WO2024217384A1 - Non-human animal modified by icos and/or icosl genes - Google Patents

Abstract

A non-human animal expressing human or chimeric humanized ICOS and/or ICOSL and a use method therefor.

Description

A non-human animal modified with ICOS and/or ICOSL gene

Priority declaration

This application claims priority to 202310408483.8 filed on April 17, 2023, 202310890285.X filed on July 19, 2023, and 202410050995.6 filed on January 12, 2024. The entire contents of the foregoing are incorporated herein by reference.

Technical Field

The present invention provides a non-human animal expressing human or chimeric (eg, humanized) ICOS and/or ICOSL protein and methods of use thereof.

background

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

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

Overview

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

In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric inducible T-cell co-stimulator (ICOS) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric ICOS protein is operably linked to at least one In some embodiments, the chimeric ICOS protein comprises a human or endogenous signal peptide, a human or humanized extracellular region, a human or humanized transmembrane region, and a human or endogenous cytoplasmic region. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOS protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human ICOS (NP_036224.1, SEQ ID NO: 2). In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOS protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-199 or 27-156 of SEQ ID NO: 2. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOS protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 13. In some embodiments, the animal is a mammal, such as a monkey, a rodent, a mouse or a rat. In some embodiments, the animal is a mouse. In some embodiments, the endogenous ICOS protein of the animal is not expressed or is expressed at a reduced level compared to ICOS in wild-type animals. In some embodiments, one or more cells of the animal express a human or chimeric ICOS protein. In some embodiments, one or more cells of the animal express a human or chimeric ICOS protein, which can bind to an endogenous ICOSL protein to induce a downstream signaling pathway. In some embodiments, one or more cells of the animal express a human or chimeric ICOS protein, which can bind to a human ICOSL protein to induce a downstream signaling pathway.

In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises a nucleotide sequence encoding a human or chimeric ICOS region at an endogenous ICOS locus replacing a nucleotide sequence encoding a corresponding region of endogenous ICOS, or a nucleotide sequence encoding a human or chimeric ICOS region is inserted into an endogenous ICOS locus. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is operably linked to an endogenous regulatory element of an endogenous ICOS locus, and one or more cells of the animal express a human or humanized ICOS protein. In some embodiments, the endogenous ICOS protein of the animal is not expressed or is expressed at a reduced level compared to ICOS in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric ICOS comprises a portion of exon 1, exon 2, exon 3, exon 4, and/or a portion of exon 5 of a human ICOS gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric ICOS comprises a portion of exon 2 and/or a portion of exon 3 of a human ICOS gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human ICOS is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 115, 10, or 62. In some embodiments, the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 1, exon 2, exon 3, exon 4, and/or a portion of exon 5 of a mouse ICOS gene. In some embodiments, the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 2 and/or a portion of exon 3 of a mouse ICOS gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is inserted into exon 1 of an endogenous ICOS locus. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is inserted into the 5' end of NM_017480.2. UTR. In some embodiments, at least 20 bp of exon 1 of the endogenous ICOS locus (such as nucleotides 46 to 103 of NM_017480.2) are deleted. In some embodiments, the nucleotide sequence of 139193 bp of nucleotides 46 to 103 and intron 1 of the endogenous ICOS locus NM_017480.2 is deleted. In some embodiments, the inserted sequence comprises all or part of the coding human ICOS signal peptide, extracellular region, transmembrane region and cytoplasmic region. In some embodiments, the amino acid sequence encoded by the inserted sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-199 of SEQ ID NO: 2. In some embodiments, the inserted sequence further comprises one or more auxiliary sequences. In some embodiments, the auxiliary sequence is a WPRE sequence and/or a PA sequence. In some embodiments, the genome of the animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 115, 10, 11, 12, 62 or 111. In some embodiments, the modified ICOS gene in the genome of the animal is homozygous and/or heterozygous for the endogenous replaced locus.

In one aspect, the invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or chimeric ICOS protein, wherein the human or chimeric ICOS protein comprises at least 50, 100, 120, 130, 140, 150, 160, 170, 180, 190 or 199 consecutive amino acids identical to the contiguous amino acid sequence of the corresponding region of a human, and the animal expresses the human or chimeric ICOS protein. In some embodiments, the chimeric ICOS protein comprises at least 50, 60, 70, 80, 90, 100, 120, 130, 140 or 141 consecutive amino acids identical to the contiguous amino acid sequence of the corresponding region of a human extracellular region and transmembrane region. In some embodiments, the human or chimeric ICOS protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-199 or 27-156 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human or chimeric ICOS protein is operably linked to an endogenous ICOS regulatory element. In some embodiments, the nucleotide sequence encoding the corresponding region of the human or chimeric ICOS can be integrated into the animal's endogenous ICOS locus. In some embodiments, the human or chimeric ICOS protein has at least one mouse ICOS activity and/or human ICOS activity.

In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein in at least one cell of the animal, at the endogenous ICOS locus of the animal, a nucleotide sequence encoding an endogenous ICOS region is replaced by a nucleotide sequence encoding a corresponding region of a human or chimeric ICOS, or a sequence encoding a human or chimeric ICOS protein is inserted into at least one cell of the animal. In some embodiments, the endogenous ICOS protein of the animal is not expressed or is expressed at a reduced level compared to ICOS in a wild-type animal. In some embodiments, the nucleotide sequence encoding the corresponding region of a human or chimeric ICOS comprises a portion of exon 1, exon 2, exon 3, exon 4, and/or exon 5 of a human ICOS gene. In some embodiments, the nucleotide sequence encoding the corresponding region of a human or chimeric ICOS comprises a portion of exon 2 and/or a portion of exon 3 of a human ICOS gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of a human or chimeric ICOS is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2 or 13. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is The nucleotide sequence of SEQ ID NO: 115, 10 or 62 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical. In some embodiments, the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 1, exon 2, exon 3, exon 4 and/or exon 5 of the mouse ICOS gene. In some embodiments, the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 2 and/or a portion of exon 3 of the mouse ICOS gene. In some embodiments, the human or chimeric ICOS protein comprises all or part of a human signal peptide, extracellular region, transmembrane region and cytoplasmic region. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is inserted into exon 1 of the endogenous ICOS locus. In some embodiments, at least 20 bp of exon 1 of the endogenous ICOS locus (such as nucleotides 46 to 103 of NM_017480.2) is deleted. In some embodiments, the endogenous ICOS locus NM_017480.2, nucleotides 46 to 103 and 13993 bp of intron 1 are deleted. In some embodiments, the nucleotide sequence encoding the corresponding region of human ICOS is operably linked to an endogenous ICOS regulatory element, such as a promoter. In some embodiments, the animal is a mammal, such as a monkey, a rodent, a mouse or a rat.

In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric ICOS, the method comprising replacing a nucleotide sequence encoding an endogenous ICOS region with a nucleotide sequence encoding a corresponding region of human ICOS at an endogenous mouse ICOS locus, or inserting a sequence encoding a human ICOS protein into an endogenous ICOS locus, to produce a genetically modified non-human animal cell expressing a human or chimeric ICOS protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human ICOS comprises a portion of exon 1, exon 2, exon 3, exon 4, and/or exon 5 of a human ICOS gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human ICOS comprises a portion of exon 2 and/or a portion of exon 3 of a human ICOS gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human ICOS is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 2 or 13. In some embodiments, the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 1, exon 2, exon 3, exon 4, and/or exon 5 of the mouse ICOS gene. In some embodiments, the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 2 and/or a portion of exon 3 of the mouse ICOS gene. In some embodiments, the nucleotide sequence encoding the human or chimeric ICOS protein is operably linked to a regulatory element of endogenous ICOS, such as a promoter. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by other genes, the human or chimeric protein being selected from at least one of ICOSL, PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28, and CTLA4. In some embodiments, the human or chimeric protein is an ICOSL protein. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by other genes, wherein the human or chimeric protein is selected from at least one of ICOSL, PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28 and CTLA4. In some embodiments, the The human or chimeric protein is ICOSL protein.

In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric inducible T-cell co-stimulator ligand (ICOSL) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric ICOSL protein is operably linked to an endogenous regulatory element (e.g., endogenous 5'UTR and/or 3'UTR) of an endogenous ICOSL locus of at least one chromosome. In some embodiments, the chimeric ICOSL protein comprises an endogenous signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOSL protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human ICOSL (NP_056074.1, SEQ ID NO: 64). In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOSL protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acid positions 2332-244 of SEQ ID NO: 64. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOSL protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 71. In some embodiments, the animal is a mammal, such as a monkey, a rodent, a mouse or a rat. In some embodiments, the animal is a mouse. In some embodiments, the endogenous ICOSL protein of the animal is not expressed or is expressed at a reduced level compared to ICOSL in wild-type animals. In some embodiments, one or more cells of the animal express the human or chimeric ICOSL protein. In some embodiments, one or more cells of the animal express the human or chimeric ICOSL protein, and the human or chimeric ICOSL protein can bind to the endogenous ICOS protein to induce downstream signaling pathways. In some embodiments, one or more cells of the animal express human or chimeric ICOSL protein, which can bind to human ICOS protein to induce downstream signaling pathways.

In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises a nucleotide sequence encoding a human or chimeric ICOSL region replacing a nucleotide sequence encoding a corresponding region of the endogenous ICOSL at an endogenous ICOSL locus. In some embodiments, the nucleotide sequence encoding the corresponding region of the human or chimeric ICOSL is operably linked to an endogenous regulatory element of the endogenous ICOSL locus, and one or more cells of the animal express a human or humanized ICOSL protein. In some embodiments, the endogenous ICOSL protein of the animal is not expressed or is expressed at a reduced level compared to ICOSL in a wild-type animal. In some embodiments, the nucleotide sequence encoding the corresponding region of the human or chimeric ICOSL comprises a portion of exon 3, exon 4, and/or exon 5 of a human ICOSL gene. In some embodiments, the nucleotide sequence encoding the corresponding region of the human ICOSL is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 69. In some embodiments, the nucleotide sequence encoding the corresponding region of the endogenous ICOSL comprises a portion of exon 3, exon 4, and/or exon 5 of a mouse ICOSL gene. In some embodiments, the modified ICOSL gene in the genome of the animal is homozygous and/or heterozygous for the endogenous replaced locus.

In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a human or chimeric ICOSL protein, wherein the human or chimeric ICOSL protein comprises at least 50, 100, 120, 130, 150, 200, 210, 213, 250, 300 or 302 consecutive amino acids identical to the corresponding region of a human, and the animal expresses the human or chimeric ICOSL protein. In some embodiments, the human or chimeric ICOSL protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 32-244 of SEQ ID NO: 64. In some embodiments, the nucleotide sequence encoding the human or chimeric ICOSL protein is operably linked to an endogenous ICOSL regulatory element. In some embodiments, the nucleotide sequence encoding the corresponding region of the human or chimeric ICOSL can be integrated into the endogenous ICOSL locus of the animal. In some embodiments, the human or chimeric ICOSL protein has at least one mouse ICOSL activity and/or human ICOSL activity.

In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein in at least one cell of the animal, at the endogenous ICOSL locus of the animal, a nucleotide sequence encoding an endogenous ICOSL region is replaced by a nucleotide sequence encoding a corresponding region of human or chimeric ICOSL. In some embodiments, the endogenous ICOSL protein of the animal is not expressed or is expressed at a reduced level compared to ICOSL in a wild-type animal. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric ICOSL comprises a portion of exon 3, exon 4, and/or a portion of exon 5 of the human ICOSL gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human or chimeric ICOSL is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 64 or 71. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric ICOSL is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence shown in SEQ ID NO: 69. In some embodiments, the nucleotide sequence encoding the endogenous ICOSL region comprises a portion of exon 3, exon 4, and/or exon 5 of the mouse ICOSL gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human ICOSL is operably linked to an endogenous ICOSL regulatory element, such as a promoter. In some embodiments, the animal is a mammal, such as a monkey, a rodent, a mouse, or a rat.

In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric ICOSL, the method comprising replacing a nucleotide sequence encoding an endogenous ICOSL region with a nucleotide sequence encoding a corresponding region of human ICOSL at an endogenous mouse ICOSL locus, producing a genetically modified non-human animal cell, the animal cell expressing a human or chimeric ICOSL protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human ICOSL comprises a portion of exon 3, exon 4 and/or exon 5 of the human ICOSL gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human ICOSL is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human ICOSL is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of positions 32-244 of SEQ ID NO: 64. In some embodiments, The nucleotide sequence encoding the endogenous ICOSL region comprises a portion of exon 3, exon 4 and/or exon 5 of the mouse ICOSL gene. In some embodiments, the nucleotide sequence encoding the human or chimeric ICOSL protein is operably linked to a regulatory element of the endogenous ICOSL, such as a promoter. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, the human or chimeric proteins being selected from at least one of ICOS, PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28 and CTLA4. In some embodiments, the human or chimeric protein is an ICOS protein. In some embodiments, the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, the human or chimeric proteins being selected from at least one of ICOS, PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28 and CTLA4. In some embodiments, the human or chimeric protein is an ICOS protein.

In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating cancer, the method comprising: 1) administering a therapeutic agent to an animal as described herein, wherein the animal suffers from cancer; 2) determining the inhibitory effect of the therapeutic agent on cancer. In some embodiments, the therapeutic agent is an anti-ICOS antibody and/or an anti-ICOSL antibody. In some embodiments, the cancer is a tumor, and the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the animal. In some embodiments, the cancer comprises injecting one or more cancer cells into the animal. In some embodiments, the cancer is leukemia, solid tumors, lymphoma, respiratory cancer, skin cancer, gastric cancer, digestive tract cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, melanoma, non-small cell lung cancer, bladder cancer, breast cancer, colorectal cancer, lung cancer, multiple myeloma, non-Hodgkin's lymphoma, prostate cancer, kidney cancer, etc.

In one aspect, the present invention provides a method for determining the effectiveness of anti-ICOS antibody and/or anti-ICOSL antibody and additional therapeutic agent in treating cancer, the method comprising: 1) administering anti-ICOS antibody and/or anti-ICOSL antibody and additional therapeutic agent to the animal described in the present application, wherein the animal has a tumor; 2) determining the inhibitory effect of anti-ICOS antibody and/or anti-ICOSL antibody and additional therapeutic agent on the tumor. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the tumor comprises one or more cancer cells injected into the animal. In some embodiments, the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the animal. In some embodiments, the cancer is leukemia, solid tumor, lymphoma, respiratory cancer, skin cancer, gastric cancer, digestive tract cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, melanoma, non-small cell lung cancer, bladder cancer, breast cancer, colorectal cancer, lung cancer, multiple myeloma, non-Hodgkin's lymphoma, prostate cancer, kidney cancer, etc.

In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating an immune disease, the method comprising: 1) administering a therapeutic agent to a non-human animal as described herein, wherein the non-human animal suffers from an immune disease; 2) determining the therapeutic effect of the therapeutic agent on the immune disease. In some embodiments, the immune disease is systemic lupus erythematosus, transplant rejection, blood disease, immune neuromuscular disease, rheumatoid arthritis, etc.

In one aspect, the present invention provides a method for determining the effectiveness of an ICOS therapeutic agent in treating inflammation, the method comprising: 1) administering a therapeutic agent to an animal described herein, wherein the animal has inflammation; 2) determining the effectiveness of the therapeutic agent in treating inflammation. In some embodiments, the inflammation is arthritis, inflammation, inflammatory bowel disease, etc.

In one aspect, the present invention provides a method for determining the toxicity of an ICOS therapeutic agent, the method comprising: 1) administering the therapeutic agent to an animal as described herein; 2) determining the effect of the therapeutic agent on the animal. In some embodiments, determining the effect of the therapeutic agent on the animal involves measuring the animal's weight, red blood cell count, hematocrit, and/or hemoglobin.

In one aspect, the present invention provides a humanized ICOS protein, wherein the humanized ICOS protein comprises all or part of a human ICOS protein. In some embodiments, the humanized ICOS protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 13.

In one aspect, the present invention provides a humanized ICOS gene, which encodes the humanized protein described in the present application. In some embodiments, the humanized ICOS gene comprises a portion of exon 1 of the human ICOS gene, all of exons 2-4, and/or a portion of exon 5, and the humanized ICOS gene further comprises a portion of exon 1 of a non-human animal ICOS gene, and/or a portion of exon 5. In some embodiments, the humanized ICOS gene comprises a portion of exon 2 of the human ICOS gene, and/or a portion of exon 3, and the humanized ICOS gene further comprises all of exon 1 of a non-human animal ICOS gene, a portion of exon 2, a portion of exon 3, and/or all of exons 4-5. In some embodiments, the humanized ICOS gene comprises a portion of exon 1 of the human ICOS gene, all of exons 2-4, and/or a portion of exon 5, and the humanized ICOS gene further comprises a portion of exon 1 of a non-human animal ICOS gene, and/or all of exons 2-5. In some embodiments, the humanized ICOS gene comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 58, 59, 60, 61, 62 or 11111, 12 or 111.

In one aspect, the present invention provides a humanized ICOSL protein, wherein the humanized ICOSL protein comprises all or part of a human ICOSL protein. In some embodiments, the humanized ICOSL protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 71.

In one aspect, the present invention provides a humanized ICOSL gene, which encodes the humanized protein described in the present application. In some embodiments, the humanized ICOSL gene comprises part of exon 3, all of exon 4, and/or part of exon 5 of the human ICOSL gene, and the humanized ICOSL gene further comprises all of exons 1-2, part of exon 3, part of exon 5, and/or all of exons 6-7 of the ICOSL gene of a non-human animal. In some embodiments, the nucleotide sequence contained in the humanized ICOSL gene is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 65, 66, 67, 68, 69 or 7070.

In one aspect, the present invention provides a cell comprising the protein and gene described in the present application.

In one aspect, the present invention provides an animal model, wherein the animal model comprises the protein and gene described in the present application.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may be used. The materials, methods, and examples are exemplary only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

Those skilled in the art can easily discern other conveniences and advantages of the present application from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic diagram comparing the mouse ICOS locus and the human ICOS locus (not to scale);

Figure 2: Schematic diagram of the humanization of the mouse ICOS locus (not to scale);

Figure 3: Schematic diagram of ICOS gene targeting strategy and targeting vector V1 design (not to scale);

Figure 4: Schematic diagram of the FRT recombination process of ICOS gene humanized mice (not to scale);

Figure 5: PCR identification results of ICOS gene humanized mice F1 generation, where M is Marker, WT is wild type control, and H ₂ O is water control;

Figure 6: RT-PCR test results, where +/+ is wild-type C57BL/6 mice, H/+ is ICOS gene humanized heterozygous mice, and H ₂ O is water control;

FIG7 : Schematic diagram of the humanization of the mouse ICOS locus (not to scale);

Figure 8: Schematic diagram of ICOS gene targeting strategy and targeting vector V2 design (not to scale);

FIG9 : Schematic diagram 2 of the FRT recombination process of ICOS gene humanized mice (not to scale);

Figure 10: Schematic diagram of ICOS gene targeting strategy and targeting vector V3 design (not to scale);

Figure 11: PCR identification results of ICOS gene humanized mice F1 generation, where M is Marker, WT is wild type control, and H ₂ O is water control;

Figure 12: Southern blot test results of ICOS gene humanized mouse F1 generation, where WT is the wild-type control;

Figure 13: RT-PCR test results, where +/+ is wild-type C57BL/6 mice, H/+ is ICOS gene humanized heterozygous mice, and H ₂ O is water control;

FIG14 : Schematic diagram of the humanization of the mouse ICOS locus (not to scale);

Figure 15: Schematic diagram of ICOS gene targeting strategy and targeting vector V4 design (not to scale);

Figure 16: Schematic diagram of the FRT recombination process of ICOS gene humanization in mice (not to scale), wherein chiExon1 from left to right is: mouse Exon1, human ICOS nucleotide sequence, WPRE sequence, PA sequence;

Figure 17: Schematic diagram of ICOS gene targeting strategy and targeting vector V5 design (not to scale);

Figure 18: PCR identification results of ICOS gene humanized mice F1 generation, where PC is the positive control and WT is Wild type control, H ₂ O is water control, M is Marker;

Figure 19: Southern blot test results of ICOS gene humanized mouse F1 generation, where WT is the wild-type control;

Figure 20: Schematic diagram comparing the mouse ICOSL locus and the human ICOSL locus (not to scale);

Figure 21: Schematic diagram of the humanization of the mouse ICOSL locus (not to scale);

Figure 22: Schematic diagram of ICOSL gene targeting strategy and targeting vector V6 design (not to scale);

Figure 23: Schematic diagram of the FRT recombination process of ICOSL gene humanized mice (not to scale);

Figure 24: Schematic diagram 2 of ICOSL gene targeting strategy and targeting vector V7 design (not to scale);

Figure 25: PCR identification results of F1 generation of ICOSL gene humanized mice, where M is Marker, PC is positive control, WT is wild type control, and H ₂ O is water control;

Figure 26: Southern blot test results of F1 generation of ICOSL gene humanized mice, where WT is the wild-type control;

Figure 27: Alignment results between the amino acid sequence of human ICOS (NP_036224.1; SEQ ID NO: 2) and the amino acid sequence of mouse ICOS (NP_059508.2; SEQ ID NO: 1);

Figure 28: Alignment results between the amino acid sequence of human ICOS (NP_036224.1; SEQ ID NO: 2) and the amino acid sequence of rat ICOS (NP_072132.1; SEQ ID NO: 114);

Figure 29: Alignment results between the amino acid sequence of human ICOSL (NP_056074.1; SEQ ID NO: 64) and the amino acid sequence of mouse ICOSL (NP_056605.1; SEQ ID NO: 63);

Figure 30: Alignment results between the amino acid sequence of human ICOSL (NP_056074.1; SEQ ID NO: 64) and the amino acid sequence of rat ICOSL (XP_006256323.1; SEQ ID NO: 9);

Figure 31: CD4/CD8 T cell proliferation rate in mouse spleen;

Figure 32 (A): IgG1 concentration in wild-type C57BL/6 mice and ICOS humanized homozygous mice at resting state;

Figure 32 (B): The concentration of IgG1 in vivo after immunization of wild-type C57BL/6 mice and ICOS humanized homozygous mice;

FIG. 32(C) : IgE concentration in vivo after immunization of wild-type C57BL/6 mice and ICOS humanized homozygous mice.

Detailed description

ICOS

In the human genome, the ICOS gene (Gene ID: 29851) contains five exons, namely exon 1, exon 2, exon 3, exon 4 and exon 5 (Figure 1). The nucleotide sequence of human ICOS mRNA is NM_012092.4, the amino acid sequence of human ICOS is NP_036224.1 (SEQ ID NO: 2). Based on the nucleotide sequence and amino acid sequence of the transcript NM_012092.4 and its encoded protein NP_036224.1, the corresponding position of each exon is as follows:

Table 1

The human ICOS gene (NCBI Gene ID: 29851) is located at positions 203936763 to 203961577 of NC_000002.12 on chromosome 2, based on transcript NM_012092.4. Among them, the 5'-UTR is located at positions 203,936,763 to 203,936,814, exon 1 is located at positions 203,936,763 to 203,936,872, intron 1 is located at positions 203,936,873 to 203,955,635, exon 2 is located at positions 203,955,636 to 203,955,971, intron 2 is located at positions 203,955,972 to 203,956,658, and exon 3 is located at positions 203,956,763 to 203,956,764. ,659 to 203,956,765, intron 3 is located at 203,956,766 to 203,957,798, exon 4 is located at 203,957,799 to 203,957,883, intron 4 is located at 203,957,884 to 203,959,585, exon 5 is located at 203,959,586 to 203,961,577, and 3'-UTR is located at 203,959,600 to 203,961,577. All relevant information about the human ICOS locus can be retrieved on the NCBI website (Gene ID: 29851). The entire content is incorporated herein by reference.

In the mouse genome, the ICOS gene (Gene ID: 54167) contains 5 exons, namely exon 1, exon 2, exon 3, exon 4 and exon 5 (Figure 1). The nucleotide sequence of mouse ICOS mRNA is NM_017480.2, and the amino acid sequence of mouse ICOS is NP_059508.2 (SEQ ID NO: 1). Based on the nucleotide sequence and amino acid sequence of the transcript NM_017480.2 and its encoded protein NP_059508.2, the corresponding position of each exon is as follows:

Table 2

The mouse ICOS gene (NCBI Gene ID: 54167) is located at positions 60999909 to 61039481 of NC_000067.7 on chromosome 1, based on transcript NM_017480.2. Among them, the 5'UTR is located at positions 61,017,086 to 61,017,117, exon 1 is located at positions 61,017,086 to 61,017,175, intron 1 is located at positions 61,017,176 to 61,032,860, exon 2 is located at positions 61,032,861 to 61,033,199, intron 2 is located at positions 61,033,200 to 61,033,768, and exon 3 is located at positions 61,033, 769 to 61,033,875, intron 3 is located at 61,033,876 to 61,034,682, exon 4 is located at 61,034,683 to 61,034,767, intron 4 is located at 61,034,768 to 61,036,859, exon 5 is located at 61,036,860 to 61,039,479, and 3'-UTR is located at 61,036,874 to 61,039,479. All relevant information about the mouse ICOS locus can be found on the NCBI (Gene ID: 54167) website. The entire content is incorporated herein by reference.

Figure 27 shows the alignment of the amino acid sequence of human ICOS (NP_036224.1; SEQ ID NO: 2) and the amino acid sequence of mouse ICOS (NP_059508.2; SEQ ID NO: 1). Therefore, the corresponding amino acid residues or regions between human and mouse ICOS can be found in Figure 27.

ICOS genes, proteins and gene loci of other species are also known in the art. For example, Gene ID of ICOS of Rattus norvegicus (rat): 64545, Gene ID of ICOS of Macaca mulatta (rhesus monkey): 705788, Gene ID of ICOS of Canis lupus familiaris (dog): 403456, and Gene ID of ICOS of Sus scrofa (pig): 733597. The relevant information of these genes (e.g., intron sequence, exon sequence and amino acid sequence) can be found in NCBI, the entire contents of which are incorporated herein by reference.

Figure 28 shows the amino acid sequence of human ICOS (NP_036224.1; SEQ ID NO: 2) and the amino acid sequence of rat ICOS (NP_072132.1; SEQ ID NO: 114). Therefore, the corresponding amino acid residues or regions between human and rat ICOS can be retrieved in Figure 11.

The present invention provides a human or chimeric (e.g., humanized) ICOS nucleotide sequence or amino acid sequence. In some embodiments, exon 1, exon 2, exon 3, exon 4, exon 5, signal peptide, cell cycle inhibitory factor, and cytokine are selected from the group consisting of: The entire nucleotide sequence of the extracellular region, transmembrane region, and/or cytoplasmic region is replaced by the corresponding nucleotide sequence of the human ICOS gene. In some embodiments, "part" of exon 1, exon 2, exon 3, exon 4, exon 5, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region of the mouse ICOS gene is replaced by the corresponding sequence of the human ICOS gene. By “portion” is meant at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 215, 250, 300, 350, 390, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 910, 920, 930, 940, 950, 960, 962, 1000, 1100, 1200, 1300, 1400, 1500 70, 80, 90, 100, 110, 120, 130, 131, 140, 150, 160, 170, 180, 190, or 200 consecutive amino acid sequences. In some embodiments, the "part" is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 100% identical to exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region. In some embodiments, the "partial" or "complete" sequence of exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, and exon 5 of the mouse ICOS gene is replaced by the "partial" or "complete" sequence of the corresponding exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, and exon 5 of the human ICOS gene. In some embodiments, the extracellular region comprises a signal peptide. In some embodiments, the extracellular region does not comprise a signal peptide.

In some embodiments, "partial" deletion of the endogenous signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, and/or exon 5.

In some embodiments, the present invention provides a genetically modified non-human animal, the genome of which includes a chimeric ICOS nucleotide sequence. In some embodiments, the protein encoded by the chimeric ICOS nucleotide sequence comprises a human or humanized signal peptide, a human or humanized extracellular region, a human or humanized transmembrane region, and a human or humanized cytoplasmic region. In some embodiments, the protein encoded by the chimeric ICOS nucleotide sequence comprises an endogenous signal peptide, a human or humanized extracellular region, a human or humanized transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the encoded protein is at least 70%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1, 2 or 13. In some embodiments, the non-human animal genome comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 58, 59, 60, 61, 62, 72, 73, 74, 75 and 111.

In some embodiments, the non-human animals described herein comprise a sequence encoding a human or humanized ICOS protein. In some embodiments, the ICOS protein comprises, from the N-terminus to the C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized ICOS protein comprises a human or humanized signal peptide. For example, the human or humanized ICOS signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 1-20 of SEQ ID NO: 2. In some embodiments, the humanized ICOS protein comprises an endogenous signal peptide. For example, the endogenous ICOS signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 1-20 of SEQ ID NO: 1. In some embodiments, the humanized ICOS protein comprises a human or humanized extracellular region. For example, the humanized extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95% or 100% identical to amino acids 21-140 or 27-140 of SEQ ID NO: 2, and in some embodiments, the humanized ICOS protein comprises at least 1, 2, 3, 4, 5 or 6 amino acids at the C-terminus of the endogenous extracellular region, such as SEQ ID NO: 1 positions 21-26. In some embodiments, the humanized ICOS protein comprises a human or humanized transmembrane region, for example, the humanized transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95% or 100% identical to amino acids 141-161 or 141-156 of SEQ ID NO: 2. In some embodiments, the humanized ICOS protein comprises an endogenous transmembrane region, for example, the endogenous ICOS transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 158-165 of SEQ ID NO: 1. In some embodiments, the humanized ICOS protein comprises a human or humanized cytoplasmic region, for example, the humanized cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 162-199 of SEQ ID NO: 2. In some embodiments, the humanized ICOS protein comprises an endogenous cytoplasmic region. For example, the endogenous ICOS cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 166-200 of SEQ ID NO: 1. In some embodiments, the humanized ICOS protein comprises an endogenous ICOS sequence of amino acids 1-26 and/or 158-200 of SEQ ID NO: 1. In some embodiments, the humanized ICOS protein comprises a human ICOS sequence of amino acids 27-156 of SEQ ID NO: 2. In some embodiments, the humanized ICOS protein comprises a human ICOS sequence of amino acids 1-199 of SEQ ID NO: 2.

In some embodiments, the non-human animals described herein comprise a human or humanized ICOS gene. In some embodiments, the humanized ICOS gene comprises, from 5' to 3': a portion of endogenous exon 1 (e.g., NM_017480.2, positions 1-45), a portion of human exon 1 (e.g., NM_012092.4, positions 53-110 nucleotide sequence), human exons 2-4, a portion of human exon 5 (e.g., NM_012092.4, positions 639-652 nucleotide sequence), a portion of endogenous exon 5 (e.g., NM_017480.2, positions 649-3254 nucleotide sequence). In some embodiments, the humanized ICOS gene comprises, from 5'-3': endogenous exon 1, part of endogenous exon 2 (e.g., nucleotide sequence at positions 104-123 of NM_017480.2), part of human exon 2 (e.g., nucleotide sequence at positions 131-446 of NM_012092.4), part of human exon 3 (e.g., nucleotide sequence at positions 447-520 of NM_012092.4), part of endogenous exon 3 (e.g., nucleotides at positions 517-549 of NM_017480.2), and endogenous exons 4-5. In some embodiments, the humanized ICOS gene comprises, from 5' to 3', part of endogenous exon 1 (e.g., nucleotide sequence 1-45 of NM_017480.2), part of human exon 1 (e.g., nucleotide sequence 53-110 of NM_012092.4), human exons 2-4, human Part of exon 5 (e.g., nucleotide sequence 639-652 of NM_012092.4), endogenous exons 2-5.

In some embodiments, humanized ICOS comprises 5 exons. In some embodiments, humanized ICOS comprises endogenous exon 1, humanized exon 2, humanized exon 3, endogenous exon 4 and/or endogenous exon 5. In some embodiments, humanized ICOS comprises humanized exon 1, human exon 2, human exon 3, human exon 4 and/or humanized exon 5. In some embodiments, humanized ICOS further comprises humanized exon 1, endogenous exon 2, endogenous exon 3, endogenous exon 4 and/or endogenous exon 5. In some embodiments, humanized ICOS does not comprise introns. In some embodiments, the humanized ICOS gene further comprises one or more auxiliary sequences (e.g., WPRE sequence and/or PA sequence). In some embodiments, the humanized ICOS gene further comprises a human or humanized 5'UTR. In some embodiments, the humanized ICOS gene further comprises a human or humanized 3'UTR. In some embodiments, the humanized ICOS gene further comprises an endogenous 5'UTR. In some embodiments, the humanized ICOS gene further comprises an endogenous 3'UTR.

In some embodiments, the genetically modified non-human animal may express human ICOS and/or chimeric (e.g., humanized) ICOS proteins, wherein the endogenous ICOS gene sequence is replaced by the human ICOS gene and/or nucleotide sequence. Further, the amino acid sequence encoded by the human ICOS gene and/or nucleotide sequence is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 2 or 13. In some embodiments, the nucleotide sequence of the ICOS gene of the endogenous non-human animal is replaced by all or part of the nucleotide sequence encoding the mature human ICOS protein.

In some embodiments, the genetically modified non-human animal expresses human ICOS and/or chimeric ICOS proteins (e.g., humanized ICOS) under endogenous promoters and/or regulatory elements. Replacement of the endogenous locus provides a non-human animal that expresses human or chimeric ICOS proteins (e.g., humanized ICOS) in the same type of cells. The genetically modified mice do not show potential diseases observed in certain other transgenic mice known in the art. The human ICOS or chimeric ICOS protein expressed in the non-human animal can maintain the function of one or more wild-type or human ICOS proteins, for example, the expressed ICOS protein can bind to human or non-human ICOSL protein. Further, in some embodiments, the genetically modified non-human animal does not express endogenous ICOS protein. In some embodiments, the genetically modified non-human animal has reduced expression of endogenous ICOS protein. The "endogenous ICOS protein" described herein refers to the ICOS protein encoded by the endogenous ICOS nucleotide sequence of the non-human animal (e.g., mouse) before genetic modification.

The genome of the non-human animal comprises a nucleotide sequence encoding an amino acid that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of human ICOS protein (NP_036224.1; SEQ ID NO: 2). In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or at least 100% identical to the nucleotide sequence of SEQ ID NO: 115, 10, 11, 12, 62 or 111.

The nucleotide sequence encoding the endogenous ICOS region in the non-human animal genome is replaced by the nucleotide sequence encoding the corresponding region of human ICOS. In some embodiments, the nucleotide sequence encoding the endogenous ICOS region is any sequence of the endogenous ICOS locus, such as exon 1, exon 2, exon 3, exon 4, exon 5, 5'UTR, 3'UTR, intron 1, intron 2, intron 3, intron 4 or any combination thereof. In some embodiments, the nucleotide sequence encoding the endogenous ICOS region is located within the endogenous ICOS regulatory region. In some embodiments, the nucleotide sequence encoding the endogenous ICOS region is exon 1, exon 2, exon 3, exon 4, and/or exon 5, or a portion thereof.

One or more cells of the genetically modified non-human animal express a human or chimeric ICOS protein (e.g., a humanized ICOS protein). In some embodiments, the human or chimeric ICOS protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 195 or 199 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 2.

In some embodiments, the genetically modified non-human animal genome contains all or part of exon 1, exon 2, exon 3, exon 4, and/or exon 5 of the human ICOS gene, or all or part of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2.

In some embodiments, the genetically modified non-human animal genome comprises a portion of exon 1 of the human ICOS gene, all of exons 2-4, and a portion of exon 5. In some embodiments, the portion of exon 1 comprises at least 5, 10, 20, 30, 40, 45, 50, 55, 58, 60, 61, 62, 63, 64, 65, 70, 75, 80, 90, 100, or 110 bp of continuous nucleotide sequence of human ICOS gene exon 1. In some embodiments, the portion of exon 1 comprises a continuous nucleotide sequence of 58 bp. In some embodiments, the portion of exon 1 comprises at least 20 bp of nucleotide sequence. In some embodiments, the portion of exon 5 comprises at least 5, 10, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1500, 1900 or 1992 bp of continuous nucleotide sequence of human ICOS gene exon 5. In some implementations, the portion of exon 5 comprises 14 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 5 includes at least 5 bp of nucleotides. Part of human ICOS gene exon 1, all of exons 2-4, and part of exon 5 include at least 500-1000, 1000-5000bp, 5000-10000bp, 10000-20000bp, or 20000-23000bp of continuous nucleotide sequences. In some embodiments, the nucleotide sequence encoding the corresponding region of human ICOS is located at nucleotide sequence 53-652 of human ICOS gene transcript NM_012092.4.

In some embodiments, the genetically modified non-human animal genome comprises a portion of exon 2 and a portion of exon 3 of the human ICOS gene. In some embodiments, the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 300, 316, or 336 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 2 comprises 316 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 2 comprises at least 150 bp of nucleotide sequence. In some embodiments, the portion of exon 3 comprises at least 5, 10, 14, 15, 20, 30, In some embodiments, the portion of exon 3 comprises a 74bp continuous nucleotide sequence. In some embodiments, the portion of exon 3 comprises at least 30bp of nucleotides. The portion of exon 2 of the human ICOS gene, the portion of exon 3 and the portion of exon 3 comprise at least 100-500bp, 500-1000bp or 1000-1100bp of continuous nucleotide sequences. In some embodiments, the nucleotide sequence encoding the corresponding region of human ICOS is located at the 131st-520th nucleotide sequence of the human ICOS gene transcript NM_012092.4.

In some embodiments, the ICOS gene of the genetically modified non-human animal is heterozygous or homozygous for the endogenous modified locus.

In some embodiments, the humanized ICOS genome comprises a 5'UTR of a human ICOS gene. In some embodiments, the humanized ICOS genome comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanized ICOS genome comprises a 3'UTR of a human ICOS gene. In some embodiments, the humanized ICOS genome comprises an endogenous (e.g., mouse) 3'UTR. Where appropriate, based on the similarity of the 5' flanking sequences, it is reasonable to infer that the mouse and human ICOS genes are similarly regulated. As described herein, a humanized ICOS mouse comprises a replacement of an endogenous mouse locus that retains mouse endogenous regulatory elements but comprises a humanized ICOS coding sequence. Expression of ICOS in a genetically modified heterozygous or homozygous mouse is completely normal.

In another aspect, the present invention provides a genetically modified non-human animal, wherein the genome of the non-human animal comprises a deletion of an endogenous ICOS gene, wherein the deletion of the endogenous ICOS gene comprises exon 1, exon 2, exon 3, exon 4, and/or exon 5, or a portion of the endogenous ICOS locus.

In some embodiments, the deletion of the endogenous ICOS gene comprises one or more exons or portions of exons selected from exon 1, exon 2, exon 3, exon 4, and/or exon 5.

In some embodiments, the endogenous ICOS gene deletion further comprises one or more introns or portions of introns, wherein the introns are selected from intron 1, intron 2, intron 3, and/or intron 4 of the ICOS gene.

In some embodiments, the deletion comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 215, 250, 300, 350, 390, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 910, 920, 930, 940, 950, 960, 962, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1750, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 10000, 15000, 19756, 20000, 30000 or 39573 bp of continuous nucleotide sequence or more.

In some embodiments, the deletion of the endogenous ICOS gene comprises at least 20, 50, 55, 58, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 393, 400, 500, 600, 603, 700, 800, 900, 1000, 1500, 2000, 3000, 3200, or 3272 bp of contiguous nucleotide sequence, or more, of exon 1, exon 2, exon 3, exon 4, and/or exon 5.

The present invention provides a humanized mouse ICOS genomic DNA sequence, a construct expressing the amino acid sequence of the humanized ICOS protein, a cell comprising the construct, and a tissue comprising the cell.

Therefore, in some embodiments, the present invention provides a chimeric (e.g., humanized) ICOS nucleotide sequence and/or amino acid sequence, wherein in some embodiments, the chimeric nucleotide sequence is homologous to mouse endogenous ICOS mRNA (e.g., NM_017480.2), mouse ICOS amino acid sequence (e.g., NP_059508.2, SEQ ID NO: 1), or a portion thereof (e.g., 5'UTR, a portion of exon 1, a portion of exon 5 and a 3'UTR, or a 5'UTR, %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the chimeric nucleotide sequence has at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a human ICOS mRNA sequence (e.g., NM_012092.4), a human ICOS amino acid sequence (e.g., NP_036224.1, SEQ ID NO: 2), or a portion thereof (e.g., a portion of exon 1, a portion of exons 2-4 and exon 5, or a portion of exon 2 and a portion of exon 3).

In some embodiments, the nucleotide sequence encoding amino acids 1-200, 27-157 or 1-19 of mouse ICOS (SEQ ID NO: 1) is replaced by the corresponding nucleotide sequence encoding amino acids 1-199 or 27-156 of human ICOS (SEQ ID NO: 2).

In some embodiments, the nucleotide sequence is operably linked to a promoter or regulatory element, such as an endogenous mouse ICOS promoter, an inducible promoter, an enhancer, and/or a mouse or human regulatory element.

In some embodiments, at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) of a chimeric nucleic acid sequence described herein differs from all or a portion of a mouse ICOS nucleotide sequence (e.g., a portion of exon 1, exons 2-4, and exon 5 of mouse ICOS gene transcript NM_017480.2, or a portion of exon 2 and a portion of exon 3 of mouse ICOS gene transcript NM_017480.2, or a portion of exon 1 of mouse ICOS gene transcript NM_017480.2).

In some embodiments, at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) of the chimeric nucleic acid sequence is identical to all or a portion of a mouse ICOS nucleotide sequence (e.g., a portion of exon 1 and a portion of exon 5 of mouse ICOS gene transcript NM_017480.2, or exon 1, a portion of exon 2, a portion of exon 3, exons 4-5, or mouse ICOS gene transcript NM_017480.2). Part of exon 1 of ICOS gene transcript NM_017480.2, exons 2-5).

In some embodiments, at least a portion of the chimeric nucleic acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) is different from all or a portion of a human ICOS nucleotide sequence (e.g., a portion of exon 1 and a portion of exon 5 of human ICOS gene transcript NM_012092.4, or exon 1, a portion of exon 2, a portion of exon 3, and exons 4-5 of human ICOS gene transcript NM_012092.4).

In some embodiments, at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) of the chimeric nucleic acid sequence is identical to all or part of a human ICOS nucleotide sequence (e.g., a portion of exon 1, exons 2-4, and a portion of exon 5 of human ICOS gene transcript NM_012092.4, or a portion of exon 2 and a portion of exon 3 of human ICOS gene transcript NM_012092.4).

In some embodiments, at least a portion of the amino acids encoded by the chimeric nucleic acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, such as continuous or non-contiguous amino acid residues) is different from all or part of the mouse ICOS protein amino acid sequence (e.g., amino acids 1-200 or 27-157 of the mouse ICOS protein sequence NP_059508.2 (SEQ ID NO: 1)).

In some embodiments, at least a portion of the amino acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, consecutive or non-consecutive amino acid residues) is identical to all or part of the amino acid sequence of the mouse ICOS protein (e.g., amino acids 1-26 and/or 158-200 of the mouse ICOS protein sequence NP_059508.2 (SEQ ID NO: 1)).

In some embodiments, at least a portion of the amino acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, consecutive or non-consecutive amino acid residues) is different from all or part of the amino acid sequence of human ICOS protein (e.g., amino acids 1-26 and/or 157-199 of human ICOS protein sequence NP_036224.1 (SEQ ID NO: 2)).

In some embodiments, at least a portion of the amino acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, consecutive or non-consecutive amino acid residues) is identical to all or part of the amino acid sequence of human ICOS protein (e.g., amino acids 1-199 or 27-156 of human ICOS protein sequence NP_036224.1 (SEQ ID NO: 2)).

The present invention also provides a humanized ICOS amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

A) the amino acid sequence shown in SEQ ID NO: 1, 2 or 13;

B) is 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: 1, 2 or 13;

C) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is capable of hybridizing with a nucleotide sequence encoding an amino acid as set forth in SEQ ID NO: 1, 2 or 13 under low stringency conditions or under stringent conditions;

D) differs from the amino acid sequence of SEQ ID NO: 1, 2 or 13 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; or

E) An amino acid sequence as shown in SEQ ID NO: 1, 2 or 13, including substitution, deletion and/or insertion of one or more amino acid residues.

The present invention also provides a humanized ICOS amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

A) the amino acid sequence shown in positions 1-199 or 27-156 of SEQ ID NO: 2;

B) is 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 at positions 1-199 or 27-156;

C) differs from the amino acid sequence of SEQ ID NO: 2 at positions 1-199 or 27-156 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

D) An amino acid sequence that is the same as that shown in SEQ ID NO: 2 at positions 1-199 or 27-156, including substitution, deletion and/or insertion of one or more amino acid residues.

The present invention also provides a humanized ICOS amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

A) the amino acid sequence shown in positions 1-26 and/or 158-200 of SEQ ID NO: 1;

B) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence set forth in positions 1-26 and/or 158-200 of SEQ ID NO: 1;

C) differs from the amino acid sequence of SEQ ID NO: 1 at positions 1-26 and/or 158-200 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

D) An amino acid sequence comprising substitution, deletion and/or insertion of one or more amino acid residues as shown in positions 1-26 and/or 158-200 of SEQ ID NO: 1.

The present invention also provides a humanized ICOS nucleotide (eg, DNA or RNA) sequence, wherein the nucleotide sequence comprises any one of the following groups:

A) a nucleic acid sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 58, 59, 60, 61, 62 or 111, or a nucleic acid sequence encoding a humanized mouse ICOS homologous amino acid sequence;

B) can be combined with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, A nucleic acid sequence that hybridizes with the nucleotide sequence shown in 12, 58, 59, 60, 61, 62 or 111;

C) a nucleic acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 90% homology to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 58, 59, 60, 61, 62 or 111;

D) the amino acid sequence it encodes is 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: 1, 2 or 13;

E) the encoded amino acid sequence differs from the amino acid sequence of SEQ ID NO: 1, 2 or 13 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; or

F) The amino acid sequence encoded by it is the same as that shown in SEQ ID NO: 1, 2 or 13, including the amino acid sequence of substitution, deletion and/or insertion of one or more amino acid residues.

The present invention further provides a humanized mouse ICOS genomic DNA sequence. The DNA sequence is obtained by reverse transcription of mRNA transcribed from the ICOS genomic DNA sequence, and is consistent with or complementary to a DNA sequence homologous to the sequence shown in SEQ ID NO: 115, 10, 11, 12, 62 or 111.

ICOSL

In the human genome, the ICOSL gene (Gene ID: 23308) contains 7 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and exon 7 (Figure 20). The nucleotide sequence of human ICOSL mRNA is NM_015259.6, and the amino acid sequence of human ICOSL is NP_056074.1 (SEQ ID NO: 64). The corresponding positions of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_015259.6 and its encoded protein NP_056074.1 are as follows:

Table 3

The human ICOSL gene (NCBI Gene ID: 23308) is located at positions 44216981 to 44240943 of NC_000021.9 on chromosome 21, based on transcript NM_015259.6. Among them, the 5′-UTR is located at positions 44,240,943 to 44,240,818, exon 1 is located at positions 44,240,943 to 44,240,804, intron 1 is located at positions 44,240,803 to 44,238,489, exon 2 is located at positions 44,238,488 to 44,238,448, intron 2 is located at positions 44,238,447 to 44,237,218, exon 3 is located at positions 44,237,217 to 44,236,867, intron 3 is located at positions 44,236,866 to 44,235,563, and exon 4 is located at positions 44,235,569. ,562 to 44,235,272, intron 4 is at positions 44,235,271 to 44,231,445, exon 5 is at positions 44,231,444 to 44,231,280, intron 5 is at positions 44,231,279 to 44,230,090, exon 6 is at positions 44,230,089 to 44,230,054, intron 6 is at positions 44,230,053 to 44,229,045, exon 7 is at positions 44,229,044 to 44,222,991, and the 3’-UTR is at positions 44,229,033 to 44,222,991. All relevant information about the human ICOSL locus can be retrieved on the NCBI website (Gene ID: 23308). The entire content is incorporated herein by reference.

In the mouse genome, the ICOSL gene (Gene ID: 50723) contains 7 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and exon 7 (Figure 20). The nucleotide sequence of mouse ICOSL mRNA is NM_015790.3, and the amino acid sequence of mouse ICOSL is NP_056605.1 (SEQ ID NO: 63). Based on the nucleotide sequence and amino acid sequence of the transcript NM_015790.3 and its encoded protein NP_056605.1, the corresponding position of each exon is as follows:

Table 4

The mouse ICOSL gene (NCBI Gene ID: 50723) is located at positions 77904921 to 77915359 of NC_000076.7 on chromosome 10, based on transcript NM_015790.3. Among them, the 5'-UTR is located at positions 77,905,136 to 77,905,358, exon 1 is located at positions 77,905,136 to 77,905,369, and intron 1 is located at positions 77,905,370 to 77,906,994, exon 2 is at position 77,906,995 to 77,907,113, intron 2 is at position 77,907,114 to 77,907,571, exon 3 is at position 77,907,572 to 77,907,925, intron 3 is at position 77,907,926 to 77,909,540, exon 4 is at position 77,909,541 to 77,909,834, intron 4 is at position 77,909,835 to 77,911, 038, exon 5 is located at positions 77,911,039 to 77,911,188, intron 5 is located at positions 77,911,189 to 77,912,977, exon 6 is located at positions 77,912,978 to 77,913,007, intron 6 is located at positions 77,913,008 to 77,913,726, exon 7 is located at positions 77,913,727 to 77,915,359, and 3'-UTR is located at positions 77,913,738 to 77,915,359. All relevant information about the mouse ICOSL locus can be found on the NCBI (Gene ID: 50723) website. The entire content is incorporated herein by reference.

Figure 29 shows the alignment of the amino acid sequence of human ICOSL (NP_056074.1; SEQ ID NO: 64) and the amino acid sequence of mouse ICOSL (NP_056605.1; SEQ ID NO: 63). Therefore, the corresponding amino acid residues or regions between human and mouse ICOSL can be found in Figure 29.

ICOSL genes, proteins and gene loci of other species are also known in the art. For example, Rattus norvegicus (rat) ICOSL has Gene ID: 499415. The relevant information of these genes (such as intron sequences, exon sequences and amino acid sequences) can be found in NCBI, the entire contents of which are incorporated herein by reference.

Figure 30 shows the amino acid sequence of human ICOSL (NP_056074.1; SEQ ID NO: 64) and the amino acid sequence of rat ICOSL (XP_006256323.1; SEQ ID NO: 9). Therefore, the corresponding amino acid residues or regions between human and rat ICOSL can be retrieved in Figure 30.

The present invention provides a human or chimeric (e.g., humanized) ICOSL nucleotide sequence or amino acid sequence. In some embodiments, the entire nucleotide sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region of the mouse ICOSL gene is replaced by the corresponding nucleotide sequence of the human ICOSL gene. In some embodiments, "part" of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region of the mouse ICOSL gene is replaced by the corresponding sequence of the human ICOSL gene. The term "portion" refers to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 215, 250, 300, 350, 390, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 910, 920, 930, 940, 950, 960, 1000, 1100, 1200, 1300, 1400 , 1500, 1600, 1750, 1800, 1900, 2000, 3000, 3400, 3455, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or 10439 bp of continuous nucleotide sequence, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 19, 20, 30, 40, 50, 55, 58, 60, 70, 80, 90, 100, 110, 120, 150, 200, 210, 212, 250, 300, 310, 320 or 322 continuous amino acid sequence. In some embodiments, the "portion" is related to exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron The identity of the polypeptide of the present invention to the polypeptide of the present invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 100%. In some embodiments, "partial" or "complete" sequences of Exon 1, Intron 1, Exon 2, Intron 2, Exon 3, Intron 3, Exon 4, Intron 4, Exon 5, Intron 5, Exon 6, Intron 6, and Exon 7 of the mouse ICOSL gene are replaced by "partial" or "complete" sequences of the corresponding Exon 1, Intron 1, Exon 2, Intron 2, Exon 3, Intron 3, Exon 4, Intron 4, Exon 5, Intron 5, Exon 6, Intron 6, and Exon 7 of the human ICOSL gene. In some embodiments, the extracellular region comprises a signal peptide. In some embodiments, the extracellular region does not comprise a signal peptide.

In some embodiments, "partial" deletion of the endogenous signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, and/or exon 7.

In some embodiments, the present invention provides a genetically modified non-human animal, the genome of which comprises a chimeric ICOSL nucleotide sequence. In some embodiments, the protein encoded by the chimeric ICOSL nucleotide sequence comprises an endogenous signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the protein encoded thereby is at least 70%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 63, 64 or 71. In some embodiments, the genome of the non-human animal comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95% or 100% identical to the nucleotide sequence of SEQ ID NO: 65, 66, 67, 68, 69, 70, 89, 90, 91 and 92.

In some embodiments, the non-human animals described herein comprise a sequence encoding a human or humanized ICOSL protein. In some embodiments, the ICOSL protein comprises, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized ICOSL protein comprises a human or humanized signal peptide, for example, a human or humanized ICOSL signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95% or 100% identical to amino acids 1-18 of SEQ ID NO: 64. In some embodiments, the humanized ICOSL protein comprises an endogenous signal peptide, for example, an endogenous ICOSL signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95% or 100% identical to amino acids 1-46 of SEQ ID NO: 63. In some embodiments, the humanized ICOSL protein comprises a human or humanized extracellular region, for example, the humanized extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 32-244 of SEQ ID NO: 64. In some embodiments, the humanized ICOSL protein comprises an endogenous extracellular region, for example, the endogenous ICOSL extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 47-56 and 269-277 of SEQ ID NO: 63. In some embodiments, the humanized ICOSL protein comprises a human or humanized transmembrane region, for example, the humanized transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 257-277 of SEQ ID NO: 64. In some embodiments, the humanized The ICOSL protein comprises an endogenous transmembrane region, for example, the endogenous ICOSL transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 278-298 of SEQ ID NO: 63. In some embodiments, the humanized ICOSL protein comprises a human or humanized cytoplasmic region, for example, the humanized cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 278-302 of SEQ ID NO: 64. In some embodiments, the humanized ICOSL protein comprises an endogenous cytoplasmic region. For example, the endogenous ICOSL cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 299-322 of SEQ ID NO: 63. In some embodiments, the humanized ICOSL protein comprises an endogenous ICOSL sequence of amino acids 1-56 and/or 269-322 of SEQ ID NO: 63. In some embodiments, the humanized ICOSL protein comprises a human ICOSL sequence of amino acids 32-244 of SEQ ID NO: 64.

In some embodiments, the non-human animals described herein comprise a human or humanized ICOSL gene. In some embodiments, the humanized ICOSL gene comprises, from 5' to 3': endogenous exons 1-2, part of endogenous exon 3 (e.g., NM_015790.3, 288-325), part of human exon 3 (e.g., NM_015259.6, 220-532 nucleotide sequence), human exon 4, part of human exon 5 (e.g., NM_015259.6, 824-858 nucleotide sequence), part of endogenous exon 5 (e.g., NM_015790.3, 962-1085), endogenous exons 6-7.

In some embodiments, the humanized ICOSL comprises 7 exons. In some embodiments, the humanized ICOSL comprises endogenous exon 1, endogenous exon 2, humanized exon 3, human exon 4, humanized exon 5, endogenous exon 6, and/or endogenous exon 7. In some embodiments, the humanized ICOSL gene further comprises a human or humanized 5'UTR. In some embodiments, the humanized ICOSL gene further comprises a human or humanized 3'UTR. In some embodiments, the humanized ICOSL gene further comprises an endogenous 5'UTR. In some embodiments, the humanized ICOSL gene further comprises an endogenous 3'UTR.

In some embodiments, the genetically modified non-human animal may express human ICOSL and/or chimeric (e.g., humanized) ICOSL protein, wherein the endogenous ICOSL gene sequence is replaced by a human ICOSL gene and/or nucleotide sequence. Further, the amino acid sequence encoded by the human ICOSL gene and/or nucleotide sequence is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 100% identical to SEQ ID NO: 64 or 71. In some embodiments, the nucleotide sequence of the ICOSL gene of the endogenous non-human animal is replaced by all or part of the nucleotide sequence encoding the mature human ICOSL protein.

In some embodiments, the genetically modified non-human animal expresses human ICOSL and/or chimeric ICOSL protein (e.g., humanized ICOSL) under endogenous promoters and/or regulatory elements. Replacement of the endogenous locus provides a non-human animal that expresses human or chimeric ICOSL protein (e.g., humanized ICOSL) in the same type of cells. The genetically modified mice do not develop potential diseases observed in certain other transgenic mice known in the art. The human ICOSL or chimeric ICOSL protein expressed in the non-human animal can maintain one or more functions of the wild-type or human ICOSL protein, for example, the expressed The ICOSL protein can bind to human or non-human ICOSL proteins. Further, in some embodiments, the genetically modified non-human animal does not express endogenous ICOSL protein. In some embodiments, the genetically modified non-human animal has reduced expression of endogenous ICOSL protein. The "endogenous ICOSL protein" described herein refers to the ICOSL protein encoded by the endogenous ICOSL nucleotide sequence of the non-human animal (e.g., mouse) before genetic modification.

The genome of the non-human animal comprises a nucleotide sequence encoding an amino acid that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of human ICOSL protein (NP_056074.1; SEQ ID NO: 64). In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or at least 100% identical to the nucleotide sequence of SEQ ID NO: 69 or 70.

The nucleotide sequence encoding the endogenous ICOSL region in the non-human animal genome is replaced by the nucleotide sequence encoding the corresponding region of human ICOSL. In some embodiments, the nucleotide sequence encoding the endogenous ICOSL region is any sequence of the endogenous ICOSL locus, such as exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, 5'UTR, 3'UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6 or any combination thereof. In some embodiments, the nucleotide sequence encoding the endogenous ICOSL region is located within the endogenous ICOSL regulatory region. In some embodiments, the nucleotide sequence encoding the endogenous ICOSL region is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and/or exon 7, or a portion thereof.

One or more cells of the genetically modified non-human animal express a human or chimeric ICOSL protein (e.g., a humanized ICOSL protein). In some embodiments, the human or chimeric ICOSL protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 210, 213, 250, 300, or 302 consecutive amino acids of the amino acid sequence of SEQ ID NO: 64.

In some embodiments, the genome of the genetically modified non-human animal contains all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and/or exon 7 of the human ICOSL gene, or all or part of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2.

In some embodiments, the genetically modified non-human animal genome comprises a portion of exon 3, all of exon 4, and a portion of exon 5 of the human ICOSL gene. In some embodiments, the portion of exon 3 comprises at least 5, 10, 20, 30, 40, 45, 50, 55, 58, 60, 65, 70, 80, 90, 100, 200, 300, 310, 313, 350, or 351 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 3 comprises 313 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 3 comprises at least 150 bp of nucleotide sequence. In some embodiments, the portion of exon 5 comprises at least 5, 10, 14, 15, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 165 bp of continuous nucleotide sequence. In some implementations, the portion of exon 5 comprises 35 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 5 comprises at least 10 bp of nucleotides. The portion of exon 3, the entirety of exon 4, and the portion of exon 5 of the human ICOSL gene comprises at least 500-1000, 1000-5000 bp, or 5000-6000 bp continuous nucleotide sequence. In some embodiments, the nucleotide sequence encoding the corresponding region of human ICOSL is located at the nucleotide sequence 220-858 of the human ICOSL gene transcript NM_015259.6.

In some embodiments, the ICOSL gene of the genetically modified non-human animal is heterozygous or homozygous for the endogenous modified locus.

In some embodiments, the humanized ICOSL genome comprises a 5'UTR of a human ICOSL gene. In some embodiments, the humanized ICOSL genome comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanized ICOSL genome comprises a 3'UTR of a human ICOSL gene. In some embodiments, the humanized ICOSL genome comprises an endogenous (e.g., mouse) 3'UTR. Where appropriate, based on the similarity of the 5' flanking sequences, it can be reasonably inferred that the mouse and human ICOSL genes are similarly regulated. As described herein, humanized ICOSL mice comprise a replacement of an endogenous mouse locus that retains mouse endogenous regulatory elements but comprises a humanized ICOSL coding sequence. Expression of ICOSL in genetically modified heterozygous or homozygous mice is completely normal.

In another aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises a deletion of an endogenous ICOSL gene, wherein the deletion of the endogenous ICOSL gene comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and/or exon 7, or a portion of the endogenous ICOSL locus.

In some embodiments, the deletion of the endogenous ICOSL gene comprises one or more exons or portions of exons selected from exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7.

In some embodiments, the endogenous ICOSL gene deletion further comprises one or more introns or portions of introns selected from intron 1, intron 2, intron 3, intron 4, intron 5, and/or intron 6 of the ICOSL gene.

In some embodiments, the deletion comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 215, 250, 300, 350, 390, 400, 450, 500, 550, 600, 650, 700, 750 , 800, 850, 900, 910, 920, 930, 940, 950, 960, 962, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1750, 1800, 1900, 2000, 3000, 3455, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or 10439 bp of continuous nucleotide sequence or more.

In some embodiments, the deletion of the endogenous ICOSL gene comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 636, 650, 700, 800, 900, 1000, 1500, 2000, 3000, 2700, or 2748 bp of contiguous nucleotide sequence, or more, of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7.

The present invention provides a humanized mouse ICOSL genomic DNA sequence, a construct expressing the amino acid sequence of the humanized ICOSL protein; a cell comprising the construct; and a cell comprising the cell. Weaving.

Therefore, in some embodiments, the present invention provides a chimeric (e.g., humanized) ICOSL nucleotide sequence and/or amino acid sequence, wherein in some embodiments, the chimeric nucleotide sequence is homologous to mouse endogenous ICOSL mRNA (e.g., NM_015790.3), mouse ICOSL amino acid sequence (e.g., NP_056605.1, SEQ ID NO: 63), or a portion thereof (e.g., 5'UTR, exons 1-2, portions of exon 3). %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the chimeric nucleotide sequence has at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a human ICOSL mRNA sequence (e.g., NM_015259.6), a human ICOSL amino acid sequence (e.g., NP_056074.1, SEQ ID NO: 64), or a portion thereof (e.g., a portion of exon 3, a portion of exon 4 and a portion of exon 5).

In some embodiments, the nucleotide sequence encoding amino acids 57-268 of mouse ICOSL (SEQ ID NO: 63) is replaced by the corresponding nucleotide sequence encoding amino acids 32-244 of human ICOSL (SEQ ID NO: 64).

In some embodiments, the nucleotide sequence is operably linked to a promoter or regulatory element, such as an endogenous mouse ICOSL promoter, an inducible promoter, an enhancer, and/or a mouse or human regulatory element.

In some embodiments, at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) of a chimeric nucleic acid sequence described herein differs from all or a portion of a mouse ICOSL nucleotide sequence (e.g., a portion of exon 3, exon 4, and a portion of exon 5 of mouse ICOSL gene transcript NM_015790.3).

In some embodiments, at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) of the chimeric nucleic acid sequence is identical to all or part of a mouse ICOSL nucleotide sequence (e.g., exons 1-2, a portion of exon 3, a portion of exon 5, and exons 6-7 of mouse ICOSL gene transcript NM_015790.3).

In some embodiments, at least a portion of the chimeric nucleic acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) differs from all or a portion of a human ICOSL nucleotide sequence (e.g., exons 1-2, a portion of exon 3, a portion of exon 5, and exons 6-7 of the human ICOSL gene transcript NM_015259.6).

In some embodiments, at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) of the chimeric nucleic acid sequence is identical to all or part of a human ICOSL nucleotide sequence (e.g., a portion of exon 3, exon 4, and a portion of exon 5 of the human ICOSL gene transcript NM_015259.6).

In some embodiments, at least a portion of the amino acids encoded by the chimeric nucleic acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, such as continuous or non-contiguous amino acid residues) is different from all or part of the mouse ICOSL protein amino acid sequence (e.g., amino acids 57-268 of the mouse ICOSL protein sequence NP_056605.1 (SEQ ID NO: 63)).

In some embodiments, at least a portion of the amino acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, consecutive or non-consecutive amino acid residues) is identical to all or part of the amino acid sequence of the mouse ICOSL protein (e.g., amino acids 1-56 and/or 269-322 of the mouse ICOSL protein sequence NP_056605.1 (SEQ ID NO: 63)).

In some embodiments, at least a portion of the amino acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, consecutive or non-consecutive amino acid residues) is different from all or part of the amino acid sequence of human ICOSL protein (e.g., amino acids 1-31 and 245-302 of human ICOSL protein sequence NP_056074.1 (SEQ ID NO: 64)).

In some embodiments, at least a portion of the amino acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, consecutive or non-consecutive amino acid residues) is identical to all or part of the amino acid sequence of human ICOSL protein (e.g., amino acids 32-244 of human ICOSL protein sequence NP_056074.1 (SEQ ID NO: 64)).

The present invention also provides a humanized ICOSL amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

A) the amino acid sequence shown in SEQ ID NO: 63, 64 or 71;

B) is 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: 63, 64 or 71;

C) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is capable of hybridizing under low stringency conditions or stringent conditions with a nucleotide sequence encoding the amino acid set forth in SEQ ID NO: 63, 64 or 71;

D) differs from the amino acid sequence of SEQ ID NO: 63, 64 or 71 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

E) with respect to SEQ ID NO: 63, 64 or 71, including substitution, deletion and/or insertion of one or more amino acids The amino acid sequence of the residues.

The present invention also provides a humanized ICOSL amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

A) the amino acid sequence shown in SEQ ID NO: 64, positions 32-244;

B) is 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: 64 at positions 32-244;

C) differs from the amino acid sequence of SEQ ID NO: 64 at positions 32-244 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

D) An amino acid sequence similar to that shown in SEQ ID NO: 64, positions 32-244, including substitution, deletion and/or insertion of one or more amino acid residues.

The present invention also provides a humanized ICOSL amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

A) the amino acid sequence shown in positions 1-56 and/or 269-322 of SEQ ID NO: 63;

B) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence set forth in positions 1-56 and/or 269-322 of SEQ ID NO: 63;

C) differs from the amino acid sequence of SEQ ID NO: 63 at positions 1-56 and/or 269-322 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

D) An amino acid sequence comprising substitution, deletion and/or insertion of one or more amino acid residues as shown in positions 1-56 and/or 269-322 of SEQ ID NO: 63.

The present invention also provides a humanized ICOSL nucleotide (eg, DNA or RNA) sequence, wherein the nucleotide sequence comprises any one of the following groups:

A) a nucleic acid sequence as shown in SEQ ID NO: 65, 66, 67, 68, 69, 70, 89, 90, 91 or 92, or a nucleic acid sequence encoding a humanized mouse ICOSL homologous amino acid sequence;

B) a nucleic acid sequence that can hybridize to the nucleotide sequence shown in SEQ ID NO: 65, 66, 67, 68, 69, 70, 89, 90, 91 or 92 under low stringency conditions or stringent conditions;

C) a nucleic acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 90% homology to the nucleotide sequence shown in SEQ ID NO: 65, 66, 67, 68, 69, 70, 89, 90, 91 or 92;

D) the amino acid sequence it encodes is 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: 63, 64 or 71;

E) The amino acid sequence encoded by the amino acid sequence differs from the amino acid sequence of SEQ ID NO: 63, 64 or 71 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or not more than 1 amino acid; or

F) The amino acid sequence encoded by it is the same as that shown in SEQ ID NO: 63, 64 or 71, including the amino acid sequence of substitution, deletion and/or insertion of one or more amino acid residues.

The present invention further provides a humanized mouse ICOSL genomic DNA sequence. The DNA sequence is obtained by reverse transcription of the mRNA transcribed therefrom, and is consistent with or complementary to a DNA sequence homologous to the sequence shown in SEQ ID NO: 69 or 70.

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

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

The invention also provides cells, tissues and animals (e.g., mice) comprising the nucleotide sequences described herein, as well as cells, tissues and animals (e.g., mice) expressing human or chimeric (e.g., humanized) ICOS and/or ICOSL at an endogenous non-human ICOS and/or ICOSL locus.

Genetically modified non-human animals

The "genetically modified non-human animal" of the present invention refers to a non-human animal with exogenous DNA in at least one chromosome in the genome of the animal. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal have exogenous DNA. The cells with exogenous DNA can be various cells, for example, endogenous cells, somatic cells, immune cells, T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells, germ cells, blastocysts or endogenous tumor cells. In some embodiments, a genetically modified non-human animal is provided, the animal comprising a modified endogenous ICOS and/or ICOSL locus, comprising an exogenous sequence (e.g., a human sequence), for example, replacing one or more non-human sequences with one or more human sequences, or inserting one or more human and/or non-human sequences. Animals are generally able to pass genetic modifications to offspring through germline transmission.

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

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

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

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

Animals with humanized ICOS locus

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

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

In some embodiments, the humanized ICOS protein comprises a polypeptide sequence of 5-199 amino acids (continuous or non-continuous) identical to the human ICOS protein. In some embodiments, the length of the polypeptide sequence is 5-199, 10-130, or 10-199 amino acids. In some embodiments, the humanized ICOS gene comprises a nucleotide sequence of 20-24815 bp (continuous or non-continuous) identical to the human ICOS gene. In some embodiments, the nucleotide sequence is 20-600 bp, 20-1077 bp, or 20-22785 bp.

In some embodiments, the ICOS extracellular region is human or humanized. In some embodiments, the ICOS signal peptide is human or humanized. In some embodiments, the ICOS cytoplasmic region is human or humanized. In some embodiments, the ICOS transmembrane region is human or humanized. In some embodiments, both the ICOS extracellular region and the transmembrane region are human or humanized. In some embodiments, both the ICOS signal peptide and the cytoplasmic region are endogenous.

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

The genetically modified animal may express human ICOS and/or chimeric (e.g., humanized) ICOS at an endogenous mouse locus, wherein the endogenous mouse ICOS gene has been replaced or inserted with a gene for human ICOS and/or a nucleotide sequence encoding a region of a human ICOS sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% identical to a human ICOS sequence. In various embodiments, the endogenous non-human animal ICOS locus is modified with a human all or part of a nucleic acid sequence that encodes a mature ICOS protein.

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

The genome of the animal comprises a nucleotide sequence encoding at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of human ICOS (NP_036224.1; SEQ ID NO: 2). In some embodiments, The genome comprises a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 115, 10, 11, 12, 62 or 111. In some embodiments, the genome comprises a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to NM_012092.4 positions 53-652 or 131-520.

The genome of the genetically modified animal can include replacing a sequence encoding a region of an endogenous ICOS locus with a sequence encoding a corresponding region of human ICOS. In some embodiments, the replaced sequence is any sequence of the endogenous ICOS locus, such as exon 1, exon 2, exon 3, exon 4, exon 5, 5'UTR, 3'UTR, intron 1, intron 2, intron 3, intron 4, or any combination thereof. In some embodiments, the replaced sequence is within the regulatory region of the endogenous ICOS gene. In some embodiments, the replaced sequence is a portion of exon 1, exons 2-4, and a portion of exon 5 of the endogenous mouse ICOS locus. In some embodiments, the replaced sequence is a portion of exon 2 and a portion of exon 3 of the endogenous mouse ICOS locus. In some embodiments, the replaced sequence is a portion of exon 1 of the endogenous mouse ICOS locus.

The genetically modified animal may have one or more cells expressing human or chimeric ICOS (e.g., humanized ICOS) having, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the signal peptide comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the signal peptide of human ICOS. In some embodiments, the signal peptide of humanized ICOS has a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids (e.g., continuous or non-continuous) that is consistent with the human ICOS signal peptide. In some embodiments, the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the extracellular region of human ICOS. In some embodiments, the extracellular region of humanized ICOS has a sequence of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 113, 114, 115, 116 or 120 amino acids (continuous or non-continuous) that are identical to the extracellular region of human ICOS. The amino acids are identical to at least 1, 2, 3, 4, 5 or 6 amino acids at the N-terminus in the extracellular region of endogenous ICOS (e.g., mouse ICOS). In some embodiments, the extracellular region described herein includes a signal peptide. In some embodiments, the extracellular region described herein does not include a signal peptide. Because in many cases, the sequences of human ICOS and non-human ICOS (e.g., mouse ICOS) are different, antibodies that bind to human ICOS do not necessarily have the same affinity as non-human ICOS or have the same effect on non-human ICOS. Therefore, transgenic animals with human or humanized extracellular regions and/or transmembrane regions can be used to better evaluate the effects of anti-human ICOS antibodies in animal models.

In some embodiments, the transmembrane region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the transmembrane region of human ICOS. In some embodiments, the transmembrane region of humanized ICOS has a sequence of at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids (contiguous or non-contiguous) that are identical to the transmembrane region of human ICOS. In some embodiments, the transmembrane region of humanized ICOS has a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, In some embodiments, the cytoplasmic region of humanized ICOS has at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids (continuous or non-continuous) that are identical to the endogenous ICOS transmembrane region. In some embodiments, the cytoplasmic region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the cytoplasmic region of human ICOS. In some embodiments, the cytoplasmic region of humanized ICOS has a sequence of at least 5, 10, 11, 12, 13, 15, 20, 25, 30, 35, 36, 37 or 38 amino acids (continuous or non-continuous) that are identical to the cytoplasmic region of human ICOS. In some embodiments, the cytoplasmic region of humanized ICOS has at least 5, 10, 11, 12, 13, 15, 20, 25, 30, 31, 32, 33, 34, or 35 amino acids (contiguous or non-contiguous) identical to the cytoplasmic region of endogenous ICOS (e.g., mouse ICOS).

In some embodiments, the entire extracellular region, the entire transmembrane region, and the entire cytoplasmic region of the humanized ICOS described herein are derived from human ICOS sequences. In some embodiments, part of the extracellular region, part of the transmembrane region, and the entire cytoplasmic region of the humanized ICOS described herein are derived from endogenous ICOS sequences.

In some embodiments, the genome of the animal comprises: a portion of exon 1, exon 2, exon 3, exon 4, and/or exon 5 of the human ICOS gene; or a nucleotide sequence encoding amino acids 1-199 or 21-199 of SEQ ID NO: 2. In some embodiments, the genome of the animal comprises: a portion of exon 2 and/or a portion of exon 3 of the human ICOS gene; or a nucleotide sequence encoding amino acids 27-156 of SEQ ID NO: 2.

In some embodiments, the genome of the genetically modified animal includes a portion of exon 1, all of exons 2-4, and a portion of exon 5 of the human ICOS gene. In some embodiments, the portion of exon 1 includes at least 5, 10, 20, 30, 40, 45, 50, 55, 58, 60, 61, 62, 63, 64, 65, 70, 75, 80, 90, 100, or 110 bp of nucleotides. In some embodiments, the portion of exon 1 includes a 58 bp continuous nucleotide sequence. In some embodiments, the portion of exon 1 includes at least 20 bp of nucleotide sequence. In some embodiments, the portion of exon 5 includes at least 5, 10, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1500, 1900, or 1992 bp of nucleotides. In some embodiments, the portion of exon 5 includes a 14 bp continuous nucleotide sequence. In some embodiments, the portion of exon 5 includes at least 5 bp of nucleotides.

In some embodiments, the genetically modified non-human animal genome comprises a portion of human ICOS gene exon 2 and a portion of exon 3. In some embodiments, the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 300, 316 or 336bp continuous nucleotide sequence. In some embodiments, the portion of exon 2 comprises a continuous nucleotide sequence of 316bp. In some embodiments, the portion of exon 2 comprises a nucleotide sequence of at least 150bp. In some embodiments, the portion of exon 3 comprises at least 5, 10, 14, 15, 20, 30, 40, 50, 60, 70, 74, 75, 80, 90, 100, 105 or 107bp continuous nucleotide sequence. In some implementations, the portion of exon 3 comprises a 74bp continuous nucleotide sequence. In some embodiments, the portion of exon 3 comprises at least 30bp of nucleotides.

In some embodiments, the genome of the genetically modified animal includes a portion of exon 1 and a portion of exon 5 of an endogenous ICOS gene (e.g., mouse ICOS). In some embodiments, the portion of exon 1 includes at least 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, 101, 102, or 103 nucleotides. In some embodiments, the portion of exon 1 includes 45 nucleotides. In some embodiments, the portion of exon 1 includes at least 20 bp of nucleotides. In some embodiments, the portion of exon 5 includes at least 10, 20, 30, 40, 45, 50, 51, 52, 53, 54, 55, 60, 70, 80, 500, 550, 555, 558, 600, 800, 1000, 1500, 2000, 2500, 2600, 2606, or 2620 nucleotides. In some embodiments, the portion of exon 5 includes 2606 nucleotides. In some embodiments, the portion of exon 5 includes at least 1000 bp of nucleotides.

In some embodiments, the genome of the genetically modified animal comprises exon 1, a portion of exon 2, a portion of exon 3, and exons 4-5 of an endogenous ICOS gene (e.g., mouse ICOS). In some embodiments, the portion of exon 2 comprises at least 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 335, or 339 nucleotides. In some embodiments, the portion of exon 2 comprises 20 nucleotides. In some embodiments, the portion of exon 2 comprises at least 10 bp of nucleotides. In some embodiments, the portion of exon 3 comprises at least 10, 20, 30, 33, 40, 45, 50, 55, 60, 70, 80, 90, 105, 106, or 107 nucleotides. In some embodiments, the portion of exon 3 comprises 33 nucleotides. In some embodiments, the portion of exon 3 comprises at least 15 bp of nucleotides.

In some embodiments, a non-human animal has a nucleotide sequence encoding a chimeric human/non-human ICOS polypeptide at an endogenous ICOS locus that expresses functional ICOS on the surface of cells of the animal. In some embodiments, the human portion of the chimeric human/non-human ICOS polypeptide may comprise an amino acid sequence encoded by a portion of exon 1, exons 2-4, and/or a portion of exon 5 of a human ICOS gene. In some embodiments, the human portion of the chimeric human/non-human ICOS polypeptide may comprise an amino acid sequence encoded by a portion of exon 2 and/or a portion of exon 3 of a human ICOS gene. In some embodiments, the human portion of the chimeric human/non-human ICOS polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:2.

In addition, the modified gene in the genome of the modified animal is homozygous or heterozygous for the endogenous replaced locus. In a specific embodiment, the modified ICOS gene in the genome is homozygous or heterozygous for the endogenous replaced locus.

In some embodiments, the humanized ICOS locus comprises a human 5'UTR. In some embodiments, the humanized ICOS locus comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'UTR. Where appropriate, it is reasonable to assume that based on the similarity of the 5' flanking sequences of the mouse and human ICOS genes, they appear to be similarly regulated. As indicated in the application, humanized ICOS mice comprising insertions or substitutions in the endogenous mouse ICOS locus, which retain mouse regulatory elements but comprise humanization of the ICOS coding sequence, do not exhibit disease Both humanized ICOS heterozygous and homozygous genetically modified mice were normal.

In another aspect, the present invention also provides a genetically modified non-human animal, whose genome comprises a disruption of the animal's endogenous ICOS gene, wherein the disruption of the endogenous ICOS gene comprises a deletion of exon 1, exon 2, exon 3, exon 4, and/or exon 5, or a partial deletion thereof.

In some embodiments, the disruption of the endogenous ICOS gene further comprises the deletion of one or more introns selected from intron 1, intron 2, intron 3, and intron 4.

In some embodiments, the deletion may include deleting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 215, 250, 300, 350, 390, 400, 450, 500, 550, 600, 650, 700, 750 , 800, 850, 900, 910, 920, 930, 940, 950, 960, 962, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1750, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 10000, 15000, 19756, 20000, 30000 or 39573 bp or more nucleotides.

In some embodiments, the disruption of the endogenous ICOS gene comprises a deletion of at least 20, 50, 55, 58, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 393, 400, 500, 600, 603, 700, 800, 900, 1000, 1500, 2000, 3000, 3200, or 3272 bp nucleotides of exon 1, exon 2, exon 3, exon 4, and/or exon 5.

Animals with humanized ICOSL locus

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

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

In some embodiments, the humanized ICOSL protein comprises a polypeptide sequence of 5-302 amino acids (continuous or non-continuous) identical to the human ICOSL protein. In some embodiments, the length of the polypeptide sequence is 5-302, 10-213, or 10-302 amino acids. In some embodiments, the humanized ICOSL gene comprises a nucleotide sequence of 20-23963 bp (continuous or non-continuous) identical to the human ICOSL gene. In some embodiments, the nucleotide sequence is 20-3455 bp.

In some embodiments, the ICOSL extracellular region is human or humanized. In some embodiments, the ICOSL signal peptide is human or humanized. In some embodiments, the ICOSL cytoplasmic region is human or humanized. In some embodiments, the ICOSL transmembrane region is human or humanized. In some embodiments, the ICOSL signal peptide, transmembrane region, and cytoplasmic region are endogenous.

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

The genetically modified animal may express human ICOSL and/or chimeric (e.g., humanized) ICOSL at an endogenous mouse locus, wherein the endogenous mouse ICOSL gene has been replaced or inserted with a gene for human ICOSL and/or a nucleotide sequence encoding a region of a human ICOSL sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 100% identical to a human ICOSL sequence. In various embodiments, the endogenous non-human animal ICOSL locus is modified with a nucleic acid sequence that comprises all or part of a human nucleic acid sequence encoding a mature ICOSL protein.

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

The genome of the animal comprises a nucleotide sequence encoding an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human ICOSL (NP_056074.1; SEQ ID NO: 64). In some embodiments, the genome comprises a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 69 or 70. In some embodiments, the genome comprises a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to positions 220-858 of NM_015259.6.

The genome of the genetically modified animal may include replacing a sequence encoding a region of the endogenous ICOSL locus with a sequence encoding a corresponding region of human ICOSL. In some embodiments, the replaced sequence is an endogenous ICOSL gene. In some embodiments, the replaced sequence is a portion of exon 3, exon 4, exon 5, exon 6, exon 7, 5'UTR, 3'UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, or any combination thereof. In some embodiments, the replaced sequence is within the regulatory region of the endogenous ICOSL gene. In some embodiments, the replaced sequence is a portion of exon 3, exon 4, and a portion of exon 5 of the endogenous mouse ICOSL locus.

The genetically modified animal may have one or more cells expressing human or chimeric ICOSL (e.g., humanized ICOSL) having, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the signal peptide comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the signal peptide of human ICOSL (e.g., amino acids 1-18 of SEQ ID NO: 64). In some embodiments, the signal peptide of humanized ICOSL has a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 46 amino acids (e.g., continuous or non-continuous) that is identical to the endogenous ICOSL signal peptide. In some embodiments, the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the extracellular region of human ICOSL. In some embodiments, the extracellular region of humanized ICOSL has a sequence of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 213, 215, 220, 230, 235 or 238 amino acids (continuous or non-continuous) identical to the extracellular region of human ICOSL. In some embodiments, the extracellular region of humanized ICOSL has a sequence of at least 10, 15, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 213, 215, 220, 230 or 231 amino acids (continuous or non-continuous) identical to the extracellular region of endogenous ICOSL. In some embodiments, the extracellular region described herein includes a signal peptide. In some embodiments, the extracellular region described herein does not include a signal peptide. Because in many cases, human ICOSL and non-human ICOSL (e.g., mouse ICOSL) sequences are different, antibodies that bind to human ICOSL do not necessarily have the same affinity or effect on non-human ICOSL. Therefore, transgenic animals with human or humanized extracellular regions can be used to better evaluate the effects of anti-human ICOSL antibodies in animal models.

In some embodiments, the transmembrane region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the transmembrane region of human ICOSL (e.g., amino acids 257-277 of SEQ ID NO: 64). In some embodiments, the transmembrane region of humanized ICOSL has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids (contiguous or non-contiguous) that are identical to the transmembrane region of endogenous ICOSL. In some embodiments, the cytoplasmic region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the cytoplasmic region of human ICOSL (e.g., amino acids 278-302 of SEQ ID NO: 64). In some embodiments, the cytoplasmic region of humanized ICOSL has at least 5, 10, 11, 12, 13, 15, 20, 21, 22, 23, or 24 amino acids (contiguous or non-contiguous) identical to the cytoplasmic region of endogenous ICOSL (e.g., mouse ICOSL).

In some embodiments, the entire signal peptide, the entire transmembrane region, and the entire cytoplasmic region of the humanized ICOSL described herein are derived from the endogenous ICOSL sequence.

In some embodiments, the genome of the animal includes: a portion of exon 3, exon 4, and/or a portion of exon 5 of the human ICOSL gene; or a nucleotide sequence encoding amino acids 32-244 of SEQ ID NO: 64.

In some embodiments, the genome of the genetically modified animal comprises a portion of exon 3, all of exon 4, and a portion of exon 5 of the human ICOSL gene. In some embodiments, the portion of exon 3 comprises at least 5, 10, 20, 30, 40, 45, 50, 55, 58, 60, 65, 70, 80, 90, 100, 200, 300, 310, 313, 350, or 351 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 3 comprises 313 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 3 comprises at least 150 bp of nucleotide sequence. In some embodiments, the portion of exon 5 comprises at least 5, 10, 14, 15, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 165 bp of continuous nucleotide sequence. In some implementations, the portion of exon 5 comprises 35 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 5 comprises at least 10 bp of nucleotides.

In some embodiments, the genome of the genetically modified animal comprises exons 1-2, a portion of exon 3, a portion of exon 5, and exons 6-7 of an endogenous ICOSL gene (e.g., mouse ICOSL). In some embodiments, the portion of exon 3 comprises at least 1, 2, 3, 4, 5, 6, 10, 20, 30, 35, 38, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 350, or 354 nucleotides. In some embodiments, the portion of exon 3 comprises 38 nucleotides. In some embodiments, the portion of exon 3 comprises at least 15 bp of nucleotides. In some embodiments, the portion of exon 5 comprises at least 10, 20, 30, 40, 45, 50, 51, 52, 53, 54, 55, 60, 70, 80, 90, 100, 110, 120, 124, 125, 130, 140, or 150 nucleotides. In some embodiments, the portion of exon 5 includes 124 nucleotides. In some embodiments, the portion of exon 5 includes at least 50 bp of nucleotides.

In some embodiments, the non-human animal has a nucleotide sequence encoding a chimeric human/non-human ICOSL polypeptide at an endogenous ICOSL locus, and the animal expresses functional ICOSL on the surface of cells. In some embodiments, the human portion of the chimeric human/non-human ICOSL polypeptide may comprise an amino acid sequence encoded by a portion of exon 3, exon 4, and/or a portion of exon 5 of a human ICOSL gene. In some embodiments, the human portion of the chimeric human/non-human ICOSL polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 64.

In addition, the modified gene in the genome of the modified animal is homozygous or heterozygous for the endogenous replaced locus. In a specific embodiment, the modified ICOSL gene in the genome is homozygous or heterozygous for the endogenous replaced locus.

In some embodiments, the humanized ICOSL locus comprises a human 5'UTR. In some embodiments, the humanized ICOSL locus comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'UTR. Where appropriate, it is reasonable to assume that based on the similarity of the 5' flanking sequences of the mouse and human ICOSL genes, they appear to be similarly regulated. As indicated in the application, a humanized ICOSL mouse comprising an insertion or substitution in the endogenous mouse ICOSL locus that retains the mouse regulatory elements but comprises a humanization of the ICOSL coding sequence does not represent Both humanized ICOSL heterozygous and homozygous genetically modified mice were normal.

In another aspect, the present invention also provides a genetically modified non-human animal, whose genome comprises a disruption of the animal's endogenous ICOSL gene, wherein the disruption of the endogenous ICOSL gene comprises a deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or a partial deletion thereof.

In some embodiments, the disruption of the endogenous ICOSL gene further comprises a deletion of one or more introns selected from intron 1, intron 2, intron 3, intron 4, intron 5, and intron 6.

In some embodiments, the deletion may include deleting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 215, 250, 300, 350, 390, 400, 450, 500, 550, 600, 650, 700, 750 , 800, 850, 900, 910, 920, 930, 940, 950, 960, 962, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1750, 1800, 1900, 2000, 3000, 3455, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or 10439 bp of continuous nucleotide sequence or more.

In some embodiments, the disruption of the endogenous ICOSL gene comprises a deletion of at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 636, 650, 700, 800, 900, 1000, 1500, 2000, 3000, 2700, or 2748 bp of nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7.

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

In one aspect, the animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the family Muridae. In one embodiment, the genetically modified animal is from a family selected from the family Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny mice, crested rats), Myrmionidae (climbing mice, rock mice, tailed rats, Madagascar rats, and mice), Spiny Dormouse (e.g., spiny dormouse), and Myrmionidae (e.g., mole rats, bamboo rats, and zokors). In a specific embodiment, the gene The modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny mice, and crested rats. In one embodiment, the genetically modified mouse is from a member of the family Muridae. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

In some embodiments, the animal is a mouse of the C57BL strain selected from the group consisting of C57BL/a, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/min, C57BL6J, C57B1/6ByJ, C57BL/6NJ, C57BL/10, C57BL10SnSn, C57BL/10Cr, and C57BL/Ola. In some embodiments, the mouse is a 129 strain selected from 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the BL/6 strain. In some embodiments, the mouse is a BALB strain, such as a BALB/c strain. In some embodiments, the mouse is a hybrid of the BALB strain and another strain. In some embodiments, the mouse is from a hybrid strain (e.g., 50% BALB/c-50% 12954/Sv; or 50% C57BL/6-50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse with 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, C57BL6, C57L/6J, C57BL/6ByJ, C5C57BL/6NJ. Mice of C57BL/10, C57BL/10ScSn, C57BL (C57BL/10Cr and C57BL/Ola), C58, CBA/Br, CBA/Ca, CBA/J, CBA/st or CBA/H strains.

In some embodiments, the animal is a rat. The rat can be selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a hybrid of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

The animal may have one or more other genetic modifications and/or other modifications that are suitable for the specific purpose of preparing a humanized animal. For example, a suitable mouse for maintaining a xenograft (e.g., a human cancer or tumor) may have one or more modifications that damage, inactivate, or destroy all or part of the immune system of a non-human animal. Damage, inactivation, or destruction of the immune system of a non-human animal may include, for example, chemical means (e.g., administration of toxins), physical means (e.g., irradiation of the animal), and/or genetic modification (e.g., knocking out one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD/SCID mice, IL2Rγ knockout mice, NOD/SCID/γc ^null mice (Ito, M. et al., NOD/SCID/γc ^null mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100(9): 3175-3182, 2002), nude mice, and Rag1 and/or Rag2 knockout mice. These mice can be optionally irradiated or otherwise treated to destroy one or more immune cell types. Therefore, in various embodiments, a genetically modified mouse is provided, which can include humanization of at least a portion of the endogenous non-human OSMR, OSM, IL31RA and/or IL31 loci, and also includes modifications that damage, inactivate or partially destroy the immune system (or one or more cell types of the immune system) of the non-human animal. In some embodiments, the mouse modification type is selected from the group consisting of NOD mice, SCID mice, NOD/SCID mice, IL-2Rγ knockout mice, NOD/SCID/γc ^null mice, nude mice, Rag1 and/or Rag2 knockout mice, NOD Prkdc ^scid IL-2Rγ ^null mice, NOD Rag 1 ^-/- IL2rg ^-/- (NRG) mice, Rag2 ^-/- IL2rg ^-/- (RG) mice, and combinations thereof. These transgenic animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety. In some embodiments, the mouse may include replacing all or part of the mouse endogenous mature ICOS and/or ICOSL coding sequences with all or part of the human mature ICOS and/or ICOSL coding sequences, respectively.

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

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

In some embodiments, the non-human mammal expresses a protein encoded by a humanized ICOS and/or ICOSL gene.

In addition, the present invention also provides a non-human mammal model carrying a tumor, characterized in that the non-human mammal model is obtained by the method described herein. In some embodiments, the non-human mammal is a rodent (eg, mouse).

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

The present invention provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal, a genetically modified non-human animal, the genome of which comprises DNA of human or humanized ICOS and/or ICOSL is provided.

In some embodiments, a non-human mammal comprises a gene construct described herein (e.g., a gene construct as shown in Figures 2, 3, 4, 7, 8, 9, 10, 14, 15, 16, 17, 21, 22, 23, and 24). In some embodiments, a non-human mammal expressing a human or humanized ICOS and/or ICOSL protein is provided. In some embodiments, a tissue that specifically expresses a human or humanized ICOS and/or ICOSL protein is provided.

In some embodiments, the expression of human or humanized ICOS protein in non-human animals is controllable. Inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System/Tet-On System) or the tamoxifen system (Tamoxifen System).

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

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

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

There are many analytical methods that can be used to detect exogenous DNA, including nucleic acid level methods (including the use of reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein level methods (including histochemical analysis, immunoblot analysis and in vitro binding studies). In addition, the expression level of the target gene can be quantified by the ELSA method well known to those skilled in the art. Many standard analytical methods can be used to achieve quantitative detection. For example, transcript levels can be detected using RT-PCR and hybridization methods, including RNase protection assays, Southern Blots, and RNA dot hybridization analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blots can also be used to detect the presence of human or humanized ICOS and/or ICOSL proteins.

Carrier

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

In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000067.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000067.7.

In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 61014085 to 61017117 of NCBI Accession No. NC_000067.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 61036874 to 61040481 of NCBI Accession No. NC_000067.7.

In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be changed is selected from the nucleotide sequence of positions 61029033 to 61032880 of NCBI accession number NC_000067.7; c) the DNA fragment homologous to the 3' end of the switch region to be changed The DNA fragment was selected from the nucleotide sequence at positions 61035125 to 61039380 with NCBI accession number NC_000067.7.

In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 61032025 to 61032880 of NCBI Accession No. NC_000067.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 61033843 to 61035091 of NCBI Accession No. NC_000067.7.

In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 61013786 to 61017117 of NCBI Accession No. NC_000067.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 61017369 to 61021784 of NCBI Accession No. NC_000067.7.

In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 61015718 to 61017117 with NCBI Accession No. NC_000067.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 61017369 to 61018575 with NCBI Accession No. NC_000067.7.

In some embodiments, the length of the genomic nucleotide sequence selected by the targeting vector may exceed about 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb.

In some embodiments, the switch region to be altered is located on exons 1 to 5 of the ICOS gene of a non-human animal.

In some embodiments, the switch region to be altered is located on exons 2 to 3 of the ICOS gene of a non-human animal.

In some embodiments, the switch region to be altered is located in exon 1 and/or intron 1 of the ICOS gene of a non-human animal (eg, positions 46-103 of NM_017480.2).

In some embodiments, the 5’ arm sequence is a nucleotide sequence as shown in SEQ ID NO: 3; the 3’ arm sequence is a nucleotide sequence as shown in SEQ ID NO: 4. In some embodiments, the 5’ arm sequence is a nucleotide sequence as shown in SEQ ID NO: 5; the 3’ arm sequence is a nucleotide sequence as shown in SEQ ID NO: 6. In some embodiments, the 5’ arm sequence is a nucleotide sequence as shown in SEQ ID NO: 7; the 3’ arm sequence is a nucleotide sequence as shown in SEQ ID NO: 8. In some embodiments, the 5’ arm sequence is a nucleotide sequence as shown in SEQ ID NO: 58; the 3’ arm sequence is a nucleotide sequence as shown in SEQ ID NO: 59. In some embodiments, the 5’ arm sequence is a nucleotide sequence as shown in SEQ ID NO: 60; the 3’ arm sequence is a nucleotide sequence as shown in SEQ ID NO: 61.

In some embodiments, the 5' arm is a nucleotide having at least 90% homology with NCBI accession number NC_000067.7, and further preferably, the 5' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 3, 5, 7, 58 or 60. In some embodiments, the 3' arm is a nucleotide having at least 90% homology with NCBI accession number NC_000067.7, and further preferably, the 3' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 4, 6, 8, 59 or 61.

In some embodiments, the targeting vector comprises a human sequence (e.g., positions 203936815 to 203959599 of NC_000002.12, or positions 203955656 to 203956732 of NC_000002.12, or positions 53 to 652 of NM_012092.4). For example, the targeting region in the targeting vector includes: a partial or complete nucleotide sequence of the human ICOS gene, Preferred are exon 1, exon 2, exon 3, exon 4, and/or exon 5 of the human ICOS gene. In some embodiments, the nucleotide sequence of the humanized ICOS gene encodes all or part of the human ICOS protein, and the protein number of NCBI is NP_036224.1 (SEQ ID NO: 2). In some embodiments, the protein encoded by the nucleotide sequence of the humanized ICOS gene is SEQ ID NO: 13.

The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the ICOS gene, and the sgRNA is unique on the target sequence of the gene to be changed, and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'; and in some embodiments, the targeting site of the sgRNA in the mouse ICOS gene is located in exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, upstream of exon 1 of the mouse ICOS gene, or downstream of exon 5.

In some embodiments, the targeting sequence is shown as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 76, 77, 78, 79 and 80. Therefore, the present invention provides sgRNA sequences for constructing genetically modified animal models. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 37 and 39. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 38 and 40. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 41 and 43. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 42 and 44. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 77 and 79. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 78 and 80.

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

In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000076.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000076.7.

In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 77903264 to 77907609 of NCBI Accession No. NC_000076.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 77911065 to 77914575 of NCBI Accession No. NC_000076.7.

In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 77906310 to 77907609 of NCBI Accession No. NC_000076.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 77911065 to 77912364 of NCBI Accession No. NC_000076.7.

In some embodiments, the length of the genomic nucleotide sequence selected by the targeting vector can exceed about 0.8 kb, 1 kb, 1.5kb, 2kb, 2.5kb, 3kb, 3.5kb, 4kb, 4.5kb.

In some embodiments, the switch region to be altered is located on exons 3 to 5 of the ICOSL gene of a non-human animal.

In some embodiments, the 5' arm sequence is a nucleotide sequence as shown in SEQ ID NO: 65; the 3' arm sequence is a nucleotide sequence as shown in SEQ ID NO: 66. In some embodiments, the 5' arm sequence is a nucleotide sequence as shown in SEQ ID NO: 67; the 3' arm sequence is a nucleotide sequence as shown in SEQ ID NO: 68.

In some embodiments, the 5' arm is a nucleotide having at least 90% homology with NCBI accession number NC_000076.7, and further preferably, the 5' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 65 or 67. In some embodiments, the 3' arm is a nucleotide having at least 90% homology with NCBI accession number NC_000076.7, and further preferably, the 3' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 66 or 68.

In some embodiments, the targeting vector comprises a human sequence (e.g., positions 44231410 to 44237179 of NC_000021.9). For example, the targeting region in the targeting vector includes: a portion or all of the nucleotide sequence of the human ICOSL gene, preferably exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of the human ICOSL gene. In some embodiments, the nucleotide sequence of the humanized ICOSL gene encodes all or part of the human ICOSL protein, and the protein number of NCBI is NP_056074.1 (SEQ ID NO: 64). In some embodiments, the protein encoded by the nucleotide sequence of the humanized ICOSL gene is SEQ ID NO: 71.

The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the ICOSL gene, and the sgRNA is unique on the target sequence of the gene to be changed, and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'; and in some embodiments, the targeting site of the sgRNA in the mouse ICOSL gene is located in exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, upstream of exon 1 of the mouse ICOSL gene, or downstream of exon 7.

In some embodiments, the targeting sequence is shown as SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101 and 102. Therefore, the present invention provides sgRNA sequences for constructing genetically modified animal models. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 95 and 97. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 96 and 98. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 99 and 101. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 100 and 102.

In some embodiments, the targeting vector further comprises one or more marker genes. For example, a positive screening marker gene or a negative screening marker gene. In some embodiments, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo. In some embodiments, the coding gene for the negative screening marker is a coding gene for the diphtheria toxin A subunit. (DTA).

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

The present invention also relates to a cell comprising a targeting vector as described above.

In addition, the present invention also provides a non-human mammalian cell having any of the above-mentioned targeting vectors and one or more in vitro transcripts of the constructs described herein. In some embodiments, the cell contains Cas9 mRNA or its in vitro transcript.

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

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

Methods for constructing genetically modified non-human animals

Genetically modified non-human animals can be prepared by several techniques known in the art, 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. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR/Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR-Cas9 genome editing is used to produce genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in Yin et al., "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

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

Figures 3, 8, 10, 15 and 17 show the humanization targeting strategy of the mouse ICOS locus. The targeting vector comprises a vector consisting of a 5' homology arm, a human or humanized ICOS gene fragment and a 3' homology arm. The process involves replacing the endogenous corresponding ICOS sequence with a human or humanized ICOS sequence using homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs or CRISPR) can result in DNA double-strand breaks, and homologous recombination is used to replace the mouse endogenous ICOS sequence with a human or humanized ICOS sequence.

In some embodiments, the non-human animal is constructed by introducing any of the following nucleotide sequences into the ICOS locus of a non-human animal:

A) a portion of the human ICOS gene, preferably comprising all or part of exons 1 to 5 of the human ICOS gene, more preferably comprising one, two or more exons of exons 1 to 5 of the human ICOS gene, and even more preferably comprising part of exon 1, all of exons 2 to 4 and part of exon 5 of the human ICOS gene, wherein the portion of exon 1 comprises at least 20 to at least 110 bp (e.g., 20, 30, 40, 45, 50, 55, 58, 60, 61, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1500, 1900 or 1992 bp). The portion of the human ICOS gene preferably comprises all or part of exons 1 to 5 of the human ICOS gene, and further preferably comprises a portion of exon 2 and a portion of exon 3 of the human ICOS gene, wherein the portion of exon 2 comprises a nucleotide sequence of at least 20 to at least 316 bp (e.g., 5, 10, 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 300, 316 or 336 bp), the portion of exon 3 comprises a nucleotide sequence of at least 10 to at least 107 bp (e.g., 5, 10, 14, 15, 20, 30, 40, 50, 60, 70, 74, 75, 80, 90, 100, 105 or 107 bp), and further preferably comprising the nucleotide sequence shown in SEQ ID NO: 115, 10 or 62; or, comprising at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to the nucleotide sequence shown in SEQ ID NO: 115, 10 or 62; or, comprising no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 115, 10 or 62; or, comprising the nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO: 115, 10 or 62, including substitution, deletion and/or insertion of one or more nucleotides;

B) all or part of a nucleotide sequence encoding a human ICOS protein, preferably comprising all or part of a nucleotide sequence encoding a signal peptide, an extracellular region, a cytoplasmic region and/or a transmembrane region of a human ICOS protein, further preferably comprising all or part of a nucleotide sequence encoding an extracellular region and/or a transmembrane region of a human ICOS protein, more preferably comprising a nucleotide sequence encoding at least 50 consecutive amino acids in the extracellular region and/or a nucleotide sequence encoding at least 10 consecutive amino acids in the transmembrane region of a human ICOS protein, further preferably comprising a nucleotide sequence encoding the amino acids shown at positions 1-199 or 27-156 of SEQ ID NO:2; or, comprising a nucleotide sequence encoding amino acids having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to the amino acid sequence shown at positions 1-199 or 27-156 of SEQ ID NO:2; or, comprising a nucleotide sequence encoding amino acids having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to the amino acid sequence shown at positions 1-199 or 27-156 of SEQ ID NO:2; NO:2, positions 1-199 or 27-156, differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises a nucleotide sequence encoding the same amino acid sequence as SEQ ID NO:2, positions 1-199 or 27-156, including Nucleotide sequences that substitute, delete and/or insert one or more amino acid residues;

C) a nucleotide sequence encoding a human or humanized ICOS protein; or,

D) Nucleotide sequence of human or humanized ICOS gene.

Preferably, the non-human animal is constructed using the above-mentioned targeting vector.

Preferably, the non-human animal further comprises other gene modifications, more preferably, the other genes are selected from at least one of ICOSL, PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28, and CTLA4.

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

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

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

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

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

Therefore, in some embodiments, the method for preparing a genetically modified humanized animal includes replacing the nucleic acid sequence encoding the endogenous ICOS region with the nucleotide sequence encoding the corresponding region of human ICOS at the endogenous ICOS locus (or site). The replaced sequence may include regions (e.g., part or all of regions) of exon 1, exon 2, exon 3, exon 4, and/or exon 5 of the human ICOS gene. In some embodiments, the sequence includes a portion of exon 1, exons 2-4, and exon 5 of the human ICOS gene (e.g., the nucleotide sequence of positions 53-652 of NM_012092.4). In some embodiments, the sequence includes a portion of exon 1 of the endogenous ICOS gene and a portion of exon 5 (e.g., the nucleotide sequence of positions 1-45 and 649-3254 of NM_017480.2). In some embodiments, the sequence includes a portion of exon 2 of the human ICOS gene and a portion of exon 3 (e.g., the nucleotide sequence of positions 131-520 of NM_012092.4). In some embodiments, the sequence includes a portion of exon 1 and a portion of exon 5 of an endogenous ICOS gene (eg, nucleotide sequences at positions 1-123 and 517-3254 of NM_017480.2).

In some embodiments, the method may include inserting a nucleic acid sequence encoding a human ICOS region into an endogenous ICOS locus (or site). The inserted sequence may include regions (e.g., part or all of) exon 1, exon 2, exon 3, exon 4, and/or exon 5 of the human ICOS gene. In some embodiments, the sequence comprises In some embodiments, the sequence comprises a portion of exon 1, exons 2-4, and a portion of exon 5 of the human ICOS gene (e.g., the nucleotide sequence of positions 53-652 of NM_012092.4). In some embodiments, the sequence does not contain introns. In some embodiments, the sequence comprises the entirety of the signal peptide, extracellular region, transmembrane region, and cytoplasmic region of human ICOS (e.g., amino acids 1-199 of SEQ ID NO: 2). In some embodiments, the endogenous ICOS locus (or site) is exon 1 and/or intron 1 of mouse ICOS. In some embodiments, the sequence is inserted after the 5'UTR of the mouse ICOS gene, and 1-251 bp of nucleotides are deleted (e.g., inserted after the 5'UTR of NM_017480.2, and nucleotides 46-103 of exon 1 and 193 bp of nucleotides downstream of exon 1 are deleted).

In some embodiments, methods of modifying the ICOS locus of a mouse to express a chimeric human/mouse ICOS polypeptide may comprise replacing a nucleotide sequence encoding mouse ICOS at an endogenous mouse ICOS locus with a nucleotide sequence encoding human ICOS, thereby generating a chimeric human/mouse ICOS sequence. In some embodiments, the methods may comprise inserting a nucleotide sequence encoding a chimeric human/mouse ICOS at an endogenous mouse ICOS locus, thereby generating a chimeric human/mouse ICOS sequence.

The present invention also provides a method for establishing an ICOS gene humanized animal model, comprising the following steps:

(a) providing a cell (e.g., a fertilized egg cell) according to the method described herein;

(b) culturing the cells in a liquid culture medium;

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

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

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

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

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

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

In some embodiments, the method for preparing a genetically modified animal comprises modifying the coding framework of the ICOS gene of a non-human animal, for example, by replacing a nucleic acid sequence (e.g., a DNA or cDNA sequence) encoding an endogenous ICOS region with a nucleotide sequence encoding a corresponding region of human ICOS under the control of an endogenous regulatory element of the ICOS gene of the non-human animal. One or more functional region sequences of the ICOS gene of a non-human animal can be knocked out or inserted into a sequence, so that the endogenous ICOS protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the modified ICOS gene of a non-human animal can be all or part of the nucleotide sequence of exon 1 to exon 5 of the ICOS gene of a non-human animal.

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

In some embodiments, a method for preparing a transgenic animal comprises:

(1) providing a plasmid comprising a human ICOS gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target endogenous ICOS;

(2) providing one or more guide RNAs (sgRNAs) targeting the endogenous ICOS gene;

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

(4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express the humanized ICOS protein;

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

In some embodiments, the fertilized egg is modified by CRISPR with sgRNA targeting a 5'-terminal targeting site and a 3'-terminal target site.

In some embodiments, the sequence encoding the humanized ICOS protein is operably linked to endogenous regulatory elements at the endogenous ICOS locus.

In some embodiments, the genetically modified animal does not express endogenous ICOS protein.

In some embodiments, a method for preparing a transgenic animal comprises:

(1) providing a plasmid comprising a human or chimeric ICOS gene fragment, the plasmid being flanked by 5' homology arms and 3' homology arms, wherein the 5' and 3' homology arms target endogenous ICOS;

(2) providing one or more guide RNAs (sgRNAs) targeting the endogenous ICOS gene;

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

In some embodiments, the nucleotide sequence encoding the endogenous ICOSL region in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding the corresponding region of human ICOSL. The expression level of the source ICOSL protein is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

Figures 22 and 24 show the humanization targeting strategy of the mouse ICOSL locus. The targeting vector comprises a vector consisting of a 5' homology arm, a human or humanized ICOSL gene fragment and a 3' homology arm. The process involves replacing the endogenous corresponding ICOSL sequence with the human or humanized ICOSL sequence using homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double-strand breaks, and homologous recombination is used to replace the mouse endogenous ICOSL sequence with the human or humanized ICOSL sequence.

In some embodiments, the non-human animal is constructed by introducing any of the following nucleotide sequences into the ICOSL locus of the non-human animal:

A) a portion of the human ICOSL gene, preferably comprising all or part of exons 1 to 7 of the human ICOSL gene, further preferably comprising one, two or more exons of exons 1 to 7 of the human ICOSL gene, and further preferably comprising part of exon 3, all of exon 4 and part of exon 5 of the human ICOSL gene, wherein the portion of exon 3 comprises a nucleotide sequence of at least 20 to at least 351 bp (e.g., 20, 30, 40, 45, 50, 55, 58, 60, 65, 70, 80, 90, 100, 200, 300, 310, 313, 350 or 351 bp), and the portion of exon 5 comprises at least 10 to at least 165 bp (e.g., 10, 14, 15, 20, 30, 35, 40, 50, 60, 70 , 80, 90, 100, 110, 120, 130, 140, 150, 160 or 165 bp), further preferably comprising the nucleotide sequence shown in SEQ ID NO: 69; or, comprising at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to the nucleotide sequence shown in SEQ ID NO: 69; or, comprising no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 69; or, comprising the nucleotide sequence shown in SEQ ID NO: 69, including substitution, deletion and/or insertion of one or more nucleotides;

B) all or part of a nucleotide sequence encoding a human ICOSL protein, preferably comprising all or part of a nucleotide sequence encoding a signal peptide, an extracellular region, a cytoplasmic region and/or a transmembrane region of a human ICOSL protein, further preferably comprising all or part of a nucleotide sequence encoding the extracellular region of a human ICOSL protein, more preferably comprising a nucleotide sequence encoding at least 100 consecutive amino acids in the extracellular region of a human ICOSL protein, further preferably comprising a nucleotide sequence encoding the amino acids shown at positions 1-302 or 32-244 of SEQ ID NO:64; or, comprising a nucleotide sequence encoding amino acids having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to the amino acid sequence shown at positions 1-302 or 32-244 of SEQ ID NO:64; or, comprising a nucleotide sequence encoding an amino acid having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to the amino acid sequence shown at positions 1-302 or 32-244 of SEQ ID NO:64; The amino acid sequence of NO:64 at positions 1-302 or 32-244 differs by no more than 10, 9, 8, or a nucleotide sequence encoding 7, 6, 5, 4, 3, 2 or no more than 1 amino acid residue; or, a nucleotide sequence encoding the same as shown in positions 1-302 or 32-244 of SEQ ID NO: 64, including substitution, deletion and/or insertion of one or more amino acid residues;

C) a nucleotide sequence encoding a human or humanized ICOSL protein; or,

D) Nucleotide sequence of human or humanized ICOSL gene.

Preferably, the non-human animal is constructed using the above-mentioned targeting vector.

Preferably, the non-human animal further comprises other gene modifications, more preferably, the other genes are selected from at least one of ICOS, PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28, and CTLA4.

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

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

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

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

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

Therefore, in some embodiments, the method for preparing a genetically modified humanized animal comprises replacing a nucleic acid sequence encoding an endogenous ICOSL region with a nucleotide sequence encoding a corresponding region of human ICOSL at an endogenous ICOSL locus (or site). The replaced sequence may include regions (e.g., part or all of) exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of a human ICOSL gene. In some embodiments, the sequence includes a portion of exon 3, exon 4, and a portion of exon 5 of a human ICOSL gene (e.g., the nucleotide sequence of positions 220-858 of NM_015259.6).

In some embodiments, the method of modifying the ICOSL locus of a mouse to express a chimeric human/mouse ICOSL polypeptide may comprise replacing a nucleotide sequence encoding mouse ICOSL at an endogenous mouse ICOSL locus with a nucleotide sequence encoding human ICOSL, thereby generating a chimeric human/mouse ICOSL encoding sequence. In some embodiments, the method may comprise inserting a nucleotide sequence encoding a chimeric human/mouse ICOSL at an endogenous mouse ICOSL locus, thereby generating a chimeric human/mouse ICOSL encoding sequence.

The present invention also provides a method for establishing an ICOSL gene humanized animal model, comprising the following steps:

(a) providing a cell (e.g., a fertilized egg cell) according to the method described herein;

(b) culturing the cells in a liquid culture medium;

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

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

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

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

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

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

In some embodiments, the method for preparing a genetically modified animal comprises modifying the coding frame of the ICOSL gene of a non-human animal, for example, by replacing a nucleic acid sequence (e.g., a DNA or cDNA sequence) encoding an endogenous ICOSL region with a nucleotide sequence encoding a corresponding region of human ICOSL under the control of an endogenous regulatory element of the ICOSL gene of the non-human animal. For example, one or more functional region sequences of the ICOSL gene of the non-human animal can be knocked out or a sequence can be inserted, so that the endogenous ICOSL protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the ICOSL gene of the modified non-human animal can be all or part of the nucleotide sequence of exon 1 to exon 7 of the ICOSL gene of the non-human animal.

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

In some embodiments, a method for preparing a transgenic animal comprises:

(1) providing a plasmid comprising a human ICOSL gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target endogenous ICOSL;

(2) providing one or more guide RNAs (sgRNAs) targeting the endogenous ICOSL gene;

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

(4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express humanized ICOSL protein;

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

In some embodiments, the fertilized egg is modified by CRISPR with sgRNA targeting a 5'-terminal targeting site and a 3'-terminal target site.

In some embodiments, the sequence encoding the humanized ICOSL protein is operably linked to endogenous regulatory elements at the endogenous ICOSL locus.

In some embodiments, the genetically modified animal does not express endogenous ICOSL protein.

In some embodiments, a method for preparing a transgenic animal comprises:

(1) providing a plasmid comprising a human or chimeric ICOSL gene fragment, the plasmid being flanked by 5' homology arms and 3' homology arms, wherein the 5' and 3' homology arms target endogenous ICOSL;

(2) providing one or more guide RNAs (sgRNAs) targeting the endogenous ICOSL gene;

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

Use of genetically modified non-human animals

Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence or inserting a homologous or orthologous human gene or human sequence into a non-human animal at an endogenous non-human animal locus and under the control of an endogenous promoter and/or regulatory element can produce a non-human animal with qualities and characteristics that may be significantly different from a typical knockout plus transgenic animal. In a typical knockout plus transgenic animal, the endogenous locus is removed or destroyed, and a full human transgene is inserted into the genome of the animal and may be randomly integrated into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of a human gene and/or protein assay and/or functional assay. In a human transgene, the upstream and/or downstream of the human sequence provides suitable support for expression and/or regulation of the transgene.

In some cases, transgenes with human regulatory elements are expressed in a non-physiological or otherwise unsatisfactory manner, and may actually be harmful to the animal. The present invention demonstrates that replacement or insertion of human sequences at endogenous loci under the control of endogenous regulatory elements to produce humanized animals provides physiologically appropriate expression patterns and levels that are meaningful and appropriate in the context of the physiology of the humanized animal with respect to the physiology of the replaced gene.

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

The present invention also provides a use of the ICOS and/or ICOSL gene-modified non-human animal, or a non-human animal obtained by any of the above construction methods.

In some embodiments, the application comprises:

A) Application in the development of products involving immunological processes related to ICOS and/or ICOSL in human cells;

B) Application as a model system for pharmacological, immunological, microbiological and medical research related to ICOS and/or ICOSL;

C) applications involving the production and use of animal experimental disease models for etiological studies related to ICOS and/or ICOSL and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of modulators of the human ICOS and/or ICOSL signaling pathway; or

E) To study the gene functions of ICOS and/or ICOSL, to study the drugs and efficacy targeting human ICOS and/or ICOSL target sites, and to study the application of drugs in inflammation and immune-related diseases related to ICOS and/or ICOSL.

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

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

In some embodiments, a tumor comprises one or more cancer cells injected into an animal (e.g., a human or mouse cancer cell line). In some embodiments, the antibody activates the ICOS signaling pathway. In some embodiments, the antibody does not activate the ICOS signaling pathway.

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

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

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

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

In some embodiments, therapeutic agents (e.g., antibodies targeting ICOS and/or ICOSL; or drugs targeting the ICOS signaling pathway) are designed to treat various autoimmune diseases, including rheumatoid arthritis, Crohn's disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease (IBD), ulcerative colitis, or scleroderma. Therefore, the methods described herein can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting ICOS and/or ICOSL; or drugs targeting the ICOS signaling pathway) in suppressing immune responses. In some embodiments, the immune diseases described herein are graft-versus-host disease (GVHD), psoriasis, allergies, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological diseases, etc. In some embodiments, therapeutic agents (e.g., antibodies targeting ICOS and/or ICOSL; or drugs targeting the ICOS signaling pathway) are designed to treat various inflammations, such as viral inflammation. In some embodiments, the inflammation described herein includes acute inflammation and chronic inflammation. Specifically, inflammation includes, but is not limited to, degenerative inflammation, exudative inflammation (e.g., serous inflammation, fibrin inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (e.g., tuberculosis, syphilis, leprosy, or lymphogranuloma). In some embodiments, the inflammation described herein includes infection, and infection refers to local tissue and systemic inflammatory responses caused by bacteria, viruses, fungi, parasites, and/or other pathogens that invade the human body.

The present invention also provides a method for determining the toxicity of a therapeutic agent (e.g., an anti-ICOS and/or ICOSL antibody). The method comprises administering a therapeutic agent to a non-human animal as described above, and assessing the animal's weight change, red blood cell count, hematocrit, and/or hemoglobin. In some embodiments, the antibody can reduce red blood cells (RBC), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the animal's body weight is at least 5%, 10%, 20%, 30%, or 40% less than a control group (e.g., the average body weight of an animal not treated with the antibody).

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

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

The present invention also provides an animal model generated by the method described herein to screen, validate, evaluate or study ICOS and/or or ICOSL gene function, human ICOS and/or ICOSL antibodies, drugs or effectiveness targeting human ICOS and/or ICOSL sites, drugs for immune-related diseases and anti-tumor drugs.

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

Non-human animal models with two or more human or chimeric genes

The present invention also provides a method for generating a transgenic animal model having two or more human or chimeric genes. The animal may comprise a human or chimeric ICOS and/or ICOSL gene and a sequence encoding an additional human or chimeric protein. In some embodiments, the animal comprises a human or humanized ICOS and ICOL gene.

In some embodiments, the additional gene is a non-human animal modified with at least one gene of PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28, CTLA4. In some embodiments, the non-human animal further expresses at least one of human or humanized PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28, CTLA4 proteins.

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

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

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

In some embodiments, the other genetically modified non-human animals include non-human animals humanized with one or a combination of two or more of the genes PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28, and CTLA4, some of which are described, for example, in PCT/CN2017/090320, PCT/CN2017/099574, PCT/CN2021/119112, PCT/CN2017/099575, PCT/CN2023/073036, PCT/CN2022/127313, PCT/CN2022/113594, PCT/CN2022/120819, PCT/CN2018/091846, PCT/CN2017/099577; each of which is incorporated herein by reference in its entirety.

In some embodiments, humanization is performed directly on non-human animals with human or chimeric PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28, CTLA4 gene modifications.

Since these proteins may involve different mechanisms, a combination therapy targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are ongoing and show good results. Multigene modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, for example, anti-ICOS antibodies (optionally, anti-ICOSL antibodies), and additional therapeutic agents for treating cancer or immune diseases (e.g., asthma or atopic dermatitis). The method includes administering an anti-ICOS antibody (optionally, an anti-ICOSL antibody) and an additional therapeutic agent to an animal, wherein the animal has a tumor or an immune disease, and determining the effect of the combined therapy on the immune tumor or immune disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28, CTLA4. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., nivolumab) or an anti-PD-L1 antibody. In some embodiments, the non-human animal described above further comprises a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the tumor described above comprises one or more tumor cells expressing PD-L1 and/or PD-L2.

In some embodiments, the combination therapy is used to treat various cancers described herein, e.g., breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, or cancer. In some embodiments, the combination therapy is designed to treat immune disorders described herein, e.g., psoriasis.

In some embodiments, the methods described herein can be used to evaluate combined treatment with some other methods. Methods for treating cancer that can be used alone or in combination with the methods described herein include, for example, treating a subject with chemotherapy, for example, camphor alkali, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chlorbutiazine, thiophene, nitrosourea, dacrynic acid, daunorubicin, bleomycin, primycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatin, 5-fluorouric acid, vincristine, vinblastine and/or methotrexate. Alternatively, in addition, the method can include performing surgery on the subject to remove at least a portion of the cancer, for example, removing a portion or all of a tumor from a patient.

DETAILED DESCRIPTION

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

Materials and methods

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

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

BamHI, BstEII, AseI, ScaI, XmnI, and NcoI enzymes were purchased from NEB with catalog numbers: R3136S, R3162S, R0526S, R3122S, R0194S, and R3193S, respectively;

Brilliant Violet 510 ^TM anti-mouse CD45 Antibody was purchased from Biolegend, catalog number: 103138;

FITC anti-mouse CD19 Antibody was purchased from Biolegend, catalog number: 115506;

APC Armenian Hamster IgG Isotype Ctrl Antibody was purchased from Biolegend, catalog number: 400912;

Zombie NIR ^TM Fixable Viability Kit was purchased from Biolegend, catalog number: 423106;

Purified anti-mouse CD16/32 Antibody was purchased from Biolegend, catalog number: 101302;

CD278 (ICOS) Monoclonal Antibody (C398.4A), APC, eBioscience ^TM were purchased from eBioscience, catalog number: 17-9949-82;

FITC Rat Anti-Mouse CD3 Antibody was purchased from BD Pharmingen, catalog number: 561798;

Brilliant Violet 650 ^TM anti-mouse CD19 Antibody was purchased from Biolegend, catalog number: 115541;

PE anti-mouse CD275 (B7-H2, B7-RP1, ICOS Ligand) Antibody was purchased from Biolegend, catalog number: 107405;

PE Rat IgG2a, κIsotype Ctrl Antibody was purchased from Biolegend, catalog number: 400508;

R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγfragment specific was purchased from Jacksonimmuno, with the catalog number: 109-115-098;

Human IgG2, Kappa isotype control was purchased from CrownBio, catalog number: C0002;

Alexa 700 anti-mouse CD3 Antibody was purchased from Biolegend, catalog number: 100216;

Brilliant Violet 650 ^TM anti-mouse Ly-6G Antibody was purchased from Biolegend with the catalog number: 127641.

Example 1 ICOS gene humanized mice

Mouse ICOS gene (NCBI Gene ID: 54167, Primary source: MGI: 1858745, UniProt ID: Q9WVS0, located at positions 60999909 to 61039481 on chromosome 1 NC_000067.7, based on transcript NM_017480.2 and its encoded protein NP_059508.2 (SEQ ID NO: 1)) and human ICOS A comparison diagram of the gene (NCBI Gene ID: 29851, Primary source: HGNC:5351, UniProt ID: Q9Y6W8, located at positions 203936763 to 203961577 of chromosome 2 NC_000002.12, based on the transcript NM_012092.4 and its encoded protein NP_036224.1 (SEQ ID NO: 2)) is shown in Figure 1.

In order to achieve the purpose of the present invention, a nucleotide sequence encoding human ICOS protein can be introduced into the mouse endogenous ICOS locus. Specifically, using gene editing technology, under the control of the mouse ICOS gene regulatory element, the mouse exon 1 partial sequence to the exon 5 partial sequence coding region (e.g., start codon ATG to stop codon TAA) of about 22.8 kb was replaced with the mouse exon 1 partial sequence to the exon 5 partial sequence coding region (e.g., start codon ATG to stop codon TAA) of about 19.8 kb, and the schematic diagram of the humanized ICOS locus was obtained as shown in Figure 2, thereby achieving the humanization of the mouse ICOS gene.

According to Figure 2, a targeting strategy as shown in Figure 3 was further designed, which shows that the V1 targeting vector contains homology arm sequences upstream and downstream of the mouse ICOS gene, and an A fragment containing a human ICOS gene fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence of positions 61014085 to 61017117 of NCBI accession number NC_000067.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence of positions 61036874 to 61040481 of NCBI accession number NC_000067.7. The nucleotide sequence of the human ICOS gene fragment (SEQ ID NO: 115) is identical to the nucleotide sequence of positions 203936815 to 203959599 of NCBI accession number NC_000002.12; the connection design of the upstream of the human ICOS fragment sequence and the mouse is: (SEQ ID NO: 14), wherein the last "C" in the sequence " CAGAC " is the last nucleotide of the mouse, the sequence The first "A" in is the first nucleotide of the human sequence. The downstream connection of the human ICOS fragment sequence and the mouse is designed as: (SEQ ID NO: 15), wherein the last "A" in the sequence " TATAAA " is the last nucleotide of the human sequence, and the sequence The first "G" in is the first nucleotide of the mouse sequence.

The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the human ICOS gene is designed as follows: (SEQ ID NO: 16), wherein the last "T" in the sequence " GG TCT " is the last nucleotide of the human ICOS gene, and the sequence The first "A" in is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the human ICOS gene is designed as: (SEQ ID NO: 17), wherein the last "C" in the sequence " GAGCC " is the last nucleotide of the Neo box, and the sequence The first "C" in is the first nucleotide of the human ICOS gene. The mRNA sequence of the modified humanized mouse ICOS is shown in SEQ ID NO: 11, and the expressed protein sequence is shown in SEQ ID NO: 2.

The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified by sequencing is electroporated and transfected into the embryonic stem cells of C57BL/6 mice, and the obtained cells are screened using the positive clone screening marker gene to screen the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to the technology known in the art, and the obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white) can be produced. F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. Positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene (see Figure 4 for the schematic diagram of this process), and then ICOS gene humanized homozygous mice can be obtained by mating with each other.

The PCR method can be used to identify the somatic cell genotype of F1 generation mice (primers are shown in Table 5). The exemplary identification results of F1 generation mice are shown in Figure 9. Five mice numbered F1-01 to F1-05 are positive mice.

Table 5 Primer sequences and recombinant fragment sizes for PCR detection of F1 genotypes

The expression of mRNA in ICOS gene humanized mice can be detected by RT-PCR. Specifically, 1 female C57BL/6 mouse (+/+) aged 6 weeks and 1 ICOS gene humanized heterozygote (H/+) prepared in this embodiment were selected, and thymus tissue was obtained after euthanasia by cervical dislocation. RT-PCR detection was performed using the primer sequences shown in Table 6, and the detection results are shown in Figure 6. It can be seen from the figure that only mouse ICOS mRNA was detected in wild-type C57BL/6 mice, and human ICOS mRNA was not detected; human ICOS mRNA was only detected in ICOS gene humanized heterozygote mice.

Table 6 RT-PCR primer sequences and target fragment sizes

GSK3359609 (VH and VL sequences are shown in SEQ ID NO:56 and SEQ ID NO:57, respectively) was developed by GlaxoSmithKline (GSK) and is an agonist antibody targeting the human ICOS target.

In addition, the expression of human ICOS (hICOS) protein in ICOS humanized mice can be detected by conventional methods such as flow cytometry. Specifically, one 8-week-old male C57BL/6 mouse (+/+) and one 17-week-old male ICOS gene humanized heterozygote (H/+) prepared in this embodiment were selected, and anti-mouse CD3e antibody (7.5ug/200uL) was injected intraperitoneally. The spleen tissue of the mouse was collected after 24 hours of stimulation. In addition, human peripheral blood cells (PBMC) and human and mouse ovarian cells (hCHO and mCHO) were taken, and Brilliant Violet 510 ^™ anti-mouse CD45 Antibody, FITC anti-Mouse CD19 Antibody, APC Armenian Hamster IgG Isotype Ctrl Antibody, Zombie NIR ^™ Fixable Viability Kit, Purified anti-mouse CD16/32Antibody, human-mouse cross antibody CD278 (ICOS) Monoclonal Antibody, anti-human ICOS antibody GSK3359609analog and the like were used for identification and staining, and then flow cytometry was performed. The test results are shown in Table 7.

Table 7 Flow cytometry detection results

As can be seen from Table 7, when the human-mouse cross antibody CD278 (ICOS) Monoclonal Antibody was used, ICOS protein was detected in the spleen cells of wild-type C57BL/6 mice and ICOS humanized hybrid mice, human PBMC cells, and human and mouse CHO cells due to the cross-reaction of the antibody, but when the specific anti-human ICOS antibody GSK3359609analog was used, the expression of human ICOS protein was only detected in the spleen cells of ICOS humanized hybrid mice, human PBMC cells and human CHO cells.

In addition, we used a similar method to detect the expression of ICOS protein in ICOS humanized homozygous mice. Consistent with the above results, the expression of human ICOS protein was only detected in the spleen cells of ICOS humanized homozygous mice.

Flow cytometry was further used to detect the proliferation of CD4 T cells and CD8 T cells and ICOS expression in ICOS humanized mice. Specifically, one 14-week-old wild-type C57BL/6 mouse, one male ICOS humanized heterozygous mouse, and one female ICOS humanized homozygous mouse were selected and euthanized by cervical dislocation. The spleen cells of the mice were obtained and stained with anti-mCD3e (mCD3-ε), or anti-mCD3e and anti-mCD28, or anti-mCD3e and human The cells were stimulated with ICOSL recombinant protein for 72 hours. Anti-mouse ICOS antibody (Anti-mICOS), anti-human ICOS antibody (Anti-hICOS), anti-mouse CD4 antibody, and anti-mouse CD8 antibody were used to detect ICOS expression and CD4/CD8 T cell proliferation by flow cytometry.

The results are shown in Table 8 and Figure 31. Mouse ICOS expression can be detected in wild-type mice and ICOS humanized heterozygous mice, and human ICOS is only detected in ICOS humanized heterozygous mice and ICOS humanized homozygous mice (Table 8). The use of anti-mouse CD3e and human ICOSL (hICOSL) stimulation can promote the proliferation and activation of CD4+T cells and CD8+T cells in ICOS humanized heterozygous mice and ICOS humanized homozygous mice. The proliferation and activation effect is comparable to that of anti-mCD3e (mCD3ε) and anti-mCD28 (Figure 31). This shows that the ICOS-ICOSL signaling pathway in the modified ICOS gene humanized mice is normal.

Table 8 ICOS protein expression flow cytometry detection results

Example 2 ICOS gene humanized mice (II)

In order to achieve the humanization of the mouse ICOS gene, the mouse exon 2 partial sequence to the exon 3 partial sequence (about 1 kb) can also be replaced with the exon 2 partial sequence to the exon 3 partial sequence (about 1.1 kb) containing the human ICOS gene, and the schematic diagram of the humanized ICOS locus is shown in Figure 7. According to Figure 7, the targeting strategy shown in Figure 8 is further designed, which shows the homology arm sequences upstream and downstream of the mouse ICOS gene on the targeting vector, as well as the human ICOS gene fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 5) is the same as the nucleotide sequence of positions 61029033 to 61032880 of NCBI accession number NC_000067.7, and the downstream 3' homology arm sequence (SEQ ID NO: 6) is the same as the nucleotide sequence of positions 61035125 to 61039380 of NCBI accession number NC_000067.7. The nucleotide sequence of the human ICOS fragment (SEQ ID NO: 10) is consistent with the NCBI accession number NC_000002.12, No. 2039 The nucleotide sequences from position 55656 to position 203956732 are identical; the connection design of the upstream of the human ICOS fragment sequence and the mouse is: (SEQ ID NO: 18), wherein the last "C" in the sequence " CGGC C " is the last nucleotide of the mouse sequence, and the sequence The first "A" in is the first nucleotide of the human sequence. The downstream connection of the human ICOS fragment sequence and the mouse is designed as: (SEQ ID NO: 19), wherein the "G" in the sequence " TTTTG " is the last nucleotide of the human sequence, and the sequence The first "G" in is the first nucleotide of the mouse sequence.

The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the mouse gene is designed as follows: (SEQ ID NO: 20), wherein the "G" in the sequence " TTACG " is the last nucleotide of the mouse sequence, and the sequence The "G" in the Neo box is the first nucleotide; the connection between the 3' end of the Neo box and the mouse gene is designed as: (SEQ ID NO: 21), wherein the last "C" in the sequence " GATCC " is the last nucleotide of the Neo box, and the sequence The first "T" in is the first nucleotide of the mouse. The mRNA sequence of the modified humanized ICOS mouse is shown in SEQ ID NO: 12, and the expressed protein sequence is shown in SEQ ID NO: 13.

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

In addition, the CRISPR/Cas9 system can be used for gene editing, and a targeting strategy as shown in FIG10 is further designed, which shows the homology arm sequences upstream and downstream of the mouse ICOS gene and the human ICOS fragment on the targeting vector V3. The schematic diagram of the constructed humanized ICOS locus is shown in FIG2. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 7) is identical to the nucleotide sequence 61032025 to 61032880 of the NCBI accession number NC_000067.7. The nucleotide sequence of the human ICOS fragment (SEQ ID NO: 10) is the same as the nucleotide sequence of the 203955656 to 203956732 positions of the NCBI accession number NC_000002.12. The mRNA sequence of the modified humanized ICOS mouse is shown in SEQ ID NO: 12, and the expressed protein sequence is shown in SEQ ID NO: 13.

Conventional methods can be used to construct the targeting vector, such as enzyme digestion, ligation, direct synthesis, etc. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector that is verified to be correct by sequencing is used for subsequent experiments.

The target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize target sites. The target sequence of an exemplary sgRNA on the ICOS gene is as follows:

sgRNA1 target site (SEQ ID NO: 35): 5’-TCAATGGCTCGGCCGATCATAGG-3’;

sgRNA2 target site (SEQ ID NO: 36): 5’-AGGGTGTGCAGCTTTCGTTGTGG-3’;

The activity of sgRNA was detected by UCA kit. After confirming that it could mediate efficient cutting efficiency, restriction sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 9. After annealing, the annealing products were connected to pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain expression vectors pT7-ICOS-1 and pT7-ICOS-2.

Table 9 Sequence table of sgRNA1 and sgRNA2

The pT7-sgRNA vector is synthesized by a plasmid synthesis company as a fragment DNA containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 45) and connected to the backbone vector (source: Takara, item number 3299) through enzyme digestion (EcoRI and BamHI) in sequence. After sequencing verification by a professional sequencing company, the results showed that the target plasmid was obtained. Take the pronuclear fertilized eggs of mice, such as C57BL/6 mice, and use a microinjector to mix the in vitro transcription products of the pT7-ICOS-1 and pT7-ICOS-2 plasmids (using the Ambion in vitro transcription kit, transcribe according to the instructions), the targeting vector and Cas9 mRNA, and then inject them into the cytoplasm or nucleus of the mouse fertilized egg. According to the "Mouse Embryo Operation Experiment Manual (Third Edition)" (Andras Nagy, Chemical Industry Press, 2006) was used for microinjection of fertilized eggs, the injected fertilized eggs were transferred to culture medium for short-term culture, and then transplanted into the oviduct of the recipient mother mouse for development, and the obtained mice (F0 generation) were hybridized and self-fertilized to expand the population and establish a stable ICOS gene humanized mouse strain. PCR technology can be used to screen F0 generation positive mice, and the primers are shown in Table 10.

Table 10 Primer sequences and recombinant fragment sizes for PCR detection of F0 genotypes

The ICOS gene humanized mice identified as positive in F0 were mated with wild-type mice to obtain F1 mice. The somatic cell genotype of F1 mice was identified by PCR (primers are shown in Table 10), and the identification results of exemplary F1 mice are shown in Figure 11. The clones identified as positive by PCR were then subjected to Southern blot detection (cell DNA was digested with BamHI or BstEII and hybridized with two probes, and the probes and target fragment lengths are shown in Table 11) to confirm whether there was random insertion. The exemplary results are shown in Figure 12. Combined with the PCR and sequencing results, 11 mice numbered F1-18, F1-19, F1-21, F1-30, F1-32, F1-33, F1-34, F1-37, F1-38, F1-39 and F1-44 had no random insertion. This shows that the present method can be used to construct ICOS gene humanized mice that can be stably propagated and have no random insertion.

Table 11 Length of specific probes and target fragments

A Probe(5’)-F：5’-TGTTTTGACAACTTTTCTCACTAAT-3’(SEQ ID NO: 50);

A Probe(5’)-R: 5’-TGCAAAACCAAACCTTAAATCTTAG-3’(SEQ ID NO: 51);

3’Probe-F: 5’-GATTTAGGTGGATTCTGGGGGGAGG-3’ (SEQ ID NO: 52);

3’Probe-R: 5’-CATCCATGCAGTGATTCCATGACAA-3’ (SEQ ID NO: 53);

The expression of mRNA in ICOS gene humanized mice can be detected by RT-PCR. Specifically, 1 female C57BL/6 mouse (+/+) aged 7 weeks and 1 female ICOS gene humanized heterozygote (H/+) aged 8 weeks prepared in this embodiment were selected, and anti-mouse CD3e antibody (7.5ug/200uL) was injected intraperitoneally. After 24 hours of stimulation, thymus tissue was taken and RT-PCR detection was performed using the primer sequences shown in Table 12. The detection results are shown in Figure 13. As can be seen from the figure, only mouse ICOS mRNA was detected in wild-type C57BL/6 mice, and human ICOS mRNA was not detected; human ICOS mRNA was only detected in ICOS gene humanized heterozygote mice.

Table 12 RT-PCR primer sequences and target fragment sizes

Similar to Example 1, flow cytometry can be used to detect the expression of humanized ICOS protein in ICOS humanized homozygous mice. Specifically, 8-week-old male C57BL/6 mice (+/+) and 7-8-week-old male ICOS gene humanized homozygous mice (H/H) prepared in this embodiment were selected, and anti-mouse CD3e antibody (7.5ug/200uL) was injected intraperitoneally. The spleen tissue of the mice was collected after 24 hours of stimulation. In addition, human peripheral blood cells (PBMC) and human and mouse CHO cells (hCHO and mCHO) were taken as controls, and flow cytometry was performed using the above antibodies. The test results are shown in Table 13.

Table 13 Flow cytometry detection results

As can be seen from Table 13, when human-mouse cross-antibodies were used, ICOS protein was detected in spleen cells of C57BL/6 mice and ICOS humanized homozygous mice, human PBMC cells, and human and mouse CHO cells, but when the specific anti-human ICOS antibody GSK3359609analog was used, the expression of humanized ICOS protein was only detected in spleen cells of ICOS humanized homozygous mice, human PBMC cells, and human CHO cells.

Example 3 Verification of ICOS/ICOSL signaling pathway in humanized ICOS mice

ICOS is a T cell co-stimulatory molecule that is essential for the effective interaction between T cells and B cells and the normal antibody response to T cell-dependent antigens. Studies have shown that homozygous mice with ICOS gene deletion show reduced basal IgG1 levels, impaired interactions between T cells and B cells, and defects in immunoglobulin class switching (including the production of IgE that mediates allergies) (Dong, C., Juedes, AE, Temann, UA, Shresta, S., Allison, JP, Ruddle, NH, & Flavell, RA (2001). ICOS co-stimulatory receptor is essential for T-cell In order to detect the ICOS/ICOSL signaling pathway in ICOS humanized mice, we used ELISA to measure the IgG1 and IgE levels in the serum of wild-type C57BL/6 mice and ICOS humanized homozygous mice before and after immunization.

Specifically, five wild-type C57BL/6 mice (G1), humanized ICOS homozygous mice prepared in Example 1 (G2), and ICOS humanized homozygous mice prepared in Example 2 (G3) were selected. Peripheral blood of the mice was obtained, and the concentration of IgG1 in the serum of wild-type C57BL/6 mice (G1) and ICOS humanized homozygous mice (G2, G3) was detected using Mouse IgG1 ELISA Kit. After that, the mice were sensitized by intraperitoneal injection of 20 μg ovalbumin (OVA) twice (each time with an interval of 6-7 days). Six days after the second sensitization, 0.5% OVA aerosol was used for immunization. Two days later, peripheral blood of the mice was obtained, and the concentrations of IgG1 and IgE in the serum of wild-type C57BL/6 mice and ICOS humanized homozygous mice after immunization were detected using Mouse anti-OVA-IgG1 ELISA Kit and Mouse Serum Anti-OVA IgE Antibody Assay Kit.

The test results are shown in Figure 32. The test results show that the basal serum IgG1 level of ICOS humanized homozygous mice (G2, G3) before immunization is not lower than that of wild-type C57BL/6 mice, but after OVA immunization, the concentration levels of IgG1 and IgE produced in ICOS humanized homozygous mice are similar to or slightly higher than those of wild-type C57BL/6 mice, which indicates that ICOS humanized homozygous mice can produce IgG normally, and the immune protein conversion is normal, and the ICOS/ICOSL signaling pathway in their bodies functions normally.

Example 4 ICOS gene humanized mouse (III)

In order to achieve the humanization of the mouse ICOS gene, the coding region sequence (about 0.6 kb) containing the start codon to the stop codon of the human ICOS gene can also be used to replace the mouse exon 1 partial sequence to the intron 1 partial sequence (about 0.25 kb), and the schematic diagram of the humanized ICOS locus is shown in Figure 14. According to Figure 14, the targeting strategy shown in Figure 15 is further designed, which shows the homology arm sequences upstream and downstream of the mouse ICOS gene on the targeting vector, and the A2 fragment containing the human ICOS nucleotide sequence. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 58) is the same as the nucleotide sequence of positions 61013786 to 61017117 of NCBI accession number NC_000067.7, and the downstream 3' homology arm sequence (SEQ ID NO: 59) is the same as the nucleotide sequence of positions 61017369 to 61021784 of NCBI accession number NC_000067.7. The nucleotide sequence of the human ICOS fragment (SEQ ID NO: 62) is identical to the nucleotide sequence at positions 53-652 of NCBI accession number NM_012092.4; the connection upstream of the human ICOS fragment sequence and the mouse sequence is designed as: (SEQ ID NO: 72), wherein the last "C" in the sequence " CAGAC " is the last nucleotide of the mouse sequence, and the sequence The first "A" in is the first nucleotide of the human sequence. The connection between the downstream of the human ICOS sequence and the WPRE sequence is designed as: (SEQ ID NO: 73), wherein the last "A" in the sequence " TATAA " is the last nucleotide of the human sequence, and the sequence The first "A" in is the first nucleotide of the WPRE sequence.

The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the PA sequence is designed as follows: (SEQ ID NO: 74), wherein the "A" in the sequence " GGGGA " is the last nucleotide of the PA sequence, and the sequence The "C" in the Neo box is the first nucleotide; the connection between the 3' end of the Neo box and the mouse gene is designed as: (SEQ ID NO: 75), wherein the last "C" in the sequence " GATCC " is the last nucleotide of the Neo box, and the sequence The "A" in is the first nucleotide of the mouse. The mRNA sequence of the modified humanized ICOS mouse is shown in SEQ ID NO: 111, and the expressed protein sequence is shown in SEQ ID NO: 2.

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

In addition, the CRISPR/Cas9 system can be used for gene editing, and the targeting strategy shown in FIG17 is further designed, which shows the homology arm sequences upstream and downstream of the mouse ICOS gene on the targeting vector V5, and the A3 fragment containing the human ICOS nucleotide sequence. The schematic diagram of the constructed humanized ICOS locus is shown in FIG14. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 60) is identical to the nucleotide sequence of positions 61015718 to 61017117 of NCBI accession number NC_000067.7, and the downstream 3' homology arm sequence (SEQ ID NO: 61) is identical to the nucleotide sequence of positions 61017369 to 61018575 of NCBI accession number NC_000067.7. The human ICOS nucleotide sequence (SEQ ID NO: 62) is identical to the nucleotide sequence of positions 53 to 652 of NCBI accession number NM_012092.4. The mRNA sequence of the modified humanized ICOS mouse is shown in SEQ ID NO: 111, and the expressed protein sequence is shown in SEQ ID NO: 2.

The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion, ligation, direct synthesis, etc. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector that is verified by sequencing is used for subsequent experiments. Test.

The target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize target sites. The target sequence of an exemplary sgRNA on the ICOS gene is as follows:

sgRNA3 target site (SEQ ID NO: 76): 5’-GGCACTGAAGCTCACGGCTCAGG-3’;

The activity of sgRNA was detected by UCA kit. After confirming that it could mediate efficient cutting efficiency, restriction sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 14. After annealing, the annealing product was connected to pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain the expression vector pT7-ICOS-3.

Table 14 sgRNA3 sequence list

The pT7-sgRNA vector was synthesized by a plasmid synthesis company to contain a fragment DNA (SEQ ID NO: 45) containing a T7 promoter and sgRNA scaffold, and then connected to a backbone vector (source: Takara, item number 3299) by restriction digestion (EcoRI and BamHI) in sequence. After sequencing verification by a professional sequencing company, the results showed that the target plasmid was obtained. Take a mouse pronuclear fertilized egg, such as a C57BL/6 mouse, and use a microinjector to pre-mix the in vitro transcription product of the pT7-ICOS-3 plasmid (using the Ambion in vitro transcription kit, according to the instructions for transcription), the targeting vector, and Cas9 mRNA, and then inject it into the mouse fertilized egg or cell nucleus. According to the method in the Mouse Embryo Operation Manual (3rd Edition) (Andras Nagy, Chemical Industry Press, 2006), the fertilized eggs were microinjected, the injected fertilized eggs were transferred to the culture medium for short-term culture, and then transplanted into the oviduct of the recipient mother mouse for development. The obtained mice (F0 generation) were hybridized and self-fertilized to expand the population and establish a stable ICOS gene humanized mouse strain. The F0 generation positive mice can be screened by PCR technology, and the primers are shown in Table 15.

The ICOS gene humanized mice identified as positive in F0 were mated with wild-type mice to obtain F1 mice. The somatic cell genotype of the F1 mice was identified by PCR, and the primers are shown in Table 14. The identification results of exemplary F1 mice are shown in Figure 18, and all 15 mice numbered F1-01 to F1-15 were positive.

Table 15 Primer sequences and recombinant fragment sizes for PCR detection of F1 genotypes

The clones identified as positive by PCR were then subjected to Southern blot detection (cell DNA was digested with AseI or NcoI and hybridized with two probes, and the probes and target fragment lengths are shown in Table 16) to confirm whether there was random insertion. The exemplary results are shown in Figure 19. Combining the PCR and sequencing results, 10 mice numbered F1-01, F1-02, F1-03, F1-06, F1-07, F1-08, F1-09, F1-10, F1-11 and F1-12 had no random insertion. This shows that the present method can be used to construct ICOS gene humanized mice that can be stably propagated and have no random insertion.

Table 16 Length of specific probes and target fragments

3’Probe-F: 5’-GGAAACTAAACACTCATGGGTAG-3’ (SEQ ID NO: 85);

3’Probe-R: 5’-CAAAGTAGTCTTCCTAAAGCCAG-3’ (SEQ ID NO: 86);

WPRE-F: 5’-GTGGATACGCTGCTTTAATGCC-3’ (SEQ ID NO: 87);

WPRE-R: 5’-AAGGGAGATCCGACTCGTCT-3’ (SEQ ID NO: 88);

Example 5 ICOSL gene humanized mice

Mouse ICOSL gene (NCBI Gene ID: 50723, Primary source: MGI: 1354701, UniProt ID: Q9JHJ8, located at positions 77904921 to 77915359 on chromosome 10 NC_000076.7, based on transcript NM_015790.3 and its encoded protein NP_056605.1 (SEQ ID NO: 63)) and human ICO A comparison diagram of the SL gene (NCBI Gene ID: 23308, Primary source: HGNC:17087, UniProt ID: O75144, located at positions 44216981 to 44240943 of chromosome 21 NC_000021.9, based on transcript NM_015259.6 and its encoded protein NP_056074.1 (SEQ ID NO: 64)) is shown in Figure 20.

In order to achieve the purpose of the present invention, a nucleotide sequence encoding a human ICOSL protein can be introduced into the mouse endogenous ICOSL locus, so that the mouse expresses a human or humanized ICOSL protein. Specifically, using gene editing technology, under the control of the mouse ICOSL gene regulatory element, a partial sequence of exon 3 to a partial sequence of exon 5 of about 5.8 kb of the human ICOSL gene is used to replace a partial sequence of exon 3 to a partial sequence of exon 5 of about 3.5 kb of the mouse, and a schematic diagram of the humanized ICOSL locus is obtained as shown in Figure 21, thereby achieving humanization of the mouse ICOSL gene.

According to Figure 21, a targeting strategy as shown in Figure 22 was further designed, which shows the homology arm sequences upstream and downstream of the mouse IC OSL gene on the V6 targeting vector, and the A4 fragment containing the human ICOSL gene fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 65) is identical to the nucleotide sequence of positions 77903264 to 77907609 of NCBI accession number NC_000076.7, and the downstream 3' homology arm sequence (SEQ ID NO: 66) is identical to the nucleotide sequence of positions 77911065 to 77914575 of NCBI accession number NC_000076.7. The nucleotide sequence of the human ICOSL gene fragment (SEQ ID NO: 69) is identical to the nucleotide sequence of positions 44231410 to 44237179 of NCBI accession number NC_000021.9; the connection design of the upstream of the human ICOSL fragment sequence and the mouse is: (SEQ ID NO: 89), wherein the last "C" in the sequence " GCAGC " is the last nucleotide of the mouse, the sequence The first "G" in is the first nucleotide of the human sequence. The downstream connection of the human ICOSL fragment sequence and the mouse is designed as: (SEQ ID NO: 90), wherein the last "G" in the sequence " CAGAG " is the last nucleotide of the human sequence, and the sequence The first "A" in is the first nucleotide of the mouse sequence.

The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the human ICOSL gene is designed as follows: (SEQ ID NO: 91), wherein the last "G" in the sequence " GTCTG " is the last nucleotide of the human ICOS L gene, and the sequence The "G" in is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the human ICOSL gene is designed as: (SEQ ID NO: 92), wherein the last "T" in the sequence " GTACT " is the last nucleotide of the Neo box, and the sequence The "A" in is the first nucleotide of the human ICOSL gene. The mRNA sequence of the modified humanized mouse ICOSL is shown in SEQ ID NO: 70, and the expressed protein sequence is shown in SEQ ID NO: 71.

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

In addition, the CRISPR/Cas9 system can be used for gene editing, and a targeting strategy as shown in FIG24 is further designed, which shows the homology arm sequences upstream and downstream of the mouse ICOSL gene on the targeting vector V7, as well as the human ICOSL fragment. The schematic diagram of the humanized ICOSL locus after construction is shown in FIG20. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 67) is identical to the nucleotide sequence of positions 77906310 to 77907609 of the NCBI accession number NC_000076.7, and the downstream 3' homology arm sequence (SEQ ID NO: 68) is identical to the nucleotide sequence of positions 77906310 to 77907609 of the NCBI accession number NC_000076.7. The nucleotide sequence of the human ICOSL fragment (SEQ ID NO: 69) is identical to the nucleotide sequence of positions 44231410 to 44237179 of NCBI accession number NC_000021.9. The mRNA sequence of the transformed humanized ICOSL mouse is shown in SEQ ID NO: 70, and the expressed protein sequence is shown in SEQ ID NO: 71.

The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion, ligation, direct synthesis, etc. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector that is verified to be correct by sequencing is used for subsequent experiments.

The target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize target sites. The target sequence of an exemplary sgRNA on the ICOSL gene is as follows:

sgRNA4 target site (SEQ ID NO: 93): 5’-GGACAGTTCCTACAAGAACAGGG-3’;

sgRNA5 target site (SEQ ID NO: 94): 5’-ATGTGGTGCCTGTGAACCCGAGG-3’;

The activity of sgRNA was detected by UCA kit. After confirming that it could mediate efficient cutting efficiency, restriction sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 17. After annealing, the annealing products were connected to pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain expression vectors pT7-ICOSL-1 and pT7-ICOSL-2.

Table 17 sgRNA4 and sgRNA5 sequence list

The pT7-sgRNA vector is synthesized by a plasmid synthesis company as a fragment DNA containing a T7 promoter and sgRNA scaffold (SEQ ID NO: 45) and connected to a backbone vector (source: Takara, item number 3299) by enzyme digestion (EcoRI and BamHI) in sequence. It is sequenced and verified by a professional sequencing company, and the results show that the target plasmid has been obtained. Take a mouse pronuclear fertilized egg, such as a C57BL/6 mouse, and use a microinjector to mix the in vitro transcription products of the pT7-ICOSL-4 and pT7-ICOSL-5 plasmids (using the Ambion in vitro transcription kit, transcribe according to the instructions), the targeting vector and Cas9mRNA, and then inject them into the cytoplasm or nucleus of the mouse fertilized egg. Microinjection of fertilized eggs is performed according to the method in the "Mouse Embryo Operation Experiment Manual (Third Edition)" (Andras Nagy, Chemical Industry Press, 2006). Injection The fertilized eggs are transferred to the culture medium for short-term culture, and then transplanted into the oviduct of the recipient mother mouse for development. The obtained mice (F0 generation) are hybridized and self-fertilized to expand the population and establish a stable ICOSL gene humanized mouse strain. The PCR technology can be used to screen the F0 generation positive mice, and the primers are shown in Table 18.

Table 18 Primer sequences and recombinant fragment sizes for PCR detection of F0 genotypes

The ICOSL gene humanized mice identified as positive in F0 were mated with wild-type mice to obtain F1 mice. The somatic cell genotype of the F1 mice was identified by PCR, and the primers were shown in Table 18. The identification results of exemplary F1 mice are shown in Figure 25. The four mice numbered F1-01 to F1-04 were all positive mice.

The clones identified as positive by PCR were then subjected to Southern blot detection (cell DNA was digested with ScaI or XmnI and hybridized with two probes, the probes and target fragment lengths are shown in Table 19) to confirm whether there was random insertion. The exemplary results are shown in Figure 26. Combining the PCR and sequencing results, the four mice numbered F1-01, F1-02, F1-03 and F1-04 had no random insertion. This shows that the present method can be used to construct ICOSL gene humanized mice that can be stably propagated and have no random insertion.

Table 19 Length of specific probes and target fragments

A Probe(3’)-F: 5’-CCTGAAGGAAGCCGTTTTGATT-3’(SEQ ID NO: 107);

A Probe(3’)-R: 5’-TGAACACTTGTCCACGATGTGA-3’(SEQ ID NO: 108);

5’Probe-F: 5’-TAAAGTGAGTCCGTTCGCTGTT-3’ (SEQ ID NO: 109);

5’Probe-R: 5’-TGGCCTTTGAGGTAACACACAT-3’ (SEQ ID NO: 110);

Prezalumab is a human monoclonal antibody targeting human ICOSL, jointly developed by Amgen and AstraZeneca for the treatment of psoriasis, Sjögren's syndrome and other diseases. Its VH and VL sequences are shown in SEQ ID NO: 112 and SEQ ID NO: 113.

Flow cytometry can be used to detect the expression of humanized ICOSL protein in ICOSL humanized heterozygous mice. Specifically, 1 female C57BL/6 mouse (+/+) aged 6 weeks and 1 female ICOSL gene humanized heterozygous mouse (H/+) aged 6 weeks prepared in this embodiment were selected, and the spleen was taken after euthanasia by cervical dislocation. Brilliant Violet 510 ^TM anti-mouse CD45 Antibody, FITC Rat Anti-Mouse CD3 Antibody, Brilliant Violet 650 ^TM anti-mouse CD19 Antibody, PE anti-mouse CD275 (B7-H2, B7-RP1, ICOS Ligand) Antibody, PE Rat IgG2a, κIsotype Ctrl Antibody, R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγfragment specific, Zombie NIR ^TM Fixable Viability Kit, Purified anti-mouse CD16/32Antibody, Human IgG2, Kappa isotype control and ICOSL prezalumab-anaolg hIgG2 were used for detection. The results are shown in Table 20. The results showed that the expression of mouse ICOSL was detected in C57BL/6 mice (+/+) and ICOSL gene humanized heterozygous mice (H/+), and the expression of human ICOSL was detected in ICOSL gene humanized heterozygous mice (H/+), proving that human ICOSL protein can be successfully expressed in humanized heterozygous ICOSL gene mice.

Table 20 Flow cytometry detection results

Example 6 ICOS and ICOSL dual gene humanized mice

The ICOS humanized mice prepared in Examples 1, 2 and 4 above were mated with the ICOSL humanized mice prepared in Example 5 to obtain ICOS and ICOSL double-gene humanized mice.

Flow cytometry can be used to detect the expression of humanized ICOS and ICOSL proteins in ICOS and ICOSL double gene humanized homozygous mice. Specifically, 12-week-old female C57BL/6 mice (+/+) and 8-week-old female ICOS and ICOSL double gene humanized homozygous mice (H/H) prepared in this embodiment were selected, and anti-mouse CD3e antibody (7.5ug/200uL) was injected intraperitoneally. After 24 hours of stimulation, the spleen tissue of the mice was collected and Brilliant Violet 510 ^TM anti-mouse CD45 Antibody, Alexa Fluor 500 TM anti-mouse CD45 Antibody, and Alexa Fluor 500 TM anti-mouse CD45 Antibody were used. 700 anti-mouse CD3 Antibody, FITC anti-mouse CD19 Antibody, Brilliant Violet 650 ^TM anti-mouse Ly-6G Antibody, Zombie NIR ^TM Fixable Viability Kit, Purified anti-mouse CD16/32 Antibody, specific anti-human ICOS antibody GSK3359609 anaolg and specific anti-human ICOSL antibody ICOSL Prezalumab-anaolg hIgG2 were used for detection.

The results are shown in Table 21. The expression of humanized ICOS protein and humanized ICOSL protein was detected only in ICOS and ICOSL double-gene humanized homozygous mice, proving that humanized ICOS and ICOSL proteins can be successfully expressed in ICOS and ICOSL double-gene humanized homozygous mice.

Table 21 Flow cytometry detection results

Example 7 Pharmacodynamic Model

ICOS humanized mice, ICOSL humanized mice and/or ICOS/ICOSL double-gene humanized mice prepared by the present method can be used to evaluate the efficacy of modulators targeting human ICOS and/or ICOSL. For example, ICOS humanized homozygous mice, ICOSL humanized homozygous mice and/or ICOS/ICOSL double-gene humanized homozygous mice are subcutaneously inoculated with colon cancer cells MC38, and after the tumor volume grows to about 100 mm ³ , they are divided into a control group or a treatment group according to the tumor volume. The treatment group is injected with an antibody drug targeting human ICOS and/or ICOSL, and the control group is injected with an equal volume of normal saline. The tumor volume is measured regularly and the weight of the mice is weighed. By comparing the weight changes of the mice and the tumor volume, the safety and in vivo efficacy of the antibody drug in humanized ICOS and/or ICOSL mice can be effectively evaluated.

Example 8 Preparation of double-gene or multi-gene humanized mice

The humanized ICOS and/or ICOSL gene mice prepared by the present method can also be used to prepare multi-humanized mouse models. For example, in the above-mentioned Example 1, the embryonic stem cells used for microinjection can be selected from mice modified with genes containing PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28, CTLA4, etc. Alternatively, on the basis of humanized ICOS and/or ICOSL mice, mouse ES embryonic stem cells can be separated and gene recombination targeting technology can be used to obtain double humanized or multi-humanized mouse models. The homozygous or heterozygous ICOS and/or ICOSL mice obtained by the present method can also be mated with other gene-modified mice, and their offspring can be screened. According to Mendel's genetic law, there is a certain probability that multi-gene mice modified with humanized ICOS and/or ICOSL genes and other genes can be obtained, and then the heterozygotes can be mated with each other to obtain homozygous modified with double genes or multiple genes.

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

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

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

Some of the nucleotides involved in the present invention are as follows:

SEQ ID NO: 115 V1 human ICOS sequence 22785 bp NC_000002.12, positions 203936815 to 203959599

Claims (135)

A genetically modified non-human animal, characterized in that the genome of the animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric inducible T-cell co-stimulator (ICOS) protein. The animal of claim 1, wherein the nucleotide sequence encoding the human or chimeric ICOS protein is operably linked to an endogenous regulatory element (e.g., endogenous 5'UTR and/or 3'UTR) of an endogenous ICOS locus on at least one chromosome. The animal according to claim 1 or 2, characterized in that the chimeric ICOS protein comprises a human or endogenous signal peptide, a human or humanized extracellular region, a human or humanized transmembrane region and a human or endogenous cytoplasmic region. The animal according to claim 1 or 2, characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOS protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human ICOS (NP_036224.1, SEQ ID NO: 2). The animal according to claim 1 or 2, characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOS protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acids at positions 1-199 or 27-156 of SEQ ID NO: 2. The animal according to claim 1 or 2, characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOS protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 13. The animal according to any one of claims 1-6, characterized in that the animal is a mammal, such as a monkey, a rodent, a mouse or a rat. The animal according to claim 7, characterized in that the animal is a mouse. The animal according to any one of claims 1 to 8, characterized in that the endogenous ICOS protein in the animal is not expressed or the expression level is reduced compared with ICOS in wild-type animals. The animal according to any one of claims 1-9, wherein one or more cells of the animal express human or chimeric ICOS protein. The animal according to any one of claims 1-10, characterized in that one or more cells of the animal express human or chimeric ICOS protein, and the human or chimeric ICOS protein can bind to endogenous ICOSL protein to induce downstream signaling pathways. The animal according to any one of claims 1-10, characterized in that one or more cells of the animal express human or chimeric ICOS protein, and the human or chimeric ICOS protein can bind to the human ICOSL protein to induce downstream signaling pathways. A genetically modified non-human animal, characterized in that the genome of the animal comprises a nucleotide sequence encoding a human or chimeric ICOS region at an endogenous ICOS locus replacing a nucleotide sequence encoding a corresponding region of the endogenous ICOS, or a nucleotide sequence encoding a human or chimeric ICOS region is inserted into the endogenous ICOS locus. The animal of claim 13, wherein the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is operably linked to an endogenous regulatory element of an endogenous ICOS locus, and one or more cells of the animal express the human or humanized ICOS protein. The animal according to claim 13 or 14, characterized in that the endogenous ICOS protein of the animal is not expressed or the expression level is reduced compared with ICOS in wild-type animals. The animal according to any one of claims 13 to 15, characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOS comprises a portion of exon 1, exon 2, exon 3, exon 4 and/or a portion of exon 5 of the human ICOS gene. The animal according to any one of claims 13 to 15, characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOS comprises a portion of exon 2 and/or a portion of exon 3 of the human ICOS gene. The animal according to any one of claims 13-17 is characterized in that the nucleotide sequence encoding the corresponding region of human ICOS is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence shown in SEQ ID NO: 115, 10 or 62. The animal according to any one of claims 13-18, characterized in that the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 1, exon 2, exon 3, exon 4 and/or exon 5 of the mouse ICOS gene. The animal according to any one of claims 13-18, characterized in that the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 2 and/or a portion of exon 3 of the mouse ICOS gene. The animal according to any one of claims 13 to 15, characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is inserted into exon 1 of the endogenous ICOS locus. The animal according to any one of claims 13-15, characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is inserted after the 5'UTR of NM_017480.2. The animal according to claim 21 or 22, characterized in that at least 20 bp of exon 1 of the endogenous ICOS locus (such as nucleotides 46 to 103 of NM_017480.2) is deleted. The animal according to any one of claims 21-23, characterized in that the nucleotide sequence of nucleotides 46 to 103 of the endogenous ICOS locus NM_017480.2 and 193 bp of intron 1 are deleted. The animal according to any one of claims 21-24, characterized in that the inserted sequence comprises all or part of the sequence encoding the human ICOS signal peptide, extracellular region, transmembrane region and cytoplasmic region. The animal of claim 25, wherein the amino acid sequence encoded by the inserted sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to amino acids 1-199 of SEQ ID NO: 2. 100%. The animal according to claim 25 or 26, characterized in that the insertion sequence further comprises one or more auxiliary sequences. The animal according to claim 27, characterized in that the auxiliary sequence is a WPRE sequence and/or a PA sequence. The animal according to any one of claims 13-28, characterized in that the nucleotide sequence contained in the genome of the animal is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 115, 10, 11, 12, 62 or 111. The animal according to any one of claims 13-29, characterized in that the modified ICOS gene in the animal's genome is homozygous and/or heterozygous for the endogenous replaced locus. A non-human animal, characterized in that the animal comprises at least one cell that comprises a nucleotide sequence encoding a human or chimeric ICOS protein, wherein the human or chimeric ICOS protein comprises at least 50, 100, 120, 130, 140, 150, 160, 170, 180, 190 or 199 consecutive amino acids that are identical to the corresponding region of a human, and the animal expresses the human or chimeric ICOS protein. The animal of claim 31, wherein the chimeric ICOS protein comprises at least 50, 60, 70, 80, 90, 100, 120, 130, 140 or 141 consecutive amino acids that are identical to the corresponding consecutive amino acid sequences of the human extracellular region and transmembrane region. The animal of claim 31 or 32, wherein the human or chimeric ICOS protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-199 or 27-156 of SEQ ID NO: 2. The animal according to any one of claims 31-33, characterized in that the nucleotide sequence encoding the human or chimeric ICOS protein is operably linked to an endogenous ICOS regulatory element. The animal according to any one of claims 31-34, characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOS can be integrated into the endogenous ICOS locus of the animal. The animal according to any one of claims 31-35, characterized in that the human or chimeric ICOS protein has at least one mouse ICOS activity and/or human ICOS activity. A method for constructing a genetically modified non-human animal, characterized in that in at least one cell of the animal, at the animal's endogenous ICOS locus, the nucleotide sequence encoding the endogenous ICOS region is replaced by a nucleotide sequence encoding the corresponding region of human or chimeric ICOS, or a sequence encoding human or chimeric ICOS protein is inserted into at least one cell of the animal. The method according to claim 37, characterized in that the endogenous ICOS protein of the animal is not expressed or is expressed at a reduced level compared to ICOS in wild-type animals. The method according to claim 37 or 38 is characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOS comprises a portion of exon 1, exon 2, exon 3, exon 4 and/or exon 5 of the human ICOS gene. The method according to claim 37 or 38, characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOS comprises a portion of exon 2 and/or a portion of exon 3 of the human ICOS gene. The method according to any one of claims 37-40 is characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 2 or 13. The method according to any one of claims 37-41 is characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence shown in SEQ ID NO: 115, 10 or 62. The method according to any one of claims 37-42 is characterized in that the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 1, exon 2, exon 3, exon 4 and/or exon 5 of the mouse ICOS gene. The method according to any one of claims 37-42, characterized in that the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 2 and/or a portion of exon 3 of the mouse ICOS gene. The method according to claim 37, characterized in that the human or chimeric ICOS protein comprises all or part of the human signal peptide, extracellular region, transmembrane region and cytoplasmic region. The method according to claim 37 or 45, characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOS is inserted into exon 1 of the endogenous ICOS locus. The animal according to claim 45 or 46, characterized in that at least 20 bp of exon 1 of the endogenous ICOS locus (such as nucleotides 46 to 103 of NM_017480.2) is deleted. The animal according to any one of claims 45-47, characterized in that the nucleotide sequence of nucleotides 46 to 103 of the endogenous ICOS locus NM_017480.2 and 193 bp of intron 1 are deleted. The method according to any one of claims 37-48, characterized in that the nucleotide sequence encoding the corresponding region of human ICOS is operably linked to an endogenous ICOS regulatory element, such as a promoter. The method according to any one of claims 37-49 is characterized in that the animal is a mammal, such as a monkey, a rodent, a mouse or a rat. A method for constructing a genetically modified non-human animal cell expressing human or chimeric ICOS, the method comprising replacing a nucleotide sequence encoding an endogenous ICOS region with a nucleotide sequence encoding a corresponding region of human ICOS at an endogenous mouse ICOS locus. The sequence encoding the human ICOS protein is replaced, or a sequence encoding the human ICOS protein is inserted into the endogenous ICOS locus to generate a genetically modified non-human animal cell that expresses the human or chimeric ICOS protein. The method according to claim 51 is characterized in that the nucleotide sequence encoding the corresponding region of human ICOS comprises a portion of exon 1, exon 2, exon 3, exon 4 and/or exon 5 of the human ICOS gene. The method according to claim 51 is characterized in that the nucleotide sequence encoding the corresponding region of human ICOS comprises a portion of exon 2 and/or a portion of exon 3 of the human ICOS gene. The method according to any one of claims 51-53 is characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human ICOS is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 2 or 13. The method according to any one of claims 51-54 is characterized in that the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 1, exon 2, exon 3, exon 4 and/or exon 5 of the mouse ICOS gene. The method according to any one of claims 51-54 is characterized in that the nucleotide sequence encoding the endogenous ICOS region comprises a portion of exon 2 and/or a portion of exon 3 of the mouse ICOS gene. The method according to any one of claims 51-56, characterized in that the nucleotide sequence encoding the human or chimeric ICOS protein is operably linked to a regulatory element of endogenous ICOS, such as a promoter. The method according to any one of claims 51-57 is characterized in that the non-human animal is a mouse. The animal according to any one of claims 1-36, characterized in that the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins are selected from at least one of ICOSL, PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28 and CTLA4. The animal of claim 59, wherein the human or chimeric protein is ICOSL protein. The method according to any one of claims 37-58 is characterized in that the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins are selected from at least one of ICOSL, PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28 and CTLA4. The animal of claim 61, wherein the human or chimeric protein is ICOSL protein. A genetically modified non-human animal, characterized in that the genome of the animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric inducible T-cell co-stimulator ligand (ICOSL) protein. The animal of claim 1, wherein the nucleotide sequence encoding the human or chimeric ICOSL protein is operably linked to an endogenous regulatory element (e.g., endogenous 5'UTR and/or 3'UTR) of an endogenous ICOSL locus on at least one chromosome. The animal of claim 63 or 64, wherein the chimeric ICOSL protein comprises an endogenous signal peptide, human or humanized extracellular region, endogenous transmembrane region and endogenous cytoplasmic region. The animal according to claim 63 or 64, characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOSL protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human ICOSL (NP_056074.1, SEQ ID NO: 64). The animal according to claim 63 or 64, characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOSL protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acids at positions 32-244 of SEQ ID NO: 64. An animal according to claim 63 or 64, characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric ICOSL protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 71. The animal according to any one of claims 63-68, characterized in that the animal is a mammal, such as a monkey, a rodent, a mouse or a rat. The animal of claim 69, wherein the animal is a mouse. The animal according to any one of claims 63-70, characterized in that the endogenous ICOSL protein in the animal is not expressed or the expression level is reduced compared with ICOSL in wild-type animals. The animal of any one of claims 63-71, wherein one or more cells of the animal express human or chimeric ICOSL protein. The animal according to any one of claims 63-72, characterized in that one or more cells of the animal express human or chimeric ICOSL protein, and the human or chimeric ICOSL protein can bind to endogenous ICOS protein to induce downstream signaling pathways. The animal according to any one of claims 63-72, characterized in that one or more cells of the animal express human or chimeric ICOSL protein, and the human or chimeric ICOSL protein can bind to the human ICOS protein to induce downstream signaling pathways. A genetically modified non-human animal, characterized in that the genome of the animal comprises a nucleotide sequence encoding a human or chimeric ICOSL region at the endogenous ICOSL locus replacing a nucleotide sequence encoding a corresponding region of the endogenous ICOSL. The animal of claim 75, wherein the nucleotide sequence encoding the corresponding region of human or chimeric ICOSL is operably linked to an endogenous regulatory element of an endogenous ICOSL locus, and one or more cells of the animal express human or humanized ICOSL protein. The animal according to claim 75 or 76, characterized in that the endogenous ICOSL protein of the animal is not expressed or the expression level is reduced compared to ICOSL in wild-type animals. The animal according to any one of claims 75-77, characterized in that the encoding human or chimeric ICOSL corresponding The nucleotide sequence of the region includes a portion of exon 3, a portion of exon 4 and/or a portion of exon 5 of the human ICOSL gene. The animal according to any one of claims 75-78 is characterized in that the nucleotide sequence encoding the corresponding region of human ICOSL is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence shown in SEQ ID NO: 69. The animal according to any one of claims 75-79, characterized in that the nucleotide sequence encoding the endogenous ICOSL region comprises a portion of exon 3, exon 4 and/or exon 5 of the mouse ICOSL gene. The animal according to any one of claims 75-80, characterized in that the modified ICOSL gene in the genome of the animal is homozygous and/or heterozygous for the endogenous replaced locus. A non-human animal, characterized in that the animal comprises at least one cell encoding a nucleotide sequence of a human or chimeric ICOSL protein, wherein the human or chimeric ICOSL protein comprises at least 50, 100, 120, 130, 150, 200, 210, 213, 250, 300 or 302 consecutive amino acids identical to the corresponding region of a human, and the animal expresses the human or chimeric ICOSL protein. The animal of claim 82, wherein the human or chimeric ICOSL protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 32-244 of SEQ ID NO: 64. The animal of claim 82 or 83, wherein the nucleotide sequence encoding the human or chimeric ICOSL protein is operably linked to an endogenous ICOSL regulatory element. The animal according to any one of claims 82-84, characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOSL can be integrated into the endogenous ICOSL locus of the animal. The animal according to any one of claims 82-85, wherein the human or chimeric ICOSL protein has at least one of mouse ICOSL activity and/or human ICOSL activity. A method for constructing a genetically modified non-human animal, characterized in that in at least one cell of the animal, at the animal's endogenous ICOSL locus, a nucleotide sequence encoding an endogenous ICOSL region is replaced by a nucleotide sequence encoding a corresponding region of a human or chimeric ICOSL. The method of claim 87, wherein the endogenous ICOSL protein of the animal is not expressed or is expressed at a reduced level compared to ICOSL in wild-type animals. The method according to claim 87 or 88, characterized in that the nucleotide sequence encoding the corresponding region of human or chimeric ICOSL comprises a portion of exon 3, exon 4 and/or exon 5 of the human ICOSL gene. The method according to any one of claims 87-89 is characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human or chimeric ICOSL is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 64 or 71. The method according to any one of claims 87-90, characterized in that the coding human or chimeric ICOSL corresponding The nucleotide sequence of the region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:69. The method according to any one of claims 87-91, characterized in that the nucleotide sequence encoding the endogenous ICOSL region comprises a portion of exon 3, exon 4 and/or exon 5 of the mouse ICOSL gene. The method according to any one of claims 87-92 is characterized in that the nucleotide sequence encoding the corresponding region of human ICOSL is operably linked to an endogenous ICOSL regulatory element, such as a promoter. The method according to any one of claims 87-93 is characterized in that the animal is a mammal, such as a monkey, a rodent, a mouse or a rat. A method for constructing a genetically modified non-human animal cell expressing human or chimeric ICOSL, the method comprising replacing a nucleotide sequence encoding an endogenous ICOSL region at an endogenous mouse ICOSL locus with a nucleotide sequence encoding a corresponding region of human ICOSL, thereby producing a genetically modified non-human animal cell expressing a human or chimeric ICOSL protein. The method according to claim 95, characterized in that the nucleotide sequence encoding the corresponding region of human ICOSL comprises a portion of exon 3, exon 4 and/or exon 5 of the human ICOSL gene. The method according to claim 95 or 96 is characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human ICOSL is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 64. The method according to any one of claims 95-97 is characterized in that the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human ICOSL is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence at positions 32-244 of SEQ ID NO: 64. The method according to any one of claims 95-98, characterized in that the nucleotide sequence encoding the endogenous ICOSL region comprises a portion of exon 3, exon 4 and/or exon 5 of the mouse ICOSL gene. The method according to any one of claims 95-99, characterized in that the nucleotide sequence encoding the human or chimeric ICOSL protein is operably linked to a regulatory element of endogenous ICOSL, such as a promoter. The method according to any one of claims 95-100 is characterized in that the non-human animal is a mouse. The animal according to any one of claims 63-86, characterized in that the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins are selected from at least one of ICOS, PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28 and CTLA4. The animal of claim 102, wherein the human or chimeric protein is ICOS protein. The method according to any one of claims 87-101 is characterized in that the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins are selected from at least one of ICOS, PD-1, PD-L1, GLP1R, OX40, NKP46, IL36R, HER2, TROP2, CD28 and CTLA4. The animal of claim 104, wherein the human or chimeric protein is ICOS protein. A method for determining the effectiveness of a therapeutic agent in treating cancer, characterized in that the method comprises: 1) administering a therapeutic agent to an animal as described in any one of claims 1-36, 59-60, 63-86 and 102-103, wherein the animal has cancer; 2) Determine the inhibitory effect of therapeutic agents on cancer. The method according to claim 106, characterized in that the therapeutic agent is an anti-ICOS antibody and/or an anti-ICOSL antibody. The method according to claim 106 or 107 is characterized in that the cancer is a tumor, and the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the animal. The method according to any one of claims 106-108 is characterized in that the cancer comprises injecting one or more cancer cells into the animal. The method according to any one of claims 106-109 is characterized in that the cancer is leukemia, solid tumor, lymphoma, respiratory system cancer, skin cancer, gastric cancer, digestive tract cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, melanoma, non-small cell lung cancer, bladder cancer, breast cancer, colorectal cancer, lung cancer, multiple myeloma, non-Hodgkin's lymphoma, prostate cancer, kidney cancer, etc. A method for determining the effectiveness of anti-ICOS antibodies and/or anti-ICOSL antibodies and additional therapeutic agents in treating cancer, characterized in that the method comprises: 1) administering an anti-ICOS antibody and/or an anti-ICOSL antibody and an additional therapeutic agent to an animal of any one of claims 1-36, 59-60, 63-86 and 102-103, wherein the animal has a tumor; 2) Determine the inhibitory effect of anti-ICOS antibody and/or anti-ICOSL antibody and additional therapeutic agents on tumors. The method according to claim 111, characterized in that the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. The method of claim 111 or 1112, wherein the tumor comprises one or more cancer cells injected into the animal. The method according to any one of claims 111-113 is characterized in that the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the animal. The method according to any one of claims 111-114 is characterized in that the cancer is leukemia, solid tumor, lymphoma, respiratory system cancer, skin cancer, gastric cancer, digestive tract cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, melanoma, non-small cell lung cancer, bladder cancer, breast cancer, colorectal cancer, lung cancer, multiple myeloma, non-Hodgkin's lymphoma, prostate cancer, kidney cancer, etc. A method for determining the effectiveness of a therapeutic agent in treating an immune disease, characterized in that the method comprises: 1) administering a therapeutic agent to a non-human animal as described in any one of claims 1-36, 59-60, 63-86 and 102-103, wherein the non-human animal suffers from an immune disease; 2) Determine the therapeutic effect of therapeutic agents on immune diseases. The method according to claim 116 is characterized in that the immune disease is systemic lupus erythematosus, transplant rejection, blood disease, immune neuromuscular disease, rheumatoid arthritis, etc. A method for determining the effectiveness of an ICOS therapeutic agent in treating inflammation, characterized in that the method comprises: 1) administering a therapeutic agent to an animal as described in any one of claims 1-36, 59-60, 63-86 and 102-103, wherein the animal has inflammation; 2) Determine the effectiveness of therapeutic agents in treating inflammation. The method according to claim 118 is characterized in that the inflammation is arthritis, inflammation, inflammatory bowel disease, etc. A method for determining the toxicity of an ICOS therapeutic agent, characterized in that the method comprises: 1) administering a therapeutic agent to an animal as described in any one of claims 1-36, 59-60, 63-86 and 102-103; 2) Determine the effect of a therapeutic agent on an animal. The method of claim 120, wherein determining the effect of the therapeutic agent on the animal involves measuring the animal's body weight, red blood cell count, hematocrit and/or hemoglobin. A humanized ICOS protein, characterized in that the humanized ICOS protein comprises all or part of the human ICOS protein. The humanized ICOS protein according to claim 122 is characterized in that the identity of the humanized ICOS protein to the amino acid sequence shown in SEQ ID NO: 2 or 13 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. A humanized ICOS gene, characterized in that the humanized ICOS gene encodes the humanized protein according to any one of claims 122-123. The humanized ICOS gene according to claim 124 is characterized in that the humanized ICOS gene comprises a portion of exon 1, all of exons 2-4, and/or a portion of exon 5 of the human ICOS gene, and the humanized ICOS gene further comprises a portion of exon 1 and/or a portion of exon 5 of a non-human animal ICOS gene. The humanized ICOS gene according to claim 124, characterized in that the humanized ICOS gene comprises a portion of exon 2 and/or a portion of exon 3 of the human ICOS gene, and the humanized ICOS gene further comprises the entirety of exon 1, a portion of exon 2, a portion of exon 3, and/or the entirety of exons 4-5 of the ICOS gene of a non-human animal. department. The humanized ICOS gene according to claim 124 is characterized in that the humanized ICOS gene comprises part of exon 1, all of exons 2-4, and/or part of exon 5 of the human ICOS gene, and the humanized ICOS gene further comprises part of exon 1 and/or all of exons 2-5 of a non-human animal ICOS gene. The humanized ICOS gene according to any one of claims 124-127 is characterized in that the nucleotide sequence comprised by the humanized ICOS gene is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 58, 59, 60, 61, 62 or 111. A humanized ICOSL protein, characterized in that the humanized ICOSL protein comprises all or part of a human ICOSL protein. The humanized ICOSL protein according to claim 129 is characterized in that the humanized ICOSL protein has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% with the amino acid sequence shown in SEQ ID NO: 71. A humanized ICOSL gene, characterized in that the humanized ICOSL gene encodes the humanized protein according to any one of claims 129-130. The humanized ICOSL gene according to claim 131, characterized in that the humanized ICOSL gene comprises part of exon 3, all of exon 4, and/or part of exon 5 of the human ICOSL gene, and the humanized ICOSL gene further comprises all of exons 1-2, part of exon 3, part of exon 5, and/or all of exons 6-7 of the ICOSL gene of a non-human animal. The humanized ICOSL gene according to claim 131 or 132 is characterized in that the nucleotide sequence comprised by the humanized ICOSL gene is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 65, 66, 67, 68, 69 or 70. A cell, characterized in that it contains the proteins described in claims 122-123, 129-130 and the genes described in claims 124-127, 131-133. An animal model, characterized in that the animal model comprises the proteins described in claims 122-123, 129-130 and the genes described in claims 124-127, 131-133.