Phe Ala Gln Ala Gln Gln Gln Gln Leu 35 40 45 Pro Leu Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His 50 55 60 Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala 65 70 75 80 Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu 85 90 95 Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu 100 105 110 Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu 115 120 125 Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg 130 135 140 Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro 145 150 155 160 Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu 165 170 175 Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg 180 185 190 Pro <210> 3 <211> 4385 <212> DNA <213> Artificial Sequence <400> 3 agactgctgg gctagagagg aaagcccagc gtattgtagt aggagtgtta ggttaggact 60 tagcaccaac cttatatcc aacatggccc cagcccctca ttgaggaatt ctagcaggca 120 ggaagacttg gggggatgag ggggacagga caggaccaat tcacccctga ccacaccagc 180 atccctcccc cagagaaatc taagcaggga ctccacccct gagccacgcc cccaaggcca 240 gtggatgagt gtggatgaat gggcaacact tgccaactct atactgctct agcttccatc 300 ctcaccactg caaataataa tataataat aataataa aacatgatgt aaatcattgg 360 tacatggatt ctttataaag ttcaaagtcc tccagcagtt aatttcctag tgtactgcat 420 agtttatgt caatacaaat agggttggac aaggcatgac tgcatttcct tgattaatgt 480 tcaatgtagg agggcccagc acacaacatt tccatggaga tggtcctaag ccctacaaga 540 atgtaggctg agcaagtcag taagccagag atccccctag gctcctgctt cagtcctgcc 600 ttctctcgat gatgaggaac tataaagagg aaaaatcct tccctctcca aggtcacggt 660 gttcatcaac aataaaacct ctagcatgaa ccacatacat tagatgcctc aaagccccca 720 aaatggagct gaagaaatga gttaggtatg agcaccggct gggactctcg ctgactctgg 780 agggctccat ttcagctagg ctgatcgatg agctgcggca atcctcctgt ctctgcttct 840 cagcaggcta ccattacaga caggcactga tgaaccccac tttcaggtat caaactcaga 900 gttcccattc ttgcttgatg gggcctgtac cagtaacttt aattttttt cagaccttat 960 tttcaatgat gattcttaaa cactttaacc ttccttatag cccaccaccc accagaggta 1020 gtgggaaaga aaggatacag gagaagtgga cctgtttaga aaggttcttt ggagcaactc 1080 ctgcctgtgt tgtcaggaaa gaagttcaca ggttagcagc cgcagcttga tccactcaca 1140 aaccttcac ggacacacca gcagtccagt ttggtagagt caagatagca aacaggaatg 1200 aatccgaagt agtggctcta cctagcacta ggagaagctc tctctcagct gtgcctcttt 1260 cagggaagtt aagaacaatt catcagaccc acaggcgttg tacagctagc tccttaagcc 1320 tagctccgcc tctgtcactg tccatcaagc actttttttt ttttcattt ttcgaaacag 1380 ggttctctg tgtagccctg gctgtcctgg aactcattttt atagaccagg ctggcctcga 1440 actcagaaat ccgcctgcct ctgcctccca agtgctgggt gtaaaggcat gggccaccac 1500 gactggctca tcaagtcctt tttataccct ccaaacctca catgttctcc aagggtcttg 1560 cctcaacact ccgccaatca gcatgagtcc gaggaagctg caagaaactg cagcacacca 1620 ccagaagact tttggtgctc ttttctctc tatagagtcc ctgtgagacc ccgacttaga 1680 gcgtttctcc ctgggaggta aagcccctga atgttcccc aagcctgttt tccctgatct 1740 ctaaaaatac agccagaaag aagctctttg tcctctacag ccagcaaaaa gccttttgt 1800 ttctatacct tataaccaga gatacgataa ttgtaatttt accaaggaca cctggcagag 1860 aagagctgta gccaactctc ctcccaactc ctcccaactc ctcccaattc ctcagctagc 1920 tgttaaaagc cccctacggt taccactcag ggtggaactc ccctgccctg cgcagcgaag 1980 aggagttcgt cctcagctag ctgataataa aacctcttgc agtttgcatc aggtgtggtt 2040 ttctcaagtc attggggtgc tggccagctc atcccgggac ttgagcggag gcccagcttc 2100 aggggtctta catcccgaca aaggcagcat gttgttagca aagatccttc atcacgtgtc 2160 ctttcacgtg cttgctttag tagaacatgc cctctgctgt gtctgcttca gctaaacgtt ccttcaggag tctgccttag tctatcacct gtgtccactt caagaaaaca ctccttcact tgtctgccct agcaaaacac caccaacaca actgactttc caagaaccc ctaagtttcc acttcagggc ctgtatccac tgagccatat ccccagccta cttcatattt tttaaagggt ttgttttgtt ttaatgtatg tgtggggggg cgatatgtgc acaggtgccc atggaaacca gagttgccaa atactcctag aactggaatt tcagatcgtt ttaaaccatt tgacatgggt gctgggaagt gagccctctc tctgggatag acacactcaa cctctgagat gtctctccaa ccccatgtta atctcactat acctgaattg caggagatta gatccagggg ccactgacca acatgttctc cccaaaacag aatctgaagg tggtggtggc ctctagctgg gccagctcaa acccatttca gaggcaacct ctccctggta ccctctcctg tagtagcat ctcaacttcg gaaaaacttg agcttgtaga tgtgttccaa catttcatca aacagtctgg atcctgggta aaatagactt ccgtgaagaa cacacagcac tcttggggtg cccagagaag atccagaaga gaggacagca gagacaggag agatacaagc cccaggcctg ccaggctggg gcagctgctg 2940 cccagctcgg cacctcctgc ctcaagtgtt ttctcgcctc tgccagttct aagtaaactc 3000 tcagctgctc actctgctac cgtatctcat gtggcaaaga ggctagaaca aagtggaggc 3060 cacctgaaag caccaagcct ggttgtctgc ctgttcattc tttattgtct gacagcttcg 3120 tatatttgca aaaatgaatt gtagtcatga ccaccccctt tcccctctcc agtccactcc 3180 cctccttgtc ctgctgaaca ttttcttcca agcaagttct gctccccacc cctgtaatcg 3240 ccactgcctt acaggctttc gagtctcttt ctgtgtgggc tgcactgaat catgggcagg 3300 aggctggttt tctggggcaa tggcaactta ctggtggcca caacactgaa gaaagtgacc 3360 ttcctttccc agtggccaga actgcccaga aagcctcagg gaggggcaga atctcatggc 3420 ccagccccta tcaaggccaa accattcggg gacatcttgt gccaattttc acagctgagt 3480 taagtgtagg agtgcaatgg ctgtgtccac tttcaggtag gatcattgtc tcagtctccg 3540 ggtgacatgg atggggatgg gggtggggat cagcagtagc taatcaatat gctagctatc 3600 atattgagac caagccaagt gtggtggctc acaccagcaa tctagtagtc ctgcagtcag 3660 aaggatgtga gaggccagcc tgggctacat gacaagtttg acacctgcct gagctacttg 3720 aggagctgaa ggtcaccatg gacaccgtgg caaatactaa gccagcctgg gctctacggt 3780 gagttccaat gtgagctgtt tataatggcc ccccatggaa gagcctacaa tcgtgtccaa 3840 atattttggg aagcacaagg gattcctgga tgcggatgct tcctgatttc ctagcattca 3900 gcccaatact gaacctctaa tagatgctca gtgaatggtt tgtcgaatga atgaatgaat 3960 gaatgggcac cgagcagcag gagcaggggc tgaatgccca atcggaagct taaatattct 4020 gaagcaggga caggcctgct tcagtttgtc tgtgggatgg cagaaggtgc caaaagctcc 4080 aggggatttc cctgccctcc gagaagaggc ccagttcttc ccctgcatcg gacatccccg 4140 aggttctaag ggcaggtcaa ggcaggcaga agcttcaaaa gctcggctga ggaggctaca 4200 gcttcccgct gccttcaggc cgctgcttcc gtgcagggat gccggaggaa ggtcgccctt 4260 gcccctgggt tcgctggagc gggaccgcgt tccagcgcca atggccatgg ctgctgctgg 4320 tggtgtttat tactgtgttt tgctgttggt ttcattgtag cggactactc agtaagcagc 4380 aacag 4385 <210> 4 <211> 4068 <212> DNA <213> Artificial Sequence <400> 4 tactatggag gaggcctgtg cagaagctat aggcctctag gtgggaagtg agcactctaa 60 gcttgacctt caaagctccc tgtgataggt cacagctagg ccccacctcc aaagggtcca 120 catggaccct agattcaaac ctgaacattc caatcctggt cccaataggc ccatgggcat 180 ctcacagtac aaatgtattt aattcaaatt caacaccctc caccccttaa caccttcaat 240 gtctttcaaa agactgagtt cagaggcagc agaaacagct cagcagttag agcaccagct 300 gctcttgcag aggacctggg tttggttcca gcacctatag ggcagctcac agccagctct 360 aactccagct ccagggatcc cacactccgt tctggcctct gtgggcacag tgcacacatg 420 ttgcacagac agacatgaag gcaaaacacc catgcacaga aaaaatttta aaaacagaga 480 ggcctgtcta acagtctaaa ccaactaatc cttaactcgg gggggggggg ggagggcact 540 atgaaactag aatccatcaa ggttttccg tggtagaaaa taaatcatcc aactgtcaag 600 tggggaggat ggaataggga ggcagatgag gccgaggcaa ggtgaaaccc cagcattgca 660 agttttgctc cctctactcc aaacctggtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 720 tgtgtgtgtg tgtttaaatg tcaaagggtt aaattgacag acaaaagttc attaaacaga 780 cagctaacct agcagacatc ctacctccct gacagcttct ttttgtgctc cagagaacag 840 aacacagcac ttacagtttg cacgagggat gaagctgggt gttgttcaga gccaggatct 900 gtccaggagc cagaggctta ctcttcagtc tagactggct ggctagtgag ctgccagggt 960 ccctcctgtg tccacctccc cagcatggga cacagacagg catgccatca gacacaaagt 1020 ttacatggct gccggagctc tgaacgcagg tttcccctga ctgtcttgtt cctatccaac 1080 ggtctgtcca attccaatgt gtgaactctg ggaacacgtt catttaggct gtagcaaata 1140 ttcacaggca gaaacggtga gtggactata aggaaactct gttctctgct atgcaaacca 1200 taaaaggatg tgttcttaag taaaataaa atgacctcag tggaaatata ttgtgacggg 1260 aaaggactga tttcaggt ttgagcacgg ggggtattat aaagctgcta gggggaatct 1320 ccaccatcca cataggatga tgtcacagg tgcagccttg aaacttcccc ttcaatgaca 1380 ctcaactgtg tagtacttg atttatgta ctaagtgaca actgagagga gacctttaat 1440 cccagcactt gggaggcaga ggcaggcaga tctgaattca aagccagcct gatctacaga 1500 gtgattttca gggctataca gtgaaacctt gtgagagctg gggaagcgat gctactctt 1560 ccagaggacc tggtttgag tccccgtgct ttatggactg aaccatgcct ggacctttaa 1620 ggttcattc ccctcaggat gtaggctcca gcttattct gtttctgaag gtctgtgcgt 1680 aataagttgg gcatcatggc acacaccgt catcccaaca tcccaggaa gctgaggcag 1740 gaggtttgca agtttgaggc caatcttagc tacatagtgt gctcttatct ggaaaaaaaaaa 1800 aaaaacagt gggaatggga gggagaggaa gaagggggt gtagggg aggagagatag 1860 ggagatgggg ggcggcatg gcagggaga gagagagaag gagaatggc gagggaatg 1920s caccacctct ctgttcgact ctattgttg ttccgggtct catttacaa acagaatagt 1980 tacacatatg tgcttttgaa actcgggatc tggacacaca cactgcacaa tggtcacttc 2040 tattcaacaa gttgaggtct cccgtctcct cagttgaccg tttctcctgc ctgcatgttt 2100 ctctttctgt ggaaatgcta tcaacagtcg atattattac cattgttact gctgaaagtt 2160 cctacagtct cggtcagggc ctccaactga ctcagataca caaccacaac ttctgagtaa 2220 ggtctcacac atttcacagg ggtctaggga gttcagcctt cttatttccc acgtctaagt 2280 aacaatatgc agtagcatcg ttcagggctg aataacactt cattgggtct ggatacccca 2340 ttagttgatt tggctagagc tgaaaataga ggatttcatt atgacatgtt cctacatggg 2400 gctcacattc acccccacaa tattctcttg tccccccttc ctcctacccc cttccccttt 2460 cctctaggcc tcatcaaatt gccccttctt gtatatttct ctctccaaat gcctaatatt 2520 agagaaaagg atggctttgt ctgagtctgt ctatgtctgt cttgttttaa taaaatggcc 2580 tctgtttcta tccataatag aaacatgatt taattccttt ttaaaaggtt ttaaagaatg 2640 tgatttattt attattgatg tgtatgtgat tctcacagtg catgtgcagg tccagggata 2700 gaacctagag tctgccttta cccactggat cagctcaatg cccccccccc accttttctt 2760 taacaggttt tgcacagtcc agtctggcct cacttcactc ttttttacag ttcaataaga 2820 cagcttcatt ttgtgcaatg tgtatctaga ccaaattttc tttttttgaa gacttattta 2880 tgtgtgaaag cccatagaag ccagaaatgg atatagtaac ctctggggag ggagttacag 2940 atggttgtga gtgccagtga gtgcagaatc gaattcagtc ctctgcaaga gctgcccagt 3000 gcgctaacca ctgggccact gctccagcct ctcagccttt tttaagagtc atcctcacag 3060 atatgaggtt tgttgggact ctggttagca ctttctgcca ttgctgcagt tggactcctt 3120 ttgcatactc tctgcccatt tctagtgctt acgaaacaga ttgaacttca ccaagaaagg 3180 aattttgctt ctactgaaaa gacaagccag ggcagcctct ctggggggtc ccattcttca 3240 gagcccaaaa ttaggacggt ggtgtgacct cagcatggac ccaaagaatg ttcaactgac 3300 tcaggacaga cacctgctca tggtactcac tgctgctccc ttcgcagtag ccaagaagtg 3360 tgccagcctg tgtgcctaac agcagattcg tgatgaggaa aacgtgacag cctggtggga 3420 atttctctat ttttgcaggc aatggagaca ggtaatgatg taagctgttg ctaggtccat 3480 gagaagagac tagtcacaat tctattccat tgatatatgt gtctatgcat atatccacgc 3540 ctcagtgttt attttgtctt tgagatagat gagagacaca cagagaatga agtatctatg 3600 gatggataaa tgaatgatag acagacagac aaacagatag atacctagat acatacatag 3660 acacagatat atagatatat agatagatct tagatagata catagatcaa aagacaggtg 3720 atagatgatg atagatatat agacagataa taatacatat agatgcatac atacacatat 3780 atagagagat acatacatac atacatacat atacatacat atgtgcatag ttacatacat 3840 ggaaacatag ataataattg tcctataaga cagcctgggc tcacctccac catcctgttt 3900 tcataagtga acttccagct ttcaactgtt ttctgatgga tttatcttga ttttgtttgt 3960 ttatttgacc aattggttgg ttgagcaact tttgagacaa ggtctcatgt agtctaagcc 4020 tcaaactatc tgtaaagctg aatacatcct tcaactcctg cttcttcc 4068 <210> 5 <211> 4842 <212> DNA <213> Artificial Sequence <400> 5 ctgccgctcg agtcacttgg ggtgagttga gatgaaag tgggaagaa aacatagaga 60 ggcgcgtgac cgaaaagaca gatgagatg ggtacaaaga ggccagagag gaagatctgg 120 taggcagag acagagacca gacagggag gcgaggcggg gaccaggctg cccggtgtag 180 gggctacgag acaggcagcc ctgccaggag gtacagggag atcccgggat gggaaggta 240 ggcacacatg gaatggaag atgactcggc tctgtgttc ccccggcagg ctgactcaga 300 ggctgctggg ggctcacaa ggctgggcgt gggggctcc tggggctcc taggacggga 360 tggccccagc cactcgctcc gggtggggga ggggtccct tggggaccgc gccgggcgcc 420 tttgcagcgt agagagtccg ctgcgcgcgg tgctcgcg cccagtgaca tccaggaaaa 480 cgattcggga aacgaagaag ttctttgaa ggtctcgact tcacgttccc cgctggttca 540 gacctgcttc ctctttaaga agtcttaaga gtaaaaaaaa aaaatgaa ataaaatcac 600 cagtgcgcgc cgtgggatga gaggtggaaa ggaggatgga cagagaaag agagctcctg 660 gcacagggga cacatagaac ctctctgctt acgtccgtgc cctgttttct ggtctttctct 720 tccagtggga cgtagctgag ctgcagctga atcacacagg taacacgggg gacgtggagg 780 gacgggaga agagaggca cagagagaga aggagga ggtagaaga caagtgggga 840 Gagagagagagagagagagagagagagagagggagggaggga 900 gggaaacgga gagggggaaaaaaaaggaaaaaaaaaaaaggggaaaaagg 960 Gagggagag ahaaaaaaa gagagagag aggagag ggagagacg gagagagaga 1020 agaaaaaaaahaaaaaaaahaaaaaahaaaaaahaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa hasa havea havea have been acaggcgggg gagagaaaaaaaaagggaaaaaaaaagggg 1140 atagaggaag agagataaa acgagagag gcgacctggc gtggttgctg gagcctgtaa 1200 1260 cctggccaac atggtgaaac cccgtctcta ctaaaattac aaaaattagc cgggtgtggt 1320 ggcaggcacc tgtaatccca gctactcagg agggccgaggc aggagaatcg cctgaacctg 1380 ggaggcagaa gttgcagtga gccaagataa tggcactgct ctccagcctg ggtgacagag 1440 ggagattctg tcaataat aaaaaaag aaaaaaaaaaaaaaaaaaaaaaaah saying Gaaaaaaaaaaaaaaaaaaaaaaaagg out out from outside 1560 Aaaaaaagh aaaaaaaaaaaaaaaaaaaaaaaaaaaggca 1620 Gagagagagagagagagagagagagagagagagagagagagagagggagg 1680 aaaaaaaaagggggggggaaaagagatc attcccaagaaaaaaaaaaagggaa 1740 acgagagaga gagagagg agagagagcg agcaggtttg tgttctctgg catccactac 1800 ctttctctcc ctttccctct agaatcaaa 1860 ggggttttgt tagtaacga aaacgctcac aggaaggct acccttccctc cattcatgct 1920 acagcacta attackacatc tgcagtgggc agcactgtt ccggtactgc agtcgcgcac 1980 aaaacagaca gaaatccctg cctggtgagc ttccctcta ctatcactcc taccgaggag 2040 gagatgacca aaaaaaaaaacgaacatg tgaaaaacag agcaatgcca cgtagagtaa 2100 aacaggccaa gagcaggtttt tagagaaaaaacaggcaag gaggtgggtg gggaatccgg 2160 gcatgccagg gttggggatt ttcatttaaa gtggagtcgc ggcggtcggg tgggggggtg 2220 gtatctgagt gggtggcatt ggcaagaagc cttgagggag aagaaatgaa cagttctgct 2280 ctaggtggaa gtctctacct cctcccagcc tctctcgaac gcttcttcct cttctgtttt 2340 tccacggttc atccccagag actaaaatta ctgcacgctt gcgtgttttg ttccttccat 2400 tttgtaatgg aagggacctt tgttgctctc tcttctcctt gggtctctga cccctgatat 2460 atcttccccc cctccccggc ctttgccata tttttcctgt aatttggtct tgaggcccct 2520 ttctggagag tggctgtcca gccccacatc gatggggcac caggggtggt ggccacaggt 2580 gttgacagtg tctttcacag gacagctctt tcttctggtg gatgatatca tgcctaagtg 2640 tgtgacaggg gaccaggtgt cactcttaca ggaaatttgc aaagcctggt aaacaccctt 2700 ggggctcctg tttgaccccg ttcagttcct tcctaacaag atagccagtc tccaggagag 2760 ccttgagtga ggaggagagt gaggctcagg tgtgtcagtc agataagttg cggagacagg 2820 agacgctgag atgaaacacg tgaaatatag ccgaggacct tggctcccgg ctgtgatccc 2880 agcgctttgg gaggctgagg tgggaggatc gcttgagccc aggagttcta gactatgttt 2940 cttgggaaac atagcaagac caccatctct agaacaaaat tttgaaaatt agctggtcat 3000 ggtggcgcat gccacctccc agtgactcgg gaggctgagg tgggagatag cttgagcccc 3060 agaggctgag gctgcagtga gctatgattg caccactgca ctccagcctg ggtgacagga 3120 ccagaccctg tctctaaaga aaataaagat cctagagaga agagggccaa cgggaaggta 3180 ggggagctct ctgagacaca cactcaacca gcgggtggaa agcaagaaaa aaaaagggtc 3240 ctgggagcca aagcttttat tggcgttcag ggtgtgaccc gagcaggcgt cccttgggga 3300 attctctcgt gcactctctt gcgcgctgtc tcgtgcgcac gctgtctctc tcgtgctgtc 3360 tgtctcactc tctcaggcac tctctcgctc actcccgtgc actctcgagc tctgtagctc 3420 tcatcctctc tcacacacac actctcccat gcactctctc gagctcactc tcacactctg 3480 tctcgtgcac tctctctcaa gctctctcgt cctctctcac acacacactc tctctcttgc 3540 tctctctctc atgctgtctc tcgtactctc tcctgctctc tcgtgctctc tctcactctc 3600 tcgtgcactc tccctcaagc tctctcgtcc tctctctcac actctctcgc tgtctcatgc 3660 tctctctccc tcatgatctc tctctctctc atgctgtctc ttgcactctc tcctgctctc 3720 tcgtgctctc tcgcactctc tcgtgcactc tccctcaagc tctctcgtcc tctcacacac 3780 tctctcgctc tctcatgctc tctctctcat gctctctgtc gcactctctc gtgcactctc 3840 gtcctctctc acacatacac tctctctctt gctctctctc tgcattctct ctctatatat 3900 ctctccctcc ccctctatct ttacccttgt ctgtttctgt tttttttttt tttttttttg 3960 tctctctctc tcctggtgtc tctctctccc tgtctctacc cctttcccct tacccctctc 4020 tccttccctt tagatctcta accttctatc cccctccctt ttttttgaga cagggtttct 4080 gtgaccaagg ctggagtgca gtggtgtgat catagctcac tgcagtctca gcctcccagg 4140 ctcaagcgat cctccagcct cagcctccca agagtagcca ggaccagagg cgtgcaccac 4200 cacacctggc tgacttttta ttttttgtag agatggggtc tcgccctgtt gtccaggctg 4260 gtcttgaact gctgacctca ggtgatccac ccgccttggc ctcctaaact gttaggattt 4320 caggcatgag ccaccacgtc caacctattc cttgatctct aactctcgat ctctatatct 4380 gactttctct atgaccccct gtgcctcagt ttccctaaac ctccatcctc tcatccctct 4440 aaccctgtgc ccccaggacc tcagcaggac cccaggctat actggcaggg gggcccagca 4500 ctgggccgct ccttcctgca tggaccagag ctggacaagg ggcagctacg tatccatcgt 4560 gatggcatct acatggtaca catccaggtg acgctggcca tctgctcctc cacgacggcc 4620 tccaggcacc accccaccac cctggccgtg ggaatctgct ctcccgcctc ccgtagcatc 4680 agcctgctgc gtctcagctt ccaccaaggt tgtaccattg cctcccagcg cctgacgccc 4740 ctggcccgag gggacacact ctgcaccaac ctcactggga cacttttgcc ttcccgaaac 4800 actgatgaga ccttctttgg agtgcagtgg gtgcgcccct ga 4842 <210> 6 <211> 80 <212> DNA <213> Artificial Sequence <400> 6 ttggtttcat tgtagcggac tactcagtaa gcagcaacag ctgccgctcg agtcacttgg 60 ggtgagttga gatggaaaag 80 <210> 7 <211> 80 <212> DNA <213> Artificial Sequence <400> 7 tgatgagacc ttctttggag tgcagtgggt gcgcccctga ccacaactcc aggatgactt gtgaattt tttttctttt <210> 8 <211> 80 <212> DNA <213> Artificial Sequence <400> 8 taaaatttta attctgttct tagtaacact cctcacgcag cacccagtgg atatcgaatt ccgaagttcc tattctctag <210> 9 <211> 121 <212> DNA <213> Artificial Sequence <400> 9 gagttccta ttctctagaa agtatagga cttcatcagt caggtacata atggtggatc cactagttct agagcggccg ctactatgga ggaggcctgt gcagaagcta tagcctcta g 121 <210> 10 <211> 842 <212> DNA / RNA <213> Artificial Sequence <400> 10 60. gaggtgcca aaagctccag gggatttccc tgccctccga gaagaggccc agttcttccc ctgcatcgga catccccgag gttctaaggg caggtcaagg caggcagaag cttcaaaagc 120 tcggctgagg aggctacagc ttcccgctgc cttcaggccg ctgcttccgt gcagggatgc 180 cggaggaagg tcgcccttgc ccctgggttc gctggagcgg gaccgcgttc cagcgccaat 240 ggccatggct gctgctggtg gtgtttatta ctgtgttttg ctgttggttt cattgtagcg 300 gactactcag taagcagcaa cagctgccgc tcgagtcact tgggtgggac gtagctgagc 360 tgcagctgaa tcacacagga cctcagcagg accccaggct atactggcag gggggcccag 420 cactgggccg ctccttcctg catggaccag agctggacaa ggggcagcta cgtatccatc 480 gtgatggcat ctacatggta cacatccagg tgacgctggc catctgctcc tccacgacgg 540 cctccaggca ccaccccacc accctggccg tgggaatctg ctctcccgcc tcccgtagca 600 tcagcctgct gcgtctcagc ttccaccaag gttgtaccat tgcctcccag cgcctgacgc 660 ccctggcccg aggggacaca ctctgcacca acctcactgg gacacttttg ccttcccgaa 720 acactgatga gaccttcttt ggagtgcagt gggtgcgccc ctgaccacaa ctccaggatg 780 acttgtgaat attttttttc ttttcaagtt ctacgtattt ataaatgtat atagtacaca 840 ta 842 <210> 11 <211> 195 <212> PRT <213> Artificial Sequence <400> 11 Met Pro Glu Glu Gly Arg Pro Cys Pro Trp Val Arg Trp Ser Gly Thr 1 5 10 15 Ala Phe Gln Arg Gln Trp Pro Trp Leu Leu Leu Val Val Phe Ile Thr 20 25 30 Val Phe Cys Cys Trp Phe His Cys Ser Gly Leu Leu Ser Lys Gln Gln 35 40 45 Gln Leu Pro Leu Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu 50 55 60 Asn His Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly 65 70 75 80 Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly 85 90 95 Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val 100 105 110 Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr 115 120 125 Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu 130 135 140 Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu 145 150 155 160 Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr 165 170 175 Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp 180 185 190 Val Arg Pro 195 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <400> 12 cactgccacggaattcttgaggtga 25 <210> 13 <211> twenty four <212> DNA <213> Artificial Sequence <400> 13 tcattctgtc ttttcggtca cgcg 24 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 gctcgactag agcttgcgga 20 <210> 15 <211> twenty one <212> DNA <213> Artificial Sequence <400> 15 acagtctgga cctccgaggc a 21 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <400> 16 agaagcaact tgcagaaggg acagg 25 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <400> 17 tttagaggcc ctcggagaatt ggtct 25 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <400> 18 tctcctctct ctttgttggc tctgg 25 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <400> 19 aaggccttct agcacaggatctcca 25 <210> 20 <211> twenty one <212> DNA <213> Artificial Sequence <400> 20 ggatcggcca ttgaacaaga t 21 <210> twenty one <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty one cagaagaact cgtcaagaag gc 22 <210> twenty two <211> 25 <212> DNA <213> Artificial Sequence <400> twenty two gtctgtggga tggcagaagg tgcca 25 <210> twenty three <211> 25 <212> DNA <213> Artificial Sequence <400> twenty three ctcatgccac ccttcccact acttc 25 <210> twenty four <211> 26 <212> DNA <213> Artificial Sequence <400> twenty four ccacaggtgt tgacagtgtc tttcac 26 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <400> 25 tctagagatg gtggtcttgc tatgttt 27 <210> 26 <211> twenty four <212> DNA <213> Artificial Sequence <400> 26 tacaacccag ctccctctca gcac 24 <210> 27 <211> 25 <212> DNA <213> Artificial Sequence <400> 27 gggaccagga ttggaatgtt caggt 25 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <400> 28 gacaagcgtt agtaggcaca tatac 25 <210> 29 <211> twenty four <212> DNA <213> Artificial Sequence <400> 29 gctccaattt cccacaacat tagt 24 <210> 30 <211> twenty three <212> DNA <213> Artificial Sequence <400> 30 tgctggtggt gtttattact gtg 23 <210> 31 <211> twenty one <212> DNA <213> Artificial Sequence <400> 31 cagggtgagg ttggtacaga g 21 <210> 32 <211> twenty one <212> DNA <213> Artificial Sequence <400> 32 tagcggacta ctcagtaagc a 21 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <400> 33 cagtgtttcg ggaaggcaaa 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 accacagtcc atgccatcac 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <400> 35 tacagcaaca gggtggtgga 20

Claims

1. A method for constructing a non-human animal with humanized CD70 gene, characterized in that: The non-human animal expresses humanized CD70 protein in vivo; The non-human animal has reduced or absent endogenous CD70 protein expression; The construction method comprises introducing a nucleotide sequence encoding a portion of the extracellular region of the human CD70 protein into the CD70 gene locus of a non-human animal; The part of the extracellular region of the human CD70 protein is shown in SEQ ID NO: 2, positions 48-193; The amino acid sequence of the humanized CD70 protein is shown in SEQ ID NO: 11; The introduction of the non-human animal CD70 locus is to replace the corresponding region of the non-human animal; The non-human animal is a mouse.

2. The construction method according to claim 1, characterized in that: The genome of the non-human animal contains part of the human CD70 gene or a humanized CD70 gene.

3. The construction method according to claim 2, characterized in that: The humanized CD70 gene encodes the humanized CD70 protein.

4. The construction method according to claim 2, characterized in that: The portion of the human CD70 gene includes a portion of exon 1, all of exon 2, and a portion of exon 3 of the human CD70 gene, wherein the portion of exon 1 contains at least 10bp of nucleotide sequence, and the portion of exon 3 contains at least 100bp of nucleotide sequence.

5. The construction method according to claim 4, characterized in that: The portion of the human CD70 gene also includes introns 1-2 and / or introns 2-3.

6. The construction method according to claim 2, characterized in that: The portion of the human CD70 gene comprises the nucleotide sequence shown in SEQ ID NO:

5.

7. The construction method according to claim 2, characterized in that: The humanized CD70 gene comprises a portion of the human CD70 gene and a portion of the non-human animal CD70 gene.

8. The construction method according to claim 7, characterized in that: The portion of the non-human animal CD70 gene includes a portion of exon 1 and / or a portion of exon 3 of the non-human animal CD70 gene.

9. The construction method according to claim 7, characterized in that: The portion of the non-human animal CD70 gene comprises the nucleotide sequence encoding positions 1-49 of SEQ ID NO:

1.

10. The construction method according to claim 2, characterized in that: The mRNA transcribed from the humanized CD70 gene comprises the nucleotide sequence shown in SEQ ID NO:

10.

11. The construction method according to claim 1, characterized in that: in, The nucleotide sequence encoding part of the extracellular region of human CD70 protein is regulated by endogenous regulatory elements in non-human animals.

12. The construction method according to claim 1, characterized in that: A portion of exon 1, the entirety of exon 2, and a portion of exon 3 of the non-human animal CD70 gene are replaced.

13. The construction method according to claim 12, characterized in that: Introns 1-2 and / or introns 2-3 of the non-human animal are also replaced.

14. The construction method according to claim 1, characterized in that: Use targeting vectors for non-human animal construction.

15. The construction method according to claim 14, characterized in that: The targeting vector comprises a donor nucleotide sequence, and the donor nucleotide sequence comprises a nucleotide sequence encoding a portion of the extracellular region of the human CD70 protein.

16. The construction method according to claim 14, characterized in that: The targeting vector further comprises a 5' arm and / or a 3' arm; the 5' arm is as shown in SEQ ID NO: 3; The 3' arm is shown in SEQ ID NO:

4.

17. The construction method according to claim 1, characterized in that: The construction method includes mating a non-human animal with a humanized CD70 gene with other gene-modified non-human animals, in vitro fertilization or direct gene editing, and screening to obtain a multi-gene modified non-human animal.

18. The construction method according to claim 17, characterized in that: The other genes are selected from at least one of PD-1, PD-L1, CD27, CD40, OX40, 4-1BB or LAG3.

19. The construction method according to claim 2 or 17, characterized in that: The portion of the human CD70 gene or the humanized CD70 gene or other gene is homozygous or heterozygous for the endogenous modified locus.

20. A humanized CD70 protein, characterized in that: The amino acid sequence of the humanized CD70 protein is shown in SEQ ID NO:

11.

21. A humanized CD70 gene, characterized in that: The humanized CD70 gene encodes the humanized CD70 protein according to claim 20.

22. The humanized CD70 gene according to claim 21, characterized in that The humanized CD70 gene comprises a portion of the human CD70 gene.

23. The humanized CD70 gene according to claim 22, characterized in that: The portion of the human CD70 gene includes a portion of exon 1, all of exon 2, and a portion of exon 3 of the human CD70 gene, wherein the portion of exon 1 contains at least 10bp of nucleotide sequence, and the portion of exon 3 contains at least 100bp of nucleotide sequence.

24. The humanized CD70 gene according to claim 23, characterized in that The portion of the human CD70 gene also includes introns 1-2 and / or introns 2-3.

25. The humanized CD70 gene according to claim 22, characterized in that: The portion of the human CD70 gene comprises the nucleotide sequence shown in SEQ ID NO:

5.

26. The humanized CD70 gene according to claim 21, characterized in that The humanized CD70 gene also includes a portion of a non-human animal CD70 gene.

27. The humanized CD70 gene according to claim 26, characterized in that The portion of the non-human animal CD70 gene includes a portion of exon 1 and / or a portion of exon 3 of the non-human animal CD70 gene.

28. The humanized CD70 gene according to claim 26, characterized in that The portion of the non-human animal CD70 gene comprises the nucleotide sequence encoding positions 1 to 49 of SEQ ID NO:

1.

29. The humanized CD70 gene according to claim 21, characterized in that The mRNA transcribed from the humanized CD70 gene comprises the nucleotide sequence shown in SEQ ID NO:

10.

30. A targeting vector, characterized in that The targeting vector comprises a donor nucleotide sequence, a 5' arm and a 3' arm; The donor nucleotide sequence is a nucleotide sequence encoding part of the extracellular region of human CD70 protein; The part of the extracellular region of the human CD70 protein is shown in SEQ ID NO: 2, positions 48-193; The 5' arm is a DNA fragment homologous to the 5' end of the switch region to be changed; The 3' arm is a DNA fragment homologous to the 3' end of the switch region to be changed; The switch region to be changed is located on exons 1 to 3 of the CD70 gene of a non-human animal; The non-human animal is a mouse.

31. The targeting vector according to claim 30, characterized in that The donor nucleotide sequence comprises part of exon 1, all of exon 2 and part of exon 3 of the human CD70 gene, wherein the part of exon 1 comprises at least 10 bp of nucleotide sequence, and the part of exon 3 comprises at least 100 bp of nucleotide sequence.

32. The targeting vector according to claim 30, characterized in that The donor nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:

5.

33. The targeting vector according to claim 30, characterized in that The 5' arm sequence is shown in SEQ ID NO:3; the 3' arm sequence is shown in SEQ ID NO:

4.

34. A cell, tissue or organ, characterized in that: The cell, tissue or organ expresses the humanized CD70 protein according to claim 20, or the genome of the cell, tissue or organ contains the humanized CD70 gene according to any one of claims 21-29, or the cell, tissue or organ is derived from a non-human animal obtained by the construction method according to any one of claims 1-19; The cells are incapable of developing into animal individuals.

35. A tumor tissue after tumor bearing, characterized in that: The tumor tissue expresses the humanized CD70 protein described in claim 20, or the genome of the tumor tissue contains the humanized CD70 gene described in any one of claims 21-29, or the tumor tissue is derived from a non-human animal obtained by the construction method described in any one of claims 1-19.

36. A use of the humanized CD70 protein of claim 20, the humanized CD70 gene of any one of claims 21-29, or a non-human animal obtained by the construction method of any one of claims 1-19, characterized in that: The applications described include: A) Application in product development involving CD70-related immune processes in human cells; B) Application as a model system related to CD70 in pharmacology, immunology, microbiology and medical research; C) applications involving the production and use of animal experimental disease models for the study of CD70-related etiology; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human CD70 signaling pathway regulators, where the application is for non-therapeutic purposes; or E) Studying the function of CD70 gene, studying drugs and drug efficacy targeting human CD70 target sites, and studying the application of CD70-related immune-related disease drugs and anti-tumor drugs, wherein the application is for non-therapeutic purposes.

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