Humanized SIRPA-IL15 knock-in mouse and method of use thereof

Abstract

To provide: genetically modified non-human animals expressing human SIRPα and human IL-15 from the non-human animal genome; methods for making non-human animals expressing human SIRPα and human IL-15 from the non-human animal genome; and methods for using non-human animals expressing human SIRPα and human IL-15 from the non-human animal genome.SOLUTION: A genetically modified non-human animal comprises: a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, where the sequence encodes a human SIRPα protein and is operably linked to a SIRPα gene promoter; and a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, where the sequence encodes a human IL-15 protein and is operably linked to an IL-15 gene promoter; where the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein.SELECTED DRAWING: Figure 7A

Description

[Technical Field]

[0001] cross reference This application claims the interests of U.S. Provisional Patent Application No. 62 / 146,938, filed on 13 April 2015, No. 62 / 148,667, filed on 16 April 2015, and No. 62 / 287,842, filed on 27 January 2016, the disclosures of each of these applications incorporated herein by reference in their entirety.

[0002] Field of Invention This invention relates to the field of genetically modified non-human animals. [Background technology]

[0003] Introduction Genetically modified non-human animals, such as humanized mice, enable the modeling and study of human diseases in vivo, and therefore hold great promise in translational research. Over the past decade, significant progress has been made in the development of humanized mice by genetically inserting human genes essential for the proper development and function of human immune cells within the mouse body. However, several limitations still restrict the usefulness of humanized mice in translational research. In particular, the development and survival of human T cells are not optimal.

[0004] While bone marrow-liver-thymus (BLT) models have been shown to improve intestinal T cell rearrangement in NS / NSG-BLT mice (Denton PW, Nochi T, Lim A et al. Mucosal Immunol 2012;5:555-566 (Non-Patent Literature 1), Nochi T, Denton PW, Wahl A et al. Cell Rep 2013;3:1874-1884 (Non-Patent Literature 2)), these mice have been shown to develop graft-versus-host disease, resulting in large-scale infiltration of immune cells in multiple tissues (Greenblatt MB, Vrbanac V, Tivey T et al. PLoS One 2012;7:e44664 (Non-Patent Literature 3)). Therefore, current humanized mouse models are still insufficient to properly develop and function human T cells. In particular, the absence of human tissue-resident memory T cells hinders the development and testing of more efficient immune strategies for inducing sustained mucosal immunity against pathogens such as HIV using humanized mice as a preclinical tool.

[0005] Obtaining genetically modified non-human animals that generate human tissue-resident T cells is considered useful for better understanding the development and survival of human tissue-resident T cells and for providing models to test novel immunization strategies that induce sustained T cell-dependent mucosal immunity. Such mouse models can also be used to study the interaction between human tissue-resident immune cells and the gut microbiota, for example, how the microbiota can shape the development and survival of human immune cells in the small and colon.

[0006] Furthermore, non-human animal models of human natural killer (NK) cell development and function are needed in this field. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Denton PW, Nochi T, Lim A et al. Mucosal Immunol 2012;5:555-566 [Non-Patent Document 2] Nochi T, Denton PW, Wahl A et al.Cell Rep 2013;3:1874-1884 [Non-Patent Document 3] Greenblatt MB, Vrbanac V, Tivey T et al.PLoS One 2012;7:e44664 [Overview of the project]

[0008] overview This invention provides genetically modified non-human animals that express human SIRPα and human IL-15 from a non-human animal genome. It also provides a method for producing non-human animals that express human SIRPα and human IL-15 from a non-human animal genome, as well as a method for using non-human animals that express human SIRPα and human IL-15 from a non-human animal genome. These animals and methods are expected to have many applications in the art, such as modeling the development and function of human T cells and / or natural killer (NK) cells; modeling human pathogen infection of human T cells and / or NK cells; in vivo screening of drugs that inhibit infection by pathogens that activate, induce, and / or target T cells and / or NK cells; in vivo screening of drugs that modulate the development and / or function of human T cells and / or NK cells, for example, in a healthy or diseased state; in vivo screening of drugs that are toxic to human T cells and / or NK cells; in vivo screening of drugs that prevent, mitigate, or reverse the toxic effects of toxic substances on human T cells and / or NK cells; in vivo screening of candidate T cell-inducible vaccines; and in vivo and in vitro screening of drugs that inhibit tumor growth and / or infection by activation of antibody-dependent cell-mediated cytotoxicity (ADCC) processes mediated by NK cells.

[0009] In a first embodiment, the present disclosure provides a genetically modified non-human animal having a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal which encodes a human SIRPα protein and is operably linked to the SIRPα gene promoter, and a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal which encodes a human IL-15 protein and is operably linked to the IL-15 gene promoter, and which expresses the human SIRPα protein and the human IL-15 protein.

[0010] The SIRPα gene promoter can be an endogenous non-human SIRPα gene promoter. For example, the SIRPα gene promoter can be an endogenous non-human SIRPα gene promoter within a non-human animal SIRPα locus. If the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter within a non-human animal SIRPα locus, the genetically modified non-human animal may contain a null mutation in the non-human SIRPα gene within the non-human animal SIRPα locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4. In another such embodiment, the genetically modified non-human animal is heterozygous with respect to an allele having a nucleic acid sequence encoding the human SIRPα protein. In yet another such embodiment, the genetically modified non-human animal is homozygous with respect to an allele having a nucleic acid sequence encoding the human SIRPα protein.

[0011] In another embodiment of the first embodiment, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genome coding sequence and a human SIRPα genome non-coding sequence.

[0012] In another embodiment of the first embodiment, or in a further embodiment of any of the above embodiments, the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. In one such embodiment, the functional fragment has an extracellular domain of human SIRPα, for example, an extracellular domain comprising at least amino acids 28-362 of SEQ ID NO:12.

[0013] In another embodiment of the first embodiment, or in a further embodiment of any of the above embodiments, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter. In one such embodiment, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within a non-human animal IL-15 locus. In one embodiment, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within a non-human animal IL-15 locus, and the genetically modified non-human animal contains a null mutation in the non-human IL-15 gene within the non-human animal IL-15 locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8. In another such embodiment, the genetically modified non-human animal is heterozygous with respect to an allele having a nucleic acid sequence encoding the human IL-15 protein. In another such embodiment, the genetically modified non-human animal is homozygous with respect to an allele having a nucleic acid sequence encoding the human IL-15 protein.

[0014] In another embodiment of the first embodiment, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0015] In another embodiment of the first embodiment, or in a further embodiment of any of the above embodiments, the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0016] In another embodiment of the first embodiment, or in a further embodiment of any of the above embodiments, the genetically modified non-human animal is immunodeficient. For example, in one embodiment, the genetically modified non-human animal has a Rag2 gene knockout. In another embodiment, the genetically modified non-human animal has an IL2rg gene knockout, or both a Rag2 gene knockout and an IL2rg gene knockout.

[0017] In another embodiment of the first embodiment, or in a further embodiment of any of the above embodiments, the non-human animal is a mammal. In one such embodiment, the mammal is a rodent, such as a mouse.

[0018] In another embodiment of the first embodiment, or in a further embodiment of any of the above embodiments, the genetically modified non-human animal has engraftment of human hematopoietic cells. In one such embodiment, the genetically modified non-human animal has infection with a human pathogen. In one embodiment, the genetically modified non-human animal has infection with a human pathogen, and the human pathogen activates, induces, and / or targets T cells and / or natural killer (NK) cells. In another embodiment, the genetically modified non-human animal has infection with a human pathogen, the human pathogen is a pathogen that affects the human intestine (e.g., by infection). In one such embodiment, the human pathogen is human rotavirus. In another embodiment, the genetically modified non-human animal has infection with a human pathogen, the pathogen affects the human lungs (e.g., by infection). In one such embodiment, the human pathogen is influenza virus. In another embodiment, the genetically modified non-human animal has infection with a human pathogen, the pathogen affects the human liver (e.g., by infection). In yet another embodiment, the genetically modified non-human animal has human hematopoietic cells and tumors, such as human tumors, such as transplanted human tumors.

[0019] In a second aspect, the disclosure provides an in vivo model comprising a genetically modified non-human animal having a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human SIRPα protein and is operably linked to the SIRPα gene promoter, a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human IL-15 protein and is operably linked to the IL-15 gene promoter, and engraftment of human hematopoietic cells, wherein the genetically modified non-human animal (i) expresses human SIRPα protein and human IL-15 protein, and (ii) harbors human intraepithelial lymphocytes (IELs) in the small intestine and Peyer's patches of the genetically modified non-human animal.

[0020] In one embodiment of the second aspect, a genetically modified non-human animal is infected with a human pathogen, such as an intestinal pathogen. In one such embodiment, the intestinal pathogens include Campylobacter jejuni, Clostridium difficile, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, human rotavirus, Listeria monocytogenes, Norwalk virus, Salmonella enterica, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Yersinia pestis, and Yersinia enterocolitica. The species selected is Helicobacter pylori (Helicobacter enterocolitica).

[0021] In another embodiment of the second embodiment, or in a further embodiment of any of the above embodiments, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter. In one such embodiment, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter within the non-human animal SIRPα locus. In one embodiment, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter within the non-human animal SIRPα locus, and the genetically modified non-human animal contains a null mutation in the non-human SIRPα gene within the non-human animal SIRPα locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4. In another such embodiment, the genetically modified non-human animal is heterozygous with respect to an allele having a nucleic acid sequence encoding the human SIRPα protein. In another such embodiment, the genetically modified non-human animal is homozygous with respect to an allele having a nucleic acid sequence encoding the human SIRPα protein.

[0022] In another embodiment of the second embodiment, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human SIRPα protein has a human SIRPα genome encoding and non-coding sequence.

[0023] In another embodiment of the second embodiment, or in a further embodiment of any of the above embodiments, the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. In one such embodiment, the functional fragment has an extracellular domain of human SIRPα, for example, an extracellular domain comprising amino acids 28-362 of SEQ ID NO:12.

[0024] In another embodiment of the second embodiment, or in a further embodiment of any of the above embodiments, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter. In one such embodiment, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within a non-human animal IL-15 locus. In one embodiment, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within a non-human animal IL-15 locus, and the genetically modified non-human animal contains a null mutation in the non-human IL-15 gene within the non-human animal IL-15 locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8. In one such embodiment, the genetically modified non-human animal is heterozygous with respect to an allele having a nucleic acid sequence encoding the human IL-15 protein. In another such embodiment, the genetically modified non-human animal is homozygous with respect to an allele having a nucleic acid sequence encoding the human IL-15 protein.

[0025] In another embodiment of the second embodiment, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0026] In another embodiment of the second aspect, or in a further embodiment of any of the above embodiments, the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0027] In another embodiment of the second embodiment, or in a further embodiment of any of the above embodiments, the genetically modified non-human animal is immunodeficient. For example, in one embodiment, the genetically modified non-human animal has a Rag2 gene knockout. In another embodiment, the genetically modified non-human animal has an IL2rg gene knockout, or both a Rag2 gene knockout and an IL2rg gene knockout.

[0028] In another embodiment of the second aspect, or in a further embodiment of any of the above embodiments, the non-human animal is a mammal. In one such embodiment, the mammal is a rodent, such as a mouse.

[0029] In a third aspect, the disclosure provides an in vivo model comprising a genetically modified non-human animal having a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human SIRPα protein and is operably linked to the SIRPα gene promoter, a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human IL-15 protein and is operably linked to the IL-15 gene promoter, and engraftment of human hematopoietic cells, wherein the genetically modified non-human animal (i) expresses human SIRPα protein and human IL-15 protein, and (ii) harbors human intraepithelial lymphocytes (IELs) in the lungs of the genetically modified non-human animal.

[0030] In one embodiment of the third aspect, a genetically modified non-human animal is infected with a human pathogen, such as a lung pathogen. In one such embodiment, the lung pathogens include Streptococcus pyogenes, Haemophilus influenza, Corynebacterium diphtheria, SARS coronavirus, Bordetella pertussis, Moraxella catarrhalis, influenza viruses (A, B, C), coronaviruses, adenoviruses, RSV, parainfluenza virus, mumps virus, Streptococcus pneumoniae, Staphylococcus aureus, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Mycoplasma pneumoniae. The selection is made from Mycobacterium tuberculosis, Chlamydia pneumoniae, Blastomyces dermatitidis, Cryptococcus neoformans, and Aspergillus fumigatus.

[0031] In another embodiment of the third aspect, or in a further embodiment of any of the above embodiments, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter. In one such embodiment, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter within the non-human animal SIRPα locus. In one embodiment, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter within the non-human animal SIRPα locus, and the genetically modified non-human animal contains a null mutation in the non-human SIRPα gene within the non-human animal SIRPα locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4. In another such embodiment, the genetically modified non-human animal is heterozygous with respect to an allele having a nucleic acid sequence encoding the human SIRPα protein. In another such embodiment, the genetically modified non-human animal is homozygous with respect to an allele having a nucleic acid sequence encoding the human SIRPα protein.

[0032] In another embodiment of the third aspect, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genome coding sequence and a human SIRPα genome non-coding sequence.

[0033] In another embodiment of the third aspect, or in a further embodiment of any of the above embodiments, the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. In one such embodiment, the functional fragment has an extracellular domain of human SIRPα, for example, an extracellular domain comprising at least amino acids 28-362 of SEQ ID NO:12.

[0034] In another embodiment of the third aspect, or in a further embodiment of any of the above embodiments, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter. In one such embodiment, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter located within the non-human animal IL-15 locus.

[0035] In one embodiment, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within the non-human animal IL-15 locus, and the genetically modified non-human animal contains a null mutation in the non-human IL-15 gene at the non-human animal IL-15 locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8. In another such embodiment, the genetically modified non-human animal is heterozygous with respect to an allele having a nucleic acid sequence encoding the human IL-15 protein. In yet another such embodiment, the genetically modified non-human animal is homozygous with respect to an allele having a nucleic acid sequence encoding the human IL-15 protein.

[0036] In another embodiment of the third aspect, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0037] In another embodiment of the third aspect, or in a further embodiment of any of the above embodiments, the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0038] In another embodiment of the third aspect, or in a further embodiment of any of the above embodiments, the genetically modified non-human animal is immunodeficient. For example, in one embodiment, the genetically modified non-human animal has a Rag2 gene knockout. In another embodiment, the genetically modified non-human animal has an IL2rg gene knockout, or both a Rag2 gene knockout and an IL2rg gene knockout.

[0039] In another embodiment of the third aspect, or in a further embodiment of any of the above embodiments, the non-human animal is a mammal. In one such embodiment, the mammal is a rodent, such as a mouse.

[0040] In a fourth aspect, the Disclosure provides a method for determining the efficacy of a candidate T cell-inducing vaccine, the method comprising administering the candidate T cell-inducing vaccine to a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system and has (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human SIRPα protein and is operably linked to the SIRPα gene promoter, (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human IL-15 protein and is operably linked to the IL-15 gene promoter, and (iii) engraftment of human hematopoietic cells, wherein the genetically modified non-human animal expresses human SIRPα protein and human IL-15 protein, the method also comprising challenging the genetically modified non-human animal with a human pathogen and determining whether the candidate T cell-inducing vaccine induces a T cell-mediated immune response in the genetically modified non-human animal.

[0041] In one embodiment of the fourth aspect, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter. In one such embodiment, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter within the non-human animal SIRPα locus. In one embodiment, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter within the non-human animal SIRPα locus, and the genetically modified non-human animal contains a null mutation in the non-human SIRPα gene within the non-human animal SIRPα locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4. In another such embodiment, the genetically modified non-human animal is heterozygous with respect to an allele having a nucleic acid sequence encoding the human SIRPα protein. In another such embodiment, the genetically modified non-human animal is homozygous with respect to an allele having a nucleic acid sequence encoding the human SIRPα protein.

[0042] In another embodiment of the fourth aspect, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genome coding sequence and a human SIRPα genome non-coding sequence.

[0043] In another embodiment of the fourth aspect, or in a further embodiment of any of the above embodiments, the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. In one such embodiment, the functional fragment has an extracellular domain of human SIRPα, for example, an extracellular domain comprising at least amino acids 28-362 of SEQ ID NO:12.

[0044] In another embodiment of the fourth aspect, or in a further embodiment of any of the above embodiments, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter. In one such embodiment, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within the non-human animal IL-15 locus. In one embodiment, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within the non-human animal IL-15 locus, and the genetically modified non-human animal contains a null mutation in the non-human IL-15 gene within the non-human animal IL-15 locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8. In another such embodiment, the genetically modified non-human animal is heterozygous with respect to an allele having a nucleic acid sequence encoding the human IL-15 protein. In another such embodiment, the genetically modified non-human animal is homozygous with respect to an allele having a nucleic acid sequence encoding the human IL-15 protein.

[0045] In another embodiment of the fourth aspect, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0046] In another embodiment of the fourth aspect, or in a further embodiment of any of the above embodiments, the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0047] In another embodiment of the fourth aspect, or in a further embodiment of any of the above embodiments, the genetically modified non-human animal has a Rag2 gene knockout.

[0048] In another embodiment of the fourth aspect, or in a further embodiment of any of the above embodiments, the genetically modified non-human animal has an IL2rg gene knockout.

[0049] In another embodiment of the fourth aspect, or in a further embodiment of any of the above embodiments, the genetically modified non-human animal is a mammal such as a rodent, for example, a mouse.

[0050] In a fifth aspect, the Disclosure provides a method for identifying agents that inhibit infection by pathogens that activate, induce, and / or target human T cells and / or natural killer (NK) cells, the method comprising administering the agent to a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system and comprises (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes the human SIRPα protein and is operably linked to the SIRPα gene promoter, and (ii) the genetically modified non-human animal A nucleic acid sequence incorporated into the genome of an organism, which encodes the human IL-15 protein and is operablely linked to the IL-15 gene promoter; (iii) engraftment of human hematopoietic cells; and (iv) infection by a pathogen that activates, induces and / or targets human T cells and / or natural killer cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein; the method also includes determining whether the agent reduces the amount of the pathogen in the non-human animal infected with the pathogen.

[0051] In one embodiment of the fifth aspect, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter. In one such embodiment, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter within the non-human animal SIRPα locus. In one embodiment, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter within the non-human animal SIRPα locus, and the genetically modified non-human animal contains a null mutation in the non-human SIRPα gene within the non-human animal SIRPα locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4. In another such embodiment, the genetically modified non-human animal is heterozygous with respect to an allele having a nucleic acid sequence encoding the human SIRPα protein. In another such embodiment, the genetically modified non-human animal is homozygous with respect to an allele having a nucleic acid sequence encoding the human SIRPα protein.

[0052] In another embodiment of the fifth aspect, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genome coding sequence and a human SIRPα genome non-coding sequence.

[0053] In another embodiment of the fifth aspect, or in a further embodiment of any of the above embodiments, the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. In one such embodiment, the functional fragment has an extracellular domain of human SIRPα, for example, an extracellular domain comprising amino acids 28-362 of SEQ ID NO:12.

[0054] In another embodiment of the fifth aspect, or in a further embodiment of any of the above embodiments, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter. In one such embodiment, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within a non-human animal IL-15 locus. In one embodiment, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within a non-human animal IL-15 locus, and the genetically modified non-human animal contains a null mutation in the IL-15 gene within the non-human animal IL-15 locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8. In another such embodiment, the genetically modified non-human animal is heterozygous with respect to an allele having a nucleic acid sequence encoding the human IL-15 protein. In another such embodiment, the genetically modified non-human animal is homozygous with respect to an allele having a nucleic acid sequence encoding the human IL-15 protein.

[0055] In another embodiment of the fifth aspect, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0056] In another embodiment of the fifth aspect, or in a further embodiment of any of the above embodiments, the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0057] In another embodiment of the fifth aspect, or in a further embodiment of any of the above embodiments, the genetically modified non-human animal has a Rag2 gene knockout.

[0058] In another embodiment of the fifth aspect, or in a further embodiment of any of the above embodiments, the genetically modified non-human animal has an IL2rg gene knockout.

[0059] In another embodiment of the fifth aspect, or in a further embodiment of any of the above embodiments, the genetically modified non-human animal is a mammal such as a rodent, for example, a mouse.

[0060] In a sixth aspect, the Disclosure provides a method for producing a non-human animal expressing human IL-15 protein and human SIRPα protein, the method comprising introducing a nucleic acid sequence encoding human IL-15 protein and operably ligated to an IL-15 gene promoter sequence into the genome of a first non-human animal, introducing a nucleic acid sequence encoding human SIPRα protein and operably ligated to a SIRPα promoter sequence into the genome of a second non-human animal, and producing a third non-human animal having the nucleic acid sequence encoding human IL-15 protein and the nucleic acid sequence encoding human SIRPα protein, wherein the third non-human animal expresses human IL-15 protein and human SIPRα protein.

[0061] In one embodiment of the sixth aspect, the introduction step includes generating a non-human animal from pluripotent stem cells possessing nucleic acids encoding human IL-15 or human SIRPα.

[0062] In another embodiment of the sixth aspect, or in a further embodiment of the above aspect, the first animal is a different animal from the second animal, and the step of producing the third animal includes mating the first and second animals.

[0063] In another embodiment of the sixth aspect, the first animal and the second animal are identical, the step of introducing into the genome of the first animal includes contacting the first pluripotent stem cells with a nucleic acid sequence encoding human IL-15 protein to obtain second pluripotent stem cells, the step of introducing into the genome of the second animal includes contacting the second pluripotent stem cells with a nucleic acid sequence encoding human SIRPα protein to obtain third pluripotent stem cells, and the third non-human animal is produced from the third pluripotent stem cells.

[0064] In another version of the sixth aspect of the disclosure, the present disclosure provides a method for producing a non-human animal expressing human IL-15 protein and human SIRPα protein, the method comprising introducing a nucleic acid sequence encoding human SIPRα protein and operably ligated to a SIPRα gene promoter sequence into the genome of a first non-human animal, introducing a nucleic acid sequence encoding human IL-15 protein and operably ligated to an IL-15 promoter sequence into the genome of a second non-human animal, and producing a third non-human animal having a nucleic acid sequence encoding human IL-15 protein and a nucleic acid sequence encoding human SIRPα protein, wherein the third non-human animal expresses human IL-15 protein and human SIPRα protein.

[0065] In yet another embodiment of the sixth aspect, the first animal and the second animal are identical, the step of introducing into the genome of the first animal includes contacting the first pluripotent stem cells with a nucleic acid sequence encoding human SIRPα to obtain second pluripotent stem cells, the step of introducing into the genome of the second animal includes contacting the second pluripotent stem cells with a nucleic acid sequence encoding human IL-15 protein to obtain third pluripotent stem cells, and the third non-human animal is produced from the third pluripotent stem cells.

[0066] In another embodiment of the sixth aspect, or in a further embodiment of any of the above embodiments, the pluripotent stem cells are ES cells or iPS cells.

[0067] In another embodiment of the sixth aspect, or in a further embodiment of any of the above embodiments, the pluripotent stem cells are deficient in Rag2.

[0068] In another embodiment of the sixth aspect, or in a further embodiment of any of the above embodiments, the pluripotent stem cells are deficient in IL2rg.

[0069] In another embodiment of the sixth aspect, or in a further embodiment of any of the above embodiments, the third non-human animal is deficient in either or both Rag2 and IL2rg.

[0070] In another embodiment of the sixth aspect, or in a further embodiment of any of the above embodiments, the IL-15 promoter sequence is a human IL-15 promoter sequence.

[0071] In another embodiment of the sixth aspect, or in a further embodiment of any of the above embodiments, the IL-15 promoter sequence is an endogenous non-human animal IL-15 promoter sequence.

[0072] In another embodiment of the sixth aspect, or in a further embodiment of any of the above embodiments, the incorporation results in the substitution of a non-human IL-15 gene within a non-human IL-15 locus.

[0073] In another embodiment of the sixth aspect, or in a further embodiment of any of the above embodiments, the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0074] In a seventh aspect, the disclosure provides a method for engrafting a genetically modified non-human animal expressing human IL-15 protein, the method comprising transplanting a cell population containing human hematopoietic cells into a genetically modified non-human animal prepared by the method of the sixth aspect or any embodiment thereof. In one such embodiment, the transplantation includes tail vein injection, fetal liver injection, or post-orbital injection.

[0075] In another embodiment of the seventh aspect, or in a further embodiment of any of the above embodiments, a genetically modified non-human animal is subjected to a sublethal dose of irradiation before transplantation.

[0076] In another embodiment of the seventh aspect, or in a further embodiment of any of the above embodiments, the human hematopoietic cells are CD34+ cells.

[0077] In another embodiment of the seventh aspect, or in a further embodiment of any of the above embodiments, the human hematopoietic cells are derived from fetal liver, adult bone marrow, or umbilical cord blood.

[0078] In the eighth aspect, the Disclosure provides a method for determining the effectiveness of a candidate therapeutic antibody or antigen-binding protein in killing target cells, the method comprising administering the candidate therapeutic antibody or antigen-binding protein to a genetically modified non-human animal, the genetically modified non-human animal lacking an endogenous immune system and having (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human SIRPα protein and is operably linked to the SIRPα gene promoter, (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human IL-15 protein and is operably linked to the IL-15 gene promoter, and (iii) engraftment of human hematopoietic cells, wherein the genetically modified non-human animal expresses human SIRPα protein and human IL-15 protein, and the method also comprises determining whether the candidate therapeutic antibody or antigen-binding protein modulates NK cell-mediated antibody-dependent cytotoxicity against target cells in the genetically modified non-human animal.

[0079] In a ninth aspect, the Disclosure provides a method for determining the effectiveness of a candidate therapeutic antibody or antigen-binding protein in killing target cells, the method comprising isolating NK cells from a genetically modified non-human animal, the genetically modified non-human animal lacking an endogenous immune system and having (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human SIRPα protein and is operably linked to the SIRPα gene promoter, (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human IL-15 protein and is operably linked to the IL-15 gene promoter, and (iii) engraftment of human hematopoietic cells, wherein the genetically modified non-human animal expresses human SIRPα protein and human IL-15 protein, the method also comprising contacting the isolated NK cells with the candidate therapeutic antibody or antigen-binding protein and target cells, and measuring the antibody or antigen-binding protein-dependent cytolytic activity of the isolated NK cells against the target cells.

[0080] In a tenth aspect, the Disclosure provides a method for screening candidate therapeutic antibodies or antigen-binding proteins for improving their effectiveness in killing target cells, the method comprising administering the candidate therapeutic antibody or antigen-binding protein to a genetically modified non-human animal, the genetically modified non-human animal lacking an endogenous immune system and having (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human SIRPα protein and is operably linked to the SIRPα gene promoter, (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes human IL-15 protein and is operably linked to the IL-15 gene promoter, and (iii) engraftment of human hematopoietic cells, wherein the genetically modified non-human animal expresses human SIRPα protein and human IL-15 protein, and the method also comprises determining whether the candidate therapeutic antibody or antigen-binding protein shows improved effectiveness in killing target cells in the genetically modified non-human animal.

[0081] In any one embodiment of the eighth, ninth, and tenth aspects, the target cells are one or more of tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells. [Invention 1001] Genetically modified non-human animals, A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, and A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, encoding the human IL-15 protein and operably linked to the IL-15 gene promoter. A genetically modified non-human animal having the above characteristics and expressing the human SIRPα and the human IL-15 protein. [Invention 1002] The genetically modified non-human animal of the present invention 1001, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter. [Invention 1003] The genetically modified non-human animal of the present invention 1002, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter located within the SIRPα locus of a non-human animal. [Invention 1004] A genetically modified non-human animal according to the present invention 1003, comprising a null mutation in the non-human SIRPα gene within the SIRPα locus of the aforementioned non-human animal. [Invention 1005] The genetically modified non-human animal of the present invention 1004, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4. [Invention 1006] A genetically modified non-human animal according to the present invention 1004, which is heterozygous with respect to the allele having the nucleic acid sequence encoding the human SIRPα protein. [Invention 1007] A genetically modified non-human animal according to the present invention 1004, which is homozygous with respect to the allele having the nucleic acid sequence encoding the human SIRPα protein. [Invention 1008] A genetically modified non-human animal according to any of the invention items 1001 to 1007, wherein the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. [Invention 1009] The aforementioned functional fragment comprises the extracellular domain of human SIRPα, wherein the genetically modified non-human animal according to the present invention 1008. [Invention 1010] A genetically modified non-human animal according to any of the invention items 1001 to 1009, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter. [Invention 1011] The genetically modified non-human animal of the present invention 1010, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within the IL-15 locus of a non-human animal. [Invention 1012] A genetically modified non-human animal according to the present invention 1011, comprising a null mutation in the non-human IL-15 gene within the aforementioned non-human animal IL-15 gene locus. [Invention 1013] The genetically modified non-human animal of the present invention 1012, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8. [Invention 1014] A genetically modified non-human animal according to the present invention 1012, which is heterozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein. [Invention 1015] A genetically modified non-human animal according to the present invention 1012, which is homozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein. [Invention 1016] A genetically modified non-human animal according to any of the inventions 1001 to 1015, wherein the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence. [Invention 1017] A genetically modified non-human animal according to any of the invention items 1001 to 1016, wherein the human IL-15 protein is a functional fragment of the full-length human IL-15 protein. [Invention 1018] A non-human animal that is immunodeficient and has been genetically modified according to any of the invention items 1001 to 1017. [Invention 1019] A genetically modified non-human animal according to the present invention 1018, having a Rag2 gene knockout. [Invention 1020] A genetically modified non-human animal according to invention 1018 or 1019, having an IL2rg gene knockout. [Invention 1021] A non-human animal that is a mammal and is genetically modified according to any of the invention items 1001 to 1020. [Invention 1022] The aforementioned mammal is a rodent, a genetically modified non-human animal according to the present invention 1021. [Invention 1023] The aforementioned rodent is a mouse, a genetically modified non-human animal according to the present invention 1022. [Invention 1024] A genetically modified non-human animal according to any of the invention items 1001 to 1023, having the ability to engraft human hematopoietic cells. [Invention 1025] A genetically modified non-human animal according to the present invention 1024, which is infected by a human pathogen. [Invention 1026] A genetically modified non-human animal according to the present invention 1025, wherein the human pathogen activates, induces, and / or targets T cells and / or natural killer (NK) cells. [Invention 1027] The aforementioned human pathogen is a pathogen that infects the human intestine, a genetically modified non-human animal according to the present invention 1025. [Invention 1028] A genetically modified non-human animal according to Invention 1027, wherein the aforementioned human pathogen is human rotavirus. [Invention 1029] A genetically modified non-human animal according to the present invention 1025, wherein the pathogen infects the human lungs. [Invention 1030] A genetically modified non-human animal according to Invention 1029, wherein the aforementioned human pathogen is an influenza virus. [Invention 1031] A step of introducing a nucleic acid sequence encoding human SIRPα protein, operably linked to the SIRPα gene promoter sequence, into the genome of a first non-human animal. The process of introducing a nucleic acid sequence encoding the human IL-15 protein, operably linked to an IL-15 promoter sequence, into the genome of a second non-human animal, and A step to produce a third non-human animal that possesses the nucleic acid sequence encoding the human IL-15 protein and the nucleic acid sequence encoding the human SIRPα protein, and that expresses the human IL-15 protein and the human SIPRα protein. A method for producing non-human animals that express human IL-15 protein and human SIRPα protein, including the following. [Invention 1032] The method of the present invention 1031, wherein the introduction step includes producing a non-human animal from pluripotent stem cells possessing the nucleic acid encoding human IL-15 or human SIRPα. [Invention 1033] The method of the present invention 1031 or 1032, wherein the first animal is a different animal from the second animal, and the step of producing the third animal includes mating the first and second animals. [Invention 1034] The method of the present invention 1031, wherein the first animal and the second animal are the same, the step of introducing into the genome of the first animal includes contacting the nucleic acid sequence encoding the human SIRPα protein with the first pluripotent stem cell to obtain a second pluripotent stem cell, the step of introducing into the genome of the second animal includes contacting the nucleic acid sequence encoding the human SIRPα protein with the second pluripotent stem cell to obtain a third pluripotent stem cell, and the third non-human animal is produced from the third pluripotent stem cell. [Invention 1035] The method according to any one of items 1031 to 1034 of the present invention, wherein the pluripotent stem cells are ES cells or iPS cells. [Invention 1036] The method according to any one of the present invention 1031 to 1034, wherein the pluripotent stem cells lack Rag2. [Invention 1037] The method according to any one of the present invention 1031 to 1036, wherein the pluripotent stem cells lack IL2rg. [Invention 1038] The method according to any one of the invention 1031 to 1037, wherein the third non-human animal lacks one or both of Rag2 and IL2rg. [Invention 1039] The method according to any one of the present invention 1031 to 1038, wherein the IL-15 promoter sequence is a human IL-15 promoter sequence. [Invention 1040] The method according to any one of the present invention 1031 to 1038, wherein the IL-15 promoter sequence is an endogenous non-human animal IL-15 promoter sequence. [Invention 1041] Any method 1031 to 1038 of the present invention, wherein the integration results in the substitution of the non-human IL-15 gene within the non-human IL-15 locus. [Invention 1042] The method according to any one of the present invention 1031 to 1041, wherein the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence. [Invention 1043] A method for engraftment into a genetically modified non-human animal expressing human IL-15 protein, comprising the step of transplanting a population of cells including human hematopoietic cells into the genetically modified non-human animal prepared by any of the methods 1031 to 1042 of the present invention. [Invention 1044] The method of the present invention 1043, wherein the transplantation step includes injection into the tail vein, injection into the fetal liver, or injection into the posterior orbit. [Invention 1045] The method according to the present invention 1043 or 1044, wherein the genetically modified non-human animal is subjected to a sublethal dose of radiation before transplantation. [Invention 1046] The method according to any of items 1043 to 1045 of the present invention, wherein the human hematopoietic cells are CD34+ cells. [Invention 1047] The method according to any one of items 1043 to 1046 of the present invention, wherein the human hematopoietic cells are derived from fetal liver, adult bone marrow, or umbilical cord blood. [Invention 1048] A method for engraftment into a genetically modified non-human animal expressing human IL-15 protein, comprising the step of transplanting a population of cells including human hematopoietic cells into a genetically modified non-human animal according to any of invention 1018 to 1023. [Invention 1049] An animal engraftment model including a genetically modified non-human animal, wherein the genetically modified non-human animal is A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, encoding the human IL-15 protein and operably linked to the IL-15 gene promoter, and Engraftment of human hematopoietic cells The animal engraftment model having, wherein the genetically modified non-human animal (i) expresses the human SIRPα protein and the human IL-15 protein, and (ii) harbors human intraepithelial lymphocytes (IELs) in the small intestine and Peyer's patches of the genetically modified non-human animal. [Invention 1050] A model of the present invention 1048, wherein the genetically modified non-human animal is infected by a human pathogen. [Invention 1051] A model of Invention 1049 in which the human pathogen is an intestinal pathogen. [Invention 1052] The aforementioned intestinal pathogens include Campylobacter jejuni, Clostridium difficile, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, human rotavirus, Listeria monocytogenes, Norwalk virus, Salmonella enterica, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Yersinia pestis, Yersinia enterocolitica, and Helicobacter pylori. A model of the present invention 1050, selected from pylori. [Invention 1053] An animal engraftment model including a genetically modified non-human animal, wherein the genetically modified non-human animal is A nucleic acid sequence incorporated into the genome of the aforementioned genetically modified non-human animal, encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, encoding the human IL-15 protein and operably linked to the IL-15 gene promoter, and Engraftment of human hematopoietic cells The animal engraftment model having, where the genetically modified non-human animal (i) expresses the human SIRPα protein and the human IL-15 protein, and (ii) harbors human intraepithelial lymphocytes (IELs) in the lungs of the genetically modified non-human animal. [Invention 1054] A model of the present invention 1053, wherein the genetically modified non-human animal is infected by a human pathogen. [Invention 1055] A model of the present invention 1054, wherein the human pathogen is a lung pathogen. [Invention 1056] The aforementioned lung pathogens include Streptococcus pyogenes, Haemophilus influenza, Corynebacterium diphtheria, SARS coronavirus, Bordetella pertussis, Moraxella catarrhalis, influenza viruses (A, B, C), coronaviruses, adenoviruses, RSV, parainfluenza virus, mumps virus, Streptococcus pneumoniae, Staphylococcus aureus, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae, and Mycobacterium tuberculosis. A model of the present invention 1055, selected from tuberculosis, Chlamydia pneumoniae, Blastomyces dermatitidis, Cryptococcus neoformans, and Aspergillus fumigatus. [Invention 1057] A step of administering a drug to a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system, (i) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, (ii) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, encoding the human IL-15 protein and operably linked to the IL-15 gene promoter, (iii) Engraftment of human hematopoietic cells, and (iv) Infection by pathogens that activate, induce, and / or target human T cells and / or natural killer cells The genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein, and the administration step, A step to determine whether the drug reduces the amount of the pathogen in non-human animals infected with the pathogen. A method for identifying drugs that inhibit infection by pathogens that activate, induce, and / or target human T cells and / or natural killer (NK) cells. [Invention 1058] A step of administering a candidate therapeutic antibody or antigen-binding protein to a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system, (i) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, (ii) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, encoding the human IL-15 protein and operably linked to the IL-15 gene promoter, and (iii) Engraftment of human hematopoietic cells The genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein, and the administration step, The process of determining whether the candidate therapeutic antibody or antigen-binding protein is activated in the antibody-dependent cytotoxicity of NK cells against target cells in the genetically modified non-human animal. A method for determining the effectiveness of a candidate therapeutic antibody or antigen-binding protein in the killing of target cells via NK cells, including the method for determining the effectiveness of such a antibody. [Invention 1059] The method of the present invention 1058, wherein the target cells are selected from the group consisting of tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells. [Invention 1060] The method of the present invention 1059, wherein the target cells are tumor cells. [Invention 1061] The method of the present invention 1060, wherein the tumor cells are B-cell lymphoma cells. [Invention 1062] A model of antibody-dependent cytotoxicity mediated by NK cells, including a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system, A nucleic acid sequence incorporated into the genome of the aforementioned genetically modified non-human animal, encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, encoding the human IL-15 protein and operably linked to the IL-15 gene promoter, and Engraftment of human hematopoietic cells The genetically modified non-human animal has the following characteristics: (i) expresses the human SIRPα protein and the human IL-15 protein; (ii) possesses human lymphocytes; and (iii) possesses target cells selected from the group consisting of tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells. The aforementioned model. [Invention 1063] A model of the present invention 1062, wherein the target cells are tumor cells. [Invention 1064] A model of the present invention 1063, wherein the tumor cells are B-cell lymphoma cells. [Invention 1065] A model of the present invention 1063 or 1064 comprising an exogenous candidate therapeutic antibody or antigen-binding protein. [Invention 1066] A model according to any of the invention 1062 to 1065, wherein the genetically modified non-human animal possesses human intraepithelial lymphocytes (IELs) in the small intestine and Peyer's patches of the genetically modified non-human animal. [Invention 1067] A model according to any of the invention 1062 to 1066, wherein the genetically modified non-human animal possesses human intraepithelial lymphocytes (IELs) in the lungs of the genetically modified non-human animal.

[0082] A patent or application file shall include at least one color drawing. Upon request and payment of the required fees, the Patent Office shall provide a copy of the published patent or patent application, including the color drawing(s). [Brief explanation of the drawing]

[0083] [Figure 1] This diagram provides a schematic representation of the substitution of the mouse SIRPα gene with a human SIRPα sequence. The upper panel shows the mouse SIRPα locus, indicating the relative positions of exons 1-8. The lower panel provides a schematic representation of the final targeted allele containing human exons 2-4. The encoding chimeric protein has an extracellular region corresponding to amino acids 28-362 of the wild-type human SIRPα protein fused to the intracellular portion of the mouse SIRPα protein. The diagonally striped regions represent the inserted human sequence. [Figure 2] This diagram provides a schematic representation of the genomic substitution targeting the mouse IL-15 gene achieved in mouse 2. The hollowed-out areas represent the inserted human sequence. [Figure 3A] This graph shows the hIL-15 gene expression in various tissues of non-engrafted SRG (human SIRPα, Rag KO, IL-2rg KO) and SRG-15 (human SIRPα, Rag KO, IL-2rg KO, human IL-15 (mouse 1)) mice. The Y-axis represents the level of hIL-15 mRNA relative to the housekeeping gene HPrt. [Figure 3B] This document provides graphs showing human hIL-15 gene expression in various tissues of non-engrafted RG (Rag KO, IL-2rg KO) and non-engrafted SRG-15 (human SIRPα, Rag KO, IL-2rg KO, human IL-15) mice (mouse #1 and mouse #2 in the figure). [Figure 4]This shows the serum levels of human IL-15 protein in SRG, SRG IL-15h / m(mouse 2), and SRG IL-15h / h(mouse 2) mice after challenge with poly(I:C). [Figure 5A] This report provides graphs illustrating the efficient engraftment of human hematopoietic cells in the blood of NSG, SRG, and SRG-15 (mouse 2) mice 12–14 weeks after engraftment. All data are presented as mean ± sem. Statistical analysis was performed using independent two-tailed Mann-Whitney U tests (*P<0.05, **P<0.01, ****P<0.0001). [Figure 5B] This provides graphs showing the number of human CD45+ cells in the bone marrow, spleen, lymph nodes, liver, and lungs of SRG and SRG-15 (mouse 2) at 14 weeks after engraftment. [Figure 6A] This provides plots showing the frequency of human T and NK cell occurrence in the bone marrow (BM), liver, and lungs of SRG and SRG-15 mice (mouse 1). [Figure 6B] This provides graphs showing the frequency of human NK cell appearance in various tissues in SRG and SRG-15 mice (mouse 1). [Figure 6C] This paper provides plots and graphs showing the maturation of human NK cells in the liver of SRG and SRG-15 mice (mouse 1). [Figure 6D] This plot shows that human CD56dimCD16+ NK cells express high levels of human killer cell suppressor receptors in the spleen of SRG-15 mice. [Figure 7A] This report provides graphs showing the frequency of human NK cells in the blood of NSG, SRG, and SRG-15 (mouse 2) mice 10-12 weeks after engraftment. All data are presented as mean ± sem. Statistical analysis was performed using the independent two-tailed Mann-Whitney U test (*P<0.05, **P<0.01, ****p<0.0001). [Figure 7B]This graph shows the percentage of human NKp46+ cells in the spleen 14 weeks after engraftment of SRG, SRG-15h / m, and SRG-15h / h. All data are shown as mean ± sem. Statistical analysis was performed using the independent two-sided Mann-Whitney U test (*P<0.05, **P<0.01, ****p<0.0001). [Figure 7C] This report provides plots showing the frequency of human NK cells in the blood, spleen (SP), liver, and lungs of SRG and SRG-15 (mouse 2) mice at 14 weeks post-engraftment. All data are presented as mean ± sem. Statistical analysis was performed using independent two-tailed Mann-Whitney U tests (*P<0.05, **P<0.01, ****p<0.0001). [Figure 7D] This report provides graphs showing the frequency of human NK cells in the spleen (SP), liver, and lungs of SRG and SRG-15 (mouse 2) mice at 14 weeks post-engraftment. All data are presented as mean ± sem. Statistical analysis was performed using independent two-tailed Mann-Whitney U tests (*P<0.05, **P<0.01, ****p<0.0001). [Figure 8] This document provides a plot (left) showing the distribution of human T cells and NK cells in the blood of SRG and SRG-15 (mouse 2) mice (gated to human CD45+ cells (hematopoietic cells) and NKp46+ cells (NK cells)), and a graph (right) showing the percentage of NKp46+ cells among hCD45+ cells in the blood of engrafted SRG-15 mice. [Figure 9A] This paper provides plots showing the distribution of NK cells and T cells within the spleen, as well as graphs showing the percentage and number of NKp46+ cells in the spleen of SRG-15 mice (mouse 2) engrafted with CD34+ huHSCs, compared to SRG mice engrafted with CD34+ huHSCs. [Figure 9B] This provides graphs showing the human immune cell composition in the blood of NSG (n=5), SRG (n=19), and SRG-15 (mouse 2) mice (n=39) 10-12 weeks after engraftment. [Figure 9C]This shows the number of human CD45+ cells in the thymus of SRG and SRG-15 (mouse 2) mice 14 weeks after engraftment. [Figure 9D] This provides representative flow cytometry plots of hCD45+ cells in the thymus of SRG and SRG-15 (mouse 2) mice. [Figure 9E] This graph shows the composition of hCD45+ cells in the thymus of SRG (n=8) and SRG-15 (2 mice) (n=4) mice 14 weeks after engraftment. [Figure 10A] This provides plots showing the frequency of CD56brightCD16- and CD56dimCD16+ NK cell subsets in the blood and spleen of SRG and SRG-15 (mouse 2) mice 7 weeks after engraftment. [Figure 10B] This provides graphs showing the frequency of CD56brightCD16- and CD56dimCD16+ NK cell subsets in the blood and spleen of SRG and SRG-15 (mouse 2) mice 7 weeks after engraftment. [Figure 10C] This document provides plots and graphs showing the expression of killer inhibitory receptors (KIRs) in NK cell subsets in humans and SRG-15 mice (mouse 2). [Figure 11] Two plots (upper left and upper right) are provided showing the distribution of CD16+ vs. CD16- NK cells in the blood of SRG-15 mice (mouse 2) compared to PBMC samples. A graph (lower panel) is also provided showing the ratio of CD16+ vs. CD16- NKp46+ cells in the blood of either SRG-15 mice (mouse 2) or PBMC-derived samples. [Figure 12] This report provides graphs showing the development of human NK cells in the bone marrow of SRG and SRG-15 (mouse 2) mice at 7 weeks post-engraftment. All data are presented as mean ± sem. Statistical analysis was performed using independent two-tailed Mann-Whitney U tests (*P<0.05, **P<0.01, ****p<0.0001). [Figure 13A]This provides graphs showing the frequency of human T cell appearance in various tissues in SRG and SRG-15 mice (Mouse 1). (K / μl = 1000 cells / μl). [Figure 13B] This paper provides plots and graphs showing the human CD8+ T cell phenotype in the blood and liver of SRG and SRG-15 mice (mouse 1). [Figure 14A] This provides plots and graphs showing the expression of the tissue resident marker CD69 in lung CD8+ T cells of SRG and SRG-15 (mouse 1) mice. [Figure 14B] This provides plots and graphs showing the expression of the tissue resident marker CD69 in hepatic CD8+ T cells of SRG and SRG-15 (mouse 1) mice. [Figure 15A] This provides graphs showing the frequency of hCD3+ T cell appearance in the spleen, lungs, and liver of SRG and SRG-15 (mouse 2) mice 16 weeks after engraftment. [Figure 15B] This document provides graphs showing the CD4 / CD8 ratio in the spleen, lungs, and liver of SRG and SRG-15 (mouse 2) mice 16 weeks after engraftment. [Figure 16A] This provides a plot showing the frequency of human mucosal lamina propria lymphocytes (LPLs) in the colon of SRG and SRG-15 (mouse 1) mice. [Figure 16B] This provides a graph showing the frequency of human mucosal lamina propria lymphocytes (LPLs) in the colon of SRG and SRG-15 (mouse 1) mice. [Figure 17A] Figures 17B-17C show the efficient engraftment of human intraepithelial lymphocytes (IELs) in the small intestine of 16-week-old SRG-15 mice (mouse 1). Figure 17A provides plots and graphs showing human CD45+ cells and CD8+ T cells in the IEL fraction of SRG and SRG-15 (mouse 1) mice. [Figure 17B] Images of immunohistochemical staining of hCD45 in the small intestine of 16-week-old SRG and SRG-15 (mouse 1) mice are provided. [Figure 17C]This provides plots showing the phenotypic characteristics of human CD8+ T cells in the spleen and small intestine of SRG-15 mice (mouse 1). [Figure 18A] This provides representative FACS plots showing mouse and human CD45+ cells in the IEL fraction of SRG and SRG-15 (mouse 2) mice 16 weeks after engraftment. [Figure 18B] This document provides graphs comparing the number of human IELs in the small intestine of SRG mice with that of SRG-15 (mouse 2) mice, and the number of human LPLs in the large intestine of SRG mice with that of SRG-15 (mouse 2) mice. All data are presented as mean ± sem. Statistical analysis was performed using the independent two-tailed Mann-Whitney U test (***p<0.001). [Figure 18C] This provides a plot showing the composition of hCD3+ cells in the small intestine of SRG-15 mice (mouse 2). It is a representative FACS plot from one of eight SRG-15 mice (mouse 2). [Figure 18D] This provides graphs showing the phenotypic characteristics of hCD3+ hCD8+ T cells in the spleen and small intestine of SRG-15 mice (mouse 2). [Figure 18E] Images of immunohistochemical staining for hCD8 in the small intestine of SRG and SRG-15 (mouse 2) mice are provided. Arrows indicate hCD8+IEL. The images are representative of three mice per group. [Figure 19A] This paper provides plots and graphs showing the distribution and number of hCD45+ cells in the intraepithelial lymphocyte population of SRG and SRG-15 mice, as well as the relative percentages of NK cells and T cells in the hCD45+ cell population within the intraepithelial lymphocyte population of SRG and SRG-15(mouse 2) mice. [Figure 19B] This document provides plots and graphs showing the distribution and proportion of CD16+ and CD16- NK cells in intraepithelial lymphocytes of SRG-15 mice (mouse 2), compared to those in blood and spleen. [Figure 19C] This document provides plots and graphs showing the distribution and number of human IELs and human mucosal lamina propria lymphocytes (LPLs) in SRG and SRG-15 (mouse 2) mice. [Figure 20A] This provides plots and graphs showing the presence of recognizable Peyer's patches, mainly containing hCD45+ cells, in SRG-15 mice (mouse 2). [Figure 20B] This provides plots and graphs showing the presence of recognizable Peyer's patches, mainly containing hCD45+ cells, in SRG-15 mice (mouse 2). [Figure 21A] This provides a timeline for cohousing and fecal sample collection for sequencing of the gut microbiota. [Figure 21B] This diagram shows the relative abundance of mouse bacteria in the intestines of non-engrafted, engrafted SRG, and SRG-15 (mouse 1) mice. [Figure 22] This paper demonstrates the functional relevance of human tissue-resident T cells in SRG-15 mice. More specifically, it provides a graph showing the functional relevance of human IELs in clearing acute rotavirus infection. [Figure 23A] This provides ViSNE plots showing CyTOF-based analysis of 42 parameters of CD56brightCD16- and CD56dimCD16+ NK cell subsets in human (n=20) and SRG-15 mice (2 mice) (n=9). Each dot represents a single cell. [Figure 23B] This provides ViSNE plots showing the expression intensities of eight select markers on CD56brightCD16-NK cells in humans (n=20) and SRG-15 mice (2 mice) (n=9). [Figure 23C] This is a ViSNE plot showing the expression intensities of eight select markers on CD56dimCD16+ NK cells in humans (n=20) and SRG-15 mice (n=9). [Figure 24A] This document provides a graph comparing the percentage of CD69+ circulating NK cells before and after poly-IC injection in SRG and SRG-15 (mouse 2) mice. [Figure 24B]This document provides graphs showing IFNγ production from NK cells derived from SRG and SRG-15 (mouse 2) after in vitro stimulation with poly(I:C) or human IL-12p70. Mouse-derived NK cells are compared with healthy human PBMC-derived NK cells. All samples are normalized by NK cell count. [Figure 24C] This graph shows the cytolytic ability of SRG and SRG-15(mouse 2) mouse-derived spleen NK cells against either HLA class I-deficient K562 cells (left) or Raji cells in the absence (upper right) or presence (lower right) of anti-CD20 antibody. SRG-15#1 and SRG-15#2 represent two different NK cell preparations derived from SRG-15(mouse 2) littermates. [Figure 25A] This graph shows that human NK cells in SRG-15 mice (mouse 2) inhibit tumor growth after rituximab (RTX) treatment. All data are shown as mean ± sem. Statistical analysis was performed using the independent two-sided Mann-Whitney U test (***p<0.001). [Figure 25B] This paper provides plots and graphs showing the frequency of human NK cells and T cells in human tumor xenografts from untreated (n=5) and RTX-treated SRG-15 mice (n=1). All data are presented as mean ± sem. Statistical analysis was performed using the independent two-sided Mann-Whitney U test (***p<0.001). [Figure 25C] This paper provides plots and graphs showing the human NK cell subsets in the blood and tumors of untreated (n=2) and RTX-treated SRG-15 mice (n=1). All data are presented as mean ± sem. Statistical analysis was performed using the independent two-sided Mann-Whitney U test (***p<0.001). [Modes for carrying out the invention]

[0084] Detailed explanation Before describing the methods and compositions of the present invention, it should be understood that the present invention is not limited to the specific methods or compositions described and is therefore subject to various modifications. It should also be understood that the scope of the present invention is limited only by the appended claims, and that the terms used herein are merely for describing specific forms and are not intended to limit them.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which this invention pertains. Any methods and substances similar to or equivalent to those described herein may be used in carrying out or testing the invention, but specific methods and substances are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or substances that refer to the publications relating thereto. In the event of any conflict, it should be understood that this disclosure takes precedence over any disclosure in the referencing publications.

[0086] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features and may be readily separated from or combined with features of any of several other embodiments without departing from the scope or spirit of the invention. Any method described may be carried out in the order of referenced events or in any other logically possible order.

[0087] As used herein and in the appended claims, it should be noted that the singular forms "a," "an," and "the" encompass multiple referents unless otherwise explicitly indicated by the context. For example, a reference to "cell" includes multiple such cells, and a reference to "protein" includes one or more proteins and their equivalents known to those skilled in the art.

[0088] The publications discussed herein are provided only for disclosures prior to the filing date of this application. Nothing in this specification shall be construed as admitting that the present invention is not prior to such publications.

[0089] This invention provides genetically modified non-human animals that express human SIRPα and human IL-15 from a non-human animal genome. It also provides a method for producing non-human animals that express human SIRPα and human IL-15 from a non-human animal genome, as well as a method for using non-human animals that express human SIRPα and human IL-15 from a non-human animal genome. These animals and methods are expected to have many applications in the art, such as modeling the development and function of human T cells and / or natural killer (NK) cells; modeling human pathogen infection, such as human pathogen infection of specific tissues, such as the human gut, lung, or liver; modeling human pathogen infection of human T cells and / or NK cells; in vivo screening of drugs that inhibit infection by pathogens that activate, induce, and / or target T cells and / or NK cells, such as in vivo screening of drugs that modulate the development and / or function of human T cells and / or NK cells in a healthy or diseased state; in vivo screening of drugs that are toxic to human T cells and / or NK cells; in vivo screening of drugs that prevent, mitigate, or reverse the toxic effects of toxic substances on human T cells and / or NK cells; in vivo screening of candidate T cell-inducible vaccines; and in vivo and in vitro screening of drugs that inhibit tumor growth and / or infection by activation of antibody-dependent cell-mediated cytotoxicity (ADCC) processes mediated by NK cells.

[0090] Humanized SIRPα Non-human animals In some aspects of this disclosure, humanized SIRPα non-human animals are provided. Humanized SIRPα non-human animal, or “SIRPα non-human animal,” means a non-human animal possessing a nucleic acid sequence encoding the human SIRPα protein. As used herein, “human SIRPα protein” means wild-type (or naturally occurring) human SIRPα protein or a variant of wild-type (or naturally occurring) human SIRPα protein, which retains one or more signaling and / or receptor functions of the wild-type human SIRPα protein. As used herein, the term “variant” defines either a spontaneously occurring genetic mutant of an isolated human polypeptide or nucleic acid sequence, or a variation of a human polypeptide or nucleic acid sequence prepared by recombination, each containing one or more mutations in comparison to the corresponding wild-type human nucleic acid or polypeptide sequence. For example, such mutations may be one or more amino acid substitutions, additions, and / or deletions. The term “variant” also includes human homologs and orthologues. In some embodiments, the mutant polypeptide of the present invention has 70% or more identity with wild-type human polypeptide, for example, 75%, 80%, or 85% or more identity, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with wild-type human polypeptide.

[0091] The degree of identity between two sequences may be determined using any convenient technique in the art, such as sequence alignment using publicly available software. Mutations can be introduced using standard molecular biology techniques such as site-directed mutagenesis, PCR-induced mutagenesis, and directional evolution. Those skilled in the art will recognize that one or more nucleic acid substitutions can be introduced without altering the amino acid sequence, and one or more amino acid mutations can be introduced without altering the functional properties of human proteins.

[0092] Conservative amino acid substitutions can be generated in human proteins to create human protein variants. A conservative amino acid substitution, as recognized in this field, refers to the substitution of one amino acid for another amino acid with similar properties. For example, each amino acid may be referred to as having one or more of the following properties: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic, and hydrophilic. A conservative substitution is the substitution of one amino acid having a specific structural or functional characteristic with another amino acid having the same property. Examples of acidic amino acids include aspartic acid and glutamic acid; basic amino acids include histidine, lysine, and arginine; aliphatic amino acids include isoleucine, leucine, and valine; aromatic amino acids include phenylalanine, glycine, tyrosine, and tryptophan; polar amino acids include aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, and tryptophan. Conservative substitutions include substitutions between amino acids within each group. Amino acids may also be referred to in terms of their relative size, with alanine, cysteine, aspartic acid, glycine, asparagine, proline, threonine, serine, and valine all generally considered small in size.

[0093] Human variants may include synthetic amino acid analogs, amino acid derivatives, and / or non-standard amino acids, and examples, but not limited to, include α-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxyphenylalanine, diencholic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, methylhistidine, and ornithine.

[0094] Human variants are typically encoded by nucleic acids that have high identity with the nucleic acid encoding the wild-type human protein. The complement of the nucleic acid encoding the human variant specifically hybridizes with the nucleic acid encoding the wild-type human protein under high stringency conditions. Nucleic acids encoding human variants can be generated by isolation, recombination, or synthesis using well-known methods. Furthermore, the term "human SIRPα protein" encompasses fragments of the wild-type human SIRPα protein (or its variants) that retain one or more signaling and / or receptor functions of the wild-type human SIRPα protein, such as the extracellular domain of the human SIRPα protein.

[0095] Furthermore, the term "human SIRPα protein" encompasses fusion proteins, i.e., chimeric proteins, that possess one or more fragments of wild-type human SIRPα protein (or its variants) while retaining one or more signaling and / or receptor functions of wild-type human SIRPα protein. Fusion proteins containing one or more fragments of wild-type human SIRPα protein (or its variants) in combination with, for example, one or more non-human peptides or polypeptides may also be referred to herein as humanized SIRPα proteins. Therefore, for example, a protein having the amino acid sequence of the extracellular domain of wild-type human SIRPα protein fused with the signaling domain of wild-type mouse SIRPα protein is included in the term "human SIRPα protein."

[0096] In some cases, the human SIRPα protein according to this disclosure has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity with respect to amino acids 28-362 of SEQ ID NO:12.

[0097] Therefore, the nucleic acid sequence encoding the human SIRPα protein is a polynucleotide having a coding sequence for the human SIRPα protein, such as the wild-type human SIRPα protein, a variant of the wild-type human SIRPα protein, a fragment of the wild-type human SIRPα protein (or its variant) that retains one or more signaling and / or receptor functions of the wild-type human SIRPα protein, or a fusion protein, i.e., a chimeric protein, that has one or more fragments of the wild-type human SIRPα protein (or its variant) and also retains one or more signaling and / or receptor functions of the wild-type human SIRPα protein.

[0098] SIRPα (also known as "signal-regulating protein α" and "CD172A" in humans) is a member of the signal-regulating protein (SIRP) family and also belongs to the immunoglobulin superfamily. SIRPα has been shown to improve cell engraftment in immunodeficient mice (Strowig et al. Proc Natl Acad Sci USA 2011;108:13218-13223). The polypeptide sequence of wild-type human SIRPα and the nucleic acid sequence encoding wild-type human SIRPα can be found in Genbank accessions NM_001040022.1 (mutant 1), NM_001040023.1 (mutant 2), and NM_080792.2 (mutant 3). The SIRPα gene is conserved in at least chimpanzees, rhesus monkeys, dogs, cattle, mice, rats, and chickens. The genomic locus encoding the wild-type human SIRPα protein can be found on the human genome at chromosome 20, NC_000020.11 (1894117-1939896). The protein sequence is encoded by exons 1–8 of this locus. Therefore, in some embodiments, the nucleic acid sequence containing the coding sequence for human SIRPα includes one or more exons 1–8 of the human SIRPα gene. In some examples, the nucleic acid sequence also includes aspects of the human SIRPα genomic locus, e.g., introns, 3' and / or 5' untranslated sequences (UTRs). In some examples, the nucleic acid sequence includes the entire region of the human SIRPα genomic locus. In some examples, the nucleic acid sequence includes exons 2–4 of the human SIRPα genomic locus.

[0099] In the humanized SIRPα non-human animal of the present invention, the nucleic acid sequence encoding the human SIRPα protein is operably ligated to one or more regulatory sequences of the SIRPα gene, for example, regulatory sequences of the SIRPα gene of a non-human animal. The SIRPα regulatory sequences of a non-human animal, such as a mouse, are sequences of the non-human animal SIRPα genomic locus that regulate the expression of non-human animal SIRPα, such as a 5' regulatory sequence, such as the SIRPα promoter, the SIRPα 5' untranslated region (UTR), a 3' regulatory sequence, such as the 3'UTR, and an enhancer.

[0100] A “promoter” or “promoter sequence” refers to a DNA regulatory region that can bind to RNA polymerase in a cell and initiate the transcription of a downstream (3' direction) coding sequence. The promoter sequence binds to the transcription initiation site at its 3' end and extends upstream (5' direction) to contain the minimum number of bases or elements necessary to initiate transcription at a detectable level above the background. Within the promoter sequence, the transcription initiation site and a protein-binding domain responsible for RNA polymerase binding are found. Eukaryotic promoters often contain, but not always, “TATA” and “CAT” boxes. This disclosure is of particular interest to DNA regulatory elements, such as promoters, that promote the transcription of human proteins in the same spatial and temporal expression patterns as those observed for the corresponding endogenous proteins—that is, in the same cells and tissues and over the same period of time.

[0101] Mouse SIRPα is located on chromosome 2, NC_000068.7 (129592606-129632228), and the mouse SIRPα coding sequence can be found in Genbank accession numbers NM_007547.4 (isoform 1), NM_001177647.2 (isoform 2), NM_001291019.1 (isoform 3), NM_001291020.1 (isoform 3), NM_001291021.1 (isoform 4), and NM_001291022.1 (isoform 5). The regulatory sequence of mouse SIRPα is well defined in the art and can be easily identified using in silico methods, for example by referring to the above Genbank accession numbers on the UCSC Genome Browser on the World Wide Web, or by experimental methods described in the art. In some cases, for example, when the nucleic acid sequence encoding the human SIRPα protein is located at the mouse SIRPα genome locus, the regulatory sequences that operably ligate to the human SIRPα coding sequence are endogenous to the mouse genome, i.e., native, meaning they existed in the mouse genome before the incorporation of the human nucleic acid sequence.

[0102] In some examples, humanized SIRPα non-human animals, such as mice, are generated by the random incorporation or insertion of a human nucleic acid sequence encoding the human SIRPα protein (including the fragments described above), i.e., a "human SIRPα nucleic acid sequence" or "human SIRPα sequence," into the genome. Typically, in such embodiments, the location of the nucleic acid sequence encoding the human SIRPα protein in the genome is not known. In other examples, humanized SIRPα non-human animals are generated by targeted incorporation or insertion of the human SIRPα nucleic acid sequence into the genome, such as homologous recombination. In homologous recombination, a polynucleotide is inserted into a target locus in the host genome, while simultaneously removing host genomic material, e.g., 50 base pairs (bp) or larger, 100 bp or larger, 200 bp or larger, 500 bp or larger, 1 kB or larger, 2 kB or larger, 5 kB or larger, 10 kB or larger, 15 kB or larger, 20 kB or larger, or 50 kB or larger, from the target locus. Therefore, for example, when creating a humanized SIRPα mouse possessing a nucleic acid sequence encoding the human SIRPα protein by targeting the mouse SIRPα locus with a human SIRPα nucleic acid sequence, some or all of the mouse sequence at the SIRPα locus, such as exons and / or introns, may be replaced with the human SIRPα nucleic acid sequence. In some such examples, the human SIRPα nucleic acid sequence is incorporated into the mouse SIRPα locus, thereby regulating the expression of the human SIRPα sequence by a native, i.e., endogenous regulatory sequence within the mouse SIRPα locus. In other words, the regulatory sequence(s) to which the nucleic acid sequence encoding the human SIRPα protein is operably linked are the native SIRPα regulatory sequences within the mouse SIRPα locus.

[0103] In some cases, the incorporation of a human SIRPα sequence does not affect the transcription of the gene into which it is incorporated. For example, when a human SIRPα sequence is incorporated as an intein into a coding sequence, or when the human SIRPα sequence contains a 2A peptide, the human SIRPα sequence is transcribed and translated simultaneously with the gene into which it is incorporated. In other cases, the incorporation of a human SIRPα sequence inhibits the transcription of the gene into which it is incorporated. For example, during homologous recombination, some or all of the coding sequence at the target locus may be removed and the human SIRPα sequence transcribed in its place. In some such cases, the incorporation of a human SIRPα sequence results in the generation of a null mutation, and therefore a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of that gene. This can result in the complete absence of a gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product. At the phenotypic level, a null allele is indistinguishable from a deletion at an entire locus.

[0104] In some cases, a humanized SIRPα non-human animal, such as a mouse, possesses one copy of the nucleic acid sequence encoding the human SIRPα protein. For example, the non-human animal may be heterozygous with respect to the nucleic acid sequence. In other words, one allele at the locus contains the nucleic acid sequence, and the other is an endogenous allele. For example, as described above, in some cases, the human SIRPα nucleic acid sequence is incorporated into the SIRPα locus of a non-human animal, such as a mouse, thereby generating a null allele in the non-human animal SIRPα. In some such embodiments, the humanized SIRPα non-human animal may be heterozygous with respect to the nucleic acid sequence encoding human SIRPα, i.e., the humanized SIRPα non-human animal possesses one null allele (the allele containing the nucleic acid sequence) and one endogenous SIRPα allele (wild type or otherwise) in the non-human animal SIRPα. In other words, the non-human animal possesses SIRPα h / mThis refers to a non-human animal, where "h" represents the allele containing the human sequence and "m" represents the endogenous allele. In other examples, humanized SIRPα contains two copies of the nucleic acid sequence encoding the human SIRPα protein. For example, a non-human animal, such as a mouse, may be homozygous with respect to the nucleic acid sequence, i.e., both alleles at the locus in the diploid genome contain the nucleic acid sequence, i.e., humanized SIRPα in a non-human animal has two null alleles (alleles containing the nucleic acid sequence) in the non-human animal SIRPα. In other words, a non-human animal may have SIRPα h / h It is a non-human animal.

[0105] In some embodiments, humanized SIRPα non-human animals, such as mice, have other genetic modifications. In some embodiments, humanized SIRPα non-human animals are immunodeficient animals. For example, humanized SIRPα non-human animals may possess at least one null allele in the Rag2 gene ("recombinant activator gene 2," the coding sequence of its mouse gene can be found at Genbank accession number NM_009020.3). In some embodiments, humanized SIRPα non-human animals possess two null alleles in Rag2. In other words, humanized SIRPα non-human animals are homozygous null with respect to Rag2. As another example, humanized SIRPα non-human animals possess at least one null allele in the IL2rg gene ("interleukin 2 receptor γ," also known as common γ chain or γC, the coding sequence of its mouse gene can be found at Genbank accession number NM_013563.3). In some embodiments, humanized SIRPα non-human animals possess two null alleles for IL2rg. In other words, humanized SIRPα non-human animals have a homozygous null for IL2rg, i.e., IL2rg - / - (Alternatively, if the IL2rg gene is located on the X chromosome, as in mice, then IL2rg Y / - In some embodiments, SIRPα non-human animals possess a null allele for both Rag2 and IL2rg, i.e., it is Rag2 - / - IL2rg - / -(Alternatively, if the IL2rg gene is located on the X chromosome, as in mice, Rag2 - / - IL2rg Y / - Other gene modifications are also being considered. For example, a humanized SIRPα non-human animal may include modifications in other genes related to the development and / or function of hematopoietic cells and the immune system, e.g., substituting one or more other non-human animal genes with nucleic acid sequences encoding human orthologs. In addition to or instead of this, a humanized SIRPα non-human animal may include modifications in genes related to the development and / or function of other cells and tissues, e.g., genes associated with human disorders or diseases, or, in the case of modifications in non-human animals, e.g., mice, genes that provide models of human disorders and diseases.

[0106] Humanized IL-15 non-human animals In some aspects of this disclosure, a humanized IL-15 non-human animal is provided. A humanized IL-15 non-human animal, or "IL-15 non-human animal," means a non-human animal possessing a nucleic acid sequence encoding the human IL-15 protein. As used herein, "human IL-15 protein" means a wild-type (or natural) human IL-15 protein or a variant of the wild-type (or natural) human IL-15 protein that retains one or more signaling functions of the wild-type (or natural) human IL-15 protein, for example, a protein that can stimulate (or signal via) the human IL-15 receptor and / or can bind to the human IL-15 receptor α subunit of the human IL-15 receptor and / or can bind to IL-2Rβ / IL-15Rβ and the common γ chain (γc). The term “human IL-15 protein” also encompasses fragments of wild-type human IL-15 protein (or its variants) that retain one or more signaling functions of wild-type human IL-15 protein, for example, that can stimulate (or signal through) the human IL-15 receptor and / or bind to the human IL-15 receptor α subunit of the human IL-15 receptor and / or bind to IL-2Rβ / IL-15Rβ and the common γ chain (γc).

[0107] The term "human IL-15 protein" also includes fusion proteins, i.e., chimeric proteins, that contain one or more fragments of wild-type human IL-15 protein (or its variants) while retaining one or more signaling functions of wild-type human IL-15 protein, such as those described above. Fusion proteins containing one or more fragments of wild-type human IL-15 protein (or its variants) may also be referred to as humanized IL-15 proteins in this specification.

[0108] Therefore, the nucleic acid sequence encoding the human IL-15 protein is a polypeptide containing the coding sequence of the human IL-15 protein, that is, for example, the wild-type human IL-15 protein, a variant of the wild-type human IL-15 protein, a fragment of the wild-type human IL-15 protein (or its variant) that retains one or more signaling functions of the wild-type human IL-15 protein, or a fusion protein, i.e., a chimeric protein, that contains one or more fragments of the wild-type human IL-15 protein (or its variant) and also retains one or more signaling functions of the wild-type human IL-15 protein.

[0109] IL-15 (also known as "interleukin-15") is a cytokine that stimulates the proliferation of T lymphocytes. The polypeptide sequence and the nucleic acid sequence encoding wild-type human IL-15 can be found in Genbank accessions NM_000585.4, NP_000576.1 (isoform 1), NM_172175.2, and NP_751915.1 (isoform 2). The genomic locus encoding the wild-type human IL-15 protein can be found in the human genome on chromosome 4, NC_000004.12 (141636596-141733987). The human IL-15 locus contains eight exons, with exons 3-8 being coding exons. As such, in some embodiments, the nucleic acid sequence having the coding sequence for human IL-15 includes one or more exons 3-8 (i.e., coding exons 1-6, see Figure 2) of the human IL-15 gene. For example, various IL-15 mRNA isoforms produced by the following combinations of exon usage have been identified: exons 1-2-3-4-5-6-7-8, exons 1-3-4-5-6-7-8, or exons 1-3-4-(selective exon 5)-5-6-7-8). In some examples, the nucleic acid sequence also includes aspects of the human IL-15 genomic locus, e.g., introns, 3' and / or 5' untranslated sequences (UTRs). In some examples, the nucleic acid sequence includes the entire region of the human IL-15 genomic locus. In some examples, the nucleic acid sequence includes exons 5-8 (i.e., coding exons 3-6) of the human IL-15 genomic locus.

[0110] In some cases, the human IL-15 protein according to this disclosure has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity with respect to SEQ ID NO:31.

[0111] In the humanized IL-15 non-human animals of this application, the nucleic acid sequence encoding the human IL-15 protein is operably linked to one or more regulatory sequences of the IL-15 gene, e.g., regulatory sequences of the non-human animal IL-15 gene. The IL-15 regulatory sequences of the non-human animal, e.g., mouse, are regulatory sequences of the non-human animal IL-15 genomic locus that regulate non-human animal IL-15 expression, e.g., 5' regulatory sequences, e.g., the IL-15 promoter, the 5' untranslated region (UTR) of IL-15, 3' regulatory sequences, e.g., the 3'UTR, and enhancers. Mouse IL-15 is located on chromosome 8, NC_000074.6 (82331624-82403227, complement), and the mouse IL-15 coding sequence can be found in Genbank accession numbers NM_008357.2 (mutant 1) and NM_001254747.1 (mutant 2). The regulatory sequences of mouse IL-15 are well defined in the art and can be readily identified using in silico methods, for example by referring to the Genbank accession number on the UCSC Genome Browser at genome.ucsc.edu on the World Wide Web, or by experimental methods as described in the art. In some cases, for example, when the nucleic acid sequence encoding the human IL-15 protein is located at the mouse IL-15 genome locus, the regulatory sequences that operably ligate to the human IL-15 coding sequence are endogenous, i.e., native, meaning they existed in the mouse genome before the incorporation of the human nucleic acid sequence.

[0112] In some examples, humanized IL-15 non-human animals, such as mice, are generated by the random incorporation or insertion of a human nucleic acid sequence encoding the human IL-15 protein (including the fragments mentioned above), i.e., a "human IL-15 nucleic acid sequence" or "human IL-15 sequence," into the genome. Typically, in such embodiments, the location of the nucleic acid sequence encoding the human IL-15 protein in the genome is not known. In other examples, humanized IL-15 non-human animals are generated by targeted incorporation or insertion of the human IL-15 nucleic acid sequence into the genome, such as homologous recombination. In homologous recombination, a polynucleotide is inserted into a target locus in the host genome, while simultaneously removing host genomic material, e.g., 50 base pairs (bp) or larger, 100 bp or larger, 200 bp or larger, 500 bp or larger, 1 kB or larger, 2 kB or larger, 5 kB or larger, 10 kB or larger, 15 kB or larger, 20 kB or larger, or 50 kB or larger, from the target locus. Therefore, for example, when creating a humanized IL-15 mouse possessing a nucleic acid sequence encoding the human IL-15 protein by targeting the mouse IL-15 locus with a human IL-15 nucleic acid sequence, some or all of the mouse sequence within the IL-15 locus, such as exons and / or introns, may be replaced with the human IL-15 nucleic acid sequence. In some such examples, the human IL-15 nucleic acid sequence is incorporated into the mouse IL-15 locus, thereby regulating the expression of the human IL-15 sequence by a native, i.e., endogenous regulatory sequence within the mouse IL-15 locus. In other words, the regulatory sequence(s) to which the nucleic acid sequence encoding the human IL-15 protein is operably linked is the native IL-15 regulatory sequence within the mouse IL-15 locus.

[0113] In some examples, the integration of the human IL-15 sequence does not affect the transcription of the gene into which the human IL-15 sequence is integrated. For example, when the human IL-15 sequence is integrated into the coding sequence as an intein, or when the human IL-15 sequence contains a 2A peptide, the human IL-15 sequence is transcribed and translated simultaneously with the gene into which the human IL-15 sequence is integrated. In other examples, the integration of the human IL-15 sequence inhibits the transcription of the gene into which the human IL-15 sequence is integrated. For example, upon integration of the human IL-15 sequence by homologous recombination, part or all of the coding sequence of the integration locus may be removed and replaced with the human IL-15 sequence for transcription. In some such examples, as a result of the integration of the human IL-15 sequence, a null mutation, and thus a null allele, is generated. A null allele is a mutant copy of a gene that completely lacks the normal function of that gene. This can be the result of the complete absence of the gene product (protein, RNA) at the molecular level or the expression of a non-functional gene product. At the phenotypic level, a null allele cannot be distinguished from a deletion of the entire locus.

[0114] In some examples, a humanized IL-15 non-human animal, such as a mouse, harbors one copy of a nucleic acid sequence encoding the human IL-15 protein. For example, the non-human animal may be heterozygous with respect to the nucleic acid sequence. In other words, one allele at the locus contains the nucleic acid sequence and the other is an endogenous allele. For example, as described above, in some examples, the human IL-15 nucleic acid sequence is integrated into the IL-15 locus of a non-human animal, such as a mouse, thereby generating a null allele in the non-human animal IL-15. In some such embodiments, the humanized IL-15 non-human animal may be heterozygous with respect to the nucleic acid sequence encoding human IL-15, i.e., the humanized IL-15 non-human animal harbors one null allele (the allele having the nucleic acid sequence) and one endogenous IL-15 allele (wild-type or otherwise) in the non-human animal IL-15. In other words, the non-human animal has IL-15 h / mNon-human animals, where "h" represents the allele having the human sequence and "m" represents the endogenous allele. In other examples, humanized IL-15 contains two copies of the nucleic acid sequence encoding the human IL-15 protein. For example, a non-human animal, such as a mouse, may be homozygous with respect to the nucleic acid sequence, i.e., both alleles at the locus in the diploid genome have the nucleic acid sequence, i.e., a humanized IL-15 non-human animal has two null alleles (alleles having the nucleic acid sequence) in non-human animal IL-15. In other words, a non-human animal has IL-15 h / h It is a non-human animal.

[0115] Humanized SIRPα-IL-15 non-human animals By crossing the humanized IL-15 non-human animal described above with a humanized SIRPα non-human animal of the same species as described above, it is possible to produce a genetically modified non-human animal that expresses both human SIRPα and human IL-15. In some embodiments, such a genetically modified non-human animal is an immunodeficient animal, for example, an animal lacking an endogenous immune system as a result of having a null allele for one or both of Rag2 and IL2rg. For example, in some embodiments, the non-human animal according to this disclosure is Rag2 - / - and / or IL2rg - / - (Alternatively, if the IL2rg gene is located on the X chromosome, as in mice, Rag2 - / - and / or IL2rg Y / - ) In some embodiments, a genetically modified non-human animal, e.g., a mouse, is provided, where the genetically modified non-human animal, e.g., a mouse, is SIRPα h / m IL-15 h / m Rag2 - / - IL2rg Y / - , SIRPα h / h IL-15 h / m Rag2 - / - IL2rg Y / - , or SIRPα h / m IL-15 h / h Rag2 - / - IL2rg Y / - That is the case.

[0116] In some embodiments, the present invention provides a genetically modified non-human animal, such as a mouse, that possesses a nucleic acid sequence incorporated into the genome of a genetically modified non-human animal, which encodes the human SIRPα protein and is operably linked to the SIRPα gene promoter, and a nucleic acid sequence incorporated into the genome of a genetically modified non-human animal, which encodes the human IL-15 protein and is operably linked to the IL-15 gene promoter, and which expresses the human SIRPα protein and the human IL-15 protein.

[0117] In some embodiments, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter. In some such embodiments, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter within the non-human animal SIRPα locus. In another embodiment, the SIRPα gene promoter is a human SIRPα promoter.

[0118] In some embodiments, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter. In some such embodiments, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter within a non-human animal IL-15 locus. In another embodiment, the IL-15 promoter is a human IL-15 promoter.

[0119] In some embodiments, the genetically modified non-human animals described herein express human IL-15 mRNA in the liver, lungs, bone marrow (BM), small intestine (SI), and colon.

[0120] In some embodiments, genetically modified non-human animals expressing both human SIRPα and human IL-15 as described herein, such as mice, exhibit a higher proportion and number of human T cells and NK cells after engraftment of human hematopoietic cells, such as CD45+ cells, compared to genetically modified non-human animals expressing only human SIRPα, such as mice. In some embodiments, genetically modified non-human animals expressing both human SIRPα and human IL-15 as described herein, such as mice, exhibit a higher proportion and number of NK cells in the blood and spleen. In some embodiments, genetically modified non-human animals expressing both human SIRPα and human IL-15 as described herein, such as mice, exhibit a higher proportion and number of human NK cell subset CD56 in the blood, spleen and liver after engraftment of human hematopoietic cells, such as CD45+ cells. bright CD16 - and CD56 dim CD16 + It possesses both. In some embodiments, genetically modified non-human animals expressing both human SIRPα and human IL-15 as described herein, such as mice, exhibit a distribution of CD16+ NK cells versus CD16-NK cells in the blood similar to the distribution of CD16+ NK cells versus CD16-NK cells in PBMCs isolated from human subjects.

[0121] In some embodiments, genetically modified non-human animals expressing both human SIRPα and human IL-15 as described herein, such as mice, possess NK cells in their livers that exhibit higher CD16 and CD56 expression levels. This indicates that NK cells mature more rapidly after engraftment of human hematopoietic cells, such as CD45+ cells, compared to genetically modified non-human animals expressing only human SIRPα, such as mice.

[0122] In some embodiments, genetically modified non-human animals, such as mice, that express both human SIRPα and human IL-15 as described herein and engraft human hematopoietic cells, such as CD45+ cells, have a higher proportion of CD56-expressing cells. dim CD16 +Along with the NK cell population, the spleen contains NK cells exhibiting clearly defined levels of killer inhibitory receptors, similar to those found in NK cell subsets in human blood.

[0123] In some embodiments, as described herein, genetically modified non-human animals expressing both human SIRPα and human IL-15 and engrafted with human hematopoietic cells, such as CD45+ cells, such as mice, exhibit a higher frequency of human CD45+ and CD8+ T cells in the intraepithelial lymphocyte population compared to genetically modified non-human animals expressing only human SIRPα, such as mice. In some embodiments, as described herein, genetically modified non-human animals expressing both human SIRPα and human IL-15 and engrafted with human hematopoietic cells, such as mice, exhibit a distribution of CD16+ NK cells in the IEL comparable to that of CD16-NK cells, and a higher distribution of CD16+ NK cells in the blood and spleen than that of CD16-NK cells, which reflects normal human physiological function.

[0124] In some embodiments, genetically modified non-human animals, such as mice, that express both human SIRPα and human IL-15 and engraft human hematopoietic cells, such as CD45+ cells, as described herein, exhibit a greater number of human T cells in their lungs compared with genetically modified non-human animals, such as mice, that express only human SIRPα. In some such embodiments, such genetically modified non-human animals, such as mice, express CD69 at higher levels on human CD8+ T cells in their lungs compared with genetically modified non-human animals, such as mice, that express only human SIRPα.

[0125] In some embodiments, genetically modified non-human animals, such as mice, that express both human SIRPα and human IL-15 and engraft human hematopoietic cells, such as CD45+ cells, as described herein, exhibit higher levels of CD69 expression on human CD8+ T cells in the liver compared to genetically modified non-human animals, such as mice, that express only human SIRPα.

[0126] In some embodiments, as described herein, genetically modified non-human animals, such as mice, that express both human SIRPα and human IL-15 and engraft human hematopoietic cells exhibit recognizable Peyer's patches, which are primarily human CD45+.

[0127] Any non-human mammal may be genetically modified in accordance with this disclosure. Non-limiting examples include laboratory animals, domesticated animals, livestock, etc., such as mice, rodents, dogs, cats, pigs, horses, cattle, sheep, non-human primates, such as mice, rats, rabbits, hamsters, guinea pigs, cattle, pigs, sheep, goats, and other transgenic animal species well known in the art, particularly mammalian species. In other embodiments, the non-human animal may be a pheasant such as a chicken, turkey, quail, pheasant, or partridge; an anseriform such as a duck, goose, or swan; or a bird such as a pigeon or dove. In various embodiments, the genetically modified animal of the present invention is a mouse, rat, or rabbit.

[0128] In some embodiments, the non-human animal is a mammal. In some such embodiments, the non-human animal is, for example, a small mammal of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modified animal is derived from a family selected from Calomyscidae (e.g., mouse-like hamster), Cricetidae (e.g., hamster, New World rat and mouse, field vole), Muridae (true mouse and rat, gerbil, spiny mouse, maned mouse), Nesomyidae (tree mouse, rock mouse, naked mouse, Madagascar rat and mouse), Platacanthomyidae (e.g., spiny dormouse), and Spalacidae (e.g., mole rat, bamboo rat, and burrowing mouse). In a specific embodiment, the genetically modified rodent is selected from true mouse or rat (Muridae), gerbil, spiny mouse, and maned mouse.

[0129] In one embodiment, the genetically modified non-human animal of the present invention is a rat. In one such embodiment, the rat is selected from Wistar rat, LEA strain, Sprague Dawley strain, Fischer strain, F344, F6, and Dark Agouti. In another embodiment, the rat strain is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0130] In another embodiment, the genetically modified non-human animal of the present invention is a mouse, for example, a mouse of the C57BL strain (e.g., C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, C57BL / Ola, etc.) These include mice of the 129 strain (e.g., 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2), and mice of the BALB strain, e.g., BALB / c. See, for example, Festing et al. (1999) Mammalian Genome 10:836, and also Auerbach et al. (2000) Establishment and Chimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In another embodiment, the mice are a mixture of the above strains.

[0131] In some embodiments, the genetically modified non-human animals of the present invention are also immunodeficient. "Immunodeficiency" means having a deficiency in one or more aspects of the animal's natural or endogenous immune system, for example, the animal is deficient in one or more types of functional host immune cells, such as the number and / or function of non-human B cells, the number and / or function of non-human T cells, the number and / or function of non-human NK cells, etc.

[0132] One method for achieving immunodeficiency in the animals of the present invention is sublethal radiation. For example, neonatal offspring of genetically modified mice can be irradiated with sublethal doses, for example, 2 × 200 cGy at 4-hour intervals. Alternatively, immunodeficiency may be achieved by any one of the many gene mutations known in the art, any of which may be incorporated into the genetically modified non-human animals of the present disclosure, either alone or in combination, or used as a source of stem cells into which the gene mutations of the present disclosure can be introduced. Non-limiting examples include X-linked SCID associated with IL2rg gene mutations and characterized by lymphocyte phenotype T(-)B(+)NK(-), autosomal recessive SCID associated with Jak3 gene mutations and characterized by lymphocyte phenotype T(-)B(+)NK(-), ADA gene mutations characterized by lymphocyte phenotype T(-)B(-)NK(-), IL-7Rα chain mutations characterized by lymphocyte phenotype T(-)B(+)NK(+), and lymphocyte phenotype Examples include CD3δ or ε mutations characterized by the present-day T(-)B(+)NK(+) phenotype, RAG1 and RAG2 mutations characterized by the lymphocyte phenotype T(-)B(-)NK(+), Artemis gene mutations characterized by the lymphocyte phenotype T(-)B(-)NK(+), CD45 gene mutations characterized by the lymphocyte phenotype T(-)B(+)NK(+), and Prkdcscid mutations characterized by the lymphocyte phenotype T(-)B(-). Thus, in some embodiments, the genetically modified immunodeficient non-human animal has an IL2 receptor γ chain (I12rg y / - The animal has one or more deficiencies selected from the following: ) deficiency, Jak3 deficiency, ADA deficiency, IL7R deficiency, CD3 deficiency, RAG1 and / or RAG2 deficiency, Artemis deficiency, CD45 deficiency, and Prkdc deficiency. These and other animal models of immunodeficiency are well known to those skilled in the art, and any of them may be used to construct the immunodeficient animals of this disclosure.

[0133] In some embodiments, the genetically modified non-human animal according to the present invention can be used as a recipient of human hematopoietic cells, and human immune cells can be generated from the engrafted human hematopoietic cells. Thus, in some aspects of the present invention, the genetically modified animal of the present invention is a genetically modified immunodeficient non-human animal in which human hematopoietic cells have been engrafted.

[0134] Humanized SIRPα-IL-15 engraftment in non-human animals As described above, in some aspects of the present invention, humanized SIRPα-IL-15 non-human animals, such as mice, such as Rag2 - / - IL2rg Y / - Cells are engrafted, i.e., transplanted, into hSIRPα hIL-15 mice or hSIRPα hIL-15 mice irradiated with a sublethal dose of radiation. The cells may be mitotic cells or postmitotic cells, and include cells of interest such as pluripotent stem cells, e.g., ES cells, iPS cells, and embryonic germ cells, as well as somatic cells, e.g., fibroblasts, hematopoietic cells, neurons, muscle cells, osteocytes, vascular endothelial cells, intestinal cells, etc., and their lineage-restricted progenitor and precursor cells. Cell populations of particular interest include cell populations containing hematopoietic stem cells or hematopoietic progenitor cells, which contribute to or reconstitute the hematopoietic system of humanized SIRPα-IL-15 non-human animals, e.g., peripheral blood leukocytes, fetal hepatocytes, fetal bone, fetal thymus, fetal lymph nodes, vascularized skin, arterial segments, and purified hematopoietic stem cells, e.g., recruited HSCs or umbilical cord blood HSCs.

[0135] Any source of human hematopoietic cells, human hematopoietic stem cells (HSCs) and / or hematopoietic stem cell progenitor cells (HSPCs) that is well known in the art or described herein may be transplanted into the genetically modified immunodeficient non-human animals of this disclosure. One suitable source of human hematopoietic cells that is well known in the art is human umbilical cord blood cells, in particular CD34-positive (CD34 +These are human hematopoietic cells. Another source of human hematopoietic cells is the human fetal liver. Another source is the human bone marrow. It also includes induced pluripotent stem cells (iPSCs) and induced hematopoietic stem cells (iHSCs) produced, for example, by dedifferentiation of somatic cells using methods known in this field.

[0136] The cells may be derived from any mammalian species, such as mice, rodents, dogs, cats, horses, cattle, sheep, primates, or humans. The cells may be derived from established cell lines or primary cells, where “primary cells,” “primary cell lines,” and “primary cultures” are used interchangeably herein and refer to cells and cell cultures derived from the subject that can be grown in vitro for a limited number of passages, i.e., a limited number of divisions. For example, a primary culture may have undergone 0, 1, 2, 4, 5, 10, or 15 passages, but has not yet reached a critical stage. Typically, the primary cell lines of the present invention are maintained in vitro for fewer than 10 passages.

[0137] If the cells are primary cells, they may be collected from the individual by any convenient method. For example, cells, such as blood cells or leukocytes, may be collected by apheresis, leukocyte removal, density gradient separation, etc. Another example is that cells, such as tissues from the skin, muscle, bone marrow, spleen, liver, pancreas, lungs, intestines, or stomach, may be collected by biopsy. A suitable solution may be used for the dispersion or suspension of the collected cells. Such solutions are generally equilibrium salt solutions, such as physiological saline, PBS, or Hanks equilibrium salt solution, conveniently supplemented with fetal bovine serum or other natural developmental factors along with a low concentration, usually 5-25 mM, acceptable buffer. Convenient buffers include HEPES, phosphate buffer, and lactate buffer.

[0138] In some cases, a heterogeneous population of cells is transplanted into a humanized non-human animal, such as a mouse. In other cases, a population of cells enriched with a specific type of cell, such as progenitor cells, such as hematopoietic progenitor cells, is engrafted into a humanized non-human animal, such as a mouse. Enrichment of the cell population of interest may be carried out by any simple separation technique. For example, the target cells may be enriched by a culture method. In such a culture method, specific growth factors and nutrients are usually added to the culture to promote the survival and / or proliferation of one cell population more than others. Other culture conditions that affect survival and / or proliferation include growth on an adherent or non-adherent substrate and culture for a specific period. Such culture conditions are well known in the art. As another example, the cells of interest may be enriched by separating the target cells from an initial population by affinity separation techniques. Affinity separation techniques include magnetic separation using magnetic beads coated with affinity reagents, affinity chromatography, affinity reagents bound to a solid matrix such as a plate, "panning" with cytotoxic agents such as complement and cytotoxic agents bound to or used in combination with affinity reagents, or other simple techniques. Techniques that provide accurate separation include fluorescence-activated cell sorters that can have varying degrees of sophistication, such as multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels. Cells may be selected for dead cells using dyes associated with cell death (e.g., propidium iodide). Any technique that is not excessively harmful to the viability of the cells of interest may be used.

[0139] For example, affinity separation techniques can be used to deplete cells that are not of interest for transplantation by exposing a population to an affinity reagent that specifically recognizes and selectively binds to markers not expressed on cells of interest. For instance, to enrich a population of hematopoietic progenitor cells, cells expressing mature hematopoietic cell markers may be depleted. In addition to or instead of this, positive selection and separation may be performed by exposing a population to an affinity reagent that specifically recognizes and selectively binds to markers associated with hematopoietic progenitor cells, such as CD34 and CD133. "Selectively binding" means that a molecule preferentially binds to the target of interest over other molecules, or binds to the target with greater affinity. For example, an antibody binds to molecules containing a specific epitope and does not bind to unrelated epitopes. In some embodiments, the affinity reagent may be an antibody, i.e., an antibody specific to CD34, CD133, etc. In some embodiments, the affinity reagent may be a specific receptor or ligand for CD34, CD133, etc., such as a peptide ligand and receptor, an effector and receptor molecule, or a T cell receptor specific to CD34, CD133, etc. In some embodiments, multiple affinity reagents specific to the marker of interest may be used.

[0140] Antibodies and T cell receptors used as affinity reagents may be monoclonal or polyclonal and may be produced by transgenic animals, immunized animals, immortalized human or animal B cells, or cells transfected with DNA vectors encoding antibodies or T cell receptors. Details of antibody preparation and their suitability for use as specific binding members are well known to those skilled in the art. Of particular interest is the use of labeled antibodies as affinity reagents. These antibodies can be easily conjugated with labeling substances for separation. Labeling substances include magnetic beads that enable direct separation, biotin that can be removed by binding avidin or streptavidin to a support, and fluorescent dyes that can be used with fluorescence-activated cell sorters, which can facilitate the separation of specific cell types. Fluorescent dyes that are expected to be used include, for example, phycobilin proteins such as phycoerythrin and allophycocyanin, fluorescein, and Texas Red. Often, each antibody is labeled with a different fluorescent dye so that each marker can be sorted individually.

[0141] The initial population of cells is brought into contact with affinity reagents(s) and incubated for a sufficient time to bind to available cell surface antigens. Incubation is typically at least about 5 minutes and usually less than about 60 minutes. The antibody in the reaction mixture should be at a sufficient concentration so that separation efficiency is not limited by a lack of antibody. The appropriate concentration is determined by titration, but generally the dilution ratio of the antibody to the volume of the cell suspension is about 1:50 (i.e., 1 part antibody per 50 parts reaction volume), about 1:100, about 1:150, about 1:200, about 1:250, about 1:500, about 1:1000, about 1:2000, or about 1:5000. The medium in which the cells are suspended is any medium that maintains the viability of the cells. Preferred media are phosphate-buffered saline containing 0.1–0.5% BSA or 1–4% goat serum. Various culture media are commercially available and can be used depending on the properties of the cells. Examples include Dulbecco's modified Eagle medium (dMEM), Hanks' basic salt solution (HBSS), Dulbecco's phosphate-buffered saline (dPBS), RPMI, Iskov medium, and PBS containing 5 mM EDTA. These are often supplemented with fetal bovine serum, BSA, HSA, goat serum, etc.

[0142] Cells in a population are selected by contact with an affinity reagent and labeled, for example, by any simple affinity separation technique known in the art, or the one described above. After separation, the separated cells may be collected in any suitable medium that maintains the viability of the cells, and the medium usually has a serum cushion at the bottom of the collection tube. Various media are commercially available and may be used depending on the properties of the cells, such as dMEM, HBSS, dPBS, RPMI, and Iskoff medium, and these are often supplemented with fetal bovine serum.

[0143] A composition highly enriched with the cell type of interest, for example, hematopoietic cells, is thus achieved. The cells constitute about 70%, about 75%, about 80%, about 85%, about 90% or more of the cell composition, about 95% or more of the enriched cell composition, and preferably about 95% or more of the enriched cell composition. In other words, the composition is a substantially pure composition of the cells of interest.

[0144] Cells to be transplanted into humanized SIRPα-IL-15 non-human animals (e.g., mice) may be transplanted immediately if they are heterogeneous or enriched cell populations. Alternatively, cells may be frozen at liquid nitrogen temperature and stored for a long period, then thawed and reused. In such cases, cells are usually frozen in 10% DMSO, 50% serum, 40% buffered medium, or any other solution commonly used in the art, stored at such freezing temperatures, and thawed using methods commonly known in the art for thawing frozen cultured cells. In addition to or instead of this, cells may be cultured in vitro under various culture conditions. The culture medium may be liquid or semi-solid, such as agar or methylcellulose. Cell populations may be conveniently suspended in a suitable nutrient medium such as Iskoff modified DMEM or RPMI-1640 supplemented with, for example, fetal bovine serum (about 5-10%), L-glutamine, thiols, especially 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture may contain growth factors to which the cells respond. Growth factors, as defined herein, are molecules capable of promoting cell survival, proliferation, and / or differentiation in either a culture or intact tissue through specific effects on transmembrane receptors. Growth factors include polypeptides and non-polypeptide factors.

[0145] Cells may be genetically modified before transplantation into non-human animals, such as mice, to provide a selectable or trackable marker, induce a genetic defect in the cells (e.g., for disease modeling), repair the genetic defect, or ectopically express the gene within the cell (e.g., to determine whether such modification affects the course of the disease). Cells may be genetically modified by transduction or translocation with a suitable vector, homologous recombination, or other suitable techniques to express the gene of interest, or to block the expression of an undesirable gene using antisense mRNA, siRNA, or ribozyme. Various techniques for introducing nucleic acids into target cells are known in the art. Various techniques may be used to confirm that the cells have been genetically modified. The cell genome may be used with restriction enzyme digestion, amplified, or unamplified. Polymerase chain reaction, gel electrophoresis, restriction enzyme analysis, Southern, Northern, and Western blotting, sequencing, etc., may be used.The general methods in molecular biology and cell biochemistry disclosed herein for these and other purposes are based on: Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons) These can be found in standard textbooks such as the one from 1998, and these disclosures are incorporated herein by reference. The reagents, cloning vectors, and kits for genetic manipulation referred to herein are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0146] For example, the cells may be transplanted into a humanized SIRPα-IL-15 non-human animal, such as a mouse, by any convenient method, including injection into the liver, tail vein, or posterior orbit. Generally, about 0.5 × 10⁻⁶ cells are used. 5 ~2×10 6 A number of pluripotent or progenitor cells, e.g., about 1 × 10⁶ 5 ~1 × 10 6 Cells, or approximately 2 × 10 5 ~5×10 5Cells are transplanted. In some cases, non-human animals, such as mice, are irradiated with a non-lethal dose of radiation, and then human cells are transplanted. In other words, non-human animals, such as mice, are exposed to a non-lethal dose of radiation, for example, as is well known in the art. The engrafted humanized SIRPα-IL-15 non-human animals, such as mice, are then maintained under experimental animal housing conditions for at least one week, for example, one week or more, or two weeks or more, sometimes four weeks or more, and in some cases, six weeks or more, for example, ten weeks or fifteen weeks or more, to allow the immune system to be sufficiently reconstituted by the engrafted cells.

[0147] Humanized SIRPα-IL-15 non-human animals, e.g., mice, and humanized SIRPα-IL-15 non-human animals with engrafted human hematopoietic cells, e.g., mice, e.g., engrafted Rag2 - / - IL2rg Y / -The hSIRPα hIL-15 mouse, and other gene modifications as needed, are useful for many applications. For example, these non-human animals, e.g., mice, provide a useful system for modeling human immune diseases and human pathogens. For example, the non-human animals of the present invention, e.g., mice, are useful for modeling the development and function of human T cells and / or natural killer (NK) cells, human pathogen infection of specific tissues and / or cells, e.g., human pathogen infection of the intestines or lungs, and / or human T cells and / or NK cells' response to or from human pathogen infection. Such non-human animals are also expected to be useful in in vivo screening of drugs that inhibit infection by pathogens, e.g., pathogens that affect specific tissues or cell types (e.g., by infection), e.g., human pathogens of the intestines or lungs, e.g., human pathogens that activate, induce, and / or target T cells and / or NK cells; in vivo screening of drugs that modulate the development and / or function of human T cells and / or NK cells in healthy or diseased states; in vivo screening of drugs that are toxic to human T cells and / or NK cells; in vivo screening of drugs that prevent, mitigate, or reverse the toxic effects of toxic substances on human T cells and / or NK cells; in vivo screening of candidate T cell-inducible vaccines; and in vivo and in vitro screening of drugs that inhibit tumor growth and / or infection by activation of antibody-dependent cell-mediated cytotoxicity (ADCC) processes mediated by NK cells.

[0148] This disclosure relates to humanized SIRPα-IL-15 non-human animals, such as mice, on which human hematopoietic cells have been engrafted, such as engrafted Rag2 - / - IL2rg Y / -The hSIRPαhIL-15 mouse provides unexpected results showing that it generates tissue-resident lymphocytes, e.g., intraepithelial lymphocytes in the intestine and lungs. Therefore, this disclosure provides a novel animal model that enables the monitoring and testing of such tissue-resident lymphocytes. Such an animal model is particularly useful for modeling the immune response of tissue-resident lymphocytes, e.g., T cells and NK cells, to human pathogens affecting the intestine and / or lungs (e.g., by infection), and for screening therapeutic agents and vaccines that target such pathogens and / or induce or enhance the tissue-resident lymphocyte response. Furthermore, the presence of these tissue-resident lymphocytes can also be used to model human immune cell-driven autoimmune diseases affecting the gastrointestinal tract, such as celiac disease and IBD.

[0149] Accordingly, in some embodiments, the present disclosure provides in vivo models comprising a genetically modified non-human animal having a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes the human SIRPα protein and is operably linked to the SIRPα gene promoter. The genetically modified non-human animal also has a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes the human IL-15 protein and is operably linked to the IL-15 gene promoter. Finally, the genetically modified non-human animal has engraftment of human hematopoietic cells, where the genetically modified non-human animal (i) expresses the human SIRPα protein and the human IL-15 protein, and (ii) has human tissue-resident lymphocytes in the genetically modified non-human gut, such as intraepithelial lymphocytes (IELs). In some such embodiments, the genetically modified non-human animal is infected with a human pathogen, such as a human pathogen that affects the gut (e.g., by infection).

[0150] Human pathogens that may affect the intestines (for example, through infection) include, but are not limited to, Campylobacter jejuni, Clostridium difficile, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, human rotavirus, Listeria monocytogenes, Norwalk virus, Salmonella enterica, Sigella flexneri, Sigella sonei, Shigella shiga, Plague bacillus, Yersinia enterocolitica, and Helicobacter pylori.

[0151] In other embodiments, the disclosure provides an in vivo model comprising a genetically modified non-human animal having a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes the human SIRPα protein and is operably linked to the SIRPα gene promoter. The genetically modified non-human animal also has a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal that encodes the human IL-15 protein and is operably linked to the IL-15 gene promoter. Finally, the genetically modified non-human animal has engraftment of human hematopoietic cells, where the genetically modified non-human animal (i) expresses the human SIRPα protein and the human IL-15 protein, and (ii) has human tissue-resident lymphocytes, such as intraepithelial lymphocytes (IELs), in the lungs of the genetically modified non-human animal. In some such embodiments, the genetically modified non-human animal is infected with a human pathogen, such as a human pathogen that affects the lungs (e.g., by infection).

[0152] Human pathogens that may affect the lungs (for example, through infection) include, but are not limited to, Streptococcus pyogenes, Haemophilus influenzae, Mycobacterium diphtheriae, SARS coronavirus, Bordetella pertussis, Moraxella catarrhalis, influenza viruses (A, B, C), coronaviruses, adenoviruses, RSV, parainfluenza virus, mumps virus, Streptococcus pneumoniae, Staphylococcus aureus, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Blastomyces dermatichidis, Cryptococcus neoformans, and Aspergillus fumigatus.

[0153] Novel therapeutic agents, novel vaccines, and novel methods for testing the efficacy of therapeutic agents and vaccines are needed. Non-human animals that support efficient human T cell and NK cell engraftment, such as mice, are considered useful in identifying novel therapeutic agents and vaccines, particularly against human pathogens that infect human T cells and / or NK cells. Novel therapeutic agents and vaccines can be tested in such non-human animals, such as mice, by measuring the amount of human pathogens, such as virus, in non-human animals (in the blood or in designated tissues) in response to presumptive antiviral therapy, or by inoculating mice with a presumptive vaccine, then exposing them to an infectious dose of a human pathogen, such as HIV, and observing any changes in infectivity due to presumptive vaccination compared to unvaccinated but HIV-infected controls.

[0154] Such non-human animal models of pathogen infections, such as mouse models, are useful for research aimed at better understanding the progression of infection in humans. These mouse infection models are also useful in drug discovery to identify candidate drugs for preventing or treating infections.

[0155] The engrafted genetically modified animals of this disclosure are expected to be used in screening candidate drugs to identify drugs that treat infections caused by human pathogens, such as human T cells and / or NK cells. The terms “to treat,” “to cure,” and “to treat” are used herein to generally encompass obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a disease in a mammal, including (a) preventing the development of the disease in a subject that is susceptible to the disease but has not yet been diagnosed as diseased, (b) inhibiting the disease, i.e., preventing its development, or (c) alleviating the disease, i.e., regressing the disease.

[0156] The terms “individual,” “subject,” “host,” and “patient” are used interchangeably herein and include any mammalian subject, in particular humans, who desire diagnosis, treatment, or therapy.

[0157] Humanized SIRPα-IL-15 non-human animals engrafted with human hematopoietic cells, such as mice, also provide a useful system for screening candidate drugs for other desired in vivo activities, such as drugs that modulate (i.e., promote or suppress) the development and / or activity of human T cells and NK cells in healthy or diseased states, for example, to identify novel therapeutic agents and / or to gain a better understanding of the molecular basis of immune system development and function, drugs that are toxic to T cells and / or NK cells and their progenitor cells, as well as drugs that prevent, mitigate or reverse the toxic effects of toxic substances on T cells, NK cells and their progenitor cells, antibodies or antigen-binding proteins that mediate NK cell-dependent ADCC processes, etc. As yet another example, the genetically modified mice described herein provide, for example, an in vivo platform for screening the responsiveness of an individual's immune system to drugs, such as therapeutic agents, and by predicting the individual's responsiveness to such drugs, provide a useful system for predicting an individual's responsiveness to disease treatment.

[0158] In screening assays for biologically active drugs, humanized SIRPα-IL-15 non-human animals, such as mice, engrafted with human hematopoietic cells and, in some cases, infected with human pathogens, such as engrafted Rag2 cells, are used. - / - IL2rg Y / -Contact a candidate agent of interest to hSIRPα hIL-15 mice or to cells engrafted into humanized SIRPα-IL-15 non-human animals, such as mice, and evaluate the effect of the candidate agent by monitoring one or more output parameters. These output parameters, such as the total number of hematopoietic cells or the number of cells of a specific hematopoietic cell type, by methods well known in the art, may reflect the viability of the cells, or, for example, the amount of DNA fragmentation, the amount of cell vesiculation, the amount of phosphatidylserine on the cell surface, etc. may reflect the apoptotic state of the cells. Alternatively, or in addition, the output parameter may reflect the differentiation ability of the cells, such as the proportion of differentiated cells and the types of differentiated cells, such as T cells and / or NK cells. Alternatively, or in addition, the output parameter may reflect the function of the cells, such as cytokines and chemokines produced by the cells, the ability of the cells to extravasate towards the challenge site, the ability of the cells to regulate, i.e., promote or inhibit, the activity of other cells in vitro or in vivo. Other output parameters may reflect the degree of pathogen infection in the animal, such as the titer of the pathogen in non-human animals, such as mice.

[0159] Parameters are preferably quantifiable components of cells in a high-throughput system, particularly components for which accurate measurement is possible. Parameters can be any cellular component or cellular product, including cell surface determinants, receptors, proteins or their conformational or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids such as mRNA, DNA, etc., or moieties derived from such cellular components, or combinations thereof. Most parameters provide quantitative readout information, although in some instances semi-quantitative or qualitative results are acceptable. The readout value may have a single measurement value or may include an average value, median value or variance, etc. Characteristically, a range of parameter readout values is obtained for each parameter from multiple identical assays. Variability is expected and a range of values is obtained using standard statistical methods along with common statistical methods used to provide a single value for each of the test parameter sets.

[0160] Candidate agents of interest for screening can include known and unknown compounds of numerous chemical classes with organic molecules as the main component, such as organometallic molecules, inorganic molecules, gene sequences, vaccines, antibiotics or other agents that may have antibiotic properties, peptides, polypeptides, antibodies, antigen-binding proteins, drugs approved as pharmaceuticals for use in humans, etc. An important aspect of the present invention is to evaluate candidate agents, which includes toxicity tests and the like.

[0161] Candidate drugs include organic molecules containing functional groups necessary for structural interactions, particularly hydrogen bonding, and generally contain at least an amine, carbonyl, hydroxyl, or carboxyl group, and often at least two functional chemical groups. Candidate drugs often contain cyclic carbon or heterocyclic structures and / or aromatic or polycyclic aromatic structures substituted with one or more of the above functional groups. Candidate drugs are also found in biomolecules including peptides, polynucleotides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Candidate drugs include pharmacologically active agents, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, hormones, or hormone antagonists. Examples of pharmaceutical agents suitable for the present invention are described in “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition. Candidate agents also include toxins, as well as biological and chemical weapons agents (see, for example, Somani, SM (Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992).

[0162] Candidate drugs of interest for screening include nucleic acids, such as siRNA, shRNA, antisense molecules, or nucleic acids encoding miRNA, or polypeptides. Many vectors useful for transferring nucleic acids into target cells are available. Vectors may be maintained as plasmids, minicircle DNA, or episomes such as viral vectors derived from cytomegalovirus, adenovirus, etc., or retroviral vectors such as MMLV, HIV-1, ALV may be incorporated into the target cell genome via homologous recombination or random integration. The vector may be delivered directly to the target cells. In other words, the vector containing the nucleic acid of interest is brought into contact with pluripotent cells, thereby causing the cells to take up the vector.

[0163] Methods for contacting cells, such as cultured cells, or cells in non-human animals, such as mice, with nucleic acid vectors, such as electroporation, calcium chloride translocation, and lipofection, are well known in the art. Alternatively, the nucleic acid of interest may be provided to cells via a virus. In other words, viral particles containing the nucleic acid of interest are brought into contact with the cells. Retroviruses, such as lentiviruses, are particularly suitable for the method of the present invention. Retroviral vectors commonly used are of the "deficient" type, that is, types that cannot produce the viral proteins necessary for proliferative infection. Rather, proliferation within a packaging cell line is necessary for vector replication. To generate viral particles containing the nucleic acid of interest, the retroviral nucleic acid containing the nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins that are incorporated into the capsid, and these envelope proteins determine the specificity of the viral particles in the cell. The envelope proteins consist of at least three types: narrow-host, broad-host, and heterospecific. Retroviruses packaged with narrow-host envelope proteins, such as MMLV, can infect most mouse and rat cell types and are produced using narrow-host packaging cell lines such as BOSC23 (Pear et al. (1993) PNAS 90:8392-8396). Retroviruses with broad-host envelope proteins, such as 4070A (Danos et al., see above), can infect most mammalian cell types, including human, dog, and mouse, and are produced using broad-host packaging cell lines such as PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437), PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902), and GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with heteromorphic envelope proteins, such as AKR env, can infect most mammalian cell types except mouse cells.Using an appropriate packaging cell line, cells of interest—in some cases engrafted cells, and in some cases host cells, i.e., humanized SIRPα-IL-15—may be reliably targeted by the packaged viral particles.

[0164] Vectors used to deliver nucleic acids of interest to target cells generally contain a suitable promoter to drive the expression, i.e., transcriptional activation, of the nucleic acid of interest. This may include ubiquitous promoters, such as the CMV-β-actin promoter, or inductive promoters, such as those that are active in a specific cell population or that respond to the presence of a drug like tetracycline. The transcriptional activation is intended to increase transcription in the target cells by at least about 10 times, at least about 100 times, and more commonly, at least about 1000 times, above the basal level. Furthermore, vectors used to deliver reprogramming factors to target cells may contain genes that need to be removed later, which are removed, for example, using a recombinase system such as Cre / Lox, or by using cells that express the gene and are destroyed by a gene that enables selective toxicity, such as herpesvirus TK, bcl-xs.

[0165] Polypeptides are also included among the candidate drugs of interest for screening. If necessary, such polypeptides may be fused to polypeptide domains that enhance the solubility of the product. These domains may be linked to the polypeptide via a specified protease cleavage site, e.g., a TEV sequence cleaved by a TEV protease. The linker may also have one or more flexible sequences, e.g., 1 to 10 glycine residues. In some embodiments, cleavage of the fusion protein is carried out in a buffer that maintains the solubility of the product, for example, in the presence of 0.5 to 2 M urea, the solubility-enhancing polypeptide, and / or polynucleotides. Domains of interest include endosomal degradation domains, e.g., influenza HA domains, and other polypeptides that assist in production, e.g., IF2 domains, GST domains, GRPE domains, etc. In addition to or instead of this, such polypeptides may be formulated for improved stability. For example, the peptide may be PEGylated, where the polyethyleneoxy group provides extended lifespan in the bloodstream. Polypeptides may be fused to other polypeptides to provide further functionality and, for example, enhance in vivo stability. Generally, such fusion partners are stable plasma proteins, and when present as fusions, they can, for example, extend the in vivo plasma half-life of polypeptides, especially if such stable plasma proteins are immunoglobulin constant domains. In most cases, stable plasma proteins are typically found in multimerized forms, such as immunoglobulins or lipoproteins, where identical or different polypeptide chains are usually linked disulfide and / or non-covalently to form aggregated multi-chain polypeptides, and the fusions herein containing such polypeptides are produced and used as multimers having substantially the same structure as the stable plasma protein precursors. These multimers may be homogeneous with respect to the polypeptide agents they contain, or they may contain multiple polypeptide agents.

[0166] Candidate polypeptides may be produced from eukaryotic cells or prokaryotic cells. They may be further treated by unfolding, such as thermal denaturation or DTT reduction, or further refolded using methods known in the art. Modifications of interest that do not alter the primary sequence include chemical derivatization of polypeptides, such as acylation, acetylation, carboxylation, and amidation. Glycosylation modifications are also included, which are carried out by altering the glycosylation pattern of polypeptides, for example, during polypeptide synthesis and processing, or in a further processing step by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylation enzymes or deglycosylation enzymes. Sequences containing phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine, or phosphothreonine, are also included. Polypeptides may be modified using conventional molecular biological techniques and synthetic chemistry to improve resistance to proteolysis, optimize solubility, or make them more suitable as therapeutic agents. Such polypeptide analogs include those containing residues other than naturally occurring L-amino acids, such as D-amino acids or unnaturally synthesized amino acids. Some or all of the amino acid residues may be substituted with D-amino acids.

[0167] Candidate polypeptides may be prepared by in vitro synthesis using conventional methods known in the art. Various commercially available synthesizers, such as automated synthesizers from Applied Biosystems, Inc., Beckman, etc., are available. Natural amino acids may be substituted with non-natural amino acids using the synthesizer. The specific sequence and preparation method are determined according to convenience, cost-effectiveness, required purity, etc. Alternatively, candidate polypeptides may be isolated and purified by conventional methods of recombinant synthesis. Lysates may be prepared in the expression host, and the lysates are purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In many cases, in relation to the method of product preparation and purification, the composition used contains at least 20% by weight, more generally at least about 75% by weight, preferably at least about 95% by weight, and generally at least about 99.5% by weight of the desired product. These percentages are usually based on total protein.

[0168] In some cases, the candidate polypeptide agents to be screened are antibodies or antigen-binding proteins. The term “antibody” or “antibody moiety” is intended to include any polypeptide chain-containing molecular structure having a specific shape that fits to and recognizes an epitope, where one or more non-covalent interactions stabilize the complex between the molecular structure and the epitope. The specific or selective fit of a given structure and its specific epitope is often referred to as a “lock and key” fit. Typical antibody molecules are immunoglobulins, and all types of immunoglobulins such as IgG, IgM, IgA, IgE, IgD, etc., derived from all sources, e.g., humans, rodents, rabbits, cattle, sheep, pigs, dogs, other mammals, chickens, other birds, etc., are considered “antibodies.” The antibodies used in this invention may be either polyclonal or monoclonal antibodies. Antibodies are generally provided in the culture medium for cells. Beyond antibodies, antigen-binding proteins similarly include polypeptides designed to bind to an antigen of interest and induce a response, such as an immunological reaction. The term “antigen-binding protein” also includes antigen-binding fragments known in the art (e.g., Fab, Fab' F(ab')2, Fabc, and scFv). The terms “antibody” and “antigen-binding protein” also include one or more immunoglobulin chains or fragments that can be expressed as fusion proteins with other proteins, single-chain antibodies, and bispecific antibodies, or chemically complexed with them.

[0169] Candidate drugs may be obtained from a wide range of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for the random and directional synthesis of a wide variety of organic compounds, including biomolecules, including the expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. Furthermore, libraries and compounds prepared naturally or synthetically may be readily modified by conventional chemical, physical, and biochemical means, and combinatorial libraries may be prepared using them. Known pharmacological agents may be subjected to directional or random chemical modifications such as acylation, alkylation, esterification, and amidation to produce structural analogs.

[0170] Candidate drugs are screened for their biological activity by administering the drug to at least one, usually multiple, samples, sometimes in combination with drug-free samples. Changes in parameters in response to the drug are measured, and the results are evaluated by comparing them with reference cultures, e.g., cultures in and without the drug, cultures obtained with other drugs, etc. When screening is performed to identify candidate drugs that prevent, mitigate, or reverse the effects of a toxic substance, the screening is generally performed in the presence of the toxic substance, and the toxic substance is added at the most appropriate time for the result to be determined. For example, when testing the protective / preventive capacity of a candidate drug, the candidate drug may be added before the toxic substance, simultaneously with the candidate drug, or after treatment with the candidate drug. As another example, when testing the ability of a candidate drug to reverse the effects of a toxic substance, the candidate drug may be added after treatment with the candidate drug. As described above, in some examples, the sample is a humanized SIRPα-IL-15 non-human animal with engrafted cells, e.g., a mouse, i.e., the candidate drug is supplied to a humanized SIRPα-IL-15 non-human animal with engrafted cells, e.g., a mouse. In some cases, the sample is the cells to be engrafted, i.e., the cells are supplied with candidate drugs before transplantation.

[0171] When a candidate drug is administered directly to a non-human animal, such as a mouse, the drug may be administered by any of the many methods known in the art for administering peptides, small molecules, and nucleic acids. For example, the drug may be administered orally, mucosally, topically, intradermally, or by injection, such as intraperitoneal, subcutaneous, intramuscular, intravenous, or intracranial injection. The drug may be administered in a buffer solution or, for example, in combination with a suitable pharmaceutically acceptable vehicle to be included in any of the various formulations. A “pharmaceutically acceptable vehicle” may be a vehicle approved by a federal or state regulatory authority or listed in the United States Pharmacopeia or a generally accepted pharmacopoeia for use in mammals such as humans. The term “vehicle” refers to a diluent, adjuvant, excipient, or carrier used to formulate the compound of the present invention for administration to a mammal. Such pharmaceutical vehicles may include lipids, such as liposomes, such as liposomal dendrimers; petroleum, animal, plant-derived or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil; liquids such as water and oil containing physiological saline; acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Furthermore, auxiliaries, stabilizers, thickeners, lubricants, and colorants may be used. Pharmaceutical compositions may be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The drug may be systemic after administration, or localized by topical administration, intramural administration, or the use of implants that act to retain the active dose at the implantation site. The active drug may be formulated for immediate action or for sustained release. For some medical conditions, particularly those affecting the central nervous system, it may be necessary to formulate drugs to cross the blood-brain barrier (BBB). One strategy for BBB-crossing drug delivery involves disrupting the BBB using osmotic means such as mannitol or leukotrienes, or biochemical disruption using vasoactive substances such as bradykinin. When a composition is administered by intravascular injection, the BBB disruptor can be administered concurrently with the drug.Other strategies for crossing the blood-brain barrier (BBB) ​​involve the use of endogenous transport systems, including caveolin-1-mediated transcytosis, carrier-mediated transporters such as glucose and amino acid carriers, receptor-mediated transcytosis of insulin or transferrin, and active efflux transporters such as p-glycoprotein. Alternatively, the active transport moiety may be complexed with the therapeutic compound used in this invention to facilitate transport across the vascular endothelial wall. Alternatively, drug delivery to the back of the blood-brain barrier may be by local delivery, such as intrathecal delivery, such as via an Onmeyer reservoir (see U.S. Patents No. 5,222,982 and 5,385,582 as incorporated herein by reference), by bolus injection, such as intravitreous or intracranial injection, by continuous injection with convection, such as via cannula insertion (see, for example, U.S. Patent Application No. 20070254842 as incorporated herein by reference), or by implantation of a device that reversibly fixes the drug (see, for example, U.S. Patent Applications No. 20080081064 and 20090196903 as incorporated herein by reference).

[0172] Prior to transplantation, when supplying cells with a drug(s), the drug(s) can be conveniently added to the cell culture medium in solution or in a readily soluble form. The drug(s) may be added as an intermittent or continuous flow in a flow-through system, or a large amount of the compound may be added individually or incrementally to a static solution. In a flow-through system, two fluids are used: one is a physiologically neutral solution, and the other is a solution of the test compound(s) to be added. The first fluid is passed over the cells, and then the second fluid(s) is passed over the cells. In the single-solution method, a large amount of the test compound(s) is added to the surrounding medium volume. The overall concentration of the medium components should not change significantly with large additions, or between the two solutions in the flow-through method.

[0173] Multiple assays at different drug concentrations may be run in parallel to obtain a variety of different responses at various concentrations. As is well known in the art, the effective concentration of a drug is generally measured using a concentration range resulting from dilutions at 1:10 or other logarithmic scales. If necessary, the effective concentration may be further examined using a second series of dilutions. Typically, one of these concentrations serves as a negative control, i.e., a negative control whose concentration is zero, below the detection level of the drug, or below a drug concentration that does not produce a detectable change in phenotype.

[0174] The response of humanized SIRPα-IL-15 non-human animal cells, such as mouse cells, to a candidate drug may be analyzed at any point in time after drug treatment. For example, cells may be analyzed 1, 2, or 3 days after contact with the candidate drug, optionally 4, 5, or 6 days, optionally 8, 9, or 10 days, optionally 14 days, optionally 21 days, optionally 28 days, optionally 1 month or more, for example 2 months, 4 months, 6 months or more. In some embodiments, the analysis includes analysis at multiple time points. The selection of time points for analysis will depend on the type of analysis to be performed, as will be readily apparent to those skilled in the art.

[0175] The analysis may include measuring any of the parameters described herein or known in the art for measuring cell viability, cell proliferation, cell identity, cell morphology, and cell function, particularly those that may be related to immune system cells, such as T cells and / or NK cells. For example, flow cytometry may be used to measure the total number of hematopoietic cells or the number of cells of a particular hematopoietic cell type. The apoptotic state of cells may be determined by performing histochemistry or immunohistochemistry, such as terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) to measure DNA fragmentation, or immunohistochemistry to detect annexin V binding to phosphatidylserine on the cell surface. The proportion of differentiated cells and differentiated cell types may be evaluated using flow cytometry to determine, for example, the differentiation capacity of hematopoietic cells in the presence of a drug. ELISA, Western blotting, and Northern blotting may be performed to measure the levels of cytokines, chemokines, immunoglobulins, etc., expressed in engrafted humanized SIRPα-IL-15 non-human animals, such as mice, to evaluate the function of engrafted cells. In vivo assays for testing the function of immune cells, as well as assays related to specific diseases or disorders of interest, such as diabetes, autoimmune diseases, graft-versus-host diseases, and AMD, may be performed. See, for example, Current Protocols in Immunology (Richard Coico, ed., John Wiley & Sons, Inc. 2012) and Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), the disclosures of which are incorporated herein by reference.

[0176] Therefore, for example, engrafted humanized SIRPα-IL-15 non-human animals, such as mice, for example, engrafted Rag2 - / - IL2rg Y / -The present invention provides a method for measuring the efficacy of a drug against a human pathogen, comprising exposing hSIRPα hIL-15 mice to an effective amount of a human pathogen, i.e., an amount of the pathogen necessary to cause infection in mice, enabling the pathogen to infect the mice, measuring parameters of infection over time in the presence of the drug, and comparing the measured values ​​with those measured in engrafted humanized SIRPα-IL-15 non-human animals, such as mice, that have not been exposed to the drug. A drug is determined to be an antipathogenic drug if, after a single dose or two or more doses over a selected period, the amount of the drug in the blood or tissues of non-human animals, such as mice, is reduced by at least half.

[0177] As another example, we provide a method for determining whether a pathogen isolate or strain of interest is drug-resistant, for example, multidrug-resistant. These methods use engrafted humanized SIRPα-IL-15 non-human animals, such as mice, or engrafted Rag2. - / - IL2rg Y / -hSIRPα hIL-15 mice are exposed to an effective amount of human pathogen, i.e., an isolate or strain of interest of the pathogen in an amount sufficient to cause infection in a non-human animal, such as a mouse, thereby enabling the pathogen to infect the non-human animal. Infection parameters, such as the titer of the isolate or strain of interest in the blood or tissue of the non-human animal, the ability of the isolate or strain of interest to maintain infection in the non-human animal, or the regenerative capacity of the isolate or strain of interest in the non-human animal at a point in time after drug administration, are measured in the presence of the drug, and these measurements are compared with measurements in engrafted humanized SIRPα-IL-15 non-human animals, such as mice infected with a pathogen that has not been exposed to the drug. Examples of drugs of interest include amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime, chloramphenicol, ciprofloxacin, cotrimoxazole, ertapenem, imipenem, fluoroquinolones (e.g., ciprofloxacin, gatifloxacin, ofloxacin), streptomycin, sulfadiazine, sulfamethoxazole, tetracyclines, and combinations thereof. In certain embodiments, administration of the drug or combination of drugs is performed at least one week, ten days, two weeks, three weeks, or four weeks after exposure that induces infection with the infectious isolate or the strain of interest.

[0178] Furthermore, humanized SIRPα-IL-15 non-human animals (e.g., mice) and humanized SIRPα-IL-15 non-human animals (e.g., mice) with engrafted human hematopoietic cells, for example, engrafted Rag2 - / - IL2rg Y / - hSIRPα hIL-15 mice, and non-human animals with other genetic modifications as needed, are useful for studying antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells (e.g., human NK cells). Such animals are also useful models for testing the ability of therapeutic drug candidates, such as antigen-binding proteins or antibodies, designed to target various cells (e.g., tumor or infected cells) or infectious pathogens and activate NK cell pathways involved in killing such cells or infectious pathogens.

[0179] It is widely known that one of the underlying mechanisms of monoclonal antibody therapy is the activation of NK cells by binding to the NK cell Fc receptor CD16 (Fcγ receptor IIIA). To improve ADCC, attempts have been made to increase the affinity of various known monoclonal candidates (e.g., rituximab) to FcγRIIIA (e.g., Bowles et al. Blood 2006;108:2648-2654, Garff-Tavernier et al. Leukemia 2011;25:202-209). As shown herein, non-human animals engrafted with humanized SIRPα-IL-15 produce human NK cells capable of mediating ADCC, and therefore these animals present a useful in vivo model for studying the ADCC mechanism and screening various therapeutic candidates.

[0180] Therefore, the engrafted humanized SIRPα-IL-15 non-human animals and cells isolated therefrom, such as human NK cells, may be used in screening methods designed to identify agents that enhance the antibody-dependent cellular cytotoxicity (ADCC) activity of the engrafted cell type in humanized non-human animals or cells, such as human NK cells. For example, a suitable method involves administering an agent to an engrafted humanized SIRPα-IL-15 non-human animal and measuring the in vivo effect of the agent on the antibody-dependent cellular cytotoxicity (ADCC) activity of the engrafted cell type within the humanized non-human animal. In one embodiment, such an effect results in an increase in tumor death, for example, of a human tumor, such as a transplanted tumor. In another embodiment, such an effect increases the killing of infected cells, such as virus-infected or bacterially-infected cells. In yet another embodiment, such an effect results in an increase in the killing of bacteria, fungi, or parasites. In various embodiments, the agent is an antibody or an antigen-binding protein. In some embodiments, the antibody or antigen-binding protein is designed to target an antigen expressed on human tumor cells. In some embodiments, the antibody or antigen-binding protein is designed to target an antigen expressed on virus-infected or bacterially-infected cells. In some embodiments, the antibody or antigen-binding protein is designed to target an antigen of bacteria, fungi, or parasites. In some embodiments, human cells, such as human NK cells, are isolated from an engrafted humanized SIRPα-IL-15 non-human animal and contacted in vitro with an agent, such as an antibody or antigen-binding protein, and target cells (e.g., tumor cells), and an in vitro method is provided for measuring the efficacy of the agent in mediating the killing of the target cells. Subsequently, the effect of the agent on the cytolytic activity of human cells, such as human NK cells, can be measured.

[0181] Other uses of the mice of the present invention are described elsewhere in this specification. Further applications of the genetically modified mice and engrafted mice described in this disclosure will be apparent to those skilled in the art upon reading this disclosure.

[0182] Method for Producing Genetically Modified Non-Human Animals of the Present Invention In some embodiments of the present invention, a method for producing non-human animals of the present disclosure is provided. In carrying out the method of the present invention, a non-human animal is produced that possesses a nucleic acid sequence incorporated into the genome of a genetically modified non-human animal, which encodes the human SIRPα protein and is operably linked to a SIRPα gene promoter, such as an endogenous non-human SIRPα gene promoter, and a nucleic acid sequence incorporated into the genome of a genetically modified non-human animal, which encodes the human IL-15 protein and is operably linked to an IL-15 gene promoter, such as an endogenous non-human IL-15 gene promoter.

[0183] The creation of non-human animals possessing a nucleic acid sequence encoding human SIRPα protein and operably ligated to the SIRPα promoter, and / or a nucleic acid sequence encoding human IL-15 protein and operably ligated to the IL-15 promoter, may be achieved using any simple method for creating genetically modified animals, such as those known in the art or described herein.

[0184] For example, nucleic acids encoding human SIRPα protein or human IL-15 protein may be incorporated into a recombinant vector in a form suitable for insertion into the genome of a host cell and expression of the human protein in a non-human host cell. In various embodiments, the recombinant vector includes one or more regulatory sequences operably ligated to the nucleic acid encoding the human protein in a manner that enables mRNA transcription from the nucleic acid and translation of mRNA into a human protein, as described above. It will be understood that the design of the vector may depend on factors such as the selection of the host cell to be transfected and / or the amount of human protein to be expressed.

[0185] Next, a human nucleic acid sequence may be introduced into animal cells using one of various methods to create a genetically modified animal that expresses human genes. Such techniques are well known in the art and include, but are not limited to, pronuclear microinjection, embryonic stem cell transformation, homologous recombination, and knock-in techniques. Methods for generating usable genetically modified animals include those described in Sundberg and Ichiki (2006, Genetically Engineered Mice Handbook, CRC Press), Hofker and van Deursen (2002, Genetically modified Mouse Methods and Protocols, Humana Press), Joyner (2000, Gene Targeting: A Practical Approach, Oxford University Press), Turksen (2002, Embryonic stem cells: Methods and Protocols in Methods Mol Biol., Humana Press), Meyer et al. (2010, Proc.Nat.Acad.Sci.USA 107:15022-15026), and Gibson (2004, A Primer of Genome Science 2 nd This includes, but is not limited to, the following: Sunderland ed., Massachusetts: Sinauer, U.S. Patent No. 6,586,251, Rathinam et al. (2011, Blood 118:3119-28), Willinger et al. (2011, Proc Natl Acad Sci USA, 108:2390-2395), Rongvaux et al. (2011, Proc Natl Acad Sci USA, 108:2378-83), and Valenzuela et al. (2003, Nat Biot 21:652-659).

[0186] For example, the genetically modified animals of the present invention can be produced by introducing nucleic acids encoding human proteins into oocytes, for example, by microinjection, and generating the oocytes in the body of a female livestock. In a preferred embodiment, nucleic acids are injected into oocytes after fertilization. Post-fertilization oocytes can be collected from superovulating females the day after mating and injected with an expression construct. The injected oocytes are cultured overnight or directly transplanted into the fallopian tubes of pseudopregnant females 0.5 days after mating. Methods for superovulation, oocyte collection, injection of expression constructs, and embryo transfer are well known in the art and are described in Manipulating the Mouse Embryo (2002, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press). The presence of the introduced nucleic acids can be evaluated in offspring by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blotting, etc.).

[0187] As another example, a construct containing a nucleic acid sequence encoding a human protein may be transfused into stem cells (e.g., ES cells or iPS cells) using well-known methods such as electroporation, calcium phosphate precipitation, or lipofection. The presence of the introduced nucleic acid can then be evaluated by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing) or protein analysis (e.g., ELISA, Western blotting). Cells determined to have incorporated the expression construct can then be introduced into preimplantation embryos. For a detailed description of methods known in the art that are useful for the compositions and methods of the present invention, see Nagy et al., (2002, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press), Nagy et al. (1990, Development 110:815-821), U.S. Patent No. 7,576,259, U.S. Patent No. 7,659,442, U.S. Patent No. 7,294,754, and Kraus et al. (2010, Genesis 48:394-399).

[0188] In a preferred embodiment, the method for producing genetically modified animals described herein involves using a targeting construct prepared using VELOCIGENE® technology, introducing the construct into ES cells, and then introducing the target ES cell clone into a mouse embryo using VELOCIMOUSE® technology, as described in the examples.

[0189] Genetically modified progenitor animals can be crossed with additional animals that also possess genetic modifications. For example, a humanized SIRPα non-human animal can be crossed with a humanized IL-15 non-human animal of the same species to produce the hSIRPα-hIL-15 non-human animal described herein. Genetically modified animals possessing nucleic acids encoding the human protein(s) of this disclosure can be further crossed with knockout animals, such as non-human animals lacking one or more proteins, for example, animals that do not express one or more of the genes, such as Rag2-deficient animals and / or Il2rg-deficient animals.

[0190] As described above, in some embodiments, the genetically modified non-human animals of the present invention are immunodeficient animals. A genetically modified non-human animal that is immunodeficient and possesses one or more human proteins, such as hSIRPα and / or hIL-15, may be produced using any simple method for producing genetically modified animals that is well known in the art or described herein. For example, the production of a genetically modified immunodeficient animal can be achieved by introducing a nucleic acid encoding a human protein into oocytes or stem cells possessing an allele of a mutated SCID gene, which, if homozygous, produces immunodeficiency as described in more detail in the examples above and herein. Next, the modified oocytes or ES cells are used to produce mice, for example, using a method known in the art as described herein, and bred to produce immunodeficient mice having the desired genetic modification. As another example, genetically modified non-human animals can be created in an immunocompetent environment, crossed with animals carrying alleles of a mutant gene that causes immunodeficiency in hemizygous or homozygous cases, and then the offspring can be crossbred to create immunodeficient animals that express at least one human protein of interest.

[0191] In some embodiments, genetically modified non-human animals are treated to eliminate endogenous hematopoietic cells that may be present in the genetically modified non-human animals. In one embodiment, the treatment includes irradiating the genetically modified non-human animals. In a specific embodiment, neonatal genetically modified mouse pups are irradiated with a sublethal dose. In a specific embodiment, the pups are irradiated with 2 × 200 cGy of radiation at 4-hour intervals.

[0192] Various embodiments of the present invention provide genetically modified animals having human nucleic acids in substantially all of their cells, as well as genetically modified animals having human nucleic acids in some but not all of their cells. In some examples, such as targeted recombination, one copy of human nucleic acid is incorporated into the genome of the genetically modified animal. In other examples, such as random integration, multiple copies of human nucleic acids, either adjacent or distant from each other, may be incorporated into the genome of the genetically modified animal.

[0193] Therefore, in some embodiments, the genetically modified non-human animal of the present invention may be an immunodeficient animal having a genome containing a nucleic acid encoding a human polypeptide that is operably linked to a corresponding non-human animal promoter, wherein the animal expresses the encoded human polypeptide. In other words, the genetically modified immunodeficient non-human animal of the present invention has a genome containing a nucleic acid encoding at least one human polypeptide, the nucleic acid being operably linked to a corresponding non-human promoter and polyadenylation signal, and the animal expresses the encoded human polypeptide.

[0194] Reagents, equipment, and kits Reagents, apparatus, and kits for carrying out one or more of the above methods are also provided. The reagents, apparatus, and kits of the present invention may be subject to significant modification.

[0195] In some embodiments, the reagent or kit comprises one or more agents for use in the method described herein. For example, the kit may contain humanized SIRPα-IL-15 in non-human animals, such as mice, such as Rag2. - / -IL2rg Y / - The kit may include hSIRPα hIL-15 mice. This kit may include reagents for mating humanized SIRPα-IL-15 non-human animals, e.g., mice, e.g., primers, and in some cases, reagents for genotyping humanized SIRPα-IL-15 non-human animals, e.g., mice. The kit may include reagents for preparing populations of human hematopoietic cells or enriched populations of human hematopoietic progenitor cells for transplantation into humanized SIRPα-IL-15 non-human animals, e.g., mice, or populations of human-derived hematopoietic cells or enriched populations of hematopoietic cells for transplantation into humanized SIRPα-IL-15 non-human animals, e.g., mice. Other reagents may include, for example, candidate drugs, reagents for measuring the viability and / or function of hematopoietic cells or differentiated immune cells (e.g., T cells and / or NK cells) in the presence / absence of one or more antibodies specific to markers expressed on different types of hematopoietic cells or differentiated immune cells (e.g., T cells and / or NK cells), or reagents for detecting specific cytokines, chemokines, etc. Other reagents may include culture media, culture supplements, and matrix compositions.

[0196] In addition to the components described above, the kit of the present invention further includes instructions for carrying out the method of the present invention. These instructions may be present in the kit of the present invention in various forms in which one or more may be present in the kit. One possible form in which these instructions may be present is as printed information on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, within the kit packaging, within the accompanying documentation of the packaging, etc. Yet another means may be a computer-readable medium on which the information is recorded, such as a disk or CD. Yet another possible means may be a website address that may be used to access information from a remote location via the Internet. Any convenient means may be present in the kit.

[0197] Exemplary, non-limiting aspects of this disclosure The embodiments of the present invention described above may be useful on their own or in combination with one or more other embodiments or forms. Without limiting the foregoing, certain non-limiting embodiments of the disclosure are provided below, numbered 1 to 167. As will be apparent to those skilled in the art in reading this disclosure, each of the individually numbered embodiments may be used individually or in combination with any of the preceding or following individually numbered embodiments. This is intended to provide support for all such combinations of embodiments, and is not limited to the combinations of embodiments expressly provided below. 1. Genetically modified non-human animals, A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, wherein the nucleic acid sequence encodes the human SIRPα protein and is operably linked to the SIRPα gene promoter, and The genetically modified non-human animal possesses a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the nucleic acid sequence encoding the human IL-15 protein and operably linked to the IL-15 gene promoter, and the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein.

[0198] 2. The genetically modified non-human animal according to 1, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter.

[0199] 3. The genetically modified non-human animal according to 2, wherein the SIRPα gene promoter is the endogenous non-human SIRPα gene promoter located within the SIRPα locus of a non-human animal.

[0200] 4. The genetically modified non-human animal according to 3, wherein the non-human SIRPα gene within the SIRPα locus of the non-human animal contains a null mutation.

[0201] 5. The genetically modified non-human animal according to 4, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4.

[0202] 6. The genetically modified non-human animal according to 4, wherein the genetically modified non-human animal is heterozygous for the allele having the nucleic acid sequence encoding the human SIRPα protein.

[0203] 7. The genetically modified non-human animal according to 4, wherein the genetically modified non-human animal is homozygous for the allele having the nucleic acid sequence encoding the human SIRPα protein.

[0204] 8. A genetically modified non-human animal according to any one of claims 1 to 7, wherein the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genome coding sequence and a human SIRPα genome non-coding sequence.

[0205] 9. A genetically modified non-human animal according to any one of items 1 to 8, wherein the human SIRPα protein is a functional fragment of the full-length human SIRPα protein.

[0206] 10. The genetically modified non-human animal according to 9, wherein the functional fragment comprises the extracellular domain of human SIRPα.

[0207] 11. The genetically modified non-human animal described in 10, wherein the extracellular domain contains amino acids 28-362 of SEQ ID NO:12.

[0208] 12. A genetically modified non-human animal according to any one of items 1 to 11, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter.

[0209] 13. The genetically modified non-human animal according to 12, wherein the IL-15 gene promoter is the endogenous non-human IL-15 gene promoter within the non-human animal IL-15 gene locus.

[0210] 14. A genetically modified non-human animal according to 13, comprising a null mutation in the non-human IL-15 gene within the non-human animal IL-15 gene locus.

[0211] 15. The genetically modified non-human animal according to 14, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8.

[0212] 16. The genetically modified non-human animal according to 14, wherein the genetically modified non-human animal is heterozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein.

[0213] 17. The genetically modified non-human animal according to 14, wherein the genetically modified non-human animal is homozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein.

[0214] 18. A genetically modified non-human animal according to any one of claims 1 to 17, wherein the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0215] 19. A genetically modified non-human animal according to any one of items 1 to 18, wherein the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0216] 20. A genetically modified non-human animal according to any one of items 1 to 19, wherein the genetically modified non-human animal is immunodeficient.

[0217] 21. The genetically modified non-human animal according to 20, wherein the genetically modified non-human animal has a Rag2 gene knockout.

[0218] 22. The genetically modified non-human animal according to 20 or 21, wherein the genetically modified non-human animal has an IL2rg gene knockout.

[0219] 23. A genetically modified non-human animal as described in any one of items 1 to 22, wherein the non-human animal is a mammal.

[0220] 24. A genetically modified non-human animal as described in 23, wherein the mammal is a rodent.

[0221] 25. The genetically modified non-human animal described in 24, wherein the rodent is a mouse.

[0222] 26. The genetically modified non-human animal according to any one of items 1 to 25, wherein the genetically modified non-human animal has engraftment of human hematopoietic cells.

[0223] 27. The genetically modified non-human animal described in 26, wherein the genetically modified non-human animal is infected with a human pathogen.

[0224] 28. The genetically modified non-human animal according to 27, wherein the human pathogen activates, induces, and / or targets T cells and / or natural killer (NK) cells.

[0225] 29. The genetically modified non-human animal described in 27, wherein the human pathogen is a pathogen that infects the human intestine.

[0226] 30. A genetically modified non-human animal as described in 29, wherein the human pathogen is human rotavirus.

[0227] 31. A genetically modified non-human animal as described in 27, in which the pathogen infects the human lungs.

[0228] 32. A genetically modified non-human animal as described in 31, wherein the human pathogen is an influenza virus.

[0229] 33. An animal engraftment model including a genetically modified non-human animal, wherein the genetically modified non-human animal is A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, wherein the nucleic acid sequence encodes the human SIRPα protein and is operably linked to the SIRPα gene promoter, A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, wherein the nucleic acid sequence encodes the human IL-15 protein and is operably linked to the IL-15 gene promoter, and An animal engraftment model comprising the engraftment of human hematopoietic cells, wherein the genetically modified non-human animal (i) expresses the human SIRPα protein and the human IL-15 protein, and (ii) possesses human intraepithelial lymphocytes (IELs) in the small intestine and Peyer's patches of the genetically modified non-human animal.

[0230] 34. The model described in 33, wherein the genetically modified non-human animal is infected by a human pathogen.

[0231] 35. The model described in 34, wherein the human pathogen is an intestinal pathogen.

[0232] 36. The model described in 35, wherein the intestinal pathogen is selected from Campylobacter jejuni, Clostridium difficile, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, human rotavirus, Listeria monocytogenes, Norwalk virus, Salmonella enterica, Sigella flexneri, Sigella sonei, Shigella shiga, Pest bacillus, Yersinia enterocolitica, and Helicobacter pylori.

[0233] 37. The model described in any one of items 33 to 36, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter.

[0234] 38. The model according to 37, wherein the SIRPα gene promoter is the endogenous non-human SIRPα gene promoter located within the SIRPα locus of a non-human animal.

[0235] 39. The model according to 38, wherein the genetically modified non-human animal contains a null mutation in the non-human SIRPα gene within the SIRPα locus of the non-human animal.

[0236] 40. The model described in 39, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4.

[0237] 41. The model according to 39, wherein the genetically modified non-human animal is heterozygous with respect to the allele having the nucleic acid sequence encoding the human SIRPα protein.

[0238] 42. The model according to 39, wherein the genetically modified non-human animal is homozygous with respect to the allele having the nucleic acid sequence encoding the human SIRPα protein.

[0239] 43. The model according to any one of items 33 to 42, wherein the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genome coding sequence and a human SIRPα genome non-coding sequence.

[0240] 44. The model described in any one of items 33 to 43, wherein the human SIRPα protein is a functional fragment of the full-length human SIRPα protein.

[0241] 45. The model according to 44, wherein the functional fragment comprises the extracellular domain of human SIRPα.

[0242] 46. ​​The model described in 45, wherein the extracellular domain has amino acids 28-362 of SEQ ID NO:12.

[0243] 47. The model described in any one of items 33 to 46, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter.

[0244] 48. The model according to 47, wherein the IL-15 gene promoter is the endogenous non-human IL-15 gene promoter located within the IL-15 locus of a non-human animal.

[0245] 49. The model according to 48, wherein the genetically modified non-human animal contains a null mutation in the non-human IL-15 gene within the IL-15 locus of the non-human animal.

[0246] 50. The model described in 49, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8.

[0247] 51. The model according to 48, wherein the genetically modified non-human animal is heterozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein.

[0248] 52. The model according to 48, wherein the genetically modified non-human animal is homozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein.

[0249] 53. The model according to any one of items 33 to 52, wherein the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0250] 54. The model described in any one of items 33 to 53, wherein the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0251] 55. A model described in any one of items 33 to 54, wherein the genetically modified non-human animal is immunodeficient.

[0252] 56. The model according to 55, wherein the genetically modified non-human animal has a Rag2 gene knockout.

[0253] 57. The model according to 55 or 56, wherein the genetically modified non-human animal has an IL2rg gene knockout.

[0254] 58. A model according to any one of items 33 to 57, wherein the non-human animal is a mammal.

[0255] 59. The model described in 58, wherein the mammal is a rodent.

[0256] 60. The model described in 59, wherein the rodent is a mouse.

[0257] 61. An animal engraftment model including a genetically modified non-human animal, wherein the genetically modified non-human animal is A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, wherein the nucleic acid sequence encodes the human SIRPα protein and is operably linked to the SIRPα gene promoter, A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, wherein the nucleic acid sequence encodes the human IL-15 protein and is operably linked to the IL-15 gene promoter, and An animal engraftment model comprising the engraftment of human hematopoietic cells, wherein the genetically modified non-human animal (i) expresses the human SIRPα protein and the human IL-15 protein, and (ii) possesses human intraepithelial lymphocytes (IELs) in the lungs of the genetically modified non-human animal.

[0258] 62. The model described in 61, wherein the genetically modified non-human animal is infected by a human pathogen.

[0259] 63. The model described in 62, wherein the human pathogen is a lung pathogen.

[0260] 64. The model described in 63, wherein the lung pathogen is selected from Streptococcus pyogenes, Haemophilus influenzae, Mycobacterium diphtheriae, SARS coronavirus, Bordetella pertussis, Moraxella catarrhalis, influenza viruses (A, B, C), coronaviruses, adenoviruses, RSV, parainfluenza virus, mumps virus, Streptococcus pneumoniae, Staphylococcus aureus, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Blastomyces dermatichidis, Cryptococcus neoformans, and Aspergillus fumigatus.

[0261] 65. The model described in any one of items 61 to 64, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter.

[0262] 66. The model according to 65, wherein the SIRPα gene promoter is the endogenous non-human SIRPα gene promoter located within the SIRPα locus of a non-human animal.

[0263] 67. The model according to 66, wherein the genetically modified non-human animal contains a null mutation in the non-human SIRPα gene within the SIRPα locus of the non-human animal.

[0264] 68. The model described in 67, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4.

[0265] 69. The model according to 67, wherein the genetically modified non-human animal is heterozygous with respect to the allele having the nucleic acid sequence encoding the human SIRPα protein.

[0266] 70. The model according to 67, wherein the genetically modified non-human animal is homozygous with respect to the allele having the nucleic acid sequence encoding the human SIRPα protein.

[0267] 71. The model according to any one of items 61 to 70, wherein the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genome coding sequence and a human SIRPα genome non-coding sequence.

[0268] 72. The model described in any one of items 61 to 71, wherein the human SIRPα protein is a functional fragment of the full-length human SIRPα protein.

[0269] 73. The model according to 72, wherein the functional fragment comprises the extracellular domain of human SIRPα.

[0270] 74. The model described in 73, wherein the extracellular domain has amino acids 28-362 of SEQ ID NO:12.

[0271] 75. The model described in any one of items 61 to 74, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter.

[0272] 76. The model according to 75, wherein the IL-15 gene promoter is the endogenous non-human IL-15 gene promoter located within the IL-15 locus of a non-human animal.

[0273] 77. The model according to 76, wherein the genetically modified non-human animal contains a null mutation in the non-human IL-15 gene within the IL-15 locus of the non-human animal.

[0274] 78. The model described in 77, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8.

[0275] 79. The model according to 77, wherein the genetically modified non-human animal is heterozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein.

[0276] 80. The model according to 77, wherein the genetically modified non-human animal is homozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein.

[0277] 81. The model according to any one of items 61 to 80, wherein the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0278] 82. The model described in any one of items 61 to 80, wherein the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0279] 83. A model described in any one of items 61 to 82, wherein the genetically modified non-human animal is immunodeficient.

[0280] 84. The model described in 83, wherein the genetically modified non-human animal has a Rag2 gene knockout.

[0281] 85. The model according to 83 or 84, wherein the genetically modified non-human animal has an IL2rg gene knockout.

[0282] 86. A model according to any one of items 61 to 85, wherein the non-human animal is a mammal.

[0283] 87. The model described in 86, wherein the mammal is a rodent.

[0284] 88. The model described in 87, wherein the rodent is a mouse.

[0285] 89. A method for determining the effectiveness of a candidate T cell-inducing vaccine, wherein the method is The method involves administering a candidate T-cell-inducing vaccine to a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system, (i) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the nucleic acid sequence encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, (ii) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, which encodes the human IL-15 protein and is operablely linked to the IL-15 gene promoter, and (iii) Engraftment of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein, and is administered as described above. To challenge the aforementioned genetically modified non-human animals with human pathogens, and The determination method, comprising determining whether the candidate T cell-inducible vaccine induces a T cell-mediated immune response in the genetically modified non-human animal.

[0286] 90. The method according to 89, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter.

[0287] 91. The method according to 90, wherein the SIRPα gene promoter is the endogenous non-human SIRPα gene promoter located within the SIRPα locus of a non-human animal.

[0288] 92. The method according to 91, wherein the genetically modified non-human animal contains a null mutation in the non-human SIRPα gene within the SIRPα locus of the non-human animal.

[0289] 93. The method according to 92, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4.

[0290] 94. The method according to 92, wherein the genetically modified non-human animal is heterozygous with respect to the allele having the nucleic acid sequence encoding the human SIRPα protein.

[0291] 95. The method according to 92, wherein the genetically modified non-human animal is homozygous with respect to the allele having the nucleic acid sequence encoding the human SIRPα protein.

[0292] 96. The method according to any one of claims 89 to 95, wherein the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genome coding sequence and a human SIRPα genome non-coding sequence.

[0293] 97. The method according to any one of claims 89 to 96, wherein the human SIRPα protein is a functional fragment of full-length human SIRPα protein.

[0294] 98. The method according to 97, wherein the functional fragment comprises the extracellular domain of human SIRPα.

[0295] 99. The method according to 98, wherein the extracellular domain has amino acids 28-362 of SEQ ID NO:12.

[0296] 100. The method according to any one of claims 89 to 99, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter.

[0297] 101. The method according to 100, wherein the IL-15 gene promoter is the endogenous non-human IL-15 gene promoter located within the IL-15 locus of a non-human animal.

[0298] 102. The method according to 101, wherein the genetically modified non-human animal contains a null mutation in the non-human IL-15 gene within the IL-15 locus of the non-human animal.

[0299] 103. The method according to 102, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8.

[0300] 104. The method according to 101, wherein the genetically modified non-human animal is heterozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein.

[0301] 105. The method according to 101, wherein the genetically modified non-human animal is homozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein.

[0302] 106. The method according to any one of claims 89 to 105, wherein the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0303] 107. The method according to any one of claims 89 to 106, wherein the human IL-15 protein is a functional fragment of full-length human IL-15 protein.

[0304] 108. The method according to any one of claims 89 to 107, wherein the genetically modified non-human animal has a Rag2 gene knockout.

[0305] 109. The method according to any one of claims 89 to 108, wherein the genetically modified non-human animal has an IL2rg gene knockout.

[0306] 110. The method according to any one of items 89 to 109, wherein the genetically modified non-human animal is a mammal.

[0307] 111. The method according to 110, wherein the mammal is a rodent.

[0308] 112. The method according to 111, wherein the rodent is a mouse.

[0309] 113. A method for identifying drugs that inhibit infection by pathogens that activate, induce, and / or target human T cells and / or natural killer (NK) cells, wherein the method is This involves administering drugs to genetically modified non-human animals, The aforementioned genetically modified non-human animals lack an endogenous immune system, (i) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the nucleic acid sequence encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, (ii) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the nucleic acid sequence encoding the human IL-15 protein and operably linked to the IL-15 gene promoter, (iii) Engraftment of human hematopoietic cells, and (iv) Infection by a pathogen that activates, induces and / or targets human T cells and / or natural killer cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein, and the administration of the pathogen, The identification method includes determining whether the drug reduces the amount of the pathogen in a non-human animal infected with the pathogen.

[0310] 114. The method according to 113, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter.

[0311] 115. The method according to 114, wherein the SIRPα gene promoter is the endogenous non-human SIRPα gene promoter located within the SIRPα locus of a non-human animal.

[0312] 116. The method according to 115, wherein the genetically modified non-human animal contains a null mutation in the non-human SIRPα gene within the SIRPα locus of the non-human animal.

[0313] 117. The method according to 116, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse SIRPα exons 2-4.

[0314] 118. The method according to 116, wherein the genetically modified non-human animal is heterozygous with respect to the allele having the nucleic acid sequence encoding the human SIRPα protein.

[0315] 119. The method according to 116, wherein the genetically modified non-human animal is homozygous with respect to the allele having the nucleic acid sequence encoding the human SIRPα protein.

[0316] 120. The method according to any one of claims 113 to 119, wherein the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genome coding sequence and a human SIRPα genome non-coding sequence.

[0317] 121. The method according to any one of items 113 to 120, wherein the human SIRPα protein is a functional fragment of full-length human SIRPα protein.

[0318] 122. The method according to 121, wherein the functional fragment comprises the extracellular domain of human SIRPα.

[0319] 123. The method according to 122, wherein the extracellular domain has amino acids 28-362 of SEQ ID NO:12.

[0320] 124. The method according to any one of claims 113 to 123, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter.

[0321] 125. The method according to 124, wherein the IL-15 gene promoter is the endogenous non-human IL-15 gene promoter located within the IL-15 locus of a non-human animal.

[0322] 126. The method according to 125, wherein the genetically modified non-human animal contains a null mutation in the non-human IL-15 gene within the IL-15 locus of the non-human animal.

[0323] 127. The method according to 126, wherein the genetically modified non-human animal is a mouse, and the null mutation is a deletion of at least mouse IL-15 exons 5-8.

[0324] 128. The method according to 125, wherein the genetically modified non-human animal is heterozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein.

[0325] 129. The method according to 125, wherein the genetically modified non-human animal is homozygous with respect to the allele having the nucleic acid sequence encoding the human IL-15 protein.

[0326] 130. The method according to any one of claims 113 to 129, wherein the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0327] 131. The method according to any one of items 113 to 130, wherein the human IL-15 protein is a functional fragment of full-length human IL-15 protein.

[0328] 132. The method according to any one of claims 113 to 131, wherein the genetically modified non-human animal has a Rag2 gene knockout.

[0329] 133. The method according to any one of claims 113 to 132, wherein the genetically modified non-human animal has an IL2rg gene knockout.

[0330] 134. The method according to any one of items 113 to 133, wherein the genetically modified non-human animal is a mammal.

[0331] 135. The method according to 134, wherein the mammal is a rodent.

[0332] 136. The method according to 135, wherein the rodent is a mouse.

[0333] 137. A method for producing non-human animals that express human IL-15 protein and human SIRPα protein, The introduction of a nucleic acid sequence encoding human SIRPα protein into the genome of a first non-human animal, wherein the sequence encoding human SIRPα protein is operably linked to a SIRPα gene promoter sequence. The introduction of a nucleic acid sequence encoding human IL-15 protein into the genome of a second non-human animal, wherein the sequence encoding human IL-15 protein is operably linked to an IL-15 promoter sequence, and A method for producing a third non-human animal having the nucleic acid sequence encoding the human IL-15 protein and the nucleic acid sequence encoding the human SIRPα protein, wherein the third non-human animal expresses the human IL-15 protein and the human SIPRα protein.

[0334] 138. The method according to 137, wherein the introduction step comprises generating a non-human animal from pluripotent stem cells possessing the nucleic acid encoding human IL-15 or human SIRPα.

[0335] 139. The method according to 137 or 138, wherein the first animal is a different animal from the second animal, and the step of producing the third animal includes mating the first and second animals.

[0336] 140. The method according to 137, wherein the first animal and the second animal are the same, the step of introducing into the genome of the first animal comprises contacting the first pluripotent stem cells with the nucleic acid sequence encoding the human SIRPα protein to obtain second pluripotent stem cells, the step of introducing into the genome of the second animal comprises contacting the second pluripotent stem cells with the nucleic acid sequence encoding the human SIRPα protein to obtain third pluripotent stem cells, and the third non-human animal is produced from the third pluripotent stem cells.

[0337] 141. The method according to any one of items 137 to 140, wherein the pluripotent stem cells are ES cells or iPS cells.

[0338] 142. The method according to any one of items 137 to 140, wherein the pluripotent stem cells lack Rag2.

[0339] 143. The method according to any one of items 137 to 142, wherein the pluripotent stem cells are deficient in IL2rg.

[0340] 144. The method according to any one of items 137 to 143, wherein the third non-human animal is deficient in one or both Rag2 and IL2rg.

[0341] 145. The method according to any one of claims 137 to 144, wherein the IL-15 promoter sequence is a human IL-15 promoter sequence.

[0342] 146. The method according to any one of claims 137 to 144, wherein the IL-15 promoter sequence is an endogenous non-human animal IL-15 promoter sequence.

[0343] 147. The method according to any one of items 137 to 144, wherein the insertion replaces the non-human IL-15 gene within the non-human IL-15 locus.

[0344] 148. The method according to any one of claims 137 to 147, wherein the nucleic acid sequence encoding the human IL-15 protein comprises a human IL-15 genome coding sequence and a human IL-15 genome non-coding sequence.

[0345] 149. A method for engrafting a genetically modified non-human animal expressing human IL-15 protein, The engraftment method comprising transplanting a population of cells, including human hematopoietic cells, into the genetically modified non-human animal prepared by any one of the methods described in items 137 to 148.

[0346] 150. The method according to 149, wherein the transplantation includes injection into the tail vein, injection into the fetal liver, or injection into the posterior orbit.

[0347] 151. The method according to 149 or 150, wherein the genetically modified non-human animal is subjected to a sublethal dose of radiation before transplantation.

[0348] 152. The method according to any one of items 149 to 151, wherein the human hematopoietic cells are CD34+ cells.

[0349] 153. The method according to any one of items 149 to 151, wherein the human hematopoietic cells are derived from fetal liver, adult bone marrow, or umbilical cord blood.

[0350] 154. A method for determining the effectiveness of a candidate therapeutic antibody or antigen-binding protein in killing target cells, The method involves administering the candidate therapeutic antibody or antigen-binding protein to a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system, (i) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the nucleic acid sequence encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, (ii) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, which encodes the human IL-15 protein and is operablely linked to the IL-15 gene promoter, and (iii) Engraftment of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein, and the administration of the above, The determination method, comprising determining whether the candidate therapeutic antibody or antigen-binding protein modulates NK cell-mediated antibody-dependent cytotoxicity against the target cells in the genetically modified non-human animal.

[0351] 155. A method for determining the effectiveness of a candidate therapeutic antibody or antigen-binding protein in killing target cells, wherein the method is The method involves isolating NK cells from a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system, (i) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the nucleic acid sequence encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, (ii) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, which encodes the human IL-15 protein and is operablely linked to the IL-15 gene promoter, and (iii) Engraftment of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein, and is isolated. The isolated NK cells are brought into contact with the candidate therapeutic antibody or antigen-binding protein and the target cells, and The determination method comprising measuring the antibody or antigen-binding protein-dependent cell lysis activity of the isolated NK cells against the target cells.

[0352] 156. A screening method for candidate therapeutic antibodies or antigen-binding proteins for improving the effectiveness of killing target cells, The method involves administering the candidate therapeutic antibody or antigen-binding protein to a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system, (i) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the nucleic acid sequence encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, (ii) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, which encodes the human IL-15 protein and is operablely linked to the IL-15 gene promoter, and (iii) Engraftment of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein, and the administration of the above, The screening method, comprising determining whether the candidate therapeutic antibody or antigen-binding protein shows improved efficacy when killing the target cells in the genetically modified non-human animal.

[0353] 157. The method according to any one of claims 154 to 156, wherein the target cells are selected from the group consisting of tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells.

[0354] 158. A method for determining the effectiveness of a candidate therapeutic antibody or antigen-binding protein in the NK cell-mediated death of target cells, The method involves administering the candidate therapeutic antibody or antigen-binding protein to a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system, (i) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the nucleic acid sequence encoding the human SIRPα protein and operably linked to the SIRPα gene promoter, (ii) A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, which encodes the human IL-15 protein and is operablely linked to the IL-15 gene promoter, and (iii) Engraftment of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein, and the administration of the above, The determination method, comprising determining whether the candidate therapeutic antibody or antigen-binding protein modulates (e.g., activates) the antibody-dependent cytotoxicity of NK cells against the target cells in the genetically modified non-human animal.

[0355] 159. The method according to 158, wherein the target cells are selected from the group consisting of tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells.

[0356] 160. The method according to claim 159, wherein the target cell is a tumor cell.

[0357] 161. The method according to claim 160, wherein the tumor cells are B-cell lymphoma cells.

[0358] 162. A model of antibody-dependent cytotoxicity mediated by NK cells, including a genetically modified non-human animal, wherein the genetically modified non-human animal lacks an endogenous immune system, A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, wherein the nucleic acid sequence encodes the human SIRPα protein and is operably linked to the SIRPα gene promoter, A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, wherein the nucleic acid sequence encodes the human IL-15 protein and is operably linked to the IL-15 gene promoter, and The model comprises the engraftment of human hematopoietic cells, wherein the genetically modified non-human animal (i) expresses the human SIRPα protein and the human IL-15 protein, (ii) possesses human lymphocytes, and (iii) possesses target cells selected from the group consisting of tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells.

[0359] 163. The model according to claim 162, wherein the target cell is a tumor cell.

[0360] 164. The model according to claim 163, wherein the tumor cells are B-cell lymphoma cells.

[0361] 165. The model according to claim 163 or claim 164, wherein the model comprises an exogenous candidate therapeutic antibody or antigen-binding protein.

[0362] 166. The model according to any one of claims 162 to 165, wherein the genetically modified non-human animal possesses human intraepithelial lymphocytes (IELs) in the small intestine and Peyer's patches of the genetically modified non-human animal.

[0363] 167. The model according to any one of claims 162 to 166, wherein the genetically modified non-human animal possesses human intraepithelial lymphocytes (IELs) in the lungs of the genetically modified non-human animal. [Examples]

[0364] The following examples are presented solely to provide a complete disclosure and explanation of the manufacturing method and use method of the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all that should be performed, i.e., that no other experiments should be conducted. While efforts have been made to maintain accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), some degree of experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or close to it.

[0365] Example 1: Preparation of humanized SIRPα (SRG) knock-in mice We created a human SIRPα knock-in mouse expressing the extracellular domain of human SIRPα that is operablely linked to the mouse SIRPα promoter (see Figure 1). Human SIRPα is known to exist in at least 10 allele forms. In this particular example, human SIRPα mutant 1 is used to humanize the mouse endogenous SIRPα gene.

[0366] Materials and methods Genetic background Rag2 - / - Il2rg Y / - Knock-in mice with a 129xBalb / c(N2) count encoding human SIRPα were generated using the VELOCIGENE® technology, as detailed below. The mice were maintained under conditions free from specific pathogens and in the presence of continuous treatment of drinking water with enrofloxacin (Baytril, 0.27 mg / mL).

[0367] Targeted vectors for humanizing the extracellular region of the SIRP (e.g., SIRPα) gene were constructed using VELOCIGENE® technology (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659).

[0368] In short, the mouse bacterial artificial chromosome (BAC) clone bMQ-261H14 was modified to delete the sequence containing exons 2-4 of the endogenous SIRPα gene, and exons 2-4 of the human SIRPα gene were inserted using the human BAC clone CTD-3035H21. The genomic DNA (approximately 8555 bp) corresponding to exons 2-4 of the endogenous SIRPα gene was replaced in BAC clone bMQ-261H14 with a DNA fragment of approximately 8581 bp containing exons 2-4 of the human SIRPα gene from BAC clone CTD-3035H21. Sequence analysis of the human SIRPα allele contained in BAC clone CTD-3035H21 revealed the allele corresponding to human variant 1. A neomycin cassette adjacent to the LoxP site was added to the end of the approximately 8581 bp human DNA fragment containing exons 2-4 of the human SIRPα gene (Figure 1 (bottom panel)).

[0369] Homology arms were obtained from the 5' and 3' positions of exons 2 and 4 of mouse BAC DNA, respectively. These were attached to a human fragment-neomycin cassette of approximately 8581 bp. A final targeting vector for humanizing the endogenous SIRPα gene was constructed, consisting of a 5' homology arm containing 19 kb of the 5' end of exon 2 of the endogenous SIRPα gene in mouse DNA, an approximately 8581 bp DNA fragment containing human SIRPα exons 2-4, a neomycin cassette adjacent to the loxP site, and a 3' homology arm containing 21 kb of the 3' end of exon 4 of the endogenous SIRPα gene in mouse DNA, in sequence from 5' to 3'. Targeted insertion of the targeting vector placed the neomycin cassette in the fifth intron between exons 4 and 5 of the mouse SIRPα gene. The targeting vector was digested with SwaI to form a linear chain, and then used for homologous recombination in bacterial cells to achieve targeted substitution from mouse SIRPα gene exons 2-4 to human SIRPα gene exons 2-4 (Figure 1 (bottom panel)).

[0370] Using the target BAC DNA (shown above), Rag2 - / - IL2rg Y / - Mouse ES cells were electroporated, and modified ES cells were created by substituting endogenous mouse SIRPα gene exons 2-4 with a genomic fragment containing human SIRPα gene exons 2-4. Positive ES cells possessing the genomic fragment containing human SIRPα gene exons 2-4 were identified by quantitative PCR using the TAQMAN® probe (Lie and Petropoulos, 1998. Curr. Opin. Biotechnology 9:43-48). The nucleotide sequence across the upstream insertion site had the following sequence, indicating that the endogenous mouse sequence upstream of the insertion site (included in parentheses below) is continuously linked to the human SIRPα genomic sequence present at the insertion site. The nucleotide sequence extending to the downstream insertion site at the 5' end of the TIFF0007839229000001.tif34145 neomycin cassette had the following sequence, which indicates that the human SIRPα genome sequence is continuously followed by the cassette sequence downstream of the insertion site (the sequence shown in parentheses below, including the loxP sequence shown in italics). The nucleotide sequence extending to the downstream insertion site at the 3' end of the neomycin cassette TIFF0007839229000002.tif26155 had the following sequence, indicating that the cassette sequence is continuous with the 3' side of exon 4 of the endogenous SIRPα gene in the mouse genome sequence (shown in parentheses below). Next, the positive ES cell clones were used for transplantation into female mice using the VELOCIMOUSE® method (see, for example, U.S. Patent No. 7,294,754 and Poueymirou et al. 2007, F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses, Nature Biotech. 25(1):91-99) to generate a population of offspring in which human SIRPα gene exons 2-4 were inserted into the mouse endogenous SIRPα gene.

[0371] The targeted ES cells described above were used as donor ES cells and introduced into 8-cell stage mouse embryos using the VELOCIMOUSE® method (described above). By genotyping using a modified allele assay (Valenzuela et al., described above) that detected the presence of the human SIRPα gene sequence, mice possessing humanized endogenous SIRPα gene exons 2-4 were identified.

[0372] To remove any loxed neomycin cassette introduced by a targeted vector that is not removed, for example, at the ES cell stage or in embryo, mice possessing a humanized SIRPα gene construct (i.e., mice with human SIRPα exons 2-4 in the mouse SIRPα gene) can be crossed with Cre-deficient mouse strains (see, for example, International Patent Application Publication WO2009 / 114400). Neomycin cassettes are retained within the mice as needed. Heterozygotes are crossed to obtain homozygous Sirpα mice.

[0373] result Mice carrying nucleic acids encoding the humanized version of the mouse SIRPα gene (SRG mice) exhibit physiological expression of the humanized SIRPα protein (data not shown). These mice also show engraftment of human immune cells in the spleen, peripheral lymph nodes (LNs), and thymus comparable to that of NOD scidγ (NSG) mice (data not shown).

[0374] Example 2: Humanized SRG IL-I5 h / h (SRG-15) Knock-in mouse creation The cytokine IL-15 is involved in the development and memory of mouse NK cells and CD8 + It has been shown to be important for T cell differentiation and maintenance. To investigate the effects of human IL-15 on the development, differentiation, and maintenance of human immune cells in the context of animal models, human IL-15 human SIRPα knock-in mice were created, as detailed below. Figure 2 shows a schematic diagram of the IL-15 knock-in construct.

[0375] Materials and methods Using VELOCIGENE® genetic engineering technology, mouse ES cells are modified, and at the endogenous mouse IL-15 locus, the mouse IL-15 gene sequence, which is under the control of the mouse IL-15 regulatory element, is replaced with the human IL-15 gene sequence, thereby producing a humanized locus as shown in Figure 2. Rag2 - / - Il2rg Y / -Knock-in mice were generated with a 129xBalb / c gene background and possessing human IL-15. Figure 2 does not show the upstream region (with respect to the transcription direction of the IL-15 gene) of the mouse gene's 5' untranslated exons (exons 1 and 2), and coding exon 1 (exon 3) in Figure 2 shows a small untranslated region (hollow) upstream of the coding exon. Except as described below, mouse coding exons 1 and 2 (exons 3 and 4) were retained, while mouse coding exons 3-6 (exons 5-8) were replaced with human coding exons 3-6 (exons 5-8). At the downstream end, human coding exon 6 (exon 8) is followed by a stop codon and a human 3'-UTR, and further downstream, a human sequence found downstream of the human 3'UTR. For selection purposes, a selection cassette (with loxP introduced for removal by Cre) was incorporated. The humanized locus in Figure 2 expresses a fully human, mature IL-15 protein.

[0376] Specifically, we performed homologous recombination (BHR) using standard bacterial homologous recombination (BHR) techniques (see, for example, Valenzuela et al. (2003) above) and gap repair BHR to construct large targeting vectors (LTVECs) containing the human IL-15 gene sequence for targeting the mouse IL-15 locus. Linear fragments were generated by ligating PCR-generated homology boxes to the cloned cassettes, followed by isolation of the ligation products on a gel, and electroporation was performed on BHR-competent bacteria containing the target bacterial artificial chromosome (BAC). Mouse BAC PRCI23-203P7 was used as the source of the mouse sequence, and human BAC RP11-103B12 was used as the source of the human IL-15 gene sequence. After the selection step, clones that underwent accurate recombination were identified by PCR and restriction enzyme analysis across the novel junction. LTVECs containing homology arms and the human IL-15 gene sequence were then constructed.

[0377] The mouse IL-15 gene (mouse GeneID: 103014, RefSeq transcript: NM_008357.2, ensemble eID: 16168) was modified using genomic coordinates GRCM38:ch8:82331173-82343471 (minus strand) for deletions and GRCh37:ch4:142642924-142655819 (plus strand) for substitutions. 12,299 nucleotides of the mouse sequence were replaced with 12,896 nucleotides of the human sequence. The above mouse IL-15 sequence substitutions are illustrated in Figure 2.

[0378] LTVEC, containing the humanized IL-15 gene, had an upstream mouse targeting arm of approximately 13 kb adjacent to the MluI site and a downstream mouse targeting arm of 27 kb adjacent to the AscI site. For electroporation, LTVEC was linearized using MluI and AscI.

[0379] The LTVEC nucleotide sequences across the mouse / human 5' and human / mouse 3' junctions after LTVEC construction are shown in Table 1 below. SEQ ID NO:4 indicates the upstream junction (with respect to the direction of IL-15 gene transcription) between the mouse and human sequences, and the sequence shown begins with the mouse sequence in uppercase, followed by the AsisI restriction site in lowercase, followed by the human IL-15 nucleic acid sequence in uppercase. SEQ ID NO:5 indicates the downstream human IL-15 coding and non-coding sequences (human 3'UTR in bold italics) in uppercase, followed by the XhoI site in lowercase, followed by the lox site (uppercase, in bold italics), followed by the downstream neo selection cassette (uppercase), which extends 2.6kb downstream (not shown). SEQ ID NO:6 is a nucleic acid sequence indicating the junction between the downstream portion of the neo selection cassette (uppercase) and the lox region (uppercase and bold italics), followed by the NheI region (lowercase), then the mouse sequence downstream of the humanized portion (uppercase), and the selection cassette extends another 2.6kb upstream.

[0380] (Table 1) Conjugation sequences of the humanized IL-15 gene locus TIFF0007839229000004.tif235158TIFF0007839229000005.tif220158

[0381] Mouse ES cells were electroporated with an LTVEC construct and grown on selective medium. These were then used as donor ES cells to generate humanized IL-15 mice with human sequence substitutions at the endogenous mouse IL-15 locus, as shown in Figure 2. Following electroporation of the ES cells, a native allele deletion assay (see, for example, Valenzuela et al. (2003) above) was performed to detect deletions of the endogenous IL-15 sequence caused by targeting.

[0382] Precisely targeted ES cells were further electroporated with a transient Cre expression vector to remove the Neo drug selection cassette.

[0383] Donor mouse ES cells containing the humanized IL-15 locus were introduced into early mouse embryos using the VELOCIMOUSE® method (Poueymirou et al. (2007) F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses, Nat Biotechnol 25:91-99). Heterozygous mice were obtained, and heterozygotes were crossed to obtain homozygotes for humanized IL-15. Two versions of humanized IL-15 mice were created (referred to herein as Mouse 1 and Mouse 2). Further analysis revealed that Mouse 1 version had exon duplication in its genome. In Mouse 2, the endogenous mouse IL-15 locus was replaced with a human sequence as shown in Figure 2.

[0384] Human IL-15 mRNA levels were measured as follows. Reverse transcription (RT)-qPCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) and the SYBR® FAST universal qPCR kit (KAPA Biosystems). Sequence-specific oligonucleotide primers were designed using Primer3 software and synthesized by Sigma-Aldrich. The following primers were used: Mouse HPRT forward: TIFF0007839229000006.tif4128, Mouse HPrt Reverse: TIFF0007839229000007.tif4128, Human Il15 forward: TIFF0007839229000008.tif4128, Human Il15 Reverse: TIFF0007839229000009.tif4128. Relative expression values ​​were calculated using the threshold cycle comparison method and normalized to mouse HPRT.

[0385] (1) Both are Rag2 - / - Il2rg Y / - By crossing a mouse with a background and human SIRPα substitution with a mouse with human IL-15 substitution, or (2) Rag2 - / - Il2rg Y / - ES cells with background and human SIRPα substitution were introduced with a large targeting vector containing human IL-15 (as described in Example 1), and both human IL-15 and SIRPα gene substitutions, as well as Rag2, were targeted using the VELOCIMOUSE® method. - / - Il2rg Y / - SRG-15 mice are generated by creating mice from ES cells containing [specific trait]. Homozygotes are obtained by mating heterozygous mice.

[0386] result As shown in Figures 3A and 3B, high levels of human IL-15 mRNA expression were found in the liver, lungs, bone marrow (BM), small intestine (SI), and colon of non-engrafted SRG-15 mouse 1. Similar high levels of human IL-15 mRNA were found in the liver, lungs, and small intestine of non-engrafted SRG-15 mouse 2 (Figure 3B). As shown in Figure 4, even upon stimulation with poly(I:C), high levels of human IL-15 protein could be detected in the serum of SRG-15 mouse 2, in which endogenous mouse exons were replaced with human exons 5-8.

[0387] Example 3: Engraftment of SRG-15 mice Materials and methods The following procedure is used to engraft the huHSCs into SRG and SRG-15 mice. Newborn mice 3-5 days after birth are given a sublethal dose of 160 cGy of radiation without anesthesia and returned to their mothers for rest. 4-12 hours after radiation, 25 μl of CD34+ huHSCs in PBS are transplanted into the liver (ih) of these newborns using a 30G needle.

[0388] result To evaluate the effects of human IL-15 on immune cell development, human CD45 in NSG, SRG, and SRG-15 mice was used. + We compared cell engraftment. Efficient engraftment of human hematopoietic cells in the blood of NSG, SRG, and SRG-15 (mouse 2) mice was observed 12–14 weeks after engraftment, as shown in Figure 5A. Figure 5B shows a comparison of engraftment demonstrated by the number of human CD45+ cells in the BM, spleen, LN, liver, and lungs of SRG and SRG-15 (mouse 2) mice 14 weeks after engraftment.

[0389] In mouse 1, human CD45 +While there were no differences in cell engraftment, as shown in Figures 6A and 6B, higher proportions and numbers of human NK cells were found in various tissues of SRG-15 mice compared to SRG mice. IL-15 is important not only for NK cell development and survival but also for maturation. As shown in Figure 6C, human NK cells in the liver of SRG-15 mice (mouse 1) expressed higher levels of CD16 and CD56, indicating that NK cell maturation was more advanced in SRG-15 mice compared to SRG mice. Human NK cell subset, CD56 bright CD16 - and CD56 dim CD16 + Both were found to be present in the blood, spleen, and liver of SRG-15 mice, as shown in Figure 6D (spleen) (and data not shown). Furthermore, as shown in Figure 6D, analysis of two human NK cell subsets in the spleen of SRG-15 mice (mouse 1) revealed that these subsets had clear levels of expression of killer inhibitory receptors as well as CD56 dim CD16 + The NK cell population was found to contain a higher proportion of CD158-expressing cells. This result is similar to what is found in a subset of NK cells in human blood (data not shown).

[0390] In SRG-15 mouse 2, efficient engraftment of human NK cells was observed in lymphoid and non-lymphoid tissues, as shown in Figures 7A-7D. Figures 7A and 7B show the percentage of NK cells in the blood and spleen, respectively. Figures 7C and 7D show the frequency of human NK cell appearance in the blood, spleen (SP), liver, and lungs of SRG and SRG-15 (mouse 2) mice 14 weeks after engraftment. Further data showing the distribution and percentage of NK cells in the blood and spleen of SRG and SRG-15 (mouse 2) mice from different experiments are shown in Figures 8 and 9A, respectively. Figure 9B shows the increase in hNKp46 fragments of hCD45+ cells in the blood of SRG-15 mouse (mouse 2). Figures 9C-9E show the relative number, distribution, and composition of hCD45+ cells in the thymus of SRG and SRG-15 (mouse 2) mice.

[0391] We characterized the NK cell subsets of humans and SRG-15 mice (mouse 2). As shown in Figures 10A and 10B, the blood and spleen of SRG-15 mice showed a higher proportion of hCD-56 compared to SRG mice. bright hCD16 - and hCD56 dim hCD16 + Elevated levels of both were observed. Similar to humans, expression of killer inhibitory receptors (KIRs) was observed in the NK cell subset of SRG-15 mice (mouse 2) (Figure 10C). Figure 10C shows CD56bright CD16 - NK cells (left box in each plot) and CD56dim CD16 + The right-hand boxes in each plot show NK cells. The histograms in the lower panel show CD158 expression in their subsets. CD158 (KIR2D) on the SRG-15 mouse NK cell subset is similar to that observed in human PBMC-derived NK cells.

[0392] The distribution of human NK cells in the blood of SRG-15 mice was compared with the distribution of human NK cells in the blood of two healthy human donors. Peripheral blood mononuclear cells (PBMCs) were isolated from the pia mater of two separate donors via Ficoll-Paque (obtained from BioreclamationIVT, Westbury, NY). As a result, a higher proportion of NK cells in the blood was observed in engrafted SRG-15 mice compared to human donor-derived PBMCs. However, physiologically equivalent distributions were observed between cytotoxic (CD16+) NK cells and IFN-g producing (CD16-) NK cells (Figure 11).

[0393] Finally, analysis of the bone marrow of SRG and SRG-15 (mouse 2) showed that the development of human NK cells in SRG-15 mice was higher than in SRG mice (Figure 12).

[0394] The effect of human IL-15 on human T cell development in SRG-15 mice was also evaluated. Comparison between SRG-15 (mouse 1) mice and SRG mice revealed that the effect of human IL-15 on the proportion, number, and / or ratio of T cells varied depending on the tissue (Figure 13A). There was no difference in lymph node size and number between SRG and SRG-15 mice at 16 weeks post-engraftment, which confirmed that the number of human T cells in the lymph nodes of SRG and SRG-15 (mouse 1) mice was equivalent (Figure 13A). Figure 13B shows human CD8 in the blood and liver of SRG and SRG-15 mice (mouse 1). + The T cell phenotype was observed, and hCD62L was detected in both the blood and liver of SRG-15 mice (mouse 1). - The cells show higher levels compared to SRG mice. Further data characterizing T cells in SRG-15 mice (mouse 1) compared to SRG mice are shown in Figures 14A and 14B, which show CD8 levels in the lungs (14A) and livers (14B) of SRG and SRG-15 mice. + The expression of the tissue resident marker CD69 in T cells is shown. The above data provides evidence of increased effector tissue resident T cells in SRG-15 mice.

[0395] For mouse 2, as shown in Figures 15A and 15B, hCD3 was found in the spleen, lungs, and liver. + The frequency of T cell appearance was evaluated at 16 weeks post-engraftment compared to SRG mice.

[0396] Example 4: Generation of human tissue-resident lymphocytes in SRG-15 mice Since IL-15 has been shown to be produced by epithelial cells of the intestine and lung, and may play an important role in the development and survival of human tissue-resident T cells and NK cells, we analyzed human tissue-resident T and NK cells in SRG and SRG-15 mice.

[0397] Materials and methods Newborn mice at 3 to 5 days after birth are irradiated with a dose less than the lethal dose of 160 cGy without anesthesia and then returned to their mothers for rest. Four to twelve hours after irradiation, CD34+ huHSCs in 25 μl of PBS are transplanted into the livers (i.h.) of these newborn mice using a 30G needle.

[0398] Results As shown in Figure 17A, isolates of the intraepithelial lymphocyte population from the small intestine of mouse 1 in the steady state showed a higher frequency of human CD45 + cells in SRG-15 mice compared to SRG mice. As shown in Figure 17B, the results of immunohistochemical analysis showed that human CD45 + NK cells were located in the epithelial cell layer of the small intestine of SRG-15 mice (mouse 1) (as indicated by the arrow in Figure 17B), while few intraepithelial lymphocytes were found in SRG mice. Human CD8 + IELs in SRG-15 mice showed high expression with respect to CD69, a common marker for tissue-resident T cells. In contrast to human IELs (Sathaliyawala T, Kubota M, Yudanin N et al. Immunity 2013;38:187-197), only a subset of human CD8 + IELs in SRG-15 mice expressed the tissue-resident marker CD103 (Figure 17C). As shown in Figures 16A and 16B, there was little difference in the number of lamina propria cells in the steady-state colon between SRG mice and SRG-15 mice, so the increased human CD8 + IEL phenotype in SRG-15 mice (mouse 1) was specific. In addition to the increase in human T cell numbers in the lungs of SRG-15 mouse 1 shown in Figure 13A, as shown in Figure 14A, the expression of CD69 on human CD8 + T cells in the lungs of SRG-15 mice was also found to be higher compared to SRG mice. Furthermore, Figure 14B shows that the level of hCD69 expressed by CD8 + T cells in the liver of SRG-15 mouse 1 is higher compared to SRG mice.

[0399] Similar to Mouse 1 engrafted with SRG-15, in Mouse 2 engrafted with SRG-15, by FACS analysis, it was revealed that the proportion of human CD45 + cells in the IEL fraction of SRG-15 mice was higher than that of SRG mice (Figure 18A). Furthermore, although the number of LPLs did not significantly change between SRG and SRG-15 (Mouse 2) mice, a significant increase in IELs was observed in SRG-15 (Mouse 2) mice compared to SRG mice (Figure 18B). The composition of hCD3+ cells in the small intestine of SRG-15 mice (Mouse 2) is shown in Figure 18C, indicating that the proportion of hCD8+ is higher than that of hCD4+ cells. The phenotypic characteristics of hCD3+ hCD8+ T cells in the spleen and small intestine of SRG-15 mice (Mouse 2) are shown in Figure 18D. As shown in Figure 18E, the results of immunohistochemical analysis showed that human CD8 + IELs were located in the epithelial cell layer of the small intestine of SRG-15 mice (Mouse 2) (as indicated by the arrow in Figure 18E), while few intraepithelial lymphocytes were found in SRG mice.

[0400] As described above with respect to Figures 18A and 18B, SRG-15 engrafted mouse 2 showed greater reconstitution of human intraepithelial lymphocytes (IELs) in the gut-associated lymphoid tissue (GALT) compared to SRG mice (Figures 19A and 19C). Interestingly, the majority of the observed human lymphocytes were human NK cells. As expected from normal human GALT physiology, the majority of NK cells in the blood and spleen of SRG-15 mouse 2 were cytotoxic NK cells (CD16+), while the distribution of CD16+ NK cells and CD16- NK cells was similar in IELs (Figure 19B). There was no change in the number of lamina propria lymphocytes between engrafted SRG and SRG-15 mice (Figure 19C). Unlike engrafted SRG-15 mouse 1, a higher proportion of human CD3+ CD8+ IELs in engrafted SRG-15 mouse 2 expressed the human CD103 marker. Peyer's patches were completely absent in SRG mice, but they were present in SRG-15 mice 2, where human lymphocytes were dominant, as shown in Figures 20A and 20B.

[0401] Example 5: Determination of the functional role of human tissue-resident T cells in SRG-15 mice infected with the virus. To investigate whether tissue-resident T cells in SRG-15 mice have a functional relevance while maintaining homeostasis, human CD8 cells were introduced into SRG-15 mice. + We determined whether an increase in the number of IELs induces characteristic changes in the composition of the mouse gut microbiota.

[0402] Materials and methods Newborn mice, 3 to 5 days old, are irradiated with a sublethal dose of 160 cGy without anesthesia and returned to their mothers for rest. 4 to 12 hours after irradiation, CD34+ huHSCs in 25 μl of PBS are transplanted into the liver (ih) of these newborns using a 30G needle.

[0403] Four weeks after engraftment, SRG-15 mice were co-contained with SRG and donor Balb / c mice for four weeks to equalize the gut microbiota between the different strains. The mice were then isolated, fecal samples were collected, and analyzed by 16S rRNA sequencing. Figure 21A shows the timeline for co-containment and fecal sample collection for gut microbiota sequencing.

[0404] result As shown in 21B, with respect to mouse 1, the results showed no significant change between engrafted SRG-15 mice and SRG mice after co-accommodation, and this is related to human CD8 + This shows that even when IEL is generated, it does not induce significant changes during the steady state. Further experiments will be conducted to determine if there is enough CD8 to eliminate acute rotavirus infection. + We determined whether IEL could eliminate rotavirus infection in engrafted SRG-15 mice. As shown in Figure 22, the results showed that acute rotavirus infection could be eliminated in engrafted SRG-15 mice, but not in non-engrafted SRG mice.

[0405] Example 6: Analysis of NK cell subsets in SRG-15 mice (mouse 2) and humans We characterized the NK cell subset in SRG-15(mouse2) mice with respect to various phenotypic markers and compared them to those in humans.

[0406] Materials and methods Generally, as described in Yao et al. J. of Immunological Methods 415(2014)1-5, NK cell subsets were detected via time-of-flight cytometry (CyTOF) and analyzed using ViSNE (el-AD et al. Nat. Biotechnol. 2013 Jun;31(6):545-52 doi:10.1038 / nbt.2594. Epub 2013 May 19).

[0407] result Figure 23A shows the CD56 levels in humans (n=20) and SRG-15 mice (2 mice) (n=9). bright CD16 - and CD56 dim CD16 + This provides a ViSNE plot showing a CyTOF-based analysis of 33 parameters of an NK cell subset. Each dot represents a single cell.

[0408] Figure 23B shows the CD56 levels in humans (n=20) and SRG-15 mice (2 mice) (n=9). bright CD16 - This provides a ViSNE plot showing the expression intensity of eight select markers on NK cells.

[0409] Figure 23C shows the CD56 levels in humans (n=20) and SRG-15 mice (n=9). dim CD16 + This is a ViSNE plot showing the expression intensity of eight select markers on NK cells. This multidimensional single-cell analysis of 33 key molecules in human NK cells demonstrates that human NK cells generated in SRG-15 mice are remarkably equivalent to human NK cells in healthy individuals.

[0410] Example 7: Cytotoxic capacity of NK cells derived from SRG-15 mice Materials and methods To investigate in vitro NK cell toxicity, spleen NK cells isolated from human HSC-engrafted SRG and SRG-15 mice (Mouse 2) were treated overnight with human IL-2. The following day, the NK cells were cultured with CFSE-labeled NK-sensitive K562 target cells at various effector-to-target ratios (E:T). After 5 hours of co-culture, K562 cell death was measured by FACS analysis of the survival dye Topro3 uptake by K562 cells (K562 cells were gated to CFSE+ cells, and then the positivity rate for Topro3 was analyzed).

[0411] Furthermore, to investigate antibody-dependent cytotoxicity (ADCC) in vitro, spleen NK cells isolated from human HSC-engrafted SRG and SRG-15 mice were treated overnight with human IL-2. The following day, the NK cells were cultured with CFSE-labeled Raji target cells at various effector-to-target ratios (E:T). Raji cells were pre-treated with anti-CD20 (rituximab) or control IgG. After co-culture for 5 hours, Raji cell death was measured by FACS analysis of the survival dye Topro3 uptake by Raji cells (gated to CFSE+ cells, then the positivity rate for Topro3 was analyzed).

[0412] To investigate in vivo NK cell activation, 50 μg of poly-IC was intraperitoneally injected into human HSC-engrafted SRG and SRG-15 mice (Mouse 2). Pre-blood sampling was performed on the mice (before poly-IC injection) and again 18 hours after poly-IC injection. The expression of the activation marker CD69 before and after poly-IC administration was analyzed by FACS in human CD45+ NKp46+ (NK cells).

[0413] result In a classical NK cytotoxicity test, classical NK-targeted HLA class I-deficient K562 cells were killed by activated NK cells derived from SRG or SRG-15 mice (mouse 2). As shown in Figure 24C (left), splenic NK cells derived from SRG and SRG-15 mice showed comparable cytolytic ability to K562 cells when normalized for number.

[0414] NK cells typically play a role in anti-CD20 antibody-mediated ADCC against B-cell leukemia and lymphoma (see, e.g., J. Golay et al. Haematologica 2003;88:1002-12). To demonstrate the ability of SRG-15 engrafted mouse-derived NK cells to promote anti-CD20-mediated ADCC, both SRG and SRG-15 mouse-derived spleen NK cells were tested and, after normalization for cell number, were found to exhibit comparable antibody-dependent cell-mediated cytotoxicity (ADCC) activity to anti-CD20-treated Raji cells (Figure 24C (right)).

[0415] For example, as shown in Figures 8 and 9, significant upregulation of NK cells was observed in both the spleen and blood of SRG-15 animals. The activating ability of NK cells in SRG-15 mice was tested by measuring CD69 marker activation after poly-IC injection. As shown in Figure 24A, the percentage of NK cells positive for the activation marker CD69 was increased in SRG-15 mice compared to SRG mice. Since SRG-15 NK cells showed comparable ADCC mediation to SRG NK cells under normalized conditions in vitro, the ability of SRG-15 NK cells to exhibit a highly activated phenotype in vivo, as well as the ability to exhibit a large number of NK cells in SRG-15 mice, suggests that SRG-15 mice may be a suitable in vivo model for studying human NK cell ADCC.

[0416] Example 8: IFNγ production by SRG and SRG-15-derived NK cells Materials and methods NK cells were isolated from pooled splenocytes (3 spleens per group) derived from SRG or SRG-15 mice, and these NK cells were isolated using the EasySep Human NK Concentration Kit (StemCell Technologies, catalog number 19055).

[0417] NK cells were also isolated from healthy human PBMCs. NK cells were treated overnight with 10 ng / mL human IL-2. The following day, cells were stimulated overnight with 10 ng / mL human IL-12p70 or 2 mg / mL poly(I:C), or left untreated. The following day, the supernatant was collected, and IFNg levels were evaluated using the Human IFNg Quantikine ELISA Kit (R&D Systems, catalog number DIF50). NK cell purity was analyzed by FACS and by IFNg levels normalized as picograms (pg) produced by individual NK cells. Statistical analysis was performed using the ANOVA test.

[0418] result As shown in Figure 24B, SRG and SRG-15-derived NK cells secrete IFNγ to a similar degree, but when treated with IL-12p70, the secretion is less than that of human PBMC-derived NK cells.

[0419] Example 9: Human NK cells inhibit tumor growth in SRG-15 mice. We investigated the ability of human NK cells to infiltrate human tumor xenografts in SRG-15 mice (mouse 2) and inhibit tumor growth.

[0420] Materials and methods Rituximab was administered intraperitoneally every other day (started 14 days after subcutaneous injection of 5 million Raji cells). Tumor growth was evaluated by caliper measurement, and the volume was calculated using the following formula: Tumor volume = 0.5 × (length × width). ∧ 2) Data were pooled from two independent experiments. Statistical analysis was performed using the independent two-tailed Mann-Whitney U test to compare untreated SRG-15 mice with engrafted grafts with RTX-treated SRG-15 mice with engrafted grafts. * P<0.05).

[0421] Subcutaneous tumors were crushed and digested with collagenase D (1 hour, 37°C). The recovered cells, including tumor cells and immune cells, were analyzed using an LSRII flow cytometer.

[0422] result As shown in Figure 25A, human NK cells from SRG-15 mice inhibit tumor growth after rituximab (RTX) treatment. Figure 25B shows the frequency of human NK cells and T cells in human tumor xenografts from untreated (n=5) and RTX-treated SRG-15 mice (n=1). Figure 25C shows the human NK cell subsets in the blood and tumors of untreated (n=2) and RTX-treated SRG-15 mice (n=1).

[0423] Example 10: Additional materials and methods used in relation to the above examples Human CD34 + Cell isolation and injection. Human CD34 +Cell isolation and injection were performed according to the method described, for example, Rongvaux A, Willinger T, Martinek J et al. Nat Biotechnol 2014;32:364-372.

[0424] Flow cytometry analysis of human cell populations. Flow cytometry analysis of human cell populations was performed according to the methods described in Strowig T, Rongvaux A, Rathinam C et al. Proc Natl Acad Sci USA 2011;108:13218-13223 and Rongvaux A, Willinger T, Martinek J et al. Nat Biotechnol 2014;32:364-372.

[0425] Histology. The tissue was fixed overnight in 4% paraformaldehyde, transferred to 70% ethanol, and embedded in paraffin.

[0426] Quantitative RT-PCR. Quantitative RT-PCR was performed according to the method described in Rongvaux A, Willinger T, Martinek J et al. Nat Biotechnol 2014;32:364-372.

[0427] 16S rRNA sequencing was performed according to the method described in Palm NW, de Zoete MR, Cullen TW et al. Cell 2014;158:1000-1010.

[0428] Viral infection. Rotavirus and influenza virus were obtained and used in the method of the present invention.

[0429] Statistical analysis. Statistical significance was calculated using independent two-tailed Student t-tests with Prism6 software (GraphPad).

[0430] FACS antibodies were obtained from BD Biosciences and BioLegend.

[0431] The above merely describes the principles of the present invention. Those skilled in the art will understand that various configurations embodying the principles of the present invention and falling within its spirit and scope can be devised, even if not explicitly described or illustrated herein. Furthermore, all embodiments and conditional statements referred to herein are intended primarily to assist the reader in understanding the principles of the present invention and the concepts contributing to the advancement of the art by the inventors, and should be interpreted as not limiting the reader to such specifically listed embodiments and conditions. Moreover, all descriptions herein referring to the principles, aspects, and embodiments of the present invention, and their specific examples, are intended to encompass both their structural and functional equivalents. Furthermore, such equivalents are intended to encompass both currently known equivalents and those to be developed in the future, i.e., any developed elements that perform equivalent functions regardless of their structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied in the appended claims.

[0432] Additional sequence information Locus NM_001040022 4201bp mRNA Linear Primate March 15, 2015 Definition: Homo sapiens signal regulatory protein α (SIRPA), transcript variant 1, mRNA Accession number NM_001040022 Version number NM_001040022.1 GI:91105763 Origin: Homo sapiens (human) TIFF0007839229000010.tif86128TIFF0007839229000011.tif235125TIFF0007839229000012.tif235125TIFF0007839229000013.tif235125TIFF0007839229000014.tif39128TIFF0007839229000015.tif74128 Locus NM_001040023 4109bp mRNA Linear Primate March 15, 2015 Definition: Homo sapiens signal regulatory protein α (SIRPA), transcript variant 2, mRNA Accession number NM_001040023 Version number NM_001040023.1 GI:91105766 Origin: Homo sapiens (human) TIFF0007839229000016.tif74128TIFF0007839229000017.tif235125TIFF0007839229000018.tif235125TIFF0007839229000019.tif235125TIFF0007839229000020.tif27128TIFF0007839229000021.tif74128 Locus NM_080792 3868bp mRNA Linear Primate March 15, 2015 Definition: Homo sapiens signal regulatory protein α (SIRPA), transcript variant 3, mRNA Accession number NM_080792 NM_004648 Version number NM_080792.2 GI:91105786 Origin: Homo sapiens (human) TIFF0007839229000022.tif86128TIFF0007839229000023.tif235125TIFF0007839229000024.tif235125TIFF0007839229000025.tif207125TIFF0007839229000026.tif74128 Locus NM_007547 4031bp mRNA Linear Rodents February 15, 2015 Definition: Mus musculus signal regulatory protein α (Sirpa), transcript variant 1, mRNA Accession number NM_007547 NM_011208 Version number NM_007547.4 GI:597084939 Origin: Mus musculus (house mouse) TIFF0007839229000027.tif146128TIFF0007839229000028.tif235125TIFF0007839229000029.tif235125TIFF0007839229000030.tif183128TIFF0007839229000031.tif78128 Locus NM_001177647 3377bp mRNA Linear Rodents February 15, 2015 Definition: Mus musculus signal regulatory protein α (Sirpa), transcript variant 3, mRNA Accession number NM_001177647 Version number NM_001177647.2 GI:597436949 Origin: Mus musculus (house mouse) TIFF0007839229000032.tif170128TIFF0007839229000033.tif235125TIFF0007839229000034.tif235125TIFF0007839229000035.tif27128TIFF0007839229000036.tif50128 Locus NM_001291019 4043bp mRNA Linear Rodents February 15, 2015 Definition: Mus musculus signal regulatory protein α (Sirpa), transcript variant 4, mRNA Accession number NM_001291019 XM_006498985 Version number NM_001291019.1 GI:597436868 Origin: Mus musculus (house mouse) TIFF0007839229000037.tif114146TIFF0007839229000038.tif235146TIFF0007839229000039.tif235125TIFF0007839229000040.tif147128TIFF0007839229000041.tif82128 Locus NM_001291020 3845bp mRNA Linear Rodents February 15, 2015 Definition: Mus musculus signal regulatory protein α (Sirpa), transcript variant 5, mRNA Accession number NM_001291020 XM_006498984 Version number NM_001291020.1 GI:597436945 Keywords RefSeq Origin: Mus musculus (house mouse) TIFF0007839229000042.tif198146TIFF0007839229000043.tif235125TIFF0007839229000044.tif235125TIFF0007839229000045.tif27128TIFF0007839229000046.tif82128 Locus NM_001291021 3389bp mRNA Linear Rodents February 15, 2015 Definition: Mus musculus signal regulatory protein α (Sirpa), transcript variant 6, mRNA Accession number NM_001291021 XM_006498987 Version number NM_001291021.1 GI:597436920 Origin: Mus musculus (house mouse) TIFF0007839229000047.tif86128TIFF0007839229000048.tif235125TIFF0007839229000049.tif235125TIFF0007839229000050.tif111128TIFF0007839229000051.tif50128 Locus NM_001291022 3020bp mRNA Linear Rodents February 15, 2015 Definition: Mus musculus signal regulatory protein α (Sirpa), transcript variant 7, mRNA Accession number NM_001291022 Version number NM_001291022.1 GI:597436963 Origin: Mus musculus (house mouse) TIFF0007839229000052.tif27128TIFF0007839229000053.tif235125TIFF0007839229000054.tif235125TIFF0007839229000055.tif99128TIFF0007839229000056.tif34128 Locus NM_009020 3393bp mRNA Linear Rodents February 15, 2015 Definition: Mus musculus, recombinant activator gene 2 (Rag2), mRNA Accession number NM_009020 Version number NM_009020.3 GI:144227233 Origin: Mus musculus (house mouse) TIFF0007839229000057.tif65128TIFF0007839229000058.tif235125TIFF0007839229000059.tif235125TIFF0007839229000060.tif135128TIFF0007839229000061.tif82128 Locus NM_013563 1612bp mRNA Linear Rodents February 15, 2015 Definition: Mus musculus, interleukin-2 receptor γ chain (Il2rg), mRNA Accession number NM_013563 Version number NM_013563.3 GI:118129799 Origin: Mus musculus (house mouse) TIFF0007839229000062.tif206125TIFF0007839229000063.tif111131TIFF0007839229000064.tif58128 Locus NM_000585 2012bp mRNA Linear Primate March 15, 2015 Definition: Homo sapiens, interleukin-15 (IL-15), transcript variant 3, mRNA Accession number NM_000585 Version number NM_000585.4 GI:323098327 Origin: Homo sapiens (human) TIFF0007839229000065.tif27128 TIFF0007839229000066.tif235125 TIFF0007839229000067.tif135128 TIFF0007839229000068.tif26128 Locus NM_172175 2333bp mRNA Linear Primate March 15, 2015 Definition: Homo sapiens, interleukin-15 (IL-15), transcript variant 2, mRNA Accession number NM_172175 Version number NM_172175.2 GI:323098328 Origin: Homo sapiens (human) TIFF0007839229000069.tif27128TIFF0007839229000070.tif235125TIFF0007839229000071.tif195133TIFF0007839229000072.tif26128 Locus NM_008357 1297bp mRNA Linear Rodents February 15, 2015 Definition: Mus musculus, interleukin-15 (Il15), transcript variant 1, mRNA Accession number NM_008357 Version number NM_008357.2 GI:363000959 Origin: Mus musculus (house mouse) TIFF0007839229000073.tif206125TIFF0007839229000074.tif51128TIFF0007839229000075.tif26128 Locus NM_001254747 1287bp mRNA Linear Rodents February 15, 2015 Definition: Mus musculus, interleukin-15 (Il15), transcript variant 2, mRNA Accession number NM_001254747 Version number NM_001254747.1 GI:363000983 Origin: Mus musculus (house mouse) TIFF0007839229000076.tif110128TIFF0007839229000077.tif147128TIFF0007839229000078.tif26128

[0433] Sequence information SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. Yale Plaza Institute for Research in Biomedicine (IRB) <120> HUMANIZED SIRPA-IL15 KNOCKIN MICE AND METHODS OF USE THEREOF <150> US62 / 146938 <151> 2015-04-13 <150> US62 / 148667 <151> 2015-04-16 <150> US62 / 287842 <151> 2016-01-27 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 200 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 1 agctctccta ccactagact gctgagaccc gctgctctgc tcaggactcg atttccagta 60 cacaatctcc ctctttgaaa agtaccacac atcctggggt gctcttgcat ttgtgtgaca 120 ctttgctagc caggctcagt cctgggttcc aggtggggac tcaaacacac tggcacgagt 180 ctacattgga tattcttggt 200 <210> 2 <211> 199 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 2 gctccccatt cctcactggc ccagcccctc ttccctactc tttctagccc ctgcctcatc 60 tccctggctg ccattgggag cctgccccac tggaagccag tcgagataac ttcgtataat 120 gtatgctata cgaagttata tgcatggcct ccgcgccggg ttttggcgcc tcccgcgggc 180 gcccccctcc tcacggcga 199 <210> 3 <211> 200 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 3 cattctcagt attgttttgc caagttctaa ttccatcaga cctcgacctg cagcccctag 60 ataacttcgt ataatgtatg ctatacgaag ttatgctagc tgtctcatag aggctggcga 120 tctggctcag ggacagccag tactgcaaag agtatccttg ttcatacctt ctcctagtgg 180 ccatctccct gggacagtca 200 <210> 4 <211> 208 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 4 atccatttag cctttctctg atcactaagt tggacagttg gacagtcttc ctcaaattag 60 cttagactat caaaattata ctgtattttt ggtatttcca gcgatcgctt cagttacaag 120 gctgttgaat gcacagaagc aaggataaca ctgatttttt cactggtcag aataaaaatt attgattgct cttttgctta tagtattc <210> 5 <211> 2028 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 5 aatgtaacag aatctggatg caaagaatgt gaggaactgg aggaaaaaaa tattaaaga 120. tttttgcaga gttttgtaca tattgtccaa atgttcatca acacttcttg attgcaattg attcttttta aagtgtttct gttattaaca aacatcactc tgctgcttag acataacaaa acactcggca tttcaaatgt gctgtcaaaa caagtttttc tgtcaagaag atgatcagac cttggatcag atgaactctt aagaatgaag gcagaaaaat gtcattgagt aatatagtga ctatgaactt ctctcagact tactttactc atttttttaa tttattattg aaattgtaca tatttgtgga father atgttgaata aaaatatgta caagtgttgt tttttaagtt gcactgatat tttacctctt attgcaaaat agcatttgtt taagggtgat agtcaaatta tgtattggtg gggctgggta ccaatgctgc aggtcaacag ctatgctggt aggctcctgc 540 cagtgtggaa ccactgacta ctggctctca ttgacttcct tactaagcat agcaaacaga 600 ggaagaattt gttatcagta agaaaaagaa gaactatatg tgaatcctct tctttatact 660 gtaatttagt tattgatgta taaagcaact gttatgaaat aaagaaattg caataactgg 720 catataatgt ccatcagtaa atcttggtgg tggtggcaat aataaacttc tactgatagg 780 tagaatggtg tgcaagcttg tccaatcacg gattgcaggc handicapgcggc ccaggacaac 840 tttgaatgtg gcccaacaca aattcataaa ctttcataca tctcgttttt agctcatcag 900 ctatcattag cggtagtgta tttaaagtgt ggcccaagac aattcttctt attccaatgt 960 ggcccaggga aatcaaaaga ttggatgccc ctggtataga aaactaatag tgacagtgtt 1020 catatttcat gctttcccaa atacaggtat tttattttca cattcttttt gccatgttta 1080 tataataata aagaaaaacc ctgttgattt gttggagcca ttgttatctg acagaaaata 1140 attgtttata ttttttgcac tacactgtct aaaattagca agctctcttc taatggaact 1200 gtaagaaaga tgaaatattt ttgttttatt ataaattta ttcaccttaa ttctggtaat 1260 actcactgag tgactgtggg gtgggaaatg atctcttaag aatttgattt cttcttattc 1320 catagtacaa actcgttctc tgttgaaaca ttcttctatc accccagtgc cctatccatg 1380 tacatgtgtt cttattgctc tagtcaaacg gtgcttataa atatctttca gaaagtttag 1440 gagaaatctg tatcctattt gacttccaat aatcatgtat tggctgtcag cttcttacct 1500 actctcagtc cagagaaata gtatttggca gccactcttt aaagtttatg ggttgtggat 1560 tgtggcggtt gatttatttt ttttatttca attgggatag aatttttaa tatacctgta 1620 ttttgtttt gttttatgta gcttttctat tagggagagt aggaaaagtg caccattttc 1680 ttctctaaat ttccagtcca gtctttaggg gaatgttagt cttcctgaga tgggggaagg 1740 aaaatcataa tgccagtcac tttgcaaata atattttata gtgataaatg gttcattttg 1800 gttacatagg catacaagtg ggcttaaaac ttggaattta ccagggctca aaattaaaat 1860 tcttacatta gttactcgat atggatcgct tcagttgatc ttagaaaact caaggcatag 1920 atctgcaacc tcgagataac ttcgtataat gtatgctata cgaagttata tgcatggcct 1980 ccgcgccggg ttttggcgcc tcccgcgggc gcccccctcc tcacggcg 2028 <210> 6 <211> 200 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 6 cattctcagt attgttttgc caagttctaa ttccatcaga cctcgacctg cagcccctag 60 ataacttcgt ataatgtatg ctatacgaag ttatgctagc gtgatagtcc ttcacggaaa 120 gtacaagaat acacagaaaa ctgctgttta cattagtctt tcacgttttt attttattct 180 cacaaatttt aatgcaatac 200 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic oligonucleotide <400> 7 agggatttga atcacgtttg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic oligonucleotide <400> 8 tttactggca acatcaacag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic oligonucleotide <400> 9 gcccagggaa atcaaaagat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic oligonucleotide <400> 10 tggctccaac aaatcaacag 20 <210> 11 <211> 4201 <212> DNA <213> Homo sapiens <400> 11 tccggcccgc acccaccccc aagaggggcc ttcagctttg gggctcagag gcacgacctc 60 ctggggaggg ttaaaaggca gacgcccccc cgccccccgc gcccccgcgc cccgactcct 120 tcgccgcctc cagcctctcg ccagtgggaa gcggggagca gccgcgcggc cggagtccgg 180 aggcgagggg aggtcggccg caacttcccc ggtccacctt aagaggacga tgtagccagc 240 tcgcagcgct gaccttagaa aaacaagttt gcgcaaagtg gagcggggac ccggcctctg 300 ggcagccccg gcggcgcttc cagtgccttc cagccctcgc gggcggcgca gccgcggccc 360 atggagcccg ccggcccggc ccccggccgc ctcgggccgc tgctctgcct gctgctcgcc 420 gcgtcctgcg cctggtcagg agtggcgggt gagggaggc tgcaggtgat tcagcctgac 480 aagtccgtgt tggttgcagc tggagagaca gccactctgc gctccactgc gacctctctg 540 atccctgtgg ggcccatcca gtggttcaga ggagctggac caggccggga attaatctac 600 aatcaaaaag aaggccactt cccccgggta acaactgttt cagacctcac aaagaagaac 660 aacatggact tttccatccg catcggtaac atcaccccag cagatgccgg cacctactac 720 tgtgtgaagt tccggaaagg gagccccgat gacgtggagt ttaagtctgg agcaggcact 780 gagctgtctg tgcgcgccaa accctctgcc cccgtggtat cgggccctgc ggcgagggcc 840 acacctcagc acacagtgag cttcacctgc gagtcccacg gcttctcacc cagagacatc 900 accctgaaat ggttcaaaaa tgggaatgag ctctcagact tccagaccaa cgtggacccc gtaggagaga gcgtgtccta cagcatccac agcacagcca aggtggtgct gacccgcgag gacgttcact ctcaagtcat ctgcgaggtg gcccacgtca ccttgcaggg ggaccctctt 1080 cgtgggactg ccaacttgtc tgagaccatc cgagttccac ccaccttgga ggttactcaa cagcccgtga gggcagaga ccaggtga gtcacctgcc aggtgagga gttctacccc cagagactac agctgacctg gttggagaat ggaaacgtgt cccggacaga aacggcctca accgttacag agaacaagga tggtacctac aactggatga gctggctcct ggtgaatgta tctgcccaca gggatgatgt gaagctcacc tgccaggtgg agcatgacgg gcagccagcg gtcagcaaaa gccatgacct gaaggtctca gcccacccga aggagcaggg ctcaaatacc gccgctgaga acactggatc taatgaacgg aacatctata ttgtggtggg tgtggtgtgc accttgctgg tggccctact gatggcggcc ctctacctcg tccgaatcag acagaagaaa gcccagggct ccacttcttc tacaaggttg catgagcccg agaagaatgc agaagaat acacaggaca caaatgatat cacatatgca gacctgaacc tgcccaaggg gaagaagcct 1680 gctccccagg ctgcggagcc caacaaccac acggagtatg ccagcattca gaccagcccg 1740 cagcccgcgt cggaggacac cctcacctat gctgacctgg acatggtcca cctcaaccgg 1800 acccccaagc agccggcccc caagcctgag ccgtccttct cagagtacgc cagcgtccag 1860 gtcccgagga agtgaatggg accgtggttt gctctagcac ccatctctac gcgctttctt 1920 gtcccacagg gagccgccgt gatgagcaca gccaacccag ttcccggagg gctggggcgg 1980 tgcaggctct gggacccagg ggccagggtg gctcttctct ccccacccct cttggctct 2040 ccagcacttc ctgggcagcc acggccccct ccccccacat tgccacatac ctggaggctg 2100 acgttgccaa accagccagg gaaccaacct gggaagtggc cagaactgcc tggggtccaa 2160 gaactcttgt gcctccgtcc atcaccatgt gggttttgaa gaccctcgac tgcctccccg 2220 atgctccgaa gcctgatctt ccagggtggg gaggagaaaa tcccacctcc cctgacctcc 2280 accacctcca ccaccaccac caccaccacc accaccacta ccaccaccac ccaactgggg 2340 ctagagtggg gaagatttcc cctttagatc aaactgcccc ttccatggaa aagctggaaa 2400 aaaactctgg aacccatatc caggcttggt gaggttgctg ccaacagtcc tggcctcccc 2460 catccctagg ctaaagagcc atgagtcctg gaggaggaga ggacccctcc caaaggactg 2520 gagacaaaac cctctgcttc cttgggtccc tccaagactc cctggggccc aactgtgttg 2580 ctccacccgg acccatctct cccttctaga cctgagcttg cccctccagc tagcactaag 2640 caacatctcg ctgtggacgc ctgtaaatta ctgagaaatg tgaaacgtgc aatcttgaaa 2700 ctgaggtgtt agaaaacttg atctgtggtg ttttgttttg ttttttttct taaaacaaca 2760 gcaacgtgat cttggctgtc tgtcatgtgt tgaagtccat ggttgggtct tgtgaagtct 2820 gaggtttaac agtttgttgt cctggaggga ttttcttaca gcgaagactt gagttcctcc 2880 aagtcccaga accccaagaa tgggcaagaa ggatcaggtc agccactccc tggagacaca 2940 gccttctggc tgggactgac ttggccatgt tctcagctga gccacgcggc tggtagtgca 3000 gccttctgtg accccgctgt ggtaagtcca gcctgcccag ggctgctgag ggctgcctct 3060 tgacagtgca gtcttatcga gacccaatgc ctcagtctgc tcatccgtaa agtggggata 3120 gtgaagatga cacccctccc caccacctct cataagcact ttaggaacac acagagggta 3180 gggatagtgg ccctggccgt ctatcctacc cctttagtga ccgcccccat cccggctttc 3240 tgagctgatc cttgaagaag aaatcttcca tttctgctct caaaccctac tgggatcaaa 3300 ctggaataaa ttgaagacag ccagggggat ggtgcagctg tgaagctcgg gctgattccc 3360 cctctgtccc agaaggttgg ccagagggtg tgacccagtt accctttaac ccccaccctt 3420 ccagtcgggt gtgagggcct gaccgggccc agggcaagca gatgtcgcaa gccctattta 3480 ttcagtcttc actataactc ttagagttga gacgctaatg ttcatgactc ctggccttgg 3540 gatgcccaag ggatttctgg ctcaggctgt aaaagtagct gagccatcct gcccattcct 3600 ggaggtccta caggtgaaac tgcaggagct cagcatagac ccagctctct gggggatggt 3660 cacctggtga tttcaatgat ggcatccagg aattagctga gccaacagac catgtggaca 3720 gctttggcca gagctcccgt gtggcatctg ggagccacag tgacccagcc acctggctca 3780 ggctagttcc aaattccaaa agattggctt gtaaaccttc gtctccctct cttttaccca 3840 gagacagcac atacgtgtgc acacgcatgc acacacacat tcagtatttt aaaagaatgt 3900 tttcttggtg ccattttcat tttattttat ttttaattc ttggaggggg aaataaggga 3960 ataaggccaa ggaagatgta tagctttagc tttagcctgg caacctggag aatccacata 4020 ccttgtgtat tgaaccccag gaaaaggaag aggtcgaacc aaccctgcgg aaggagcatg 4080 gtttcaggag tttattttaa gactgctggg aaggaaacag gccccatttt gtatatagtt 4140 gcaacttaaa ctttttggct tgcaaaatat ttttgtaata aagatttctg ggtaataatg 4200 at 4201 <210> 12 <211> 504 <212> PRT <213> Homo sapiens <400> 12 Put Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys 1 5 10 15 Leu Leu Leu Wing Wing Ser Cys Wing Trp Ser Gly Val Wing Gly Glu Glu 20 25 30 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 35 40 45 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly 50 55 60 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr 65 70 75 80 Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 85 90 95 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr 100 105 110 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 115 120 125 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 130 135 140 Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala 145 150 155 160 Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser 165 170 175 Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser 180 185 190 Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser 195 200 205 Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser 210 215 220 Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu 225 230 235 240 Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu 245 250 255 Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr 260 265 270 Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu 275 280 285 Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu 290 295 300 Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val 305 310 315 320 Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp 325 330 335 Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His 340 345 350 Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn 355 360 365 Glu Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu Leu Val 370 375 380 Ala Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln Lys Lys 385 390 395 400 Ala Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn 405 410 415 Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu 420 425 430 Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn 435 440 445 Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser 450 455 460 Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg 465 470 475 480 Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu Tyr 485 490 495 Ala Ser Val Gln Val Pro Arg Lys 500 <210> 13 <211> 4109 <212> DNA <213> Homo sapiens <400> 13 ctctctggcc gccctggct ttatttcg cgcgcttggg gtctctccca gtctccgtct 60 ctccatttct cctggggggc ggggaggggg ggtctccaaa aaccgcggcg gcggcggcgg 120 ccgctccagg cgcccgttcc ggagtcgggg ggaggcccag ccgggagggg ggaagggggg 180 gagccttagt cattccccg ctccagcctg ctcccgcccg agcgcgcact cacggcgct 240 ctccctccctc gctccgcagc cgcggcccat ggagcccgcc ggcccggccc ccggccgcct 300 cgggccgctg ctctgcctgc tgctcgcgc gtcctgcgcc tggtcaggag tggcgggtga 360 ggaggagctg caggtgattc agcctgacaa gtccgtgttg gttgcagctg gagagacagc 420 cactctgcgc tgcactgcga cctctctgat ccctgtgggg cccatccagt ggttcagagg 480 agctggacca ggccgggaat taatctacaa tcaaaaagaa ggccacttcc cccgggtaac 540 aactgtttca gacctcacaa agagaaacaa catggacttt tccatccgca tcggtaacat 600 caccccagca gatgccggca cctactactg tgtgaagttc cggaaaggga gccccgatga 660 cgtggagttt aagtctggag caggcactga gctgtctgtg cgcgccaaac cctctgcccc 720 cgtggtatcg ggccctgcgg cgagggccac acctcagcac acagtgagct tcacctgcga 780 gtcccacggc ttctcaccca gagacatcac cctgaaatgg ttcaaaaatg ggaatgagct ctcagacttc cagaccaacg tggaccccgt aggagagagc gtgtcctaca gcatccacag cacagccaag gtggtgctga cccgcgagga cgttcactct caagtcatct gcgaggtggc ccacgtcacc ttgcaggggg accctcttcg tgggactgcc aacttgtctg agaccatccg agttccaccc accttggagg ttactcaaca gcccgtgagg gcagagaacc aggtgaatgt cacctgccag gtgaggaagt tctaccccca gagactacag ctgacctggt tggagaatgg aaacgtgtcc cggacagaaa cggcctcaac cgttacagag aacaaggatg gtacctacaa ctggatgagc tggctcctgg tgaatgtatc tgcccacagg gatgatgtga agctcacctg 1260. ccaggtggag catgacgggc agccagcggt cagcaaaagc catgacctga aggtctcagc ccacccgaag gagcagggct caataccgc cgctgagaac actggatcta atgaacggaa catctatatt gtggtgggtg tggtgtgcac cttgctggtg gccctactga tggcggccct 1440 ctacctcgtc cgaatcagac agaagaaagc ccagggctcc acttcttcta caaggttgca 1500 tgagccccgag aagaatgcca gagaaataac acaggacaca aatgatatca catatgcaga 1560 cctgaacctg cccaagggga agaagcctgc tccccaggct gcggagccca acaaccacac 1620 ggagtatgcc agcattcaga ccagcccgca gcccgcgtcg gaggacaccc tcacctatgc 1680 tgacctggac atggtccacc tcaaccggac ccccaagcag ccggccccca agcctgagcc 1740 gtccttctca gagtacgcca gcgtccaggt cccgaggaag tgaatgggac cgtggtttgc 1800 tctagcaccc atctctacgc gctttcttgt cccacaggga gccgccgtga tgagcacagc 1860 caacccagtt cccggagggc tggggcggtg caggctctgg gacccagggg ccagggtggc 1920 tctctctcc ccacccctcc ttggctctcc agcacttcct gggcagccac ggccccctcc 1980 ccccacattg ccacatacct ggaggctgac gttgccaaac cagccaggga accaacctgg 2040 gaagtggcca gaactgcctg gggtccaaga actcttgtgc ctccgtccat caccatgtgg 2100 gttttgaaga ccctcgactg cctccccgat gctccgaagc ctgatcttcc agggtgggga 2160 ggagaaaatc ccacctcccc tgacctccac cacctccacc accaccacca ccaccaccac 2220 caccactacc accaccaccc aactggggct agagtgggga agatttcccc tttagatcaa 2280 actgcccctt ccatggaaaa gctggaaaaa aactctggaa cccatatcca ggcttggtga 2340 ggttgctgcc aacagtcctg gcctccccca tccctaggct aaagagccat gagtcctgga 2400 ggaggagagg acccctccca aaggactgga gacaaaaccc tctgcttcct tgggtccctc 2460 caagactccc tggggcccaa ctgtgttgct ccacccggac ccatctctcc cttctagacc 2520 tgagcttgcc cctccagcta gcactaagca acatctcgct gtggacgcct gtaaattact 2580 gagaaatgtg aaacgtgcaa tcttgaaact gaggtgttag aaaacttgat ctgtggtgtt 2640 ttgttttgtt ttttttctta aaacaacagc aacgtgatct tggctgtctg tcatgtgttg 2700 aagtccatgg ttgggtcttg tgaagtctga ggtttaacag tttgttgtcc tggagggatt 2760 ttcttacagc gaagacttga gttcctccaa gtcccagaac cccaagaatg ggcaagaagg 2820 atcaggtcag ccactccctg gagacacagc cttctggctg ggactgactt ggccatgttc 2880 tcagctgagc cacgcggctg gtagtgcagc cttctgtgac cccgctgtgg taagtccagc 2940 ctgcccaggg ctgctgaggg ctgcctcttg acagtgcagt cttatcgaga cccaatgcct 3000 cagtctgctc atccgtaaag tggggatagt gaagatgaca cccctcccca ccacctctca 3060 taagcacttt aggaacacac agagggtagg gatagtggcc ctggccgtct atcctacccc 3120 tttagtgacc gcccccatcc cggctttctg agctgatcct tgaagaagaa atcttccatt 3180 tctgctctca aaccctactg ggatcaaact ggaataaatt gaagacagcc agggggatgg 3240 tgcagctgtg aagctcgggc tgattccccc tctgtcccag aaggttggcc agagggtgtg 3300 acccagttac cctttaaccc ccacccttcc agtcgggtgt gagggcctga ccgggcccag 3360 ggcaagcaga tgtcgcaagc cctatttatt cagtcttcac tataactctt agagttgaga 3420 cgctaatgtt catgactcct ggccttggga tgcccaaggg atttctggct caggctgtaa 3480 aagtagctga gccatcctgc ccattcctgg aggtcctaca ggtgaaactg caggagctca 3540 gcatagaccc agctctctgg gggatggtca cctggtgatt tcaatgatgg catccaggaa 3600 ttagctgagc caacagacca tgtggacagc tttggccaga gctcccgtgt ggcatctggg 3660 agccacagtg acccagccac ctggctcagg ctagttccaa attccaaaag attggcttgt 3720 aaaccttcgt ctccctctct tttacccaga gacagcacat acgtgtgcac acgcatgcac 3780 acacacattc agtattttaa aagaatgttt tcttggtgcc attttcattt tattttatt 3840 tttaattctt ggaggggaa ataagggaat aaggccaagg aagatgtata gctttagctt 3900 tagcctggca acctggagaa tccacatacc ttgtgtattg aaccccagga aaaggaagag 3960 gtcgaaccaa ccctgcggaa ggagcatggt ttcaggagtt tattttaaga ctgctgggaa 4020 ggaaacaggc cccattttgt atatagttgc aacttaaact ttttggcttg caaaatattt 4080 ttgtaataaa gatttctggg taataatga 4109 <210> 14 <211> 3868 <212> DNA <213> Homo sapiens <400> 14 cgctcgctcg cagagaagcc gcggcccatg gagcccgccg gcccggcccc cggccgcctc 60 gggccgctgc tctgcctgct gctcgccgcg tcctgcgcct ggtcaggagt ggcgggtgag 120 gaggagctgc aggtgattca gcctgacaag tccgtgttgg ttgcagctgg agagacagcc 180 actctgcgct gcactgcgac ctctctgatc cctgtggggc ccatccagtg gttcagagga 240 gctggaccag gccgggaatt aatctacaat caaaaagaag gccacttccc ccgggtaaca 300 actgtttcag acctcacaaa gagaaacaac atggactttt ccatccgcat cggtaacatc 360 accccagcag atgccggcac ctactactgt gtgaagttcc ggaaagggag ccccgatgac 420 gtggagttta agtctggagc aggcactgag ctgtctgtgc gcgccaaacc ctctgccccc 480 gtggtatcgg gccctgcggc gagggccaca cctcagcaca cagtgagctt cacctgcgag 540 tcccacggct tctcacccag agacatcacc ctgaaatggt tcaaaaatgg gaatgagctc 600 tcagacttcc agaccaacgt ggaccccgta ggagagagcg tgtcctacag catccacagc 660 acagccaagg tggtgctgac ccgcgaggac gttcactctc aagtcatctg cgaggtggcc 720 cacgtcacct tgcaggggga ccctcttcgt gggactgcca acttgtctga gaccatccga 780 gttccaccca ccttggaggt tactcaacag cccgtgaggg cagagaacca ggtgaatgtc 840 acctgccagg tgaggaagtt ctacccccag agactacagc tgacctggtt ggagaatgga 900 aacgtgtccc ggacagaaac ggcctcaacc gttacagaga acaaggatgg tacctacaac 960 tggatgagct ggctcctggt gaatgtatct gcccacaggg atgatgtgaa gctcacctgc 1020 caggtggagc atgacgggca gccagcggtc agcaaaagcc atgacctgaa ggtctcagcc 1080 cacccgaagg agcagggctc aaataccgcc gctggagaaca ctggatctaa tgaacggaac 1140 atctatattg tggtgggtgt ggtgtgcacc ttgctggtgg ccctactgat ggcggccctc 1200 tacctcgtcc gaatcagaca gaaaagcc cagggctcca cttcttctac aaggttgcat 1260 1320 ctgaacctgc ccaaggggaa gaagcctgct ccccaggctg cggagcccaa caaccacag 1380 gagtatgcca gcattcagac cagcccgcag cccgcgtcgg aggacaccct cacctatgct 1440 gacctggaca tggtccacct caaccggacc cccaagcagc cggcccccaa gcctgagccg 1500 tccttctcag agtacgccag cgtccaggtc ccgaggaagt gaatgggacc gtggtttgct 1560 ctagcaccca tctctacgcg ctttcttgtc ccacagggag ccgccgtgat gagcacagcc 1620 aacccagttc ccggagggct ggggcggtgc aggctctggg acccaggggc cagggtggct 1680 cttctctccc cacccctcct tggctctcca gcacttcctg ggcagccacg gccccctccc 1740 cccacattgc cacatacctg gaggctgacg ttgccaaacc agccagggaa ccaacctggg 1800 aagtggccag aactgcctgg ggtccaagaa ctcttgtgcc tccgtccatc accatgtggg 1860 ttttgaagac cctcgactgc ctccccgatg ctccgaagcc tgatcttcca gggtggggag 1920 gagaaaatcc cacctcccct gacctccacc acctccacca ccaccaccac caccaccacc 1980 accactacca ccaccaccca actggggcta gagtggggaa gatttcccct ttagatcaaa 2040 ctgccccttc catggaaaag ctggaaaaaa actctggaac ccatatccag gcttggtgag 2100 gttgctgcca acagtcctgg cctcccccat ccctaggcta aagagccatg agtcctggag 2160 gaggagagga cccctcccaa aggactggag acaaaaccct ctgcttcctt gggtccctcc 2220 aagactccct ggggcccaac tgtgttgctc cacccggacc catctctccc ttctagacct 2280 gagcttgccc ctccagctag cactaagcaa catctcgctg tggacgcctg taaattactg 2340 2400 tgttttgttt tttttcttaa aacaacca acgtgatctt ggctgtctgt catgtgttga 2460 agtccatggt tgggtcttgt gaagtctgag gtttaacagt ttgttgtcct ggagggattt 2520 tcttacagcg aagacttgag ttcctccaag tcccagaacc ccaagaatgg ccaagaagga 2580 tcaggtcagc cactccctgg agacacagcc ttctggctgg gactgacttg gccatgttct 2640 cagctgagcc acgcggctgg tagtgcagcc ttctgtgacc ccgctgtggt aagtccagcc 2700 tgcccagggc tgctgagggc tgcctcttga cagtgcagtc ttatcgagac ccaatgcctc 2760 agtctgctca tccgtaaagt ggggatagtg aagatgacac ccctccccac cacctctcat 2820 aagcacttta ggaacaca gagggtaggg atagtggccc tggccgtcta tcctacccct 2880 ttagtgaccg cccccatccc ggctttctga gctgatcctt gaagaaaa tcttccattt 2940 ctgctctcaa accctactgg gatcaaactg gaataaattg aagacagcca gggggatggt 3000 gcagctgtga agctcgggct gattccccct ctgtcccaga aggttggcca gagggtgtga 3060 cccagttacc ctttaacccc cacccttcca gtcgggtgtg agggcctgac cgggcccagg 3120 gcaagcagat gtcgcaagcc ctatttattc agtcttcact ataactctta gagttgagac 3180 gctaatgttc atgactcctg gccttgggat gcccaaggga tttctggctc aggctgtaaa 3240 agtagctgag ccatcctgcc cattcctgga ggtcctacag gtgaaactgc aggagctcag 3300 catagaccca gctctctggg ggatggtcac ctggtgattt caatgatggc atccaggaat 3360 tagctgagcc aacagaccat gtggacagct ttggccagag ctcccgtgtg gcatctggga 3420 gccacagtga cccagccacc tggctcaggc tagttccaaa ttccaaaaga ttggcttgta 3480 aaccttcgtc tccctctctt ttacccagag acagcacata cgtgtgcaca cgcatgcaca 3540 cacacattca gtattttaaa agaatgtttt cttggtgcca ttttcatttt attttatttt 3600 ttaattcttg gagggggaaa taagggaata aggccaagga agatgtatag ctttagcttt 3660 agcctggcaa cctggagaat ccacatacct tgtgtattga accccaggaa aaggaagagg 3720 tcgaaccaac cctgcggaag gagcatggtt tcaggagttt atttaagac tgctgggaag 3780 gaaacaggcc ccattttgta tatagttgca acttaaactt tttggcttgc aaaatatttt 3840 tgtaataaag atttctgggt aataatga 3868 <210> 15 <211> 4031 <212> DNA <213> Mus musculus <400> 15 cgggaaggtg cgggcgcgag gagggcgc tcggcgggc cgccctcgcg ctggcctcgc 60 gacggctccg cacagcccgc actcgctctg cgagctgtc ccgctcgcg ttgctctccg 120 atctccgtcc ccgctccctc tccctctc tctccccctc tttccttctc cctcgctatc 180 cgctcccccg cccccgtgcc tctggctctg cgcctggctc cctcgggtcc gctcccctt 240 cccgccggcc tgcccggcg tcacgctccc ggagtctccc cgctcggcgg cgtctcattg 300 tgggagggg tcagatcacc ccgccgggcg gtggcgctgg ggggcagcgg aggggggg 360 gccttagtcg ttcgcccgcg ccgcccgccc gcctgccgag cgcgctcacc gcgctctcc 420 ctccttgctc tgcagccgcg gcccatggag cccgccggcc cggcccctgg ccgcctaggg 480 ccgctgctgc tctgcctgct gctctccgcg tcctgtttct gtacaggagc cacggggaag 540 gaactgaagg tgactcagcc tgagaaatca gtgtctgttg ctgctgggga ttcgaccgtt 600 ctgaactgca ctttgacctc cttgttgccg gtgggaccca ttaggtggta cagaggagta 660 gggccaagcc ggctgttgat ctacagtttc gcaggagaat acgttcctcg aattagaaat 720 gtttcagata ctactaagag aaacaatatg gacttttcca tccgtatcag taatgtcacc 780 ccagcagatg ctggcatcta ctactgtgtg aagttccaga aaggatcatc agagcctgac 840 acagaaatac aatctggagg gggaacagag gtctatgtac tcgccaaacc ttctccaccg 900 gaggtatccg gcccagcaga caggggcata cctgaccaga aagtgaactt cacctgcaag 960 tctcatggct tctctccccg gaatatcacc ctgaagtggt tcaaagatgg gcaagaactc 1020 caccccttgg agaccaccgt gaaccctagt ggaaagaatg tctcctacaa catctccagc 1080 acagtcaggg tggtactaaa ctccatggat gttaattcta aggtcatctg cgaggtagcc 1140 cacatcacct tggatagaag ccctcttcgt gggattgcta acctgtctaa cttcatccga 1200 gtttcaccca ccgtgaaggt cacccaacag tccccgacgt caatgaacca ggtgaacctc 1260 acctgccggg ctgagaggtt ctaccccgag gatctccagc tgatctggct ggagaatgga 1320 aacgtatcac ggaatgacac gcccaagaat ctcacaaaga acacggatgg gacctataat 1380 tacacaagct tgttcctggt gaactcatct gctcatagag aggacgtggt gttcacgtgc 1440 caggtgaagc acgaccaaca gccagcgatc acccgaaacc ataccgtgct gggatttgcc 1500 cactcgagtg atcaagggag catgcaaacc ttccctgata ataatgctac ccacaactgg 1560 aatgtcttca tcggtgtggg cgtggcgtgt gctttgctcg tagtcctgct gatggctgct 1620 ctctacctcc tccggatcaa acagaagaaa gccaaggggt caacatcttc cacacggttg 1680 cacgagcccg agaagaacgc cagggaaata acccagatcc aggacacaaa tgacatcaac 1740 gacatcacat acgcagacct gaatctgccc aaagagaaga agcccgcacc ccgggcccct 1800 gagcctaaca accacacaga atatgcaagc attgagacag gcaaagtgcc taggccagag 1860 gataccctca cctatgctga cctggacatg gtccacctca gccgggcaca gccagccccc 1920 aagcctgagc catctttctc agagtatgct agtgtccagg tccagaggaa gtgaatgggg 1980 ctgtggtctg tactaggccc catccccaca agttttcttg tcctacatgg agtggccatg 2040 acgaggacat ccagccagcc aatcctgtcc ccagaaggcc aggtggcacg ggtcctagga 2100 ccaggggtaa gggtggcctt tgtcttccct ccgtggctct tcaacacctc ttgggcaccc 2160 acgtcccctt cttccggagg ctgggtgttg cagaaccaga gggcgaactg gagaaagctg 2220 cctggaatcc aagaagtgtt gtgcctcggc ccatcactcg tgggtctgga tcctggtctt 2280 ggcaacccca ggttgcgtcc ttgatgttcc agagcttggt cttctgtgtg gagaagagct 2340 caccatctct acccaacttg agctttggga ccagactccc tttagatcaa accgccccat 2400 ctgtggaaga actacaccag aagtcagcaa gttttcagcc aacagtgctg gcctccccac 2460 ctcccaggct gactagccct ggggagaagg aaccctctcc tcctagacca gcagagactc 2520 cctgggcatg ttcagtgtgg ccccacctcc cttccagtcc cagcttgctt cctccagcta 2580 gcactaactc agcagcatcg ctctgtggac gcctgtaaat tattgagaaa tgtgaactgt 2640 gcagtcttaa agctaaggtg ttagaaaatt tgatttatgc tgtttagttg ttgttgggtt 2700 tcttttcttt ttaatttctt tttctttttt gatttttttt ctttccctta aaacaacagc 2760 agcagcatct tggctctttg tcatgtgttg aatggttggg tcttgtgaag tctgaggtct 2820 aacagtttat tgtcctggaa ggattttctt acagcagaaa cagatttttt tcaaattccc 2880 agaatcctga ggaccaagaa ggatccctca gctgctactt ccagcaccca gcgtcactgg 2940 gacgaaccag gccctgttct tacaaggcca catggctggc cctttgcctc catggctact 3000 gtggtaagtg cagccttgtc tgacccaatg ctgacctaat gttggccatt ccacattgag 3060 gggacaaggt cagtgatgcc ccccttcact cacaagcact tcagaggcat gcagagagaa 3120 gggacactcg gccagctctc tgaggtaatc agtgcaagga ggagtccgtt ttttgccagc 3180 aaacctcagc aggatcacac tggaacagaa cctggtcata cctgtgacaa cacagctgtg 3240 agccagggca aaccacccac tgtcactggc tcgagagtct gggcagaggc tctgaccctc 3300 caccctttaa actggatgcc ggggcctggc tgggcccaat gccaagtggt tatggcaacc 3360 ctgactatct ggtcttaaca tgtagctcag gaagtggagg cgctaatgtc cccaatccct 3420 ggggattcct gattccagct attcatgtaa gcagagccaa cctgcctatt tctgtaggtg 3480 cgactgggat gttaggagca cagcaaggac ccagctctgt agggctggtg acctgatact 3540 tctcataatg gcatctagaa gttaggctga gttggcctca ctggcccagc aaaccagaac 3600 ttgtctttgt ccgggccatg ttcttgggct gtcttctaat tccaaagggt tggttggtaa 3660 agctccaccc ccttctcctc tgcctaaga catcacatgt gtatacacac acgggtgttat 3720 agatgagtta aaagaatgtc ctcgctggca tcctaatttt gtcttaagtt tttttggagg 3780 gggggggggggggggggtgtgtgtgtggct ttaaccaggc agcctggggg 3840 ctcccaagcc tatggaaccc tggtacaaag aagagaacag aagcgccctg tgaggagtgg 3900 gatttgtttt tctgtagacc agatgagaag gaacaggcc ctgttttgta catagttgca 3960 actaaaaatt tttggcttgc aaaatatttt tgtaataaag atttctgggt aacaataaaa 4020 aaaaaaaaaaaaa a 4031 <210> 16 <211> 509 <212> PRT <213> Mus musculus <400> 16 Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Leu 1 5 10 15 Cys Leu Leu Leu Ser Ala Ser Cys Phe Cys Thr Gly Ala Thr Gly Lys 20 25 30 Glu Leu Lys Val Thr Gln Pro Glu Lys Ser Val Ser Val Ala Ala Gly 35 40 45 Asp Ser Thr Val Leu Asn Cys Thr Leu Thr Ser Leu Leu Pro Val Gly 50 55 60 Pro Ile Arg Trp Tyr Arg Gly Val Gly Pro Ser Arg Leu Leu Ile Tyr 65 70 75 80 Ser Phe Ala Gly Glu Tyr Val Pro Arg Ile Arg Asn Val Ser Asp Thr 85 90 95 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn Val Thr 100 105 110 Pro Ala Asp Ala Gly Ile Tyr Tyr Cys Val Lys Phe Gln Lys Gly Ser 115 120 125 Ser Glu Pro Asp Thr Glu Ile Gln Ser Gly Gly Gly Thr Glu Val Tyr 130 135 140 Val Leu Ala Lys Pro Ser Pro Pro Glu Val Ser Gly Pro Ala Asp Arg 145 150 155 160 Gly Ile Pro Asp Gln Lys Val Asn Phe Thr Cys Lys Ser His Gly Phe 165 170 175 Ser Pro Arg Asn Ile Thr Leu Lys Trp Phe Lys Asp Gly Gln Glu Leu 180 185 190 His Pro Leu Glu Thr Thr Val Asn Pro Ser Gly Lys Asn Val Ser Tyr 195 200 205 Asn Ile Ser Ser Thr Val Arg Val Val Leu Asn Ser Met Asp Val Asn 210 215 220 Ser Lys Val Ile Cys Glu Val Ala His Ile Thr Leu Asp Arg Ser Pro 225 230 235 240 Leu Arg Gly Ile Ala Asn Leu Ser Asn Phe Ile Arg Val Ser Pro Thr 245 250 255 Val Lys Val Thr Gln Gln Ser Pro Thr Ser Met Asn Gln Val Asn Leu 260 265 270 Thr Cys Arg Ala Glu Arg Phe Tyr Pro Glu Asp Leu Gln Leu Ile Trp 275 280 285 Leu Glu Asn Gly Asn Val Ser Arg Asn Asp Thr Pro Lys Asn Leu Thr 290 295 300 Lys Asn Thr Asp Gly Thr Tyr Asn Tyr Thr Ser Leu Phe Leu Val Asn 305 310 315 320 Ser Ser Ala His Arg Glu Asp Val Val Phe Thr Cys Gln Val Lys His 325 330 335 Asp Gln Gln Pro Ala Ile Thr Arg Asn His Thr Val Leu Gly Phe Ala 340 345 350 His Ser Ser Asp Gln Gly Ser Met Gln Thr Phe Pro Asp Asn Asn Ala 355 360 365 Thr His Asn Trp Asn Val Phe Ile Gly Val Gly Val Ala Cys Ala Leu 370 375 380 Leu Val Val Leu Leu Met Ala Ala Leu Tyr Leu Leu Arg Ile Lys Gln 385 390 395 400 Lys Lys Ala Lys Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu 405 410 415 Lys Asn Ala Arg Glu Ile Thr Gln Ile Gln Asp Thr Asn Asp Ile Asn 420 425 430 Asp Ile Thr Tyr Ala Asp Leu Asn Leu Pro Lys Glu Lys Lys Pro Ala 435 440 445 Pro Arg Ala Pro Glu Pro Asn Asn His Thr Glu Tyr Ala Ser Ile Glu 450 455 460 Thr Gly Lys Val Pro Arg Pro Glu Asp Thr Leu Thr Tyr Ala Asp Leu 465,470,475,480 Asp Met Val His Leu Ser Arg Ala Gln Pro Ala Pro Lys Pro Glu Pro 485 490 495 Ser Phe Ser Glu Tyr Ala Ser Val Gln Val Gln Arg Lys 500 505 <210> 17 <211> 3377 <212> DNA <213> Mus musculus <400> 17 cgggaaggtg cgggcgcgag gagggcgc tcggcgggc cgccctcgcg ctggcctcgc 60 gacggctccg cacagcccgc actcgctctg cgagctgtc ccgctcgcg ttgctctccg 120 atctccgtcc ccgctccctc tccctctc tctccccctc tttccttctc cctcgctatc 180 cgctcccccg cccccgtgcc tctggctctg cgcctggctc cctcgggtcc gctcccctt 240 cccgccggcc tgcccggcg tcacgctccc ggagtctccc cgctcggcgg cgtctcattg 300 tgggaggggg tcagatcacc ccgccgggcg gtggcgctgg ggggcagcgg agggggaggg 360 gccttagtcg ttcgcccgcg ccgcccgccc gcctgccgag cgcgctcacc gccgctctcc 420 ctccttgctc tgcagccgcg gcccatggag cccgccggcc cggcccctgg ccgcctaggg 480 ccgctgctgc tctgcctgct gctctccgcg tcctgtttct gtacaggagc cacggggaag 540 gaactgaagg tgactcagcc tgagaaatca gtgtctgttg ctgctgggga ttcgaccgtt 600 ctgaactgca ctttgacctc cttgttgccg gtgggaccca ttaggtggta cagaggagta 660 gggccaagcc ggctgttgat ctacagtttc gcaggagaat acgttcctcg aattagaaat 720 gtttcagata ctactaagag aaacaatatg gacttttcca tccgtatcag taatgtcacc 780 ccagcagatg ctggcatcta ctactgtgtg aagttccaga aaggatcatc agagcctgac 840 acagaaatac aatctggagg gggaacagag gtctatgtac tcgataataa tgctacccac 900 aactggaatg tcttcatcgg tgtgggcgtg gcgtgtgctt tgctcgtagt cctgctgatg 960 gctgctctct acctcctccg gatcaaacag aagaaagcca aggggtcaac atcttccaca 1020 cggttgcacg agcccgagaa gaacgccagg gaaataaccc agatccagga cacaaatgac 1080 atcaacgaca tcacatacgc agacctgaat ctgcccaaag...

Claims

1. Rodent embryonic stem (ES) cells, in their genome, A signal-regulating protein α (SIRPα) gene comprising a nucleic acid sequence encoding a signal-regulating protein α (SIRPα) protein, which is operably linked to the endogenous rodent SIRPα gene promoter at the rodent signal-regulating protein α (SIRPα) gene locus in the genome of the ES cell, wherein the SIRPα gene comprises a partial substitution of the endogenous rodent SIRPα coding sequence, and the encoded SIRPα protein comprises a human SIRPα extracellular domain, has wild-type human SIRPα receptor function, and comprises the signal transduction domain of the endogenous rodent SIRPα protein, and An interleukin-15 (IL-15) gene comprising a nucleic acid sequence encoding the IL-15 (IL-15) protein, wherein the nucleic acid sequence comprises exons 5-8 of the human IL-15 gene, the nucleic acid sequence is operably linked to the endogenous rodent IL-15 gene promoter at the rodent IL-15 locus in the genome of the ES cell, the IL-15 gene comprises substitutions of part or all of the endogenous rodent IL-15 coding sequence, and the encoded IL-15 protein has wild-type human IL-15 signaling function. including, The aforementioned rodent ES cells.

2. The rodent ES cell according to claim 1, wherein the SIRPα gene comprises exon 1 of the rodent SIRPα gene, exons 2, 3, and 4 of the human SIRPα gene, and exons 5, 6, 7, and 8 of the rodent SIRPα gene.

3. The rodent ES cell according to claim 1, wherein it is homozygous with respect to the SIRPα gene.

4. The rodent ES cell according to claim 1, wherein the IL-15 gene comprises exons 3 and 4 of the rodent IL-15 gene.

5. The rodent ES cell according to claim 1, wherein it is homozygous with respect to the IL-15 gene.

6. The SIRPα gene comprises substitutions of exons 2, 3, and 4 of the rodent SIRPα gene with exons 2, 3, and 4 of the human SIRPα gene at the endogenous rodent SIRPα locus, The IL-15 gene includes substitutions of exons 5, 6, 7, and 8 of the rodent IL-15 gene with exons 5, 6, 7, and 8 of the human IL-15 gene at the endogenous rodent IL-15 locus. Rodent ES cells according to claim 1.

7. The SIRPα gene comprises exon 1 of the rodent SIRPα gene, exons 2, 3, and 4 of the human SIRPα gene, and exons 5, 6, 7, and 8 of the rodent SIRPα gene; The IL-15 gene comprises exons 3 and 4 of the rodent IL-15 gene; The rodent ES cells are homozygous with respect to the SIRPα gene; and The aforementioned rodent ES cells are homozygous for the IL-15 gene. Rodent ES cells according to claim 6.

8. Rodent ES cells according to claim 1, comprising Rag2 gene knockout.

9. Rodent ES cells according to claim 1, comprising IL2rg gene knockout.

10. Rodent ES cells according to claim 1, comprising Rag2 gene knockout and IL2rg gene knockout.

11. Rodent ES cells according to claim 7, comprising Rag2 gene knockout and IL2rg gene knockout.

12. A rodent ES cell according to any one of claims 1 to 11, which is a mouse ES cell.

13. A rodent embryo comprising rodent ES cells according to any one of claims 1 to 11.

14. A mouse embryo comprising mouse ES cells as described in claim 12.

15. A method for producing a genetically modified rodent, comprising the step of producing a genetically modified rodent from a rodent embryo as described in claim 13.

16. A method for producing a genetically modified mouse, comprising the step of producing a genetically modified mouse from a mouse embryo as described in claim 14.

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