Humanized Mouse Models

Abstract

The human immune system is complex, and so are its associated diseases. Thus, human diseases (e.g., cancer) are often difficult to characterize and effectively treat. Provided herein are immunodeficient transgenic mice for use as humanized mouse models. Said humanized mouse models can further be transplanted with pathological tissues or cells (e.g., human cancer cells). Also provided herein are methods for evaluating disease progression, immune response, and the effectiveness of proposed therapeutic agents using said humanized mouse models.

Description

[Background technology]

[0001] background Human immune system is complex, and its related diseases are also complex.Therefore, human diseases (such as cancer) are often difficult to characterize and effectively treat.There is a continuing need for animal models that allow the separation of aspects of immune response, and provide methods and compositions that are useful, for example, for identifying effective medical and pharmaceutical treatments. Summary of the Invention [Means for solving the problem]

[0002] overview The present disclosure provides, in some aspects, an immunodeficient transgenic mouse comprising a transgene encoding human interleukin-3 (IL-3), a transgene encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), a transgene encoding human stem cell factor (SCF), and a transgene encoding human IL-15. As described herein, the immunodeficient transgenic mouse represents an improved humanized mouse model, at least in part due to its physiological properties, which more closely mimic human physiological properties over previous humanized mouse models. Humanization of the immunodeficient transgenic mouse provided herein allows for disease modeling and evaluation of therapeutic agents (e.g., candidate therapeutic agents). Unexpectedly, data provided herein show that exposure of immunodeficient mice to myeloablative treatments (e.g., irradiation) enhances immunoglobulin production, independent of humanization. Interestingly, different doses of irradiation lead to different outcomes. Mice exposed to 50 cGy of irradiation, for example, produced the highest levels of human immunoglobulin of any dose tested.

[0003] Some aspects provide a method of generating a mouse model of human humoral and cellular immunity comprising: (a) providing an immunodeficient mouse containing a transgene encoding human interleukin-3 (IL-3), a transgene encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), a transgene encoding human stem cell factor (SCF), and a transgene encoding human IL-15; (b) administering a myeloablative treatment to the immunodeficient mouse; and (c) administering human peripheral blood mononuclear cells (hPBMCs) to the immunodeficient mouse.

[0004] In some embodiments, the myeloablative treatment is irradiation.

[0005] In some embodiments, the irradiation is gamma irradiation (cGy). In some embodiments, the myeloablative treatment is ≦150 cGy. In some embodiments, the myeloablative treatment is ≦100 cGy. In some embodiments, the myeloablative treatment is about 50 to about 100 cGy. In some embodiments, the myeloablative treatment is 40 to 60 cGy. In some embodiments, the myeloablative treatment is about 50 cGy.

[0006] In some embodiments, 1×10 7 Fewer than 0.5×10 hPBMCs are administered to the immunodeficient mouse. 6 pieces~5×10 6 The hPBMCs are administered to the immunodeficient mice.

[0007] In some embodiments, administration of the hPBMCs is within 3 days of administration of the myeloablative treatment. In some embodiments, administration of the hPBMCs is on the same day as administration of the myeloablative treatment. In some embodiments, administration of the hPBMCs is within 6 hours of administration of the myeloablative treatment.

[0008] In some embodiments, the method further comprises administering to the immunodeficient mouse a human pathological cell.

[0009] In some embodiments, the method further comprises administering to the immunodeficient mouse a human therapeutic or prophylactic agent (or candidate human therapeutic).

[0010] In some embodiments, the human therapeutic agent (or candidate human therapeutic agent) is selected from a human immunomodulatory agent. In some embodiments, the human therapeutic agent (or candidate human therapeutic agent) is selected from a monoclonal antibody. In some embodiments, the human immunomodulatory agent is selected from a cell therapy, optionally a T cell therapy.

[0011] In some embodiments, the method further comprises administering to the immunodeficient mouse a human therapeutic or prophylactic agent that is a vaccine.

[0012] In some embodiments, the vaccine is a protein antigen or a nucleic acid encoding a protein antigen, optionally a cancer antigen or a pathogenic antigen.

[0013] In some embodiments, administration of the human therapeutic agent (or candidate human therapeutic agent) is within 30 days of administration of the myeloablative treatment. In some embodiments, administration of the human therapeutic agent (or candidate human therapeutic agent) is within 28 days of administration of the myeloablative treatment. In some embodiments, administration of the human therapeutic agent (or candidate human therapeutic agent) is within 21 days of administration of the myeloablative treatment. In some embodiments, administration of the human therapeutic agent (or candidate human therapeutic agent) is within 14 days of administration of the myeloablative treatment.

[0014] In some embodiments, the method further includes assaying a sample from the mouse for one or more human cytokines. The sample can be, for example, a blood sample.

[0015] In some embodiments, the one or more human cytokines are selected from interferon gamma (IFN-γ), interleukin (IL)-2, IL-4, IL-6, IL-10, and tumor necrosis factor alpha (TNFα).

[0016] In some embodiments, the method further includes assaying a sample from the mouse for one or more human immunoglobulins. The sample can be, for example, a blood sample.

[0017] In some embodiments, the one or more human immunoglobulins are selected from IgM, IgA, IgG, optionally IgG1, IgG2, IgG3, and IgG4.

[0018] In some embodiments, the method further comprises assaying a sample from the mouse, optionally a blood sample, for one or more anti-drug antibodies.

[0019] Some aspects provide a mouse model of human humoral and human cellular immunity generated by a method of any one of the preceding aspects and / or embodiments.

[0020] Another aspect provides an immunodeficient mouse comprising a transgene encoding human interleukin-3 (IL-3), a transgene encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), a transgene encoding human stem cell factor (SCF), and a transgene encoding human IL-15.

[0021] In some embodiments, the immunodeficient mouse has a non-obese diabetic genetic background.

[0022] In some embodiments, the immunodeficient mouse is a mouse with a severe combined immunodeficiency mutation (Prkdc scid).

[0023] In some embodiments, the immunodeficient mouse is a mouse with a null allele of the IL2 receptor common gamma chain (IL2rg null ).

[0024] In some embodiments, the immunodeficient mouse is a NOD-scid IL2Rgamma null Has a genetic background.

[0025] In some embodiments, the immunodeficient mouse has been exposed to a myeloablative treatment. In some embodiments, the myeloablative treatment is irradiation.

[0026] In some embodiments, the cells of the immunodeficient mice produce human IgM, human IgG1, human IgG2, human IgG3, and human IgG4.

[0027] In some embodiments, the cells of the immunodeficient mouse produce 6000 to 16000 μl / mL of human IgG. In some embodiments, the cells of the immunodeficient mouse produce 1000 to 2000 μl / mL of human IgM. In some embodiments, the cells of the immunodeficient mouse produce 1500 to 2500 μl / mL of human IgG1. In some embodiments, the cells of the immunodeficient mouse produce 500 to 1500 μl / mL of human IgG2. In some embodiments, the cells of the immunodeficient mouse produce 150 to 205 μl / mL of human IgG3. In some embodiments, the cells of the immunodeficient mouse produce 50 to 1000 μl / mL of human IgG4.

[0028] In some embodiments, the immunodeficient mouse is + , CD4 + , CD8 + and CD19 + Generate cells. [Brief description of the drawings]

[0029] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A-B] Figures 1A-1F show a comparison of human CD45+ engraftment in NSG-SGM3 and NSG-SGM3xIL15 mice transplanted with CD34+ hematopoietic stem cells (HSCs) from three different donors. Figures 1A-1C show the frequency of CD45+ cells from three different donors, while Figures 1D-1F show the corresponding CD45+ cells / microliter from the three donors. [Figure 1C-D] Same as above. [Figure 1E-F] Same as above. [Figure 2A] Figures 2A-2F show a comparison of human natural killer (NK), T, B, and myeloid cells over time in NSG-SGM3 and NSG-SGM3xIL15 mice transplanted with CD34+ HSCs from three different donors. Figures 2A-2C highlight the percentages of various human immune cells in NSG-SGM3 mice, and Figures 2D-2F highlight the percentages of various human immune cells in NSG-SGM3xIL15 mice. Arrows point to NK cell data. [Figure 2B-C] Same as above. [Fig. 2D-E] Same as above. [Figure 2F] Same as above. [Figure 3A-B] Figures 3A-3F show a comparison of human NK cells over time in NSG-SGM3 and NSG-SGM3xIL15 mice transplanted with CD34+ HSCs from three different donors. Figures 3A-3C show NK cells as a percentage of CD45+ cells (cell frequency) and Figures 3D-3F show NK cells / microliter. [Figure 3C-D] Same as above. [Figure 3E-F] Same as above. [Figure 4A-B]Figures 4A-4F show a comparison of tumor growth kinetics over time in NSG®, NSG-SGM3 and NSG-SGM3xIL15 mice engrafted with CD34+ HSCs and implanted orthotopically with human cell line-derived xenografts (CDX; MDA-MB-231, Figures 4A and 4D) or subcutaneously with patient-derived xenografts (PDX). The two patient-derived xenografts (PDX) used were: PS4050 (Figures 4B and 4E) or LG1306 (Figures 4C and 4F). Figures 4A-4C show the average tumor volume of the three xenograft models and Figures 4D-4F show the individual tumor sizes over time. [Figure 4C-D] Same as above. [Figure 4E-F] Same as above. [Diagram 5] Figures 5A-5B show the frequency of human regulatory T (Treg) cells in the blood of NSG-SGM3 and NSG-SGM3xIL15 mice transplanted with CD34+ HSCs (Figure 5A) and PD-1 expression on CD4+ and CD8+ T cells in the spleen (Figure 5B). [Figure 6A-B] Figures 6A-6C show a comparison of leukocytes (Figure 6A), human myeloid cells (Figure 6B), and human NK cells (Figure 6C) in NSG-SGM3 and NSG-SGM3xIL15 mice transplanted with 5 x 106 human peripheral blood mononuclear cells (PBMCs) per mouse. [Figure 6C] Same as above. [Figure 7A] 7A-7C show a comparison of activated T cells in the spleens of NSG-SGM3 and NSG-SGM3xIL15 mice engrafted with 5×10 6 human PBMCs per mouse. [Figure 7B-C] Same as above. [Figure 8A] Figures 8A-8C show a comparison of human CD45+ cells (Figure 8A), human myeloid cells (Figure 8B), and human NK cells (Figure 8C) in NSG-SGM3 and NSG-SGM3xIL15 mice engrafted with 1 x 106 or 5 x 106 human PBMCs per mouse using two different irradiation doses (150 and 175 cGy). [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A-B] Figures 9A-9C show a comparison of TNF (Figure 9A), IL-6 (Figure 9B), and IL-2 (Figure 9C) levels in NSG-SGM3 and NSG-SGM3xIL15 mice engrafted with 5 x 106 human PBMCs per mouse. [Figure 9C] Same as above. [Figure 10A] Figures 10A-10C show a comparison of IFN-γ (Figure 10A), TNF (Figure 10B), and IL-6 (Figure 10C) levels in NSG-SGM3 and NSG-SGM3xIL15 mice engrafted with 1 x 106 or 2 x 106 human PBMCs per mouse using two different radiation doses (150 and 175 cGy). [Figure 10B-C] Same as above. [Figure 11] Figures 11A-11B show a comparison of humanization in NSG-SGM3xIL15 mice transplanted with PBMCs from a single donor with or without irradiation. Figure 11A shows human CD45+ cells as cells / μL at 7, 14 and 21 days post-transplant. Figure 11B shows human CD45+ cells as a percentage of total viable cells at 7, 14 and 21 days post-transplant. [Figure 12] Figures 12A-12B show a comparison of humanization in NSG-SGM3xIL15 mice transplanted with PBMCs from two different donors, with or without irradiation. Figure 12A shows human CD45+ cells as a percentage of total live cells at 14 and 21 days post-transplant. Figure 12B shows human CD45+ cells as cells / μL at 14 and 21 days post-transplant. [Figure 13] FIG. 13 shows a comparison of circulating human IgG levels in NSG-SGM3xIL15 mice 14 and 21 days after transplantation of PBMC from three different donors with or without irradiation. [Figure 14]14A-14B show a comparison of human Ig isotypes in serum of NSG-SGM3xIL15 mice 14 days (FIG. 14A) and 21 days (FIG. 14B) after transplantation of PBMCs derived from a single donor with or without irradiation. [Figure 15] Figures 15A-15B clearly show a comparison of human immune cell populations in NSG-SGM3xIL15 mice 14 days (Figure 15A) and 21 days (Figure 15B) after transplantation of PBMCs derived from a single donor with or without irradiation. [Figure 16] 16A-16B show a comparison of humanized (FIG. 16A) and circulating human immunoglobulin G (hIgG) levels (FIG. 16B) in PBMC-engrafted NSG-SGM3xIL15 mice at various radiation doses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description The present disclosure provides, in some embodiments, an immune-deficient mouse model expressing human IL-3, human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), human SCF, and human IL-15. In some embodiments, the mouse is generated by crossing an NSG-SGM3 mouse with an NSG-IL-15 mouse. The mouse is, in some embodiments, humanized with CD34+ hematopoietic stem cells (HSC) or peripheral blood mononuclear cells (PBMC), and unexpectedly has a more physiological and expanded humanized immune profile relative to either of the two parental strains. For example, as described herein, when the mouse model is humanized with PBMC, a higher level of human immune cells (e.g., CD45+ cells) is generated than either of the parental strains. In this way, it is surprisingly possible to generate immune cells from a small number of patient PBMCs (e.g., 1×10 6To a lesser extent, patient PBMCs can be used to humanize the mice. This is important since patient PBMCs are often present in very low numbers, especially early in the disease. The human immune cell populations have higher levels of activated T cells and higher levels of natural killer (NK) cells in the blood in the mouse models provided herein versus the parental lineage. Similarly, human physiological levels of T regulatory cells and PD-1+ T cells are generated in the mice, thereby increasing the sensitivity for screening immunomodulatory agents (e.g., checkpoint inhibitor drugs). In some embodiments, the mouse models contain significantly more human T cells, human B cells, and / or human NK cells in the peripheral blood versus humanized NSG-SGM3 mice.

[0031] Also provided herein are methods of generating the mouse models, humanizing the mouse models, and using the mouse models to evaluate, for example, disease progression (e.g., cancer and / or other diseases of the human immune system) and therapeutic agents (e.g., candidate therapeutic agents). The models provided herein, in some embodiments, are further engrafted with cells (e.g., mammalian (e.g., human) cells), such as human diseased cells (e.g., human cancer cells). In some embodiments, the human cells are derived from patient-derived xenografts (PDX) or human cell lines (e.g., human cancer cell lines). Diseased cells and tissues are associated with unique genetic profiles used herein to generate mouse models that can be utilized, for example, to explore genetic components that may underlie certain diseases (e.g., cancer, autoimmune diseases, and other inflammatory diseases). By replicating the human immune system, these models can also be used in some embodiments to evaluate toxicity (e.g., cytokine release syndrome) and other side effects of certain therapeutic agents aimed at preventing or treating disease.

[0032] The animal model may be, but is not limited to, a non-human mammal, a rodent (e.g., mouse, rat, or hamster), or a livestock animal (e.g., pig, cow, chicken, or goat) model. In some embodiments, the animal is a rodent. In some embodiments, the animal is a mouse.

[0033] For simplicity, reference will be made herein to "mouse" and "mouse model" (e.g., surrogate for the human condition). It should be understood that these terms may be used interchangeably throughout the specification to encompass "rodent" and "rodent model," including mice, rats and other rodent species, unless otherwise stated.

[0034] It can also be understood that the standard genetic nomenclature used herein provides a unique identification for different rodent strains, and the strain symbol conveys basic information about the type of strain or stock used and the genetic content of the strain. The rules for symbolizing strains and stocks are published by the International Committee on Standardized Genetic Nomenclature for Mice, which are available online at the Mouse Genome Database (MGD; informatics.jax.org) and have also been published in print (Lyon et al. 1996). The strain symbol typically includes a Laboratory Registration Code (Lab Code). The registration is maintained at the Institute for Laboratory Animal Research (ILAR) of the National Academy of Sciences (Washington, DC). The Lab Code can be obtained electronically at the ILAR website (nas.edu / cls / ilarhome.nsf). See also Davisson MT, Genetic and Phenotypic Definition of Laboratory Mice and Rats / What Constitutes an Acceptable Genetic-Phenotypic Definition, National Research Council (US) International Committee of the Institute for Laboratory Animal Research. Washington (DC): National Academies Press (US); 1999.

[0035] The mouse model of disease can be modified to allow evaluation of disease. Any system (e.g., immune, respiratory, nervous, or circulatory), organ (e.g., blood, heart, blood vessel, spleen, thymus, lymph node, or lung), tissue (e.g., epithelial tissue, connective tissue, muscle tissue, and nervous tissue), or cell type (e.g., lymphocyte or macrophage), either independently or in combination, can be modified to allow disease study in the model provided herein.

[0036] Three conventional methods used for the generation of transgenic mice include DNA microinjection (Gordon and Ruddle, Science 1981: 214: 1244-124, incorporated herein by reference), embryonic stem cell-mediated gene transfer (Gossler et al., Proc. Natl. Acad. Sci. 1986, 83: 9065-9069, incorporated herein by reference), and retrovirus-mediated gene transfer (Jaenisch, Proc. Natl. Acad. Sci. 1976, 73: 1260-1264, incorporated herein by reference), any of which may be used as provided herein. For example, genome editing methods using clustered regularly interspace palindromic repeats (CRISPR / Cas) nucleases, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs) are described elsewhere herein.

[0037] Following delivery of the nucleic acid into a fertilized embryo (e.g., a single cell embryo (e.g., a zygote) or a multicellular embryo (e.g., a post-zygote developmental stage (e.g., a blastocyst)), the fertilized embryo is transferred to a pseudopregnant female, which subsequently gives birth to an offspring. The presence or absence of nucleic acid encoding a human FcRn and / or a chimeric IgG antibody can be confirmed, for example, using any number of genotyping methods (e.g., sequencing and / or genomic PCR).

[0038] New mouse models can also be created by breeding parental lines, as described in the examples herein.With available mutants, knockout, knock-in, transgenic, Cre-lox, Tet-inducible system, and other mouse lineage variants, multiple mutations and transgenes can be combined to generate new mouse models.Multiple mouse lines can be bred together to generate double, triple, or even quadruple and more advanced multiple mutant / transgenic mice.

[0039] In some embodiments, parent mice are bred to generate F1 mice.Parent mice can be, for example, homozygous, heterozygous, hemizygous, or homozygous null at a particular allele.Homozygous describes the genotype of two identical alleles at a given locus, heterozygous describes the genotype of two different alleles at a locus, hemizygous describes the genotype of only a single copy of a particular gene in an otherwise diploid organism, and homozygous null refers to an otherwise diploid organism in which both copies of a gene are missing.

[0040] Immunodeficiency Mouse Model In some embodiments, provided herein is an immunodeficient mouse model comprising a transgene encoding human interleukin-3 (IL-3), a transgene encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), a transgene encoding human stem cell factor (SCF), and a transgene encoding human IL-15.

[0041] As known in the art, immunodeficient mice have impaired or disrupted immune systems, such as specific deficiencies in MHC class I, II, or both, B-cell or T-cell deficiencies, natural killer cell deficiencies, myeloid deficiencies (e.g., granulocyte and / or monocyte deficiencies), and immunodeficiencies resulting from gene knockdown of cytokines, cytokine receptors, TLR receptors, and various transducers and transcription factors of signal transduction pathways. Immunodeficient mouse models include single gene mutation models (e.g., nude mouse (nu) and severe combined immunodeficient (scid) strains, non-obese diabetic (NOD) strains, RAG (recombinant activating gene) strains with targeted gene deletions, and various hybrids generated by crossing double and triple mutant mouse strains with additional deficiencies in innate and adaptive immunity.

[0042] Non-limiting examples of spontaneous and transgenic immunodeficient mouse models include the following mouse strains: Nude (nu) [Flanagan SP. Genet Res 1966; 8: 295-309; and Nehls M et al. Nature 1994; 372: 103-7]; Scid (scid) [Bosma GC et al. Nature 1983; 301:527-30; Mosier DE et al. Nature 1988; 335: 256-9; and Greiner DL et al. Stem Cells 1998; 16: 166-77]; NOD [Kikutani H et al. Adv Immunol 1992; 51: 285-322; and Anderson MS et al. Ann Rev Immunol 2005; 23: 447-85]; RAG1 and RAG2 (rag) [Mombaerts P et al. Cell 1992; 68: 869-77; Shinkai U et al. Cell 1992; 68: 855-67]; NOD-scid [Greiner DL et al. 1998; Shultz LD et al. J Immunol 1995; 154: 180-91; Melkus MW et al. Nature Med 2006; 12: 1316-22; and Denton PW et al. PLoS Med 2008; 4(12): e357]; IL2rg null [DiSanto JP et al. Proc Natl Acad Sci USA 1995; 92: 377-81]; · B2m null [Christianson SW et al. J Immunol 1997;158:3578-86]; NOD-scid IL2rγ null [Shultz LD et al. Nat Rev Immunol 2007;7:118-30; Ito M et al. Blood 2002;100:3175-82; Ishikawa I et al. Blood 2005;106:1565-73; and Macchiarini F et al. J Exp Med 2005;202:1307-11]; NOD-scid B2m null [Shultz et al. 2007; Shultz LD et al. Transplantation 2003; 76: 1036-42; Islas-Ohlmayer MA et al. J Virol 2004; 78:13891-900; and Macchiarini et al. 2005]; HLA transgenic mice [Grusby MJ et al. Proc Natl Acad Sci USA 1993; 90(9): 3913-7; and Roy CJ et al. Infect Immun 2005; 73(4): 2452-60]. See also, e.g., Belizario JE The Open Immunology Journal, 2009; 2:79-85; ·NOG mice (NOD.cg-Prkdc scid Il2rg tm1Sug ) [Shultz LD et al. Nat Rev Immunol 2007;7:118-30]; and ·BRG mice (BALB / c; 129S4- Rag2tm1.1Flv ) [Song J et al. Cell Host Microbe 2010; 8(4): 369-76; Goldman JP et al. Br J Haematol. 1998; 103: 335-342].

[0043] In some embodiments, provided herein is an immunodeficient mouse model having a non-obese diabetic (NOD) mouse genotype. The NOD mouse (e.g., Jackson Labs Stock #001976, NOD-Shi LtJ NOD mice are a polygenic mouse model of autoimmune (e.g., type 1) diabetes characterized by hyperglycemia and insulitis, leukocyte infiltration of islet cells. The NOD mice are hypoinsulinemic and hyperglucagonemic and exhibit selective destruction of islet β cells. A major component of diabetes susceptibility in NOD mice is the unique MHC haplotype. NOD mice also exhibit multiple abnormal immune phenotypes, including defective antigen-presenting cell immunoregulatory function, defects in regulating the T lymphocyte repertoire, defective NK cell function, defective cytokine production from macrophages (Fan et al., 2004), and impaired wound healing. They also lack the hemolytic complement, C5. NOD mice are also severely deaf. Various mutations causing immune deficiencies, targeted mutations in cytokine genes, as well as transgenes affecting immune function have been backcrossed into the NOD inbred background.

[0044] In some aspects of the present disclosure, the immunodeficient mice provided herein based on a NOD background are NOD-Cg.-Prkdc scid IL2rg tm1wJl / SzJ (NSG®), NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl / SzJ(NRG), and NOD.Cg-Prkdc scid Il2rg tm1Sug / ShiJic(NOG). Other immunodeficient mouse strains are contemplated herein.

[0045] In some embodiments, the immunodeficiency mouse model based on a NOD background is NOD-Cg.-Prkdc scid IL2rg tm1wJl / SzJ(NSG TM ) genetic background. TM The NSG mice (e.g., Jackson Labs Stock No.: #005557) are immunodeficient mice that lack mature T cells, B cells, and NK cells, are defective in multiple cytokine signaling pathways, and have numerous defects in innate immunity (see, e.g., Shultz, Ishikawa, & Greiner, 2007; Shultz et al., 2005; and Shultz et al., 1995, each of which is incorporated herein by reference). TM The mice were derived from the NOD mouse strain NOD / ShiLtJ (see, e.g., Makino et al., 1980, which is incorporated herein by reference) and were engineered to express Prkdc scid mutation (also called "severe combined immunodeficiency" or "scid" mutation) and Il2rg tm1Wjl Includes targeted mutations. Prkdc scid The mutation is a loss-of-function (null) mutation in the mouse homolog of the human PRKDC gene - this mutation essentially eliminates adaptive immunity (see, e.g., (Blunt et al., 1995; Greiner, Hesselton, & Shultz, 1998), each of which is incorporated herein by reference). tm1Wjl The mutation is a null mutation in the gene encoding the interleukin 2 receptor gamma chain (IL2Rγ, homologous to IL2RG in humans), which blocks NK cell differentiation, thereby removing a roadblock that prevents efficient engraftment of primary human cells (Cao et al., 1995; Greiner et al., 1998; and Shultz et al., 2005, each of which is incorporated herein by reference). In some embodiments, the mouse model comprises NSG-SGM3 mice crossed with NSG-IL-15 mice.

[0046] In some embodiments, the immunodeficient mouse model has the NRG genotype. The NRG mouse (e.g., Jackson Labs Stock #007799) is severely immunodeficient. This mouse harbors two mutations on a NOD / ShiLtJ genetic background; a targeted knockout mutation in recombination activating gene 1 (Rag1) and a complete null allele of the IL2 receptor common gamma chain (IL2rg null ) above. Rag1 null The mutation causes mice to be deficient in B and T cells, and null The mutations impair cytokine signaling through multiple receptors, resulting in a deficiency in functional NK cells. The severe immunodeficiency of NRG mice is due to the expression of human CD34 + This allows for highly efficient humanization by transplantation of hematopoietic stem cells (HSCs) and patient-derived xenografts (PDXs).The immunodeficient NRG mice are more resistant to irradiation and genotoxic drugs than mice with a scid mutation in the DNA repair enzyme Prkdc.

[0047] In some embodiments, the immunodeficient mouse model is a NOG mouse. The NOG mouse (Ito M et al., Blood 2002) is an extremely severe combined immunodeficient (scid) mouse established by combining NOD / scid mice and IL-2 receptor-gamma chain knockout (IL2rγKO) mice (Ohbo K. et al., Blood 1996). The NOG mouse lacks T cells and B cells, lacks natural killer (NK) cells, shows reduced dendritic cell function and reduced macrophage function, and lacks complement activity.

[0048] In some embodiments, the immunodeficient mouse model has NCG genotype. The NCG mouse (e.g., Charles River Stock #572) was created by sequential CRISPR / Cas9 editing of Prkdc and Il2rg loci in NOD / Nju mice, generating mice that are coisogenic to NOD / Nju. The NOD / Nju has a mutation in the Sirpa (SIRPα) gene that allows transplantation of foreign hematopoietic stem cells. The Prkdc knockout generates a SCID-like phenotype that lacks proper T and B cell formation. The knockout of the Il2rg gene further exacerbates the SCID-like phenotype while also causing a reduction in NK cell generation.

[0049] In some embodiments, immunodeficient mouse models are provided herein that are deficient (e.g., lack) MHC class I, MHC class II, or MHC class I and MHC class II. Mice that are deficient in MHC class I and / or MHC class II do not express the same levels of MHC class I proteins (e.g., α-microglobulin and β2-microglobulin (B2M)) and / or MHC class II proteins (e.g., α-chain and β-chain) or do not have the same levels of MHC class I and / or MHC class II protein activity as non-immunodeficient (e.g., MHC class I / II wild type) mice. In some embodiments, the expression or activity of MHC class I and / or MHC class II proteins is reduced (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more) relative to non-immunodeficient mice. MHC class I, MHC class II, and immunodeficient mice deficient in MHC class I and MHC class II are described in International Publication No. WO 2018 / 209344, the contents of which are incorporated herein by reference. In some embodiments, the immunodeficient mice are H2-K1 tmlBpe , H2-Ab1 emlMvw , and H2-D1 tmlBpeThe present invention includes one or more alleles selected from the following:

[0050] In some embodiments, the immunodeficient mouse model further comprises an inactivated mouse Flt3 allele.In some embodiments, the immunodeficient mouse model expresses a human FLT3L transgene.In some embodiments, the immunodeficient mouse model further comprises an inactivated mouse Flt3 allele and expresses a human FLT3L transgene, for example, as described in WO 2020 / 168029 (the contents of which are incorporated herein by reference).

[0051] NSG-SGM3 mice are a derivative of the NSG mouse NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav / MloySzJ (Jackson Laboratory Stock No: 013062). Transgenic NSG-SGM3 mice express three human cytokines: human interleukin-3 (IL-3), human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), and human stem cell factor (SCF). NSG-SGM3 mice combine the characteristics of severely immunodeficient NSG mice with expression of human cytokines, IL-3, GM-CSF, and SCF, which support stable engraftment of myeloid lineages and regulatory T cell populations.

[0052] NSG-IL-15 mice, NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(IL15)1Sz / SzJ (Jackson Laboratory Stock No: 030890) expresses human IL15 and is combined with severely immunodeficient NOD scidγ (NSG) mice. Expression of human IL15, in some embodiments, enhances the development of human NK cells in immunodeficient mice transplanted with human stem cells.

[0053] Thus, the transgenic mice described herein can be generated by breeding an immunodeficient transgenic mouse containing a transgene encoding human interleukin-3 (IL-3), a transgene encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), a transgene encoding human stem cell factor (SCF), and a transgenic mouse containing a transgene encoding human IL-15. In some embodiments, the transgenic mice described herein are generated by breeding NSG-SGM3 mice with NSG-IL-15 mice.

[0054] Humanized Mouse Models In some embodiments, humanized immunodeficient mouse models and methods for generating said models are provided herein. Immunodeficient mice transplanted with functional human cells and / or tissues are referred to as "humanized mice". As used herein, the terms "humanized mouse", "humanized immunodeficient mouse", "humanized immunodeficient mouse" and plural versions thereof are used interchangeably to refer to immunodeficient mice humanized by transplantation of functional human cells and / or tissues. For example, mouse models can be transplanted with human hematopoietic stem cells (HSCs) (e.g., CD34+ HSCs) and / or human peripheral blood mononuclear cells (PMBCs). In some embodiments, mouse models are transplanted with human tissues (e.g., pancreatic islets, liver, skin, and / or solid or blood cancers). In other embodiments, mouse models can be genetically modified to convert endogenous mouse genes to human homologs (see, e.g., Pearson, et al., Curr Protoc Immunol., 2008, Chapter: Unit-15.21).

[0055] Humanized mice are generated by starting with an immunodeficient mouse (e.g., 2-week-old, 3-week-old, 4-week-old, 5-week-old, 6-week-old, 7-week-old, 8-week-old, 9-week-old, 10-week-old, or older) and, if necessary, depleting and / or suppressing any remaining mouse immune cells (e.g., chemically or by irradiation). That is, successful survival of the human immune system in the immunodeficient mouse may require suppression of the mouse's immune system to prevent GVHD (graft-versus-host disease) rejection. After the immune system of the immunodeficient mouse is sufficiently suppressed, the mouse is engrafted with human cells (e.g., HSCs and / or PBMCs). As used herein, "engrafting" refers to the process of migrating and incorporating human cells into an existing tissue of interest in vivo. With respect to the humanized immunodeficient mouse, the engrafted human cells provide functional mature human cells (e.g., immune cells). The model has a specific time window of approximately 4-5 weeks after transplant before GVHD sets in. To increase the longevity of the model, double knockout mice lacking functional MHC I and MHC II, as described above, can be used.

[0056] In some embodiments, the transplanted human cells (e.g., HSCs or PMBCs) for humanization are human leukocyte antigen (HLA)-matched to the human cells (e.g., human cancer cells) of the mouse model. HLA-matched refers to cells that express the same major histocompatibility complex (MHC) genes. Transplanting HLA-matched human xenografts and human immune cells into mice reduces or prevents, for example, the immunogenicity of human immune cells. In some embodiments, the humanized mice provided in the present disclosure are transplanted with human PMBCs or human HSCs that are HLA-matched to PDXs or human cancer cell lines.

[0057] In some embodiments, the human cells (e.g., HSCs or PMBCs) transplanted for humanization are not HLA-matched to the human cells (e.g., human cancer cells) of the mouse model. That is, in some embodiments, the humanized mice provided in the present disclosure are transplanted with human PMBCs or human HSCs that are not HLA-matched to the PDX or human cancer cell line.

[0058] Bone marrow destruction As described above, in some embodiments, the immunodeficient mouse is treated (e.g., chemically or with irradiation) to deplete and / or suppress any remaining mouse immune cells. In some embodiments, the immunodeficient mouse is treated only chemically or only with irradiation. In other embodiments, the immunodeficient mouse is treated both chemically and with irradiation.

[0059] In some embodiments, the immunodeficient mice are administered a myeloablative agent, i.e., a chemical agent that suppresses or depletes mouse immune cells. Examples of myeloablative agents include busulfan, dimethyl mileran, melphalan, and thiotepa.

[0060] In some embodiments, immunodeficient mice are irradiated prior to transplantation of human cells (e.g., human HSCs and / or PMBCs). Irradiation of immunodeficient mice destroys mouse immune cells in the peripheral blood, spleen, and bone marrow, which is believed to promote engraftment of human cells (e.g., human HSCs and / or PMBCs) (e.g., by increasing human cell survival factors), as well as the expansion of other immune cells. Irradiation also shortens the time it takes to accumulate the number of human immune cells required to "humanize" the mouse model.

[0061] Immunodeficient mice (e.g., NSG TMFor mice), this preparation is commonly accomplished via whole-body gamma irradiation. Irradiators can vary in size depending on their intended use. Animals are generally irradiated for short periods of time (less than 15 minutes). The amount of time spent inside the irradiator varies depending on the radioisotope decay chart, the dose required, and the ionizing energy source (i.e., X-rays vs. gamma rays, for which cesium or cobalt sources are required).

[0062] Myeloablative radiation doses are typically 700-1300 cGy, however the data provided herein clearly indicates that in some embodiments, lower doses (e.g., 50-200 cGy (e.g., about 50 cGy, about 100 cGy, about 150 cGy, or about 200 cGy) may be used. As an example, the mice may be irradiated with 50 cGy, 75 cGy, 100 cGy, 125 cGy, 150 cGy, 175 cGy, or 200 cGy.

[0063] In some embodiments, the dose is about 1 cGy, about 2 cGy, about 3 cGy, about 4 cGy, about 5 cGy, about 10 cGy, about 20 cGy, about 100 cGy, about 125 cGy, about 150 cGy, about 175 cGy, about 200 cGy, about 300 cGy, about 400 cGy, about 500 cGy, about 600 cGy, about 700 cGy, about 800 cGy, about 900 cGy, about 1000 cGy, about 1100 cGy, about 125 cGy, about 150 cGy, about 175 cGy, about 200 cGy, about 300 cGy, about 400 cGy, about 500 cGy, about 600 cGy, about 700 cGy, about 800 cGy, about 1500 cGy, about 1600 cGy, about 1750 cGy, about 1800 cGy, about 1900 cGy, about 2000 cGy, about 2100 cGy, about 2200 cGy, about 2300 cGy, about 2400 cGy, about 2500 cGy, about 2600 cGy, about 2700 cGy, about 2800 cGy, about 2900 cGy, about 3000 cGy, about 3100 cGy, about 3200 cGy, about 3300 cGy, about 3400 cGy, about 3500 cGy, about 3600 cGy, about 3750 cGy, about 380 Gy, about 900 cGy, about 1000 cGy, about 1100 cGy, about 1200 cGy, or about 1300 cGy, or between any of the two described doses described herein (e.g., 50-200 cGy, 100-300 cGy, 200-500 cGy, 600-1000 cGy, or 700-1300 cGy).

[0064] In some embodiments, the immunodeficient mice are irradiated or otherwise exposed to myeloablative treatment about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, or more prior to transplantation of human HSCs and / or PMBCs. In some embodiments, the immunodeficient mice are transplanted with human HSCs and / or PMBCs on the same day as irradiation or other myeloablative treatment. In some embodiments, the immunodeficient mice are transplanted with human HSCs and / or PMBCs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, or 18 days after irradiation or other myeloablative treatment.

[0065] Transplantation (humanization) As described above, in some embodiments, the irradiated immunodeficient mouse is transplanted with HSCs and / or PBMCs to humanize the mouse. Transplantation refers to the process of human cells migrating and incorporating into existing target tissues in vivo. The PBMCs are transplanted after irradiation and before transplantation of human pathological cells (e.g., human cancer cells), after irradiation and at the same time as transplantation of human pathological cells, or after irradiation and transplantation of human pathological cells.

[0066] Peripheral blood mononuclear cells (PBMCs) are peripheral blood cells with round nuclei. These mononuclear blood cells recirculate between tissues and blood and are important components of the immune system, fighting infections and adapting to invaders. There are two major types of mononuclear cells: lymphocytes and monocytes. The lymphocyte population of PBMCs typically includes T cells, B cells, and NK cells.

[0067] PBMCs can be isolated, for example, from a whole blood sample (e.g., Ficoll gradient). In some embodiments, PBMCs from a subject (e.g., a human subject) currently or previously diagnosed with cancer or an autoimmune disease can be used.

[0068] Hematopoietic stem cells (HSCs) are stem cells that give rise to other blood cells during a process called hematopoiesis. Hematopoietic stem cells give rise to various types of blood cells in lineages called myeloid and lymphoid. Both the myeloid and lymphoid lineages are involved in dendritic cell formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes to platelets. Lymphoid cells include T cells, B cells, natural killer cells, and innate lymphoid cells.

[0069] Methods for transplanting HSCs and / or hPBMCs into immunodeficient mice to obtain humanized mouse models include, but are not limited to, intraperitoneal or intravenous injections (Shultz et al., J Immunol, 2015, 174:6477-6489; Pearson et al., Curr Protoc Immunol. 2008; 15-21; Kim et al., AIDS Res Hum Retrovirus, 2016, 32(2): 194-2020; Yaguchi et al., Cell & Mol Immunol, 2018, 15:953-962). In some embodiments, the mice are transplanted with 1.0×10 6 pieces~3.0×10 7 In some embodiments, the mice are transplanted with 1×10 HSCs and / or hPBMCs. 7 In some embodiments, the mice are transplanted with less than 1×10 HSCs and / or hPBMCs. 6 Less than 5 x 10 6 In some embodiments, the mice are transplanted with about 2×10 HSCs and / or hPBMCs. 6 pcs or 1×10 6In some embodiments, the mouse is transplanted with 25,000 to 100,000 HSCs and / or hPBMCs (e.g., 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000 or more HSCs and / or hPBMCs).

[0070] For example, the mouse above was 1 × 10 5 pieces~1×10 6 pieces, 1×10 5 pieces~1×10 7 pieces, 1×10 5 pieces~1×10 8 pieces, 1×10 6 pieces~1×10 7 pieces, 1×10 6 pieces~1×10 8 In some embodiments, the mice may be transplanted with 1×10 hPBMCs. 6 pieces~2×10 6 pieces, 1×10 6 pieces~3×10 6 pieces, 1×10 6 pieces~4×10 6 pieces, 1×10 6 pieces~5×10 6 pieces, 1×10 6 pieces~6×10 6 pieces, 1×10 6 pieces~7×10 6 pieces, 1×10 6 pieces~8×10 6 pieces, 1×10 6 pieces~9×10 6 pieces, 2×10 6 pieces~3×10 6 pieces, 2×10 6 pieces~4×10 6 pieces, 2×10 6 pieces~5×10 6 pieces, 2×10 6 pieces~6×10 6 pieces, 2×10 6 pieces~7×106 pieces, 2×10 6 pieces~8×10 6 pieces, 2×10 6 pieces~9×10 6 pieces, 3×10 6 pieces~4×10 6 pieces, 3×10 6 pieces~5×10 6 pieces, 3×10 6 pieces~6×10 6 pieces, 3×10 6 pieces~7×10 6 pieces, 3×10 6 pieces~8×10 6 pcs, or 3×10 6 pieces~9×10 6 hPBMCs are transplanted.

[0071] In some embodiments, the mouse is 5 pieces~1×10 6 pieces, 1×10 5 pieces~1×10 7 pieces, 1×10 5 pieces~1×10 8 pieces, 1×10 6 pieces~1×10 7 pieces, 1×10 6 pieces~1×10 8 In some embodiments, the mouse is transplanted with 1×10 HSCs. 6 pieces~2×10 6 pieces, 1×10 6 pieces~3×10 6 pieces, 1×10 6 pieces~4×10 6 pieces, 1×10 6 pieces~5×10 6 pieces, 1×10 6 pieces~6×10 6 pieces, 1×10 6 pieces~7×10 6 pieces, 1×10 6 pieces~8×10 6 pieces, 1×10 6 pieces~9×10 6 pieces, 2×10 6 pieces~3×10 6 pieces, 2×10 6 pieces~4×10 6 pieces, 2×106 pieces~5×10 6 pieces, 2×10 6 pieces~6×10 6 pieces, 2×10 6 pieces~7×10 6 pieces, 2×10 6 pieces~8×10 6 pieces, 2×10 6 pieces~9×10 6 pieces, 3×10 6 pieces~4×10 6 pieces, 3×10 6 pieces~5×10 6 pieces, 3×10 6 pieces~6×10 6 pieces, 3×10 6 pieces~7×10 6 pieces, 3×10 6 pieces~8×10 6 pcs, or 3×10 6 pieces~9×10 6 Each patient is transplanted with HSCs.

[0072] In some embodiments, the mouse is 7 A dose of less than 1 x 10 hPBMCs (e.g., 1 x 10 6 pieces~approx. 5×10 6 hPBMCs, 1 x 10 6 pieces~approx. 4×10 6 hPBMCs, 1 x 10 6 pieces~approx. 3×10 6 hPBMCs, approximately 5 x 10 6 hPBMCs, approximately 4 x 10 6 hPBMCs, approximately 3 x 10 6 hPBMCs, approximately 2 x 10 6 hPBMCs, or approximately 1 x 10 6 hPBMCs) are transplanted.

[0073] In some embodiments, the mouse is 7 A dose of less than 1 x 10 HSCs (e.g., 1 x 10 6 pieces~approx. 5×10 6 HSC, 1 x 10 6 pieces~approx. 4×10 6 HSC, 1 x 10 6 pieces~approx. 3×106 HSCs, approximately 5 x 10 6 HSCs, approximately 4 x 10 6 HSCs, approximately 3 x 10 6 HSCs, approximately 2 x 10 6 HSCs, or approximately 1 x 10 6 Each patient is transplanted with 1 HSC.

[0074] As described herein, in some embodiments, transplantation of HSCs and / or PBMCs results in transgenic mice that contain more human CD45+ cells in the peripheral blood (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40% or more increase) relative to humanized NSG-SGM3 control mice. In embodiments, the transgenic mice contain significantly more human NK cells (e.g., 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more increase (measured as percentage of CD45+ cells)) in the peripheral blood relative to humanized NSG-SGM3 control mice.

[0075] In some embodiments, transplantation of PBMCs results in transgenic mice that contain more human myeloid cells in the peripheral blood (e.g., 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2% or more increase (measured as percentage of CD45+ cells)) relative to humanized NSG-SGM3 control mice.

[0076] In some embodiments, the transgenic mice contain more activated human T cells relative to humanized NSG-SGM3 control mice. In some embodiments, the activated T cells are present in the spleen of the transgenic mice. The term "T cell activation" refers to the mechanism of T cell activation that may vary slightly between different types of T cells. However, the "two-signal model" in CD4+ T cells is applicable for most types of T cells. More specifically, activation of CD4+ T cells typically occurs via engagement of both the T cell receptor and CD28 on the T cell surface by major histocompatibility-encoded antigen-presenting molecules, and their bound antigenic peptides and B7 family members on the surface of antigen-presenting cells (APCs), respectively. Both cell-cell contacts are generally required for the generation of an effective immune response. For example, in the absence of CD28 costimulation, T cell receptor signaling alone can result in T cell anergy. Further signaling pathways downstream from both CD28 and the T cell receptor involve many additional proteins known to those skilled in the art. Activation of T cells can be determined by cytokine release and / or cell proliferation, in particular proliferation of T cells.

[0077] Human immune cells The mouse model provided herein supports the expansion, development, and maturation of a full repertoire of human immune cells, including cells of the innate and adaptive immune systems. There are two major lineages of human immune cells: lymphoid and myeloid. Progenitor cells of the lymphoid lineage develop into B cell precursors, natural killer cells, and T cell precursors. B cell precursors continue to develop into either memory B cells or plasma cells, while T cell precursors continue to develop into memory T cells, cytotoxic T cells, or helper T cells. Progenitor cells of the myeloid lineage develop into white blood cells (e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes). Monocytes further develop into dendritic cells (e.g., cDC1 or cDC2) and macrophages.

[0078] The human immune system includes the innate and adaptive immune systems. The innate immune system includes the mononuclear phagocyte system of macrophages, dendritic cells, and monocytes, natural killer cells, mast cells, gamma delta T cells, natural killer T cells, and granulocytes (basophils, eosinophils, and neutrophils). The adaptive immune system includes humoral immunity (also called B cell immunity), which includes antibody-producing B cells, and cellular immunity (also called T cell immunity), which includes CD4+ and CD8+ T cells, natural killer cells, and gamma delta T cells.

[0079] T cells play a key role in the adaptive immune system and are generally identified by CD3 expression, detecting antigens via the T cell receptor (TCR), which recognizes peptides presented by the major histocompatibility complex (MHC). Circulating tumor cell antigens are delivered to lymph nodes where they are displayed to CD4+ and CD8+ T cells (also known as T helper cells and cytotoxic T cells, respectively). After activation, T helper cells release a variety of cytokines, including IFNγ. Cytotoxic T cells recognize cells expressing tumor-specific antigens and kill them via perforin- or granzyme-induced apoptosis.

[0080] Macrophages are also cells of the innate immune system, identified by the expression of CD68 and MHCII and the lack of CD11c. They specialize in phagocytosis and secrete cytokines that affect the immune response. Macrophages are generally classified as pro-inflammatory (M1-like) or anti-inflammatory (M2-like). M1-like macrophages are identified by the expression of CD80, CD86, or iNOS, and promote anti-tumor immune responses by phagocytosis of malignant cells and generation of T-cell activating ligands. Conversely, M2-like macrophages are identified by the expression of CD163 or CD206, and may promote tumor growth through the secretion of immunosuppressive cytokines (e.g., IL-10) and by promoting Th2 responses. M2 macrophages may also express the immunosuppressive enzyme arginase, which depletes arginine from the tumor microenvironment, resulting in reduced T-cell proliferation and function.

[0081] Natural killer (NK) cells represent the major innate immune cell type. They recognize and kill cancer by detecting downregulation of MHC class I on tumor cells and / or upregulation of ligands on tumor cells that bind to activating receptors on NK cells. NK cells are generally identified by the combination of CD56 and CD16, and the lack of CD3 expression.

[0082] Some aspects of the disclosure provide mouse models that support the expansion, development, and / or maturation of cells of the adaptive and innate immune systems. In some embodiments, the mouse models support the expansion, development, and / or maturation of human CD33+ myeloid cells. In some embodiments, the mouse models support the expansion, development, and / or maturation of human CD45+ immune cells. In some embodiments, the mouse models support the expansion, development, and / or maturation of human CD19+ B cells (e.g., human B cells that produce circulating immunoglobulin (Ig)). In some embodiments, the mouse models support the expansion, development, and / or maturation of human CD3+ T cells. In some embodiments, the mouse models support the expansion, development, and / or maturation of human NK cells.

[0083] B cell antibody class switching The mouse model provided herein, in some embodiments, supports the expansion of mature human B cells capable of immunoglobulin (Ig) class switching, which is the biological mechanism by which a B cell's Ig production is altered from one type to another, e.g., from isotype IgM to isotype IgG. Class switching occurs after activation of the mature B cell through its membrane-bound antibody molecule (or B cell receptor) to produce different classes of antibodies, all of which have the same variable domains as the original antibodies generated in the immature B cell during the process of V(D)J recombination, but have distinct constant domains in their heavy chains.

[0084] The term "Ig" as used herein refers to a region of an immunoglobulin that exists as a separate structural entity as recognized by those skilled in the art of protein structure. Ig domains typically have a characteristic β-sandwich fold topology. There are five distinct antibody classes in humans, including IgA (which includes subclasses IgA1 and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The classical antibody structural unit typically comprises a tetramer. Each tetramer typically consists of two identical pairs of polypeptide chains, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. The distinguishing feature between these antibody classes is their constant region, although more subtle differences may exist in the V region.

[0085] The term "IgG" as used herein refers to a polypeptide belonging to the antibody class substantially encoded by the recognized immunoglobulin gamma gene. In humans, this class includes the subclasses or isotypes IgG1, IgG2, IgG3, and IgG4. IgG antibodies are tetrameric proteins composed of two heavy chains and two light chains. The IgG heavy chain contains four immunoglobulin domains (referred to as heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3, respectively) linked from N-terminus to C-terminus in the order VH-CH1-CH2-CH3 (also referred to as VH-C.gamma.1-C.gamma.2-C.gamma.3, respectively referring to the heavy chain variable domain, constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3 domain). The IgG light chain is composed of two immunoglobulin domains (referred to as light chain variable domain and light chain constant domain, respectively) linked from N-terminus to C-terminus in the order VL-CL.

[0086] Naive mature B cells produce both IgM and IgD, which are the first two heavy chain segments in the immunoglobulin locus. After activation by antigen, these B cells proliferate. When these activated B cells encounter specific signaling molecules through their CD40 and cytokine receptors, both of which are regulated by T helper cells, they undergo antibody class switching to produce IgG, IgA or IgE antibodies. During class switching, the constant region of the immunoglobulin heavy chain changes, but the variable region does not, and thus the antigenic specificity remains the same. This allows different daughter cells derived from the same activated B cell to produce antibodies of different subclasses or isotypes.

[0087] In some embodiments, the amount of IgG produced in the mouse models provided herein is within the physiological range found in humans, where the normal physiological range of IgG in adults is 6,000-16,000 μg / mL.

[0088] In some embodiments, the mouse models provided herein produce IgG subclasses (i.e., IgG1, IgG2, IgG3, and / or IgG4). In some embodiments, the amount of IgG1, IgG2, IgG3, and / or IgG4 produced is within the physiological range found in humans, where the normal physiological ranges of IgG subclasses in adults are as follows: 2.0-8.00 mg / mL IgG1; 1.15-5.70 mg / mL IgG2; 0.24-1.25 mg / mL IgG3; and 0.052-1.25 mg / mL IgG4.

[0089] In some embodiments, the amount of IgM and / or IgA produced in the mouse models provided herein is within the physiological range found in humans, where the normal physiological range in adults is 0.4-2.5 mg / mL IgM and 0.8-3.0 mg / mL IgA.

[0090] Mammalian cell lines and patient-derived xenografts The mouse models of disease provided herein, in some embodiments, are engrafted with tissue or cells (living cells), e.g., mammalian cells (e.g., cell lines or derived from a parent (e.g., a human or canine patient-derived xenograft). As used herein, "mammal" includes, but is not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates (e.g., monkeys, chimpanzees and monkeys), and particularly humans. In some embodiments, the mouse models are engrafted with human tissue (cells). In other embodiments, the mouse models are engrafted with canine cells.

[0091] The cells may be pathological cells, in some embodiments, such as cancer cells, cells involved in autoimmunity, or cells involved in other inflammatory diseases. Other pathological cells are contemplated herein (e.g., cells obtained from patients with cardiovascular disease, metabolic disease, etc.).

[0092] In some embodiments, the mouse model is implanted with human cancer cells. The human cancer cells can be derived from a single source or from multiple sources (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten sources). The human cancer cells can be tumor cells (e.g., cells derived from malignant tumors), patient-derived xenografts (PDXs) (e.g., tumor tissue from humans implanted in mouse models), or human cancer cell lines (e.g., human cancer cell cultures developed from a single cell). A tumor is a mass of tissue formed by the abnormal proliferation of cells (e.g., cancer cells). Non-limiting examples of common human cancers include bladder cancer, brain cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer, testicular cancer, and thyroid cancer. Other cancer cell types are contemplated herein (see, eg, cancer.gov / types).

[0093] In some embodiments, the human cancer cells can be, for example, circulating tumor cells from a primary tumor or a secondary tumor. A primary tumor is a tumor (e.g., a lung cancer tumor or a breast cancer tumor) that grows at the anatomical site where the tumor originated. A secondary tumor is a tumor of the same type as the primary tumor (e.g., a lung cancer tumor or a breast cancer tumor) but that is formed at a secondary anatomical site distant from the primary tumor.

[0094] In some embodiments, the mouse model is implanted with a patient-derived xenograft (PDX). A PDX is, for example, a tumor tissue from a human or other mammal that is implanted in the mouse model of the present disclosure. A PDX as used herein can be obtained directly from a subject or from a PDX repository. Non-limiting examples of PDX repositories include the Jackson Laboratories Mouse Models of Human Cancer Database (Krupke, DM, et al., "Mouse Models of Human Cancer Database", Nat Rev Cancer, 2008 8(6): 459-65), the Dana Farber Cancer Institute Patient-Derived Tumor Xenograft Database, and the Charles River Patient-Derived Xenograft Model Database. A disease model can include at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) PDXs.

[0095] PDX can have any human tumor origin.For example, PDX can be derived from: bladder tumor, brain tumor, breast tumor, colorectal tumor, endometrial tumor, kidney tumor, leukemia, liver tumor, lung tumor, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic tumor, prostate tumor, sarcoma, skin cancer, testicular cancer, or thyroid tumor.Other tumor types are contemplated herein (see, for example, cancer.gov / types).

[0096] In some embodiments, the mouse model is implanted with human cancer cells derived from a human cancer cell line. A human cancer cell line is a human cancer cell culture that has developed from a single cell. In some embodiments, the human cancer cell line is immortalized. Immortalized cells divide and grow indefinitely. The human cancer cell line can be derived from any human cancer. For example, the human cancer cell line can be derived from: bladder tumor (HTB-9, HTB-3, CRL-2169), breast tumor (e.g., Hs.281.T, Hs 5788st, UACC-812, MCF 10A, or MDA-MB-157), brain tumor (SW 1088, U138, Daoy, LN-228), colorectal tumors (HT29, SW480, SW1116, Caco-2), endometrial tumors (Ishikawa), kidney tumors (Caki-1, 769-P), leukemias (MOLT-3, TALL-104, AML-193, Jurkat, Mo-B), liver tumors (e.g., SNU-182, Hs.817.T, NCI-H735 or THLE-3), lung tumors (e.g., NCI-H838, HCC827, NCI-H1666, SW 1573, ChaGo-K-1, A549, or NCI-H1555), melanoma (SK-MEL-3, A375-P, MNT-1), non-Hodgkin's lymphoma (e.g., GA-10, NCI-BL2171, HH, or Toledo), ovarian tumor (SK-OV-3, PA-1, Caov-3, SW 636), pancreatic tumor (Capan-2, Panc 10.05, CFPAC-1, SQ 1990), prostate tumor (VCaP, C4-2B, LNCaP, PC-3), sarcoma (HS 822.T, SK-LMS-1, A-204), skin (TE 354.T, Hs 456.Bt), testicular (Cates-1B, Hs 1. Tes), or thyroid (MDA-T120, MDA-T41, SW-579). The mouse models herein can be implanted with human cancer cell lines derived from a single cell line or from multiple cell lines (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 cell lines).

[0097] Additionally, any of the mouse models provided herein may include a combination of human cancer cell types, optionally a combination of sources (e.g., PDX sources and / or cell lines).

[0098] In some embodiments, human cancer cells are genetically engineered to express detectable biomolecules, for example, so that the cells can be monitored in vivo or analyzed ex vivo. Detectable biomolecules, as provided herein, refer to biomolecules in human cancer cells that can be detected using conventional or non-conventional assays. Non-limiting examples of detectable biomolecules include fluorescent proteins (e.g., green fluorescent protein, yellow fluorescent protein, or red fluorescent protein), antigens (e.g., hemagglutinin or human leukocyte antigen), and enzymes (e.g., β-galactosidase or luciferase). Other detectable biomolecules are contemplated herein.

[0099] Methods for obtaining human cancer cells and PDXs include, but are not limited to, biopsy (e.g., hollow needle, excision, incision, or brush), resection (e.g., of a tumor), and sample (e.g., a blood sample) collection followed by a sorting step to isolate human cancer cells from other cells (e.g., human non-cancer cells, non-human cells, or cell fragments).

[0100] In some embodiments, the mouse model is transplanted with human cells from a subject with an autoimmune disease. Autoimmune diseases arise from and are directed against the subject's own tissues. Examples of autoimmune diseases include, but are not limited to: arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), psoriasis, dermatitis (including atopic dermatitis);Chronic idiopathic urticaria (including chronic autoimmune urticaria), polymyositis / dermatomyositis, toxic epidermal necrolysis, systemic sclerosis and sclerosis, inflammatory bowel disease (IBD) and related responses (Crohn's disease, ulcerative colitis), as well as IBD co-isolated with pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, and / or episcleritis), respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)), meningitis, IgE-mediated diseases (e.g., anaphylaxis and allergic rhinitis), encephalitis (e.g., rheumatoid arthritis, rheumatoid arthritis, and rheumatoid arthritis), and other conditions that may be of concern include chronic idiopathic urticaria (including chronic autoimmune urticaria), polymyositis / dermatomyositis, toxic epidermal necrolysis, systemic sclerosis and sclerosis, inflammatory bowel disease (IBD) and related responses (Crohn's disease, ulcerative colitis), as well as IBD co-isolated with pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, and / or episcleritis), respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)), meningitis, IgE-mediated diseases (e.g., anaphylaxis and allergic rhinitis), encephalitis (e.g., rheumatoid arthritis, rheumatoid arthritis, and rheumatoid arthritis). Smussen's encephalitis), uveitis, colitis (e.g., microscopic colitis and collagenous colitis), glomerulonephritis (GN) (e.g., membranous GN, idiopathic membranous GN, membranoproliferative GN (MPGN) (including types I and II), and rapidly progressive GN), allergic conditions, eczema, asthma, conditions involving T cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) (e.g., cutaneous SLE, lupus (nephritis, encephalitis, pediatric, non-small cell lung cancer, pulmonary edema ... renal, discoid, alopecia), juvenile onset diabetes mellitus, multiple sclerosis (MS) (e.g., spino-optical MS), allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatous diseases (including Wegener's granulomatosis), granulocytopenia, vasculitis (large vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vasculitis (including Kawasaki disease and polyarteritis nodosa), vasculitis), CNS vasculitis, and ANCA-associated vasculitis (e.g., Churg-Strauss vasculitis or syndrome (CSS)), aplastic anemia, Coombs positive anemia, Diamond-Blackfan anemia, immune hemolytic anemia (including autoimmune hemolytic anemia (AIHA)), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, CNS inflammatory diseases, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease,disease), antiphospholipid syndrome, allergic neuritis, Behcet's disease, Castleman syndrome, Goodpasture's syndrome, Lambert-Eaton myasthenic syndrome, Raynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection (including rejection resulting from conditioning with high panel reactive antibody titers, IgA deposition in tissues, and kidney, liver, intestine, heart, etc.), graft-versus-host disease (GVHD), pemphigoid bullous, pemphigus (pemphigus vulgaris, pemphigus foliaceus, and pemphigus mucus-membrane pemphigoid), autoimmune polyendocrinopathy, Reiter's disease, stiff-man syndrome, immune complex nephritis, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), thrombocytopenia (e.g., as occurs in patients with myocardial infarction) (including autoimmune thrombocytopenia), autoimmune diseases of the testes and ovaries (including autoimmune orchitis and autoimmune oophoritis), primary hypothyroidism; autoimmune endocrine diseases (autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune polyglandular syndrome (or polyglandular endocrinopathy syndrome) type 1 diabetes mellitus (also known as insulin-dependent diabetes mellitus (IDDM)), including pediatric IDDM, and Sheehan's syndrome; autoimmune hepatitis, lymphoid interstitial pneumoniapneumonitis (HIV), bronchiolitis obliterans for NSIP (non-transplant), Guillain-Barré syndrome, Buerger's disease (IgA nephropathy), primary biliary cirrhosis, celiac sprue (gluten enteropathy), refractory sprue co-isolated with dermatitis herpetiformis, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus-myoclonus OMS, polychondritis (e.g., refractory polychondritis), pulmonary alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis, monoclonal gammopathy of undetermined / unknown significance (MGUS), peripheral neuropathy, paraneoplastic syndromes, ion channel disorders (e.g., epilepsy, migraine, arrhythmias, myopathy, deafness, blindness, periodic paralysis, and ion channel disorders of the CNS; autism, inflammatory myopathies, and focal segmental glomerulosclerosis (FSGS).

[0101] The humanized immunodeficiency mouse model of the disease provided herein is transplanted with human cells, such as human PBMCs, human HSCs, and / or human cancer cells derived from human cancer cell lines or human PDXs, as discussed above. Transplantation refers to the process of human cells moving and incorporating into existing tissues of interest in vivo.

[0102] Any tissue in the mouse can be a target tissue for transplantation of human cells (e.g., human PBMC, HSC, or cancer cells). The target tissue for transplantation is the tissue into which the human cells (e.g., human PBMC, HSC, or cancer cells) migrate and integrate. The target tissue for transplantation may depend on the disease to be tested. Non-limiting examples of target tissues for transplantation of human cells (e.g., human PBMC, HSC, or cancer cells) include lung, trachea, liver, bone marrow, brain, blood, digestive tissue, skin, stomach, small intestine, large intestine, and pancreas. Other target tissues are contemplated herein. In some embodiments, human cells engraft into one target tissue, while in some embodiments, human cells engraft into one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) target tissues.

[0103] In some embodiments, the human cells (e.g., human PBMC, HSC, or cancer cells) are delivered to mice using single cell suspensions. Single cell suspensions are suspensions of cells that lack detectable levels of cell debris and cell aggregates. Single cell suspensions allow for separation of cells from tissues (e.g., connective tissues) and maximize the efficiency of using human cells, including but not limited to transplantation into model animals (e.g., mouse models), flow cytometry, human cell culture (e.g., immortalization), and human cell labeling. The method for preparing human cell single cell suspensions depends on the origin of the cells (e.g., freshly isolated from humans, PDX, derived from cell lines). Methods for preparing single cell suspensions include, but are not limited to, dissociation (e.g., enzymatic, mechanical), purification (e.g., magnetically activated cell sorting or activated cell sorting), commercial kits (e.g., Miltenyi Biotec or StemCell), centrifugation (e.g., at >300×g), and filtration (e.g., cell strainers).

[0104] For example, the mouse above has a resolution of 1.0×10 5 pieces~2.0×10 7In some embodiments, the mice may be implanted with 1.0×10 human cells (e.g., human PBMCs, HSCs, or cancer cells). 5 pieces, 2.0×10 5 pieces, 3.0×10 5 pieces, 4.0×10 5 pieces, 5.0×10 5 pieces, 6.0×10 5 pieces, 7.0×10 5 pieces, 8.0×10 5 pieces, 9.0×10 5 pieces, 1.0×10 6 pieces, 2.0×10 6 pieces, 3.0×10 6 pieces, 4.0×10 6 pieces, 5.0×10 6 pieces, 6.0×10 6 pieces, 7.0×10 6 pieces, 8.0×10 6 pieces, 9.0×10 6 pieces, 1.0×10 7 pcs, or 2.0×10 7 Human cells (e.g., human PBMCs, HSCs, or cancer cells) are transplanted.

[0105] The human cells (e.g., human PBMCs, HSCs, or cancer cells) can be delivered to the mice via injection (e.g., tail vein, retroorbital, intravenous, intracardiac, or intraarterial) or implantation (e.g., subcutaneous, intraperitoneal, intrafemoral, intratibial, or intramuscular). In some embodiments, the delivery is via subcutaneous implantation. Other delivery methods are contemplated herein.

[0106] As described herein, in some embodiments, transplantation of HSCs or PBMCs and xenografts (e.g., human xenograft tissue or cells) results in transgenic mice that contain fewer human regulatory T (Treg) cells in the peripheral blood (e.g., a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, or more reduction (measured as percentage of CD45+ cells)) relative to humanized NSG-SGM3 control mice. In some embodiments, the transgenic mice comprise fewer PD-1+ T (CD4 and / or CD8) cells in peripheral blood (e.g., a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more decrease (measured as a percentage of CD4 and / or CD8 T cells)) relative to humanized NSG-SGM3 control mice.

[0107] In some embodiments, the exponential growth phase of the tumor is delayed; i.e., it begins 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, or more days after tumor implantation.

[0108] How to use The humanized transgenic immunodeficient mouse model of disease provided herein can be used for any of many applications.For example, the humanized immunodeficient model (e.g., immunodeficient mouse model) can be used to evaluate disease onset, evaluate disease progression, and / or test the extent to which a candidate preventive or therapeutic agent (e.g., a candidate therapeutic agent) affects human immune system (e.g., whether it induces cytokine release syndrome).

[0109] Assessment of disease behavior The immunodeficiency model (e.g., mouse model) provided herein can be used to evaluate disease behavior. Disease behavior refers to the changes that occur in the host (e.g., immunodeficiency mouse model) due to disease (e.g., cancer, autoimmunity, and / or inflammation). Non-limiting examples of disease behavior include disease spread to multiple tissues (e.g., metastasis), disease symptom production, and the production of genes and / or proteins that are important for disease progression.

[0110] In some embodiments, methods are provided herein for assessing disease spread to multiple tissues in a host (e.g., immunodeficient mouse model). The multiple tissues can be multiple instances of the same type of tissue (e.g., lung tissue) or multiple different tissue types (e.g., lung tissue, blood, liver, or brain). Any method can be used to assess disease spread, including but not limited to obtaining multiple tissue samples and verifying the presence (e.g., immunohistochemistry) of pathological cells and / or tissues; and performing in vivo imaging system (IVIS) (e.g., with fluorescent markers).

[0111] In some embodiments, methods are provided herein for evaluating the generation and progression of disease symptoms.Disease symptoms and disease progression may depend on the disease and host (e.g., humanized immunodeficiency mouse model).Desirably, the disease symptoms and disease progression in the model provided in this disclosure mimic the disease symptoms and disease progression in humans.Non-limiting examples of disease symptoms and disease progression include fever, lethargy, weight loss, diarrhea, myalgia, cough, dyspnea, vomiting, seizures, ataxia, blood pressure changes, proteinuria, hematuria, loss of fur, edema, erythema, dermatitis, and dehydration.Any method can be used to evaluate the generation and progression of disease symptoms. Non-limiting examples of methods used to assess the production of disease symptoms and disease progression include measuring body temperature, monitoring sleep / wake cycles, monitoring activity, measuring weight, assessing solid and liquid excretion, monitoring respiration and assessing pulmonary function, monitoring blood pressure and cardiac function, measuring the presence of protein and blood in the urine, measuring blood cytokine levels, assessing changes in skin or mucous membrane thickness, measuring blood ion concentrations, and any disease activity index (DAI) assessment.

[0112] Assessing the impact of disease on the human immune system The humanized immunodeficient mouse model provided herein can be used to evaluate the effect of disease on the human immune system.Non-limiting examples of the effect of disease on the human immune system include regulated human immune cell production and cytokine release.

[0113] In some embodiments, a method for evaluating regulated human immune cell production is provided herein. Regulated human immune cell production can be increased human immune cell production (e.g., compared to a control) or decreased human immune cell production (e.g., compared to a control). The control can be a humanized immune deficiency model (e.g., a mouse model) without a disease. Any human immune cell production in a humanized immune deficiency model (e.g., a mouse model) can be regulated by disease. Non-limiting examples of human immune cells whose production can be regulated by disease include hematopoietic stem cells (e.g., surface marker CD34+), T cells (e.g., surface marker CD3+, CD4+, CD8+), B cells (e.g., surface marker CD19+, CD20+), natural killer cells, plasma cells, immunoglobulins, neutrophils, monocytes, dendritic cells, and cytokines (e.g., IL-2, IL-4, or IL-6).

[0114] In some embodiments, the mouse model can be used to assess cytokine release syndrome (CRS), a systemic inflammatory response in a subject characterized by hypertension, fever and / or rigors, among others, potentially resulting in death. Cytokine storm (CRS) is likely caused by an uncontrolled positive feedback loop between cytokines and immune cells, resulting in highly elevated levels of various cytokines. Although these terms may vary somewhat in extent, they are all a result of an unacceptably high release of cytokines by a subject as a result of administration of a certain immunomodulatory drug (e.g., an antibody) to the subject. The subject responds to the treatment by releasing unacceptably high levels of cytokines. The humanized mouse model described herein can be used as a drug testing platform to identify potential drug candidates that induce cytokine release from a large number of clinically relevant drug candidates, and the method of the present invention thus represents a robust predictive assay for drug immunotoxicity testing, providing a link between preclinical and clinical trials. Thus, in one aspect, the present disclosure relates to a method for determining whether an immunomodulatory drug causes immunotoxicity in humans. In some embodiments, levels of certain cytokines (e.g., IFN-γ, IL-2, IL-4, IL-6, IL-10, and / or TNF) are measured as part of an evaluation of the potential immunotoxicity of drug candidates.

[0115] Any method for measuring human immune cell production can be used to evaluate regulated human immune cell production.Non-limiting examples of methods for measuring human immune cell production include flow cytometry, fluorescence-activated cell sorting (FACS), RT-PCR of human immune cell surface markers and cytokines, and ELISA.

[0116] Some aspects provide a method to evaluate anti-drug antibodies (ADA) associated with human therapeutic agents using the mouse models described herein. ADA can induce unwanted side effects, especially in biotechnology-derived drugs (e.g., therapeutic antibodies and growth factors). Thus, ADA is increasingly the subject of regulatory scrutiny using immunogenicity safety testing. ADA resulting in significant changes in toxicity, pharmacokinetics, and efficacy has been observed in preclinical and clinical trials. These effects may result from the generation of drug-induced (neutralizing) autoantibodies against, for example, erythropoietin (EPO), factor VIII (FVIII), or insulin, and may be the cause of allergic reactions or even anaphylactic shock. In conclusion, testing for ADA has become inevitable for bioengineered drugs, including biosimilars. Adverse immunological reactions can vary widely depending on how the active ingredient is organized, generated, and applied. For example, the development of anti-Fc antibodies, anti-idiotypic antibodies, or antibodies against glycosylated antigens may appear. Detection and characterization assays for ADA must therefore be developed, customized and optimized for each drug.

[0117] The humanized immunodeficient mouse models provided herein are, in some aspects, used as in vivo models to assess ADA, eg, the long-term development of ADA.

[0118] In some embodiments, the mouse models used to assess ADA undergo a myeloablative procedure (eg, irradiation or chemical ablation).

[0119] In some embodiments, mouse models used to assess ADA are engrafted with huPBMCs (eg, targeted drug naive huPBMCs), as discussed elsewhere herein.

[0120] Then, after transplantation, targeted drugs can be administered. The term "targeted drug" encompasses human therapeutic modalities (e.g., drugs) that induce humoral responses. Non-limiting examples include therapeutic antibodies, recombinant protein therapeutics (e.g., growth factors), cell-based therapies (e.g., chimeric antigen receptor (CAR)-T cells, TCR-engineered T cells, tumor-infiltrating lymphocytes (TIL), and regulatory T cells (Treg) therapies), DNA-based (e.g., genes, antisense oligonucleotides) therapies, and RNA-based (e.g., RNAi and mRNA) therapies.

[0121] In some embodiments, the targeted drug is administered to the mouse about 4-10 days after administration of hPBMCs. For example, the targeted drug may be administered 4, 5, 6, 7, 8, 9, or 10 days after administration of hPBMCs. In some embodiments, the targeted drug is administered to the mouse at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 days after administration of hPBMCs. In some embodiments, a single dose of the targeted drug is administered. In other embodiments, multiple doses (e.g., 2-10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) of the targeted drug are administered (e.g., weekly, biweekly, triweekly, or monthly). In some embodiments, the targeted drug is administered according to a standardized administration schedule.

[0122] Anti-drug antibodies may be evaluated over a period of time, for example, from about 7 to about 270 days. In some embodiments, ADA is evaluated by collecting blood samples from the mice and characterizing plasma and / or B cell function. In some embodiments, flow cytometry or ELISA assays are used to evaluate blood samples (or other biological samples) for antibody recognition and / or neutralization of the targeted drug. The first blood draw prior to targeted drug administration may, for example, serve as a control. In some embodiments, anti-drug Ig levels increase over time.

[0123] Analysis of ADA may include characterization of ADA titer, neutralization capacity, binding affinity, isotyping, and other properties. There are several isotypes of ADA. For example, IgM ADA may be an early marker of ADA formation. And the presence of IgE antibodies may indicate an allergic reaction to the target drug. In addition, measurement of IgG subclasses may be supportive regarding the biological activity of ADA, because in humans, IgG1 and IgG3 are primarily involved in complement activation and are more prone to NK cell recognition. Measurement of ADA binding affinity is also beneficial for ADA response interpretation.

[0124] Evaluation of prophylactic and therapeutic agents The immune deficiency model (e.g., mouse model) provided in this disclosure is used to evaluate prophylactic and therapeutic agents for preventing or treating disease or disease progression. A prophylactic agent is a substance (e.g., a drug or protein) that prevents or reduces the risk of disease, or prevents or reduces the risk of disease occurrence (disease progression). A therapeutic agent is a substance (e.g., a drug or protein) that treats disease (e.g., treats symptoms associated with disease). Therapeutic agents include palliative agents, which are substances (e.g., drugs or proteins) that improve one or more symptoms of disease.

[0125] In some embodiments, the humanized transgenic mouse is administered an agent that activates human T cells. In some embodiments, the transgenic mouse is then administered a therapeutic agent, such as an anti-cancer agent or an anti-autoimmune disease agent, such as an anti-inflammatory agent, a corticosteroid, or an immunosuppressant. In further embodiments, the efficacy of the therapeutic agent is measured.

[0126] With respect to disease prevention, it should be understood that a prophylactically effective amount of an agent need not completely eradicate the disease, but should reduce or prevent the progression (e.g., metastasis) of the disease. In some embodiments, a prophylactically effective amount of an agent reduces disease progression in the subject by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. It should be understood that a therapeutic agent (e.g., a palliative agent) need not affect diseased tissues or cells, but should alleviate at least one symptom of the disease, thus potentially reducing the short-term or long-term systemic effects of the disease.

[0127] The prophylactic and / or therapeutic agents can be delivered by any method. Non-limiting examples of methods for delivering prophylactic and / or therapeutic agents include inhalation (e.g., nasal or tracheal), injection (e.g., intravenous, intraarterial, intramuscular, or intracranial), and ingestion (e.g., tablet or liquid).

[0128] In some embodiments, the agent is a vaccine (e.g., a human vaccine). The vaccine can be, for example, a cancer vaccine or an infectious disease vaccine. In some embodiments, the agent is, for example, a protein antigen derived from a pathogen (e.g., a virus or a bacterium). In other embodiments, the agent is a nucleic acid. Non-limiting examples of nucleic acid vaccines include RNA (e.g., mRNA) or DNA that encodes a protein antigen.

[0129] In some embodiments, the candidate drug is an analgesic, antipyretic, anti-inflammatory, or immunosuppressant, including, but not limited to, NSAIDs, steroids, diuretics, statins, and beta-blockers.

[0130] Any combination of preventive and / or therapeutic agents provided herein may also be administered to an immunodeficiency model (e.g., mouse) inoculated with pathological cells and / or tissue. In some embodiments, an immunodeficiency model with pathological cells and / or tissue is administered one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more preventive agents. In some embodiments, an immunodeficiency model with pathological cells and / or tissue (e.g., mouse) is administered one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more therapeutic (e.g., palliative) agents. In some embodiments, immune deficiency models (e.g., mice) with diseased cells and / or tissues are administered one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more prophylactic and therapeutic agents.

[0131] Any effective amount of prophylactic and / or therapeutic agent can be administered to a subject (e.g., an immunodeficiency model or a human patient). An effective amount is a dose (e.g., mg, mg / mL, or mg / kg) that prevents or reduces the risk of disease or disease progression and / or treats the disease. Any dosing regimen can be used to administer prophylactic and / or therapeutic agents to a subject. Non-limiting examples of dosing regimens include 1 dose per day to 24 doses per day, 1 dose per week to 7 doses per week, 1 dose per month to 30 doses per month, 1 dose every 2 months to 1 dose per year, 1 dose per year to 1 dose per 10 or more years.

[0132] The effectiveness of the prophylactic and / or therapeutic agents can be evaluated by any method. Non-limiting examples of evaluating the effectiveness of the prophylactic and / or therapeutic agents include monitoring the reduction of symptoms associated with the disease, reduction of tumor volume, reduction of metastasis, etc.

[0133] In some embodiments, the present specification further provides a method for monitoring disease or disease progression prevention or treatment in immunodeficiency model.Prevention or treatment of disease and disease progression can be monitored by any method.Non-limiting examples of monitoring prevention or treatment of disease or disease progression include measuring body temperature, measuring body weight, monitoring movement, and / or evaluating sleep / wake cycle.

[0134] In some embodiments, a method for monitoring the whole body function of an immunodeficiency model with disease is provided herein. Whole body function refers to the productivity of organ systems in an immunodeficiency model with diseased cells and / or tissues. Any organ system in the model (e.g., respiratory system, cardiac system, digestive system, renal system, endocrine system, or nervous system) can be monitored in the method provided herein. Non-limiting examples of monitoring whole body function include: respiratory function measurement (e.g., spirometry, lung volume and airway resistance, diffusion capacity, blood gas analysis, or cardiopulmonary exercise test), cardiac function (e.g., cardiac catheterization, pulsed Doppler measurement of blood pressure, Doppler blood flow test, peripheral vascular stiffness and flow velocity), kidney / renal function (proteinuria, creatinine level, BUN), liver function (albumin, ALT, AST, bilirubin), and neurological function (e.g., patch clamp, functional MRI, gait analysis, or balance test).

[0135] In some embodiments, the mouse model provided herein is used to evaluate the cytotoxicity of an agent. Examples of such methods are known and described, for example, in International Application No. WO2018195027A1. As one non-limiting example, the immunodeficient mouse model of the present disclosure can be transplanted with human HSCs or PBMCs and then administered, for example, a therapeutic agent. The blood levels of various cytokines (e.g., IFN-γ, IL-2, IL-4, IL-6, IL-10, and / or TNF) can be evaluated. Determining the levels of such cytokines can indicate the likelihood that the therapeutic agent will induce severe cytokine release syndrome in human subjects. Other methods of evaluating cytotoxicity are also contemplated herein.

[0136] Nucleic Acids: Manipulation and Delivery The mouse models described herein comprise a nucleic acid encoding human interleukin-3 (IL-3), a nucleic acid encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), a nucleic acid encoding human stem cell factor (SCF), and a nucleic acid encoding human IL-15. In some embodiments, the mouse models comprise a transgene encoding human IL-3, a transgene encoding human GM-CSF, a transgene encoding human SCF, and a transgene encoding human IL-15 integrated into the mouse genome.

[0137] The nucleic acids provided herein are engineered in some embodiments. Engineered nucleic acids are nucleic acids that do not occur in nature (e.g., at least two nucleotides covalently linked together and optionally containing a phosphodiester bond, also referred to as a phosphodiester backbone). Engineered nucleic acids include recombinant and synthetic nucleic acids. Recombinant nucleic acids are molecules that are constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., human and mouse). Synthetic nucleic acids are molecules that are amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that have been chemically modified or otherwise altered but can base pair (bind) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules that result from the replication of any of the above.

[0138] Engineered nucleic acids can include DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or hybrid molecules, for example, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural), and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine.

[0139] In some embodiments, the nucleic acid is complementary DNA (cDNA), which is synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0140] The engineered nucleic acids of the present disclosure can be generated using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the nucleic acids are generated using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, DG et al. Nature Methods, 343-345, 2009; and Gibson, DG et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON ASSEMBLY® typically uses three enzyme activities in a single tube reaction: a 5' exonuclease, a 3' extension activity of DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity partially chews back the 5' terminal sequence, exposing complementary sequences for annealing. Polymerase activity then fills the gaps in the annealed domains. DNA ligase then seals the nicks and covalently links the DNA fragments together. The overlapping sequences of the adjacent fragments are much longer than those used in Golden Gate Assembly, resulting in a higher percentage of correct assembly. Other methods of generating engineered nucleic acids can be used according to the present disclosure.

[0141] A gene is a distinct sequence of nucleotides, the order of which determines the order of monomers in a polynucleotide or polypeptide. A gene typically encodes a protein. A gene can be endogenous (naturally occurring in a host organism) or exogenous (transferred into a host organism, naturally or via genetic engineering). An allele arises by mutation and is one of two or more alternative forms of a gene found at the same locus on a chromosome. A gene, in some embodiments, includes a promoter sequence, a coding region (e.g., exon), a non-coding region (e.g., intron), and a regulatory region (also referred to as a regulatory sequence).

[0142] A mouse containing a human gene is considered to contain a human transgene. A transgene is a gene that is exogenous to the host organism. That is, a transgene is a gene that has been transferred into the host organism, either naturally or via genetic engineering. A transgene does not naturally occur in the host organism (the organism that contains the transgene, e.g., a mouse).

[0143] A promoter is a nucleotide sequence at which RNA polymerase binds to the initial transcript (e.g., ATG). A promoter is typically located immediately upstream (at its 5' end) from the transcription start site. In some embodiments, the promoter is an endogenous promoter. An endogenous promoter is a promoter that is naturally present in the host animal.

[0144] An open reading frame is a contiguous stretch of codons that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide, e.g., a protein. An open reading frame is operably linked to a promoter if the promoter controls transcription of the open reading frame.

[0145] Exons are regions of a gene that code for amino acids. Introns (and other non-coding DNA) are regions of a gene that do not code for amino acids.

[0146] The nucleotide sequence encoding the product (e.g., a protein) has a length of 200 base pairs (bp) to 100 kilobases (kb) in some embodiments. The nucleotide sequence has a length of at least 10 kb in some embodiments. For example, the nucleotide sequence may have a length of at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, or at least 35 kb. In some embodiments, the nucleotide sequence has a length of 10 to 100 kb, 10 to 75 kb, 10 to 50 kb, 10 to 30 kb, 20 to 100 kb, 20 to 75 kb, 20 to 50 kb, 20 to 30 kb, 30 to 100 kb, 30 to 75 kb, or 30 to 50 kb.

[0147] Any one of the nucleic acids provided herein may have a length of 200bp to 500kb, 200bp to 250kb, or 200bp to 100kb. The nucleic acid, in some embodiments, has a length of at least 10kb. For example, the nucleic acid may have a length of at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 35kb, at least 50kb, at least 100kb, at least 200kb, at least 300kb, at least 400kb, or at least 500kb. In some embodiments, the nucleic acid has a length of 10 to 500kb, 20 to 400kb, 10 to 300kb, 10 to 200kb, or 10 to 100kb. In some embodiments, the nucleic acid has a length of 10-100 kb, 10-75 kb, 10-50 kb, 10-30 kb, 20-100 kb, 20-75 kb, 20-50 kb, 20-30 kb, 30-100 kb, 30-75 kb, or 30-50 kb. The nucleic acid may be circular or linear.

[0148] The nucleic acids described herein include modifications in some embodiments. Modification, in terms of nucleic acids, is any manipulation of said nucleic acids relative to their corresponding wild-type nucleic acids (e.g., naturally occurring nucleic acids). Genomic modification is thus any manipulation of nucleic acids in a genome (e.g., in coding, non-coding, and / or regulatory regions) relative to their corresponding wild-type nucleic acids in a genome (e.g., naturally occurring (unmodified) nucleic acids). Non-limiting examples of nucleic acid (e.g., genomic) modifications include deletions, insertions, "indels" (deletions and insertions), and substitutions (e.g., point mutations). In some embodiments, deletions, insertions, indels, or other modifications in a gene result in frameshift mutations such that the gene no longer encodes a functional product (e.g., a protein). Modifications also include chemical modifications, e.g., chemical modifications of at least one nucleic acid base. Methods of nucleic acid modification, e.g., those that result in gene inactivation, are known and include, but are not limited to, RNA interference, chemical modification, and gene editing (e.g., using recombinases or other programmable nuclease systems, e.g., CRISPR / Cas, TALEN, and / or ZFNs).

[0149] Loss-of-function mutation, as known in the art, produces gene products that have little or no function.Null mutation is one type of loss-of-function mutation, and produces gene products that have no function.In some embodiments, the inactivated allele is a null allele.Other examples of loss-of-function mutation include missense mutation and frameshift mutation.

[0150] A nucleic acid (e.g., an allele or alleles of a gene) can be modified so that it does not produce detectable levels of a functional gene product (e.g., a functional protein). Thus, an inactivated allele is an allele that does not produce detectable levels of a functional gene product (e.g., a functional protein). A detectable level of protein is any level of protein that is detected using a standard protein detection assay (e.g., flow cytometry and / or ELISA). In some embodiments, an inactivated allele is not transcribed. In some embodiments, an inactivated allele does not encode a functional protein.

[0151] Vectors used for delivery of nucleic acids include minicircles, plasmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes. However, it should be understood that a vector may not be required. For example, a circularized or linearized nucleic acid may be delivered to an embryo without its vector backbone. The vector backbone is small (approximately 4 kb), but the donor DNA to be circularized may range, for example, from >100 bp to 50 kb.

[0152] Methods for delivering nucleic acid to mouse embryos for the generation of transgenic mice include electroporation (see, e.g., Wang W et al. J Genet Genomics 2016;43(5):319-27; WO 2016 / 054032; and WO 2017 / 124086, each of which is incorporated by reference herein), DNA microinjection (see, e.g., Gordon and Ruddle, Science 1981: 214: 1244-124, incorporated by reference herein), embryonic stem cell-mediated gene transfer (see, e.g., Gossler et al., Proc. Natl. Acad. Sci. 1986; 83: 9065-9069, incorporated by reference herein), and retroviral-mediated gene transfer (see, e.g., Jaenisch, Proc. Natl. Acad. Sci. 1976; 73:1260-1264, incorporated herein by reference), any of which may be used as provided herein.

[0153] Genome editing The engineered nucleic acids (e.g., guide RNAs, donor polynucleotides, and other nucleic acid coding sequences) can be introduced, for example, into the genome of an embryo or cell (e.g., stem cell) using any suitable method. The present application contemplates the use of various gene editing techniques to introduce nucleic acids into the genome of a cell or embryo, for example, to generate a transgenic rodent. Non-limiting examples include programmable nuclease-based systems, such as clustered regularly interspaced short palindromic repeats (CRISPR) systems, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). See, e.g., Carroll D Genetics. 2011; 188(4): 773-782; Joung JK et al. Nat Rev Mol Cell Biol. 2013; 14(1): 49-55; and Gaj T et al. Trends Biotechnol. 2013 Jul; 31(7): 397-405, each of which is incorporated by reference herein.

[0154] In some embodiments, the CRISPR system is used to edit the genome of mouse embryos provided herein.See, for example, Harms DW et al., Curr Protoc Hum Genet. 2014; 83: 15.7.1-15.7.27; and Inui M et al., Sci Rep. 2014; 4: 5396 (each of which is incorporated herein by reference).For example, Cas9 mRNA or protein, one or more guide RNAs (gRNAs) and / or donor nucleic acid can be directly delivered (e.g., injected or electroporated) into mouse embryos at one cell (zygote) stage or later stage to promote homology directed repair (HDR), for example, to introduce engineered nucleic acid (e.g., donor nucleic acid) into genome.

[0155] The CRISPR / Cas system is a naturally occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA targeting platform for gene editing. Engineered CRISPR systems contain two major components: a guide RNA (gRNA) and a CRISPR-associated endonuclease (e.g., Cas protein). The gRNA is a short synthetic RNA composed of a scaffold sequence for nuclease binding and a user-defined nucleotide spacer (e.g., about 15-25 nucleotides, or about 20 nucleotides) that defines the genomic target (e.g., gene) to be modified. Thus, one can alter the genomic target of the Cas protein by simply altering the target sequence present in the gRNA. In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes (NGG PAM) or Staphylococcus aureus (NNGRRT or NNGRR(N) PAM), although other Cas9 homologs, orthologs, and / or variants (e.g., evolved versions of Cas9) may be used as provided herein. Further non-limiting examples of RNA-guided nucleases that can be used as provided herein include Cpf1 (TTN PAM); SpCas9 D1135E variant (NGG (reduced NAG binding) PAM); SpCas9 VRER variant (NGCG PAM); SpCas9 EQR variant (NGAG PAM); SpCas9 VQR variant (NGAN or NGNG PAM); Neisseria meningitidis (NM) Cas9 (NNNNGATT PAM); Streptococcus thermophilus (ST) Cas9 (NNAGAAW PAM); and Treponema denticola (TD) Cas9 (NAAAAC). In some embodiments, the CRISPR-associated endonuclease is selected from Cas9, Cpf1, C2c1, and C2c3. In some embodiments, the Cas nuclease is Cas9.

[0156] Guide RNA comprises at least the spacer sequence that hybridizes (binds) to target nucleic acid sequence and the CRISPR repeat sequence that binds endonuclease and guides the endonuclease to target nucleic acid sequence.As will be understood by those skilled in the art, each gRNA is designed to comprise the spacer sequence that is complementary to its genome target sequence.See, for example, Jinek et al., Science, 2012; 337: 816-821 and Deltcheva et al., Nature, 2011; 471: 602-607 (each of which is incorporated herein by reference).

[0157] In some embodiments, the RNA-guided nuclease and gRNA are complexed to form a ribonucleoprotein (RNP) prior to delivery to the embryo.

[0158] The concentration of the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease can vary. In some embodiments, the concentration is 100ng / μl to 1000ng / μl. For example, the concentration can be 100ng / μl, 150ng / μl, 200ng / μl, 250ng / μl, 300ng / μl, 350ng / μl, 400ng / μl, 450ng / μl, 500ng / μl, 550ng / μl, 600ng / μl, 650ng / μl, 700ng / μl, 750ng / μl, 800ng / μl, 850ng / μl, 900ng / μl, 950ng / μl, or 1000ng / μl. In some embodiments, the concentration is between 100 ng / μl and 500 ng / μl, or between 200 ng / μl and 500 ng / μl.

[0159] The concentration of gRNA can also vary. In some embodiments, the concentration is 200ng / μl to 2000ng / μl. For example, the concentration can be 200ng / μl, 300ng / μl, 400ng / μl, 500ng / μl, 600ng / μl, 700ng / μl, 800ng / μl, 900ng / μl, 1000ng / μl, 1100ng / μl, 1200ng / μl, 1300ng / μl, 1400ng / μl, 1500ng / μl, 1600ng / μl, 1700ng / μl, 1700ng / μl, 1900ng / μl, or 2000ng / μl. In some embodiments, the concentration is 500ng / μl to 1000ng / μl. In some embodiments, the concentration is 100ng / μl to 1000ng / μl. For example, the concentration can be 100ng / μl, 150ng / μl, 200ng / μl, 250ng / μl, 300ng / μl, 350ng / μl, 400ng / μl, 450ng / μl, 500ng / μl, 550ng / μl, 600ng / μl, 650ng / μl, 700ng / μl, 750ng / μl, 800ng / μl, 850ng / μl, 900ng / μl, 950ng / μl, or 1000ng / μl.

[0160] In some embodiments, the ratio of the concentration of the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease to the concentration of the gRNA is 2: 1. In other embodiments, the ratio of the concentration of the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease to the concentration of the gRNA is 1:1.

[0161] Donor nucleic acid typically comprises a sequence of interest flanked by homology arms. A homology arm is a region of ssDNA that is homologous to a region of genomic DNA located at a genomic locus. One homology arm is located on the left (5') side of the genomic region of interest (into which the sequence of interest is introduced) (left homology arm), and the other homology arm is located on the right (3') side of the genomic region of interest (right homology arm). These homology arms allow homologous recombination between the ssDNA donor and the genomic locus, resulting in the insertion of the sequence of interest into the genomic locus of interest (e.g., via CRISPR / Cas9-mediated homology-directed repair (HDR)).

[0162] The homology arms can vary in length. For example, each homology arm (left homology arm and right homology arm) can have a length of 20 nucleotide bases to 1000 nucleotide bases. In some embodiments, each homology arm has a length of 20 to 200 nucleotide bases, 20 to 300 nucleotide bases, 20 to 400 nucleotide bases, 20 to 500 nucleotide bases, 20 to 600 nucleotide bases, 20 to 700 nucleotide bases, 20 to 800 nucleotide bases, or 20 to 900 nucleotide bases. In some embodiments, each homology arm has a length of 20 nucleotide bases, 30 nucleotide bases, 40 nucleotide bases, 50 nucleotide bases, 60 nucleotide bases, 70 nucleotide bases, 80 nucleotide bases, 90 nucleotide bases, 100 nucleotide bases, 150 nucleotide bases, 200 nucleotide bases, 250 nucleotide bases, 300 nucleotide bases, 350 nucleotide bases, 400 nucleotide bases, 450 nucleotide bases, 500 nucleotide bases, 550 nucleotide bases, 600 nucleotide bases, 650 nucleotide bases, 700 nucleotide bases, 750 nucleotide bases, 800 nucleotide bases, 850 nucleotide bases, 900 nucleotide bases, 950 nucleotide bases, or 1000 nucleotide bases. In some embodiments, the length of one homology arm is different from the length of the other homology arm. For example, one homology arm may have a length of 20 nucleotide bases and the other homology arm may have a length of 50 nucleotide bases. In some embodiments, the donor DNA is single-stranded. In some embodiments, the donor DNA is double stranded. In some embodiments, the donor DNA is modified, for example, via phosphorothioation. Other modifications may be made.

[0163] Further embodiments The present disclosure provides, in some aspects, an immunodeficient transgenic mouse comprising a transgene encoding human interleukin-3 (IL-3), a transgene encoding human GM-CSF, a transgene encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), a transgene encoding human stem cell factor (SCF), and a transgene encoding human IL-15.

[0164] In some embodiments, the transgenic mouse has a non-obese genetic background. In some embodiments, the transgenic mouse has a severe combined immunodeficiency mutation (Prkdc scid In some embodiments, the transgenic mouse has a null allele of the IL2 receptor common gamma chain (IL2rg null In some embodiments, the transgenic mouse has a NOD-scid IL2Rgamma gene. null Has a genetic background.

[0165] In some embodiments, the transgenic mouse expresses human CD34 + Engrafted with hematopoietic stem cells (HSCs). In some embodiments, the transgenic mice are engrafted with human peripheral blood mononuclear cells (PBMCs).

[0166] In some embodiments, the peripheral blood of the transgenic mouse is human CD45 + In some embodiments, the transgenic mouse comprises circulating human CD3 cells. + T cells, human CD19 + B cells, and human CD33+ myeloid cells.

[0167] In some embodiments, the transgenic mice optionally contain human NK cells at levels significantly higher than those in humanized NSG-SGM3 control mice.

[0168] In some embodiments, the peripheral blood of the transgenic mice contains human regulatory T (Treg) cells at levels significantly lower than those in humanized NSG-SGM3 control mice.

[0169] In some embodiments, the spleens of the transgenic mice express human PD-1 at levels significantly lower than in humanized NSG-SGM3 control mice. + Includes T cells.

[0170] In some embodiments, the CD34 + Human HSCs are transplanted via tail vein injection. In some embodiments, the CD34 + Human HSCs are transplanted into the mice at about 4 weeks of age. In some embodiments, about 5×10 4 CD34 + Human HSCs are transplanted into the mouse. In some embodiments, the CD34 + The human HSCs are transplanted following total body irradiation of the mouse. In some embodiments, total body irradiation of the mouse comprises a dose of about 50-150 cGy. In some embodiments, total body irradiation of the mouse comprises a dose of about 100 cGy.

[0171] In some embodiments, the mouse is + The percentage of natural killer (NK) cells reaches about 2-10% at about 28 weeks after HSC transplantation. In some embodiments, the percentage of human T regulatory cells in the peripheral blood of the mouse reaches about 2-8% at about 16 weeks after HSC transplantation. In some embodiments, the percentage of T regulatory cells in the peripheral blood of the mouse reaches about 5% at about 16 weeks after HSC transplantation. In some embodiments, PD-1 expression on splenic T cells of the mouse reaches about 15-25% at about 16 weeks after HSC transplantation. In some embodiments, PD-1 expression on splenic T cells of the mouse reaches about 20% at about 16 weeks after HSC transplantation.

[0172] In some embodiments, the mouse is inoculated with a human patient-derived xenograft (PDX), where the HSC and the PDX are HLA-mismatched. In some embodiments, the inoculation comprises subcutaneous or orthotopic implantation. In some embodiments, the human PDX is transfected with the CD34 + About two weeks after transplantation of human HSCs, the mice are inoculated. In some embodiments, the human PDXs are transfected with the CD34 + The mice are inoculated about 12 weeks after transplantation with human HSCs. In some embodiments, the human PDX is derived from a primary patient sample. In some embodiments, the human PDX is derived from an archived tumor sample that has been passaged as a xenograft for at least one generation. In some embodiments, the human PDX is a xenograft derived from ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), bladder cancer, lymphoma (e.g., AML, CML, ALL, CLL, DLBCL (diffuse large B-cell lymphoma)), breast cancer (e.g., triple-negative breast cancer (TNBC)), brain cancer, pancreatic cancer, prostate cancer, colon cancer, colorectal cancer, endometrial cancer, gastric / GIST cancer, hepatocellular carcinoma, kidney cancer / renal cancer, skin cancer (e.g., melanoma), soft tissue cancer, sarcoma, or cancer cell line. In some embodiments, about 5×10 6 The above human PDX of cells are seeded.

[0173] In some embodiments, the exponential growth phase of the tumor occurs at least 40 days after implantation of the tumor. In some embodiments, the tumor volume is greater than or equal to 600 mm by 50 days after implantation of the tumor. 3 is less than.

[0174] In some embodiments, the mouse is administered an anti-cancer compound, hi some embodiments, the anti-cancer compound is 5-FU, Avastin, cisplatin, carboplatin, pembrolizumab, docetaxel, or a combination thereof.

[0175] In some embodiments, the mouse further comprises transplanted human peripheral mononuclear blood cells (PMBCs). In some embodiments, the human PMBCs are transplanted via tail vein injection. ... about 1×10 6 ~5×10 6 Human PBMCs of cells are transplanted. In some embodiments, about 5×10 6 The human PBMC of cells are transplanted. In some embodiments, about 2×10 6 The human PBMC of cells are transplanted. In some embodiments, about 1×10 6 The human PBMCs of cells are transplanted. In some embodiments, the human PBMCs are derived from a patient.

[0176] In some embodiments, the human PBMCs are transplanted into the mouse at about 4 weeks of age. In some embodiments, the human PBMCs are transplanted following total body irradiation of the mouse. In some embodiments, the total body irradiation of the mouse comprises a dose of about 50-200 cGy. In some embodiments, the total body irradiation of the mouse comprises a dose of about 100 cGy. In some embodiments, the total body irradiation of the mouse comprises a dose of about 150-175 cGy.

[0177] In some embodiments, the mouse is + The percentage of human CD45 cells reaches about 15-35% about 8 days after PBMC transplantation. + The percentage of myeloid cells reaches about 0.1-0.2% about 8 days after PBMC transplantation. In some embodiments, the percentage of human CD45 cells in the peripheral blood of the mice is about 0.1-0.2%. +The percentage of NK cells reaches about 5-10% about 8 days after PBMC transfer. In some embodiments, the activated splenic CD4 + The percentage of T cells reaches about 55-70% about 8 days after PBMC transfer. In some embodiments, the activated splenic CD8 + The percentage of T cells reaches approximately 80-90% approximately 8 days after PBMC transfer.

[0178] In some embodiments, the mouse is transplanted with pathological human cells. In some embodiments, the pathological human cells are selected from blood cells, muscle cells, and neuronal cells. In some embodiments, the pathological human cells are tumor cells. In some embodiments, the tumor cells are primary tumor cells. In some embodiments, the pathological human cells are cancerous cells. In some embodiments, the pathological human cells are non-cancerous cells. In some embodiments, the pathological human cells and the PBMCs are autologous. In some embodiments, the PBMCs and the human immune cells are allogeneic.

[0179] In some embodiments, the mouse is further administered a candidate agent for treating cytokine release syndrome (CRS). In some embodiments, circulating levels of a cytokine selected from the group consisting of interleukin (IL)-6, tumor necrosis factor (TNF), and IL-2 are measured.

[0180] Further embodiments of the present disclosure are encompassed by the following numbered paragraphs: 1. An immunodeficient transgenic mouse containing a transgene encoding human interleukin-3 (IL-3), a transgene encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), a transgene encoding human stem cell factor (SCF), and a transgene encoding human IL-15. 2. The transgenic mouse of paragraph 1, wherein the transgenic mouse has a non-obese diabetic genetic background. 3. The transgenic mice are those expressing a severe combined immunodeficiency mutant (Prkdc scid 3. The transgenic mouse of paragraph 1 or 2, comprising: 4. The transgenic mouse is a mouse that has a null allele of the IL2 receptor common gamma chain (IL2rg null ). The transgenic mouse of any one of the preceding paragraphs, 5. The transgenic mouse is a NOD-scid IL2Rgamma null A transgenic mouse according to any one of the preceding paragraphs, having a genetic background. 6. The transgenic mouse expresses human CD34 + The transgenic mouse of any one of the preceding paragraphs, engrafted with hematopoietic stem cells (HSCs). 7. The peripheral blood of the transgenic mice was cultured with human CD45 + 7. The transgenic mouse of paragraph 6, comprising the cell. 8. The transgenic mouse of paragraph 6 or 7, wherein the transgenic mouse comprises circulating human T cells, human B cells, and human myeloid cells. 9. The transgenic mouse of any one of paragraphs 6 to 8, wherein the transgenic mouse contains significantly more human NK cells relative to a humanized NSG-SGM3 control mouse. 10. The transgenic mouse of any one of paragraphs 6 to 9, wherein the transgenic mouse is transplanted with human xenograft tissue. 11. The transgenic mouse of paragraph 10, wherein the transgenic mouse comprises significantly fewer human regulatory T (Treg) cells in the peripheral blood relative to a humanized NSG-SGM3 control mouse. 12. The transgenic mice express significantly less human PD-1 than humanized NSG-SGM3 control mice.+ 12. The transgenic mouse of paragraph 10 or 11, comprising a T cell. 13. The transgenic mouse of any one of paragraphs 1 to 5, wherein the transgenic mouse is transplanted with human peripheral blood mononuclear cells (PBMCs). 14. The transgenic mouse of paragraph 13, wherein the transgenic mouse comprises circulating human T cells, human B cells, and human NK cells. 15. The transgenic mice had significantly more human CD45 in the peripheral blood than humanized NSG-SGM3 control mice. + 15. The transgenic mouse of paragraph 13 or 14, comprising the cell. 16. The transgenic mouse of any one of paragraphs 13 to 15, wherein the transgenic mouse contains significantly more human myeloid cells in the peripheral blood relative to a humanized NSG-SGM3 control mouse. 17. The transgenic mouse of any one of paragraphs 13 to 15, wherein the transgenic mouse contains significantly more human NK cells in the peripheral blood relative to a humanized NSG-SGM3 control mouse. 18. The transgenic mouse of any one of paragraphs 13 to 15, wherein the transgenic mouse contains significantly more activated human T cells in the spleen relative to a humanized NSG-SGM3 control mouse. 19. A method for generating a transgenic mouse, said method comprising: (a) an immunodeficient transgenic mouse comprising a transgene encoding human interleukin-3 (IL-3), a transgene encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), and a transgene encoding human stem cell factor (SCF); (b) a transgenic mouse comprising a transgene encoding human IL-15; The method comprises the step of breeding. 20. The method of paragraph 19, wherein the immunodeficient transgenic mouse of (a) has an NSG-SGM3 genetic background. 21. A method comprising the step of administering human xenograft tissue or cells, optionally pathological human xenograft tissue or cells, to a transgenic mouse described in any one of paragraphs 1 to 5. 22. Administering a dose of human peripheral blood mononuclear cells (PBMCs) to the transgenic mouse of any one of paragraphs 1 to 5, wherein the dose is 1×10 7 The method of claim 1, wherein the number of PBMCs is less than 100. 23. The dose is about 1×10 6 ~Approx. 5×10 6 23. The method of paragraph 22, wherein the PBMCs are 24. The dose is about 2×10 6 24. The method of paragraph 23, wherein the PBMCs are 25. The dose is about 1×10 6 24. The method of paragraph 23, wherein the PBMCs are 26. The method of any one of paragraphs 22 to 25, wherein the PBMCs are administered intravenously. 27. The method of any one of paragraphs 22 to 26, further comprising administering to the transgenic mouse an agent that activates human T cells. 28. The method of any one of paragraphs 22 to 27, further comprising administering a candidate therapeutic agent to the transgenic mouse. 29. The method of paragraph 28, wherein the putative therapeutic agent is an anti-cancer agent or an immunomodulatory agent. 30. The method of paragraph 29, wherein the immunomodulatory agent is an anti-inflammatory agent, a corticosteroid, or an immunosuppressant. 31. The method of any one of paragraphs 28 to 30, further comprising a step of assaying cytokine levels in the immunodeficient transgenic mouse and / or a step of assaying the effectiveness of the therapeutic agent. 32. The method of paragraph 31, wherein the cytokine assayed is at least one cytokine selected from the group consisting of IFN-γ, IL-2, IL-4, IL-6, IL-10, and TNF.

[0181] Further aspects, advantages and / or other features of the exemplary embodiments of the present disclosure will become apparent in view of the following detailed description, taken in conjunction with the accompanying drawings. It should be apparent to those skilled in the art that the detailed embodiments provided herein are merely exemplary and illustrative, and not limiting. Many embodiments of its modifications are indicated to fall within the scope of the present disclosure and its equivalents.

[0182] The following examples are provided to further illustrate various non-limiting embodiments and techniques of the method of the present invention, including the experiments carried out in developing the method of the present invention. However, it should be understood that these examples are meant to be illustrative and do not limit the scope of the claims. As will be apparent to those skilled in the art, many variations and modifications are intended to be encompassed within the spirit and scope of the present disclosure. EXAMPLES

[0183] Working Example Example 1 - NSG-SGM3xIL15 mice humanized with CD34 hematopoietic stem cells (HSC) NSG-SGM3 and NSG-SGM3xIL15 mice (4 weeks old; N=5 / group) were irradiated with 100 cGy and injected intravenously with 50,000 CD34+ HSCs derived from umbilical cord blood. Three donors were used. Human CD45+ cell levels were monitored in peripheral blood by flow cytometry for up to 32 weeks. Counting beads were added and absolute cell counts were calculated. The human CD45+ engraftment time kinetics and engraftment levels are shown in Figures 1A-1C (frequency) and Figures 1D-1F (cells per microliter of blood). Overall, the kinetics and levels were similar between the two mouse strains.

[0184] Next, levels of human immune cells were measured in the peripheral blood of NSG-SGM3 and NSG-SGM3xIL15 mice at 4, 6, 12, 16, 20, 24, and 28 weeks after transplantation of CD34+ HSCs. The mice underwent the same humanization procedure described above. The results are shown in Figures 2A-2C (NSG-SGM3 mice) and 2D-2F (NSG-SGM3xIL15 mice). Total NK cell levels (arrows) were higher in NSG-SGM3xIL15 mice than in NSG-SGM3 mice, and engraftment levels were well maintained over the entire 28-week time course for all three donors. Levels of circulating CD3+ T, CD19+ B, and CD33+ myeloid cells were similar between the two strains.

[0185] Human NK cell engraftment was further investigated using the same protocol and donors as above. The total NK cell frequencies from the three donors are shown in Figures 3A-3C, and the total NK cells per microliter of blood from the three donors are shown in Figures 3D-3F. During the 28-week time course, significantly higher levels of human NK cells were found in NSG-SGM3xIL15 mice compared to NSG-SGM3 mice. Specifically, the human NK cell frequencies were 0.1-1% in NSG-SGM3 mice and approximately 2-10% in SGM3xIL15 mice.

[0186] We also examined the frequency of regulatory T (Treg) cells in the peripheral blood of NSG-SGM3 and NSG-SGM3xIL15 mice transplanted with HSCs as described above, and PD-1 expression on CD4+T and CD8+T cells in the spleen. As shown in Figure 5A, physiological levels of Treg were observed in the blood of NSG-SGM3xIL15 mice, but higher levels were observed in NSG-SGM3 mice (20% vs. 5% for CD45). Regarding PD-1 expression on T cells, Figure 5B shows that PD-1 expression on CD4T and CD8T cells was higher in the spleen of NSG-SGM3 mice than in NSG-SGM3xIL15 mice (60% vs. 20%).

[0187] Thus, overall, the NSG-SGM3xIL15 mice humanized with HSCs clearly exhibited characteristics that more closely resemble the human immune system compared to the NSG-SGM3 mice.

[0188] Example 2 - NSG-SGM3xIL15 mice humanized with CD34 hematopoietic stem cells (HSC) and engrafted with patient-derived xenografts (PDX) NSG-SGM3xIL15, NSG-SGM3 and NSG mice were humanized with HSCs as described in Example 1. The mice were then subcutaneously implanted with a cell line-derived xenograft (MDA-MB-231) or one of two patient-derived xenografts (PS4050 or LG1306). Each line of mice was implanted with xenografts from one to three donors, as shown in Figures 4A-4F. Tumor volumes were measured during the time course experiments. Figures 4A-4C show the average tumor volumes of the three xenograft models, and Figures 4E-4F show the individual tumor sizes of the three models. The growth kinetics of all three xenograft models was significantly delayed in NSG-SGM3xIL15 mice. This is consistent with a stronger allogeneic response to the tumors due to an enhanced human immune system. Moreover, the results are consistent with the data in Figure 5A (Treg frequency) and Figure 5B (PD-1 expression on T cells), as NSG-SGM3xIL15 mice appear to have more activated T cells and more abundant NK cells.

[0189] Example 3 - NSG-SGM3xIL15 mice humanized with peripheral blood mononuclear cells (PBMC) NSG-SGM3xIL15 and NSG-SGM3 mice were humanized with human peripheral blood mononuclear cells (PBMCs) and humanization was confirmed. Briefly, the mice were irradiated (100 cGy) and on day 0, human PBMCs were administered intravenously (5 × 10 6 On day 8, blood and spleen samples were collected for analysis via flow cytometry. The results are shown in Figures 6A-6C (blood) and Figures 7A-7C (spleen).6 NSG-SGM3xIL15 mice transplanted with PBMCs show better humanization (as indicated by higher hCD45+; Figure 6A) and higher myeloid cell levels (Figure 6B) compared to NSG-SGM3 mice. Furthermore, the NSG-SGM3xIL15 mice showed significantly higher natural killer (NK) cell levels compared to NSG-SGM3 mice (Figure 6C). Taken together, the data clearly show that NSG-SGM3xIL15 mice transplanted with PBMCs may be a useful model for investigating myeloid and / or NK cells. In spleen samples, NSG-SGM3xIL15 mice showed higher activated T cell levels compared to NSG-SGM3 mice (Figures 7A-7C), suggesting that NSG-SGM3xIL15 may be used as a model for cytokine release syndrome, because T cell activation leads to T cell-associated cytokine release syndrome.

[0190] The use of low levels of PBMC for humanization was investigated. Briefly, NSG-SGM3xIL15 and NSG-SGM3 mice were irradiated (150 cGy or 175 cGy) and on day 0, human PBMC were administered intravenously (1 × 10 6 / mouse or 2 × 10 6 On day 12, blood samples were collected for analysis via flow cytometry. The results are shown in Figures 8A-8C, where NSG-SGM3xIL15 mice received 1 x 10 6 This clearly shows that humanization can be achieved with approximately 10 PBMC / mouse. This is in contrast to the amount of PBMC typically used (e.g., 10 to 20 × 10 6 PBMCs). 6 pcs or 2×10 6 With this PBMC humanization, NSG-SGM3xIL15 mice showed better humanization (Figure 8A; human CD45+ cells), higher myeloid cell levels (Figure 8B), and expanded NK cell populations (Figure 8C) in the blood, similar to the pattern seen with the higher PBMC doses (compare Figures 7A-7C).

[0191] Example 4 - NSG-SGM3xIL15 mice humanized with peripheral blood mononuclear cells (PBMC) - Toxicity induction NSG-SGM3xIL15 and NSG-SGM3 mice were humanized with human PBMCs and then administered various drugs to induce cytokine release syndrome (CRS). Briefly, mice were irradiated (100 cGy) and administered PBS, OKT3, anti-CD28, or CD19xCD3 BiTE. Serum cytokine levels were measured at this time point and the resulting levels of selected cytokines are shown in Figures 9A-9C. Overall, NSG-SGM3xIL15 mice showed higher cytokine levels in the circulation when administered OKT3 and anti-CD28, which are known to induce cytokine release, compared to NSG-SGM3 mice.

[0192] The use of lower levels of PBMC for humanization was also tested. Briefly, NSG-SGM3xIL15 and NSG-SGM3 mice were irradiated (150 cGy or 175 cGy) and administered human PBMC intravenously (1×10 6 / mouse or 2 × 10 6 On day 12, mice were administered PBS or OKT3 and serum cytokine levels were analyzed. The results are shown in Figures 10A-10C and clearly show that the model still captures human cytokine release with a larger difference between drug (OKT3) and control (PBS). Even with lower PBMC humanization, NSG-SGM3xIL15 mice show a trend for higher cytokine release than NSG-SGM3 mice. This suggests that NSG-SGM3xIL15 mice may capture cytokine release syndrome with a significantly lower number of PBMCs.

[0193] Example 5 - Irradiated and non-irradiated SGM3xIL15 mice humanized with PBMC NSG-SGM3xIL15 mice were either irradiated with 100 cGy or not. Approximately 4 hours later, mice were transplanted with 4 million PBMCs from donor 3769. Mice were bled once a week and stained using an anti-human CD45 antibody assay. Absolute cell counts were determined by adding counting beads immediately prior to flow cytometry analysis. Irradiation resulted in increased humanization and acceleration of circulating hCD45+ cells (Figures 11A-11B); however, irradiation shortened the lifespan of the mice. hCD45+ cells in non-irradiated mice expanded until day 49, reaching similar levels observed in irradiated mice at day 21 (data not shown). These results indicate that irradiation enhanced short-term humanization of hCD45 cells compared to non-irradiation.

[0194] Example 6 - Irradiated and non-irradiated SGM3xIL15 mice humanized with PBMC from two donors NSG-SGM3xIL15 mice were either irradiated with 100 cGy or not and transplanted with 4 million PBMCs from donors 0595 and 3769. Mice were bled once a week and stained using an anti-human CD45 antibody assay. Absolute cell counts were determined by adding counting beads immediately prior to flow cytometry analysis. As shown in Figures 12A-12B, irradiated mice clearly showed enhanced humanization on days 14 and 21 compared to non-irradiated mice. Humanization followed a similar pattern in both donors, although there was some donor variability. In irradiated mice, slightly higher engraftment levels were observed for donor 3769 than donor 0595. Donor 3769 also showed higher engraftment numbers than donor 0595 in non-irradiated mice.

[0195] Example 7 - Circulating human IgG levels in irradiated and non-irradiated NSG-SGM3xIL15 mice humanized with PBMCs from three donors NSG-SGM3xIL15 mice were either irradiated with 100 cGy or not and transplanted with 4 million PBMCs from donors 0364, 0595, and 3769. Mice were bled once a week and analyzed for circulating hIgG levels. All three groups of mice with donor PBMCs clearly showed increased IgG levels over time, with irradiated mice producing significantly higher IgG levels at day 14. As shown in Figure 13, higher IgG levels were observed at both time points measured in irradiated mice (days 14, 21) when compared to non-irradiated mice. Irradiated donor 0595 mice reached near-physiologic levels of human IgG as early as day 14 (normal adult IgG levels: 600-1600 mg / dL (6000-16000 μg / mL)). Irradiated mice for donors 0364 and 3769 also showed enhanced IgG production when compared to their non-irradiated counterparts on day 14. Although there was donor variability in the need for irradiation, in all cases irradiation improved IgG production.

[0196] Example 8 - Human Ig isotypes in serum of irradiated or non-irradiated NSG-SGM3xIL15 mice humanized with PBMC NSG-SGM3xIL15 mice were either irradiated with 100 cGy or not, transplanted with 4 million PBMCs from donor 0595, and bled once a week. Serum was analyzed for human IgM, IgA, and the four IgG subclasses: IgG1, IgG2, IgG3, IgG4. As shown in Figures 14A-14B, irradiation enhanced Ig levels in serum to near-physiological levels for IgM, IgG1, IgG2, IgG3, and IgG4 (normal adult ranges are 2.80-8.00 mg / ml for IgG1; 1.15-5.70 mg / mL for IgG2; 0.24-1.25 mg / mL for IgG3; 0.052-1.25 mg / mL for IgG4; and 0.4-2.5 mg / mL for IgM). Of the isotypes measured, only one (IgA) did not result in a clear increase in Ig levels in peripheral blood (normal adult range for IgA is 0.8-3.0 mg / mL).

[0197] Example 9 - Human immune cell populations in irradiated and non-irradiated NSG-SGM3xIL15 mice humanized with PBMC NSG-SGM3xIL15 mice were either irradiated with 100 cGy or not and approximately 4 hours later were transplanted with 4 million PBMCs from donor 0595. Mice were bled once a week and stained using anti-human CD45, CD3, CD4, CD8, CD19, CD56, CD14 and CD16 antibody assays. Absolute cell counts were determined by adding counting beads immediately prior to flow cytometry analysis. As shown in Figures 15A-15B, irradiation resulted in increased hCD45+ cells in the circulation, as well as expansion of B and T cells, while CD56+ and CD14+ numbers remained the same. Unlike CD3+ T cells, whose expansion was observed in non-irradiated cells in peripheral blood, little or no expansion of CD19+ B cells was observed.

[0198] Example 10 - Radiation dose affects humanized and circulating hIgG levels in PBMC-engrafted NSG-SGM3xIL15 mice NSG-SGM3xIL15 mice received radiation doses between 0 and 200 cGy (0 cGy, 50 cGy, 100 cGy, 150 cGy, or 200 cGy) and were transplanted with 4 million PBMCs from donor 3769. Mice were bled weekly and analyzed for hCD45+ cells and circulating hIgG levels.

[0199] All radiation doses clearly demonstrated increased hCD45+ cells and IgG levels over time. As shown in Figures 16A-16B, different radiation doses produced different outcomes. Radiation doses of 150 and 200 cGy significantly enhanced humanization on day 14 compared to other conditions, but produced poor immunoglobulin production compared to 50 and 100 cGy. 100 cGy enhanced humanization over 50 cGy on days 14 and 28, but did not improve immunoglobulin production, whereas 50 cGy produced the highest immunoglobulin levels of any dose tested. Higher radiation doses produced increased hCD45 cells first, but also resulted in animals meeting humane euthanasia endpoint criteria before peak hIgG levels could be obtained. Conversely, non-irradiated mice had a longer life span and delayed hCD45 expansion, but non-irradiated hIgG levels did not reach those of the 50-100 cGy group, even though comparable humanized levels were observed. As shown in Figures 16A-16B, with irradiation, immunoglobulin levels were enhanced over non-humanized mice (2000 μg / ml at SD 48) that reached the same hCD45+ cell levels over time (3500 μg / ml at day 28 for 50 cGy; 2500 μg / ml at day 21 for 100 cGy) (SD 21 for 100 cGy, SD 28 for 50 cGy, and SD 48 for 0 cGy). These results clearly show that irradiation enhances immunoglobulin production independently of humanization.

[0200] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may include the entire document.

[0201] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0202] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0203] In the claims and the above specification, all transitional phrases (e.g., "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," etc.) are to be understood to mean open ended, i.e., including, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the U.S. Patent Office Manual of Patent Examining Procedure Section 2111.03.

[0204] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0205] When a range of values ​​is provided, each value between the upper and lower ends of the range is specifically contemplated and described herein.

Claims

1. 1. A method for generating a mouse model of human humoral immunity and human cellular immunity, said method comprising: (a) providing an immunodeficient mouse comprising a transgene encoding human interleukin-3 (IL-3), a transgene encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), a transgene encoding human stem cell factor (SCF), and a transgene encoding human IL-15; (b) administering a myeloablative treatment to said immunodeficient mouse; and (c) administering human peripheral blood mononuclear cells (hPBMCs) to the immunodeficient mouse; The method includes:

2. 10. The method of claim 1, wherein the myeloablative treatment is irradiation, optionally wherein the irradiation is gamma irradiation (cGy), and further optionally wherein the myeloablative treatment is ≦150 cGy.

3. 1 x 10 ７ 2. The method of claim 1, wherein fewer than 10 hPBMCs are administered to the immunodeficient mouse.

4. 2. The method of claim 1, wherein the administration of the hPBMCs is within 3 days of the administration of the myeloablative treatment.

5. The method of claim 1, further comprising administering human pathological cells to said immunodeficient mouse.

6. The method of claim 1, further comprising administering to the immunodeficient mouse a human therapeutic or prophylactic agent.

7. 7. The method of claim 6, wherein the human therapeutic agent is selected from a human immunomodulatory agent, optionally a monoclonal antibody or cell therapy, and further optionally a T cell therapy.

8. 7. The method of claim 6, wherein the human prophylactic agent is a vaccine, optionally a protein antigen or a nucleic acid encoding a protein antigen, optionally a cancer antigen or a pathogenic antigen.

9. The method described in claim 6, wherein the administration of the human therapeutic agent is within 30 days of the administration of the myeloablative treatment.

10. 7. The method of claim 6, further comprising assaying a sample from the mouse, optionally a blood sample, for one or more human cytokines, one or more human immunoglobulins, and / or one or more anti-drug antibodies.

11. the one or more human cytokines are selected from interferon gamma (IFN-γ), interleukin (IL)-2, IL-4, IL-6, IL-10, and tumor necrosis factor alpha (TNFα); and / or the one or more human immunoglobulins are selected from IgM, IgA, IgG, optionally IgG1, IgG2, IgG3, and IgG4; The method of claim 10.

12. An immunodeficient mouse comprising a transgene encoding human interleukin-3 (IL-3), a transgene encoding human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), a transgene encoding human stem cell factor (SCF), and a transgene encoding human IL-15.

13. The immunodeficient mouse of claim 12 , wherein the immunodeficient mouse has a non-obese diabetic (NOD) genetic background.

14. The immunodeficient mice were harboring severe combined immunodeficiency mutant (Prkdc ｓｃｉｄ 13. The immunodeficient mouse of claim 12, having

15. The immunodeficient mice were bred with a null allele of the IL2 receptor common γ chain (IL2rg ｎｕｌｌ 13. The immunodeficient mouse of claim 12, having

16. The immunodeficient mice were NOD-scid IL2Rgamma ｎｕｌｌ The immunodeficient mouse of claim 12 having a genetic background.

17. The immunodeficient mouse of claim 12, wherein the immunodeficient mouse is subjected to myeloablative treatment, optionally irradiation.

18. The immunodeficient mouse of claim 12, wherein cells of the immunodeficient mouse produce human IgM, human IgG1, human IgG2, human IgG3, and human IgG4.

19. The cells of the immunodeficient mouse are 6000 to 16000 μg / mL human IgG, 1000-2000 μl / mL human IgM, 1500-2500 μl / mL human IgG1, 500-1500 μl / mL human IgG2, 150-205 μl / mL human IgG3, and / or 50-1000 μl / mL human IgG4 The immunodeficient mouse of claim 18,

20. The immunodeficient mice were cultured using human CD3 ＋ , CD4 ＋ , CD8 ＋ and CD19 ＋ The immunodeficient mouse of claim 12, which produces cells.

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