Method for constructing a fap gene humanized non-human animal and applications

Abstract

The application provides a method for constructing a FAP gene humanized non-human animal, a nucleotide sequence encoding a human FAP protein is introduced into a non-human animal genome by homologous recombination, the animal can normally express the human or humanized FAP protein, and can be used as an animal model for researching a human FAP signal mechanism, screening drugs for tumor and immune related diseases, and has important application value for new drug research and development of an immune target. The application also provides a humanized FAP protein, a humanized FAP gene, a targeting vector of the FAP gene, a FAP gene humanized non-human animal, a FAP gene humanized cell and application thereof in the field of biological medicine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a method for constructing a FAP gene humanized non-human animal and application thereof in the field of biological medicine. BACKGROUND

[0002] Fibroblast activation protein (FAP) is a type II membrane-bound serine protease, which is active in homodimer form but not in monomeric form. FAP is a cell surface protein that is expressed at low levels in most normal adult tissues but is overexpressed in common cancers, particularly on cancer-associated fibroblasts that form the tumor stroma, which is essential for tumor growth. High expression of FAP on cancer-associated fibroblasts is often associated with poor prognosis of solid tumors. In histopathological studies, FAP-positive cancer-associated fibroblasts were found in more than 90% of epithelial tumors, while normal tissue fibroblasts rarely or even do not express FAP, which makes FAP a potential target for imaging and treatment of various malignancies.

[0003] Currently, the main treatment strategies targeting FAP include monoclonal antibodies, FAP enzyme activity inhibitors, FAP-activated targeted prodrugs, and DNA vaccines. Sibrotuzumab, a monoclonal antibody developed by Boehringer Ingelheim, was the first FAP antibody to enter clinical trials in 2002, and the project was suspended as it did not reach the expected endpoint. Roche has multiple FAP antibody research pipelines, mainly including FAP-OX40 bispecific antibodies, FAP antibody-4-1BBL, and FAP antibody-IL-2 for tumor treatment, and FAP antibody-IL-10 for the treatment of intestinal inflammation IBD. FAP antibody-4-1BBL is currently in clinical phase I / II. Basically, they are all in the preclinical development stage. FAP antibody drugs and FAP enzyme activity inhibitors have limited effect in current clinical research.

[0004] Disease models in experimental animals are indispensable research tools for studying the causes and pathogenesis of human diseases, developing prevention and treatment technologies, and developing therapeutic drugs. Common experimental animals include mice, rats, guinea pigs, hamsters (mice), rabbits, dogs, monkeys, pigs, fish, etc. However, there are still many differences between human and animal genes and protein sequences, and many human proteins cannot bind to animal homologous proteins to produce biological activity, resulting in many clinical trial results not consistent with animal experiment results. A large number of clinical researches urgently need better animal models.

[0005] However, due to the differences between animals and humans in physiology and pathology, and the complexity of genes, for example, the consistency of human and mouse FAP proteins is 89.488%, how to construct an "effective" humanized animal model for new drug research and development is still the biggest challenge.

[0006] In view of the complex mechanism of FAP and the great application value in the field of tumor treatment, there is an urgent need in the art to develop a non-human animal model related to the FAP signal pathway for further exploring the related biological characteristics of FAP, improving the effectiveness of preclinical drug efficacy tests, improving the success rate of research and development, making preclinical tests more effective and minimizing research and development failures. In addition, the non-human animal obtained by the method can also be mated with other gene humanized non-human animals to obtain a multi-gene humanized animal model for screening and evaluating the drug efficacy of human drugs and combination drugs targeting the signal pathway. The present application has broad application prospects in academic and clinical research. SUMMARY

[0007] The present application replaces the homologous genes of the animal genome with human normal or mutant genes to establish normal or mutant gene animal models that are closer to human physiological or disease characteristics. Gene humanized animals not only have important application value themselves, such as improving and upgrading cell or tissue transplantation humanized animal models through gene humanization, but more importantly, due to the insertion of human gene fragments, the animal can express or partially express human proteins, which can serve as a target for drugs that can only recognize human protein sequences, providing the possibility for screening of anti-human antibodies and other drugs at the animal level.

[0008] In a first aspect of the present application, a humanized FAP protein is provided, which comprises all or part of a human FAP protein.

[0009] Preferably, the humanized FAP protein comprises all or part of the extracellular region, transmembrane region and / or cytoplasmic region of a human FAP protein.

[0010] Further preferably, the humanized FAP protein comprises all or part of the extracellular region of a human FAP protein.

[0011] In a specific embodiment of the present application, the humanized FAP protein comprises at least 20 to at least 760 amino acid sequences of a human FAP protein, such as at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735, 740, 750, 760 continuous amino acid sequences.

[0012] In one embodiment of the application, the humanized FAP protein comprises an extracellular region of a human FAP protein of at least 20 to at least 735, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735 contiguous amino acid sequences.

[0013] In one embodiment of the application, the humanized FAP protein comprises an extracellular region of a human FAP protein with 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids removed from the N-terminus.

[0014] Further preferred, the humanized FAP protein comprises a portion of the extracellular region of a non-human animal endogenous FAP protein.

[0015] Preferably, the portion of the extracellular region of a non-human animal endogenous FAP protein comprises the amino acid sequence set forth in positions 26-31 of SEQ ID NO: 1; or, comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence set forth in positions 26-31 of SEQ ID NO: 1.

[0016] In one embodiment of the application, the humanized FAP protein comprises 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus of the extracellular region of a non-human animal endogenous FAP protein.

[0017] In some embodiments, further preferred, the humanized FAP protein further comprises all or a portion of the transmembrane region and / or the cytoplasmic region of a non-human animal endogenous FAP protein.

[0018] Further preferred, the transmembrane region of a non-human animal endogenous FAP protein comprises the amino acid sequence set forth in positions 5-25 of SEQ ID NO: 1; or, comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence set forth in positions 5-25 of SEQ ID NO: 1.

[0019] Further preferred, the cytoplasmic region of a non-human animal endogenous FAP protein comprises the amino acid sequence set forth in positions 1-4 of SEQ ID NO: 1; or, comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence set forth in positions 1-4 of SEQ ID NO: 1.

[0020] Preferably, the part of the human FAP protein and the part of the endogenous FAP protein of the non-human animal are directly linked or linked via a linker, preferably a peptide linker.

[0021] In some embodiments, the humanized FAP protein comprises the amino acid sequence from position 32 to position 760 of the human FAP protein and the rest of the amino acid sequence is the amino acid sequence of the endogenous FAP protein of the non-human animal.

[0022] In one embodiment of the present application, the humanized FAP protein comprises, in the order from N-terminus to C-terminus, the entire cytoplasmic region, the entire transmembrane region, and a part of the extracellular region of the endogenous FAP protein of the non-human animal, and a part of the extracellular region of the human FAP protein.

[0023] Preferably, the humanized FAP protein comprises the amino acid sequence set forth in SEQ ID NO: 2 from position 32 to position 760; or, comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 2 from position 32 to position 760; or, comprises an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 2 from position 32 to position 760 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or, comprises an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 2 from position 32 to position 760 by including substitution, deletion, and / or insertion of one or more amino acid residues.

[0024] Preferably, the humanized FAP protein further comprises the amino acid sequence set forth in SEQ ID NO: 1 from position 1 to position 31; or, comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1 from position 1 to position 31; or, comprises an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 1 from position 1 to position 31 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or, comprises an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 1 from position 1 to position 31 by including substitution, deletion, and / or insertion of one or more amino acid residues.

[0025] In one embodiment of the present application, the humanized FAP protein comprises the amino acid sequence set forth in SEQ ID NO: 2 from position 32 to position 760 and the amino acid sequence set forth in SEQ ID NO: 1 from position 1 to position 31.

[0026] Preferably, the humanized FAP protein comprises all or part of the amino acid sequence encoded by the human FAP gene.

[0027] Preferably, the humanized FAP protein comprises an amino acid sequence encoded by all or part of the exons 1 to 26 of the human FAP gene; further preferably comprises an amino acid sequence encoded by one, two or more than three of the exons 1 to 26 of the human FAP gene. Further preferably, comprises an amino acid sequence encoded by all or part of the exons 3 to 26 of the human FAP gene; still further preferably comprises an amino acid sequence encoded by one, two or more than three of the exons 3 to 26 of the human FAP gene. Still further preferably, comprises an amino acid sequence encoded by part of the exon 3, all of the exons 4 to 25 and part of the exon 26. Wherein, the part of the exon 3 of the human FAP gene comprises at least 50bp to at least 99bp, for example 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or at least 99bp of continuous nucleotide sequence, or, the part of the exon 3 of the human FAP gene comprises the nucleotide sequence of the coding region; the part of the exon 26 of the human FAP gene comprises at least 50bp to at least 397bp, for example 50, 60, 70, 80, 90, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 397bp of continuous nucleotide sequence, or, the part of the exon 26 of the human FAP gene comprises the nucleotide sequence of the coding region.

[0028] In one embodiment of the present application, the humanized FAP protein comprises the extracellular region of a human or humanized FAP protein. Preferably, the humanized FAP protein comprises the transmembrane region of a human or humanized FAP protein, preferably further comprising the cytoplasmic region of a human or humanized FAP protein.

[0029] Preferably, the extracellular region of the humanized FAP protein comprises all or a portion of the extracellular region of a human FAP protein, preferably the extracellular region of a human FAP protein comprising at least 100 to at least 735, e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 720, 728, 729, 730, or 735 contiguous amino acids; further preferably the extracellular region of a human FAP protein comprising 729 contiguous amino acids; more preferably the extracellular region of the humanized FAP protein comprises the amino acid sequence set forth in SEQ ID NO: 2 from the 32nd to the 760th amino acid; or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 2 from the 32nd to the 760th amino acid; or an amino acid sequence differing by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid from the amino acid sequence set forth in SEQ ID NO: 2 from the 32nd to the 760th amino acid; or an amino acid sequence comprising one or more amino acid residues that are substituted, deleted, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2 from the 32nd to the 760th amino acid.

[0030] Further preferably, the humanized FAP extracellular region further comprises all or a portion of the extracellular region of a non-human animal endogenous FAP protein, preferably the extracellular region of a non-human animal endogenous FAP protein comprising at least 1 to at least 736, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 736 contiguous amino acids; further preferably the extracellular region of a non-human animal endogenous FAP protein comprising 6 contiguous amino acids; more preferably the humanized FAP extracellular region comprises the amino acid sequence set forth in SEQ ID NO: 1 from the 26th to the 31st amino acid; or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1 from the 26th to the 31st amino acid; or an amino acid sequence differing by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid from the amino acid sequence set forth in SEQ ID NO: 1 from the 26th to the 31st amino acid; or an amino acid sequence comprising one or more amino acid residues that are substituted, deleted, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1 from the 26th to the 31st amino acid.

[0031] In one embodiment of the present application, the humanized FAP ectodomain comprises an ectodomain of human FAP protein of 729 contiguous amino acids, and an ectodomain of non-human endogenous FAP protein of 6 contiguous amino acids; preferably, the humanized FAP ectodomain comprises the amino acid sequence set forth in SEQ ID NO: 2 from position 32 to position 760 and the amino acid sequence set forth in SEQ ID NO: 1 from position 26 to position 31; wherein the portion of the ectodomain of human FAP protein (SEQ ID NO: 2 from position 32 to position 760) and the portion of the ectodomain of non-human endogenous FAP protein (SEQ ID NO: 1 from position 26 to position 31) are directly connected or connected through a linker, preferably a peptide linker.

[0032] Preferably, the humanized FAP transmembrane domain comprises all or part of the transmembrane domain of human FAP protein, preferably at least 1 to at least 21, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous amino acid sequence of the transmembrane domain of human FAP protein; further preferably, the humanized FAP transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 2 from position 5 to position 25; or, comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 2 from position 5 to position 25; or, comprises an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 2 from position 5 to position 25 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence that includes substitution, deletion and / or insertion of one or more amino acid residues from the amino acid sequence set forth in SEQ ID NO: 2 from position 5 to position 25.

[0033] Preferably, the humanized FAP cytoplasmic domain comprises all or part of the cytoplasmic domain of a human FAP protein, preferably, comprises at least 1 to at least 4, e.g., 1, 2, 3, or 4 contiguous amino acid sequences of the cytoplasmic domain of a human FAP protein; further preferably, the humanized FAP cytoplasmic domain comprises the amino acid sequence set forth in positions 1-4 of SEQ ID NO: 2; or, comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence set forth in positions 1-4 of SEQ ID NO: 2; or, comprises an amino acid sequence that differs from the amino acid sequence set forth in positions 1-4 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or, comprises an amino acid sequence that is identical to the amino acid sequence set forth in positions 1-4 of SEQ ID NO: 2, including substitution, deletion, and / or insertion of one or more amino acid residues.

[0034] Preferably, the humanized FAP protein comprises any one of the following groups:

[0035] 1) a human or humanized FAP extracellular domain, a transmembrane domain of a non-human animal endogenous FAP protein, and, a cytoplasmic domain of a non-human animal endogenous FAP protein;

[0036] 2) a human or humanized FAP extracellular domain, a transmembrane domain of a non-human animal endogenous FAP protein, and, a cytoplasmic domain of a non-human animal endogenous FAP protein;

[0037] 3) a human or humanized FAP extracellular domain, a transmembrane domain of a non-human animal endogenous FAP protein, and, a cytoplasmic domain of a non-human animal endogenous FAP protein;

[0038] 4) a human or humanized FAP extracellular domain, a transmembrane domain of a non-human animal endogenous FAP protein, and, a cytoplasmic domain of a non-human animal endogenous FAP protein;

[0039] 5) a human or humanized FAP extracellular domain, a transmembrane domain of a non-human animal endogenous FAP protein, and, a cytoplasmic domain of a non-human animal endogenous FAP protein;

[0040] 6) a human or humanized FAP extracellular domain, a transmembrane domain of a non-human animal endogenous FAP protein, and, a cytoplasmic domain of a non-human animal endogenous FAP protein;

[0041] 7) a human or humanized FAP extracellular domain, a transmembrane domain of a non-human animal endogenous FAP protein, and, a cytoplasmic domain of a non-human animal endogenous FAP protein.

[0042] In one embodiment of the present application, the humanized FAP protein comprises a cytoplasmic region of the endogenous FAP protein of a non-human animal (preferably, positions 1-4 of SEQ ID NO: 1), a transmembrane region of the endogenous FAP protein of a non-human animal (preferably, positions 5-25 of SEQ ID NO: 1), a portion of the extracellular region of the endogenous FAP protein of a non-human animal (preferably, positions 26-31 of SEQ ID NO: 1), and a portion of the extracellular region of a human FAP protein (preferably, positions 32-760 of SEQ ID NO: 2).

[0043] In one embodiment of the present application, the humanized FAP protein comprises a humanized FAP protein in which the amino acid sequence of positions 32-761 of the endogenous FAP protein of a non-human animal (SEQ ID NO: 1) is replaced, preferably, the humanized FAP protein comprises a humanized FAP protein in which the corresponding portion of the endogenous FAP protein of a non-human animal is replaced with the amino acid sequence of positions 32-760 of a human FAP protein (SEQ ID NO: 2).

[0044] In one embodiment of the present application, the amino acid sequence of the humanized FAP protein comprises any one of the following groups:

[0045] A) all or part of SEQ ID NO: 11;

[0046] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 11;

[0047] C) 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 of SEQ ID NO: 11; or

[0048] D) an amino acid sequence as set forth in SEQ ID NO: 11, including substitution, deletion, and / or insertion of one or more amino acid residues.

[0049] Preferably, the amino acid sequence of the humanized FAP protein derived from a human FAP protein is as set forth in SEQ ID NO: 2 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0050] Preferably, the amino acid sequence in the humanized FAP protein derived from the endogenous FAP protein of a non-human animal is as set forth in SEQ ID NO: 1 or has at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0051] Preferably, the non-human animal can be selected from the group consisting of rodents, zebrafish, pigs, chickens, rabbits, monkeys, and any other non-human animal that can be genetically edited to produce a humanized gene.

[0052] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. More preferably, the rodent is a rat or a mouse.

[0053] 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. More preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Still more preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null mouse, NOD-Rag 1 - / - -IL2rg - / - mouse, Rag 2 - / - -IL2rg - / - mouse, NOD / SCID mouse, or nude mouse.

[0054] Preferably, the humanized FAP protein comprises at least 200, 300, 400, 500, 600, 700, 720, or 729 amino acids that are identical to the corresponding amino acid sequence of a human FAP protein. Further preferably, the at least 200, 300, 400, 500, 600, 700, 720, or 729 amino acids are derived from the extracellular region of the humanized FAP protein.

[0055] In one embodiment, the humanized FAP protein comprises at least 729 amino acids that are identical to the corresponding amino acid sequence of a human FAP protein.

[0056] In a second aspect, the present application provides a nucleic acid encoding the humanized FAP protein as described above.

[0057] In a third aspect, the present application provides a humanized FAP gene.

[0058] Preferably, the humanized FAP gene comprises a portion of a human FAP gene.

[0059] Preferably, the humanized FAP gene encodes a humanized FAP protein as described above.

[0060] Preferably, the humanized FAP gene comprises all or a portion of exons 1 to 26 of a human FAP gene. Preferably comprises one, two or more than two of exons 1 to 26 of a human FAP gene. Further preferably comprises all or a portion of exons 3 to 26 of a human FAP gene. Preferably comprises one, two or more than two of exons 3 to 26 of a human FAP gene. More preferably comprises a portion of exon 3, all of exons 4 to 25 and a portion of exon 26 of a human FAP gene. Preferably further comprises intron 3-4 and / or intron 25-26. Wherein, the portion of exon 3 of a human FAP gene comprises at least 50bp to at least 99bp, for example 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or at least 99bp of contiguous nucleotide sequence; or, the portion of exon 3 of a human FAP gene comprises nucleotide sequence of coding region; the portion of exon 26 of a human FAP gene comprises at least 50bp to at least 397bp, for example 50, 60, 70, 80, 90, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 397bp of contiguous nucleotide sequence, or, the portion of exon 26 of a human FAP gene comprises nucleotide sequence of coding region.

[0061] In some embodiments, the humanized FAP gene comprises the nucleotide sequence set forth in SEQ ID NO: 5; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 5; or, comprises a nucleotide sequence differing from the nucleotide sequence set forth 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, comprises a nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 5 with one or more nucleotides of substitution, deletion and / or insertion.

[0062] Preferably, the humanized FAP gene comprises the nucleotide sequence set forth in SEQ ID NO: 6 and / or SEQ ID NO: 7; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 6 and / or SEQ ID NO: 7; or, comprises a nucleotide sequence that differs from the nucleotide sequence set forth in SEQ ID NO: 6 and / or SEQ ID NO: 7 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 the nucleotide sequence set forth in SEQ ID NO: 6 and / or SEQ ID NO: 7 with one or more nucleotides of substitution, deletion, and / or insertion.

[0063] Preferably, the humanized FAP gene further comprises a portion of the non-human animal endogenous FAP gene, which portion of the non-human animal endogenous FAP gene is all or part of exons 1-26 of the non-human animal endogenous FAP gene; preferably all or part of exons 1-3 and all or part of exon 26 of the non-human animal endogenous FAP gene; in some embodiments, the portion of the non-human animal endogenous FAP gene is all of exons 1-2, the 2-3 intron, part of exon 3, and part of exon 26 of the non-human animal endogenous FAP gene.

[0064] In an embodiment of the present application, the humanized FAP gene comprises, in order from 5’ to 3’, a portion of the non-human animal endogenous FAP gene (preferably all of exons 1-2, part of exon 3, preferably further comprising the 2-3 intron), a portion of the human FAP gene (preferably part of exon 3, all of exons 4-25, and part of exon 26, preferably further comprising the 3-4 intron and / or the 25-26 intron), and a portion of the non-human animal endogenous FAP gene (preferably part of exon 26 of the non-human animal endogenous FAP gene).

[0065] In an embodiment of the present application, the humanized FAP gene comprises, in order from 5’ to 3’, a portion of the non-human animal endogenous FAP gene (preferably all of exons 1-2, part of exon 3, preferably further comprising the 2-3 intron), SEQ ID NO: 5 or a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 from positions 32-760, and a 3’ UTR of the non-human animal endogenous FAP gene.

[0066] In some embodiments, the humanized FAP gene further comprises a resistance gene; preferably, the resistance gene is located between the 25th and 26th exon of the humanized FAP gene; preferably, the humanized FAP gene further comprises two same-directionally arranged Frt recombination sites flanking the resistance gene. In one embodiment, the resistance gene is a neomycin phosphotransferase-encoding sequence Neo.

[0067] Preferably, the humanized FAP gene comprises a nucleotide sequence encoding all or part of a human FAP protein, further preferably a nucleotide sequence encoding all or part of an extracellular region, a transmembrane region and / or a cytoplasmic region of a human FAP protein, more further preferably a nucleotide sequence encoding all or part of an extracellular region of a human FAP protein.

[0068] Still further preferably, the humanized FAP gene comprises a nucleotide sequence encoding at least 20 to at least 735, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735 consecutive amino acid sequences of an extracellular region of a human FAP protein.

[0069] Still further preferably, the humanized FAP gene comprises a nucleotide sequence encoding an extracellular region of a human FAP protein with 0-10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids removed from the N-terminus. More further preferably, the humanized FAP gene comprises a nucleotide sequence encoding the amino acid sequence set forth in positions 32-760 of SEQ ID NO: 2; or, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the nucleotide sequence encoding the amino acid sequence set forth in positions 32-760 of SEQ ID NO: 2; or, a nucleotide sequence differing from the nucleotide sequence encoding the amino acid sequence set forth in positions 32-760 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or, a nucleotide sequence having the nucleotide sequence encoding the amino acid sequence set forth in positions 32-760 of SEQ ID NO: 2, including one or more nucleotides of substitution, deletion, and / or insertion.

[0070] Preferably, the humanized FAP gene comprises a nucleotide sequence encoding all or part of the endogenous FAP protein of a non-human animal, further preferably comprises a nucleotide sequence encoding part of the extracellular region, all of the transmembrane region and all of the cytoplasmic region of the endogenous FAP protein of a non-human animal, wherein the part of the extracellular region of the endogenous FAP protein of a non-human animal comprises at least the N-terminal 0-10 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) contiguous amino acids of the extracellular region of the endogenous FAP protein of a non-human animal, more preferably comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 at positions 1-31; or comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 at positions 1-31; or comprises a nucleotide sequence differing by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide from the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 at positions 1-31; or comprises a nucleotide sequence having the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 at positions 1-31, including substitution, deletion and / or insertion of one or more nucleotides.

[0071] In one embodiment of the present application, the humanized FAP gene comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 at positions 1-4, 1-25, 5-25 or 1-31.

[0072] Preferably, the humanized FAP gene is regulated by a regulatory element in the non-human animal, further preferably, the regulatory element is endogenous or exogenous.

[0073] Preferably, the regulatory element comprises a promoter.

[0074] Preferably, the humanized FAP gene comprises a 5' UTR and / or a 3' UTR of the endogenous FAP gene of a non-human animal.

[0075] Preferably, the non-human animal can be selected from rodents, zebrafish, pigs, chickens, rabbits, monkeys and any other non-human animal that can be genetically edited to produce a humanized gene.

[0076] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. More preferably, the rodent is a rat or a mouse.

[0077] 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. More preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Still more preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null mouse, NOD-Rag 1 - / - -IL2rg - / - mouse, Rag 2 - / - -IL2rg - / - mouse, NOD / SCID mouse or nude mouse.

[0078] In one embodiment of the present application, the mRNA transcribed from the humanized FAP gene comprises one of the following groups:

[0079] (a) all or part of the nucleotide sequence as shown in SEQ ID NO: 10;

[0080] (b) a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence as shown in SEQ ID NO: 10;

[0081] (c) a nucleotide sequence differing from the nucleotide sequence as shown in SEQ ID NO: 10 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or

[0082] (d) a nucleotide sequence as shown in SEQ ID NO: 10, including substitution, deletion and / or insertion of one or more nucleotides.

[0083] Preferably, the humanized FAP gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor can be a conventional inducer or repressor. In one embodiment of the present application, the specific inducer is selected from the Tet-Off System / Tet-On System or the Tamoxifen System.

[0084] In a fourth aspect of the present application, a targeting vector targeting the endogenous FAP gene of a non-human animal is provided, wherein the targeting vector comprises any one of the following groups:

[0085] A) a portion of the human FAP gene, preferably comprising all or part of exons 1 to 26 of the human FAP gene, preferably comprising one, two or more than two of exons 1 to 26 of the human FAP gene in combination, further preferably comprising all or part of exons 3 to 26 of the human FAP gene, more preferably comprising part of exon 3, all of exons 4 to 25 and part of exon 26 of the human FAP gene, preferably comprising the 3-4 intron and / or the 25-26 intron, wherein the portion of exon 3 of the human FAP gene comprises a contiguous nucleotide sequence of at least 50 bp to at least 99 bp, e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or at least 99 bp; or, the portion of exon 3 of the human FAP gene comprises a nucleotide sequence of the coding region; the portion of exon 26 of the human FAP gene comprises a contiguous nucleotide sequence of at least 50 bp to at least 397 bp, e.g. 50, 60, 70, 80, 90, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 397 bp, or, the portion of exon 26 of the human FAP gene comprises a nucleotide sequence of the coding region; further more preferably comprising the nucleotide sequence set forth in SEQ ID NO: 5; or, comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 5; or, comprising no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference to the nucleotide sequence set forth in SEQ ID NO: 5; or, comprising a nucleotide sequence having the sequence set forth in SEQ ID NO: 5 with one or more nucleotides substituted, deleted and / or inserted, including

[0086] B) a nucleotide sequence encoding all or part of a human FAP protein; preferably comprising a nucleotide sequence encoding all or part of an extracellular, transmembrane and / or cytoplasmic region of a human FAP protein; further preferably comprising a nucleotide sequence encoding all or part of an extracellular region of a human FAP protein; more preferably comprising a nucleotide sequence encoding at least 20 to at least 760, e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735, 740, 750, 760 contiguous amino acid sequence of an extracellular region of a human FAP protein; further more preferably comprising a nucleotide sequence encoding at least 20 to at least 735, e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735 contiguous amino acid sequence of an extracellular region of a human FAP protein; again preferably comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760, or, comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760; or, comprising a nucleotide sequence which differs from the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprising a nucleotide sequence which is identical to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760, including one or more nucleotide substitutions, deletions and / or insertions;

[0087] C) a nucleotide sequence encoding a human or humanized FAP protein; or,

[0088] D) a nucleotide sequence of a human or humanized FAP gene.

[0089] Preferably, any of the nucleotide sequences of A) to D) is a donor DNA sequence.

[0090] In one embodiment of the application, the targeting vector comprises a nucleotide sequence as set forth in SEQ ID NO: 6 and / or SEQ ID NO: 7.

[0091] In one embodiment of the application, the targeting vector comprises a nucleotide sequence as set forth in SEQ ID NO: 8 and / or SEQ ID NO: 9.

[0092] In some embodiments, the targeting vector further comprises a resistance gene; preferably, the resistance gene is located between the 25th and 26th exon of the human FAP gene; preferably, the targeting vector further comprises two Frt recombination sites in the same orientation flanking the resistance gene. In one embodiment, the resistance gene is a neomycin phosphotransferase coding sequence Neo.

[0093] Preferably, the targeting vector further comprises a 5' arm and / or a 3' arm.

[0094] The 5' arm (or 5' homology arm) is a DNA fragment homologous to the 5' end of the conversion region to be altered. It is selected from nucleotides of 100-10000 in length of the genomic DNA of the endogenous FAP gene of the non-human animal; preferably, the 5' arm has at least 90% homology to the nucleotides of NCBI Accession No. NC_000068.8; further preferably, the 5' arm sequence comprises SEQ ID NO: 3.

[0095] The 3' arm (or 3' homology arm) is a DNA fragment homologous to the 3' end of the conversion region to be altered. It is selected from nucleotides of 100-10000 in length of the genomic DNA of the endogenous FAP gene of the non-human animal; preferably, the 3' arm has at least 90% homology to the nucleotides of NCBI Accession No. NC_000068.8; further preferably, the 3' arm sequence comprises SEQ ID NO: 4.

[0096] Preferably, the conversion region to be altered is located on the 1st to 26th exon of the endogenous FAP gene of the non-human animal, further preferably on the 3rd to 26th exon of the endogenous FAP gene of the non-human animal.

[0097] Preferably, the targeting vector further comprises a non-human animal 3' UTR.

[0098] Preferably, the targeting vector further comprises a non-human animal 5' UTR.

[0099] Preferably, the non-human animal can be selected from rodents, zebrafish, pigs, chickens, rabbits, monkeys, and any non-human animal that can be genetically edited to prepare a genetically humanized non-human animal.

[0100] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. More preferably, the rodent is a rat or a mouse.

[0101] 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. More preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Still more preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null mouse, NOD-Rag 1 - / - -IL2rg - / - mouse, Rag 2 - / - -IL2rg - / - mouse, NOD / SCID mouse or nude mouse.

[0102] In a fifth aspect of the present application, there is provided a sgRNA targeting an endogenous FAP gene of a non-human animal.

[0103] Preferably, the sequence of the sgRNA is on a target sequence of an endogenous FAP gene of a non-human animal to be modified.

[0104] In a sixth aspect of the present application, there is provided a DNA molecule encoding the sgRNA targeting an endogenous FAP gene of a non-human animal.

[0105] Preferably, the double strand of the DNA molecule is the upstream and downstream sequences of the sgRNA, or the forward oligonucleotide sequence or the reverse oligonucleotide sequence after adding a restriction enzyme site.

[0106] In a seventh aspect of the present application, there is provided a vector comprising the sgRNA targeting an endogenous FAP gene of a non-human animal.

[0107] In an eighth aspect of the present application, there is provided a cell comprising the targeting vector targeting an endogenous FAP gene of a non-human animal, the sgRNA targeting an endogenous FAP gene of a non-human animal, the DNA molecule and / or the vector.

[0108] In a ninth aspect of the present application, there is provided the use of the targeting vector targeting an endogenous FAP gene of a non-human animal, the sgRNA targeting an endogenous FAP gene of a non-human animal, the DNA molecule, the sgRNA vector and / or the cell in FAP gene modification.

[0109] Preferably, the use includes but is not limited to knockout, insertion or replacement.

[0110] In a tenth aspect of the present application, there is provided a FAP gene humanized cell, wherein the cell expresses any of the humanized FAP proteins, and / or the genome of the cell comprises part of a human FAP gene or the nucleic acid or the humanized FAP gene.

[0111] Preferably, the cell further comprises other genetic modifications, preferably the other genes are selected from at least one of PD-1, PD-L1, TIGIT, CD226, CD40, CD3, CD137, CD28, 4-1BB, IL-2, OX40 or IL-10.

[0112] Preferably, the cell is incapable of developing into an animal individual.

[0113] In an eleventh aspect, the present application provides a method for preparing a FAP gene humanized cell, the method comprising introducing into a cell any one of the nucleotide sequences in the following group:

[0114] A) a portion of the human FAP gene, preferably comprising all or part of exons 1 to 26 of the human FAP gene, preferably comprising one, two or more than three of exons 1 to 26 of the human FAP gene in combination, further preferably comprising all or part of exons 3 to 26 of the human FAP gene, more preferably comprising part of exon 3, all of exons 4 to 25 and part of exon 26 of the human FAP gene, preferably further comprising intron 3-4 and / or intron 25-26, wherein the part of exon 3 of the human FAP gene comprises a contiguous nucleotide sequence of at least 50 bp to at least 99 bp, e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or at least 99 bp; or the part of exon 3 of the human FAP gene comprises a nucleotide sequence of the coding region; the part of exon 26 of the human FAP gene comprises a contiguous nucleotide sequence of at least 50 bp to at least 397 bp, e.g. 50, 60, 70, 80, 90, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 397 bp, or the part of exon 26 of the human FAP gene comprises a nucleotide sequence of the coding region; further preferably comprising the nucleotide sequence set forth in SEQ ID NO: 5; or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 5; or a nucleotide sequence differing from the nucleotide sequence set forth 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 a nucleotide sequence having the sequence set forth in SEQ ID NO: 5 with one or more nucleotides substituted, deleted and / or inserted;

[0115] B) a nucleotide sequence encoding all or part of a human FAP protein; preferably comprising a nucleotide sequence encoding all or part of an extracellular region, a transmembrane region and / or a cytoplasmic region of a human FAP protein; further preferably comprising a nucleotide sequence encoding all or part of an extracellular region of a human FAP protein; more preferably comprising a nucleotide sequence encoding at least 20 to at least 760, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735, 740, 750, 760 contiguous amino acid sequence of a human FAP protein; further more preferably comprising a nucleotide sequence encoding at least 20 to at least 735, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735 contiguous amino acid sequence of an extracellular region of a human FAP protein; again preferably comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760, or, comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760; or, comprising a nucleotide sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid difference from the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760; or, comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760, including one or more nucleotide substitutions, deletions and / or insertions;

[0116] C) a nucleotide sequence encoding a human or humanized FAP protein; or,

[0117] D) a nucleotide sequence of a human or humanized FAP gene.

[0118] In a twelfth aspect, the present application provides a non-human animal with humanized FAP gene.

[0119] Preferably, the non-human animal expresses a human or humanized FAP protein in vivo, and / or the genome of the non-human animal comprises the nucleic acid as described above, or a portion of a human FAP gene, or a humanized FAP gene.

[0120] Preferably, the humanized FAP protein is any of the humanized FAP proteins as described above.

[0121] Preferably, the humanized FAP gene is any of the humanized FAP genes as described above.

[0122] Preferably, the endogenous FAP protein expression of the non-human animal is reduced or absent.

[0123] In a specific embodiment of the present application, preferably, the non-human animal genome comprises the nucleic acid as described above or comprises the humanized FAP gene as described above.

[0124] The genome of the non-human animal comprises all or part of the human FAP gene. Preferably, the genome of the non-human animal comprises all or part of the exon 1 to 26 of the human FAP gene. Further preferably, the genome of the non-human animal comprises one, two or more than two of the exon 1 to 26 of the human FAP gene, and more preferably, the genome of the non-human animal comprises all or part of the exon 3 to 26 of the human FAP gene. More preferably, the genome of the non-human animal comprises part of the exon 3, all of the exon 4 to 25 and part of the exon 26 of the human FAP gene, and preferably further comprises the intron 3-4 and / or the intron 25-26. In the part of the exon 3 of the human FAP gene, it comprises at least 50 bp to at least 99 bp, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or at least 99 bp of continuous nucleotide sequence; or, the part of the exon 3 of the human FAP gene comprises the nucleotide sequence of the coding region. In the part of the exon 26 of the human FAP gene, it comprises at least 50 bp to at least 397 bp, such as 50, 60, 70, 80, 90, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 397 bp of continuous nucleotide sequence; or, the part of the exon 26 of the human FAP gene comprises the nucleotide sequence of the coding region.

[0125] In a more preferred specific embodiment of the present application, the genome of the non-human animal comprises the nucleotide sequence as set forth in SEQ ID NO: 5; or, comprises the nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence as set forth in SEQ ID NO: 5; or, comprises the nucleotide sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference to the nucleotide sequence as set forth in SEQ ID NO: 5; or, comprises the nucleotide sequence having the nucleotide sequence as set forth in SEQ ID NO: 5 with one or more nucleotide substitution, deletion and / or insertion.

[0126] Preferably, the non-human animal genome comprises a nucleotide sequence encoding all or part of a human or humanized FAP protein. Preferably, the nucleotide sequence comprises a nucleotide sequence encoding all or part of an extracellular, transmembrane and / or cytoplasmic region of a human or humanized FAP protein. Further preferably, the nucleotide sequence comprises a nucleotide sequence encoding all or part of an extracellular region of a human FAP protein. Still further preferably, the nucleotide sequence comprises a nucleotide sequence encoding at least 20 to at least 760, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735, 740, 750, 760 contiguous amino acids of an extracellular region of a human FAP protein; again preferably, the nucleotide sequence comprises a nucleotide sequence encoding at least 20 to at least 735, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735 contiguous amino acids of an extracellular region of a human FAP protein; still further preferably, the nucleotide sequence comprises a nucleotide sequence encoding an extracellular region of a human FAP protein with 0-10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids removed from the N-terminus.

[0127] In one embodiment of the application, the non-human animal genome comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to amino acid 760; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to amino acid 760; or, comprises a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to amino acid 760 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 is identical to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to amino acid 760, including one or more nucleotide substitutions, deletions and / or insertions.

[0128] Preferably, the non-human animal comprises a nucleotide sequence from any one of the following groups introduced into the endogenous FAP locus of the non-human animal:

[0129] A) a part of the human FAP gene, preferably all or part of exons 1 to 26 of the human FAP gene, preferably comprising a combination of one, two or more than two of exons 1 to 26 of the human FAP gene, further preferably all or part of exons 3 to 26 of the human FAP gene, still further preferably a part of exon 3, all of exons 4 to 25 and a part of exon 26 of the human FAP gene, preferably further comprising the 3-4th intron and / or the 25-26th intron, wherein the part of exon 3 of the human FAP gene comprises a contiguous nucleotide sequence of at least 50 bp to at least 99 bp, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or at least 99 bp; or the part of exon 3 of the human FAP gene comprises a nucleotide sequence of the coding region; the part of exon 26 of the human FAP gene comprises a contiguous nucleotide sequence of at least 50 bp to at least 397 bp, such as 50, 60, 70, 80, 90, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 397 bp, or the part of exon 26 of the human FAP gene comprises a nucleotide sequence of the coding region; more preferably comprising the nucleotide sequence as set forth in SEQ ID NO: 5; or a nucleotide sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence as set forth in SEQ ID NO: 5; or a nucleotide sequence which differs from the nucleotide sequence as set forth 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 a nucleotide sequence which has the nucleotide sequence as set forth in SEQ ID NO: 5 except for one or more nucleotides which are substituted, deleted and / or inserted;

[0130] B) a nucleotide sequence encoding all or part of a human FAP protein; preferably comprising a nucleotide sequence encoding all or part of an extracellular region, a transmembrane region and / or a cytoplasmic region of a human FAP protein; further preferably comprising a nucleotide sequence encoding all or part of an extracellular region of a human FAP protein; more preferably comprising a nucleotide sequence encoding at least 20 to at least 760, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735, 740, 750, 760 contiguous amino acid sequence of an extracellular region of a human FAP protein; further more preferably comprising a nucleotide sequence encoding at least 20 to at least 735, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735 contiguous amino acid sequence of an extracellular region of a human FAP protein; still further preferably comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 from amino acid 32 to amino acid 760, or, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 from amino acid 32 to amino acid 760; or, a nucleotide sequence differing by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid from a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 from amino acid 32 to amino acid 760; or, a nucleotide sequence comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 from amino acid 32 to amino acid 760, including one or more nucleotide substitutions, deletions and / or insertions;

[0131] C) a nucleotide sequence encoding a human or humanized FAP protein; or,

[0132] D) a nucleotide sequence of a human or humanized FAP gene.

[0133] Preferably, the genome of at least one cell of the non-human animal comprises a nucleotide sequence encoding a human or humanized FAP protein, a portion of a human FAP gene, or the humanized FAP gene described above.

[0134] Preferably, the nucleotide sequence encoding the human or humanized FAP protein or the nucleotide sequence of the human or humanized FAP gene is operably linked to an endogenous regulatory element at an endogenous FAP locus in the at least one chromosome.

[0135] Preferably, the non-human animal is constructed by the targeting vector described above.

[0136] Preferably, the non-human animal further comprises other genetic modifications, preferably, the other genes are selected from at least one of PD-1, PD-L1, TIGIT, CD226, CD40, CD3, CD137, CD28, 4-1BB, IL-2, OX40 or IL-10.

[0137] Preferably, the FAP gene and / or the other genes are homozygous for the endogenous modified (preferably replaced) locus.

[0138] Preferably, the FAP gene and / or the other genes are heterozygous for the endogenous modified (preferably replaced) locus.

[0139] Preferably, the non-human animal can be selected from rodent, zebrafish, pig, chicken, rabbit, monkey, and any other non-human animal that can be genetically edited to produce a humanized FAP gene.

[0140] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. More preferably, the rodent is a rat or a mouse.

[0141] 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. More preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Still more preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag1 - / - -IL2rg - / - Mouse, Rag 2 - / - -IL2rg - / - Mouse, NOD / SCID mouse or nude mouse.

[0142] In a thirteenth aspect, the present application provides a method for constructing a non-human animal with humanized FAP gene, wherein the non-human animal expresses human or humanized FAP protein in vivo, and / or the genome of the non-human animal comprises the nucleic acid, or part of human FAP gene, or humanized FAP gene as described above.

[0143] Preferably, the non-human animal has reduced or absent expression of endogenous FAP protein.

[0144] Preferably, the genome of the non-human animal comprises the humanized FAP gene as described above.

[0145] Preferably, the genome of the non-human animal comprises all or part of a human FAP gene. Preferably, the genome of the non-human animal comprises all or part of exons 1 to 26 of a human FAP gene. Preferably, the genome of the non-human animal comprises one, two or more than two of exons 1 to 26 of a human FAP gene, further preferably, the genome of the non-human animal comprises all or part of exons 3 to 26 of a human FAP gene. More preferably, the genome of the non-human animal comprises part of exon 3, all of exons 4 to 25 and part of exon 26 of a human FAP gene. Preferably, the genome of the non-human animal further comprises intron 3-4 and / or intron 25-26. Preferably, the part of exon 3 of a human FAP gene comprises a continuous nucleotide sequence of at least 50 bp to at least 99 bp, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or at least 99 bp; or, the part of exon 3 of a human FAP gene comprises a nucleotide sequence of a coding region; the part of exon 26 of a human FAP gene comprises a continuous nucleotide sequence of at least 50 bp to at least 397 bp, such as 50, 60, 70, 80, 90, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 397 bp; or, the part of exon 26 of a human FAP gene comprises a nucleotide sequence of a coding region; more preferably, the genome of the non-human animal comprises a nucleotide sequence as set forth in SEQ ID NO: 5; or, the genome of the non-human animal comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence as set forth in SEQ ID NO: 5; or, the genome of the non-human animal comprises a nucleotide sequence differing from the nucleotide sequence as set forth 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, the genome of the non-human animal comprises a nucleotide sequence having one or more nucleotides of substitution, deletion and / or insertion as compared to the nucleotide sequence as set forth in SEQ ID NO: 5.

[0146] Preferably, the genome of the non-human animal comprises a nucleotide sequence encoding all or part of a human or humanized FAP protein. Preferably, the nucleotide sequence comprises a nucleotide sequence encoding all or part of an extracellular region, a transmembrane region and / or a cytoplasmic region of a human FAP protein. Further preferably, the nucleotide sequence comprises a nucleotide sequence encoding all or part of an extracellular region of a human FAP protein. Still further preferably, the nucleotide sequence comprises a nucleotide sequence encoding at least 20 to at least 760, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735, 740, 750, 760 contiguous amino acids of an extracellular region of a human FAP protein; still more preferably, the nucleotide sequence comprises a nucleotide sequence encoding at least 20 to at least 735, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735 contiguous amino acids of an extracellular region of a human FAP protein. Still further preferably, the nucleotide sequence comprises a nucleotide sequence encoding an extracellular region of a human FAP protein with 0-10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids removed from the N-terminus.

[0147] In one embodiment of the application, the genome of the non-human animal comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 from amino acid 32 to amino acid 760, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 from amino acid 32 to amino acid 760; or a nucleotide sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid difference from the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 from amino acid 32 to amino acid 760; or a nucleotide sequence comprising the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 from amino acid 32 to amino acid 760, including one or more nucleotide substitutions, deletions and / or insertions.

[0148] Preferably, the non-human animal can be selected from the group consisting of a rodent, a zebrafish, a pig, a chicken, a rabbit, a monkey, and any other non-human animal that can be genetically edited to produce a humanized gene.

[0149] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Still further preferably, the rodent is a rat or a mouse.

[0150] 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. Still further preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Yet still further preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null mouse, NOD-Rag 1 - / - -IL2rg - / - mouse, Rag 2 - / - -IL2rg - / - mouse, NOD / SCID mouse, or nude mouse.

[0151] Preferably, the constructing method comprises introducing into the non-human animal a nucleotide sequence comprising any one of the following groups.

[0152] A) a part of the human FAP gene, preferably comprising all or part of exons 1 to 26 of the human FAP gene, preferably comprising one, two or more than three of exons 1 to 26 of the human FAP gene in combination, further preferably comprising all or part of exons 3 to 26 of the human FAP gene, more preferably comprising part of exon 3, all of exons 4 to 25 and part of exon 26 of the human FAP gene, preferably further comprising intron 3-4 and / or intron 25-26, wherein the part of exon 3 of the human FAP gene comprises a contiguous nucleotide sequence of at least 50 bp to at least 99 bp, e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or at least 99 bp; or the part of exon 3 of the human FAP gene comprises a nucleotide sequence of the coding region; the part of exon 26 of the human FAP gene comprises a contiguous nucleotide sequence of at least 50 bp to at least 397 bp, e.g. 50, 60, 70, 80, 90, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 397 bp, or the part of exon 26 of the human FAP gene comprises a nucleotide sequence of the coding region; further preferably comprising the nucleotide sequence as set forth in SEQ ID NO: 5; or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence as set forth in SEQ ID NO: 5; or a nucleotide sequence which differs from the nucleotide sequence as set forth 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 a nucleotide sequence which has the nucleotide sequence as set forth in SEQ ID NO: 5 with one or more nucleotides substituted, deleted and / or inserted, including

[0153] B) a nucleotide sequence encoding all or part of a human FAP protein; preferably comprising a nucleotide sequence encoding all or part of an extracellular region, a transmembrane region and / or a cytoplasmic region of a human FAP protein; preferably comprising a nucleotide sequence encoding all or part of an extracellular region of a human FAP protein; further preferably comprising a nucleotide sequence encoding at least 20 to at least 760, e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735, 740, 750, 760 contiguous amino acid sequence of a human FAP protein; more further preferably comprising a nucleotide sequence encoding at least 20 to at least 735, e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 725, 729, 730, 735 contiguous amino acid sequence of an extracellular region of a human FAP protein; again preferably comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760, or, comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760; or, comprising a nucleotide sequence which differs from the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprising a nucleotide sequence which is identical to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 from amino acid 32 to 760, including substitution, deletion and / or insertion of one or more nucleotides;

[0154] C) a nucleotide sequence encoding a human or humanized FAP protein; or,

[0155] D) a nucleotide sequence of a human or humanized FAP gene.

[0156] Preferably, the introduction is an insertion or a replacement.

[0157] wherein the insertion is to place the fragment of interest, such as a human FAP gene, a humanized FAP gene, a nucleotide sequence encoding a human or humanized FAP protein, a nucleotide sequence obtained by splicing a human FAP gene with a non-human animal endogenous FAP gene, between two adjacent bases without deleting nucleotides. Of course, it can also be a partial nucleotide sequence of a human FAP gene.

[0158] In some embodiments, the nucleotide sequence introduced into the FAP locus of the non-human animal further comprises a resistance gene; preferably, the resistance gene is located between the 25th and 26th exons of the human FAP gene; preferably, the nucleotide sequence further comprises two Frt recombination sites flanking the resistance gene. In one embodiment, the resistance gene is a neomycin phosphotransferase coding sequence, Neo.

[0159] Further preferably, the nucleotide sequence introduced into the FAP locus of the non-human animal further comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 8 and / or SEQ ID NO: 9, or comprises the nucleotide sequence set forth in SEQ ID NO: 8 and / or SEQ ID NO: 9.

[0160] Further preferably, the nucleotide sequence introduced into the FAP locus of the non-human animal further comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 6 and / or SEQ ID NO: 7, or comprises the nucleotide sequence set forth in SEQ ID NO: 6 and / or SEQ ID NO: 7.

[0161] Preferably, the nucleotide sequence introduced into the FAP locus of the non-human animal comprises any of A)-D) operably linked to an endogenous or exogenous regulatory element.

[0162] Preferably, the nucleotide sequence encoding the human FAP or humanized FAP protein, the portion of the human FAP gene, or the humanized FAP gene is regulated by a regulatory element in the non-human animal, which is endogenous or exogenous. Preferably, the regulatory element includes, but is not limited to, a promoter.

[0163] Preferably, the endogenous regulatory element is derived from the endogenous FAP gene of the non-human animal, and the exogenous regulatory element is derived from the human FAP gene.

[0164] Preferably, the method of construction comprises modifying the reading frame of the endogenous FAP gene of the non-human animal, and inserting a nucleotide sequence comprising the human FAP gene as described above downstream of the endogenous regulatory element of the endogenous FAP gene of the non-human animal, wherein the modification of the reading frame of the endogenous FAP gene of the non-human animal can be performed by knocking out a functional region of the endogenous FAP gene of the non-human animal or by inserting a sequence, so that the endogenous FAP protein of the non-human animal is not expressed or is expressed at a reduced level or is a non-functional protein; further preferably, the knockout is a knockout of the nucleotide sequence starting from within the 3rd exon of the endogenous FAP gene and ending within the 26th exon of the endogenous FAP gene.

[0165] Preferably, the insertion can also disrupt the coding frame of the endogenous FAP gene of the non-human animal or disrupt the coding frame of the endogenous FAP gene after the inserted sequence, and then the insertion is performed according to the specific embodiment. Alternatively, the insertion step can both make a frame shift mutation to the endogenous FAP gene and achieve the step of inserting the human sequence.

[0166] Preferably, the insertion site is after the endogenous regulatory element of the endogenous FAP gene of the non-human animal.

[0167] In one specific embodiment of the present application, the construction method comprises inserting the nucleotide sequence encoding the human or humanized FAP protein or the nucleotide sequence of the human or humanized FAP gene and / or the auxiliary sequence after the endogenous regulatory element of the endogenous FAP gene of the non-human animal, and the auxiliary sequence can be a sequence with a termination function, so that the human or humanized FAP protein is expressed in the animal model of the humanized FAP gene, and the non-human animal endogenous FAP protein is not expressed. Further preferably, the auxiliary sequence can be a WPRE and / or STOP sequence.

[0168] The replacement includes replacement at corresponding positions or replacement at non-corresponding positions. The replacement at corresponding positions not only mechanically represents the direct correspondence between the human and the non-human animal endogenous FAP gene base sites, but also includes replacement of corresponding functional regions.

[0169] Preferably, the introduction of the non-human animal endogenous FAP gene locus is to replace the corresponding region of the non-human animal.

[0170] Further preferably, the nucleotide sequence encoding the entire or part of the non-human animal endogenous FAP protein in the non-human animal genome is replaced.

[0171] Preferably, the entire or part of the 1st to 26th exon of the non-human animal endogenous FAP gene is replaced, further preferably the entire or part of the 3rd to 26th exon is replaced, more preferably part of the 3rd exon, the entire of the 4th to 25th exon and part of the 26th exon is replaced, preferably the 3rd-4th intron and / or the 25th-26th intron is also replaced.

[0172] Preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 in the non-human animal genome is replaced, further preferably the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 32-761 is replaced.

[0173] In one embodiment of the present application, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the extracellular region, transmembrane region and / or cytoplasmic region of the FAP protein in the non-human animal with a nucleotide sequence comprising all or part of the nucleotide sequence encoding the extracellular region, transmembrane region and / or cytoplasmic region of the human FAP protein.

[0174] In one embodiment of the present application, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the extracellular region of the FAP protein in the non-human animal with a nucleotide sequence comprising all or part of the nucleotide sequence encoding the extracellular region of the human FAP protein.

[0175] In one embodiment of the present application, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the extracellular region of the FAP protein in the non-human animal with a nucleotide sequence comprising all or part of the nucleotide sequence encoding the extracellular region of the human FAP protein.

[0176] In one embodiment of the present application, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the extracellular region of the FAP protein in the non-human animal with a nucleotide sequence comprising all or part of the nucleotide sequence encoding the extracellular region of the human FAP protein.

[0177] In one embodiment of the present application, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the extracellular region of the FAP protein in the non-human animal with a nucleotide sequence comprising all or part of the nucleotide sequence encoding the extracellular region of the human FAP protein.

[0178] In one embodiment of the present application, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the extracellular region of the FAP protein in the non-human animal with a nucleotide sequence comprising all or part of the nucleotide sequence encoding the extracellular region of the human FAP protein.

[0179] In one embodiment of the present application, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the extracellular region of the FAP protein in the non-human animal with a nucleotide sequence comprising all or part of the nucleotide sequence encoding the extracellular region of the human FAP protein.

[0180] In one embodiment of the present application, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the extracellular region of the FAP protein in the non-human animal with a nucleotide sequence comprising all or part of the nucleotide sequence encoding the extracellular region of the human FAP protein.

[0181] In one embodiment of the present application, the construction method comprises inserting or replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 from 32 to 761 into or in place of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 from 32 to 761 in the genome of the non-human animal.

[0182] The introduction of the non-human animal endogenous FAP gene locus is preferably insertion into the non-human animal endogenous FAP gene locus. Preferably, the coding frame of the non-human animal endogenous FAP gene is edited to reduce or delete the expression of the non-human animal endogenous FAP protein, and then the nucleotide sequence encoding the human or humanized FAP protein is inserted into the non-human animal endogenous FAP gene locus, preferably into any position of the 1st to 26th exon of the non-human animal endogenous FAP gene.

[0183] Preferably, the nucleotide sequence encoding the extracellular region of the human FAP protein is inserted after the coding region of the non-human animal endogenous FAP transmembrane region, preferably between the coding region of the non-human animal FAP transmembrane region and the extracellular region.

[0184] Preferably, the nucleotide sequence of the 3rd to 26th exon of the human FAP gene is inserted at the 3rd exon of the non-human animal endogenous FAP gene.

[0185] Preferably, the non-human animal is constructed using gene editing technology, including 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.

[0186] In one embodiment of the present application, the construction method comprises introducing the above-mentioned targeting vector into non-human animal cells (preferably embryonic stem cells), introducing positive clone cells into isolated blastocysts, transplanting into the oviduct of female non-human animals, allowing development, and identifying and screening to obtain a non-human animal with humanized FAP gene.

[0187] Preferably, to improve the recombination efficiency, the above-mentioned targeting vector targeting the non-human animal endogenous FAP gene can also be used together with the above-mentioned sgRNA to construct the non-human animal.

[0188] In one embodiment, the above-mentioned targeting vector targeting the non-human animal endogenous FAP gene, the above-mentioned sgRNA and the Cas9 protein are used to construct the non-human animal. The above-mentioned targeting vector, the above-mentioned sgRNA and the Cas9 are introduced into non-human animal cells, the cells (preferably zygotes) are cultured, and then the cultured cells are transplanted into the oviduct of female non-human animals, allowed to develop, and identified and screened to obtain a non-human animal with humanized FAP gene.

[0189] In one embodiment, the non-human animal is constructed using a targeting vector.

[0190] Preferably, the construction method comprises mating the FAP gene humanized non-human animal with other genetically modified non-human animals, in vitro fertilization or direct gene editing, and screening to obtain a multi-gene modified non-human animal.

[0191] Preferably, the other gene is at least one selected from PD-1, PD-L1, TIGIT, CD226, CD40, CD3, CD137, CD28, 4-1BB, IL-2, OX40 or IL-10.

[0192] Preferably, the non-human animal further expresses at least one of human or humanized PD-1, PD-L1, TIGIT, CD226, CD40, CD3, CD137, CD28, 4-1BB, IL-2, OX40 or IL-10 protein.

[0193] Preferably, the human or humanized FAP gene and / or other gene is homozygous for the endogenous modified (preferably replaced) locus.

[0194] Preferably, the human or humanized FAP gene and / or other gene is heterozygous for the endogenous modified (preferably replaced) locus.

[0195] Preferably, each of the plurality of modified genes in the genome of the multi-gene modified non-human animal is homozygous for the endogenous modified (preferably replaced) locus.

[0196] Preferably, each of the plurality of modified genes in the genome of the multi-gene modified non-human animal is heterozygous for the endogenous modified (preferably replaced) locus.

[0197] In a fourteenth aspect of the present application, a FAP gene deleted non-human animal is provided, wherein the non-human animal lacks all or part of exons 1-26 of the endogenous FAP gene, preferably lacks part of exons 3-26, further preferably lacks part of exon 3, all of exons 4-25 and part of exon 26, and more preferably further lacks introns 3-4 and / or introns 25-26.

[0198] In a fifteenth aspect of the present application, a construction method of a FAP gene deleted non-human animal is provided, wherein the construction method comprises using the above-mentioned targeting vector targeting the endogenous FAP gene of the non-human animal and / or the above-mentioned sgRNA to prepare the non-human animal.

[0199] In a sixteenth aspect of the present application, a FAP gene deleted cell is provided, wherein the cell has a deletion of all or part of exons 1-26 of the endogenous FAP gene, preferably a deletion of part of exons 3-26, further preferably a deletion of part of exon 3, all of exons 4-25 and part of exon 26, and more preferably a deletion of introns 3-4 and / or 25-26.

[0200] In a seventeenth aspect of the present application, a method for constructing a FAP gene deleted cell is provided, wherein the method comprises using the above-mentioned targeting vector targeting the endogenous FAP gene of a non-human animal and / or the above-mentioned sgRNA to construct the FAP gene deleted cell.

[0201] In an eighteenth aspect of the present application, a method for constructing a multi-gene modified non-human animal is provided, wherein the method comprises:

[0202] I) providing the above-mentioned non-human animal, or a non-human animal obtained by the above-mentioned method for constructing a non-human animal;

[0203] II) mating, in vitro fertilizing or directly genetically editing the non-human animal provided in step I) with another gene modified non-human animal, and screening to obtain a multi-gene modified non-human animal.

[0204] Preferably, the other gene modified non-human animal comprises at least one of a gene PD-1, PD-L1, TIGIT, CD226, CD40, CD3, CD137, CD28, 4-1BB, IL-2, OX40 or IL-10 modified non-human animal.

[0205] Preferably, the multi-gene modified non-human animal is a double gene humanized non-human animal, a triple gene humanized non-human animal, a quadruple gene humanized non-human animal, a quintuple gene humanized non-human animal, a sextuple gene humanized non-human animal, a septuple gene humanized non-human animal, an octuple gene humanized non-human animal or a nonuple gene humanized non-human animal.

[0206] Preferably, each of the plurality of humanized genes in the genome of the multi-gene modified non-human animal is homozygous for the endogenous modified (preferably replaced) locus.

[0207] Preferably, each of the plurality of humanized genes in the genome of the multi-gene modified non-human animal is heterozygous for the endogenous modified (preferably replaced) locus.

[0208] In a nineteenth aspect of the present application, a FAP gene humanized non-human animal or a multi-gene modified non-human animal obtained by the above-mentioned method for constructing a non-human animal or a progeny thereof is provided.

[0209] Preferably, the non-human animal can be selected from the group consisting of rodent, zebra fish, pig, chicken, rabbit, monkey and any other non-human animal that can be genetically edited to prepare a humanized FAP gene.

[0210] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. More preferably, the rodent is a mouse or a rat.

[0211] 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. More preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Still more preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag1 - / - -IL2rg - / - Mouse, Rag 2 - / - -IL2rg - / - Mouse, NOD / SCID mouse or nude mouse.

[0212] In a twentieth aspect of the present application, there is provided an animal model, wherein the animal model is derived from the non-human animal or the offspring thereof or the non-human animal obtained by the construction method described above. Preferably, the animal model is a tumor-bearing or inflammation animal model.

[0213] In a twenty-first aspect of the present application, there is provided a method for preparing a tumor-bearing or inflammation animal model, wherein the method comprises the step of constructing the non-human animal with humanized FAP gene or the offspring thereof or the non-human animal obtained by the construction method described above or the offspring thereof. Preferably, the method further comprises the step of implanting tumor cells.

[0214] In a twenty-second aspect of the present application, there is provided the use of the non-human animal with humanized FAP gene, the non-human animal with multiple gene modifications or the offspring thereof, or the non-human animal obtained by the construction method described above or the non-human animal with multiple gene modifications or the offspring thereof in preparing an animal model.

[0215] In a twenty-third aspect of the present application, there is provided a cell, tissue or organ, wherein the cell, tissue or organ expresses the human or humanized FAP protein, or the genome of the cell, tissue or organ comprises the nucleic acid described above or the human or humanized FAP gene described above, or the cell, tissue or organ is derived from the non-human animal described above or the non-human animal obtained by the construction method described above.

[0216] In a twenty-fourth aspect of the present application, there is provided a tumor tissue after tumor-bearing, wherein the tumor tissue expresses the human or humanized FAP protein as described above, or the genome of the tumor tissue comprises the nucleic acid as described above, or the human or humanized FAP gene as described above, or the tumor tissue is derived from the non-human animal or the offspring thereof as described above, the non-human animal obtained by the construction method as described above, or the animal model as described above.

[0217] In a twenty-fifth aspect of the present application, there is provided a genome of a non-human animal with humanized FAP gene.

[0218] Preferably, the genome comprises all or part of the human or humanized FAP gene, and / or comprises all or part of the nucleotide sequence encoding the human or humanized FAP protein.

[0219] Preferably, the humanized FAP gene is the humanized FAP gene as described above.

[0220] Preferably, the humanized FAP protein is the humanized FAP protein as described above.

[0221] Preferably, the genome comprises a genomic fragment of the human FAP gene (preferably all or part of exons 1 to 26 of the human FAP gene, further preferably all or part of exons 3 to 26 of the human FAP gene, more preferably part of exon 3, all of exons 4 to 25 and part of exon 26 of the human FAP gene, preferably further comprising introns 3-4 and / or 25-26) at the endogenous FAP locus of the non-human animal to replace a genomic fragment of the endogenous FAP gene of the non-human animal to form a modified FAP gene.

[0222] Preferably, the replaced genomic fragment of the endogenous FAP gene of the non-human animal comprises all or part of exons 1 to 26 of the endogenous FAP gene of the non-human animal, further preferably part of exons 3 to 26, more preferably part of exon 3, all of exons 4 to 25 and part of exon 26, preferably further comprising introns 3-4 and / or 25-26.

[0223] Preferably, the modified FAP gene encodes a humanized FAP protein.

[0224] Preferably, the expression of the modified FAP gene is controlled by the endogenous regulatory element of the non-human animal.

[0225] Preferably, the genome comprises a humanized endogenous FAP locus, in which a fragment of the endogenous FAP locus has been deleted and replaced with the corresponding human FAP sequence.

[0226] Preferably, the humanized FAP locus comprises an endogenous FAP promoter, wherein the human FAP sequence is operably linked to the endogenous FAP promoter.

[0227] Preferably, all or part of the 1stto 26thexons of the endogenous FAP locus (preferably all or part of the 3rdto 26thexons, further preferably part of the 3rdexon, all of the 4thto 25thexons and part of the 26thexon) has been deleted and replaced with the corresponding human FAP sequence.

[0228] In one embodiment of the present application, all or part of the nucleotide sequence encoding the extracellular region of the endogenous FAP locus has been deleted and replaced with the corresponding human FAP sequence.

[0229] In one embodiment of the present application, the transmembrane region, the cytoplasmic region, the 3'UTR and / or the 5'UTR of the endogenous FAP gene has not been deleted and has not been replaced with the corresponding human FAP sequence.

[0230] Preferably, the non-human animal can be selected from rodents, zebrafish, pigs, chickens, rabbits, monkeys and any other non-human animal that can be genetically edited to produce a humanized gene.

[0231] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. More preferably, the rodent is a rat or a mouse.

[0232] 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. More preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Still more preferably, the immunodeficient mouse is a NOD-Prkdcscid IL-2rγnull mouse, a NOD-Rag 1- / - IL2rg- / - mouse, a Rag 2- / - IL2rg- / - mouse, a NOD / SCID mouse or a nude mouse.

[0233] In a twenty-sixth aspect of the present application, there is provided a use of the humanized FAP protein, the humanized FAP gene, the non-human animal or its offspring, the cell, the non-human animal obtained by the construction method, the animal model, the cell, tissue or organ, the tumor tissue after tumor-bearing of the present application, the use comprising:

[0234] A) use in product development related to FAP-associated immune processes in human cells;

[0235] B) application in FAP-related model systems for pharmacological, immunological, microbiological and medical research;

[0236] C) application involving production and utilization of animal experimental disease models for FAP-related etiological research and / or for developing diagnostic strategies and / or for developing therapeutic strategies;

[0237] D) application in in vivo research of screening, pharmacodynamic detection, efficacy evaluation, validation or assessment of human FAP signaling pathway modulators; or,

[0238] E) application in research of FAP gene function, research of drugs targeting human FAP target sites, research of drugs for FAP-related immune-related diseases, and research of anti-tumor drugs.

[0239] In a twenty-seventh aspect of the present application, a non-human animal derived from the above, a non-human animal obtained by the above construction method, or the animal model described above is provided for screening of human FAP-specific modulators.

[0240] In a twenty-eighth aspect of the present application, a screening method for human FAP-specific modulators is provided, which comprises applying a modulator to an individual implanted with tumor cells, and detecting tumor inhibition; wherein the individual is selected from the above non-human animal or its offspring, the above non-human animal obtained by the construction method, or the animal model described above.

[0241] Preferably, the modulator is selected from CAR-T, drugs. Further preferably, the drug is an antibody.

[0242] Preferably, the modulator is a monoclonal antibody or a bispecific antibody, or a combination of two or more drugs.

[0243] Preferably, the detection comprises determining the size and / or proliferation rate of tumor cells.

[0244] Preferably, the detection method comprises vernier caliper measurement, flow cytometry detection, and / or in vivo imaging detection of animals.

[0245] Preferably, the detection comprises evaluating the individual's body weight, fat mass, activation pathway, neuroprotective activity, or metabolic changes, including changes in food consumption or water consumption.

[0246] Preferably, the tumor cells are derived from humans or non-human animals.

[0247] Preferably, the screening method of the human FAP specific modulator can be for therapeutic or non-therapeutic purposes. The method is used to screen or evaluate drugs, to detect and compare the efficacy of candidate drugs, to determine which candidate drugs can be drugs and which cannot be drugs, or to compare the efficacy sensitivity of different drugs, i.e. the therapeutic effect is not inevitable but a possibility.

[0248] In a twenty-ninth aspect of the present application, a method for evaluating a human drug screening method or intervention program is provided, wherein the human drug screening method or evaluation method comprises implanting tumor cells into an individual, administering a candidate drug to the individual implanted with tumor cells, or applying an intervention program, detecting and / or comparing the efficacy of the individual after administration of the candidate drug or application of the intervention program, or detecting and evaluating the tumor inhibition effect; wherein the individual is selected from the non-human animal described above, the non-human animal obtained by the construction method described above, the non-human animal or its offspring described above, or the tumor-bearing or inflammation model described above.

[0249] Preferably, the intervention program is selected from CAR-T, drug therapy. Further preferably, the drug includes a targeted drug, and more preferably, the targeted drug is an antigen binding protein. The antibody binding protein is an antibody.

[0250] Preferably, the tumor cells are derived from a human or a non-human animal.

[0251] Preferably, the candidate drug is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

[0252] Preferably, the detection includes measuring the size and / or proliferation rate of tumor cells; preferably, the detection method includes vernier caliper measurement, flow cytometry detection and / or animal live imaging detection.

[0253] Preferably, the detection includes evaluating the body weight, fat mass, activation pathway, neuroprotective activity or metabolic changes of the individual, and the metabolic changes include changes in food consumption or water consumption.

[0254] Preferably, the screening method of the human FAP specific modulator can be for therapeutic or non-therapeutic purposes. The method is used to screen or evaluate drugs, to detect and compare the efficacy of candidate drugs, to determine which candidate drugs can be drugs and which cannot be drugs, or to compare the efficacy sensitivity of different drugs, i.e. the therapeutic effect is not inevitable but a possibility.

[0255] In a thirty-first aspect of the present application, there is provided a use of the non-human animal derived from the above-mentioned non-human animal or its offspring, the non-human animal obtained by the above-mentioned construction method, or the animal model in the preparation of a drug for treating a tumor, an inflammation or an immune-related disease.

[0256] In a thirty-first aspect of the present application, there is provided a use of the non-human animal derived from the above-mentioned non-human animal or its offspring, the non-human animal obtained by the above-mentioned construction method, or the animal model in the preparation of a drug for treating a tumor, an inflammation or an immune-related disease.

[0257] The "immune-related disease" in the present application includes, but is not limited to, allergy, 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 disorder, etc.

[0258] The "inflammation" in the present application includes acute inflammation, and also includes chronic inflammation. Specifically, it includes, but is not limited to, metamorphic inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, purulent inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammation, and specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).

[0259] The "tumor" in the present application 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. Among them, the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the lymphoma is selected from Hodgkin'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; and the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

[0260] The FAP gene humanized non-human animal in the present application can normally express human or humanized FAP protein in vivo, and can be used for drug screening, pharmacodynamic evaluation, immune disease and tumor treatment targeting human FAP target site, so as to accelerate the new drug development process, save time and cost. It provides an effective guarantee for studying the function of FAP protein and related disease drug screening.

[0261] The "all or part", "all" of the present application refers to the whole, "part" refers to the part of the whole, or the individual that constitutes the whole.

[0262] The "humanized FAP protein" of the present application comprises a part derived from human FAP protein and a part of non-human FAP protein. Among them, the "human FAP protein" is the same as the whole human FAP protein, that is, its amino acid sequence is consistent with the full-length amino acid sequence of human FAP protein. The "part of human FAP protein" is a continuous or intermittent 5-760 amino acid sequence consistent with the amino acid sequence of human FAP protein. Preferably, it is a continuous or intermittent 10-729 or 10-735 amino acid sequence, more preferably a continuous 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 729, 735, 750, 760 amino acid sequence consistent with the amino acid sequence of human FAP protein.

[0263] The "all of the transmembrane region of human FAP protein", "all of the cytoplasmic region of human FAP protein" or "all of the extracellular region of human FAP protein" of the present application represent that their amino acid sequences are respectively consistent with the full-length amino acid sequences of the transmembrane region, cytoplasmic region or extracellular region of human FAP protein.

[0264] The "part of non-human animal endogenous FAP protein" of the present application is a continuous or intermittent 5-761 amino acid sequence consistent with the amino acid sequence of non-human animal endogenous FAP protein, preferably a continuous or intermittent 10-31, more preferably a continuous 5, 10, 20, 25, 30, 31, 50, 100, 200, 300, 400, 500, 600, 700, 750, 761 amino acid sequence consistent with the amino acid sequence of non-human animal endogenous FAP protein.

[0265] The "humanized FAP gene" of the present application comprises a part derived from human FAP gene and a part of non-human FAP gene. Among them, the "human FAP gene" is the same as the whole human FAP gene, that is, its nucleotide sequence is consistent with the full-length nucleotide sequence of human FAP gene.

[0266] In one embodiment, the "part of human FAP gene" is a contiguous or intermittent 20-72789 bp nucleotide sequence identical to the nucleotide sequence of human FAP gene, preferably 100-55641 or 100-2190 or 100-2696, more preferably 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 2190, 2696, 4000, 6000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 55641, 60000, 70000, 72000, 72789 bp nucleotide sequence identical to the nucleotide sequence of human FAP gene.

[0267] The "part of exon" of the present application means a contiguous or intermittent several, dozens or hundreds of nucleotide sequence identical to the nucleotide sequence of the whole exon. For example, the "part of exon 3" of human FAP gene comprises a contiguous or intermittent 5-99 bp, preferably 10-96 bp, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99 bp nucleotide sequence identical to the nucleotide sequence of exon 3 of human FAP gene.

[0268] The "xxth to xxxth exon" or "the whole of xxth to xxxth exon" of the present application comprises the nucleotide sequence of the exon and the intron during the exon, for example, "1st to 3rd exon" comprises the whole nucleotide sequence of 1st exon, 1st-2nd intron, 2nd exon, 2nd-3rd intron and 3rd exon.

[0269] The "xth-xxth intron" of the present application means the intron between xth exon and xxth exon. For example, "1st-2nd intron" means the intron between 1st exon and 2nd exon.

[0270] The "part of the non-human animal endogenous FAP gene" of the present application refers to a nucleotide sequence of 20-73085 bp in length, preferably 20-55014 or 20-2456 or 20-264 or 20-2193 bp in length, more preferably 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000 or 73000 nucleotides in length, which is identical to the nucleotide sequence of the non-human animal endogenous FAP gene. In one embodiment of the present application, the part of the non-human animal endogenous FAP gene comprises a part of the 3rd exon and / or a part of the 26th exon of the non-human animal endogenous FAP gene. Preferably, the part of the 3rd exon of the non-human animal endogenous FAP gene comprises at least 2 nucleotides. Preferably, the part of the 26th exon of the non-human animal endogenous FAP gene comprises at least the nucleotide sequence of the non-coding region.

[0271] The "locus" of the present application refers to the position of a gene on a chromosome in a broad sense, and refers to a DNA fragment on a certain gene in a narrow sense, which can be a gene or a part of a gene. For example, the "FAP locus" refers to a DNA fragment of an optional length on the 1st to 26th exon of the FAP gene. In one embodiment of the present application, the replaced FAP locus can be a DNA fragment of an optional length on the 1st to 26th exon of the FAP gene.

[0272] The "nucleotide sequence" of the present application comprises natural or modified ribonucleotide sequence, deoxyribonucleotide sequence. Preferably, it is DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, tRNA.

[0273] The "more than three" of the present application includes, but is not limited to, three, four, five, six, seven or eight, etc.

[0274] The "treatment" of the present application refers to slowing down, interrupting, stopping, controlling, reducing, or reversing 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 intervention to improve signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

[0275] The "cell" described herein can be a fertilized egg cell or other somatic cell, preferably including but not limited to platelets, monocytes, microglia and endothelial cells, neutrophils, activated macrophages, B cell precursors, dendritic cells, natural killer cells, late B cells or plasma cells, and the like. Thus, depending on the source of the cell, some of the cells described herein can develop into an animal individual, and some can not.

[0276] The "comprising" or "including" described herein is an open description containing the specified components or steps described, and other specified components or steps that do not materially affect. However, when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid can be composed of the sequence, or at one end or both ends of the protein or nucleic acid can have additional amino acids or nucleotides, but still have the activity described herein.

[0277] The "homology" described herein refers to the use of amino acid sequences or nucleotide sequences, and the skilled person in the art can adjust the sequence according to the actual work needs to ensure the similar structure or function with the known sequence, so that the sequence obtained by using the sequence compared with the prior art has (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%, 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.

[0278] The skilled person in the art can determine and compare the sequence elements or the degree of identity to distinguish additional mouse and human sequences.

[0279] The practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are explained fully in the literature. See, 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 (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. 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. in chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes V (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0280] In one aspect, the non-human animal is a mammal. Preferably, the non-human animal is a small mammal, such as a saltatorial rodent. In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is selected from the group consisting of a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the Muridae family. In one embodiment, the genetically modified animal is from a family selected from the group consisting of Cricetidae (e.g., Muscardinus avellanarius), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, creodonts), Muridae (mountain mice, rock mice, chinchilla, Malagasy rats and mice), Echimyidae (e.g., Dasyprocta pridgleyi), and Geomyidae (e.g., Thomasomys, Rhondinomys, and Geomyidae). In a particular embodiment, the genetically modified rodent is selected from the group consisting of a true mouse or rat (Muridae), a gerbil, a spiny mouse, and a creodont. In one embodiment, the genetically modified mouse is from a Muridae family member. In one embodiment, the animal is a rodent. In a particular 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.

[0281] In a particular embodiment, the non-human animal is a rodent that is a mouse selected from the group consisting of C57BL, C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, CBA / H strains 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, and NOD, NOD / SCID, NOD-Prkdc scidIL-2rg null Background mice.

[0282] The technical effects of the present application are beneficial in that:

[0283] Using gene editing technology, the homologous genes in the animal genome are replaced with human normal or mutant genes to establish a gene humanized animal model closer to the physiological or disease characteristics of humans, so that human proteins are expressed in vivo, which can be used as a target for drugs that can only recognize human protein sequences, and provides the possibility for screening of anti-human antibodies and other drugs at the animal level.

[0284] Using a gene humanized animal model to establish various disease models can be used for pharmacological and pharmacodynamic evaluation of anti-human antibody drugs.

[0285] The above only summarizes some aspects of the present application, and is not and should not be considered as limiting the present application in any aspect.

[0286] All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. Those skilled in the art will recognize certain changes and modifications to the present application, which are not a departure from the spirit or scope of the present application. The following examples further illustrate the present application, and are not to be considered as limiting the present application or the scope of the specific methods described herein. BRIEF DESCRIPTION OF DRAWINGS

[0287] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0288] Figure 1 : Schematic diagram of comparison between mouse FAP gene and human FAP gene locus (not to scale);

[0289] Figure 2 : Schematic diagram of humanization modification of mouse FAP gene (not to scale);

[0290] Figure 3 : Schematic diagram of FAP gene targeting strategy and targeting vector design (not to scale);

[0291] Figure 4 : Schematic diagram of Southern Blot detection of positive clones, WT is wild type;

[0292] Figure 5 : Schematic diagram of FRT recombination process of FAP gene humanized mouse (not to scale);

[0293] Figure 6 : F1 generation tail PCR identification results of FAP gene humanized mouse, wherein WT is wild type, H2O is water control, PC is positive control, and M is Marker. DETAILED DESCRIPTION

[0294] The present application will be further described in conjunction with specific embodiments, and the advantages and features of the present application will become more apparent from the description. However, these embodiments are only exemplary, and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

[0295] In each of the following embodiments, the equipment and materials are obtained from several companies as indicated below:

[0296] StuI, AvrII, BglII, DraIII, ScaI enzymes are purchased from NEB, with catalog numbers R0187S, R0174S, R0144S, R3510S, R3122S, respectively.

[0297] C57BL / 6 mice are purchased from the National Rodent Experimental Animal Seed Center of China Institute for Drug Control.

[0298] Example 1 Preparation of FAP gene humanized mice

[0299] The schematic diagram of mouse FAP gene (NCBI Gene ID: 14089, Primary source: MGI: 109608, UniProt: P97321-1, located at 62331280-62404365 of chromosome 2 NC_000068.8, based on transcript NM_007986.3 and its encoded protein NP_032012.1 (SEQ ID NO: 1)) and human FAP gene (NCBI Gene ID: 2191, Primary source: HGNC: 3590, UniProt ID: Q12884-1, located at 162170684-162243472 of chromosome 2 NC_000002.12, based on transcript NM_004460.5 and its encoded protein NP_004451.2 (SEQ ID NO: 2)) is shown in Figure 1

[0300] To achieve the purpose of the present application, a nucleotide sequence encoding human FAP protein can be introduced into the mouse endogenous FAP locus, so that the mouse expresses human or humanized FAP protein. Specifically, using gene editing technology, the partial sequence of exon 3 to the partial sequence of exon 26 of about 55.0 kb of mouse FAP gene is replaced with the partial sequence of exon 3 to the partial sequence of exon 26 of about 55.6 kb of human FAP gene under the control of mouse FAP gene regulatory elements, to obtain a humanized FAP locus, as shown in Figure 2 ​The humanization of mouse FAP gene was achieved.

[0301] The targeting strategy is shown in Figure 3 Figure 3 The targeting vector contains homologous arm sequences upstream and downstream of mouse FAP gene, and A fragment containing human FAP sequence, as shown in The last "T" in the sequence is the last nucleotide of mouse, and the "C" in the sequence CATA is the first nucleotide of human. The connection between human FAP sequence downstream and mouse is designed as The last "A" in the sequence is the last nucleotide of human, and the first "A" in the sequence ACCA is the first nucleotide of mouse sequence.

[0302] The targeting vector also includes a resistance gene for positive clone screening, i.e. neomycin phosphotransferase coding sequence Neo, and two same-direction arranged site-specific recombination system Frt recombination sites on both sides of the resistance gene, forming a Neo cassette. The connection between 5' end of the Neo cassette and human gene is designed as The "C" in the sequence is the last nucleotide of human, and the first "C" in the sequence CATC is the first nucleotide of the Neo cassette; the connection between 3' end of the Neo cassette and human gene is designed as The "C" in the sequence is the last nucleotide of the Neo cassette, and the sequence ​The first nucleotide in the human is "T". In addition, a coding gene (coding gene of diphtheria toxin A subunit (DTA)) with a negative selection marker is also constructed downstream of the 3' homologous arm of the targeting vector. The mRNA sequence of the humanized mouse FAP after modification is shown as SEQ ID NO: 10, and the expressed protein sequence is shown as SEQ ID NO: 11.

[0303] In view of the fact that human FAP has multiple subtypes or transcripts, the methods described herein can be applied to other subtypes or transcripts.

[0304] Targeting vector construction can be performed by conventional methods, such as enzyme digestion and ligation. The constructed targeting vector is subjected to preliminary verification by enzyme digestion, and then sent to a sequencing company for sequencing verification. The targeting vector that is correctly verified by sequencing is electroporated into C57BL / 6 mouse embryonic stem cells, the obtained cells are screened using a positive clone screening marker gene, and the integration of the exogenous gene is detected and confirmed using PCR and Southern Blot techniques, and the correct positive clone cells are screened, the clones that are positive by PCR are further detected by Southern Blot (cell DNA is digested with StuI, AvrII, BglII, DraIII, and ScaI enzymes and hybridized with three probes, the lengths of the probes and the target fragments are shown in Table 1), and the detection results are shown in Figure 4 As shown in Table 1, 1-A11, 1-C04, 1-C08, 1-D04, 2-C02, 2-D02, 2-E02, 2-E07, and 2-E08 are all positive clones, and the clones that are further verified by sequencing and have no random insertion are used for the next experiment.

[0305] Table 1: Lengths of specific probes and target fragments

[0306] Restriction enzyme Probe Wild type fragment Recombinant sequence fragment AvrII A1 Probe — 17.5kb BglII A1 Probe — 18.6kb DraIII A2 Probe — 20.6kb ScaI A2 Probe — 11.0kb StuI Neo Probe (3') — 13.0kb

[0307] The PCR determination includes the following primers:

[0308] A-F1: 5'-CATGGCTCTGGATTCATAGTTGGAGTC-3' (SEQ ID NO: 28),

[0309] A-R1: 5'-CATGACCCTTAAAGGGTTCATTGTCAG-3' (SEQ ID NO: 12),

[0310] A-F2: 5'-GCTCGACTAGAGCTTGCGGA-3' (SEQ ID NO: 13),

[0311] A-R2: 5'-GTACAGCCTTGGTACTGGAGTTAGAG-3' (SEQ ID NO: 14);

[0312] Southern Blot detection includes the following probe primers:

[0313] A1 Probe:

[0314] A1 Probe-F: 5'-CGTGACCTCGGTGATCAATCTC-3' (SEQ ID NO: 15),

[0315] A1 Probe-R: 5'-CCAAGATTGGCCCGCTAGGCATG-3' (SEQ ID NO: 16);

[0316] A2 Probe:

[0317] A2 Probe-F: 5'-CCACAATTCCTTATTCCTCCACC-3' (SEQ ID NO: 17),

[0318] A2 Probe-R: 5'-CTCCTATACTGTCTAATACTTCATGGAAAC-3' (SEQ ID NO: 18);

[0319] Neo Probe (3'):

[0320] Neo Probe-F: 5'-GGATCGGCCATTGAACAAGAT-3' (SEQ ID NO: 19),

[0321] Neo Probe-R: 5'-CAGAAGAACTCGTCAAGAAGGC-3' (SEQ ID NO: 20);

[0322] The correct positive clone cells screened (black mice) are introduced into the isolated blastocysts (white mice) according to the techniques known in the art, the chimeric blastocysts obtained are transferred into culture solution for short-term culture, then transplanted into the oviducts of recipient female mice (white mice), to produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and the F1 generation heterozygous mice are 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 (the process is shown in the following figure) Figure 5) and then by intercrossing, FAP gene humanized homozygous mice can be obtained. The genotype of the somatic cells of the offspring mice can be identified by PCR (the primers are shown in Table 2), and the identification results of the F1 generation mice (the Neo marker gene has been removed) are shown in Fig. 2. Figure 6 In which, the mice numbered F1-01 and F1-02 are positive heterozygous mice. This shows that using the method, FAP gene humanized mice that can be stably passaged and have no random insertion can be constructed.

[0323] Table 2: Primer names and specific sequences

[0324]

[0325]

[0326] The expression of human or humanized FAP protein in the positive mice can be detected by conventional methods, such as flow cytometry. The detection results show that murine FAP protein is detected in C57BL / 6 mice, and only humanized FAP protein can be detected in FAP homozygous mice. The results show that humanized FAP protein can be normally expressed in FAP humanized homozygous mice.

[0327] Example 2 Preparation of double humanized or multiple double humanized mice

[0328] The FAP mice prepared by the method or prepared by the method can also be used to prepare double humanized or multiple humanized mouse models. For example, in the foregoing Example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing PD-1, PD-L1, TIGIT, CD226, CD40, CD3, CD137, CD28, 4-1BB, IL-2, OX40 or IL-10 and other gene modifications, or, on the basis of humanized FAP mice, mouse ES embryonic stem cells and gene recombination targeting technology can be used to obtain FAP and other gene modified double gene or multiple gene modified mouse models. The FAP homozygous or heterozygous mice obtained by the method can also be crossed with other gene modified homozygous or heterozygous mice, and the offspring can be screened according to Mendelian inheritance law, and there is a certain probability of obtaining double gene or multiple gene modified heterozygous mice of humanized FAP and other gene modifications, and double gene or multiple gene modified homozygous mice can be obtained by intercrossing the heterozygous mice. The double gene or multiple gene modified mice can be used for in vivo pharmacodynamic verification of targeted human FAP and other gene modulators.

[0329] Example 3 Pharmacodynamic experiment

[0330] The FAP humanized mice prepared by the method can be used to evaluate the efficacy of the antibody drug targeting human FAP. For example, the FAP humanized mouse homozygote is subcutaneously inoculated with mouse colon cancer cells MC38, and when the tumor volume grows to about 100 mm 3 According to the tumor volume, the mice are divided into a control group and a treatment group, the mice in the treatment group are randomly selected to be injected with the antibody drug targeting human FAP, and the mice in the control group are injected with the same volume of normal saline. The tumor volume is measured regularly and the body weight of the mice is weighed, and the in vivo safety and in vivo efficacy of the compound can be effectively evaluated by comparing the changes in the body weight of the mice and the tumor size.

[0331] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0332] In addition, it should be noted that each specific technical feature described in the above-described specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combinations.

[0333] In addition, various different embodiments of the present application can also be combined in any manner, as long as it does not deviate from the idea of the present application, and it should also be considered as disclosed by the present application.

Claims

1. A method for constructing a non-human animal humanized for a FAP gene, characterized by, The construction method comprises introducing a nucleotide sequence encoding all or part of the extracellular region of a human FAP protein into a non-human animal endogenous FAP gene locus: The part of the extracellular region of the human FAP protein comprises an amino acid sequence shown in SEQ ID NO: 2, 32-760; The introduction into the non-human animal endogenous FAP gene locus is a replacement of a corresponding region of the non-human animal; The non-human animal expresses a humanized FAP protein in vivo; The amino acid sequence of the humanized FAP protein is shown in SEQ ID NO: 11; The non-human animal is a mouse.

2. The construction method of claim 1, wherein, The non-human animal genome comprises a humanized FAP gene.

3. The construction method of claim 1, wherein, The non-human animal endogenous FAP protein expression is reduced or deleted.

4. The construction method of claim 2, wherein, The humanized FAP gene in the non-human animal is regulated by an endogenous regulatory element.

5. The construction method of claim 1, wherein, The nucleotide sequence encoding all or part of the extracellular region of the endogenous FAP protein is replaced.

6. The construction method of claim 1, wherein, All or part of the 3rd to 26th exons of the non-human animal endogenous FAP gene are replaced.

7. The construction method of claim 1, wherein, The construction method comprises using a targeting vector to construct the non-human animal.

8. The construction method of claim 7, wherein, The targeting vector comprises a donor DNA sequence, and the donor DNA sequence comprises a nucleotide sequence encoding all or part of the extracellular region of a human FAP protein.

9. The construction method according to claim 2 or 8, characterized in that, The humanized FAP gene or the donor DNA sequence comprises a part of a human FAP gene; The part of the human FAP gene comprises a part of the 3rd exon, all of the 4th to 25th exons, and a part of the 26th exon of the human FAP gene, wherein the part of the 3rd exon of the human FAP gene comprises at least a continuous nucleotide sequence of 50 bp, and the part of the 26th exon of the human FAP gene comprises at least a nucleotide sequence of a coding region.

10. The construction method of claim 9, wherein, The part of the human FAP gene further comprises a 3-4th intron and / or a 25-26th intron.

11. The construction method of claim 9, wherein, The part of the human FAP gene comprises a nucleotide sequence shown in SEQ ID NO:

5.

12. The method of construction of claim 7, wherein, The targeting vector further comprises a 5' arm and / or a 3' arm; wherein, The 5' arm comprises SEQ ID NO: 3; The 3' arm comprises SEQ ID NO:

4.

13. The method of construction of claim 1, wherein, The construction method further comprises mating the non-human animal with a humanized FAP gene with other genetically modified non-human animals, in vitro fertilization, or direct gene editing, and screening to obtain a multi-gene modified non-human animal.

14. The construction method of claim 13, wherein, The other gene is at least one selected from PD-1, PD-L1, TIGIT, CD226, CD40, CD3, CD137, CD28, 4-1BB, IL-2, OX40, or IL-10.

15. The construction method according to claim 2 or 13, characterized in that, The humanized FAP gene and / or the other gene are homozygous for the endogenous modified locus.

16. The construction method according to claim 2 or 13, wherein, The humanized FAP gene and / or the other gene are heterozygous for the endogenous modified locus.

17. The method of construction of claim 2, wherein, The mRNA transcribed from the humanized FAP gene comprises SEQ ID NO:

10.

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

11.

19. A humanized FAP gene, characterized in that, The humanized FAP gene encodes the humanized FAP protein of claim 18.

20. The humanized FAP gene of claim 19, wherein, The humanized FAP gene comprises a part of a human FAP gene; The part of the human FAP gene comprises a part of the 3rd exon, the entire 4th to 25th exons and a part of the 26th exon of the human FAP gene, wherein the part of the 3rd exon of the human FAP gene comprises at least a continuous nucleotide sequence of 50 bp, and the part of the 26th exon of the human FAP gene comprises at least a nucleotide sequence of the coding region.

21. The humanized FAP gene of claim 20, wherein, The part of the human FAP gene comprises a nucleotide sequence as shown in SEQ ID NO:

5.

22. The humanized FAP gene of claim 20, wherein, The mRNA transcribed from the humanized FAP gene comprises SEQ ID NO:

10.

23. A targeting vector, characterized in that, The targeting vector comprises a donor DNA sequence, a 5' arm and a 3' arm; The donor DNA sequence comprises a nucleotide sequence encoding the entire or part of the extracellular region of a human FAP protein; The part of the extracellular region of the human FAP protein comprises an amino acid sequence as shown in SEQ ID NO: 2, 32-760; The targeting vector comprises a nucleotide sequence encoding the humanized FAP protein of claim 18; The 5' arm comprises SEQ ID NO: 3; The 3' arm comprises SEQ ID NO:

4.

24. The targeting vector of claim 23, wherein, The donor DNA sequence comprises a part of a human FAP gene; The part of the human FAP gene comprises a part of the 3rd exon, the entire 4th to 25th exons and a part of the 26th exon of the human FAP gene, wherein the part of the 3rd exon of the human FAP gene comprises at least a continuous nucleotide sequence of 50 bp, and the part of the 26th exon of the human FAP gene comprises at least a nucleotide sequence of the coding region.

25. The targeting vector of claim 24, wherein, The part of the human FAP gene further comprises the 3rd-4th intron and / or the 25th-26th intron.

26. The targeting vector of claim 25, wherein The part of the human FAP gene comprises a nucleotide sequence as shown in SEQ ID NO:

5.

27. A cell, tissue or organ, comprising a polynucleotide of claim 1. The cell, tissue or organ expresses the humanized FAP protein of claim 18, or the genome of the cell, tissue or organ comprises the humanized FAP gene of any one of claims 19-22, or the cell, tissue or organ is derived from the non-human animal obtained by the construction method of any one of claims 1-17, and the cell cannot develop into an animal individual.

28. The cell, tissue, or organ of claim 27, wherein, The tissue is a tumor tissue.

29. Use of the humanized FAP protein of claim 18, or the humanized FAP gene of any one of claims 19-22, or the non-human animal obtained by the construction method of any one of claims 1-17, or the cell, tissue or organ of claim 27 or 28, wherein, The use comprises: A) use in the development of products related to FAP-related immune processes involving human cells; B) use as a model system related to FAP in pharmacological, immunological, microbiological and medical research; C) use related to the production and use of animal experimental disease models for etiological research related to FAP; D) use in the screening, pharmacodynamic testing, evaluation, validation or assessment of modulators of the human FAP signaling pathway in vivo for non-diagnostic or non-therapeutic purposes; or E) use in the research of FAP gene function, the research of drugs targeting human FAP target sites, the research of drugs for FAP-related immune-related diseases and the research of anti-tumor drugs for non-diagnostic or non-therapeutic purposes.

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