Humanized SIRPα-IL15 knock-in mice and methods of use thereof

Abstract

The present invention relates to humanized SIRPα-IL15 knock-in mice and methods of using them. Genetically modified non-human animals expressing human SIRPα and human IL-15 from non-human animal genomes are provided. Methods for preparing non-human animals expressing human SIRPα and human IL-15 from non-human animal genomes, and methods of using non-human animals expressing human SIRPα and human IL-15 from non-human animal genomes are also provided. These animals and methods have many uses in the art, including, for example, modeling human T cell and / or natural killer (NK) cell development and function, modeling human pathogen infection of human T cells and / or NK cells, and various in vivo screenings.

Description

[0001] This application is a re-divisional application of a divisional application with an application date of April 12, 2016, Chinese application number 202110697073.0, and invention name “Humanized SIRPα-IL15 knock-in mice and methods of use thereof” (the original application number is 201680028455.1).

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 146,938, filed April 13, 2015; U.S. Provisional Application No. 62 / 148,667, filed April 16, 2015; and U.S. Provisional Application No. 62 / 287,842, filed January 27, 2016, the disclosures of each of which are incorporated herein by reference in their entirety. Field of the Invention

[0004] The present invention relates to the field of genetically modified non-human animals.

[0005] introduction

[0006] Genetically modified non-human animals such as humanized mice hold great prospects for translational research because they allow modeling and research of human diseases in vivo. In the past 10 years, considerable progress has been made in developing humanized mice by genetically inserting the necessary human genes for the proper development and function of human immune cells in mice. However, some restrictions still limit the effectiveness of humanized mice in translational research. Especially, the development and survival of human T cells do not reach the best.

[0007] Although it has been shown that the bone marrow-liver-thymus (BLT) model improves intestinal T cell reconstruction in NS / NSG-BLT mice (Denton PW, Nochi T, Lim A et al. Mucosal Immunol 2012; 5: 555-566, Nochi T, Denton PW, Wahl A et al. Cell Rep 2013; 3: 1874-1884), it has been shown that those mice exhibit graft-versus-host disease, resulting in a large number of immune cell infiltrations in multiple tissues (Greenblatt MB, Vrbanac V, Tivey T et al. PLoS One 2012; 7: e44664). Therefore, current humanized mouse models still lack the proper development and function of human T cells. In particular, the absence of human tissue-resident memory T cells prevents humanized mice from being used as preclinical tools for developing and testing more efficient immune strategies, which are intended to induce persistent mucosal immunity against pathogens such as HIV.

[0008] To more fully understand the development and survival of human tissue-resident T cells and to provide a model for testing novel immunization strategies for inducing persistent T cell-dependent mucosal immunity, it would be useful to have genetically modified non-human animals that produce human tissue-resident T cells. This mouse model can also be used to study the interaction of human tissue-resident immune cells with the gut microbiota, such as how the microbiota can determine the development and survival of human immune cells in the small intestine and colon.

[0009] Furthermore, there is a need in the art for non-human animal models of human natural killer (NK) cell development and function.

[0010] Overview

[0011] Provided are genetically modified non-human animals that express human SIRPα and human IL-15 from the non-human animal genome. Also provided are methods for preparing non-human animals that express human SIRPα and human IL-15 from the non-human animal genome, and methods for using non-human animals that express human SIRPα and human IL-15 from the non-human animal genome. These animals and methods have many uses in the art, including, for example, modeling human T cell and / or natural killer (NK) cell development and function; modeling human pathogen infection of human T cells and / or NK cells; in vivo screening for agents that inhibit infection caused by pathogens that activate, induce and / or target T cells and / or NK cells; in vivo screening for agents 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 for agents that are toxic to human T cells and / or NK cells; in vivo screening for agents that prevent, mitigate or reverse the toxic effects of toxic agents on human T cells and / or NK cells; in vivo screening for candidate T cell inducing vaccines; and in vivo and in vitro screening for agents that inhibit tumor growth and / or infection by activating the antibody-dependent cellular cytotoxicity (ADCC) process mediated by NK cells.

[0012] In a first aspect, the present disclosure provides a genetically modified non-human animal, comprising: a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and being operably linked to a SIRPα gene promoter; and a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and being operably linked to a IL-15 gene promoter, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein.

[0013] The SIRPα gene promoter may be an endogenous non-human SIRPα gene promoter. For example, the SIRPα gene promoter may be an endogenous non-human SIRPα gene promoter at a non-human animal SIRPα locus. When the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at a non-human animal SIRPα locus, the genetically modified non-human animal may include an invalid mutation in the non-human SIRPα gene at the non-human animal SIRPα locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the invalid mutation is a deletion of at least mouse SIRPα exons 2-4. In another such embodiment, the genetically modified non-human animal is heterozygous for an allele comprising a nucleic acid sequence encoding a human SIRPα protein. In another such embodiment, the genetically modified non-human animal is homozygous for an allele comprising a nucleic acid sequence encoding a human SIRPα protein.

[0014] In another embodiment of the first aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human SIRPα protein includes human SIRPα genomic coding sequence and non-coding sequence.

[0015] In another embodiment of the first aspect, or in further embodiments of any of the above embodiments thereof, the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. In one such embodiment, the functional fragment includes the extracellular domain of human SIRPα, for example, the extracellular domain including at least amino acids 28-362 of SEQ ID NO: 12.

[0016] In another embodiment of the first 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 at a non-human animal IL-15 locus. In one embodiment, when the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at a non-human animal IL-15 locus, the genetically modified non-human animal includes an invalid mutation in a non-human IL-15 gene at a non-human animal IL-15 locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the invalid mutation is at least the deletion of mouse IL-15 exon 5-8. In another such embodiment, the genetically modified non-human animal is heterozygous for the allele of the nucleic acid sequence including the encoding human IL-15 protein. In another such embodiment, the genetically modified non-human animal is homozygous for the allele of the nucleic acid sequence including the encoding human IL-15 protein.

[0017] In another embodiment of the first aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human IL-15 protein includes human IL-15 genomic coding sequence and non-coding sequence.

[0018] In another embodiment of the first aspect, or in further embodiments of any of the above embodiments thereof, the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0019] In another embodiment of the first aspect, or in further embodiments of any of the above embodiments thereof, the genetically modified non-human animal is immunodeficient. For example, in one embodiment, the genetically modified non-human animal includes a Rag2 gene knockout. In another embodiment, the genetically modified non-human animal includes an IL2rg gene knockout, or both a Rag2 gene knockout and an IL2rg gene knockout.

[0020] In another embodiment of the first aspect, or in further embodiments of any of the above embodiments thereof, the non-human animal is a mammal. In one such embodiment, the mammal is a rodent, such as a mouse.

[0021] In another embodiment of the first aspect, or in a further embodiment of any of the above embodiments thereof, the genetically modified non-human animal includes an implant of human hematopoietic cells. In one such embodiment, the genetically modified non-human animal includes infection by human pathogens. In one embodiment, when the genetically modified non-human animal includes infection by human pathogens, the human pathogen activates, induces and / or targets T cells and / or natural killer (NK) cells. In another embodiment, when the genetically modified non-human animal includes infection by human pathogens, the human pathogen is a pathogen that affects (e.g., by infection) the human intestine. In one such embodiment, the human pathogen is human rotavirus. In another embodiment, when the genetically modified non-human animal includes infection by human pathogens, the pathogen affects (e.g., by infection) human lungs. In one such embodiment, the human pathogen is influenza virus. In another embodiment, when the genetically modified non-human animal includes infection by human pathogens, the pathogen affects (e.g., by infection) human liver. In another embodiment, the genetically modified non-human animal includes implants and tumors of human hematopoietic cells, such as human tumors, such as transplanted human tumors.

[0022] In a second aspect, the present disclosure provides an in vivo model comprising a genetically modified non-human animal, the genetically modified non-human animal comprising: a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter; a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter; and an implant 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) includes human intraepithelial lymphocytes (IELs) in the small intestine and Peyer's patches of the genetically modified non-human animal.

[0023] In one embodiment of the second aspect, the genetically modified non-human animal comprises infection by a human pathogen, such as an enteropathogen. In one such embodiment, the enteropathogen is selected from the group consisting of 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.

[0024] In another embodiment of the second aspect, or in further embodiments of any of the above embodiments thereof, 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 at the non-human animal SIRPα locus. In one embodiment, when the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at the non-human animal SIRPα locus, the genetically modified non-human animal includes an invalid mutation in the non-human SIRPα gene at the non-human animal SIRPα locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the invalid mutation is a deletion of at least mouse SIRPα exons 2-4. In another such embodiment, the genetically modified non-human animal is heterozygous for an allele comprising a nucleic acid sequence encoding a human SIRPα protein. In another such embodiment, the genetically modified non-human animal is homozygous for an allele comprising a nucleic acid sequence encoding a human SIRPα protein.

[0025] In another embodiment of the second aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human SIRPα protein includes human SIRPα genomic coding sequence and non-coding sequence.

[0026] In another embodiment of the second aspect, or in further embodiments of any of the above embodiments thereof, the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. In one such embodiment, the functional fragment includes the extracellular domain of human SIRPα, for example, the extracellular domain including amino acids 28-362 of SEQ ID NO: 12.

[0027] In another embodiment of the second 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 at a non-human animal IL-15 locus. In one embodiment, when the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at a non-human animal IL-15 locus, the genetically modified non-human animal includes an invalid mutation in a non-human IL-15 gene at a non-human animal IL-15 locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the invalid mutation is at least the deletion of mouse IL-15 exon 5-8. In one such embodiment, the genetically modified non-human animal is heterozygous for the allele of the nucleic acid sequence including the encoding human IL-15 protein. In another such embodiment, the genetically modified non-human animal is homozygous for the allele of the nucleic acid sequence including the encoding human IL-15 protein.

[0028] In another embodiment of the second aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human IL-15 protein includes human IL-15 genomic coding sequence and non-coding sequence.

[0029] In another embodiment of the second aspect, or in further embodiments of any of the above embodiments thereof, the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0030] In another embodiment of the second aspect, or in further embodiments of any of the above embodiments thereof, the genetically modified non-human animal is immunodeficient. For example, in one embodiment, the genetically modified non-human animal includes a Rag2 gene knockout. In another embodiment, the genetically modified non-human animal includes an IL2rg gene knockout, or both a Rag2 gene knockout and an IL2rg gene knockout.

[0031] In another embodiment of the second aspect, or in further embodiments of any of the above embodiments thereof, the non-human animal is a mammal. In one such embodiment, the mammal is a rodent, such as a mouse.

[0032] In a third aspect, the present disclosure provides an in vivo model comprising a genetically modified non-human animal, the genetically modified non-human animal comprising: a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter; a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter; and an implant 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) includes human intraepithelial lymphocytes (IELs) in the lungs of the genetically modified non-human animal.

[0033] In one embodiment of the third aspect, the genetically modified non-human animal comprises infection with a human pathogen, such as a pulmonary pathogen. In one such embodiment, the pulmonary pathogen is selected from the group consisting of Streptococcus pyogenes, Haemophilus influenza, Corynebacterium diphtheria, SARS coronavirus, Bordetella pertussis, Moraxella catarrhalis, Influenza virus (A, B, C), Coronavirus, Adenovirus, Respiratory Syncytial Virus, Parainfluenza virus, Mumps virus, Streptococcus pneumoniae, Staphylococcus aureus, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae, pneumonia, Mycobacterium tuberculosis, Chlamydia Pneumoniae, Blastomyces dermatitidis, Cryptococcus neoformans, and Aspergillus fumigatus.

[0034] In another embodiment of the third aspect, or in further embodiments of any of the above embodiments thereof, 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 at the non-human animal SIRPα locus. In one embodiment, when the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at the non-human animal SIRPα locus, the genetically modified non-human animal includes an invalid mutation in the non-human SIRPα gene at the non-human animal SIRPα locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the invalid mutation is a deletion of at least mouse SIRPα exons 2-4. In another such embodiment, the genetically modified non-human animal is heterozygous for an allele comprising a nucleic acid sequence encoding a human SIRPα protein. In another such embodiment, the genetically modified non-human animal is homozygous for an allele comprising a nucleic acid sequence encoding a human SIRPα protein.

[0035] In another embodiment of the third aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human SIRPα protein includes human SIRPα genomic coding sequence and non-coding sequence.

[0036] In another embodiment of the third aspect, or in further embodiments of any of the above embodiments thereof, the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. In one such embodiment, the functional fragment includes the extracellular domain of human SIRPα, for example, the extracellular domain including at least amino acids 28-362 of SEQ ID NO: 12.

[0037] In another embodiment of the third aspect, or in a further embodiment of any of the above embodiments thereof, 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 at a non-human animal IL-15 locus.

[0038] In one embodiment, when the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at the non-human animal IL-15 locus, the genetically modified non-human animal includes an invalid 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 invalid mutation is the deletion of at least mouse IL-15 exon 5-8. In another such embodiment, the genetically modified non-human animal is heterozygous for the allele of the nucleic acid sequence including the encoding human IL-15 protein. In another such embodiment, the genetically modified non-human animal is homozygous for the allele of the nucleic acid sequence including the encoding human IL-15 protein.

[0039] In another embodiment of the third aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human IL-15 protein includes human IL-15 genomic coding sequence and non-coding sequence.

[0040] In another embodiment of the third aspect, or in further embodiments of any of the above embodiments thereof, the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0041] In another embodiment of the third aspect, or in further embodiments of any of the above embodiments thereof, the genetically modified non-human animal is immunodeficient. For example, in one embodiment, the genetically modified non-human animal includes a Rag2 gene knockout. In another embodiment, the genetically modified non-human animal includes an IL2rg gene knockout, or both a Rag2 gene knockout and an IL2rg gene knockout.

[0042] In another embodiment of the third aspect, or in further embodiments of any of the above embodiments thereof, the non-human animal is a mammal. In one such embodiment, the mammal is a rodent, such as a mouse.

[0043] In a fourth aspect, the present disclosure provides a method for determining the efficacy of a candidate T cell inducing vaccine, the method comprising: administering a candidate T cell inducing vaccine to a genetically modified non-human animal, wherein the genetically modified non-human animal is deficient in its endogenous immune system and comprises: (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter, (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter, and (iii) an implant of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein; stimulating 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.

[0044] 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 at the non-human animal SIRPα locus. In one embodiment, when the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at the non-human animal SIRPα locus, the genetically modified non-human animal includes an invalid mutation in the non-human SIRPα gene at the non-human animal SIRPα locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the invalid mutation is a deletion of at least mouse SIRPα exons 2-4. In another such embodiment, the genetically modified non-human animal is heterozygous for an allele comprising a nucleic acid sequence encoding a human SIRPα protein. In another such embodiment, the genetically modified non-human animal is homozygous for an allele comprising a nucleic acid sequence encoding a human SIRPα protein.

[0045] In another embodiment of the fourth aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human SIRPα protein includes human SIRPα genomic coding sequence and non-coding sequence.

[0046] In another embodiment of the fourth aspect, or in further embodiments of any of the above embodiments thereof, the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. In one such embodiment, the functional fragment includes the extracellular domain of human SIRPα, for example, the extracellular domain including at least amino acids 28-362 of SEQ ID NO: 12.

[0047] 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 at a non-human animal IL-15 locus. In one embodiment, when the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at a non-human animal IL-15 locus, the genetically modified non-human animal includes an invalid mutation in a non-human IL-15 gene at a non-human animal IL-15 locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the invalid mutation is at least the deletion of mouse IL-15 exon 5-8. In another such embodiment, the genetically modified non-human animal is heterozygous for the allele of the nucleic acid sequence including the encoding human IL-15 protein. In another such embodiment, the genetically modified non-human animal is homozygous for the allele of the nucleic acid sequence including the encoding human IL-15 protein.

[0048] In another embodiment of the fourth aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human IL-15 protein includes human IL-15 genomic coding sequence and non-coding sequence.

[0049] In another embodiment of the fourth aspect, or in further embodiments of any of the above embodiments thereof, the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0050] In another embodiment of the fourth aspect, or in further embodiments of any of the above embodiments thereof, the genetically modified non-human animal comprises a Rag2 gene knockout.

[0051] In another embodiment of the fourth aspect, or in further embodiments of any of the above embodiments thereof, the genetically modified non-human animal comprises an IL2rg gene knockout.

[0052] In another embodiment of the fourth aspect, or in further embodiments of any of the above embodiments thereof, the genetically modified non-human animal is a mammal, such as a rodent, eg, a mouse.

[0053] In a fifth aspect, the present disclosure provides a method for identifying an agent that inhibits infection by a pathogen that activates, induces and / or targets human T cells and / or natural killer (NK) cells, the method comprising: administering an agent to a genetically modified non-human animal, wherein the genetically modified non-human animal is defective in terms of its endogenous immune system and comprises: (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and being operably linked to a SIRPα gene promoter, (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and being operably linked to an IL-15 gene promoter, (iii) an implant 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 determining whether the agent reduces the amount of the pathogen in the non-human animal infected with the pathogen.

[0054] 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 at the non-human animal SIRPα locus. In one embodiment, when the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at the non-human animal SIRPα locus, the genetically modified non-human animal includes an invalid mutation in the non-human SIRPα gene at the non-human animal SIRPα locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the invalid mutation is a deletion of at least mouse SIRPα exons 2-4. In another such embodiment, the genetically modified non-human animal is heterozygous for an allele comprising a nucleic acid sequence encoding a human SIRPα protein. In another such embodiment, the genetically modified non-human animal is homozygous for an allele comprising a nucleic acid sequence encoding a human SIRPα protein.

[0055] In another embodiment of the fifth aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human SIRPα protein includes human SIRPα genomic coding sequence and non-coding sequence.

[0056] In another embodiment of the fifth aspect, or in further embodiments of any of the above embodiments thereof, the human SIRPα protein is a functional fragment of the full-length human SIRPα protein. In one such embodiment, the functional fragment includes the extracellular domain of human SIRPα, for example, the extracellular domain including amino acids 28-362 of SEQ ID NO: 12.

[0057] 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 at a non-human animal IL-15 locus. In one embodiment, when the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at a non-human animal IL-15 locus, the genetically modified non-human animal includes an invalid mutation in a non-human IL-15 gene at a non-human animal IL-15 locus. In one such embodiment, the genetically modified non-human animal is a mouse, and the invalid mutation is at least the deletion of mouse IL-15 exon 5-8. In another such embodiment, the genetically modified non-human animal is heterozygous for the allele of the nucleic acid sequence including the encoding human IL-15 protein. In another such embodiment, the genetically modified non-human animal is homozygous for the allele of the nucleic acid sequence including the encoding human IL-15 protein.

[0058] In another embodiment of the fifth aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human IL-15 protein includes human IL-15 genomic coding sequence and non-coding sequence.

[0059] In another embodiment of the fifth aspect, or in further embodiments of any of the above embodiments thereof, the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0060] In another embodiment of the fifth aspect, or in further embodiments of any of the above embodiments thereof, the genetically modified non-human animal comprises a Rag2 gene knockout.

[0061] In another embodiment of the fifth aspect, or in further embodiments of any of the above embodiments thereof, the genetically modified non-human animal comprises an IL2rg gene knockout.

[0062] In another embodiment of the fifth aspect, or in further embodiments of any of the above embodiments thereof, the genetically modified non-human animal is a mammal, such as a rodent, eg, a mouse.

[0063] In a sixth aspect, the present disclosure provides a method for preparing a non-human animal that expresses human IL-15 protein and human SIRPα protein, the method comprising: introducing a nucleic acid sequence encoding human IL-15 protein into the genome of a first non-human animal, wherein the sequence encoding the human IL-15 protein is operably linked to an IL-15 gene promoter sequence; introducing a nucleic acid sequence encoding human SIPRα protein into the genome of a second non-human animal, wherein the sequence encoding the human SIRPα protein is operably linked to a SIRPα promoter sequence; and preparing a third non-human animal comprising 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 SIRPα protein.

[0064] In one embodiment of the sixth aspect, the introducing step comprises producing the non-human animal from pluripotent stem cells comprising a nucleic acid encoding human IL-15 or human SIRPα.

[0065] In another embodiment of the sixth aspect, or in further embodiments of any of the above embodiments thereof, the first animal is a different animal than the second animal, and the step of preparing the third animal comprises mating the first animal and the second animal.

[0066] In another embodiment of the sixth aspect, 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 cell with a nucleic acid sequence encoding human IL-15 protein to obtain a second pluripotent stem cell, the step of introducing into the genome of the second animal comprises contacting the second pluripotent stem cell with a nucleic acid sequence encoding human SIRPα protein to obtain a third pluripotent stem cell, and the third non-human animal is prepared from the third pluripotent stem cell.

[0067] In an alternative form of the sixth aspect, the present disclosure provides a method for preparing a non-human animal that expresses human IL-15 protein and human SIRPα protein, the method comprising: introducing a nucleic acid sequence encoding human SIPRα protein into the genome of a first non-human animal, wherein the sequence encoding the human SIPRα protein is operably linked to a SIPRα gene promoter sequence; introducing a nucleic acid sequence encoding human IL-15 protein into the genome of a second non-human animal, wherein the sequence encoding the human IL-15 protein is operably linked to an IL-15 promoter sequence; and preparing a third non-human animal comprising 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.

[0068] In another embodiment of the sixth aspect, 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 cell with a nucleic acid sequence encoding human SIRPα protein to obtain a second pluripotent stem cell, the step of introducing into the genome of the second animal comprises contacting the second pluripotent stem cell with a nucleic acid sequence encoding human IL-15 protein to obtain a third pluripotent stem cell, and the third non-human animal is prepared from the third pluripotent stem cell.

[0069] In another embodiment of the sixth aspect, or in further embodiments of any of the above embodiments thereof, the pluripotent stem cells are ES cells or iPS cells.

[0070] In another embodiment of the sixth aspect, or in further embodiments of any of the above embodiments thereof, the pluripotent stem cell is deficient in Rag2.

[0071] In another embodiment of the sixth aspect, or in further embodiments of any of the above embodiments thereof, the pluripotent stem cell is deficient in IL2rg.

[0072] In another embodiment of the sixth aspect, or in further embodiments of any of the above embodiments thereof, the third non-human animal is deficient in one or both of Rag2 and IL2rg.

[0073] In another embodiment of the sixth aspect, or in further embodiments of any of the above embodiments thereof, the IL-15 promoter sequence is the sequence of a human IL-15 promoter.

[0074] In another embodiment of the sixth aspect, or in further embodiments of any of the above embodiments thereof, the IL-15 promoter sequence is the sequence of an endogenous non-human animal IL-15 promoter.

[0075] In another embodiment of the sixth aspect, or in further embodiments of any of the above embodiments thereof, the integration results in replacement of the non-human IL-15 gene at the non-human IL-15 locus.

[0076] In another embodiment of the sixth aspect, or in further embodiments of any of the above embodiments thereof, the nucleic acid sequence encoding human IL-15 protein includes human IL-15 genomic coding sequence and non-coding sequence.

[0077] In a seventh aspect, the present disclosure provides a method for implanting a genetically modified non-human animal expressing a human IL-15 protein, comprising: transplanting a cell population comprising human hematopoietic cells into the genetically modified non-human animal prepared by the method according to the sixth aspect or any embodiment thereof. In one such embodiment, the transplantation comprises tail vein injection, fetal liver injection, or retro-orbital injection.

[0078] In another embodiment of the seventh aspect, or in further embodiments of any of the above embodiments thereof, the genetically modified non-human animal is sublethally irradiated prior to transplantation.

[0079] In another embodiment of the seventh aspect, or in further embodiments of any of the above embodiments thereof, the human hematopoietic cells are CD34+ cells.

[0080] In another embodiment of the seventh aspect, or in further embodiments of any of the above embodiments thereof, the human hematopoietic cells are from fetal liver, adult bone marrow, or umbilical cord blood.

[0081] In an eighth aspect, the present disclosure provides a method for determining the efficacy 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, wherein the genetically modified non-human animal is deficient in its endogenous immune system and comprises: (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter, (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter, and (iii) an implant of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein; and determining whether the candidate therapeutic antibody or antigen-binding protein modulates NK cell-mediated antibody-dependent cellular cytotoxicity against the target cells in the genetically modified non-human animal.

[0082] In the ninth aspect, the present disclosure provides a method for determining the efficacy of a candidate therapeutic antibody or antigen-binding protein in killing target cells, comprising: isolating NK cells from a genetically modified non-human animal, wherein the genetically modified non-human animal is defective in terms of the endogenous immune system and comprises: (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and being operably linked to a SIRPα gene promoter, (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and being operably linked to an IL-15 gene promoter, and (iii) an implant of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein; contacting the isolated NK cells with the candidate therapeutic antibody or antigen-binding protein and the target cells; and determining the antibody- or antigen-binding protein-dependent cytolytic activity of the isolated NK cells against the target cells.

[0083] In the tenth aspect, the present disclosure provides a method for screening candidate therapeutic antibodies or antigen-binding proteins for improved efficacy in killing target cells, comprising: administering the candidate therapeutic antibody or antigen-binding protein to a genetically modified non-human animal, wherein the genetically modified non-human animal is deficient in its endogenous immune system and comprises: (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter, (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter, and (iii) an implant of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein; and determining whether the candidate therapeutic antibody or antigen-binding protein shows improved efficacy in killing the target cells in the genetically modified non-human animal.

[0084] In one embodiment of any one of the eighth, ninth and tenth aspects, the target cell is one or more of a tumor cell, a virally infected cell, a bacterially infected cell, a bacterial cell, a fungal cell and a parasite cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0086] Figure 1 Provides a schematic representation of the replacement of the mouse SIRPα gene with the human SIRPα sequence. Figure 1 (Top) The mouse Sirpα locus is shown indicating the relative positions of exons 1-8. Figure 1 (Bottom) A diagram showing the final targeted allele with human exons 2-4 is provided. The encoded chimeric protein has the 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 diagonal stripe shapes represent the inserted human sequence.

[0087] Figure 2 A diagram is provided illustrating targeted genomic replacement of the mouse IL-15 gene as achieved for mouse 2. The open shapes represent the inserted human sequences.

[0088] Figure 3A Provides a graph showing hIL-15 gene expression in various tissues of non-implanted 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 shows hIL-15 mRNA levels relative to the housekeeping gene Hprt.

[0089] Figure 3B A diagram is provided showing human hIL-15 gene expression in various tissues of non-implanted RG (Rag KO, IL-2rg KO) and non-implanted SRG-15 (human SIRPα, Rag KO, IL-2rg KO, human IL-15) mice (mouse #1 and mouse #2 as indicated).

[0090] Figure 4 Provided that after stimulation with poly(I:C), SRG, SRG IL-15 h / m (Mouse 2) and SRG IL-15 h / h (Mouse 2) Serum levels of human IL-15 protein in mice.

[0091] Figure 5A A graphic illustration showing efficient human hematopoietic cell engraftment in the blood of NSG, SRG and SRG-15 (mouse 2) mice 12-14 weeks after implantation is provided. All data are shown as mean ± sem. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test (*P < 0.05, **P < 0.01, ****P < 0.0001).

[0092] Figure 5B A graph is provided showing the number of human CD45+ cells in the BM, spleen, LN, liver and lung of SRG and SRG-15 (mouse 2) at 14 weeks post-implantation.

[0093] Fig. 6A Graphs showing the frequency of human T cells and NK cells in the bone marrow (BM), liver, and lung in SRG and SRG-15 mice (Mouse 1) are provided.

[0094] Figure 6B Provided is a graph showing the frequency of human NK cells in various tissues in SRG and SRG-15 mice (Mouse 1).

[0095] Figure 6C Figures and diagrams illustrating the maturation of human NK cells in the liver of SRG and SRG-15 mice (Mouse 1) are provided.

[0096] Fig.6D Provides human CD56 in the spleen of SRG-15 mice 暗淡 CD16 + Diagram showing that NK cells express high levels of human killer inhibitory receptors.

[0097] Fig. 7A A graphic diagram showing the frequency of human NK cells in the blood of NSG, SRG and SRG-15 (mouse 2) mice 10-12 weeks after implantation is provided. All data are shown as mean ± sem. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test (*P < 0.05, **P < 0.01, ****P < 0.0001).

[0098] Figure 7B The results show that at 14 weeks after implantation, SRG, SRG-15 h / m and SRG-15 h / h Human NKp46 in spleen + Graph of the percentage of cells. All data are shown as mean ± sem. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test (*P < 0.05, **P < 0.01, ****P < 0.0001).

[0099] Figure 7C Figures are provided showing the frequency of human NK cells in the blood, spleen (SP), liver, and lung of SRG and SRG-15 (mouse 2) mice 14 weeks after implantation. All data are shown as mean ± sem. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test (*P < 0.05, **P < 0.01, ****P < 0.0001).

[0100] Fig.7DA graphic illustration showing the frequency of human NK cells in the spleen (SP), liver, and lung of SRG and SRG-15 (mouse 2) mice at 14 weeks post-implantation is provided. All data are shown as mean ± sem. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test (*P < 0.05, **P < 0.01, ****P < 0.0001).

[0101] Figure 8 Provided are graphs showing the distribution of human T cells and NK cells in the blood of SRG and SRG-15 (mouse 2) mice (left side) (gated on human CD45+ cells (hematopoietic cells) and NKp46+ cells (NK cells)); and a graph showing the percentage of hCD45+ cells that are NKp46+ cells in the blood of SRG-15 mice engrafted (right side).

[0102] Fig. 9A Provided are graphs showing the distribution of NK cells and T cells in the spleen of SRG-15 mice engrafted with CD34+ huHSCs (Mouse 2) relative to SRG mice engrafted with CD34+ huHSCs and a graph showing the percentage and number of NKp46+ cells in the spleen.

[0103] Fig. 9B A graphic diagram showing the composition of human immune cells in the blood of NSG (n=5), SRG (n=19), and SRG-15 (mouse 2) mice (n=39) at 10-12 weeks post-implantation is provided.

[0104] Fig. 9C Provided are the numbers of human CD45+ cells in the thymus of SRG and SRG-15 (mouse 2) mice 14 weeks after implantation.

[0105] Fig.9D Representative flow cytometry images of hCD45+ cells in the thymus of SRG and SRG-15 (Mouse 2) mice are provided.

[0106] Fig.9E A diagram is provided showing the composition of hCD45+ cells in the thymus of SRG (n=8) and SRG-15 (mouse 2) mice (n=4) at 14 weeks post-implantation.

[0107] Fig. 10A The results show that CD56 was expressed in the blood and spleen of SRG and SRG-15 (mouse 2) mice at 7 weeks post-implantation. 明亮 CD16 - and CD56 暗淡 CD16 + Plot of the frequencies of NK cell subsets.

[0108] Fig. 10BThe results show that CD56 was expressed in the blood and spleen of SRG and SRG-15 (mouse 2) mice at 7 weeks post-implantation. 明亮 CD16 - and CD56 暗淡 CD16 + Graphical illustration of the frequencies of NK cell subsets.

[0109] Fig. 10C Figures and diagrams showing the expression of killer inhibitory receptors (KIR) on NK cell subsets in humans and SRG-15 mice (Mouse 2) are provided.

[0110] Fig.11 Provided to show that compared with PBMC samples, CD16 + NK cells relative to CD16 - Two graphs (top left and top right) showing the distribution of NK cells in the blood of SRG-15 mice (Mouse 2). Fig.11 Also provided are CD16 in blood or PBMC-derived samples obtained from SRG-15 mice (mouse 2). + NKp46 + The percentage of cells relative to CD16 - NKp46 + Graphical representation of percentage of cells (bottom).

[0111] Fig.12 A graphic diagram showing the development of human NK cells in the bone marrow of SRG and SRG-15 (mouse 2) mice 7 weeks after implantation is provided. All data are shown as mean ± sem. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test (*P < 0.05, **P < 0.01, ****P < 0.0001).

[0112] Fig.13A A graph showing the frequency of human T cells in various tissues in SRG and SRG-15 mice (Mouse 1) is provided. (K / μl = thousand cells per μl).

[0113] Fig. 13B Provides human CD8 in the blood and liver of SRG and SRG-15 mice (mouse 1) + Diagrams and illustrations of T cell phenotypes.

[0114] Fig.14A Provides lung CD8 expression in SRG and SRG-15 (mouse 1) mice + Figure and illustration of the expression of the tissue resident marker CD69 in T cells.

[0115] Fig. 14B Provides liver CD8 expression in SRG and SRG-15 (mouse 1) mice+ Figure and illustration of the expression of the tissue resident marker CD69 in T cells.

[0116] Fig.15A hCD3 in spleen, lung and liver of SRG and SRG-15 (mouse 2) mice at 16 weeks post-implantation + Graphical representation of the frequency of T cells.

[0117] Fig. 15B A graph is provided showing the CD4 / CD8 ratio in the spleen, lung and liver of SRG and SRG-15 (Mouse 2) mice at 16 weeks post-implantation.

[0118] Fig.16A A graph illustrating the frequency of human lamina propria lymphocytes (LPLs) in the colon of SRG and SRG-15 (Mouse 1) mice is provided.

[0119] Fig. 16B A graph illustrating the frequency of human lamina propria lymphocytes (LPLs) in the colon of SRG and SRG-15 (Mouse 1) mice is provided.

[0120] Fig.17A Together with Figures 17B-17C Together, we demonstrate efficient human intraepithelial lymphocyte (IEL) engraftment in the small intestine of 16-week-old SRG-15 mice (Mouse 1). Fig.17A Provided is a display of human CD45 in the IEL fraction of SRG and SRG-15 (mouse 1) mice + Diagram and illustration of cells and CD8+ T cells.

[0121] Fig. 17B Images of immunohistochemical staining for hCD45 in the small intestine of 16-week-old SRG and SRG-15 (Mouse 1) mice are provided.

[0122] Fig. 17C Provided is a display of human CD8 in the spleen and small intestine of SRG-15 mice (mouse 1) + Diagram of the phenotypic characteristics of T cells.

[0123] Fig.18A Provided are representative FACS plots showing mouse and human CD45+ cells within the IEL fraction of SRG and SRG-15 (mouse 2) mice at 16 weeks post-implantation.

[0124] Fig.18BA graph showing the number of human IELs in the small intestine of SRG relative to SRG-15(mouse 2) mice, and the number of human LPLs in the large intestine of SRG relative to SRG-15(mouse 2) mice is provided. All data are shown as mean ± sem. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test (***P < 0.001).

[0125] Fig.18C Provided are graphs showing the composition of hCD3+ cells in the small intestine of SRG-15 mice (Mouse 2). One representative FACS graph of 8 SRG-15 mice (Mouse 2).

[0126] Fig.18D A diagram showing the phenotypic characteristics of hCD3+hCD8+ T cells in the spleen and small intestine of SRG-15 mice (Mouse 2) is provided.

[0127] Fig.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. Photos are representative of 3 mice per group.

[0128] Fig.19A Provided are graphs and diagrams showing the distribution and number of hCD45+ cells in the intraepithelial lymphocyte population of SRG and SRG-15 mice, and the relative percentages of NK cells and T cells in the hCD45+ cell population in the intraepithelial lymphocyte population of SRG and SRG-15 (Mouse 2) mice.

[0129] Fig.19B Provided are graphs and diagrams showing the distribution and percentage of CD16+ and CD16- NK cells in intraepithelial lymphocytes compared to blood and spleen of SRG-15 mice (Mouse 2).

[0130] Fig.19C Figures and diagrams showing the distribution and number of human IELs and human lamina propria lymphocytes (LPLs) in SRG and SRG-15 (mouse 2) mice are provided.

[0131] Fig. 20A and 20B Figures and diagrams are provided demonstrating the presence of discernible Peyer's patches containing predominantly hCD45+ cells in SRG-15 mice (Mouse 2).

[0132] Fig.21A A timeline of cohousing and stool sample collection for gut microbiota sequencing is provided.

[0133] Fig. 21BA graphical representation showing the relative abundance of mouse bacteria in the intestines of non-implanted and implanted SRG and SRG-15 (Mouse 1) mice is provided.

[0134] Fig. 22 To illustrate the functional relevance of human tissue-resident T cells in SRG-15 mice. More specifically, Fig. 22 Provides a schematic demonstrating the functional relevance of human IELs in clearing acute rotavirus infection.

[0135] Fig.23A Provided are ViSNE images showing CyTOF-based analysis of CD56 in humans (n=20) and SRG-15 mice (mouse 2) (n=9). 明亮 CD16 - and CD56 暗淡 CD16 + Analysis of 42 parameters of NK cell subsets. Each point represents a single cell.

[0136] Fig. 23B ViSNE plots are provided showing CD56 in humans (n=20) and SRG-15 mice (mouse 2) (n=9) for the eight selected markers. 明亮 Expression intensity on CD16-NK cells.

[0137] Fig.23C ViSNE plots are provided showing CD56 in humans (n=20) and SRG-15 mice (n=9) for the eight selected markers. 暗淡 CD16 + Expression intensity on NK cells.

[0138] Fig.24A A graph is provided showing the percentage of blood NK cells that are CD69+ in SRG relative to SRG-15 (mouse 2) mice before and after injection of poly IC.

[0139] Fig. 24B A graph showing IFNγ production by SRG and SRG-15 (mouse 2) derived NK cells following in vitro stimulation with poly I:C or human IL-12p70 is provided. NK cells from mice were compared to NK cells derived from healthy human PBMCs. All samples were normalized for NK number.

[0140] Fig.24CA graph is provided showing the cytolytic capacity of splenic NK cells from SRG and SRG-15 (mouse 2) mice against class I HLA-deficient K562 cells (left) or against Raji cells in the absence (top right) or presence (bottom right) of anti-CD20 antibody. SRG-15#1 and SRG-15#2 represent two different NK cell preparations obtained from SRG-15 (mouse 2) littermates.

[0141] Fig.25A A graphic illustration showing that human NK cells in SRG-15 mice (Mouse 2) inhibited tumor growth after treatment with rituximab (RTX) is provided. All data are shown as mean ± sem. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test (***P < 0.001).

[0142] Fig.25B Figures and illustrations showing the frequency of human NK cells and T cells in human tumor xenografts of untreated (n=5) and RTX-treated SRG-15 mice (n=1) are provided. All data are shown as mean±sem. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test (***P<0.001).

[0143] Fig.25C Figures and illustrations showing human NK cell subsets in blood and tumors of untreated (n=2) and RTX-treated SRG-15 mice (n=1) are provided. All data are shown as mean±sem. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test (***P<0.001).

[0144] Details

[0145] Before describing the present methods and compositions, it should be understood that the present invention is not limited to the specific methods or compositions described, as the methods or compositions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0146] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although any method and material similar or equivalent to those methods and materials described herein can be used to implement or test the present invention, specific methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the publications cited. It should be understood that the present disclosure replaces any disclosure incorporated into the publication insofar as there is a contradiction.

[0147] As will be apparent to those skilled in the art after reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the invention. Any described method can be executed in the order of events described or in any other order that is logically possible.

[0148] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth.

[0149] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication.

[0150] Provided are genetically modified non-human animals that express human SIRPα and human IL-15 from the genome of a non-human animal. Also provided are methods for preparing non-human animals that express human SIRPα and human IL-15 from the genome of a non-human animal, and methods for using non-human animals that express human SIRPα and human IL-15 from the genome of a non-human animal. These animals and methods have many uses in the art, including, for example, modeling human T cell and / or natural killer (NK) cell development and function; modeling human pathogen infection, such as human pathogen infection of a specific tissue, such as human intestinal, lung or liver pathogen infection; modeling human pathogen infection of human T cells and / or NK cells; in vivo screening for agents that inhibit infection by pathogens that activate, induce and / or target T cells and / or NK cells; in vivo screening for agents that modulate the development and / or function of human T cells and / or NK cells, such as in a healthy or diseased state; in vivo screening for agents that are toxic to human T cells and / or NK cells; in vivo screening for agents that prevent, mitigate or reverse the toxic effects of toxic agents on human T cells and / or NK cells; in vivo screening for candidate T cell inducing vaccines; and in vivo and in vitro screening for agents that inhibit tumor growth and / or infection by activating NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) processes.

[0151] Humanized SIRPα non-human animals

[0152] In some aspects of the present disclosure, a humanized SIRPα non-human animal is provided. With respect to humanized SIRPα non-human animals or “SIRPα non-human animals”, it means a non-human animal comprising a nucleic acid sequence encoding a human SIRPα protein. As used herein, “human SIRPα protein” means a protein that is a variant of a wild-type (or natural) human SIRPα protein or a wild-type (or natural) human SIRPα protein that retains one or more signaling and / or receptor functions of a wild-type human SIRPα protein. As used herein, the term “variant” defines an isolated naturally occurring genetic mutant of a human polypeptide or nucleic acid sequence or a recombinantly prepared variation of a human polypeptide or nucleic acid sequence, each of which contains one or more mutations compared to the corresponding wild-type human nucleic acid or polypeptide sequence. For example, the mutation may be one or more amino acid substitutions, additions and / or deletions. The term “variant” also includes human homologs and orthologs. In some embodiments, a variant polypeptide of the invention has 70% or greater identity to a wild-type human polypeptide, e.g., 75%, 80% or 85% or greater identity, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a wild-type human polypeptide.

[0153] The percent identity between two sequences can be determined using any convenient technique in the art, such as sequence alignment using, for example, publicly available software. Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis, PCR-mediated mutagenesis, directed evolution, etc. One skilled in the art will recognize that one or more nucleic acid substitutions can be introduced without changing the amino acid sequence, and one or more amino acid mutations can be introduced without changing the functional properties of a human protein.

[0154] Conservative amino acid substitutions can be made in human proteins to produce human protein variants. By conservative amino acid substitutions, it is meant the substitution of one amino acid with another amino acid having similar characteristics as is known in the art. For example, each amino acid can be described as having one or more of the following characteristics: electropositivity, electronegativity, aliphatic, aromatic, polar, hydrophobic, and hydrophilic. A conservative substitution is the substitution of an amino acid having a specified structural or functional characteristic by another amino acid having the same characteristics. 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; and conservative substitutions include substitutions between amino acids within each group. Amino acids may also be described in terms of relative size, with alanine, cysteine, aspartic acid, glycine, asparagine, proline, threonine, serine, and valine all generally considered small amino acids.

[0155] Human variants may include synthetic amino acid analogs, amino acid derivatives, and / or non-standard amino acids, illustratively including, without limitation, α-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, methylhistidine, and ornithine.

[0156] Human variants will generally be encoded by nucleic acids having a high degree of identity to nucleic acids encoding wild-type human proteins. The complementary sequence of nucleic acids encoding human variants specifically hybridizes with nucleic acids encoding wild-type humans under high stringency conditions. Nucleic acids encoding human variants can be isolated using well-known methods or produced in a recombinant or synthetic manner. Also encompassed by the term "human SIRPα protein" are fragments of wild-type human SIRPα protein (or its variants) that retain one or more signal transduction and / or receptor functions of wild-type human SIRPα protein, such as the extracellular domain of human SIRPα protein.

[0157] The term "human SIRPα protein" also encompasses fusion proteins, i.e., chimeric proteins, that include one or more fragments of a wild-type human SIRPα protein (or a variant thereof) and retain one or more signaling and / or receptor functions of the wild-type human SIRPα protein. Fusion proteins comprising one or more fragments of a wild-type human SIRPα protein (or a variant thereof), for example in combination with one or more non-human peptides or polypeptides, may also be referred to herein as humanized SIRPα proteins. Thus, for example, a protein comprising an amino acid sequence in which the extracellular domain of a wild-type human SIRPα protein is fused to the signaling domain of a wild-type mouse SIRPα protein is encompassed by the term "human SIRPα protein".

[0158] In some cases, the human SIRPα protein of the present disclosure comprises 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 to amino acids 28-362 of SEQ ID NO:12.

[0159] Therefore, the nucleic acid sequence encoding the human SIRPα protein is a polynucleotide comprising the coding sequence of 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 signal transduction and / or receptor functions of the wild-type human SIRPα protein, or a fusion protein comprising one or more fragments of the wild-type human SIRPα protein (or its variant) and retaining one or more signal transduction and / or receptor functions of the wild-type human SIRPα protein, i.e., a chimeric protein.

[0160] SIRPα (also known as "signal regulatory protein α" and "CD172A" in humans) is a member of the signal regulatory 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 obtained from Genbank accession numbers NM_001040022.1 (variant 1), NM_001040023.1 (variant 2) and NM_080792.2 (variant 3). The SIRPα gene is conserved in at least chimpanzees, rhesus monkeys, dogs, cows, mice, rats and chickens. The genomic locus encoding the wild-type human SIRPα protein can be found in the human genome at chromosome 20; NC_000020.11 (1894117-1939896). The protein sequence is encoded by exons 1 to 8 at this locus. Therefore, in some embodiments, the nucleic acid sequence comprising the coding sequence of human SIRPα includes one or more of exons 1-8 of the human SIRPα gene. In some cases, the nucleic acid sequence also includes various aspects of the genomic locus of human SIRPα, such as introns, 3' and / or 5' untranslated sequences (UTR). In some cases, the nucleic acid sequence includes the entire region of the human SIRPα genomic locus. In some cases, the nucleic acid sequence includes exons 2-4 of the human SIRPα genomic locus.

[0161] In the humanized SIRPα non-human animals of the present application, the nucleic acid sequence encoding the human SIRPα protein is operably linked to one or more regulatory sequences of the SIRPα gene, such as the regulatory sequence of the SIRPα gene of the non-human animal. Non-human animal (e.g., mouse) SIRPα regulatory sequences are those sequences of the non-human animal SIRPα genomic locus that regulate the expression of non-human animal SIRPα, such as 5' regulatory sequences, such as SIRPα promoter, SIRPα 5' untranslated region (UTR), etc.; 3' regulatory sequences, such as 3'UTR; and enhancers, etc.

[0162] "Promoter" or "promoter sequence" refers to a DNA regulatory region that is capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. The promoter sequence is bounded at its 3' end by a transcription start site, and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. Within the promoter sequence will be found a transcription start site, as well as protein binding domains responsible for binding RNA polymerase. Eukaryotic promoters will often, but not always, contain a "TATA" box and a "CAT" box. Of particular interest to the present disclosure are DNA regulatory elements, such as promoters, that promote transcription of human proteins with the same spatial and temporal expression pattern as would be observed for the corresponding endogenous protein (i.e., in the same cells and tissues and at the same time).

[0163] Mouse SIRPα is located on chromosome 2; NC_000068.7 (129592606-129632228), and the mouse SIRPα coding sequence can be obtained by Genbank accession number NM_007547.4 (subtype 1), NM_001177647.2 (subtype 2), NM_001291019.1 (subtype 3), NM_001291020.1 (subtype 3), NM_001291021.1 (subtype 4), NM_001291022.1 (subtype 5). The regulatory sequence of mouse SIRPα is well determined in the art and can be easily identified using electronic methods, for example, by consulting the above Genbank accession number on the UCSC genome browser on the World Wide Web, or by experimental methods as described in the art. In some cases, such as when the nucleic acid sequence encoding human SIRPα protein is located at the mouse SIRPα genomic locus, the regulatory sequences operably linked to the human SIRPα coding sequence are endogenous or native to the mouse genome, i.e., they are present in the mouse genome prior to integration of the human nucleic acid sequence.

[0164] In some cases, a humanized SIRPα non-human animal, such as a mouse, is produced by randomly integrating or inserting a human nucleic acid sequence encoding a human SIRPα protein (including a fragment as described above), i.e., a "human SIRPα nucleic acid sequence" or a "human SIRPα sequence" into the genome. Typically, in such embodiments, the position of the nucleic acid sequence encoding the human SIRPα protein in the genome is unknown. In other cases, a humanized SIRPα non-human animal is produced by targeted integration or insertion of a human SIRPα nucleic acid sequence into the genome, such as by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at a target locus, while removing host genomic material from the target locus, such as 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more of genomic material. Thus, for example, in a humanized SIRPα mouse comprising a nucleic acid sequence encoding a human SIRPα protein produced by targeting a human SIRPα nucleic acid sequence to a mouse SIRPα locus, the human SIRPα nucleic acid sequence may replace some or all of the mouse sequences, such as exons and / or introns, at the SIRPα locus. In some of the described cases, the human SIRPα nucleic acid sequence is integrated into the mouse SIRPα locus so that the expression of the human SIRPα sequence is regulated by a natural or endogenous regulatory sequence at the mouse SIRPα locus. In other words, the regulatory sequence to which the nucleic acid sequence encoding the human SIRPα protein is operably linked is a natural SIRPα regulatory sequence at the mouse SIRPα locus.

[0165] In some cases, the integration of the human SIRPα sequence does not affect the transcription of the gene into which the human SIRPα sequence has been integrated. For example, if the human SIRPα sequence is integrated into the coding sequence as an intein, or the human SIRPα sequence includes a 2A peptide, the human SIRPα sequence will be transcribed and translated simultaneously with the gene into which the human SIRPα sequence has been integrated. In other cases, the integration of the human SIRPα sequence interrupts the transcription of the gene into which the human SIRPα sequence has been integrated. For example, after the human SIRPα sequence is integrated by homologous recombination, some or all of the coding sequences at the integration locus may be removed so that the human SIRPα sequence is transcribed instead. In some of the cases described, the integration of the human SIRPα sequence produces an invalid mutation, thereby producing an invalid allele. An invalid 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 a gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product. At the phenotypic level, an invalid allele cannot be distinguished from the absence of the entire locus.

[0166] In some cases, a humanized SIRPα non-human animal, such as a mouse, includes a copy of a nucleic acid sequence encoding a human SIRPα protein. For example, a non-human animal may be heterozygous for the nucleic acid sequence. In other words, one allele at the locus will include the nucleic acid sequence, and the other allele will be an endogenous allele. For example, as discussed above, in some cases, a human SIRPα nucleic acid sequence is integrated into a non-human animal (e.g., a mouse) SIRPα locus so that it produces an invalid allele of non-human animal SIRPα. In some of the embodiments, a humanized SIRPα non-human animal may be heterozygous for a nucleic acid sequence encoding human SIRPα, that is, a humanized SIRPα non-human animal includes an invalid allele of non-human animal SIRPα (including an allele of the nucleic acid sequence) and an endogenous SIRPα allele (wild type or another allele). In other words, the non-human animal is SIRPα h / m Non-human animals, wherein "h" represents an allele comprising a human sequence, and "m" represents an endogenous allele. In other cases, humanized SIRPα includes two copies of a nucleic acid sequence encoding a human SIRPα protein. For example, a non-human animal such as a mouse may be homozygous for the nucleic acid sequence, i.e., both alleles of a locus in a diploid genome will include the nucleic acid sequence, i.e., a humanized SIRPα non-human animal includes two null alleles of non-human animal SIRPα (including alleles of the nucleic acid sequence). In other words, the non-human animal is SIRPα h / h Non-human animals.

[0167] In some embodiments, humanized SIRPα non-human animals, such as mice, include other genetic modifications. In some embodiments, humanized SIRPα non-human animals are immunocompromised animals. For example, humanized SIRPα non-human animals may include at least one invalid allele of the Rag2 gene ("recombination activation gene 2", wherein the coding sequence of the mouse gene can be obtained by Genbank accession number NM_009020.3). In some embodiments, humanized SIRPα non-human animals include two invalid alleles of Rag2. In other words, humanized SIRPα non-human animals are homozygous for Rag2. As another example, humanized SIRPα non-human animals include at least one invalid allele of the IL2rg gene ("interleukin 2 receptor γ", also known as common γ chain or γC, wherein the coding sequence of the mouse gene can be obtained by Genbank accession number NM_013563.3). In some embodiments, humanized SIRPα non-human animals include two invalid alleles of IL2rg. In other words, the humanized SIRPα non-human animal is homozygous null for IL2rg, i.e., it is IL2rg - / - (or IL2rgY / - , wherein the IL2rg gene is located on chromosome X as in mice). In some embodiments, the SIRPα non-human animal comprises a null allele of both Rag2 and IL2rg, i.e., it is Rag2 - / - IL - / - (or Rag2 - / - IL Y / - , where the IL2rg gene is located on the X chromosome as in mice). Other genetic modifications are also contemplated. For example, humanized SIRPα non-human animals may include modifications in other genes related to the development and / or function of hematopoietic cells and the immune system, such as replacing one or more other non-human animal genes with a nucleic acid sequence encoding a human ortholog. Additionally or alternatively, humanized SIRPα non-human animals may include modifications in genes related to the development and / or function of other cells and tissues, such as genes associated with human disorders or diseases, or genes that provide models of human disorders and diseases when modified in non-human animals such as mice.

[0168] Humanized IL-15 non-human animal

[0169] In some aspects of the present disclosure, a humanized IL-15 non-human animal is provided. With respect to humanized IL-15 non-human animals or "IL-15 non-human animals", it means a non-human animal comprising a nucleic acid sequence encoding a human IL-15 protein. As used herein, "human IL-15 protein" means a protein that is a variant of a wild-type (or natural) human IL-15 protein or a wild-type (or natural) human IL-15 protein, wherein the variant retains one or more signal transduction functions of a wild-type (or natural) human IL-15 protein, such as allowing stimulation of a human IL-15 receptor (or signal transduction by a human IL-15 receptor), and / or it is capable of binding to a human IL-15 receptor α subunit of a human IL-15 receptor, and / or it is capable of binding to IL-2Rβ / IL-15Rβ and a common γ chain (γc). Also encompassed by the term "human IL-15 protein" are fragments of wild-type human IL-15 protein (or variants thereof) that retain one or more signaling functions of the wild-type human IL-15 protein, such as fragments of the human IL-15 protein that allow stimulation of the human IL-15 receptor (or signaling through the human IL-15 receptor), and / or which are capable of binding to the human IL-15 receptor α subunit of the human IL-15 receptor, and / or which are capable of binding to IL-2Rβ / IL-15Rβ and the common gamma chain (γc).

[0170] The term "human IL-15 protein" also encompasses a fusion protein, i.e., a chimeric protein, comprising one or more fragments of a wild-type human IL-15 protein (or a variant thereof) and retaining one or more signal transduction functions of the wild-type human IL-15 protein, such as those described above. A fusion protein comprising one or more fragments of a wild-type human IL-15 protein (or a variant thereof) may also be referred to herein as a humanized IL-15 protein.

[0171] Therefore, the nucleic acid sequence encoding the human IL-15 protein is a polynucleotide comprising the coding sequence of the human IL-15 protein, i.e., a 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 signal transduction functions of the wild-type human IL-15 protein, or a fusion protein comprising one or more fragments of the wild-type human IL-15 protein (or its variant) and retaining one or more signal transduction functions of the wild-type human IL-15 protein, e.g., as described above, i.e., a chimeric protein.

[0172] IL-15 (also known as "interleukin 15") is a cytokine that stimulates the proliferation of T lymphocytes. The polypeptide sequence of wild-type human IL-15 and the nucleic acid sequence encoding wild-type human IL-15 can be obtained from Genbank accession numbers NM_000585.4; NP_000576.1 (subtype 1), NM_172175.2; NP_751915.1 (subtype 2). The genomic locus encoding the wild-type human IL-15 protein can be found in the human genome at chromosome 4; NC_000004.12 (141636596-141733987). The human IL-15 locus includes 8 exons, of which exons 3-8 are coding exons. Therefore, in some embodiments, the nucleic acid sequence comprising the coding sequence of human IL-15 includes one or more of exons 3-8 of the human IL-15 gene (i.e., encoding exons 1-6, see Figure 2 ). For example, various IL-15mRNA subtypes produced by the following exon usage combinations have been identified: exon 1-2-3-4-5-6-7-8; exon 1-3-4-5-6-7-8 or exon 1-3-4-(optional exon 5)-5-6-7-8). In some cases, the nucleic acid sequence also includes various aspects of the genomic locus of human IL-15, such as introns, 3' and / or 5' untranslated sequences (UTR). In some cases, the nucleic acid sequence includes the entire region of the human IL-15 genomic locus. In some cases, the nucleic acid sequence includes exons 5-8 (i.e., encoding exons 3-6) of the human IL-15 genomic locus.

[0173] In some cases, a human IL-15 protein in accordance with the present disclosure comprises 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 to SEQ ID NO:31.

[0174] In the humanized IL-15 non-human animals of the present application, the nucleic acid sequence encoding human IL-15 protein is operably linked to one or more regulatory sequences of the IL-15 gene, such as the regulatory sequence of the IL-15 gene of the non-human animal. The non-human animal (e.g., mouse) IL-15 regulatory sequence is those sequences of the non-human animal IL-15 genomic locus regulating the expression of non-human animal IL-15, such as 5' regulatory sequences, such as IL-15 promoter, IL-15 5' untranslated region (UTR), etc.; 3' regulatory sequences, such as 3'UTR; and enhancers, etc. Mouse IL-15 is located on chromosome 8, NC_000074.6 (82331624-82403227, complementary chain), and the mouse IL-15 coding sequence can be obtained by Genbank accession number NM_008357.2 (variant 1); NM_001254747.1 (variant 2). The regulatory sequences of mouse IL-15 are well established in the art and can be readily identified using electronic methods, for example, by consulting the above Genbank accession numbers on the UCSC genome browser on the World Wide Web at genome.ucsc.edu, 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 in the mouse IL-15 genomic locus, the regulatory sequences operably linked to the human IL-15 coding sequence are endogenous or native to the mouse genome, i.e., they were present in the mouse genome prior to integration of the human nucleic acid sequence.

[0175] In some cases, a humanized IL-15 non-human animal, such as a mouse, is produced by randomly integrating or inserting a human nucleic acid sequence encoding a human IL-15 protein (including a fragment as described above), i.e., a "human IL-15 nucleic acid sequence" or a "human IL-15 sequence" into the genome. Typically, in such embodiments, the position of the nucleic acid sequence encoding the human IL-15 protein in the genome is unknown. In other cases, a humanized IL-15 non-human animal is produced by targeted integration or insertion of a human IL-15 nucleic acid sequence into the genome, such as by homologous recombination. In homologous recombination, a polynucleotide is inserted into a target locus in the host genome, while removing host genomic material, such as 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more of genomic material, from the target locus. Therefore, for example, in the humanized IL-15 mouse including the nucleic acid sequence encoding human IL-15 protein produced by making human IL-15 nucleic acid sequence target mouse IL-15 locus, human IL-15 nucleic acid sequence can replace some or all mouse sequences such as exons and / or introns at IL-15 locus. In some such cases, human IL-15 nucleic acid sequence is integrated into mouse IL-15 locus so that the expression of human IL-15 sequence is regulated by natural or endogenous regulatory sequence at mouse IL-15 locus. In other words, the regulatory sequence operably connected to the nucleic acid sequence encoding human IL-15 protein is the natural IL-15 regulatory sequence at mouse IL-15 locus.

[0176] In some cases, the integration of human IL-15 sequence does not affect the transcription of the gene into which the human IL-15 sequence has been integrated. For example, if the human IL-15 sequence is integrated into the coding sequence as an intein, or the human IL-15 sequence includes a 2A peptide, the human IL-15 sequence will be transcribed and translated simultaneously with the gene into which the human IL-15 sequence has been integrated. In other cases, the integration of human IL-15 sequence interrupts the transcription of the gene into which the human IL-15 sequence has been integrated. For example, after the human IL-15 sequence is integrated by homologous recombination, some or all of the coding sequences at the integration locus can be removed so that the human IL-15 sequence is transcribed instead. In some such cases, the integration of human IL-15 sequence produces invalid mutations, thus producing invalid alleles. Invalid alleles are mutant copies of genes that completely lack the normal function of that gene. This can be the result of the complete absence of gene products (proteins, RNA) at the molecular level, or the expression of non-functional gene products. At the phenotypic level, null alleles are indistinguishable from deletion of the entire locus.

[0177] In some cases, humanized IL-15 non-human animals, such as mice, include a copy of a nucleic acid sequence encoding a human IL-15 protein. For example, non-human animals may be heterozygous for the nucleic acid sequence. In other words, one allele at the locus will include the nucleic acid sequence, and the other allele will be an endogenous allele. For example, as discussed above, in some cases, a human IL-15 nucleic acid sequence is integrated into a non-human animal (e.g., mouse) IL-15 locus so that it produces an invalid allele of non-human animal IL-15. In some of the embodiments, humanized IL-15 non-human animals may be heterozygous for a nucleic acid sequence encoding human IL-15, that is, humanized IL-15 non-human animals include an invalid allele of non-human animal IL-15 (including an allele of the nucleic acid sequence) and an endogenous IL-15 allele (wild type or another allele). In other words, non-human animals are IL-15 h / m A non-human animal, wherein "h" represents an allele comprising a human sequence, and "m" represents an endogenous allele. In other cases, humanized IL-15 comprises two copies of a nucleic acid sequence encoding a human IL-15 protein. For example, a non-human animal such as a mouse may be homozygous for the nucleic acid sequence, i.e., both alleles of a locus in a diploid genome will comprise the nucleic acid sequence, i.e., a humanized IL-15 non-human animal comprises two null alleles of non-human animal IL-15 (including alleles of the nucleic acid sequence). In other words, a non-human animal is an IL-15 h / h Non-human animals.

[0178] Humanized SIRPα-IL-15 non-human animals

[0179] By crossing a humanized IL-15 non-human animal as described above with a humanized SIRPα non-human animal of the same species as described above, a genetically modified non-human animal expressing both human SIRPα and human IL-15 can be produced. In some embodiments, the genetically modified non-human animal is deficient in terms of the endogenous immune system, such as an immunocompromised animal, for example, due to having an ineffective allele of one or both of Rag2 and IL2rg. For example, in some embodiments, a non-human animal according to the present disclosure is Rag2 - / - and / or IL2rg - / - (or Rag2 - / - and / or IL2rg Y / - , wherein the IL2rg gene is located on chromosome X in mice). In some embodiments, a genetically modified non-human animal, such as a mouse, is provided, wherein the genetically modified non-human animal, such as a mouse, is a SIRPα h / m IL-15 h / m Rag2 - / -IL Y / - 、SIRPα h / h IL-15 h / m Rag2 - / - IL Y / - SIRPα h / m IL-15 h / h Rag2 - / - IL Y / - .

[0180] In some embodiments, a genetically modified non-human animal, such as a mouse, is provided, which includes a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and being operably linked to a SIRPα gene promoter; and a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and being operably linked to a IL-15 gene promoter, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein.

[0181] In some embodiments, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter. In some of the embodiments, the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at a non-human animal SIRPα locus. In another embodiment, the SIRPα gene promoter is a human SIRPα promoter.

[0182] In some embodiments, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter. In some of the embodiments, the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at a non-human animal IL-15 locus. In another embodiment, the IL-15 promoter is a human IL-15 promoter.

[0183] In some embodiments, a genetically modified non-human animal as described herein expresses human IL-15 mRNA in the liver, lung, bone marrow (BM), small intestine (SI), and colon.

[0184] In some embodiments, after implantation with human hematopoietic cells, such as CD45+ cells, a genetically modified non-human animal, such as a mouse, expressing both human SIRPα and human IL-15 as described herein exhibits a higher percentage and number of human T cells and NK cells compared to a genetically modified non-human animal, such as a mouse, that expresses only human SIRPα. In some embodiments, a genetically modified non-human animal, such as a mouse, that expresses both human SIRPα and human IL-15 as described herein exhibits a higher percentage and number of NK cells in the blood and spleen. In some embodiments, after implantation with human hematopoietic cells, such as CD45+ cells, a genetically modified non-human animal, such as a mouse, that expresses both human SIRPα and human IL-15 as described herein includes two human NK cell subsets in the blood, spleen, and liver, namely CD56. 明亮 CD16 - and CD56 暗淡 CD16 + In some embodiments, a genetically modified non-human animal, e.g., a mouse, expressing both human SIRPα and human IL-15 as described herein exhibits a distribution of CD16+ NK cells relative to CD16- NK cells in the blood that is similar to the distribution of CD16+ NK cells relative to CD16- NK cells in PBMCs obtained from a human subject.

[0185] In some embodiments, after implantation with human hematopoietic cells, e.g., CD45+ cells, a genetically modified non-human animal, e.g., a mouse, as described herein, expressing both human SIRPα and human IL-15 includes NK cells exhibiting higher expression levels of CD16 and CD56 in the liver of the genetically modified non-human animal, e.g., a mouse, relative to a genetically modified non-human animal, e.g., a mouse, that expresses only human SIRPα, indicating that NK cell maturation is increased.

[0186] In some embodiments, a genetically modified non-human animal, such as a mouse, expressing both human SIRPα and human IL-15 as described herein and implanted with human hematopoietic cells, such as CD45+ cells, includes NK cells in the spleen that exhibit different expression levels of killing inhibitory receptors, wherein CD56 暗淡 CD16 + The NK cell population included a high percentage of CD158-expressing cells, similar to what is seen for NK cell subsets in human blood.

[0187] In some embodiments, relative to genetically modified non-human animals such as mice that express only human SIRPα, genetically modified non-human animals such as mice that express both human SIRPα and human IL-15 as described herein and are implanted with human hematopoietic cells, such as CD45+ cells, exhibit a higher frequency of human CD45+ and CD8+T cells in the intraepithelial lymphocyte population. In some embodiments, genetically modified non-human animals such as mice that express both human SIRPα and human IL-15 as described herein and are implanted with human hematopoietic cells exhibit a comparable distribution of CD16+NK cells relative to CD16-NK cells in IELs, and more CD16+NK cells are in the blood and spleen compared to CD16-NK cells, which reflects normal human physiology.

[0188] In some embodiments, a genetically modified non-human animal, such as a mouse, expressing both human SIRPα and human IL-15 as described herein and implanted with human hematopoietic cells, such as CD45+ cells, exhibits an increased number of human T cells in the lungs relative to a genetically modified non-human animal, such as a mouse, that expresses only human SIRPα. In some such embodiments, such a genetically modified non-human animal, such as a mouse, exhibits a higher level of CD69 expression on human CD8+T cells in the lungs relative to a genetically modified non-human animal, such as a mouse, that expresses only human SIRPα.

[0189] In some embodiments, a genetically modified non-human animal, e.g., mouse, expressing both human SIRPα and human IL-15 as described herein and implanted with human hematopoietic cells, e.g., CD45+ cells, exhibits increased levels of CD69 expression on human CD8+ T cells in the liver relative to a genetically modified non-human animal, e.g., mouse, expressing only human SIRPα.

[0190] In some embodiments, a genetically modified non-human animal, such as a mouse, expressing both human SIRPα and human IL-15 as described herein and engrafted with human hematopoietic cells exhibits discernible Peyer's patches that are predominantly human CD45+.

[0191] Any non-human mammal can be genetically modified according to the present disclosure. Non-limiting examples include laboratory animals, domestic animals, livestock, and the like, such as species such as murines, rodents, canines, felines, porcines, equines, bovines, ovines, non-human primates, and the like; such as mice, rats, rabbits, hamsters, guinea pigs, cattle, pigs, sheep, goats, and other transgenic animal species, particularly mammalian species, as known in the art. In other embodiments, the non-human animal may be, for example, a bird of the Galliformes order, such as a chicken, turkey, quail, pheasant, or partridge; such as a bird of the Anseriformes order, such as a duck, goose, or swan; such as a bird of the Columbiformes order, such as a pigeon or dove. In various embodiments, the subject genetically modified animal is a mouse, a rat, or a rabbit.

[0192] In some embodiments, the non-human animal is a mammal. In some such embodiments, the non-human animal is a small mammal such as 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 Muroidea. In one embodiment, the genetically modified animal is from a family selected from the group consisting of Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, white-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormouse), and Spalacidae (e.g., mole rats, bamboo rats, and zokors). In a specific embodiment, the genetically modified rodent is selected from true mice or rats (Muridae), gerbils, spiny mice, and crested rats.

[0193] In one embodiment, the genetically modified non-human animal of the subject is a rat. In one such embodiment, the rat is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, 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.

[0194] In another embodiment, the subject genetically modified non-human animal is a mouse, e.g., 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 129 strains of mice (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); BALB strains of mice such as BALB / c; etc. See, e.g., 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 mouse is a mixture of the above-mentioned strains.

[0195] In some embodiments, the subject genetically modified non-human animal is also immunodeficient. "Immunodeficiency" includes defects in one or more aspects of the natural or endogenous immune system of the animal, such as defects in one or more types of functional host immune cells, such as defects in non-human B cell number and / or function, non-human T cell number and / or function, non-human NK cell number and / or function, etc.

[0196] One method for achieving immunodeficiency in a subject animal is sublethal radiation. For example, newborn genetically modified mouse pups can be sublethally irradiated, for example, with 2x 200 cGy at 4 hour intervals. Alternatively, immunodeficiency can be achieved by any of a number of gene mutations known in the art, any of which can be bred alone or in combination into the subject genetically modified non-human animals of the present disclosure or can be used as a source of stem cells into which the genetic modifications 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(+) ; characterized by CD3δ or ε mutations with lymphocyte phenotype T(-)B(+)NK(+); characterized by RAG1 and RAG2 mutations with lymphocyte phenotype T(-)B(-)NK(+); characterized by Artemis gene mutations with lymphocyte phenotype T(-)B(-)NK(+), characterized by CD45 gene mutations with lymphocyte phenotype T(-)B(+)NK(+); and characterized by Prkdcscid mutations with lymphocyte phenotype T(-), B(-). Therefore, in some embodiments, the genetically modified immunodeficient non-human animal has one or more selected from IL2 receptor gamma chain (Il2rg y / - ) 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 will be known to the skilled artisan, any of which can be used to generate the immunodeficient animals of the present disclosure.

[0197] In some embodiments, the genetically modified non-human animal according to the present invention is suitable for use as a recipient of human hematopoietic cells, which are capable of producing human immune cells from the implanted human hematopoietic cells. Thus, in some aspects of the present invention, the subject genetically modified animal is a genetically modified immunodeficient non-human animal implanted with human hematopoietic cells.

[0198] Implantation of humanized SIRPα-IL-15 in non-human animals

[0199] As discussed above, in some aspects of the invention, humanized SIRPα-IL-15 is expressed in a non-human animal, such as a mouse, such as Rag2 - / - IL Y / -hSIRPαhIL-15 mice or sublethally irradiated hSIRPαhIL-15 mice, implanted or transplanted with cells. The cells may be mitotic cells or post-mitotic cells, and include cells of interest such as: pluripotent stem cells such as ES cells, iPS cells, and embryonic germ cells; and somatic cells such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, vascular endothelial cells, intestinal cells, etc., and their lineage-limited progenitor cells and precursors. Cell populations of particular interest include those including hematopoietic stem cells or progenitor cells that will contribute to or reconstruct the hematopoietic system of humanized SIRPα-IL-15 non-human animals, such as peripheral blood leukocytes, fetal liver cells, fetal bones, fetal thymus, fetal lymph nodes, vascularized skin, arterial segments, and purified hematopoietic stem cells such as mobilized HSC or umbilical cord blood HSC.

[0200] Any source of human hematopoietic cells, human hematopoietic stem cells (HSCs), and / or hematopoietic stem progenitor cells (HSPCs) known in the art or described herein can be transplanted into the genetically modified immunodeficient non-human animals of the present disclosure. One suitable source of human hematopoietic cells known in the art is human umbilical cord blood cells, particularly CD34 positive (CD34 + ) cells. Another source of human hematopoietic cells is human fetal liver. Another source is human bone marrow. Also contemplated are induced pluripotent stem cells (iPSCs) and induced hematopoietic stem cells (iHSCs), for example, produced by dedifferentiation of somatic cells by methods known in the art.

[0201] Cells can be from any mammalian species such as murines, rodents, canines, felines, equines, bovines, ovines, primates, humans, etc. Cells can be from defined cell lines, or they can be primary cells, wherein "primary cells", "primary cell lines" and "primary cultures" are used interchangeably herein to refer to cells and cell cultures that have been obtained from a subject and have been sustained in vitro for a limited number of culture passages (i.e., divisions). For example, a primary culture is a culture that has been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not enough to undergo a crisis phase. Typically, primary cell lines of the present invention are maintained for less than 10 generations in vitro.

[0202] If the cells are primary cells, they can be collected from an individual by any suitable method. For example, cells, such as blood cells, such as leukocytes, can be collected by apheresis, leukocytapheresis, density gradient separation, and the like. As another example, cells can be collected by biopsy, such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach tissue, and the like. An appropriate solution can be used to disperse or suspend the collected cells. The solution will typically be a balanced salt solution, such as standard saline, PBS, Hank's balanced salt solution, and the like, in combination with an acceptable buffer at a low concentration, typically 5-25 mM, and suitably supplemented with fetal bovine serum or other naturally occurring factors. Suitable buffers include HEPES, phosphate buffers, lactate buffers, and the like.

[0203] In some cases, a heterogeneous cell colony will be transplanted into a humanized non-human animal such as a mouse. In other cases, a cell colony enriched with a specific type of cell such as a progenitor cell such as a hematopoietic progenitor cell will be implanted into a humanized non-human animal such as a mouse. The target cell colony can be enriched by any suitable separation technique. For example, the target cell can be enriched by a culture method. In such a culture method, specific growth factors and nutrients are usually added to a culture medium that promotes a cell colony to survive and / or proliferate beyond other cell colonies. Other culture conditions that affect survival and / or proliferation include growth on an adherent or non-adhesive matrix, and culture is continued for a specific duration, etc. The culture conditions are well known in the art. As another example, target cells can be enriched by affinity separation techniques from the initial population separation of target cells. Affinity separation techniques can include using magnetic beads coated with affinity reagents for magnetic separation, affinity chromatography, "panning" with affinity reagents connected to a solid matrix such as a plate, using cytotoxic agents such as complement and cytotoxins that are joined to affinity reagents or used in conjunction with affinity reagents, or other suitable techniques. Technologies that provide accurate separation include fluorescence activated cell sorters, which may have varying degrees of sophistication, such as multicolor channels, low angle and obtuse angle light scatter detection channels, impedance channels, etc. Cells may be selected relative to dead cells by employing dyes associated with dead cells (e.g., propidium iodide). Any technique that is not overly detrimental to the viability of the target cells may be employed.

[0204] For example, using affinity separation technology, cells that are not target cells for transplantation can be eliminated from the population by contacting the population with an affinity reagent, and the nucleophilic reagent specifically recognizes and selectively binds to markers that are not expressed on the target cells. For example, for a population enriched in hematopoietic progenitor cells, cells expressing mature hematopoietic cell markers can be eliminated. Additionally or alternatively, positive selection and separation can be performed by contacting the population with an affinity reagent that specifically recognizes and selectively binds to markers associated with hematopoietic progenitor cells, such as CD34, CD133, etc. With respect to "selective binding", it means that the affinity of the molecule preferentially binds to the target or binds to the target is greater than the affinity of binding to other molecules. For example, an antibody will bind to a molecule including an epitope to which it has specificity, without binding to an irrelevant epitope. 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 can be a specific receptor or ligand for CD34, CD133, etc., such as a peptide ligand and receptor; effector and receptor molecules, T cell receptors specific for CD34, CD133, etc. In some embodiments, a variety of affinity reagents specific for a target marker can be used.

[0205] Antibodies and T cell receptors suitable for use as affinity reagents may be monoclonal or polyclonal and may be produced by transgenic animals, immunized animals, immortalized human or animal B cells, cells transfected with DNA vectors encoding antibodies or T cell receptors, and the like. The 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. Suitably, these antibodies are conjugated with labels for use in separation. Labels include magnetic beads that allow direct separation; biotin that can be removed with avidin or streptavidin bound to a carrier; fluorescent dyes that can be used with a fluorescence-activated cell sorter, etc. to facilitate the separation of specific cell types. Suitable fluorescent dyes include phycobiliproteins such as phycoerythrin and allophycocyanin, fluorescein, and Texas red. Each antibody is often labeled with a different fluorescent dye to allow independent sorting for each marker.

[0206] The initial cell population is contacted with the affinity reagent and incubated for a period sufficient to bind to the available cell surface antigen. The incubation will generally be at least about 5 minutes, and generally less than about 60 minutes. It may be desirable to have sufficient antibody concentration in the reaction mixture so that the separation efficiency is not limited by the lack of antibody. The appropriate concentration is determined by titration, but the dilution of the antibody in the cell suspension volume is generally about 1:50 (i.e., 1 part antibody to 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 will be any medium that maintains the viability of the cells. A preferred medium is phosphate buffered saline containing 0.1 to 0.5% BSA or 1-4% goat serum. Various media are commercially available and can be used according to the properties of the cells, including Dulbecco's Modified Eagle Medium (dMEM), Hank's basal salt solution (HBSS), Dulbecco's phosphate-buffered saline (dPBS), RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., which are often supplemented with fetal bovine serum, BSA, HSA, goat serum, etc.

[0207] Cells that become labeled by affinity reagents in the contacted population are selected by, for example, any suitable affinity separation technique as described above or as known in the art. After separation, the separated cells can be collected in any suitable medium that maintains the viability of the cells, typically with a serum cushion at the bottom of the collection tube. Various media are commercially available and can be used according to the properties of the cells, including dMEM, HBSS, dPBS, RPMI, Iskoff's medium, etc., often supplemented with fetal bovine serum.

[0208] In this manner, a composition highly enriched in target cell types, such as hematopoietic cells, is obtained. The cells will comprise 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 will preferably comprise about 95% or more of the enriched cell composition. In other words, the composition will be a substantially pure composition of target cells.

[0209] The cells to be transplanted into humanized SIRPα-IL-15 non-human animals such as mice can be transplanted immediately regardless of whether they are heterogeneous cell populations or enriched cell populations. Alternatively, the cells can be frozen at liquid nitrogen temperature and stored for a long period of time, thawed and able to be used again. In such cases, the cells will usually be frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at the freezing temperature, and thawed in a manner as is commonly known in the art for thawing frozen cultured cells. Additionally or alternatively, cells can be cultured in vitro under various culture conditions. The culture medium may be liquid or semisolid, for example containing agar, methylcellulose, and the like. The cell population can be suitably suspended in an appropriate nutrient medium such as Iskoff's modified DMEM or RPMI-1640, usually supplemented with 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 are responsive. Growth factors as defined herein are molecules capable of promoting the survival, growth and / or differentiation of cells in culture or in intact tissues through specific actions on transmembrane receptors.Growth factors include polypeptide and non-polypeptide factors.

[0210] Cells can be genetically modified, for example, to provide selectable or traceable markers before being transplanted into SIRPα-IL-15 non-human animals such as mice, to induce genetic defects in cells (e.g., for disease modeling), to repair genetic defects or ectopically express genes in cells (e.g., to determine whether the modification will affect the course of the disease), etc. Cells can be genetically modified by transfection or transduction with a suitable vector, homologous recombination, or other appropriate techniques so that they express the target gene, or genetically modified with antisense mRNA, siRNA or ribozymes to block the expression of non-desired genes. Various techniques for introducing nucleic acids into target cells are known in the art. Various techniques can be used to prove that cells have been genetically modified. The genome of the cell can be defined and used with or without amplification. Polymerase chain reaction; gel electrophoresis; restriction analysis; Southern, Northern and Western blotting; sequencing, etc. can all be used. General methods in molecular and cellular biochemistry for these and other purposes disclosed in this application can be found in standard textbooks such as the following: Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., Cold Spring Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition (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 and Loewy, eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle and Griffiths, John Wiley & Sons 1996). 1998), the disclosure of which is incorporated herein by reference. The reagents, cloning vectors and kits used for genetic manipulations mentioned in this disclosure can be obtained from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich and ClonTech.

[0211] The cells can be transplanted into humanized SIRPα-IL-15 non-human animals such as mice by any suitable method including, for example, intrahepatic injection, tail vein injection, retro-orbital injection, etc. Typically, about 0.5 x 10 5 -2x 10 6 pluripotent cells or progenitor cells, for example about 1 x 10 5 -1x 10 6 cells or about 2 x 10 5 -5x 10 5 Cells. In some cases, non-human animals such as mice are sublethally irradiated before transplanting human cells. In other words, non-human animals such as mice are exposed to sublethal doses of radiation, such as those well known in the art. Then, non-human animals such as mice implanted with humanized SIRPα-IL-15 are maintained under laboratory animal husbandry conditions for at least 1 week, such as 1 week or longer, or 2 weeks or longer, sometimes 4 weeks or longer, and in some cases maintained for 6 weeks or longer, such as 10 weeks or longer or 15 weeks or longer, to allow the implanted cells to fully reconstruct the immune system.

[0212] Humanized SIRPα-IL-15 non-human animals such as mice and humanized SIRPα-IL-15 non-human animals such as mice implanted with human hematopoietic cells and optionally with other genetic modifications, such as implanted with Rag2 - / - IL Y / - hSIRPαhIL-15 mice are suitable for many applications. For example, these non-human animals, such as mice, provide systems suitable for modeling human immune diseases and human pathogens. For example, the subject non-human animals, such as mice, are suitable for modeling, for example, human T cell and / or natural killer (NK) cell development and function; human pathogen infection of specific tissues and / or cells, such as human pathogen infection of the intestine or lung and / or human pathogen infection of human T cells and / or NK cells, or human pathogens to human T cells and / or NK cells. The non-human animals are also suitable for in vivo screening of agents that inhibit infection by pathogens, such as pathogens that affect (e.g., by infection) specific tissues or cell types, such as human pathogens of the intestine or lung, such as human pathogens that activate, induce and / or target T cells and / or NK cells; in vivo screening of agents that regulate the development and / or function of human T cells and / or NK cells, such as in a healthy or diseased state; in vivo screening of agents that are toxic to human T cells and / or NK cells; in vivo screening of agents that prevent, mitigate or reverse the toxic effects of toxic agents on human T cells and / or NK cells; in vivo screening of candidate T cell inducing vaccines; and in vivo and in vitro screening of agents that inhibit tumor growth and / or infection by activating NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) processes.

[0213] The present disclosure provides unexpected results demonstrating that humanized SIRPα-IL-15 in non-human animals, such as mice, implanted with human hematopoietic cells, such as Rag2 - / - IL Y / - hSIRPαhIL-15 mice produce tissue-resident lymphocytes, such as intraepithelial lymphocytes, in the intestine and lungs. Therefore, the present disclosure provides previously unavailable animal models that enable monitoring and testing of such tissue-resident lymphocytes. Such animal models are particularly useful for modeling the immune response of tissue-resident lymphocytes, such as T cells and NK cells, to human pathogens that affect (e.g., by infection) the intestine and / or lungs, and for screening therapeutic agents and vaccines that target such pathogens and / or induce or improve tissue-resident lymphocyte responses. In addition, the presence of these tissue-resident lymphocytes also allows modeling of human immune cell-driven autoimmune diseases such as celiac disease and IBD that affect the gastrointestinal tract.

[0214] Therefore, in some embodiments, the present disclosure provides an in vivo model, comprising a genetically modified non-human animal, the genetically modified non-human animal comprising a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter. The genetically modified non-human animal also comprises a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter. Finally, the genetically modified non-human animal comprises an implant of artificial hematopoietic cells, wherein the genetically modified non-human animal (i) expresses human SIRPα protein and human IL-15 protein, and (ii) comprises human tissue-resident lymphocytes such as intraepithelial lymphocytes (IEL) in the intestine of the genetically modified non-human. In some such embodiments, the genetically modified non-human animal is infected with a human pathogen, such as a human pathogen that affects (e.g., by infection) the intestine.

[0215] Human pathogens that can affect (e.g., by infection) the intestine include, but are not limited to, 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.

[0216] In other embodiments, the present disclosure provides an in vivo model, including a genetically modified non-human animal, the genetically modified non-human animal including a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding human SIRPα protein and operably linked to the SIRPα gene promoter. The genetically modified non-human animal also includes a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding human IL-15 protein and operably linked to the IL-15 gene promoter. Finally, the genetically modified non-human animal includes an implant of artificial hematopoietic cells, wherein the genetically modified non-human animal (i) expresses human SIRPα protein and human IL-15 protein, and (ii) includes human tissue-resident lymphocytes such as intraepithelial lymphocytes (IEL) in the genetically modified non-human lungs. In some such embodiments, the genetically modified non-human animal is infected with a human pathogen, such as a human pathogen that affects (e.g., by infection) the lungs.

[0217] Human pathogens that can affect the lung (e.g., by infection) include, but are not limited to, Streptococcus pyogenes, Haemophilus influenzae, Corynebacterium diphtheriae, SARS coronavirus, Bordetella pertussis, Moraxella catarrhalis, influenza viruses (A, B, C), coronaviruses, adenoviruses, respiratory syncytial virus, parainfluenza viruses, mumps viruses, Streptococcus pneumoniae, Staphylococcus aureus, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Blastomyces dermatitidis, Cryptococcus neoformans, and Aspergillus fumigatus.

[0218] New therapeutic agents, new vaccines and new ways to test the efficacy of therapeutic agents and vaccines are needed. Non-human animals such as mice that support efficient human T cell and NK cell implantation will be suitable for identifying new therapeutic agents and new vaccines specifically for human pathogens that infect human T cells and / or NK cells. New therapeutic agents and new vaccines can be tested in such non-human animals such as mice in the following ways: for example, the amount of human pathogens such as viruses obtained in response to treatment with putative antiviral agents (in the blood or a given tissue) in the non-human animal is determined, or the mice are inoculated with a putative vaccine, followed by exposure to infectiously administered human pathogens such as HIV, and any infectivity changes attributed to vaccination with the putative vaccine are observed compared to controls that are not vaccinated with the vaccine but infected with HIV.

[0219] The non-human animal (e.g., mouse) model of pathogen infection is useful in research, e.g., to more fully understand the progression of human infection. The mouse model of infection is also useful in drug discovery, e.g., to identify candidate agents that protect against infection or treat infection.

[0220] The disclosed implanted genetically modified animals are suitable for screening candidate agents to identify those that will treat infections by human pathogens, such as human pathogens targeting human T cells and / or NK cells. The term "treat / treatment / treating" and the like are used herein to generally include obtaining desired pharmacological and / or physiological effects. The effect may be preventive and therapeutic in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects attributable to a disease. "Treatment" as used herein includes any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., curbing its development; or (c) alleviating the disease, i.e., causing the regression of the disease.

[0221] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and include any mammalian subject, particularly humans, in need of diagnosis, treatment, or therapy.

[0222] Humanized SIRPα-IL-15 non-human animals, such as mice, implanted with human hematopoietic cells provide a system that is also suitable for screening candidate agents for other desired activities in vivo, such as screening for agents that can modulate (i.e., promote or inhibit) the development and / or activity of human T cells and NK cells, for example in a healthy or diseased state, for example to identify novel therapeutic agents and / or generate a fuller understanding of the molecular basis of the development and function of the immune system; agents that are toxic to T cells and / or NK cells and their progenitors; and agents that prevent, mitigate or reverse the toxic effects of toxic agents on T cells, NK cells and their progenitors; antibodies or antigen binding proteins that mediate NK cell-dependent ADCC processes, etc. As another example, the genetically modified mice described herein provide a system suitable for predicting an individual's responsiveness to disease therapy, for example, by providing an in vivo platform for screening an individual's immune system's responsiveness to an agent, such as a therapeutic agent, to predict the individual's responsiveness to that agent.

[0223] In a screening assay for a bioactive agent, a humanized SIRPα-IL-15 non-human animal, such as a mouse, that has been implanted with human hematopoietic cells and, in some cases, infected with a human pathogen or cells to be implanted in the humanized SIRPα-IL-15 non-human animal, such as a mouse, is implanted with, for example, Rag2 - / - IL Y / -The hSIRPαhIL-15 mouse is contacted with a candidate target agent, and the effect of the candidate agent is evaluated by monitoring one or more output parameters. These output parameters can reflect the viability of the cell, such as the total number of hematopoietic cells or the number of cells of a specific hematopoietic cell type, or reflect the apoptotic state of the cell, such as the amount of DNA fragmentation, the amount of cell blistering, the amount of phosphatidylserine on the cell surface, etc., by methods well known in the art. Alternatively or in addition, the output parameter can reflect the differentiation ability of the cell, such as the ratio of differentiated cells and differentiated cell types such as T cells and / or NK cells. Alternatively or in addition, the output parameter can reflect the function of the cell, such as cytokines and chemokines produced by the cell, the ability of the cell to home to and extravasate to the site of excitation, the ability of the cell to regulate (i.e., promote or inhibit) the activity of other cells in vitro or in vivo, etc. Other output parameters can reflect the extent of pathogen infection in animals, such as the titer of pathogens in non-human animals such as mice, etc.

[0224] Parameter is the quantifiable component of cell, particularly the component that can be accurately measured in high-throughput system desirably.Parameter can be any cell component or cell product, including cell surface determinant, receptor, protein or its conformation or post-translational modification, lipid, carbohydrate, organic or inorganic molecule, nucleic acid such as mRNA, DNA etc. or the part or combination derived from this cell component.Although most parameters will provide quantitative reading, in some cases, semi-quantitative or qualitative result will be acceptable.Reading can comprise single measured value, or can comprise mean value, median or variance etc.As feature, parameter reading value of a certain range of each parameter will be obtained by many identical determinations.Expectation has variability, and standard statistical method will be used to obtain the value of a certain range of each in a group of test parameters, wherein conventional statistical method is used to provide single value.

[0225] Candidate target agents for screening include known and unknown compounds covering a wide range of chemical classes, mainly organic molecules that may include organometallic molecules, inorganic molecules, gene sequences, vaccines, antibiotics or other agents suspected of having antibiotic properties, peptides, polypeptides, antibodies, antigen binding proteins, agents that have been approved for drug use in humans, etc. An important aspect of the present invention is to evaluate candidate drugs, including toxicity testing; etc.

[0226] Candidate agents include organic molecules, which include functional groups necessary for structural interactions, particularly hydrogen bonding, and generally include at least amine, carbonyl, hydroxyl or carboxyl, often including at least two functional chemical groups. Candidate agents often include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted by one or more functional groups. Candidate agents are also found in biomolecules, including peptides, polynucleotides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Including pharmacologically active drugs, genetically active molecules, etc. Target compounds include chemotherapeutic agents, hormones or hormone antagonists, etc. Examples of pharmaceutical preparations suitable for the present invention are those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), 9th edition. Also included are toxins and biological and chemical warfare agents; see, e.g., Somani, SM (ed.), "Chemical Warfare Agents," Academic Press, New York, 1992.

[0227] Candidate target agents for screening also include nucleic acids, such as nucleic acids encoding siRNA, shRNA, antisense molecules or miRNA or nucleic acids encoding polypeptides. Many vectors suitable for transferring nucleic acids into target cells are available. The vector can be maintained in a free body form, such as in the form of plasmids, small ring DNA, virus-derived vectors such as cytomegalovirus, adenovirus, or they can be integrated into the target cell genome by homologous recombination or random integration, such as retrovirus-derived vectors such as MMLV, HIV-1, ALV, etc. The vector can be directly provided to the subject cell. In other words, pluripotent cells are contacted with a vector comprising the target nucleic acid so that the vector is taken up by the cell.

[0228] Methods for contacting cells, such as cultured cells or cells in non-human animals, such as mice, with nucleic acid vectors are well known in the art, such as electroporation, calcium chloride transfection, and liposome transfection. Alternatively, the target nucleic acid can be provided to the cell by a virus. In other words, the cell is contacted with a viral particle comprising the target nucleic acid. Retroviruses, such as lentiviruses, are particularly suitable for the method of the present invention. Commonly used retroviral vectors are "defective", i.e., they cannot produce the viral proteins required for proliferative infection. More specifically, the replication of the vector requires growth in a packaging cell line. To produce viral particles comprising the target nucleic acid, the retroviral nucleic acid comprising the nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins to be incorporated into the capsid, which determine the specificity of the viral particles to the cell. There are at least three types of envelope proteins, i.e., ecotropic, amphophilic, and heterotropic. Retroviruses packaged with ecotropic envelope proteins, such as MMLV, are capable of infecting most mouse and rat cell types and are produced using ecotropic packaging cell lines such as BOSC23 (Pear et al. (1993) PNAS 90:8392-8396). Retroviruses carrying amphotropic envelope proteins, such as 4070A (Danos et al., supra), are capable of infecting most mammalian cell types, including humans, dogs, and mice, and are produced using amphotropic 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); GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with heterotropic envelope proteins, such as AKR env, are capable of infecting most mammalian cell types except mouse cells. Appropriate packaging cell lines can be used to ensure that target cells (in some cases, implanted cells, in some cases, cells of the host, ie, humanized SIRPα-IL-15 host) are targeted by the packaged viral particles.

[0229] The vector for providing the target nucleic acid to the subject cell will generally include a promoter suitable for driving the expression (i.e., transcriptional activation) of the target nucleic acid. This may include ubiquitous promoters such as CMV-b-actin promoters, or inducible promoters such as promoters that are active in a specific cell population or respond to the presence of drugs such as tetracycline. Through transcriptional activation, transcription in the intended target cell will increase by at least about 10 times, at least about 100 times, more generally at least about 1000 times above the basal level. In addition, the vector for providing the subject cell with a reprogramming factor may include genes that must be subsequently removed, for example, using a recombinase system such as Cre / Lox, or cells expressing them, for example, by including genes such as herpes virus TK, bcl-xs, etc. that allow selective toxicity to be achieved to destroy.

[0230] Candidate target agents for screening also include polypeptides. Such polypeptides may be optionally fused to a polypeptide domain that increases the solubility of the product. The domain may be connected to the polypeptide through a determined protease cleavage site, such as a TEV sequence cleaved by a TEV protease. The linker may also include one or more flexible sequences, such as 1 to 10 glycine residues. In some embodiments, the fusion protein is cleaved in a buffer that maintains the solubility of the product, such as in the presence of 0.5 to 2M urea, in the presence of a polypeptide and / or polynucleotide that increases solubility, and under similar conditions. The target domain includes an endosomal solubility domain such as an influenza HA domain; and other polypeptides that contribute to the production, such as an IF2 domain, a GST domain, a GRPE domain, etc. Additionally or alternatively, the polypeptide may be formulated to achieve improved stability. For example, a peptide may be pegylated, wherein the polyethylene oxide provides an increased life span in the bloodstream. A polypeptide may be fused to another polypeptide to provide additional functionality, such as to increase in vivo stability. Typically, such fusion partners are stable plasma proteins, which can, for example, prolong the in vivo plasma half-life of a polypeptide when present in the form of a fusion, particularly where such a stable plasma protein is an immunoglobulin constant domain. In most cases, when a stable plasma protein is typically present in a polymeric form such as an immunoglobulin or lipoprotein in which the same or different polypeptide chains are typically disulfide bonded and / or non-covalently bound to form an assembled multi-chain polypeptide, the fusions herein containing the polypeptide will also be produced and employed in a polymeric form having substantially the same structure as the stable plasma protein precursor. These polymers will be homogeneous with respect to the polypeptide agents they include, or they may contain more than one polypeptide agent.

[0231] The candidate polypeptide agent may be produced by eukaryotic cells, or may be produced by prokaryotic cells. It may be further processed by unfolding, such as heat denaturation, DTT reduction, etc., and may be further refolded using methods known in the art. Target modifications that do not change the primary sequence include chemical derivatization of the polypeptide, such as acylation, acetylation, carboxylation, amidation, etc. Glycosylation modifications are also included, such as those achieved by changing its glycosylation pattern during polypeptide synthesis and processing or in other processing steps; for example, those achieved by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylases or deglycosylases. Sequences with phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, or phosphothreonine are also included. Polypeptides may have been modified using common molecular biology techniques and synthetic chemistry to improve their resistance to proteolytic degradation, or to optimize solubility properties, or to render them more suitable as therapeutic agents. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may replace some or all of the amino acid residues.

[0232] Candidate polypeptide agents can be prepared by in vitro synthesis using conventional methods as known in the art. Various commercial synthesis devices are available, such as automatic synthesizers of Applied Biosystems, Inc., Beckman, etc. By using a synthesizer, naturally occurring amino acids can be replaced with non-natural amino acids. The specific sequence and preparation method will be determined by convenience, economic factors, required purity, etc. Alternatively, candidate polypeptide agents can be separated and purified according to conventional recombinant synthesis methods. Lysates of the expression host can be prepared, and the lysates are purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography or other purification techniques. To a large extent, the composition used will include at least 20% by weight of the desired product, more generally at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, generally at least about 99.5% by weight. Typically, the percentage will be based on total protein.

[0233] In some cases, the candidate polypeptide agent to be screened is an antibody or antigen binding protein. The term "antibody" or "antibody portion" is intended to include any molecular structure containing a polypeptide chain with a specific shape that matches and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The specificity or selectivity of a given structure and its specific epitope is sometimes referred to as "lock and key" coordination. Prototype antibody molecules are immunoglobulins, and all types of immunoglobulins (IgG, IgM, IgA, IgE, IgD, etc.) from all sources such as humans, rodents, rabbits, cows, sheep, pigs, dogs, other mammals, chickens, other birds, etc. are considered "antibodies". The antibody used in the present invention can be a polyclonal antibody or a monoclonal antibody. Antibodies are generally provided in a culture medium in which cells are cultured. In addition to antibodies, antigen binding proteins encompass polypeptides that are also designed to bind to target antigens and trigger responses such as immune responses. Antigen binding fragments known in the art (including, for example, Fab, Fab', F(ab')2, Fabc and scFv) are also encompassed by the term "antigen binding protein". The terms "antibody" and "antigen binding protein" also include one or more immunoglobulin chains or fragments, single-chain antibodies and bispecific antibodies that can be chemically conjugated to other proteins or expressed as fusion proteins with other proteins.

[0234] Candidate agents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means can be used for random and directed synthesis of a wide variety of organic compounds including biomolecules, including expressing randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or easy to produce. In addition, natural or synthetically produced libraries and compounds are easy to modify by conventional chemical, physical and biochemical means, and can be used to produce combinatorial libraries. Known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc. to produce structural analogs.

[0235] Candidate agents are screened for biological activity by administering an agent to at least one and usually multiple samples, sometimes in combination with samples lacking the agent. Changes in parameters in response to the agent are measured, and the results are evaluated by comparison with reference cultures, such as the presence and absence of agents, obtained with other agents, etc. In the case where screening is performed to identify candidate agents that will prevent, mitigate or reverse the effects of toxic agents, the screening is usually performed in the presence of the toxic agent, wherein the toxic agent is added at a time that is most suitable for the results to be determined. For example, in the case of testing the protective / preventive ability of a candidate agent, the candidate agent may be added before the toxic agent, at the same time as the candidate agent, or after treatment with the candidate agent. As another example, in the case of testing the ability of a candidate agent to reverse the effects of a toxic agent, the candidate agent may be added after treatment with the candidate agent. As mentioned above, in some cases, the sample is a humanized SIRPα-IL-15 non-human animal, such as a mouse, that has been implanted with cells, i.e., a candidate agent is provided to a humanized SIRPα-IL-15 non-human animal, such as a mouse, that has been implanted with cells. In some cases, the sample is cells to be implanted, ie, the candidate agent is provided to the cells prior to transplantation.

[0236] If the candidate agent is to be administered directly to a non-human animal such as a mouse, the agent can be administered by any of the many well-known methods used in the art for administering peptides, small molecules, and nucleic acids. For example, the agent can be administered orally, transmucosally, topically, intradermally, or by injection (e.g., intraperitoneally, subcutaneously, intramuscularly, intravenously, or intracranially). The agent can be administered in a buffer, or it can be incorporated into any of a variety of preparations, for example, by combining with an appropriate pharmaceutically acceptable vehicle. A "pharmaceutically acceptable vehicle" can be a vehicle approved by a federal or state government regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in mammals such as humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which the compound of the present invention is formulated for administration to a mammal. Such pharmaceutical vehicles may be lipids, such as liposomes, such as liposomal dendrimers; liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., saline; gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants may be used. The pharmaceutical composition may be formulated into preparations in solid, semisolid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. The agent may be systemic after administration, or may be localized by using regional administration, intramural administration, or using implants that serve to maintain the active dose at the site of implantation. The active agent may be formulated to achieve immediate activity, or it may be formulated to achieve sustained release. For some conditions, particularly central nervous system conditions, it may be necessary to formulate agents that cross the blood-brain barrier (BBB). A strategy for delivering drugs across the blood-brain barrier (BBB) ​​requires biochemical destruction of the BBB by osmotic means such as mannitol or leukotrienes, or by using vasoactive substances such as bradykinin. BBB disruptors can be co-administered with agents when the composition is administered by intravascular injection. Other strategies for crossing the BBB may require the use of endogenous transport systems, including caveolin-1-mediated endocytosis, carrier-mediated transporters such as glucose and amino acid carriers, receptor-mediated insulin or transferrin endocytosis, and active efflux transporters such as p-glycoprotein. Active transport moieties may also be conjugated to therapeutic compounds for use in the present invention to promote transport across the endothelial wall of a blood vessel.Alternatively, drug delivery of an agent to reach behind the BBB can be by local delivery, such as by intrathecal delivery, such as through an Ommaya reservoir (see, e.g., U.S. Pat. Nos. 5,222,982 and 5,385,582, incorporated herein by reference); by bolus injection, such as by syringe, such as intravitreally or intracranially; by continuous infusion, such as by cannulation, such as using convection (see, e.g., U.S. App. No. 20070254842, incorporated herein by reference); or by implantation of a device to which the agent has been reversibly affixed (see, e.g., U.S. App. Nos. 20080081064 and 20090196903, incorporated herein by reference).

[0237] If the agent is provided to the cells before transplantation, the agent is suitably added to the culture medium of the cultured cells in a solution or in a readily soluble form. The agent can be added in a circulation system in the form of intermittent or continuous liquid flow, or, a single or incremental addition of a compound mass to an otherwise static solution. In the circulation system, two fluids are used, one of which is a physiologically neutral solution, and the other is the same solution to which the test compound is added. The first fluid and the second fluid are passed through the cells in sequence. In a single solution method, a mass of the test compound is added to the volume of the culture medium surrounding the cells. The total concentration of the components of the culture medium should not change significantly with the addition of a mass, or between the two solutions in a circulation method.

[0238] Multiple assays can be run in parallel with different agent concentrations to obtain differential responses to various concentrations. As known in the art, the effective concentration of an agent is determined using a range of concentrations typically produced by 1:10 or other logarithmic scale dilutions. If necessary, the concentration can be further refined with a second series of diluents. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration, or below the detection level of the agent, or at or below the concentration of the agent that does not produce a detectable phenotypic change.

[0239] The response of cells in humanized SIRPα-IL-15 non-human animals such as mice to candidate agents can be analyzed at any time after treatment with the agent. For example, cells can be analyzed 1, 2 or 3 days, sometimes 4, 5 or 6 days, sometimes 8, 9 or 10 days, sometimes 14 days, sometimes 21 days, sometimes 28 days, sometimes 1 month or longer, such as 2 months, 4 months, 6 months or longer after contact with the candidate agent. In some embodiments, the analysis includes analysis at multiple time points. The selection of the analysis time point will be based on the type of analysis to be performed, as will be readily understood by ordinary technicians.

[0240] Analysis may include measuring any parameter described herein or known in the art for measuring cell viability, cell proliferation, cell identity, cell morphology and cell function, particularly when they may involve cells of the immune system such as T cells and / or NK cells. For example, flow cytometry can be used to determine the total number of hematopoietic cells or the number of cells of a specific hematopoietic cell type. Histochemical or immunohistochemical analysis can be performed to determine the apoptotic state of the cells, such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for measuring DNA fragmentation, or immunohistochemical analysis for detecting the binding of annexin V to phosphatidylserine on the cell surface. Flow cytometry can also be used to assess the ratio of differentiated cells and differentiated cell types, such as to determine the ability of hematopoietic cells to differentiate in the presence of an agent. ELISA, Western and Northern blotting can be performed to determine the levels of cytokines, chemokines, immunoglobulins, etc. expressed in non-human animals such as mice implanted with humanized SIRPα-IL-15, for example, to assess the function of implanted cells. In vivo assays to test the function of immune cells can also be performed, as well as assays related to specific target diseases or conditions such as diabetes, autoimmune diseases, graft-versus-host disease, AMD, etc. 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.

[0241] Thus, for example, a method for determining the effect of an agent on a human pathogen is provided, comprising subjecting a non-human animal, such as a mouse, implanted with humanized SIRPα-IL-15, such as a mouse, implanted with Rag 2- / - IL Y / - hSIRPαhIL-15 mice are exposed to an effective amount of a human pathogen, the effective amount of the pathogen being the amount of the pathogen required to produce an infection in the mouse; the pathogen is allowed to infect the mouse; a parameter of the infection is measured over time in the presence of the agent; and that measurement is compared to a measurement obtained by a non-human animal, such as a mouse, that has been implanted with humanized SIRPα-IL-15 and has not been exposed to the agent. An agent is determined to be an anti-pathogenic agent if the agent reduces the amount of the agent in the blood or tissue of the non-human animal, such as a mouse, by at least half after a single administration or two or more administrations over a selected period of time.

[0242] As another example, a method for determining whether a target pathogen isolate or strain has drug resistance, such as multi-drug resistance, is provided. In these methods, a non-human animal, such as a mouse, implanted with humanized SIRPα-IL-15, such as a mouse, implanted with Rag2 - / - IL Y / - hSIRPαhIL-15 mice are exposed to an effective amount of a target human pathogen isolate or strain, wherein the effective amount of the pathogen is the amount of the pathogen required to produce an infection in the non-human animal, such as a mouse; the pathogen is allowed to infect the non-human animal; a parameter of the infection is measured in the presence of the drug at a certain time point after administration of the drug, such as the titer of the target isolate or strain in the blood or tissue of the non-human animal, the ability of the target isolate or strain to maintain infection in the non-human animal, or the ability of the target isolate or strain to reproduce in the non-human animal; and that measurement result is compared with the measurement result obtained from an implanted humanized SIRPα-IL-15 non-human animal, such as a mouse, that was infected with the pathogen but not exposed to the agent. Examples of drugs of interest include amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime, chloramphenicol, ciprofloxacin, co-trimoxazole, ertapenem, imipenem, fluoroquinolone (e.g., ciprofloxacin, gatifloxacin, ofloxacin), streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, and combinations thereof. In a specific embodiment, administration of the drug or combination of drugs is at least 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks after infection-producing exposure to the isolate or strain of interest.

[0243] In addition, humanized SIRPα-IL-15 non-human animals (eg, mice) and humanized SIRPα-IL-15 non-human animals (eg, mice) implanted with human hematopoietic cells and optionally with other genetic modifications, such as implanted Rag2 - / - IL Y / -hSIRPαhIL-15 mice are suitable for studying antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells (e.g., human NK cells). Such animals are also suitable for testing therapeutic drug candidates designed to target various cells (e.g., tumors or infected cells) or infections, such as antigen binding proteins or antibodies, to activate the ability of NK cell pathways involved in killing such cells or infections.

[0244] It is widely known that one mechanism behind monoclonal antibody therapy is that it activates NK cells by binding to the NK cell Fc receptor CD16 (Fcγ receptor IIIA). Attempts have been made to increase the affinity of various known monoclonal candidates (e.g., rituximab) for FcγRIIIA to improve ADCC (e.g., Bowles et al. Blood 2006; 108: 2648-2654; Garff-Tavernier et al. Leukemia 2011; 25: 202-209). As demonstrated herein, humanized SIRPα-IL-15 generates human NK cells capable of mediating ADCC upon implantation in non-human animals; therefore, these animals present an in vivo model suitable for studying the mechanism of ADCC and for screening various therapeutic candidates.

[0245] Therefore, non-human animals implanted with humanized SIRPα-IL-15 and cells isolated therefrom, such as human NK cells, can be used in screening methods designed to identify agents that improve the antibody-dependent cellular cytotoxicity (ADCC) activity of cell types or cells, such as human NK cells, implanted in humanized non-human animals. For example, a suitable method may include administering an agent to a non-human animal implanted with humanized SIRPα-IL-15, and determining the in vivo effect of the agent on the antibody-dependent cellular cytotoxicity (ADCC) activity of cell types implanted in humanized non-human animals. In one embodiment, the effect results in, for example, improved tumor killing of transplanted tumors, such as human tumors. In another embodiment, the effect results in improved killing of infected cells, such as virus-infected cells or bacteria-infected cells. In another embodiment, the effect results in improved killing of bacteria, fungi or parasites. In various embodiments, the agent is an antibody or an antigen-binding protein. In some embodiments, antibodies or antigen-binding proteins are designed to target antigens expressed on human tumor cells. In some embodiments, antibodies or antigen binding proteins are designed to target antigens expressed on virally infected cells or bacterially infected cells. In some embodiments, antibodies or antigen binding proteins are designed to target bacterial, fungal or parasitic antigens. In some embodiments, an in vitro method is provided, in which human cells such as human NK cells are separated from non-human animals implanted with humanized SIRPα-IL-15, and contacted with agents such as antibodies or antigen binding proteins and target cells (e.g., tumor cells) in vitro to determine the efficacy of the agent in mediating the killing of the target cells. The effect of the agent on the cytolytic activity of human cells such as human NK cells can then be determined.

[0246] Other examples of uses of the subject mice are provided elsewhere herein.Additional uses of the genetically modified and implanted mice described in this disclosure will be apparent to those of skill in the art upon reading this disclosure.

[0247] Methods for preparing subject genetically modified non-human animals

[0248] In some aspects of the present invention, methods for preparing the subject non-human animals of the present disclosure are provided. When implementing the subject methods, a non-human animal is produced, which includes a nucleic acid sequence incorporated into the genome of a genetically modified non-human animal, the sequence encoding a human SIRPα protein and 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, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter, such as an endogenous non-human IL-15 gene promoter.

[0249] The production of a non-human animal comprising a nucleic acid sequence encoding a human SIRPα protein and operably linked to a SIRPα promoter, and / or a nucleic acid sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter can be achieved using any suitable method for preparing a genetically modified animal, such as known in the art or as described herein.

[0250] For example, a nucleic acid encoding a human SIRPα protein or a human IL-15 protein can be incorporated into a recombinant vector, which is in a form suitable for insertion into the genome of a host cell and expressing a human protein in a non-human host cell. In various embodiments, the recombinant vector includes one or more regulatory sequences as described above, which are operably connected to the nucleic acid encoding the human protein in a manner that allows the nucleic acid to be transcribed into mRNA and the mRNA to be translated into a human protein. It should 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.

[0251] Any of a variety of methods can then be used to introduce human nucleic acid sequences into animal cells to produce genetically modified animals expressing 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 producing genetically modified animals that can be used include, but are not limited to, 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 2nd Edition Sunderland, Massachusetts: Sinauer), U.S. Pat. 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).

[0252] For example, nucleic acid encoding human protein can be introduced into oocytes, and the oocytes are grown in female foster animals to produce the genetically modified animals of the subject, for example, by microinjection. In a preferred embodiment, nucleic acid is injected into fertilized oocytes. Fertilized oocytes can be collected from superovulated females on the second day of mating, and expression constructs are injected. Injected oocytes are cultured overnight or directly transferred to the oviduct of 0.5 day pseudopregnant females after sexual intercourse (pc). Methods for superovulation, collection of oocytes, expression construct injections and embryo transfer are known in the art, and are described in Manipulating the Mouse Embryo (2002, A Laboratory Manual, the 3rd edition, Cold Spring Harbor Laboratory Press). The presence of nucleic acid introduced in offspring can be assessed by DNA analysis (such as PCR, Southern blotting, DNA sequencing, etc.) or by protein analysis (such as ELISA, Western blotting, etc.).

[0253] As another example, well-known methods such as electroporation, calcium phosphate precipitation, liposome transfection, etc. can be used to transfect a construct including a nucleic acid sequence encoding a human protein into a stem cell (e.g., ES cell or iPS cell). The presence of the nucleic acid introduced in the cell can be assessed by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blotting, etc.). The cell determined to have an expression construct can then be introduced into the preimplantation embryo. For a detailed description of the method for the composition and method of the present invention known in the art, 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).

[0254] In a preferred embodiment, a method of producing a genetically modified animal as described herein utilizes the use of The targeting construct prepared by the technique, the construct introduced into ES cells, and the The targeted ES cell clones were introduced into mouse embryos using the technique as described in the Examples.

[0255] The genetically modified founder animal can be mated with an additional animal carrying the genetic modification. For example, a humanized SIRPα non-human animal can be mated with a humanized IL-15 non-human animal of the same species to produce the hSIRPα-hIL-15 non-human animal described herein. A genetically modified animal carrying a nucleic acid encoding a human protein of the present disclosure can be further mated with a knockout animal, which is, for example, a non-human animal that is defective in one or more proteins, such as one or more genes that do not express it, such as a Rag2-deficient animal and / or an Il2rg-deficient animal.

[0256] As discussed above, in some embodiments, the subject genetically modified non-human animal is an immunodeficient animal. Any suitable method for producing genetically modified animals, such as those known in the art or as described herein, can be used to produce genetically modified non-human animals with immunodeficiency and including one or more human proteins, such as hSIRPα and / or hIL-15. For example, the production of genetically modified immunodeficient animals can be achieved by introducing nucleic acids encoding human proteins into oocytes or stem cells including mutant SCID gene alleles that will cause immunodeficiency when homozygous, as described in more detail above and in the working examples herein. Then, using methods such as those described herein and known in the art, mice are produced with modified oocytes or ES cells, and mated to produce immunodeficient mice including the desired genetic modifications. As another example, genetically modified non-human animals can be produced in an immunocompetent background, and hybridized with animals including mutant gene alleles that will cause immunodeficiency when hemizygous or homozygous, and the offspring are mated to produce immunodeficient animals expressing at least one target human protein.

[0257] In some embodiments, the genetically modified non-human animal is treated to eliminate endogenous hematopoietic cells that may be present in the genetically modified non-human animal. In one embodiment, the treatment includes irradiating the genetically modified non-human animal. In a specific embodiment, the newborn genetically modified mouse pup is sublethally irradiated. In a specific embodiment, the newborn pup is irradiated with 2x 200cGy at 4 hour intervals.

[0258] Various embodiments of the present invention provide genetically modified animals including human nucleic acid in their substantially whole cells, and genetically modified animals including human nucleic acid in some but not all cells thereof. In some cases, such as in the case of targeted recombination, a copy of human nucleic acid will be integrated into the genome of the genetically modified animal. In other cases, such as in the case of random integration, multiple copies of human nucleic acid adjacent to each other or alienated can be integrated into the genome of the genetically modified animal.

[0259] Thus, in some embodiments, the subject genetically modified non-human animal may be an immunodeficient animal comprising a genome comprising a nucleic acid encoding a human polypeptide operably linked to a corresponding non-human animal promoter, wherein the animal expresses the encoded human polypeptide. In other words, the subject genetically modified immunodeficient non-human animal comprises a genome comprising a nucleic acid encoding at least one human polypeptide, wherein the nucleic acid is operably linked to a corresponding non-human promoter and a polyadenylation signal, and wherein the animal expresses the encoded human polypeptide.

[0260] Reagents, Devices and Kits

[0261] Also provided are reagents, devices, and kits thereof for practicing one or more of the above methods.The subject reagents, devices, and kits thereof can vary widely.

[0262] In some embodiments, the reagents or kits will include one or more reagents for use in the methods described herein. For example, the kit may include a humanized SIRPα-IL-15 non-human animal, such as a mouse, such as Rag2 - / - IL Y / - hSIRPαhIL-15 mouse. The kit may include reagents such as primers for mating humanized SIRPα-IL-15 non-human animals such as mice, and in some cases, reagents for genotyping humanized SIRPα-IL-15 non-human animals such as mice. The kit may include human hematopoietic cells or enriched human hematopoietic progenitor cell populations for transplantation into humanized SIRPα-IL-15 non-human animals such as mice, or reagents for preparing hematopoietic cell populations or enriched hematopoietic cell populations from humans for transplantation into humanized SIRPα-IL-15 non-human animals such as mice. Other reagents may include reagents for determining the viability and / or function of hematopoietic cells or differentiated immune cells (e.g., T cells and / or NK cells), for example, in the presence / absence of candidate agents, such as one or more antibodies specific to markers expressed by different types of hematopoietic cells or differentiated immune cells (e.g., T cells and / or NK cells), or reagents for detecting specific cytokines, chemokines, and the like. Other reagents may include culture media, culture supplements, matrix compositions, and the like.

[0263] In addition to the above components, the subject test kit will further include instructions for implementing the subject method. These instructions can be present in the subject test kit in various forms, and one or more of the forms can be present in the test kit. These instructions can exist in a form adopted to be present in a suitable medium or substrate (such as one or more sheets of paper with information printed thereon), in the packaging of the test kit, in the form of printed information such as a package insert, etc. Another means will be a computer-readable medium for recording information thereon, such as a disk, CD, etc. Another means that can exist is a website address that can be used to obtain information in a distant place through the Internet. Any suitable means can be present in the test kit.

[0264] EXEMPLARY NON-LIMITING ASPECTS OF THE DISCLOSURE

[0265] The various aspects of the above-described inventive subject matter, including various embodiments, may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the previous description, certain non-limiting aspects of the present disclosure, numbered 1-167, are provided below. As will be apparent to those skilled in the art upon reading this disclosure, each individually numbered aspect may be used or combined with any previous or subsequent individually numbered aspect. This is intended to provide support for all such combinations of the various aspects, and is not limited to the combinations of the aspects explicitly provided below:

[0266] 1. A genetically modified non-human animal comprising:

[0267] a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter; and

[0268] A nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein.

[0269] 2. The genetically modified non-human animal according to 1, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter.

[0270] 3. The genetically modified non-human animal according to 2, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at the SIRPα locus of the non-human animal.

[0271] 4. The genetically modified non-human animal according to 3, comprising a null mutation in the non-human SIRPα gene at the SIRPα locus of the non-human animal.

[0272] 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.

[0273] 6. The genetically modified non-human animal according to 4, wherein the genetically modified non-human animal is heterozygous for an allele comprising the nucleic acid sequence encoding the human SIRPα protein.

[0274] 7. The genetically modified non-human animal according to 4, wherein the genetically modified non-human animal is homozygous for the allele comprising the nucleic acid sequence encoding the human SIRPα protein.

[0275] 8. The genetically modified non-human animal according to any one of 1-7, wherein the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genomic coding sequence and a non-coding sequence.

[0276] 9. The genetically modified non-human animal according to any one of 1-8, wherein the human SIRPα protein is a functional fragment of the full-length human SIRPα protein.

[0277] 10. The genetically modified non-human animal according to 9, wherein the functional fragment comprises the extracellular domain of human SIRPα.

[0278] 11. The genetically modified non-human animal according to 10, wherein the extracellular domain comprises amino acids 28-362 of SEQ ID NO:12.

[0279] 12. The genetically modified non-human animal according to any one of 1-11, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter.

[0280] 13. The genetically modified non-human animal according to 12, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at the IL-15 locus of the non-human animal.

[0281] 14. The genetically modified non-human animal according to 13, comprising a null mutation in the non-human IL-15 gene at the IL-15 locus of the non-human animal.

[0282] 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.

[0283] 16. The genetically modified non-human animal according to 14, wherein the genetically modified non-human animal is heterozygous for an allele comprising the nucleic acid sequence encoding the human IL-15 protein.

[0284] 17. The genetically modified non-human animal according to 14, wherein the genetically modified non-human animal is homozygous for the allele comprising the nucleic acid sequence encoding the human IL-15 protein.

[0285] 18. The genetically modified non-human animal according to any one of items 1 to 17, wherein the nucleic acid sequence encoding the human IL-15 protein comprises human IL-15 genomic coding sequence and non-coding sequence.

[0286] 19. The genetically modified non-human animal according to any one of 1 to 18, wherein the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0287] 20. The genetically modified non-human animal according to any one of 1-19, wherein the genetically modified non-human animal is immunodeficient.

[0288] 21. The genetically modified non-human animal according to 20, wherein the genetically modified non-human animal comprises a Rag2 gene knockout.

[0289] 22. The genetically modified non-human animal according to 20 or 21, wherein the genetically modified non-human animal comprises an IL2rg gene knockout.

[0290] 23. A genetically modified non-human animal according to any one of 1-22, wherein the non-human animal is a mammal.

[0291] 24. The genetically modified non-human animal according to 23, wherein the mammal is a rodent.

[0292] 25. The genetically modified non-human animal according to 24, wherein the rodent is a mouse.

[0293] 26. A genetically modified non-human animal according to any one of 1-25, wherein the genetically modified non-human animal comprises an implant of human hematopoietic cells.

[0294] 27. The genetically modified non-human animal according to 26, wherein the genetically modified non-human animal comprises infection by a human pathogen.

[0295] 28. A 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.

[0296] 29. The genetically modified non-human animal according to 27, wherein the human pathogen is a pathogen that infects the human intestine.

[0297] 30. The genetically modified non-human animal according to 29, wherein the human pathogen is human rotavirus.

[0298] 31. The genetically modified non-human animal according to 27, wherein the pathogen infects human lungs.

[0299] 32. The genetically modified non-human animal according to 31, wherein the human pathogen is influenza virus.

[0300] 33. An animal implant model comprising a genetically modified non-human animal, wherein the genetically modified non-human animal comprises:

[0301] a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter;

[0302] a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter; and

[0303] An implant of artificial hematopoietic cells, wherein the genetically modified non-human animal (i) expresses the human SIRPα protein and the human IL-15 protein, and (ii) comprises human intraepithelial lymphocytes (IELs) in the small intestine and Peyer's patches of the genetically modified non-human animal.

[0304] 34. The model of 33, wherein the genetically modified non-human animal comprises infection by a human pathogen.

[0305] 35. The model according to 34, wherein the human pathogen is an enteric pathogen.

[0306] 36. The model according to 35, wherein the intestinal pathogen is selected from the group consisting of 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.

[0307] 37. The model according to any one of 33-36, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter.

[0308] 38. The model according to 37, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at the SIRPα gene locus of the non-human animal.

[0309] 39. The model according to 38, wherein the genetically modified non-human animal comprises a null mutation in a non-human SIRPα gene at the non-human animal SIRPα locus.

[0310] 40. The model according to 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.

[0311] 41. The model according to 39, wherein the genetically modified non-human animal is heterozygous for an allele comprising the nucleic acid sequence encoding the human SIRPα protein.

[0312] 42. The model according to 39, wherein the genetically modified non-human animal is homozygous for an allele comprising the nucleic acid sequence encoding the human SIRPα protein.

[0313] 43. The model according to any one of items 33-42, wherein the nucleic acid sequence encoding the human SIRPα protein comprises human SIRPα genomic coding sequence and non-coding sequence.

[0314] 44. The model according to any one of items 33-43, wherein the human SIRPα protein is a functional fragment of the full-length human SIRPα protein.

[0315] 45. The model according to 44, wherein the functional fragment comprises the extracellular domain of human SIRPα.

[0316] 46. ​​The model according to 45, wherein the extracellular domain comprises amino acids 28-362 of SEQ ID NO:12.

[0317] 47. A model according to any one of 33-46, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter.

[0318] 48. The model according to 47, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at the non-human animal IL-15 locus.

[0319] 49. The model of 48, wherein the genetically modified non-human animal comprises a null mutation in a non-human IL-15 gene at the non-human animal IL-15 locus.

[0320] 50. The model of 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.

[0321] 51. The model according to 48, wherein the genetically modified non-human animal is heterozygous for an allele comprising the nucleic acid sequence encoding the human IL-15 protein.

[0322] 52. The model according to 48, wherein the genetically modified non-human animal is homozygous for an allele comprising the nucleic acid sequence encoding the human IL-15 protein.

[0323] 53. The model according to any one of items 33-52, wherein the nucleic acid sequence encoding the human IL-15 protein comprises human IL-15 genomic coding sequence and non-coding sequence.

[0324] 54. The model according to any one of 33-53, wherein the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0325] 55. A model according to any one of 33-54, wherein the genetically modified non-human animal is immunodeficient.

[0326] 56. The model of 55, wherein the genetically modified non-human animal comprises a Rag2 gene knockout.

[0327] 57. The model of 55 or 56, wherein the genetically modified non-human animal comprises an IL2rg gene knockout.

[0328] 58. The model according to any one of 33-57, wherein the non-human animal is a mammal.

[0329] 59. The model according to 58, wherein the mammal is a rodent.

[0330] 60. The model according to 59, wherein the rodent is a mouse.

[0331] 61. An animal implant model comprising a genetically modified non-human animal, wherein the genetically modified non-human animal comprises:

[0332] a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter;

[0333] a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter; and

[0334] An implant 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) comprises human intraepithelial lymphocytes (IEL) in the lungs of the genetically modified non-human animal.

[0335] 62. The model of 61, wherein the genetically modified non-human animal comprises infection by a human pathogen.

[0336] 63. The model of 62, wherein the human pathogen is a lung pathogen.

[0337] 64. A model according to 63, wherein the lung pathogen is selected from: Streptococcus pyogenes, Haemophilus influenzae, Corynebacterium diphtheriae, SARS coronavirus, Bordetella pertussis, Moraxella catarrhalis, influenza virus (A, B, C), coronavirus, adenovirus, respiratory syncytial virus, parainfluenza virus, mumps virus, Streptococcus pneumoniae, Staphylococcus aureus, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Blastomyces dermatitidis, Cryptococcus neoformans and Aspergillus fumigatus.

[0338] 65. The model according to any one of 61-64, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter.

[0339] 66. The model according to 65, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at the SIRPα locus of the non-human animal.

[0340] 67. The model according to 66, wherein the genetically modified non-human animal comprises a null mutation in a non-human SIRPα gene at the non-human animal SIRPα locus.

[0341] 68. The model of 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.

[0342] 69. The model according to 67, wherein the genetically modified non-human animal is heterozygous for an allele comprising the nucleic acid sequence encoding the human SIRPα protein.

[0343] 70. The model according to 67, wherein the genetically modified non-human animal is homozygous for an allele comprising the nucleic acid sequence encoding the human SIRPα protein.

[0344] 71. The model according to any one of items 61-70, wherein the nucleic acid sequence encoding the human SIRPα protein comprises human SIRPα genomic coding sequence and non-coding sequence.

[0345] 72. The model according to any one of items 61-71, wherein the human SIRPα protein is a functional fragment of the full-length human SIRPα protein.

[0346] 73. The model according to 72, wherein the functional fragment comprises the extracellular domain of human SIRPα.

[0347] 74. The model according to 73, wherein the extracellular domain comprises amino acids 28-362 of SEQ ID NO:12.

[0348] 75. A model according to any one of 61-74, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter.

[0349] 76. The model according to 75, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at the non-human animal IL-15 locus.

[0350] 77. The model of 76, wherein the genetically modified non-human animal comprises a null mutation in a non-human IL-15 gene at the non-human animal IL-15 locus.

[0351] 78. The model of 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.

[0352] 79. The model according to 77, wherein the genetically modified non-human animal is heterozygous for an allele comprising the nucleic acid sequence encoding the human IL-15 protein.

[0353] 80. The model according to 77, wherein the genetically modified non-human animal is homozygous for an allele comprising the nucleic acid sequence encoding the human IL-15 protein.

[0354] 81. A model according to any one of items 61-80, wherein the nucleic acid sequence encoding the human IL-15 protein comprises human IL-15 genomic coding sequence and non-coding sequence.

[0355] 82. The model according to any one of 61-80, wherein the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0356] 83. A model according to any one of 61-82, wherein the genetically modified non-human animal is immunodeficient.

[0357] 84. The model of 83, wherein the genetically modified non-human animal comprises a Rag2 gene knockout.

[0358] 85. The model of 83 or 84, wherein the genetically modified non-human animal comprises an IL2rg gene knockout.

[0359] 86. The model according to any one of 61-85, wherein the non-human animal is a mammal.

[0360] 87. The model of 86, wherein the mammal is a rodent.

[0361] 88. The model of 87, wherein the rodent is a mouse.

[0362] 89. A method for determining the efficacy of a candidate T cell inducing vaccine, the method comprising:

[0363] administering a candidate T cell inducing vaccine to a genetically modified non-human animal, wherein the genetically modified non-human animal is deficient with respect to an endogenous immune system and comprises:

[0364] (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter,

[0365] (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, said sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter, and

[0366] (iii) an implant of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein;

[0367] challenging the genetically modified non-human animal with a human pathogen; and

[0368] Determining whether the candidate T cell inducing vaccine induces a T cell-mediated immune response in the genetically modified non-human animal.

[0369] 90. The method according to 89, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter.

[0370] 91. The method according to 90, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at the SIRPα locus of the non-human animal.

[0371] 92. A method according to 91, wherein the genetically modified non-human animal comprises a null mutation in a non-human SIRPα gene at the non-human animal SIRPα locus.

[0372] 93. A 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.

[0373] 94. The method according to 92, wherein the genetically modified non-human animal is heterozygous for an allele comprising the nucleic acid sequence encoding the human SIRPα protein.

[0374] 95. The method according to 92, wherein the genetically modified non-human animal is homozygous for an allele comprising the nucleic acid sequence encoding the human SIRPα protein.

[0375] 96. The method according to any one of 89-95, wherein the nucleic acid sequence encoding the human SIRPα protein comprises a human SIRPα genomic coding sequence and a non-coding sequence.

[0376] 97. The method according to any one of 89-96, wherein the human SIRPα protein is a functional fragment of the full-length human SIRPα protein.

[0377] 98. The method according to 97, wherein the functional fragment comprises the extracellular domain of human SIRPα.

[0378] 99. The method according to 98, wherein the extracellular domain comprises amino acids 28-362 of SEQ ID NO:12.

[0379] 100. The method according to any one of 89-99, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter.

[0380] 101. The method according to 100, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at the IL-15 locus of the non-human animal.

[0381] 102. A method according to 101, wherein the genetically modified non-human animal comprises a null mutation in a non-human IL-15 gene at the non-human animal IL-15 locus.

[0382] 103. The method of 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.

[0383] 104. The method according to 101, wherein the genetically modified non-human animal is heterozygous for an allele comprising the nucleic acid sequence encoding the human IL-15 protein.

[0384] 105. The method according to 101, wherein the genetically modified non-human animal is homozygous for an allele comprising the nucleic acid sequence encoding the human IL-15 protein.

[0385] 106. The method according to any one of 89-105, wherein the nucleic acid sequence encoding the human IL-15 protein comprises human IL-15 genomic coding sequence and non-coding sequence.

[0386] 107. The method according to any one of 89-106, wherein the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0387] 108. A method according to any one of 89-107, wherein the genetically modified non-human animal comprises a Rag2 gene knockout.

[0388] 109. The method of any one of 89-108, wherein the genetically modified non-human animal comprises an IL2rg gene knockout.

[0389] 110. A method according to any one of 89-109, wherein the genetically modified non-human animal is a mammal.

[0390] 111. The method according to 110, wherein the mammal is a rodent.

[0391] 112. The method according to 111, wherein the rodent is a mouse.

[0392] 113. A method for identifying an agent that inhibits infection by a pathogen that activates, induces and / or targets human T cells and / or natural killer (NK) cells, the method comprising:

[0393] administering an agent to a genetically modified non-human animal, wherein the genetically modified non-human animal is deficient with respect to an endogenous immune system and comprises:

[0394] (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter,

[0395] (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, said sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter,

[0396] (iii) an implant of artificial hematopoietic cells, and

[0397] (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

[0398] Determining whether the agent reduces the amount of the pathogen in a non-human animal infected with the pathogen.

[0399] 114. The method according to 113, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter.

[0400] 115. The method according to 114, wherein the SIRPα gene promoter is an endogenous non-human SIRPα gene promoter at the SIRPα locus of the non-human animal.

[0401] 116. The method according to 115, wherein the genetically modified non-human animal comprises a null mutation in a non-human SIRPα gene at the non-human animal SIRPα locus.

[0402] 117. The method of 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.

[0403] 118. The method according to 116, wherein the genetically modified non-human animal is heterozygous for an allele comprising the nucleic acid sequence encoding the human SIRPα protein.

[0404] 119. The method according to 116, wherein the genetically modified non-human animal is homozygous for an allele comprising the nucleic acid sequence encoding the human SIRPα protein.

[0405] 120. The method according to any one of 113-119, wherein the nucleic acid sequence encoding the human SIRPα protein comprises human SIRPα genomic coding sequence and non-coding sequence.

[0406] 121. The method according to any one of 113-120, wherein the human SIRPα protein is a functional fragment of the full-length human SIRPα protein.

[0407] 122. The method according to 121, wherein the functional fragment comprises the extracellular domain of human SIRPα.

[0408] 123. A method according to 122, wherein the extracellular domain comprises amino acids 28-362 of SEQ IDNO:12.

[0409] 124. The method according to any one of 113-123, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter.

[0410] 125. The method according to 124, wherein the IL-15 gene promoter is an endogenous non-human IL-15 gene promoter at the IL-15 locus of the non-human animal.

[0411] 126. According to the method of 125, the genetically modified non-human animal comprises a null mutation in a non-human IL-15 gene at the non-human animal IL-15 locus.

[0412] 127. A 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.

[0413] 128. The method according to 125, wherein the genetically modified non-human animal is heterozygous for an allele comprising the nucleic acid sequence encoding the human IL-15 protein.

[0414] 129. The method according to 125, wherein the genetically modified non-human animal is homozygous for an allele comprising the nucleic acid sequence encoding the human IL-15 protein.

[0415] 130. The method according to any one of 113-129, wherein the nucleic acid sequence encoding the human IL-15 protein comprises human IL-15 genomic coding sequence and non-coding sequence.

[0416] 131. The method according to any one of 113-130, wherein the human IL-15 protein is a functional fragment of the full-length human IL-15 protein.

[0417] 132. A method according to any one of 113-131, wherein the genetically modified non-human animal comprises a Rag2 gene knockout.

[0418] 133. A method according to any one of 113-132, wherein the genetically modified non-human animal comprises an IL2rg gene knockout.

[0419] 134. A method according to any one of 113-133, wherein the genetically modified non-human animal is a mammal.

[0420] 135. The method of 134, wherein the mammal is a rodent.

[0421] 136. The method according to 135, wherein the rodent is a mouse.

[0422] 137. A method for preparing a non-human animal expressing human IL-15 protein and human SIRPα protein, comprising:

[0423] Introducing a nucleic acid sequence encoding a human SIRPα protein into the genome of a first non-human animal, wherein the sequence encoding the human SIRPα protein is operably linked to a SIRPα gene promoter sequence;

[0424] Introducing into the genome of a second non-human animal a nucleic acid sequence encoding a human IL-15 protein, wherein the sequence encoding the human IL-15 protein is operably linked to an IL-15 promoter sequence; and

[0425] A third non-human animal comprising the nucleic acid sequence encoding the human IL-15 protein and the nucleic acid sequence encoding the human SIRPα protein is prepared, wherein the third non-human animal expresses the human IL-15 protein and the human SIRPα protein.

[0426] 138. The method of 137, wherein the introducing step comprises producing a non-human animal from pluripotent stem cells comprising the nucleic acid encoding human IL-15 or human SIRPα.

[0427] 139. The method of 137 or 138, wherein the first animal is a different animal from the second animal, and the step of preparing the third animal comprises mating the first animal and the second animal.

[0428] 140. A method as described in 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 a first pluripotent stem cell with the nucleic acid sequence encoding the human SIRPα protein to obtain a second pluripotent stem cell, the step of introducing into the genome of the second animal comprises contacting the second pluripotent stem cell with the nucleic acid sequence encoding the human SIRPα protein to obtain a third pluripotent stem cell, and the third non-human animal is prepared from the third pluripotent stem cell.

[0429] 141. The method according to any one of 137-140, wherein the pluripotent stem cells are ES cells or iPS cells.

[0430] 142. The method according to any one of 137-140, wherein the pluripotent stem cell is deficient in Rag2.

[0431] 143. The method according to any one of 137-142, wherein the pluripotent stem cell is deficient in IL2rg.

[0432] 144. The method according to any one of 137-143, wherein the third non-human animal is deficient in one or both of Rag2 and IL2rg.

[0433] 145. The method according to any one of 137-144, wherein the IL-15 promoter sequence is a human IL-15 promoter sequence.

[0434] 146. A method according to any one of 137-144, wherein the IL-15 promoter sequence is an endogenous non-human animal IL-15 promoter sequence.

[0435] 147. The method according to any one of 137-144, wherein the integration results in replacement of the non-human IL-15 gene at the non-human IL-15 locus.

[0436] 148. The method according to any one of 137-147, wherein the nucleic acid sequence encoding the human IL-15 protein comprises human IL-15 genomic coding sequence and non-coding sequence.

[0437] 149. A method of implanting a genetically modified non-human animal expressing a human IL-15 protein, comprising:

[0438] The cell population comprising human hematopoietic cells is transplanted into the genetically modified non-human animal prepared by the method according to any one of 137-148.

[0439] 150. The method according to 149, wherein the transplantation comprises tail vein injection, fetal liver injection or retro-orbital injection.

[0440] 151. The method of 149 or 150, wherein the genetically modified non-human animal is sublethally irradiated prior to transplantation.

[0441] 152. The method according to any one of 149-151, wherein the human hematopoietic cells are CD34+ cells.

[0442] 153. The method according to any one of 149-151, wherein the human hematopoietic cells are from fetal liver, adult bone marrow or umbilical cord blood.

[0443] 154. A method for determining the efficacy of a candidate therapeutic antibody or antigen binding protein in killing target cells, the method comprising:

[0444] administering the candidate therapeutic antibody or antigen binding protein to a genetically modified non-human animal, wherein the genetically modified non-human animal is deficient in an endogenous immune system and comprises:

[0445] (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter,

[0446] (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, said sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter, and

[0447] (iii) an implant of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein; and

[0448] Determining whether the candidate therapeutic antibody or antigen binding protein modulates NK cell-mediated antibody-dependent cellular cytotoxicity against the target cell in the genetically modified non-human animal.

[0449] 155. A method for determining the efficacy of a candidate therapeutic antibody or antigen binding protein in killing target cells, the method comprising:

[0450] Isolating NK cells from a genetically modified non-human animal, wherein the genetically modified non-human animal is deficient in an endogenous immune system and comprises:

[0451] (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter,

[0452] (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, said sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter, and

[0453] (iii) an implant of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein;

[0454] contacting the isolated NK cells with the candidate therapeutic antibody or antigen binding protein and the target cells; and

[0455] The antibody- or antigen-binding protein-dependent cytolytic activity of the isolated NK cells against the target cells is measured.

[0456] 156. A method for screening candidate therapeutic antibodies or antigen binding proteins for improved efficacy in killing target cells, comprising:

[0457] administering the candidate therapeutic antibody or antigen binding protein to a genetically modified non-human animal, wherein the genetically modified non-human animal is deficient in an endogenous immune system and comprises:

[0458] (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter,

[0459] (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, said sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter, and

[0460] (iii) an implant of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein; and

[0461] Determining whether the candidate therapeutic antibody or antigen binding protein exhibits improved efficacy in killing the target cell in the genetically modified non-human animal.

[0462] 157. The method of any one of 154-156, wherein the target cell is selected from the group consisting of a tumor cell, a virally infected cell, a bacterially infected cell, a bacterial cell, a fungal cell, and a parasite cell.

[0463] 158. A method for determining the efficacy of a candidate therapeutic antibody or antigen binding protein in NK cell-mediated killing of a target cell, comprising:

[0464] administering the candidate therapeutic antibody or antigen binding protein to a genetically modified non-human animal, wherein the genetically modified non-human animal is deficient in an endogenous immune system and comprises:

[0465] (i) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter,

[0466] (ii) a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, said sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter, and

[0467] (iii) an implant of human hematopoietic cells, wherein the genetically modified non-human animal expresses the human SIRPα protein and the human IL-15 protein; and

[0468] Determining whether the candidate therapeutic antibody or antigen binding protein modulates (e.g., activates) NK cell antibody-dependent cellular cytotoxicity against the target cell in the genetically modified non-human animal.

[0469] 159. The method of 158, wherein the target cell is selected from the group consisting of a tumor cell, a virally infected cell, a bacterially infected cell, a bacterial cell, a fungal cell, and a parasite cell.

[0470] 160. The method of 159, wherein the target cell is a tumor cell.

[0471] 161. A method according to 160, wherein the tumor cells are B cell lymphoma cells.

[0472] 162. A model of NK cell-mediated antibody-dependent cellular cytotoxicity, comprising a genetically modified non-human animal, wherein the genetically modified non-human animal is deficient in an endogenous immune system, and comprising:

[0473] a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human SIRPα protein and operably linked to a SIRPα gene promoter;

[0474] a nucleic acid sequence incorporated into the genome of the genetically modified non-human animal, the sequence encoding a human IL-15 protein and operably linked to an IL-15 gene promoter; and

[0475] An implant of human hematopoietic cells, wherein the genetically modified non-human animal (i) expresses the human SIRPα protein and the human IL-15 protein, (ii) comprises human lymphocytes, and (iii) comprises target cells selected from the group consisting of tumor cells, virus-infected cells, bacteria-infected cells, bacterial cells, fungal cells, and parasite cells.

[0476] 163. A model according to 162, wherein the target cells are tumor cells.

[0477] 164. A model according to 163, wherein the tumor cells are B cell lymphoma cells.

[0478] 165. A model according to 163 or 164, wherein the model comprises an exogenous candidate therapeutic antibody or antigen binding protein.

[0479] 166. A model according to any one of 162-165, wherein the genetically modified non-human animal comprises human intraepithelial lymphocytes (IELs) in the small intestine and Peyer's patches of the genetically modified non-human animal.

[0480] 167. A model according to any one of 162-166, wherein the genetically modified non-human animal comprises human intraepithelial lymphocytes (IELs) in the lungs of the genetically modified non-human animal. Example

[0481] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the values ​​used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0482] Example 1: Generation of humanized SIRPα (SRG) knock-in mice

[0483] Human SIRPα knock-in mice were generated that express the extracellular domain of human SIRPα operably linked to the mouse SIRPα promoter (see Figure 1 ). It is known that human SIRPα exists in at least 10 allelic forms. In this specific embodiment, human SIRPα variant 1 was used to humanize the endogenous SIRPα gene of mice.

[0484] Materials and methods

[0485] As described in more detail below, using Technology in Rag2 - / - Iq Y / - Generation of knock-in mice encoding human SIRPα was performed in a 129xBalb / c(N2) genetic background. Mice were maintained under specific pathogen-free conditions and continuously treated with enrofloxacin in drinking water (Baytril; 0.27 mg / mL).

[0486] use Technology (see, for example, U.S. Pat. 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) was used to construct a targeting vector for humanizing the extracellular region of a SIRP (eg, SIRPα) gene.

[0487] Briefly, the mouse bacterial artificial chromosome (BAC) clone bMQ-261H14 was modified to delete the sequence containing exons 2 to 4 of the endogenous SIRPα gene, and the human BAC clone CTD-3035H21 was used to insert exons 2 to 4 of the human SIRPα gene. The genomic DNA (approximately 8555 bp) corresponding to exons 2 to 4 of the endogenous SIRPα gene in the BAC clone bMQ-261H14 was replaced by an approximately 8581 bp DNA fragment containing exons 2 to 4 of the human SIRPα gene from the BAC clone CTD-3035H21. Sequence analysis of the human SIRPα allele contained in the BAC clone CTD-3035H21 revealed that the allele corresponds to human variant 1. A neomycin cassette flanked by loxP sites was added to the end of the approximately 8581 bp human DNA fragment containing exons 2 to 4 of the human SIRPα gene ( Figure 1 (bottom)).

[0488] Upstream and downstream homology arms were obtained from mouse BAC DNA at the positions 5' and 3' of exons 2 and 4, respectively, and added to the approximately 8581 bp human fragment-neomycin cassette to produce a final targeting vector for humanizing the endogenous SIRPα gene, which contains from 5' to 3' a 5' homology arm (which contains 19 kb mouse DNA at 5' of exon 2 of the endogenous SIRPα gene), an approximately 8581 bp DNA fragment containing exons 2 to 4 of the human SIRPα gene, a neomycin cassette flanked by loxP sites, and a 3' homology arm (which contains 21 kb mouse DNA at 3' of exon 4 of the endogenous SIRPα gene). Targeted insertion of the targeting vector positions the neomycin cassette in the fifth intron between exons 4 and 5 of the mouse SIRPα gene. The targeting vector was linearized by digestion with SwaI and then used in bacterial cells to achieve targeted replacement of exons 2 to 4 of the mouse SIRPα gene with exons 2 to 4 of the human SIRPα gene by homologous recombination ( Figure 1 (bottom)).

[0489] The targeted BAC DNA (described above) was used to electroporate Rag2 - / - IL Y / - Mouse ES cells were used to generate modified ES cells, including exons 2 to 4 in the endogenous mouse SIRPα gene replaced by a genomic fragment including exons 2 to 4 of the human SIRPα gene. TMThe probe was used for quantitative PCR to identify positive ES cells containing a genomic fragment including exons 2 to 4 of the human SIRPα gene (Lie and Petropoulos, 1998. Curr. Opin. Biotechnology 9: 43-48). The nucleotide sequence spanning the upstream insertion point includes the following sequence, which indicates that the endogenous mouse sequence upstream of the insertion point (enclosed in the following brackets) is continuously connected to the human SIRPα genomic sequence present at the insertion point:

[0490] (SEQ ID NO: 1). The nucleotide sequence spanning the downstream insertion point at the 5' end of the neomycin cassette includes the following sequence, which indicates that the human SIRPα genomic sequence is adjacent to the cassette sequence (enclosed in the following brackets, where the loxP sequence is indicated in italics) downstream of the insertion point:

[0491]

[0492] (SEQ ID NO: 2). The nucleotide sequence spanning the downstream insertion point at the 3' end of the neomycin cassette includes the following sequence, which indicates that the cassette sequence is adjacent to the mouse genomic sequence (enclosed in the following brackets) 3' to exon 4 of the endogenous SIRPα gene:

[0493]

[0494] (SEQ ID NO: 3). The positive ES cell clones were then used The method (see, e.g., U.S. Pat. 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) is used to implant female mice to produce a litter of pups containing exons 2 to 4 of the human SIRPα gene inserted into the mouse's endogenous SIRPα gene.

[0495] The above-mentioned targeted ES cells are used as donor ES cells, and Methods (supra) were introduced into 8-cell stage mouse embryos. Humanized mice carrying exons 2 to 4 of the endogenous SIRPα gene were identified by genotyping using a modified version of the allelic assay (Valenzuela et al., supra) that detects the presence of human SIRPα gene sequences.

[0496] Mice carrying humanized SIRPα gene constructs (i.e., containing human SIRPα exons 2 to 4 in the mouse SIRPα gene) can be mated with a Cre-deficient mouse strain (see, e.g., International Patent Application Publication No. WO2009 / 114400) to remove any loxed neomycin cassette introduced by the targeting vector, e.g., at the ES cell stage or in the embryo that was not removed. Optionally, the neomycin cassette is retained in the mouse. To obtain homozygous Sirpα mice, heterozygotes are mated.

[0497] result

[0498] Mice (SRG mice) comprising a nucleic acid encoding a humanized form of the mouse SIRPα gene as described above exhibit physiological expression of humanized SIRPα protein (data not shown). These mice also exhibit human immune cell engraftment similar to that of NOD scidγ (NSG) mice in spleen, peripheral lymph nodes (LN) and thymus (data not shown).

[0499] Example 2: Generation of humanized SRG IL-15 h / h (SRG-15) knock-in mice

[0500] The cytokine IL-15 has been shown to be important for the development of mouse NK cells and memory CD8 + T cell differentiation and maintenance are important. To study the effects of human IL-15 on the development, differentiation and maintenance of human immune cells in the context of an animal model, human IL-15 human SIRPα knock-in mice were generated as described in more detail below. Figure 2 A schematic representation of the IL-15 knock-in construct is shown.

[0501] Materials and methods

[0502] use Genetic engineering techniques are used to modify mouse ES cells to replace the mouse IL-15 gene sequence with the human IL-15 gene sequence at the endogenous mouse IL-15 locus under the control of the mouse IL-15 regulatory element to produce Figure 2 The humanized loci shown in . Based on Rag2 - / - Iq Y / - Knock-in mice containing human Il-15 were generated in the 129xBalb / c genetic background. Figure 2 The upstream (relative to the transcription direction of the IL-15 gene) 5′ untranslated exons (exons 1 and 2) of the mouse gene are not shown; Figure 2 The coding exon 1 (exon 3) of shows a small untranslated region (not filled) upstream of the coding exon. In addition to the discussion below for mouse 1, as in Figure 2As shown in the humanization at the bottom of , mouse coding exons 1 and 2 (exons 3 and 4) are retained, while mouse coding exons 3 to 6 (exons 5-8) are replaced with human coding exons 3 to 6 (exons 5-8). At the downstream end, human coding exon 6 (exon 8) is followed by a stop codon and human 3'-UTR, and further followed by human sequences found downstream of the human 3'UTR. For selection purposes, a selection cassette (flanked with loxP to achieve removal by Cre) is included. Figure 2 The humanized locus expresses a fully human mature IL-15 protein.

[0503] Specifically, bacterial homology recombination (BHR) was performed using standard BHR techniques (see, e.g., Valenzuela et al. (2003), supra) and gap repair BHR to construct a large-scale targeting vector (LTVEC) containing the sequence of the human IL-15 gene for targeting the mouse IL-15 locus. Linear fragments were generated by connecting the homology frame generated by PCR to a cloning cassette, followed by gel separation of the ligation product and electroporation into BHR competent bacteria with a target bacterial artificial chromosome (BAC). Mouse BAC PRCI23-203P7 was used as the source of mouse sequence; human BAC RP11-103B12 was used as the source of human IL-15 gene sequence. After the selection step, PCR was performed by crossing the novel junction, and the clones of the correct recombination were identified by restriction analysis. LTVEC containing homology arms and human IL-15 gene sequence was obtained.

[0504] The mouse IL-15 gene (mouse GeneID: 103014; RefSeq transcript: NM_008357.2; ensemble eID: 16168) was modified by using the genomic coordinates GRCM38:ch 8:82331173-82343471 (minus strand) for deletion; GRCh37:ch4:142642924-142655819 (plus strand) for replacement. 12299 nucleotides of the mouse sequence were replaced by 12896 nucleotides of the human sequence. The replacement diagram of the mouse IL-15 sequence as described above is presented in Figure 2 middle.

[0505] The LTVEC including the humanized IL-15 gene has an approximately 13 kb upstream mouse targeting arm flanked by an MluI site on the upstream side, and a 27 kb downstream mouse targeting arm flanked by an AscI site on the downstream side. The LTVEC was linearized with MluI and AscI for electroporation.

[0506] After constructing LTVEC, the nucleotide sequence of LTVEC spanning the mouse / human 5' junction and the human / mouse 3' junction is shown in Table 1 below. SEQ ID NO: 4 depicts the upstream (relative to the transcription direction of the IL-15 gene) junction between the mouse sequence and the human sequence; the sequence shown starts 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 sequence and non-coding sequence in uppercase (human 3'UTR is represented by bold italics), followed by the XhoI site in lowercase, followed by the lox site (uppercase, represented by bold italics), followed by the sequence of the downstream neo selection box (uppercase), which extends 2.6kb downstream (not shown). SEQ ID NO:6 is a nucleic acid sequence depicting the junction of the downstream portion (uppercase) of the neo selection cassette, which is accompanied by a lox site (uppercase and in bold italics), followed by a NheI site (lowercase), which is followed by the mouse sequence (uppercase) downstream of the humanization; the selection cassette extends 2.6 kb further upstream.

[0507] Table 1: Junctional sequences of humanized IL-15 locus

[0508]

[0509]

[0510] Mouse ES cells are electroporated with the LTVEC construct, grown in selective medium, and used as donor ES cells to prepare proteins including Figure 2 Humanized IL-15 mice with endogenous mouse IL-15 locus replaced by human sequences are depicted in. After electroporation of ES cells, a natural allele loss assay (see, e.g., Valenzuela et al. (2003), supra) was performed to detect loss of endogenous IL-15 sequences due to targeting.

[0511] Correctly targeted ES cells were further electroporated with a transient Cre expression vector to remove the Neo drug selection cassette.

[0512] pass 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) to introduce donor mouse ES cells including humanized IL-15 locus into early mouse embryos. Heterozygous mice are obtained, and heterozygotes are mated to obtain homozygotes for humanized IL-15. Two forms of humanized IL-15 mice (referred to herein as mouse 1 and mouse 2) are produced. After further analysis, it was found that the mouse 1 form contains exon duplications in its genome. In mouse 2, the endogenous mouse IL-15 locus is replaced by a human sequence, such as Figure 2 Described in.

[0513] Human IL-15 mRNA levels were determined as follows. 7500 Fast Real-Time PCR System (Applied Biosystems) and Reverse transcription (RT)-qPCR was performed using a 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: 5'-AGGGATTTGAATCACGTTTG-3' (SEQ ID NO: 7), mouse Hprt reverse: 5'-TTTACTGGCAACATCAACAG-3' (SEQ ID NO: 8); human Il15 forward: 5'-GCCCAGGGAAATCAAAAGAT-3' (SEQ ID NO: 9), human Il15 reverse: 5'-TGGCTCCAACAAATCAACAG-3' (SEQ ID NO: 10). Relative expression values ​​were calculated using the comparative threshold cycle method and normalized to mouse Hprt.

[0514] SRG-15 mice were generated by (1) mating mice containing human SIRPα substitutions with mice containing human IL-15 substitutions, both based on Rag2 - / - Iq Y / - background, or (2) introducing a large targeting vector containing human IL-15 into the Rag2-based - / - Iq Y / - ES cells with human SIRPα replacement in the background (described in Example 1), and using The method comprises a method of replacing both the human IL-15 gene and the SIRPα gene and Rag2 - / - Iq Y / - Mice are generated from ES cells. Heterozygous mice are mated to obtain homozygosity.

[0515] result

[0516] like Figure 3A and 3B As shown in Figure 1, high expression levels of human IL-15 mRNA were found in the liver, lung, bone marrow (BM), small intestine (SI), and colon of non-implanted SRG-15 mouse 1. Similar high levels of human IL-15 mRNA were found in the liver, lung, and small intestine (SI) of non-implanted SRG-15 mouse 2. Figure 3B ).like Figure 4 As shown in , high levels of human IL-15 protein can also be detected in the serum of SRG-15 mice 2 in which human exons 5-8 replace endogenous mouse exons after stimulation with poly(I:C).

[0517] Example 3: Implantation into SRG-15 mice

[0518] Materials and methods

[0519] SRG and SRG-15 mice were implanted as follows. Newborn mice were sublethally irradiated with 160 cGy without anesthesia at 3-5 days after birth and returned to their mothers to rest. These newborns were transplanted intrahepatically (ih) with 25 μl PBS containing CD34+ huHSC using a 30G needle 4-12 hours after irradiation.

[0520] result

[0521] To evaluate the effect of human IL-15 on immune cell development, human CD45 + Efficient human hematopoietic cell engraftment was observed in the blood of NSG, SRG, and SRG-15 (mouse 2) mice 12-14 weeks after engraftment. Figure 5A as shown in . Figure 5B A comparison is provided in , which shows engraftment as evidenced by the number of human CD45+ cells in the BM, spleen, LN, liver and lung of SRG and SRG-15 (mouse 2) at 14 weeks post-implantation.

[0522] In mice 1, although human CD45 + Cell engraftment was not different, but higher percentages and numbers of human NK cells were found in various tissues in SRG-15 mice compared to SRG mice, as indicated by Fig. 6A and 6BAs described above, IL-15 is important not only for the development and survival of NK cells, but also for their maturation. Figure 6C As shown in Figure 1, human NK cells in the liver of SRG-15 mice (mouse 1) expressed higher levels of CD16 and CD56 compared to SRG mice, indicating increased NK cell maturation. 明亮 CD16 - and CD56 暗淡 CD16 + Both are present in the blood, spleen, and liver of SRG-15 mice, such as Fig.6D (spleen) (and data not shown). Fig.6D As shown in Figure 1, analysis of two human NK cell subsets in the spleen of SRG-15 mice (mouse 1) showed that they had different expression levels of killer inhibitory receptors, among which CD56 暗淡 CD16 + The NK cell population included a higher percentage of CD158 expressing cells. This is similar to what is seen for NK cell subsets in human blood (data not shown).

[0523] For SRG-15 mice 2, efficient human NK cell engraftment in both lymphoid and non-lymphoid tissues was observed, as Figures 7A-7D as shown in . Fig. 7A and 7B The percentages of NK cells in the blood and spleen are shown, respectively. Figure 7C and 7D The frequency of human NK cells in the blood, spleen (SP), liver, and lung of SRG and SRG-15 (Mouse 2) mice at 14 weeks post-implantation is shown. Additional data from different experiments showing the distribution and percentage of NK cells in the blood and spleen of SRG and SRG-15 (Mouse 2) mice, respectively, are provided in Figure 8 and 9A middle. Fig. 9B Figure 2 shows that the hNKp46 fragment is increased in hCD45+ cells in the blood of SRG-15 mice (mouse 2). Figures 9C-9E Shown are the relative number, distribution and composition of hCD45+ cells in the thymus of SRG and SRG-15 (mouse 2) mice.

[0524] Characterization of NK cell subsets in humans and SRG-15 mice (mouse 2). Fig. 10A and 10B As shown in Figure 2, hCD-56 was observed in the blood and spleen of SRG-15 mice compared with SRG mice. 明亮 hCD16 - hCD56 暗淡 hCD16 +As in humans, killer inhibitory receptors (KIR) were observed to be expressed on a subset of NK cells in SRG-15 mice (mouse 2). Fig. 10C ). Fig. 10C CD56 shows bright CD16 - NK cells (left frame of each figure) and CD56 dim CD16 + NK cells (right box of each figure). The lower graph shows CD158 expression in those subsets. CD158 (KIR2D) on NK cell subsets in SRG-15 mice is similar to that observed with human PBMC-derived NK cells.

[0525] The distribution of human NK cells in the blood of SRG-15 mice was compared to that of blood obtained from two healthy human donors. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of two individual donors (obtained from Bioreclamation VT, Westbury, NY) by Ficoll-Paque; although a greater percentage of blood NK cells was observed in SRG-15 mice engrafted compared to the percentage in PBMCs from human donors, a physiologically similar distribution of cytotoxic (CD16+) NK cells relative to IFN-g-producing (CD16-) NK cells was observed ( Fig.11 ).

[0526] Finally, analysis of the bone marrow of SRG and SRG-15 (mouse 2) revealed that human NK cell development was enhanced in SRG-15 mice relative to SRG mice ( Fig.12 ).

[0527] The effect of human IL-15 on the development of human T cells in SRG-15 mice was also evaluated. Comparison of SRG-15 (mouse 1) mice relative to SRG mice showed that the effect of human IL-15 on the percentage, number and / or ratio of T cells varied depending on the tissue ( Fig.13A ). The size and number of lymph nodes at week 16 post-implantation were not different between SRG mice and SRG-15 mice, confirming that the number of human T cells in the lymph nodes of SRG and SRG-15 (mouse 1) mice was similar ( Fig.13A ). Fig. 13B Figure 3 shows the phenotype of human CD8+ T cells in the blood and liver of SRG and SRG-15 mice (mouse 1), where hCD62L in SRG-15 mice (mouse 1) is higher in both blood and liver than in SRG mice. - The cells increased. Fig.14A and 14BAdditional data characterizing T cells in SRG-15 mice (Mouse 1) relative to SRG mice are provided in Figure 14, which shows that the tissue-resident marker CD69 is expressed in the lungs (14A) and livers (14B) of SRG and SRG-15 mice. + The above data provide evidence that effector tissue-resident T cells are increased in SRG-15 mice.

[0528] For mouse 2, hCD3 in spleen, lung, and liver was assessed at 16 weeks post-implantation relative to SRG mice. + The frequency of T cells, such as Fig.15A and 15B as shown in .

[0529] Example 4: Development of human tissue-resident lymphocytes in SRG-15 mice

[0530] Because IL-15 has been shown to be produced by epithelial cells in the intestine and lung and may play an important role in the development and survival of human tissue-resident T and NK cells, human tissue-resident T and NK cells were analyzed in SRG and SRG-15 mice.

[0531] Materials and methods

[0532] Newborn mice were sublethally irradiated with 160 cGy without anesthesia at 3-5 days after birth and returned to their mothers to rest. These newborns were transplanted intrahepatically (ih) with 25 μl PBS containing CD34+ huHSCs using a 30G needle 4-12 hours after irradiation.

[0533] result

[0534] like Fig.17A As shown in Figure 1, isolation of intraepithelial lymphocyte populations from the small intestine during steady-state conditions in mice revealed that human CD45 in SRG-15 mice was significantly higher than that in SRG mice. + The frequency of cells is higher. Fig. 17B The immunohistochemical analysis described in the + NK cells are located in the epithelial cell layer of the small intestine of SRG-15 mice (mouse 1) (as indicated by Fig. 17B Intraepithelial lymphocytes were found in SRG mice (designated by arrows in Figure 5), whereas very few intraepithelial lymphocytes were found in SRG mice. + IELs showed high expression of CD69, a typical marker of tissue-resident T cells. Compared with human IELs (Sathaliyawala T, Kubota M, Yudanin N et al. Immunity 2013; 38: 187-197), only human CD8 +IEL subsets express tissue resident marker CD103 ( Fig. 17C ).like Fig.16A and 16B As shown in Figure 1, human CD8 + The phenotype of increased IELs was specific, as there were few differences in the number of lamina propria cells in the colon during steady-state between SRG and SRG-15 mice. Fig.13A In addition to the increased number of human T cells in the lungs of SRG-15 mice as shown in Figure 1, it was also found that compared with SRG mice, the number of human CD8 + The expression of CD69 on T cells is higher, as shown by Fig.14A In addition, Fig. 14B Compared with SRG mice, hCD69 expression in the liver of SRG-15 mice was higher in CD8 + Higher levels of T cells.

[0535] Similar to SRG-15-implanted mouse 1, in SRG-15-implanted mouse 2, FACS analysis revealed that human CD45 in the IEL fraction of SRG-15 mice was significantly higher than that of SRG mice. + A higher proportion of cells ( Fig.18A In addition, although the number of LPLs did not change significantly between SRG mice and SRG-15(mouse 2) mice, a significant increase in IELs was observed in SRG-15(mouse 2) mice relative to SRG mice ( Fig.18B The composition of hCD3+ cells in the small intestine of SRG-15 mice (mouse 2) is provided in Fig.18C The phenotypic characteristics of hCD3+hCD8+ T cells in the spleen and small intestine of SRG-15 mice (mouse 2) are provided in Fig.18D In. Fig.18E The immunohistochemical analysis described in the + IELs are located in the epithelial cell layer of the small intestine of SRG-15 mice (mouse 2) (as indicated by Fig.18E ), whereas very few intraepithelial lymphocytes were found in SRG mice.

[0536] As above about Fig.18A and 18B As discussed, greater gut-associated lymphoid tissue (GALT)-resident intraepithelial human lymphocyte reconstitution (IEL) was observed in SRG-15-implanted mice 2 compared with SRG mice ( Fig.19A and 19C). Of interest, the majority of human lymphocytes observed were human NK cells. As expected for normal human GALT physiology, the majority of NK cells in both blood and spleen in SRG-15 mice 2 were cytotoxic NK cells (CD16+), while in IELs there was a similar distribution of CD16+ NK cells to CD16- NK cells ( Fig.19B There was no change in the number of lamina propria lymphocytes between SRG-implanted mice and SRG-15 mice ( Fig.19C ). Unlike SRG-15-implanted mice 1, a greater proportion of human CD3+CD8+ IELs expressed the human CD103 marker in SRG-15-implanted mice 2. Peyer's patches were completely absent in SRG mice, but they were present in SRG-15 mice 2 and were populated with human lymphocytes, as shown in Figure 2. Fig. 20A and 20B as shown in .

[0537] Example 5: Determining the functional role of human tissue-resident T cells in SRG-15 mice during viral infection

[0538] To test whether tissue-resident T cells in SRG-15 mice have functional relevance during homeostasis, we determined the expression of human CD8 + Whether the increase in the number of IELs induces characteristic changes in the composition of the mouse gut microbiota.

[0539] Materials and methods

[0540] Newborn mice were sublethally irradiated with 160 cGy without anesthesia at 3-5 days after birth and returned to their mothers to rest. These newborns were transplanted intrahepatically (ih) with 25 μl PBS containing CD34+ huHSCs using a 30G needle 4-12 hours after irradiation.

[0541] At 4 weeks post-implantation, SRG-15 mice were co-housed with SRG and donor Balb / c mice for 4 weeks to balance the gut microbiota between the different strains. The mice were then separated and fecal samples were collected and analyzed by 16S rRNA sequencing. Fig.21A Provides a timeline of co-housing and fecal sample collection for gut microbiota sequencing.

[0542] result

[0543] As described in 21B, for mouse 1, the results showed that there was no significant change between SRG-15-implanted mice and SRG mice after co-housing, indicating that the human CD8 +IELs do not induce major changes during steady-state conditions. Additional experiments were performed to determine whether CD8 + Can IELs eliminate rotavirus infection in SRG-15 mice? Fig. 22 As shown in , the results indicate that acute rotavirus infection can be cleared in SRG-15-implanted mice but not in non-SRG-implanted mice.

[0544] Example 6: Analysis of NK cell subsets in SRG-15 mice (mouse 2) and humans

[0545] Various phenotypic markers of NK cell subsets in SRG-15(mouse 2) mice were characterized and compared with humans.

[0546] Materials and methods

[0547] NK cell subsets were detected by 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) as generally described in Yao et al. J. of Immunological Methods 415 (2014) 1-5.

[0548] result

[0549] Fig.23A Provided are ViSNE images showing CyTOF-based analysis of CD56 in humans (n=20) and SRG-15 mice (mouse 2) (n=9). 明亮 CD16 - and CD56 暗淡 CD16 + Analysis of 33 parameters of NK cell subsets. Each point represents a single cell.

[0550] Fig. 23B ViSNE plots are provided showing CD56 in humans (n=20) and SRG-15 mice (mouse 2) (n=9) for the eight selected markers. 明亮 CD16 - Expression intensity on NK cells.

[0551] Fig.23C ViSNE plot showing CD56 in humans (n=20) and SRG-15 mice (n=9) for 8 selected markers 暗淡 CD16 +Expression intensity on NK cells. This multidimensional single-cell analysis of 33 key molecules of human NK cells indicated that human NK cells generated in SRG-15 mice were highly similar to those in healthy individuals.

[0552] Example 7: Cytotoxicity of NK cells from SRG-15 mice

[0553] Materials and methods

[0554] For in vitro NK cytotoxicity studies, spleen NK cells isolated from human HSC-implanted SRG and SRG-15 mice (mouse 2) were treated with human IL-2 overnight. The next day, NK cells were cultured with CFSE-labeled NK-susceptible K562 target cells at different effector to target ratios (E:T). After 5 hours of co-culture, killing of K562 cells was measured by FACS analysis of the uptake of the viability dye Topro3 by K562 cells (gating on CFSE+ cells to distinguish K562, followed by analysis of the percentage of Topro3 positivity).

[0555] In addition, for in vitro antibody-dependent cellular cytotoxicity (ADCC) studies, spleen NK cells isolated from SRG and SRG-15 mice implanted with human HSC were treated overnight with human IL-2. The next day, NK cells were cultured with CFSE-labeled Raji target cells at different effector to target ratios (E:T). Raji cells were pretreated with CD20 antibody (rituximab) or control IgG. After 5 hours of co-culture, killing of Raji cells was measured by FACS analysis of the uptake of the viability dye Topro3 by Raji cells (gating on CFSE+ cells followed by analysis of the Topro3 positive percentage).

[0556] For in vivo NK cell activation studies, SRG and SRG-15 mice implanted with human HSCs (mouse 2) were injected intraperitoneally with 50 μg of polyIC. Mice were bled pre-existing (before polyIC injection) and 18 hours after polyIC injection. Expression of the activation marker CD69 of human CD45+NKp46+ (NK cells) was analyzed by FACS before and after polyIC administration.

[0557] result

[0558] In the classical NK cytotoxicity studies, the classical NK target class I HLA-deficient K562 cells were subjected to killing by activated NK cells from SRG or SRG-15 mice (mouse 2). Fig.24C As shown in (left), splenic NK cells from SRG and SRG-15 mice displayed similar cytolytic capacity relative to K562 cells when normalized for number.

[0559] NK cells are generally responsible for anti-CD20 antibody-mediated ADCC against B-cell leukemias and lymphomas (see, e.g., J. Golay et al. Haematologica 2003; 88: 1002-12). To demonstrate that NK cells from SRG-15-implanted mice were able to promote CD20 antibody-mediated ADCC, splenic NK cells from both SRG mice and SRG-15 mice were tested and shown to exhibit similar antibody-dependent cellular cytotoxicity (ADCC) activity relative to anti-CD20-treated Raji cells when normalized for cell number ( Fig.24C (right side)).

[0560] For example, in Figure 8 and 9A -9E, there was a significant upregulation of NK cells in both the spleen and blood of SRG-15 animals. The ability to activate NK cells in SRG-15 mice was tested by measuring CD69 marker activation after injection of poly IC. Fig.24A As shown in , the percentage of NK cells positive for the activation marker CD69 was increased in SRG-15 mice relative to SRG mice. Because SRG-15 NK cells were shown to mediate ADCC similar to SRG NK cells in vitro under standardized conditions, the ability of SRG-15 NK cells to exhibit a greater activation phenotype in vivo and the greater number of NK cells in SRG-15 mice suggest that SRG-15 mice may be a suitable in vivo model for studying human NK cell ADCC.

[0561] Example 8: IFNγ production by SRG and SRG-15 derived NK cells

[0562] Materials and methods

[0563] NK cells were isolated from confluent splenocytes of SRG or SRG-15 mice (3 spleens per group) and isolated using the EasySep Human NK Enrichment Kit (StemCell Technologies; Cat. No. 19055).

[0564] NK cells were also isolated from healthy human PBMC. NK cells were treated overnight with 10ng / mL human IL-2. The next day, cells were stimulated overnight with 10ng / mL human IL-12p70 or 2mg / mL poly I:C, or kept untreated. The next day, supernatants were collected, and IFNg levels were assessed using human IFNg Quantikine ELISA kit (R&D systems; catalog number DIF50). NK cell purity was analyzed by FACS, and IFNg levels were standardized to picograms (pg) produced by individual NK cells. Statistical analysis was performed using ANOVA.

[0565] result

[0566] like Fig. 24B As shown in , after IL-12p70 treatment, SRG- and SRG-15-derived NK cells had similar IFNγ secretion, but less than that of human PBMC-derived NK cells.

[0567] Example 9: Human NK cells inhibit tumor growth in SRG-15 mice

[0568] Human NK cells were tested for their ability to infiltrate human tumor xenografts and inhibit tumor growth in SRG-15 mice (Mouse 2).

[0569] Materials and methods

[0570] Rituximab was injected intraperitoneally every other day (starting on day 14 after subcutaneous injection of 5 million Raji cells). Tumor growth was assessed by caliper measurement, and volume was calculated using the following formula: Tumor volume = 0.5 x (length x width^2). Data were pooled from 2 independent experiments. Statistical analysis was performed using an unpaired two-tailed Mann-Whitney U test comparing implanted untreated SRG-15 mice and implanted RTX-treated SRG-15 mice (*P < 0.05).

[0571] Subcutaneous tumors were crushed and digested using collagenase D (1 hour, 37°C). Recovered cells including tumor and immune cells were analyzed by LSRII flow cytometry.

[0572] result

[0573] like Fig.25A As shown in , human NK cells in SRG-15 mice inhibited tumor growth after treatment with rituximab (RTX). Fig.25B Shown are the frequencies of human NK cells and T cells in human tumor xenografts of untreated (n=5) and RTX-treated SRG-15 mice (n=1). Fig.25CHuman NK cell subsets in blood and tumors of untreated (n=2) and RTX-treated SRG-15 mice (n=1) are shown.

[0574] Example 10: Additional Materials and Methods Utilized in Connection with the Above Examples

[0575] Human CD34 + Cell isolation and injection. Human CD34 was isolated and injected according to the method described in Rongvaux A, Willinger T, Martinek J et al. Nat Biotechnol 2014; 32: 364-372. + Cell isolation and injection.

[0576] Flow cytometric analysis of human cell populations. Flow cytometric analysis of human cell populations was performed as 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.

[0577] Histology. Tissues were fixed overnight in 4% paraformaldehyde, transferred to 70% ethanol, and embedded in paraffin.

[0578] Quantitative RT-PCR. Quantitative RT-PCR was performed as described in Rongvaux A, Willinger T, Martinek J et al. Nat Biotechnol 2014; 32: 364-372.

[0579] 16S rRNA sequencing. 16S rRNA sequencing was performed as described in Palm NW, de Zoete MR, Cullen TW et al. Cell 2014; 158: 1000-1010.

[0580] Viral Infections. Rotavirus and influenza virus were obtained and used in the subject methods.

[0581] Statistical analysis: Statistical significance analysis was performed using two-tailed unpaired Student's t test using Prism 6 software (GraphPad).

[0582] FACS antibodies were obtained from BD Biosciences and BioLegend.

[0583] The foregoing only illustrates the principle of the present invention. It should be understood that those skilled in the art will be able to design various arrangements that embody the principle of the present invention, and are included in the spirit and scope of the present invention, although not explicitly described or shown herein. In addition, all examples and conditional wordings described herein are mainly intended to help readers understand the principle of the present invention and the conception contributed by the inventor to enhance the art, and should be interpreted as not being limited to the examples and conditions explicitly described. In addition, all statements herein that describe the principles, aspects and embodiments of the present invention and its specific examples are intended to cover both its structural equivalents and functional equivalents. In addition, it is intended that the equivalents include both currently known equivalents and future developed equivalents (i.e., any elements that perform the same function, regardless of structure, developed). Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. More specifically, the scope and spirit of the present invention are embodied by the appended claims.

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Claims

1. A method for preparing a genetically modified rodent, the method comprising producing a genetically modified rodent, the genome of the genetically modified rodent comprising: Humanized signal regulatory protein α ( SIRPα ) gene, the humanized SIRPα gene comprises a nucleic acid sequence encoding a humanized SIRPα (hSirpα) protein, wherein the humanized SIRPα The gene is operably linked to SIRPα Gene promoter, the SIRPα The gene promoter is a rodent in the genome of the genetically modified rodent SIRPα Endogenous rodent loci SIRPα Gene promoter, wherein the humanized SIRPα A gene comprising replacement of a portion of an endogenous rodent SIRPα coding sequence, wherein the genetically modified rodent expresses the hSIRPα protein, and wherein the hSIRPα protein has the extracellular domain of human SIRPα, wild-type human SIRPα receptor function and comprises the signaling domain of the endogenous mouse SIRPα protein; and Humanized interleukin-15 ( IL-15 ) gene, the humanized IL-15 The gene includes a nucleic acid sequence encoding human IL-15 (hIL-15) protein, wherein the nucleic acid sequence includes human IL-15 Exons 5, 6, 7 and 8 of a gene, wherein a nucleic acid sequence encoding a human IL-15 (hIL-15) protein is operably linked to IL-15 Gene promoter, the IL-15 The gene promoter is a rodent in the genome of the genetically modified rodent IL-15 Endogenous rodent loci IL-15 Gene promoter, wherein the humanized IL-15 The gene comprises a partial or complete replacement of an endogenous rodent IL-15 coding sequence, wherein the genetically modified rodent expresses the hIL-15 protein, and wherein the hIL-15 protein has wild-type human IL-15 signaling function; Wherein, the genetically modified rodent is a mouse or a rat.

2. The method according to claim 1, wherein: The humanization SIRPα Genes include rodents SIRPα Exon 1 of gene, human SIRPα Exons 2, 3, and 4 of the gene, and the rodent SIRPα Exons 5, 6, 7, and 8 of the gene.

3. The method according to claim 1, wherein: The genetically modified rodent is humanized SIRPα Genetically homozygous.

4. The method according to claim 1, wherein: The humanization IL-15 The gene includes exons 3 and 4 of the rodent IL-15 gene.

5. The method according to claim 1, wherein: The genetically modified rodent is homozygous for the humanized IL-15 gene.

6. The method according to claim 1, wherein: The humanized SIRPα gene includes a gene after replacing exons 2, 3 and 4 of the rodent SIRPα gene at the endogenous rodent SIRPα gene locus with exons 2, 3 and 4 of the human SIRPα gene; The humanization IL-15 Genes include people IL-15 Exons 5, 6, 7, and 8 of the gene are substituted at the endogenous rodent IL-15 locus IL-15 genes after exons 5, 6, 7, and 8; and The genetically modified rodent is humanized SIRPα Genetically homozygous.

7. The method according to claim 1, wherein: The genetically modified rodent is immunodeficient.

8. The method according to claim 7, wherein: The genetically modified rodents include Rag2 knockouts.

9. The method according to claim 7, wherein: The genetically modified rodents include IL2rg gene knockouts.

10. The method according to claim 7, wherein: The genetically modified rodents include Rag2 knockout and IL2rg knockout.

11. The method according to claim 7, wherein: The genetically modified rodent includes an implant of human hematopoietic cells.

12. The method according to claim 11, wherein: The genetically modified rodents include infection with human pathogens.

13. The method according to claim 12, wherein: The human pathogen is selected from the group consisting of: Human pathogens that activate, induce and / or target T cells; human pathogens that activate, induce and / or target natural killer (NK) cells; human pathogens that infect the human intestine; human pathogens that infect the human lung.

14. The method according to claim 13, wherein: The human pathogen that infects the human intestine is human rotavirus.

15. The method according to claim 13, wherein: The human pathogen that infects human lungs is influenza virus.

16. The method according to claim 7, wherein: The genetically modified rodents contain transplanted human tumors.

17. The method according to any one of claims 1 to 16, wherein: The genetically modified rodent is a mouse.

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