阅读说明：本技术 缺乏i类和ii类mhc的nsg小鼠 (NSG mice deficient in class I and class II MHC ) 是由 M·A·布雷姆 M·V·维勒斯 D·L·格雷纳 L·D·舒尔兹 于 2018-05-14 设计创作，主要内容包括：根据本发明的方面,提供了一种NOD.Cg-Prkdc<Sup>scid</Sup> Il2rg<Sup>tm1Wjl</Sup>/SzJ(NOD-scid-IL2rγ<Sup>null</Sup>,NSG)小鼠,所述小鼠被遗传修饰使得所述NSG小鼠缺乏功能性主要组织相容性复合物I(MHC I)和缺乏功能性主要组织相容性复合物II(MHC II)。根据特定的方面,遗传修饰的NSG小鼠是NOD.Cg-Prkdc<Sup>scid</Sup> H2-K1<Sup>tm1Bpe</Sup> H2-Ab1<Sup>em1Mvw</Sup> H2-D1<Sup>tm1Bpe</Sup> Il2rg<Sup>tm1Wjl</Sup>/SzJ(NSG-(K<Sup>b</Sup> D<Sup>b</Sup>)<Sup>null</Sup>(IA<Sup>null</Sup>))小鼠、NSG-RIP-DTR(K<Sup>b</Sup> D<Sup>b</Sup>)<Sup>null</Sup>(IA<Sup>null</Sup>)小鼠或NOD.Cg-B2m<Sup>tm1Unc</Sup> Prkdc<Sup>scid</Sup> H2<Sup>dlAb1-Ea</Sup> Il2rg<Sup>tm1Wjl</Sup>/SzJ(NSG-B2M<Sup>null</Sup>(IA IE<Sup>null</Sup>))小鼠。根据本文所描述的方面向遗传修饰的免疫缺陷小鼠施用人免疫细胞和/或人肿瘤细胞,并且可以使用所提供的小鼠对一种或多种测试物质进行测定。(Cg-Prkdc according to an aspect of the invention there is provided a NOD scid Il2rg tm1Wjl /SzJ(NOD‑scid‑IL2rγ null NSG) mice genetically modified such that said NSG mice lack functional major histocompatibility complex i (mhc i) and lack functional major histocompatibility complex ii (mhc ii). According to a particular aspect, the genetically modified NSG mouse is nod scid H2‑K1 tm1Bpe H2‑Ab1 em1Mvw H2‑D1 tm1Bpe Il2rg tm1Wjl /SzJ(NSG‑(K b D b ) null (IA null ) A mouse,NSG‑RIP‑DTR(K b D b ) null (IA null ) Cg-B2m mouse or NOD tm1Unc Prkdc scid H2 dlAb1‑Ea Il2rg tm1Wjl /SzJ(NSG‑B2M null (IA IE null ) Mice). Human immune cells and/or human tumor cells are administered to genetically modified immunodeficient mice according to aspects described herein, and one or more test substances can be assayed using the provided mice.)

1. Cg-Prkdc of NOD^scidIl2rg^tm1Wjl/SzJ(NOD-scid-IL2rγ^nullAn NSG) mouse genetically modified such that said NSG mouse lacks functional major histocompatibility complex i (mhc i) and lacks functional major histocompatibility complex ii (mhc ii).

2. The genetically modified NSG mouse of claim 1, wherein the mouse is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mice).

3. The genetically modified NSG mouse of claim 1, wherein the mouse is NSG-RIP-DTR (K)^bD^b)^null(IA^null) A mouse.

4. The genetically modified NSG mouse of claim 1, wherein the mouse is nod.cg-B2m^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) Mice).

5. The genetically modified NSG mouse of any of claims 1, 2, 3, or 4, further comprising a human immune cell.

6. The genetically modified NSG mouse of claim 5, wherein the human immune cell is a human peripheral blood mononuclear cell.

7. The genetically modified NSG mouse of claim 5, wherein the human immune cell is a human T cell.

8. The genetically modified NSG mouse of any of claims 1 to 7, further comprising a human tumor cell.

9. An immunodeficient mouse genetically modified such that the mouse lacks functional major histocompatibility complex I (MHC I) and lacks functional major histocompatibility complex II (MHC II), with the proviso that the immunodeficient mouse is not an NOD/Shi-scid-IL2r γ characterized by an β 2m (component of MHC I) knockout and an IA β (light chain of MHC II) knockout^nullA mouse.

10. The genetically modified immunodeficient mouse of claim 9, further comprising a human immune cell.

11. The genetically modified immunodeficient mouse of claim 10, wherein the human immune cell is a human peripheral blood mononuclear cell.

12. The genetically modified immunodeficient mouse of claim 10, wherein the human immune cell is a human T cell.

13. The genetically modified NSG mouse of any of claims 9 to 12, further comprising a human tumor cell.

14. A method of modeling the role of the human immune system in a genetically modified immunodeficient mouse, comprising:

administering a test substance to the genetically modified immunodeficient mouse of any of claims 5 to 8 or 10 to 13; and

determining the effect of the human immune system in the genetically modified immunodeficient mouse.

15. The method of claim 14, wherein the test substance is an anti-cancer agent.

16. The method of claim 14 or 15, wherein the test substance is an immunotherapeutic agent.

17. The method of claim 14, 15 or 16, wherein the test substance is an anti-cancer immunotherapeutic.

18. The method of any one of claims 14 to 17, wherein the test substance is an immune checkpoint inhibitor.

19. The method of claim 18, wherein the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

20. The method of claim 18 or 19, wherein the immune checkpoint inhibitor is alemtuzumab, avizumab, deluzumab, ipilimumab, nivolumab, or pembrolizumab, or the immune checkpoint inhibitor comprises an antigen binding fragment of any of the foregoing.

21. A genetically modified mouse substantially as described or shown herein.

22. A method of using a genetically modified mouse substantially as described or shown herein.

Technical Field

Generally described are mouse models of functional human cells and tissues. According to a particular aspect, genetically modified immunodeficient mice are provided which lack class I MHC and class II MHC. According to a further specific aspect, there is provided a genetically modified immunodeficient mouse which lacks MHC class I and MHC class II and which comprises 1) transplanted functional human T cells and 2) allogeneic or xenogeneic cells, such as tumor cells of human patient origin.

Background

Humanized mice (e.g., immunodeficient mice transplanted with functional human cells and tissues) have been widely used to model the function of human immune cells in vivo. The main limitation of studying human T cell function in such mouse models is the rapid development of Graft Versus Host Disease (GVHD), which not only shortens the experimental time window, but also confounds the analysis of human T cell function due to the ongoing potential GVHD that ultimately kills the mice. These problems have hampered the study of human T cell function.

Several attempts were made to generate humanized mouse models lacking class I or class II Major Histocompatibility Complex (MHC). For example, Vugmeyster et al disclose a mouse model lacking MHC molecules encoded by the H-2K and H-2D genes (K)^bD^b-/-Mice) (Vugmeyster et al, Proc. Natl. Acad. Sci. USA 95: 12492-. Ashizawa et al describe a humanized immunodeficient NOG mouse (NOD/Shi-scid-IL2 r. gamma^null)[NOD/Shi-Prkdc^scid-IL2rγ^null]Which knock out MHC class I/II (Ashizawa et al, Clin Cancer Res; 23(1), 149-.

There is a continuing need for mouse models of functional human cells and tissues.

Disclosure of Invention

Cg-Prkdc according to an aspect of the invention there is provided a NOD^scidIl2rg^tm1Wjl/SzJ(NOD-scid-IL2rγ^nullNSG) mice genetically modified such that said NSG mice lack functional major histocompatibility complex i (mhc i) and lack functional major histocompatibility complex ii (mhc ii). According to a particular aspect, the genetically modified NSG mouse is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse, NSG-RIP-DTR (K)^bD^b)^null(IA^null) Cg-B2m mouse or NOD^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) Mice).

Cg-Prkdc according to an aspect of the invention there is provided a NOD^scidIl2rg^tm1Wjl/SzJ(NOD-scid-IL2rγ^nullNSG) mice genetically modified such that said NSG mice lack functional major histocompatibility complex i (mhc i) and lack functional major histocompatibility complex ii (mhc ii), comprising human immune cells. According to a particular aspect, the genetically modified NSG mouse is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse comprising human immune cells, NSG-RIP-DTR (K)^bD^b)^null(IA^null) A mouse comprising human immune cells, or NOD.Cg-B2m^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) A mouse comprising human immune cells.

Cg-Prkdc according to an aspect of the invention there is provided a NOD^scidIl2rg^tm1Wjl/SzJ(NOD-scid-IL2rγ^nullNSG) mice genetically modified such that said NSG mice lack functional major histocompatibility complex i (mhc i) and lack functional major histocompatibility complex ii (mhc ii), comprising human peripheral blood mononuclear cells. According to a particular aspect, the genetically modified NSG mouse is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse comprising human peripheral blood mononuclear cells, NSG-RIP-DTR (K)^bD^b)^null(IA^null) A mouse comprising human peripheral blood mononuclear cells, or NOD.Cg-B2m^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) Mouse comprising human peripheral blood mononuclear cells.

According to aspects of the inventionCg-Prkdc is provided^scidIl2rg^tm1Wjl/SzJ(NOD-scid-IL2rγ^nullNSG) mice genetically modified such that said NSG mice lack functional major histocompatibility complex i (mhc i) and lack functional major histocompatibility complex ii (mhc ii), comprising human T cells. According to a particular aspect, the genetically modified NSG mouse is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse comprising human T cells, NSG-RIP-DTR (K)^bD^b)^null(IA^null) Cg-B2m, comprising human T cells, or NOD.^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) Mouse comprising human T cells.

Cg-Prkdc according to an aspect of the invention there is provided a NOD^scidIl2rg^tm1Wjl/SzJ(NOD-scid-IL2rγ^nullNSG) mice genetically modified such that said NSG mice lack functional major histocompatibility complex i (mhc i) and lack functional major histocompatibility complex ii (mhc ii), comprising human immune cells and human tumor cells. According to a particular aspect, the genetically modified NSG mouse is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse comprising human immune cells and human tumor cells, NSG-RIP-DTR (K)^bD^b)^null(IA^null) A mouse comprising human immune cells and human tumor cells, or NOD.Cg-B2m^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) Mice comprising human immune cells and human tumor cells.

Cg-Prkdc according to an aspect of the invention there is provided a NOD^scidIl2rg^tm1Wjl/SzJ(NOD-scid-IL2rγ^nullNSG) a mouse selected from the group consisting of,the mice are genetically modified such that the NSG mice lack functional major histocompatibility complex i (mhc i) and lack functional major histocompatibility complex ii (mhc ii), comprising human peripheral blood mononuclear cells and human tumor cells. According to a particular aspect, the genetically modified NSG mouse is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse comprising human peripheral blood mononuclear cells and human tumor cells, NSG-RIP-DTR (K)^bD^b)^null(IA^null) A mouse comprising human peripheral blood mononuclear cells and human tumor cells, or NOD.Cg-B2m^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) Mice comprising human peripheral blood mononuclear cells and human tumor cells.

Cg-Prkdc according to an aspect of the invention there is provided a NOD^scidIl2rg^tm1Wjl/SzJ(NOD-scid-IL2rγ^nullNSG) mice genetically modified such that said NSG mice lack functional major histocompatibility complex i (mhc i) and lack functional major histocompatibility complex ii (mhc ii), comprising human T cells and human tumor cells. According to a particular aspect, the genetically modified NSG mouse is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse comprising human T cells and human tumor cells, NSG-RIP-DTR (K)^bD^b)^null(IA^null) Mouse comprising human T cells and human tumor cells, or NOD.Cg-B2m^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) Mice comprising human T cells and human tumor cells.

NSG- (K) of the invention^bD^b)^null(IA^null) The mice were characterized by a clearance of human IgG administered within a period of 2 days after administration of human IgGExcept by no more than 60%, such as no more than 70%, 80%, or 90%.

An immunodeficient mouse genetically modified such that the mouse lacks functional major histocompatibility complex I (MHC I) and lacks functional major histocompatibility complex II (MHC II), with the proviso that the immunodeficient mouse is not an NOD/Shi-scid-IL2r γ characterized by an β 2m (component of MHC I) knockout and an IA β (component of MHC II) knockout^nullA mouse. According to a particular aspect, the mouse further comprises human immune cells, such as human peripheral blood mononuclear cells and such as human T cells. According to particular aspects, the mouse further comprises human immune cells, such as human peripheral blood mononuclear cells and such as human T cells, and further comprises human tumor cells.

A method of modeling the role of the human immune system in a genetically modified immunodeficient mouse is provided, comprising administering a test substance to a genetically modified immunodeficient mouse of the invention; and determining the effect of the human immune system or one or more components thereof in the genetically modified immunodeficient mouse. The test agent can be, but is not limited to, an anti-tumor antibody, an immunotherapeutic agent, an immune checkpoint inhibitor, including but not limited to a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. The test substance may be an immune checkpoint inhibitor selected from: alemtuzumab, avizumab, deluzumab, ipilimumab, nivolumab, or pembrolizumab, or an antigen-binding fragment of any of the foregoing. The test substance may be an anti-cancer agent.

A method of modeling the role of human T cells in a genetically modified immunodeficient mouse is provided, comprising administering a test substance to a genetically modified immunodeficient mouse of the invention; and determining the effect of said human T cells in said genetically modified immunodeficient mouse. The test agent can be, but is not limited to, an anti-tumor antibody, an immunotherapeutic agent, an immune checkpoint inhibitor, including but not limited to a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. The test substance may be an immune checkpoint inhibitor selected from: alemtuzumab, avizumab, deluzumab, ipilimumab, nivolumab, or pembrolizumab, or an antigen-binding fragment of any of the foregoing. The test substance may be an anti-cancer agent.

A method of modeling the effect of the human immune system or one or more components thereof in a genetically modified immunodeficient mouse is provided, wherein the genetically modified immunodeficient mouse is a NOD^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse, NSG-RIP-DTR (K)^bD^b)^null(IA^null) Cg-B2m mouse or NOD^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) A mouse, wherein the method comprises administering a test agent to the genetically modified immunodeficient mouse; and determining the effect of the human immune system or one or more components thereof in the genetically modified immunodeficient mouse. The test agent can be, but is not limited to, an anti-tumor antibody, an immunotherapeutic agent, an immune checkpoint inhibitor, including but not limited to a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. The test substance may be an immune checkpoint inhibitor selected from: alemtuzumab, avizumab, deluzumab, ipilimumab, nivolumab, or pembrolizumab, or an antigen-binding fragment of any of the foregoing. The test substance may be an anti-cancer agent.

A method of modeling the role of human leukocytes in a genetically modified immunodeficient mouse is provided, wherein the genetically modified immunodeficient mouse is NOD^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse, NSG-RIP-DTR (K)^bD^b)^null(IA^null) Cg-B2m mouse, or NOD^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) A mouse, wherein the method comprises administering a test agent to the genetically modified immunodeficient mouse; and in said genetically modified immunodeficient miceTo determine the effect of said human leukocytes. The test agent can be, but is not limited to, an anti-tumor antibody, an immunotherapeutic agent, an immune checkpoint inhibitor, including but not limited to a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. The test substance may be an immune checkpoint inhibitor selected from: alemtuzumab, avizumab, deluzumab, ipilimumab, nivolumab, or pembrolizumab, or an antigen-binding fragment of any of the foregoing. The test substance may be an anti-cancer agent.

A method of modeling the role of human PMBC in a genetically modified immunodeficient mouse is provided, wherein the genetically modified immunodeficient mouse is NOD^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse, NSG-RIP-DTR (K)^bD^b)^null(IA^null) Cg-B2m mouse, or NOD^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) A mouse, wherein the method comprises administering a test agent to the genetically modified immunodeficient mouse; and determining the effect of said human PMBC in said genetically modified immunodeficient mouse. The test agent can be, but is not limited to, an anti-tumor antibody, an immunotherapeutic agent, an immune checkpoint inhibitor, including but not limited to a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. The test substance may be an immune checkpoint inhibitor selected from: alemtuzumab, avizumab, deluzumab, ipilimumab, nivolumab, or pembrolizumab, or an antigen-binding fragment of any of the foregoing. The test substance may be an anti-cancer agent.

A method of modeling the role of human T cells in a genetically modified immunodeficient mouse is provided, wherein the genetically modified immunodeficient mouse is NOD^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ(NSG-(K^bD^b)^null(IA^null) Mouse, NSG-RIP-DTR (K)^bD^b)^null(IA^null) Cg-B2m mouse, or NOD^tm1UncPrkdc^scidH2^dlAb1-EaIl2rg^tm1Wjl/SzJ(NSG-B2M^null(IA IE^null) A mouse, wherein the method comprises administering a test agent to the genetically modified immunodeficient mouse; and determining the effect of said human T cells in said genetically modified immunodeficient mouse. The test agent can be, but is not limited to, an anti-tumor antibody, an immunotherapeutic agent, an immune checkpoint inhibitor, including but not limited to a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. The test substance may be an immune checkpoint inhibitor selected from: alemtuzumab, avizumab, deluzumab, ipilimumab, nivolumab, or pembrolizumab, or an antigen-binding fragment of any of the foregoing. The test substance may be an anti-cancer agent.

Drawings

FIGS. 1A-1C show the results in NSG- (K)^bD^b)^null(IA^null) And NSG-B2M^null(IA IE)^nullRepresentative flow cytometry plots of MHC class I and class II expression in mice. Decomposition of NSG, NSG- (K) by enzymatic and mechanical digestion^bD^b)^null(IA^null) And NSG-B2M^null(IA IE)^nullThe spleen of the mice was knocked out.

FIG. 1A is a diagram showing that monocyte-derived dendritic cells identified in live cells were CD11b +, Ly6Gdim, CD11c +, and Ly 6C-.

FIG. 1B shows the expression of H2K from individual mice^dAnd H2K^bA graph of the evaluation results of the monocyte-derived dendritic cells recovered from the line of (1). For all stains, representative stains are shown (N ═ 2).

FIG. 1C shows the expression of mouse H2 IA from each^g7And H2 IA^bA graph of the evaluation results of the monocyte-derived dendritic cells recovered from the line of (1). For all stains, representative stains are shown (N ═ 2).

FIG. 2 is a graph showing that^bD^b)^null(IA^null) And NSG-B2M^null(IA IE)^nullHalf human IgG in mouse serumGraph of the stage of decay. Mice IV were injected with 200 μ g human IgG and bled at the time points indicated to recover serum. Sera were used for ELISA analysis of circulating human IgG. The first blood draw at 2 minutes after injection was considered 100% serum IgG. Each dot represents the mean. + -. standard error of 5 male mouse IgG's at 2-3 months of age.

Figures 3A and 3B show the survival of NSG mice lacking expression of both mouse MHC class I and class II after injection of human Peripheral Blood Mononuclear Cells (PBMCs). Intravenous Injection (IV) of 10x10 into recipient mice⁶PBMCs, mice were monitored for overall health and survival.

FIG. 3A shows that when NSG and NSG- (IA) are combined^null)、NSG-(K^bD^b)^nullAnd NSG- (K)^bD^b)^null(IA^null) Graph of% survival of mice as recipient for PBMC. Data are representative of 3 independent experiments. Survival distribution among groups was examined using log rank statistics.

FIG. 3B shows the state of NSG, NSG- (IA IE)^null、NSG-B2M^nullAnd NSG-B2M^null(IA IE)^nullGraph of% survival of mice as recipient for PBMC. Data are representative of 3 independent experiments. Survival distribution among groups was examined using log rank statistics.

Figures 4A-4D show the level of human CD45+ cell chimerism in NSG mice that lack expression of both mouse MHC class I and class II following PBMC injection. IV injection of 10X10 into recipient mice⁶PBMC cells, and the level of human cell chimerism in mice was monitored by determining the proportion of human CD45+ cells in peripheral blood (fig. 4A and 4C) and spleen (fig. 4B and 4D).

FIG. 4A is a graph showing NSG, NSG- (IA) injection of PBMC over a 10 week period^null)、NSG-(K^bD^b)^nullAnd NSG- (K)^bD^b)^null(IA^null) Graph of the level of human cell chimerism monitored in the blood of mice. Data are representative of 3 independent experiments. Two-way ANOVA was used to determine significant differences between groups at each time point. Week 6; NSG vs NSG- (K)^bD^b)^nullp<0.01 and NSG vs NSG- (K)^bD^b)^null(IA^null)p<0.001；NSG-(IA^null) Compared with NSG- (K)^bD^b)^nullp<0.01, and NSG- (IA)^null) Compared with NSG- (K)^bD^b)^null(IA^null)p<0.001。

FIG. 4B is a graph showing NSG, NSG- (IA) after PBMC injection when mice are anesthetized^null)、NSG-(K^bD^b)^nullAnd NSG- (K)^bD^b)^null(IA^null) Graph of human cell chimerism levels monitored in the spleen of mice. One-way ANOVA was used to determine significant differences between groups. Denotes p<0.05, represents p<0.01。

FIG. 4C is a graph showing NSG, NSG- (IA IE) of PBMC injection over a 10 week period^null、NSG-B2M^nullAnd NSG-B2M^null(IA IE)^nullGraph of the level of human cell chimerism monitored in the blood of mice. Data are representative of 3 independent experiments. Two-way ANOVA was used to determine significant differences between groups at each time point. Week 4; NSG vs. NSG-B2M^null(IA IE)^nullp<0.01，NSG-(IA IE)^nullCompared with NSG-B2M^null(IA IE)^nullp<0.01, and NSG-B2M^nullCompared with NSG-B2M^null(IA IE)^nullp<0.05. Week 6; NSG vs. NSG-B2M^null(IA IE)^nullp<0.05，NSG-(IA IE)^nullCompared with NSG-B2M^nullp<0.05，NSG-(IA IE)^nullCompared with NSG-B2M^null(IA IE)^nullp<0.01. Week 8; NSG vs. NSG-B2M^nullp<0.001, NSG vs. NSG-B2M^null(IA IE)^nullp<0.01，NSG-(IA IE)^nullCompared with NSG-B2M^nullp<0.001，NSG-(IA IE)^nullCompared with NSG-B2M^null(IA IE)^nullp<0.01. Week 10; NSG vs. NSG-B2M^nullp<0.01, NSG vs. NSG-B2M^null(IA IE)^nullp<0.01, and NSG- (IA IE)^nullCompared with NSG-B2M^nullp<0.01，NSG-(IA IE)^nullCompared with NSG-B2M^null(IA IE)^nullp<0.01。

FIG. 4D is a graph showing NSG, NSG- (IA IE) after PBMC injection when mice are anesthetized^null、NSG-B2M^nullAnd NSG-B2M^null(IA IE)^nullGraph of human cell chimerism levels monitored in the spleen of mice. One-way ANOVA was used to determine significant differences between groups. Denotes p<0.01。

Figures 5A-5D show transplantation of human T and B cells in NSG mice lacking expression of both mouse MHC class I and class II after PBMC injection. IV injection of 10X10 into recipient mice⁶PBMC cells and levels of human CD3+ T cells (fig. 5A and 5C) and CD20+ B cells (fig. 5B and 5D) in mouse peripheral blood were monitored.

FIG. 5A shows the results obtained when NSG (N ═ 7) and NSG- (IA) are combined^null)(N＝5)、NSG-(K^bD^b)^null(N-7) and NSG- (K)^bD^b)^null(IA^null) (N-8) figure of human CD3+ cells (% of CD 45) when mice were used as recipients of PBMCs. Data are representative of 3 independent experiments. Two-way ANOVA was used to determine significant differences between groups at each time point. Denotes p<0.05。

FIG. 5B shows the results obtained when NSG (N ═ 7) and NSG- (IA) are combined^null)(N＝5)、NSG-(K^bD^b)^null(N-7) and NSG- (K)^bD^b)^null(IA^null) (N-8) figure of human CD20+ cells (% of CD 45) when mice were used as recipients of PBMCs. Data are representative of 3 independent experiments. Two-way ANOVA was used to determine significant differences between groups at each time point. Denotes p<0.05。

FIG. 5C shows the results obtained when NSG (N ═ 6), NSG- (IA IE)^null(N＝6)、NSG-B2M^null(N-5) and NSG-B2M^null(IA IE)^null(N-7) figure of human CD3+ cells (% of CD 45) when mice were used as recipients of PBMCs. Data are representative of 3 independent experiments. Two-way ANOVA was used to determine significant differences between groups at each time point. Denotes p<0.05。

FIG. 5D shows the results of the comparison of NSG (N ═ 6) and NSG- (IA IE)^null(N＝6)、NSG-B2M^null(N-5) and NSG-B2M^null(IA IE)^null(N-7) mice as PBMCHuman CD20+ cells (CD 45% of the total population). Data are representative of 3 independent experiments. Two-way ANOVA was used to determine significant differences between groups at each time point. Denotes p<0.05。

FIGS. 6A-6H show NSG, NSG- (IA) in PBMC injection^null)、NSG-(K^bD^b)^nullAnd NSG- (K)^bD^b)^null(IA^null) Phenotypic analysis of transplanted human T cells in mice. IV injection of 10X10 into recipient mice⁶PBMCs, and levels of human CD3+/CD4+ and CD3/CD8+ T cells (fig. 6A and 6D) and T cell phenotype (fig. 6B, 6C, and 6E-6H) in peripheral blood were monitored 4 weeks after injection. Data are representative of 2 independent experiments. One-way ANOVA was used to determine significant differences between groups. Denotes p<0.05, represents p<0.01, represents p<0.005 and<0.001。

fig. 6A is a graph showing the levels of CD4 and CD8T cells determined by flow cytometry and expressed as a ratio of CD4 to CD8T cells.

FIG. 6B is a graph showing NSG, NSG- (IA) for PBMC injection^null)、NSG-(K^bD^b)^nullAnd NSG- (K)^bD^b)^null(IA^null) Graph of PD-1 expressed by CD 4T cells determined by flow cytometry in mice.

FIG. 6C is a graph showing NSG, NSG- (IA) for PBMC injection^null)、NSG-(K^bD^b)^nullAnd NSG- (K)^bD^b)^null(IA^null) Graph of PD-1 expressed by CD8T cells determined by flow cytometry in mice.

Fig. 6D-6F are graphs showing representative CD4, CD8, and PD1 staining.

Fig. 6G and 6H are graphs showing CD4 and CD8T cells, respectively, evaluated by flow cytometry for expression of CD45RA and CCR 7. The percentage of T cell subpopulations is shown as CD45RA +/CCR7+ cell markers naive

CD45RA-/CCR7+ cell marker central memory, CD45RA-/CCR 7-cell markerThe cell marker is TEMRA for effector/effector memory and CD45RA +/CCR 7.

FIGS. 7A-7H show NSG, NSG- (IA IE) in PBMC injections^null、NSG-B2M^nullAnd NSG-B2M^null(IA IE)^nullPhenotypic analysis of transplanted human T cells in mice. IV injection of 10X10 into recipient mice⁶PBMCs, and levels of human CD3+/CD4+ and CD3/CD8+ T cells (fig. 7A and 7D) and T cell phenotype (fig. 7B, 7C, and 7E-7H) in peripheral blood were monitored 4 weeks after injection. Data are representative of 2 independent experiments. One-way ANOVA was used to determine significant differences between groups. Denotes p<0.05, represents p<0.01, represents p<0.005 and<0.001。

figure 7A is a graph showing CD4 and CD8T cell levels determined by flow cytometry and expressed as a ratio of CD4 to CD8T cells.

FIG. 7B is a graph showing NSG, NSG- (IA IE) for PBMC injection^null、NSG-B2M^nullAnd NSG-B2M^null(IAIE)^nullGraph of PD-1 expressed by CD4 cells determined by flow cytometry in mice.

FIG. 7C is a graph showing NSG, NSG- (IA IE) for PBMC injection^null、NSG-B2M^nullAnd NSG-B2M^null(IAIE)^nullGraph of PD-1 expressed by CD8 cells determined by flow cytometry in mice.

Fig. 7D-7F are graphs showing representative CD4, CD8, and PD1 staining.

Fig. 7G and 7H are graphs showing CD4 and CD8T cells, respectively, evaluated by flow cytometry for expression of CD45RA and CCR 7. The percentage of T cell subpopulations is shown as CD45RA +/CCR7+ cell markers naive

CD45RA-/CCR7+ cell marker is central memory, CD45RA-/CCR 7-cell marker is effector/effector memory and CD45RA +/CCR 7-cell marker is TEMRA.

FIGS. 8A-8F show NSG-RIP-DTR (K) in PBMC transplants^bD^b)^null(IA^null) Human islet (islet) allografts in miceThe rejection situation of (1). Data are representative of 2 independent experiments. T-test was used to determine significant differences between groups. Denotes p<0.05, represents p<0.01, represents p<0.005。

FIG. 8A is a graph showing NSG-RIP-DTR (K) treated with 40ng Diphtheria Toxin (DT) 6 days prior to PBMC injection^bD^b)^null(IA^null) Graph of the results of mice, followed by implantation of human islets (4000IEQ) by intrasplenic injection. At day 0, a group of mice was IP injected with 50x10⁶Personal PBMC, no treatment was performed on another group of mice. Blood glucose levels were monitored and mice with 2 consecutive measurements of blood glucose levels above 300mg/dl were considered diabetic.

Fig. 8B is a graph showing the results of monitoring the level of human cell chimerism in mice by determining the proportion of CD45+ cells in peripheral blood (6 weeks) and spleen (7 weeks).

FIGS. 8C and 8D are graphs showing CD3+/CD4+ and CD3+/CD8+ T cell levels in peripheral blood and spleen, respectively.

FIG. 8E is a graph showing circulating human C-peptide levels in plasma determined by ELISA at week 6.

Fig. 8F is a graph showing the total insulin content in the spleen of islet-transplanted mice determined by ELISA at week 7.

FIGS. 9A-9H show NSG and NSG- (K) in transplanted PBMCs^bD^b)^null(IA^null) Expression of human IL2 in mice enhanced the survival of human CD4+ tregs. To receptors NSG and NSG- (K)^bD^b)^null(IA^null) Mouse IP injection 2.5x10¹¹AAV-IL2 particles or PBS injection. Two weeks later, mice were injected Intraperitoneally (IP) with 1x10⁶And (5) PBMCs.

FIGS. 9A-9C are graphs showing the levels of human CD45+ cells (FIG. 9A), CD3+ T cells (FIG. 9B), and CD4+/CD25+/CD127-/FOXP3+ Tregs (FIG. 9C) as determined by flow cytometry. Two-way ANOVA was used to determine significant differences between groups. Denotes p <0.005, and denotes p < 0.001.

Figure 9D shows representative staining of CD4+ T cells against CD25, CD127, and FOXP3 for the indicated groups.

Fig. 9E is a graph showing survival distribution among groups monitored for% survival of recipient mice and shown using log rank statistical test.

Fig. 9F is a graph showing CD4 and CD8T cell levels determined by flow cytometry and expressed as a ratio of CD4 to CD8T cells. Filled black triangles represent NSG mice, open black triangles represent NSG mice injected with AAV-IL2, filled circles represent NSG- (K)^bD^b)^null(IA^null) Mice and open circles represent NSG- (K) injected with AAV-IL2^bD^b)^null(IA^null) A mouse.

Fig. 9G is a graph showing the results of evaluating CD45RA and CCR7 expression in CD8T cells by flow cytometry. The percentage of the T cell subpopulations is shown as CD45RA +/CCR7+ cell marker naive, CD45RA-/CCR7+ cell marker central memory, CD45RA-/CCR 7-cell marker effector/effector memory and CD45RA +/CCR 7-cell marker TEMRA. Filled black triangles represent NSG mice, open black triangles represent NSG mice injected with AAV-IL2, filled circles represent NSG- (K)^bD^b)^null(IA^null) Mice and open circles represent NSG- (K) injected with AAV-IL2^bD^b)^null(IA^null) A mouse.

Figure 9H is a graph showing granzyme B expressed by CD8T cells as determined by flow cytometry and showing representative staining. The t-test was used to determine the significant difference between mice treated with AAV-IL2 and controls. Denotes p <0.005, denotes p < 0.001. Data are representative of 3 independent experiments.

FIG. 10A is a graph showing a panel of NSG mice co-injected with PBMC and human patient-derived tumor cells and a panel of NSG- (K) co-injected with PBMC and human patient-derived tumor cells^bD^b)^null(IA^null) Graph of percent survival of mice.

FIG. 10B is a graph showing 1) NSG mice injected with human patient-derived tumor cells; 2) in NSG mice co-injected with PBMC and human patient-derived tumor cells; PBMC injected NSG- (K)^bD^b)^null(IA^null) A mouse; and co-injected with PBMC and humanNSG- (K) of patient-derived tumor cells^bD^b)^null(IA^null) Graph of tumor growth in mice.

Detailed Description

The scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. Such terms are found to be defined and used in the context of various standard references, illustratively including: sambrook and D.W.Russell, Molecular Cloning A Laboratory Manual, Cold Spring harbor Laboratory Press; 3 rd edition, 2001; (f.m. ausubel, ed., Short Protocols in molecular biology, Current Protocols; 5 th edition, 2002; alberts et al, Molecular Biology of the cell, 4 th edition, Garland, 2002; nelson and m.m.cox, Lehninger Principles of biochemistry, 4 th edition, w.h.freeman & Company, 2004; a Laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory Press; 12/15/2002, ISBN-10: 0879695919; kursad Turksen (Ed.), Embryonic stem cells: Methods and protocols in Methods mol biol.2002; 185, Humana Press; current Protocols in Stem Cell Biology, ISBN: 9780470151808; chu, e, and Devita, v.t., eds., physics' Cancer chemotherapeutics drug manual, Jones & Bartlett Publishers, 2005; m. kirkwood et al eds., Current cancer therapeutics, 4 th edition, Current Medicine Group, 2001; the Science and practice of Pharmacy, Lippincott Williams & Wilkins, 21 st edition, 2005; allen, Jr. et al, Ansel's Pharmaceutical document Forms and Drug Delivery Systems, 8 th edition, Philadelphia, PA Lippincott, Williams & Wilkins, 2004; brunton et al, Goodman & Gilman's The pharmaceutical Basis of Therapeutics, McGraw-Hill Professional, 12 th edition, 2011.

The singular terms "a" and "an" and "the" are not intended to be limiting and include the plural unless expressly stated otherwise or the context clearly dictates otherwise.

As generally used herein, the term "functional" refers to a protein, complex, cell, or other substance that retains the biological function of the corresponding native protein, complex, cell, or other substance.

Conversely, as generally used herein, the term "non-functional" refers to a protein, complex, cell, or other substance that does not retain the biological function of the corresponding native protein, complex, cell, or other substance.

The present invention provides immunodeficient mice lacking genetic modifications of MHC class I and MHC class II.

According to various aspects, there is provided a genetically modified immunodeficient mouse comprising at least one mutation in its genome, the mutation being effective to reduce or eliminate expression of functional MHC I α protein and/or to reduce or eliminate expression of functional β 2-microglobulin such that MHC I is not present or is not functional in the mouse, and comprising at least one mutation in its genome, the mutation being effective to reduce or eliminate expression of functional MHC II α protein and/or expression of functional MHC II β protein such that MHC II is not present or is not functional in the mouse.

According to various aspects, the genetically modified immunodeficient mouse is a genetically modified NSG mouse. According to aspects of the invention, NSG MHC I/II knockout mice are useful for a variety of applications, including studies of human immunity in the absence of GVHD and evaluation of antibody-based therapies.

MHC I

The terms "MHC I" and "MHC class I" are used interchangeably and refer to a complex formed by MHC I α protein and β 2-microglobulin.

MHC I α proteins include an extracellular domain (which has 3 subdomains: α 1, α 2 and α 3), a transmembrane domain and a cytoplasmic tail. α 1 and α 2 subdomains form a peptide binding cleft, while the α 3 subdomain interacts with β 2-microglobulin the terms "H2-K", "H2-D" and "H2-L" refer to the mouse MHC I α protein subclasses, all of which are encoded on mouse chromosome 17.

β 2-microglobulin is non-covalently bound to the α 3 subdomain of the MHC I α the gene encoding mouse β 2-microglobulin is encoded on chromosome 2 (Chr 2: 122147686-122153083bp, + strand, GRCm 38).

MHC II

The terms "MHC II" and "MHC class II" are used interchangeably to refer to a complex formed by two non-covalently associated proteins, MHC II α protein and MHC II β protein the terms "H-2A" and "H-2E" (commonly abbreviated as I-A and I-E, respectively) refer to the subclass of MHC II.the MHC II α and MHC II β proteins both span the plasma membrane and both contain an extracellular domain, a transmembrane domain and a cytoplasmic domain. the extracellular portion of the MHC II α protein contains MHC II α 1 and MHC II α 2 domains, and the extracellular portion of the MHC II β protein contains MHC II β 1 and MHC II β 2 domains.

The term "functional" as used herein with respect to a functional MHC I α protein, a functional β 2-microglobulin, a functional MHC II β 0 protein, a functional MHC II β 1 protein, a functional MHC I or a functional MHC II refers to an MHC I α protein, an β 2-microglobulin, an MHC II α protein, an MHC II β protein, an MHC I or an MHC II that retains the biological function of the corresponding native MHC I α protein, β 2-microglobulin, MHC II α protein, MHC II β protein, MHC I or MHC II.

In contrast, the term "non-functional" as used herein with respect to non-functional MHC I α protein, β 2-microglobulin, MHCII α protein, MHC II β protein, MHC I or MHC II refers to an MHC protein or MHC complex that does not retain the biological function of the corresponding native MHC I α protein, β 2-microglobulin, MHC II α protein, MHC II β protein, MHC I or MHC II.

As used herein, the term "native" refers to a protein or nucleic acid that is not mutated.

As used herein, the term "genetic modification" refers to a modification of genomic DNA in a mouse that disrupts the expression of at least one of functional MHC I α protein and functional β 2-microglobulin, and at least one of functional MHC II α protein and functional MHC II β protein, such that the mouse lacks functional MHC I and functional MHC II.

The term "expression" refers to the transcription of a nucleic acid sequence to produce the corresponding mRNA and/or the translation of an mRNA to produce the corresponding protein.

As used herein, the term "target gene" designates a nucleic acid sequence that defines a mouse MHC I α gene, a mouse β 2-microglobulin gene, a mouse MHC II α gene, or a mouse MHC II β gene.

Any of a variety of methods can be used to generate a genetically modified immunodeficient mouse whose genome comprises a genetic modification that disrupts expression of at least one of a functional MHC I α protein and a functional β 2-microglobulin, and at least one of a functional MHC II α protein and a functional MHC II β protein, such that the mouse lacks functional MHC I and functional MHC II.

Genetic modifications such as, but not limited to, chemical mutagenesis, radiation, homologous recombination, and transgenic expression of antisense RNA, are generated using standard methods of genetic engineering. Such techniques are well known in the art and also include, but are not limited to, prokaryotic (prokaryotic) microinjection and transformation of embryonic stem cells. Methods for producing genetically modified animals whose genomes contain genetic mutations that can be used include, but are not limited to, those described in: sundberg and t.ichiki, eds., genetic Engineered rice Handbook, CRC Press; 2006; m.h. hofker and j.vandeursen, eds., Transgenic Mouse methods and Protocols, Humana Press, 2002; joyner, Gene Targeting A Practical Approach, Oxford University Press, 2000; a Laboratory Manual, 3 rd edition, Cold Spring harbor Laboratory Press; 12/15/2002, ISBN-10: 0879695919; kursad Turksen (Ed.), Embryonic stem cells: Methods and protocols in Methods Mol biol.2002; 185, Humana Press; current Protocols in Stem Cell Biology, ISBN: 978047015180; meyer et al, PNAS USA, vol.107(34), 15022-.

According to a preferred aspect, non-endogenous MHC I or MHC II are not expressed in the genetically modified immunodeficient mice of the invention, except for the lack of functional endogenous MHC I and MHC II. In particular, according to a preferred embodiment, the human lymphocompatibility gene is absent or not expressed in the genetically modified immunodeficient mice of the invention.

As used herein, "endogenous" in relation to a gene and its encoded protein refers to the gene as it exists in its native locus in the mouse genome.

Homology-based recombinant gene modification strategies can be used to genetically modify immunodeficient mice by "knocking-out" or other mutation of genes encoding one or more endogenous proteins (e.g., at least one of the MHC I α protein and β 2-microglobulin; and at least one of the MHC II α protein and MHC II β protein).

Homology-based recombinant gene modification strategies include gene editing methods such as those using homing endonucleases, integrases, meganucleases, transposons, nuclease-mediated processes using Zinc Finger Nucleases (ZFNs), transcription activator-like (TAL), regularly clustered short palindromic repeats (CRISPR) -Cas, or drosophila recombination-associated protein (DRAP) methods. See, e.g., cerblini et al, PLoS one.2015; 10(1) e 0116032; shen et al, PLoS ONE 8(10) e 77696; and Wang et al, Protein & Cell, 2016 month 2, Vol 7, No. 2, pp 152-.

Genome editing is performed by, for example, the methods described herein and detailed in the following: sundberg and t.ichiki, eds., genetic Engineered rice Handbook, CRC Press; 2006; m.h. hofker and j.van Deursen, eds., Transgenic Mouse Methods and Protocols, Humana Press, 2002; joyner, Gene Targeting A Practical Approach, Oxford University Press, 2000; a Laboratory Manual, 3 rd edition, Cold spring harbor Laboratory Press; 12/15/2002, ISBN-10: 0879695919; kursad Turksen (Ed.), Embryonic stem cells: Methods and protocols in Methods mol. biol. 2002; 185, Humana Press; current Protocols in Stem Cell Biology, ISBN: 978047015180; meyer et al, PNAS USA,2010, vol.107(34), 15022-; and Doudna, j, et al, (eds.) CRISPR-Cas a Laboratory Manual,2016, CSHP. A brief description of several genome editing techniques is described herein.

Nuclease technology for genetic modification

Genetic modification methods, such as but not limited to nuclease gene editing techniques, can be used to introduce a desired DNA sequence into the genome at a predetermined target site, such as methods using homing endonucleases, integrases, meganucleases, transposons, nuclease-mediated processes using Zinc Finger Nucleases (ZFNs), transcription activator-like (TAL), regularly clustered short palindromic repeats (CRISPR) -Cas, or drosophila recombination-associated proteins (DRAPs). Briefly, genetic modification methods that may be used include introducing an RNA molecule encoding a target TALEN, ZFN, CRISPR, or DRAP and at least one oligonucleotide into an ES cell, iPS cell, somatic cell, zygote, or embryo, and then selecting the ES cell, iPS cell, somatic cell, zygote, or embryo having the desired genetic modification.

For example, a desired nucleic acid sequence can be introduced into the genome of a mouse at a predetermined target site by nuclease technology (such as, but not limited to, CRISPR methods, TAL (transcription activator-like effector methods), zinc finger-mediated genome editing, or DRAP) to produce a genetically modified mouse provided according to embodiments of the invention.

As used herein, in the context of nuclease gene editing technology, the terms "target site" and "target sequence" designate a nucleic acid sequence that defines a portion of a chromosomal sequence to be edited, and to which a nuclease is engineered to recognize and bind, so long as sufficient binding conditions exist.

CRISPR-Cas system

CRISPR (clustered regularly interspaced short palindromic repeats) is a locus containing multiple short direct repeats, found in the genomes of about 40% sequenced bacteria and 90% sequenced archaea, and confers resistance to foreign DNA elements, see Horvath,2010, Science,327: 167-; barrangou et al, 2007, Science,315: 1709-; and Makarova et al, 2011, Nature Reviews microbiology.9:467- -.

CRISPR repeats range in size from 24 to 48 base pairs. It usually exhibits some two-fold symmetry, which means that secondary structures such as hairpins are formed but not true palindromes. CRISPR repeats are separated by spacers of similar length.

CRISPR-associated (cas) genes are commonly associated with CRISPR repeat spacer arrays. Over 40 different Cas protein families have been described (Haft et al, 2005, PLoS Compout biol.1(6): e 60). Specific combinations of Cas genes and repeat structures have been used to define 8 CRISPR subtypes, some of which are associated with other gene modules encoding repeat associated unknown proteins (RAMP).

There are a variety of CRISPR systems in different organisms, the simplest one being the type II CRISPR system from Streptococcus pyogenes (Streptococcus pyogenes): there is only one gene encoding Cas9 protein and two RNAs, one mature CRISPR RNA (crRNA) and one partially complementary trans-acting RNA (tracrrna), which is necessary and sufficient for RNA-guided exogenous DNA silencing (gasituas et al, 2012, PNAS 109: E2579-E2586; Jinek et al, 2012, Science 337: 816-. Maturation of CrRNA requires tracrRNA and RNase III (Deltcheva et al, 2011, Nature 471: 602-607). However, this requirement can be circumvented by using engineered small guide RNAs (sgRNAs) containing engineered hairpins mimicking the tracrRNA-crRNA complex (Jinek et al, 2012, Science 337: 816-. Due to the endonuclease activity of Cas9, base pairing between the sgRNA and the target DNA results in a Double Strand Break (DSB). The binding specificity is determined by both sgRNA-DNA base pairing and the short DNA motif juxtaposed to the DNA complementary region (protospacer adjacent motif [ PAM ] sequence: NGG) (Marraffini & Sonthimer, 2010, Nature Reviews Genetics,11: 181-190). For example, CRISPR systems require a minimal set of two molecules, Cas9 protein and sgRNA, and thus can be used as host-independent gene targeting platforms. Site-selective RNA-guided genome editing, such as targeted insertion, can be performed using Cas9/CRISPR, see, e.g., Carroll,2012, Molecular Therapy 20: 1658-; chang et al, 2013, Cell Research 23: 465-; cho et al, 2013, Nature Biotechnol 31: 230-232; cong et al, 2013, Science 339: 819. sup. 823; hwang et al, 2013, Nature Biotechnol 31: 227-; jiang et al, 2013, Nature Biotechnol 31: 233-; mali et al, 2013, Science 339: 823-826; qi et al, 2013, Cell152: 1173-1183; shen et al, 2013, Cell Research 23: 720-; and Wang et al, 2013, Cell 153: 910-. In particular, Wang et al, 2013, Cell 153:910-918 describe targeted insertion of associated oligonucleotides using the CRISPR/Cas9 system.

Generating genetically modified immunodeficient mice according to the invention may comprise injecting or transfecting appropriate nucleic acids, such as expression constructs encoding cas9 and expression constructs encoding guide RNAs specific to the targeted gene for use in CRISPR, into pre-implantation embryonic or stem cells, such as embryonic stem cells (ES) or induced pluripotent stem cells (iPS). Optionally, cas9 and the guide RNA are encoded in a single expression construct.

TAL (transcription activator like) effectors

Transcription activator-like (TAL) effectors or TALEs are derived from the plant pathogen Xanthomonas, and these proteins mimic plant transcription activators and manipulate plant transcription, see Kay et al, 2007, Science,318: 648-651.

TAL effectors contain a collection of tandem repeat domains, each containing about 34 amino acids, which are critical to the DNA binding specificity of these proteins. In addition, it contains nucleic acid localization sequences and acidic transcription activation domains, for a review see Schornack et al, 2006, J.plant physiol.,163(3): 256-272; scholze and Boch,2011, Curr Opin Microbiol,14: 47-53.

The specificity of TAL effectors depends on the sequence present in the tandem repeat. The repeat sequences comprise about 102bp and are typically 91-100% homologous to each other (Bonas et al, 1989, Mol Gen Genet218: 127-. Polymorphisms in the repeat sequence are usually located at positions 12 and 13, and there appears to be a one-to-one correspondence between the identity of the hypervariable di-residues at positions 12 and 13 and the identity of consecutive nucleotides in the TAL effector target sequence, see Moscou and bogdanave 2009, Science 326: 1501; and Boch et al, 2009, Science 326: 1509-. The two hypervariable residues are called repetitive variable di-Residues (RVDs), where one RVD recognizes one nucleotide of the DNA sequence and ensures that the DNA-binding domain of each TAL effector is able to target a large recognition site with high precision (15-30 nt). Experimentally, the DNA recognition codes for these TAL-effectors have been determined such that the HD sequence at positions 12 and 13 binds to cytosine (C), NG binds to T, NI binds to A, C, G or T, NN binds to a or G and IG binds to T. These DNA binding repeats have been assembled into proteins with novel combinations and numbers of repeats to form artificial transcription factors capable of interacting with the novel sequences and activating the expression of reporter genes in plant cells (Boch et al, 2009, Science 326: 1509-. These DNA binding domains have demonstrated general applicability in the field of targeted genomic editing or targeted gene regulation in all cell types, see Gaj et al Trends in Biotechnol,2013,31(7): 397-405. Moreover, engineered TAL effectors have been shown to function in binding to foreign functional protein effector domains (e.g., nucleases) that do not naturally occur in mammalian cell native xanthomonas TAL-effectors or proteins. TAL nucleases (TALN or TALEN), Kim et al, 1996, PNAS 93: 1156-1160; christian et al, 2010, Genetics 186: 757-; li et al, 2011, Nucleic Acids Res 39: 6315-6325; and Miller et al, 2011, Nat Biotechnol 29: 143-. TALEN has been shown to cause deletion functionality via NHEJ by TALENs in rat, mouse, zebrafish, xenopus, medaka, rat and human cells, Ansai et al, 2013, Genetics,193: 739-; carlson et al, 2012, PNAS,109: 17382-; hockemeyer et al, 2011, Nature Biotechnol.,29: 731-; lei et al, 2012, PNAS,109: 17484-; moore et al, 2012, PLoS ONE,7: e 37877; stroud et al, 2013, J.biol.chem.,288: 1685-1690; sung et al, 2013, Nature Biotechnol 31: 23-24; wefers et al, 2013, PNAS 110: 3782-.

For TALENs, further described in U.S. patent nos. 8,420,782; 8,450,471, respectively; 8,450,107, respectively; 8,440,432, respectively; 8,440,431 and U.S. patent publication nos. US20130137161 and US20130137174 describe methods of making the same.

Other useful endonucleases can include, for example, HhaI, HindIII, NotI, BbvCI, EcoRI, Bg/I, and AlwI. The target specificity of TAL effectors can be enhanced by the function of some endonucleases (e.g., fokl) as dimers only. For example, in some cases, each FokI monomer can be fused to a TAL effector sequence that recognizes a different DNA target sequence, and only when the two recognition sites are in close proximity, the inactive monomers can bind together to form a functional enzyme. By requiring DNA binding to activate nucleases, highly site-specific restriction enzymes can be produced.

In some embodiments, the TALEN may further comprise a nuclear localization signal or sequence (NLS). NLS is an amino acid sequence that helps target TALEN nuclease proteins into the nucleus to introduce a double-strand break at the target sequence in the chromosome.

Nuclear localization signals are well known in the art, see, e.g., Makkerh et al, 1996, Curr biol.6: 1025-. NLS includes sequences from the SV40 large T antigen, Kalderon 1984, Cell 39: 499-; NLS from nucleoprotein, described in detail in Dingwall et al, 1988, J Cell biol.,107,841-9. In McLane and Corbett 2009, IUBMB Life,61,697-70; further examples are described in Dopie et al, 2012, PNAS,109, E544-E552.

The cleavage domain may be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases that can be the source of the cleavage domain include, but are not limited to, restriction endonucleases and homing endonucleases. See, e.g., 2002-; and Belfort et al, (1997) Nucleic Acids Res.25: 3379-3388. Other enzymes that cleave DNA are known, such as SI nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nucleases; yeast HO endonuclease. See also Linn et al, (eds.) nucleic acids, Cold spring Harbor Laboratory Press, 1993. One or more of these enzymes or functional fragments thereof may be used as a source of the cleavage domain.

Zinc finger mediated genome editing

The use of Zinc Finger Nucleases (ZFNs) for gene editing, such as targeted insertion by homology directed repair procedures, is well established. See, for example, Carbery et al, 2010, Genetics,186: 451-; cui et al, 2011, Nature Biotechnol.,29: 64-68; hauschild et al, 2011, PNAS,108: 12013-12017; orlando et al, 2010, Nucleic Acids Res.,38: e152-e 152; and Porteus & Carroll,2005, Nature Biotechnology,23: 967-.

Components of zinc finger-mediated processes include zinc finger nucleases having a DNA binding domain and a cleavage domain. This is described, for example, in the following: beerli et al, (2002) Nature Biotechnol.,20: 135-141; pabo et al, (2001) Ann. Rev. biochem.,70: 313-340; isalan et al, (2001) Nature Biotechnol.19: 656-660; segal et al, (2001) Curr Opin Biotechnol.,12: 632-637; and Choo et al, (2000) CurrOpin.struct.biol.,10: 411-416; and U.S. Pat. nos. 6,453,242 and 6,534,261. Methods for designing and selecting zinc finger binding domains of target sequences are well known in the art, see, e.g., Sera, et al, Biochemistry 2002,41, 7074-; U.S. patent nos. 6,607,882; 6,534,261 and 6,453,242.

In some embodiments, the zinc finger nuclease may further comprise a nuclear localization signal or sequence (NLS). NLS is an amino acid sequence that helps target zinc finger nuclease proteins into the nucleus to introduce double strand breaks at the target sequence in the chromosome. Nuclear localization signals are well known in the art. See, e.g., Makkerh et al, (1996) Current Biology 6: 1025-.

The cleavage domain may be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases that can be the source of the cleavage domain include, but are not limited to, restriction endonucleases and homing endonucleases. See, e.g., 2002-; and Belfort et al, (1997) Nucleic Acids Res.25: 3379-3388. Other enzymes that cleave DNA are known (e.g., SI nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). See also Linn et al, (eds.) nucleic acids, Cold spring Harbor Laboratory Press, 1993. One or more of these enzymes (or functional fragments thereof) may be used as a source of the cleavage domain. As mentioned above, the cleavage domain may also be derived from an enzyme or part thereof that requires dimerization to generate cleavage activity.

Two zinc finger nucleases may be required for cleavage, as each nuclease comprises one monomer of the active enzyme dimer. Alternatively, a single zinc finger nuclease may comprise both monomers to produce an active enzyme dimer. Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specifically binding to DNA (at a recognition site) and cleaving DNA at or near the binding site. Certain restriction enzymes (e.g., type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme fokl catalyzes double-stranded cleavage of DNA, which is 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, for example, U.S. Pat. nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al, (1992) PNAS 89: 4275-; li et al, (1993) PNAS90: 2764-; kim et al, (1994) PNAS 91: 883-887; kim et al (1994) J.biol.chem.269:31,978-31, 982. Thus, a zinc finger nuclease may comprise a cleavage domain from at least one type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. Exemplary type IIS restriction enzymes are described, for example, in International publication WO 07/014275, the entire disclosure of which is incorporated herein by reference. Other restriction enzymes also contain separable binding and cleavage domains, and these are also encompassed by the present disclosure. See, e.g., Roberts et al, (2003) Nucleic Acids Res.31: 418-420. An exemplary type IIS restriction enzyme with a cleavage domain separate from a binding domain is FokI. This particular enzyme is active as a dimer (Bitinaite et al, 1998, PNAS 95:10,570-10, 575). Thus, for the purposes of this disclosure, the portion of the FokI enzyme used in the zinc finger nuclease is considered to be a cleavage monomer. Thus, for targeting double-stranded cleavage using fokl cleavage domains, two zinc finger nucleases (each comprising a fokl cleavage monomer) can be used to reconstitute the core enzyme dimer. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two fokl cleavage monomers may also be used. In certain embodiments, the cleavage domain may comprise one or more engineered cleavage monomers that minimize or prevent homodimerization, as described, for example, in U.S. patent publication nos. 20050064474, 20060188987, and 20080131962, all of which are incorporated herein by reference in their entirety. In a non-limiting example, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of fokl are all targets that affect dimerization of the fokl cleavage half-domain. Exemplary engineered cleavage monomers of fokl that form obligate heterodimers comprise a pair, wherein the first cleavage monomer comprises mutations at amino acid residue positions 490 and 538 and the second cleavage monomer comprises mutations at amino acid residue positions 486 and 499 of fokl. Thus, in one embodiment, there is a mutation at amino acid position 490 that replaces lys (k) with glu (e); a mutation at amino acid residue 538 of ile (e) instead of lys (k); a mutation at amino acid residue 486 of gln (q) instead of glu (e); and a mutation at position 499 of Ile (I) instead of Lys (K). In particular, engineered cleavage monomers can be prepared by: engineered cleavage monomers designated "E490K: I538K" were generated in one cleavage monomer by mutating position 490 from E to K and position 538 from I to K and engineered cleavage monomers designated "Q486E: I499L" were generated in the other cleavage monomer by mutating position 486 from Q to E and position 499 from Q to E. The engineered cleavage monomers described above are obligate heterodimer mutants in which aberrant cleavage is minimized or eliminated. Engineered cleavage monomers can be prepared using suitable methods, for example, by site-directed mutagenesis of wild-type cleavage monomers (fokl), as described in U.S. patent publication No. 20050064474.

The zinc finger nucleases described above may be engineered to introduce a double strand break at the targeted integration site. The double-strand break may be located at the targeted integration site, or may be at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or 1000 nucleotides from the integration site. In some embodiments, the double-strand break may be at most 1, 2, 3, 4, 5, 10, 15, or 20 nucleotides from the integration site. In other embodiments, the double-strand break may be up to 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides from the integration site. In still other embodiments, the double-strand break may be up to 50, 100, or 1000 nucleotides from the integration site.

As described in U.S. patent nos. 6,534,643; 6,858,716 and 6,830,910 and Watt et al, 2006.

Immunodeficient mice having a genetically modified genome comprising a genetic modification that renders the mouse deficient in MHC I and MHC II can be produced by introducing a gene targeting vector into a preimplantation embryo or stem cell, such as an embryonic stem cell (ES) or an induced pluripotent stem cell (iPS).

The term "gene targeting vector" refers to a double-stranded recombinant DNA molecule that effectively recombines with and mutates a particular chromosomal locus, e.g., by insertion or substitution of a target gene.

For target gene disruption (e.g., mutation), gene targeting vectors are prepared using recombinant DNA technology and contain 5 'and 3' sequences homologous to the target gene endogenous to the stem cell. The gene targeting vector optionally and preferably also comprises a selectable marker, such as neomycin phosphotransferase, hygromycin or puromycin. One of ordinary skill in the art would be able to select sequences for inclusion in gene targeting vectors and use them using no more than routine experimentation. The gene targeting vector can be produced recombinantly or synthetically using well known methods.

For methods of injecting gene targeting vector DNA into pre-implantation embryos, the gene targeting vector is linearized prior to injection into the non-human pre-implantation embryos. Preferably, the gene targeting vector is injected into a fertilized egg. Fertilized eggs were collected from superovulated females on the day of mating (0.5dpc) and injected with the expression construct. Injected oocytes were cultured overnight or transferred directly into the oviduct of a 0.5-day p.c. pseudopregnant female. Methods for superovulation, oocyte harvesting, gene targeted injection and Embryo transfer are well known in the art and are described in Manipulating the Mouse Embryo Manual, 3 rd edition, Cold Spring Harbor Laboratory Press; 12.15.2002, ISBN-10: 0879695919. Progeny may be detected for the presence of a disruption (e.g., mutation) of the target gene by DNA analysis (e.g., PCR, Southern blot, or sequencing). Mice with a disrupted (e.g., mutated) target gene can be detected for expression of the target protein (e.g., by using ELISA or Western blot analysis) and/or expression of mRNA (e.g., by RT-PCR).

Alternatively, the gene targeting vector can be transfected into stem cells (ES cells or iPS cells) using well-known methods such as electroporation, calcium phosphate precipitation, and lipofection.

Mouse ES cells were cultured in media optimized for the particular cell line. Typically, the ES medium is Dulbecco's Modified Eagle Media (DMEM) containing 15% Fetal Bovine Serum (FBS) or a synthetic or semi-synthetic equivalent, 2nM glutamine, 1mM sodium pyruvate, 0.1mM non-essential amino acids, 50U/ml penicillin and streptomycin, 0.1mM 2-mercaptoethanol, and 1000U/ml LIF (for some cell lines with chemical differentiation inhibitors). The detailed description is well known in the art (Tremml et al, 2008, Current Protocols in Stem Cell Biology, Chapter 1: Unit 1 C.4). For a review of ES cell differentiation inhibitors, see Buehr, M, et al, (2003) genetics of experimental stem cells, genetic Transactions of the Royal Society B, Biological sciences358, 1397-1402.

Cells are screened for disruption (e.g., mutation) of the target gene by DNA analysis (e.g., PCR, Southern blot, or sequencing). Cells with the correct homologous recombination event that disrupts the target gene can be detected for expression of the target protein (e.g., by using ELISA or Western blot analysis) and/or mRNA expression (e.g., by RT-PCR). If desired, the selectable marker can be removed by treating the stem cells with Cre recombinase. After treatment with Cre recombinase, the cells are analyzed for the presence of nucleic acid encoding the target protein.

Selected stem cells with the correct genomic event to disrupt the target gene can be injected into the pre-implantation embryo. For microinjection, ES or iPS cells were made into single cells using a mixture of pancreatin and EDTA, and then resuspended in ES medium. The single cell population was selected using a drawn thin glass needle (20-25 μm inner diameter) and introduced into the blastocyst cavity (blastocoel) through the zona pellucida of the embryo using an inverted microscope equipped with a micromanipulator. As an alternative to blastocyst injection, stem cells can be injected into early embryos (e.g., 2 cells, 4 cells, 8 cells, pre-blastocysts)Morula or morula). Laser or piezoelectric pulses that drill holes to open the transparent band can be used to assist injection. About 9-10 selected stem cells (ES or iPS cells) were injected per blastocyst or 8 cell stage embryo, 6-9 stem cells per 4 cell stage embryo and about 6 stem cells per 2 cell stage embryo. After introduction of stem cells, embryos were allowed to stand at 37 ℃ in 5% CO₂、5％O₂For several hours or overnight, and subsequently transferred into pseudopregnant recipient females. In a further alternative method of stem cell injection, the morula stage embryos can be pooled with stem cells. All of these methods are well established and can be used to produce stem cell chimeras. For a more detailed description, see Manipulating the Mouse Embryo: A Laboratory Manual, 3 rd edition (A. Nagy, M. Gertsenstein, K. Vintersten, R. Behringer, Cold spring harbor Laboratory Press; 12.15.2002, ISBN-10:0879695919), Nagy et al, 1990, Development 110, 815-; US 7576259: method for making genetic modifications, US7659442, US7,294,754, Kraus et al 010, Genesis 48,394-399.

Pseudopregnant embryo recipients are prepared using methods well known in the art. Briefly, 6-8 week old fertile female mice are mated with male mice that have had vases removed or are sterile to induce a hormonal state that helps support surgical introduction of the embryo. Up to 15 blastocyst-containing stem cells were introduced into the uterine horn very close to the uterine-oviduct junction 2.5 days post-coital (dpc). For early embryos and morula, these embryos were cultured in vitro to blastocysts or implanted into the oviduct of a 0.5dpc or 1.5dpc pseudopregnant female depending on the embryo stage. Chimeric pups from transferred embryos are born 16-20 days after transfer, depending on the age of the embryo at implantation. Chimeric males were selected for reproduction. Progeny ES cell genomes can be analyzed for transmission by coat color and nucleic acid analysis (e.g., PCR, Southern blot, or sequencing). In addition, target gene expression can be analyzed, such as by protein analysis (e.g., immunoassay) or functional assays, for target mRNA or protein expression to confirm target gene disruption. Offspring with disrupted (e.g., mutated) target gene are mated with each other to produce a homozygous non-human animal with disrupted target gene. Transgenic mice are mated with immunodeficient mice to produce isogenic immunodeficient strains with disrupted target genes.

Methods of evaluating genetically modified mice to determine whether a target gene is disrupted such that the mice lack the ability to express the target gene are well known and include standard techniques such as nucleic acid assays, spectroscopic assays, immunoassays and functional assays.

One or more standards may be used to enable quantitative detection of a target protein in a sample.

Assays to assess functional target proteins can be performed in animals with putative target gene disruption. Described herein are assays for assessing the function of a target protein in animals with putative target gene disruption.

Optionally, the genetically modified immunodeficient mice according to aspects of the invention are produced by selective breeding. A first parental line of a mouse having a first desired genotype can be propagated with a second parental line of a mouse having a second desired genotype to produce progeny of the genetically modified mouse having the first and second desired genotypes. For example, an immunodeficient first mouse can be bred with a second mouse that has a disruption of the MHC I gene such that no MHC I is expressed or its expression is reduced to produce an offspring that is immunodeficient and has a disruption of the MHC I gene such that no MHC I is expressed or its expression is reduced. In a further example, NSG mice can be bred with mice in which the target gene is disrupted such that the target gene is not expressed or is reduced in expression, to produce offspring that are immunodeficient and in which the target gene is disrupted such that the target protein is not expressed or is reduced in expression.

Aspects of the invention provide a genetically modified immunodeficient mouse comprising a target gene disruption in substantially all cells, and a genetically modified mouse comprising a target gene disruption in some but not all cells.

Immunodeficiency of the invention

The term "immunodeficient non-human animal" means a non-human animal characterized by one or more of the following: lack of functional immune cells, such as T cells and B cells; DNA repair defects; a defect in gene rearrangement encoding an antigen-specific receptor on lymphocytes; lack immune function molecules such as IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA.

According to an aspect of the invention, a genetically modified immunodeficient non-human animal provided according to an aspect of the invention, the genome of which comprises a genetic modification, is a mouse, wherein the genetic modification is such that the non-human animal lacks MHC I and MHC II activity. Although described herein primarily with respect to aspects of the invention in which the genetically modified immunodeficient non-human animal is a mouse, the genetically modified immunodeficient non-human animal can also be a mammal, such as a rat, gerbil, guinea pig, hamster, rabbit, pig, sheep, or a non-human primate.

The term "immunodeficient mouse" means a mouse characterized by one or more of the following: lack of functional immune cells, such as T cells and B cells; DNA repair defects; a defect in gene rearrangement encoding an antigen-specific receptor on lymphocytes; lack immune function molecules such as IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA. Immunodeficient mice can be characterized by a defect or defects in one or more genes involved in immune function, such as Rag1 and Rag2(Oettinger, M.A et al, Science,248: 1517-.

A particularly useful strain of immunodeficient mice is NOD^scidIl2rg^tm1Wjl/SzJ, commonly referred to as NOD scid γ (NSG) mice, which is described in detail in Shultz LD et al, 2005, J.Immunol,174: 6477-89. NSG is a representative strain of mice developed in the Jackson Laboratory. Other similar mouse sub-strains may be used to prepare NSG and are intended to be included in the present invention. Other useful immunodeficient mouse strains include NOD.Cg-Rag1^tm1MomIl2rg^tm1Wjl/SzJ, see Shultz LD et al, 2008, Clin Exp Immunol 154(2):270-84, commonly referred to as NRG mice; Cg-Prkdc^scidIl2rg^tm1SugJicTac or NOD/Shi-scid-IL2rγ^nullIt is commonly referred to as a NOG mouse, as described in detail in Ito, M. et al, Blood 100, 3175-3182 (2002).

The term "Severe Combined Immunodeficiency (SCID)" means a condition characterized by a lack of T cell and B cell function.

Common forms of SCID include X-linked SCID characterized by a gamma chain gene mutation in the IL2RG gene and lymphocyte phenotype T (-) B (+) NK (-), and autosomal recessive SCID characterized by a Jak3 gene mutation and lymphocyte phenotype T (-) B (+) NK (-), ADA gene mutation and lymphocyte phenotype T (-) B (-) NK (-), IL-7R α chain mutation and lymphocyte phenotype T (-) B (+) NK (+), CD3 delta or epsilon mutation and lymphocyte phenotype T (-) B (+) NK (+), RAG1/RAG2 mutation and lymphocyte phenotype T (-) B (-) NK (+), Artemis gene mutation and lymphocyte phenotype T (-) B (-) NK (+), CD45 gene mutation and lymphocyte phenotype T (-) B (+) NK (+).

In a further aspect, the genetically modified immunodeficient mouse is deficient in its endogenous gene encoding a DNA-dependent protein kinase catalytic subunit (Prkdc) that results in the mouse expressing the deficient endogenous DNA-dependent protein kinase catalytic subunit and/or a decreased amount of endogenous DNA-dependent protein kinase catalytic subunit, or the mouse may not express the endogenous DNA-dependent protein kinase catalytic subunit at all. The immunodeficient mouse can optionally be Prkdc null such that it lacks a functional endogenous Prkdc gene.

Mice genetically modified according to aspects of the invention have mutations with severe combined immunodeficiency (Prkdc)^scid) Commonly referred to as scid mutations. The scid mutation is well known and is located on mouse chromosome 16 as described in Bosma et al, Immunogenetics 29:54-56,1989. Mice homozygous for scid mutations are characterized by a lack of functional T and B cells, lymphopenia, hypoglobulinemia, and a normal hematopoietic microenvironment. The scid mutation can be detected, for example, by detecting a marker of the scid mutation using well-known methods (e.g., PCR or flow cytometry).

According to aspects of the invention, the genetically modified mouse has a deficiency in the gamma chain of the IL2 receptor. The term "deficiency of the gamma chain of the IL2 receptor" means a reduction of the gamma chain of the IL2 receptor. The reduction of the gamma chain of the IL2 receptor may be due to gene deletion or mutation. Reduced gamma chain of the IL2 receptor may be detected, for example by detecting deletion or mutation of the gene for the gamma chain of the IL2 receptor and/or by detecting reduced expression of the gamma chain of the IL2 receptor using well known methods.

According to an aspect of the invention there is provided a genetically modified immunodeficient NSG mouse, the genome of which comprises a genetic modification, wherein the genetic modification results in the immunodeficient mouse being deficient in MHC I and MHC II such that the genetically modified immunodeficient NSG mouse is deficient in functional MHC I and deficient in functional MHC II.

According to an aspect of the invention, there is provided a genetically modified immunodeficient NRG mouse, the genome of which comprises a genetic modification, wherein the genetic modification results in the immunodeficient mouse being deficient in MHC I and MHC II such that the genetically modified immunodeficient NRG mouse is deficient in functional MHC I and deficient in functional MHC II.

According to an aspect of the invention there is provided a genetically modified immunodeficient NOG mouse, the genome of which comprises a genetic modification, wherein the genetic modification results in the immunodeficient mouse lacking MHC I and MHC II, such that the genetically modified immunodeficient NOG mouse lacks functional MHC I and lacks functional MHC II, the precursor being that the immunodeficient mouse is not an NOD/Shi-scid-IL2r γ characterized by an β 2m (component of MHC I) knockout and an IA β (component of MHC II) knockout^nullA mouse.

^{b b null null}NSG- (KD) (IA) mice

According to aspects of the invention, a genetically modified immunodeficient mouse lacking MHC class I and MHC class II is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1Wjl/SzJ (abbreviated NSG- (K)^bD^b)^null(IA^null) Mice lacking functional MHC I and lacking functional MHC II, due to homozygous null mutations (abbreviated (K) of the H2-K and H2-D MHC I α protein subclasses^bD^b)^null) So that NSG- (K)^bD^b)^null(IA^null) Mice lack functional MHCI. Homozygous null mutations (abbreviated IA) due to the H-2A subclass of MHC II^null) So that NSG- (K)^bD^b)^null(IA^null) Mice lack functional MHC II.

Although NSG- (K)^bD^b)^null(IA^null) And NSG-B2M^null(IA IE)^nullMice lack functional MHC I and MHC II, but it was not expected that human IgG was in NSG- (K)^bD^b)^null(IA^null) Clearance in mice and NSG-B2M^null(IA IE)^nullThere were significant differences in the mice. Although NSG- (K)^bD^b)^null(IA^null) Mice showed a slow pattern of human IgG clearance (similar to that observed in NSG mice; notably, NSG mice have functional MHC I and MHC II), but NSG-B2M^null(IA IE)^nullMice showed rapid clearance of IgG (see fig. 2), which makes this mouse model unsuitable for use in antibody testing. NSG- (K) of the present invention^bD^b)^null(IA^null) The mice are characterized by no more than 60%, such as no more than 70%, 80%, or 90% clearance within a period of 2 days after administration of human IgG. After about 2 weeks, in NSG- (K)^bD^b)^null(IA^null) Approximately 90% of human IgG is cleared in mice. The term "clearance" as used in relation to administration of human IgG to mice refers to the process of removing functional human IgG from the mice.

^{null null}NSG-B2M (IA IE) mice

According to aspects of the invention, the genetically modified immunodeficient mouse deficient in functional MHC I and in class I MHC and class II MHC deficient in functional MHC II is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1WjlTg (Ins2-HBEGF)6832Ugfm/Sz (abbreviated NSG-B2M)^null(IA IE)^null) Mouse due to homozygous null mutation of β 2 microglobulin (abbreviated as B2M)^null) So that NSG-B2M^null(IA IE)^nullMouse deficient in functionMHC I. Homozygous null mutations (abbreviated (IA IE) due to MHC II subclasses H-2A and H-2E^null) So that NSG-B2M^null(IA IE)^nullMice lack functional MHC II.

In NSG-B2M^null(IA IE^null) Rapid clearance of human IgG was observed in mice. After about 2 days, in NSG-B2M^null(IA IE^null) Approximately 90% of the human IgG in the mice was cleared, see figure 2.

^{b b null null}NSG-RIP-DTR (KD) mice

According to aspects of the invention, the genetically modified immunodeficient mouse deficient in functional MHC I and in class I MHC and class II MHC deficient in functional MHC II is nod^scidH2-K1^tm1BpeH2-Ab1^em1MvwH2-D1^tm1BpeIl2rg^tm1WjlTg (Ins2-HBEGF)6832Ugfm/Sz transgenic mice, abbreviated to NSG-RIP-DTR (K)^bD^b)^null(IA^null) Injection of Diphtheria Toxin (DT) into mice expressing diphtheria toxin receptor under the control of rat insulin promoter leads to death of mouse islet β cells and hyperglycemia NSG-RIP-DTR (K)^bD^b)^null(IA^null) The strain is capable of completely and specifically eliminating mouse pancreatic β cells, thereby avoiding the broad toxic effects of diabetic drugs (such as streptozotocin).

Mouse model comprising allogeneic and/or xenogeneic cells

Genetically modified immunodeficient mice according to aspects of the invention further comprise allogeneic and/or xenogeneic cells or tissues. An increased survival of the genetically modified immunodeficient mice of the invention that have been administered allogeneic and/or xenogeneic cells or tissues is observed due to the lack of functional MHC I and functional MHC II in the mice that reduces or eliminates Graft Versus Host Disease (GVHD). For example, genetically modified immunodeficient mice lacking functional MHC I and functional MHC II have an increased survival after administration of allogeneic and/or xenogeneic cells or tissue to the genetically modified immunodeficient mice compared to immunodeficient mice of the same type but not lacking functional MHC I and functional MHC II.

The invention relates to a method for treating a mammal, such as a human, comprising administering to a genetically modified immunodeficient mouse lacking functional MHC I and functional MHC II allogeneic and/or xenogeneic cells or tissues, wherein the genetically modified immunodeficient mouse lacks functional MHC I and functional MHC II.

Allogeneic and/or xenogeneic cells or tissues administered include, but are not limited to, non-human pancreatic cells, non-human islets of langerhans, non-human pancreatic β cells, stem cells such as, but not limited to, non-human CD34+ cells, non-human primary tumor cells, non-human tumor cell line cells, non-human stem cells, non-human hematopoietic cells, isolated or mixed non-human subpopulations of blood cells such as white blood cells, red blood cells, lymphocytes, monocytes, neutrophils, eosinophils, basophils, platelets, NK cells, non-human peripheral blood mononuclear cells, and combinations of two or more types of cells or tissues.

Optionally, the allogeneic and/or xenogeneic cells or tissues administered to the genetically modified immunodeficient mouse lacking functional MHC I and functional MHC II are genetically modified.

According to a particular aspect of the invention, human T cells are administered to a genetically modified mouse that is deficient in functional MHC I and functional MHC II. The human T cells may be administered as an isolated population of human T cells, as a population of human stem cells or human precursor cells that will differentiate into human T cells in mice, or as a mixed population of cells with human T cells as a subset.

According to a particular aspect of the invention, human tumor cells are administered to a genetically modified mouse that lacks functional MHC I and functional MHC II immunodeficiencies. The human tumor cells can be administered in the form of an isolated population of human tumor cells (such as, but not limited to, primary human tumor cells of human patient origin or human tumor cell line cells), or in the form of a mixed population of cells with human tumor cells as a subset.

According to a particular aspect of the invention, human tumor cells are administered to a genetically modified mouse that lacks functional MHC I and functional MHC II immunodeficiencies. The human tumor cells can be administered in the form of an isolated population of human tumor cells (such as, but not limited to, primary human tumor cells of human patient origin or human tumor cell line cells), or in the form of a mixed population of cells with human tumor cells as a subset.

Allogeneic and/or xenogeneic cells or tissues may be administered to the genetically modified immunodeficient mice of the invention by various routes, such as, but not limited to, intravenous or intraperitoneal administration.

The genetically modified immunodeficient mouse can be administered one or more times allogeneic and/or xenogeneic cells or tissues. The survival of the immunodeficient mice lacking the genetic modification of functional MHC I and functional MHC II of the present invention that have been administered allogeneic and/or xenogeneic cells or tissues is increased due to the reduction or elimination of Graft Versus Host Disease (GVHD).

According to aspects of the invention, differentiated allogeneic and/or xenogeneic cells are introduced into immunodeficient, genetically modified mice lacking functional MHC I and functional MHC II by administering one or more types of stem cells that engraft the immunodeficient, genetically modified mice and generate differentiated cells or tissues by differentiating the stem cells in the mice.

The number of allogeneic and/or xenogeneic cells is not considered limiting. Thus, although more or fewer cells may be used,however, the number of allogeneic and/or xenogeneic cells administered is typically 1x10³To 1x10⁸And (1,000 to 100,000,000).

Thus, a method according to the methods of the invention may comprise administering about 1x10 to an immunodeficient, genetically modified mouse³(1000) To about 1x10⁸(100,000,000), about 1X10⁴(10,000) to about 1x10⁸(100,000,000), about 1X10⁴(10,000) to about 1x10⁷(10,000,000), about 1X10⁵(100,000) to about 1x10⁷(10,000,000), about 1X10³(1,000) to about 1x10⁴(10,000), about 5x10³(5,000) to about 5x10⁴(50,000), about 1X10⁴(10,000) to about 1x10⁵(100,000), about 5x10⁴(50,000) to about 5x10⁵(500,000), about 1X10⁶(1,000,000) to about 1x10⁸(100,000,000), about 5x10⁶(5,000,000) to about 1x10⁸(100,000,000), about 1X10⁷(10,000,000) to about 1x10⁸(100,000,000), about 2x10⁴(20,000) to about 5x10⁵(500,000) or about 5x10⁴(50,000) to about 2x10⁵(200,000) allogeneic and/or xenogeneic cells. The method can comprise administering at least about 1x10 to an immunodeficient genetically modified mouse²(100) About 2x10²(200) About 3x10²(300) About 4x10²(400) About 5x10²(500) About 6x10²(600) About 7x10²(700) About 8x10²(800) About 9x10²(900) About 1x10³(1000) About 2x10³(2000) About 3x10³(3000)4x10³(4000) About 5x10³(5000) About 6x10³(6000) About 7x10³(7000) About 8x10³(8000) About 9x10³(9000) About 1x10⁴(10,000), about 2x10⁴(20,000), about 3X10⁴(30,000), about 4x10⁴(40,000), about 5x10⁴(50,000), about 6x10⁴(60,000), about 7x10⁴(70,000), about 8X10⁴(80,000), about 9x10⁴(90,000), about 1X10⁵(100,000), about 2x10⁵(200,000), about 3x10⁵(300,000) About 4x10⁵(400,000), about 5x10⁵(500,000), about 6x10⁵(600,000), about 7x10⁵(700,000), about 8x10⁵(800,000), about 9x10⁵(900,000), about 1x10⁶(1,000,000), about 2X10⁶(2,000,000), about 3X10⁶(3,000,000), about 4x10⁶(4,000,000), about 5X10⁶(5,000,000), about 6x10⁶(6,000,000), about 7x10⁶(7,000,000), about 8X10⁶(8,000,000), about 9x10⁶(9,000,000), about 1X10⁷(10,000,000), about 2x10⁷(20,000,000), about 3x10⁷(30,000,000), about 4x10⁷(40,000,000), about 5x10⁷(50,000,000), about 6x10⁷(60,000,000), about 7x10⁷(70,000,000), about 8x10⁷(80,000,000), about 9x10⁷(90,000,000) or about 1x10⁸(100,000,000) allogeneic and/or xenogeneic cells. One of ordinary skill in the art will be able to determine the number of allogeneic and/or xenogeneic cells to administer to a particular mouse through limited experimentation.

Administering allogeneic and/or xenogeneic cells to the mouse may comprise administering a composition comprising allogeneic and/or xenogeneic cells to the mouse. The composition may further include, for example, water, a tonicity modifier (e.g., a salt such as sodium chloride), a pH buffer (e.g., citrate), and/or a sugar (e.g., glucose).

Allogeneic and/or xenogeneic hematopoietic stem cells are transplanted into genetically modified immunodeficient animals and are characterized by the presence of differentiated allogeneic and/or xenogeneic cells (e.g., hematopoietic cells) in the genetically modified immunodeficient mice of the invention. The engraftment of allogeneic and/or xenogeneic cells can be assessed by any of a variety of methods, such as, but not limited to: flow cytometry analysis of cells in animals administered allogeneic and/or xenogeneic cells is performed at one or more time points after administration of the cells.

Tumor xenografts

Various aspects of the invention relate to the administration of xenogeneic tumor cells to the genetically modified immunodeficient mice of the invention.

The xenogeneic tumor cells administered to the genetically modified immunodeficient mice of the invention can be any of a variety of tumor cells including, but not limited to, tumor cell line cells and primary tumor cells. The xenogeneic tumour cells may be derived from any of a variety of organisms, preferably mammals, including humans, non-human primates, rats, guinea pigs, rabbits, cats, dogs, horses, cattle, goats, pigs and sheep.

According to a particular aspect of the invention, the heterologous tumor cell is a human tumor cell. According to a particular aspect of the invention, the human tumor cells are present in a sample obtained from a human, such as, but not limited to, a blood sample, a tissue sample, or a sample obtained by human tumor biopsy.

Tumor cells obtained from humans may be administered directly to the genetically modified immunodeficient mice of the invention, or may be cultured in vitro prior to administration to the genetically modified immunodeficient mice.

As used herein, the term "tumor" means cells characterized by unregulated growth, including, but not limited to, pre-neoplastic hyperproliferative, carcinoma in situ, tumors, metastases, and solid and non-solid tumors. Examples of tumors are those caused by cancer, including, but not limited to, lymphoma, leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, cancer of the adrenal gland, anal cancer, cancer of the bile duct, cancer of the bladder, cancer of the brain, breast cancer, triple negative breast cancer, cancer of the central or peripheral nervous system, cancer of the cervical, colon, colorectal, endometrial, esophageal, biliary bladder, gastrointestinal, glioblastoma, head and neck, kidney, liver, nasopharynx, nasal cavity, oropharynx, oral cavity, osteosarcoma, ovarian, pancreatic, parathyroid, pituitary, prostate, retinoblastoma, sarcoma, salivary gland, skin, small intestine, stomach, testicular, thymus, thyroid, uterine, vaginal, and vulval cancers.

Administration of tumor cells to genetically modified immunodeficient mice can be any method suitable as known in the art. For example, administration may include administering the cells into an organ, body cavity, or blood vessel, e.g., by injection or implantation, e.g., subcutaneous and/or intraperitoneal implantation. The tumor cells can be administered in the form of a tumor mass, a mass of tumor cells, or dissociated cells.

Tumor cells can be administered by various routes, such as, but not limited to, by subcutaneous injection, intraperitoneal injection, or injection into the tail vein.

Implantation of the xenogeneic tumor cells can be assessed by any of a variety of methods, including, for example, but not limited to, visual inspection of the mice for signs of tumor formation.

Any of a variety of methods can be used to measure the growth of the xenogeneic tumor, including, but not limited to, measuring in a living mouse, measuring a tumor excised from a living mouse, or measuring a tumor in situ or excised from a dead mouse. The measurements may be measured using a measuring instrument such as a caliper, using one or more imaging techniques such as ultrasonography, computed tomography, positron emission tomography, fluorescence imaging, bioluminescence imaging, magnetic resonance imaging, and combinations of any two or more of these or other tumor measurement methods. The number of tumor cells in a sample obtained from a mouse bearing xenogeneic tumor cells can be used to measure tumor growth, particularly for non-solid tumors. For example, the number of non-solid tumor cells in a blood sample can be assessed to obtain a measure of non-solid tumor growth in mice.

The number of tumor cells administered is not considered to be limiting. A single tumor cell can be expanded to a detectable tumor in a genetically modified immunodeficient animal as described herein. The number of tumor cells administered is generally in the range of 10³(1,000) to 1X10⁸(100,000,000) tumor cells, although more or less may be administered.

Thus, methods according to aspects of the invention may comprise administering about 1x10 to a genetically modified immunodeficient mouse²(100) To about 1x10⁸(100,000,000), about 1X10³(1,000) to about 1x10⁵(100,000), about 1x10⁴(10,000) to about 1x10⁶(1,000,000), about 1X10⁵(100,000) to about 1x10⁷(10,000,000), about 1X10³(1000) To about 1x10⁴(10,000), about 5x10³(5,000) to about 5x10⁴(50,000), about 1X10⁴(10,000) to about 1x10⁵(100,000), about 5x10⁴(50,000) to about 5x10⁵(500,000), about 1X10⁵(100,000) to about 1x10⁶(1,000,000), about 5X10⁵(500,000) to about 5x10⁶(5,000,000), about 1X10⁶(1,000,000) to about 1x10⁷(10,000,000), about 2x10⁴(20,000) to about 5x10⁵(500,000) or about 5x10⁴(50,000) to about 2x10⁵(200,000) heterogeneous tumor cells (e.g., human tumor cells). The methods may comprise administering at least about 1x10 to an immunodeficient QUAD mouse²(100) About 2x10²(200) About 3x10²(300) About 4x10²(400) About 5x10²(500) About 6x10²(600) About 7x10²(700) About 8x10²(800) About 9x10²(900) About 1x10³(1,000), about 2X10³(2,000), about 3X10³(3,000), about 4X10³(4000) About 5x10³(5,000), about 6X10³(6,000), about 7X10³(7,000), about 8X10³(8,000), about 9x10³(9,000), about 1X10⁴(10,000), about 2x10⁴(20,000), about 3X10⁴(30,000), about 4x10⁴(40,000), about 5x10⁴(50,000), about 6x10⁴(60,000), about 7x10⁴(70,000), about 8X10⁴(80,000), about 9x10⁴(90,000), about 1X10⁵(100,000), about 2x10⁵(200,000), about 3x10⁵(300,000), about 4x10⁵(400,000), about 5x10⁵(500,000), about 6x10⁵(600,000), about 7x10⁵(700,000), about 8x10⁵(800,000), about 9x10⁵(900,000), about 1x10⁶(1,000,000), about 2X10⁶(2,000,000), about 3X10⁶(3,000,000), about 4x10⁶(4,000,000), about 5X10⁶(5,000,000), about 6x10⁶(6,000,000), about 7x10⁶(7,000,000), about 8X10⁶(8,000,000), about 9x10⁶(9,000,000) or about 1x10⁷(10,000,000) xenogeneic tumor cells (e.g., human tumor cells). The method can include administering about 1x10 to a genetically modified immunodeficient mouse²(100) About 2x10²(200) About 3x10²(300) About 4x10²(400) About 5x10²(500) About 6x10²(600) About 7x10²(700) About 8x10²(800) About 9x10²(900) About 1x10³(1,000), about 2X10³(2,000), about 3X10³(3,000), about 4X10³(4,000), about 5X10³(5,000), about 6X10³(6,000), about 7X10³(7,000), about 8X10³(8,000), about 9x10³(9,000), about 1X10⁴(10,000), about 2x10⁴(20,000), about 3X10⁴(30,000), about 4x10⁴(40,000), about 5x10⁴(50,000), about 6x10⁴(60,000), about 7x10⁴(70,000), about 8X10⁴(80,000), about 9x10⁴(90,000), about 1X10⁵(100,000), about 2x10⁵(200,000), about 3x10⁵(300,000), about 4x10⁵(400,000), about 5x10⁵(500,000), about 6x10⁵(600,000), about 7x10⁵(700,000), about 8x10⁵(800,000), about 9x10⁵(900,000), about 1x10⁶(1,000,000), about 2X10⁶(2,000,000), about 3X10⁶(3,000,000), about 4x10⁶(4,000,000), about 5X10⁶(5,000,000), about 6x10⁶(6,000,000), about 7x10⁶(7,000,000), about 8X10⁶(8,000,000), about 9x10⁶(9,000,000), about 1X10⁷(10,000,000) or about 1x10⁸(100,000,000) heterogeneous tumor cells (e.g., human tumor cells). One of ordinary skill in the art will be able to determine the number of xenogeneic tumor cells to administer to a particular mouse through limited experimentation.

According to aspects of the invention, xenogeneic tumor cells and xenogeneic leukocytes are administered to genetically modified immunodeficient mice. The xenogeneic tumor cells and the xenogeneic leukocytes can be administered simultaneously or at different times.

According to aspects of the invention, the tumor cells are from the same species as the administered leukocytes. According to various aspects, the tumor cells and leukocytes administered to the genetically modified immunodeficient mice of the invention are both human cells.

According to aspects of the invention, a xenogeneic tumor cell and a xenogeneic T cell are administered to a genetically modified immunodeficient mouse. The xenogeneic tumor cells and the xenogeneic T cells may be administered at the same time or at different times.

According to aspects of the invention, the tumor cells are from the same species as the administered T cells. According to various aspects, the tumor cells and T cells administered to the genetically modified immunodeficient mice of the invention are both human cells.

According to aspects of the invention, a xenogeneic tumor cell and a xenogeneic PBMC are administered to a genetically modified immunodeficient mouse. The xenogeneic tumor cells and the xenogeneic PBMCs may be administered at the same time or at different times.

According to an aspect of the invention, the tumor cells are from the same species as the PBMCs to be administered. According to various aspects, the tumor cells and PBMCs administered to the genetically modified immunodeficient mice of the invention are both human cells.

Preprocessing (Conditioning)

Implanting xenogeneic cells in immunodeficient genetically modified mice according to aspects of the invention includes "pre-treating" the immunodeficient genetically modified mice prior to administration of the xenogeneic cells, for example by sub-lethal irradiation of the recipient animal with high frequency electromagnetic radiation or gamma ray radiation, or treatment with a radiation mimicking drug such as busulfan or mechlorethamine. It is believed that the pretreatment reduces the number of host immune cells (e.g., hematopoietic cells) and creates a suitable microenvironment factor for the implantation of xenogenic immune cells (e.g., without limitation, leukocytes, T cells, PBMCs, or other cells) and/or creates a suitable microenvironment for the implantation of xenogenic immune cells. Standard methods for pretreatment are well known in the art, as described herein and in J.Hayakawa et al, 2009, Stem Cells,27(1): 175-.

Methods provided according to aspects of the invention include administering xenogeneic cells (such as, but not limited to, leukocytes, T cells, PBMCs, or other cells) to an immunodeficient, genetically modified mouse without "preconditioning" the immunodeficient, genetically modified mouse prior to administration of xenogeneic immune cells (such as, but not limited to, leukocytes, T cells, PBMCs, or other cells). Methods provided according to aspects of the invention include administering xenogeneic cells (such as, but not limited to, leukocytes, T cells, PBMCs, or other cells) to an immunodeficient, genetically modified mouse without "pre-treating" the immunodeficient, genetically modified mouse with radiation or a radiation-mimicking drug prior to administration of the xenogeneic immune cells.

Measurement of

According to an aspect of the present invention there is provided a method of determining the effect of a putative therapeutic agent, comprising administering to a genetically modified immunodeficient mouse comprising allogeneic and/or xenogeneic cells or tissue an amount of a putative therapeutic agent; and measuring the effect of the putative therapeutic agent.

The putative therapeutic agent used in the methods of the invention may be any chemical entity, including, for example, synthetic or naturally occurring compounds or combinations of synthetic or naturally occurring compounds, organic or inorganic small molecules, proteins, peptides, nucleic acids, carbohydrates, oligosaccharides, lipids or combinations of any of these.

Standards suitable for the assay are well known in the art, and the standard used may be any suitable standard.

The assay results can be analyzed by any of a variety of methods using statistical analysis, examples being by parametric or nonparametric tests, analysis of variance, analysis of covariance, logistic regression for multivariate analysis, Fisher's exact test, chi-square test, Student's T test, Mann-Whitney test, Wilcoxon signature ranking test, McNemar test, Friedman test, and Page's L trend test. These and other statistical tests are well known in the art and are described in detail in Hicks, CM, Research Methods for Clinical therapeutics: Applied Project Design and catalysis, Churchill Livingstone (publisher); 5 th edition, 2009; and Freund, RJ, et al, statistical methods, Academic Press; version 3, 2010.

The methods and genetically modified immunodeficient mice provided according to aspects of the invention have a variety of uses, such as in vivo testing for substances against human cancers.

According to aspects of the invention, a method for identifying an anti-tumor activity of a test agent comprises providing a genetically modified immunodeficient mouse; administering a xenogeneic tumor cell to the genetically modified immunodeficient mouse, wherein the xenogeneic tumor cell forms a solid or non-solid tumor in the genetically modified immunodeficient mouse; administering a test substance to the genetically modified immunodeficient mouse; determining the response of the xenogeneic tumor and/or tumor cell to the test agent, wherein an inhibitory effect of the test agent on the tumor and/or tumor cell identifies the test agent as having anti-tumor activity.

According to aspects of the invention, a method for identifying an anti-tumor activity of a test agent comprises providing a genetically modified immunodeficient mouse; wherein the genetically modified immunodeficient mouse has been implanted with a xenogenic PMBC; administering a xenogeneic tumor cell to the genetically modified immunodeficient mouse, wherein the xenogeneic tumor cell forms a solid or non-solid tumor in the genetically modified immunodeficient mouse; administering a test substance to the genetically modified immunodeficient mouse; determining the response of the xenogeneic tumor and/or tumor cell to the test agent, wherein an inhibitory effect of the test agent on the tumor and/or tumor cell identifies the test agent as having anti-tumor activity.

According to aspects of the invention, a method for identifying an anti-tumor activity of a test agent comprises providing a genetically modified immunodeficient mouse; wherein the genetically modified immunodeficient mouse has been implanted with a human PMBC; administering a human tumor cell to the genetically modified immunodeficient mouse, wherein the human tumor cell forms a solid or non-solid tumor in the genetically modified immunodeficient mouse; administering a test substance to the genetically modified immunodeficient mouse; determining the response of the human tumor and/or tumor cell to the test agent, wherein an inhibitory effect of the test agent on the tumor and/or tumor cell identifies the test agent as having anti-tumor activity.

According to aspects of the invention, there is provided an anti-tumor agent for identifying a test substanceThe genetically modified immunodeficient mouse used in the determination of tumor Activity is NSG- (K)^bD^b)^null(IA^null) A mouse; or NSG-B2M^null(IA IE^null) A mouse.

As used herein, the term "inhibitory effect" means the effect of a test substance to inhibit one or more of tumor growth, tumor cell metabolism, and tumor cell division.

Determining the response of the xenogeneic tumor and/or tumor cell to a test substance comprises comparing the response to a standard to determine the effect of the test substance on the xenogeneic tumor cell according to aspects of the methods of the invention, wherein an inhibitory effect of the test substance on the xenogeneic tumor cell identifies the test substance as an anti-tumor composition. Standards are well known in the art and the standard used may be any suitable standard. In one example, the standard is a compound known to have an anti-tumor effect. In another example, no treatment of a comparable heterogeneous tumor provides a basal level indication of growth of the treatment-free tumor for comparison of the effect of the test substance. The standard may be a reference level of expected tumor growth previously determined in a comparable individual mouse or a comparable population of mice and saved in print or electronic matrix for recall and comparison to assay results.

The assay results can be determined by any of a variety of methods using statistical analysis to determine whether a test substance has an inhibitory effect on a tumor, exemplified by parametric or nonparametric tests, analysis of variance, analysis of covariance, logistic regression for multivariate analysis, Fisher's exact test, chi-square test, Student's T test, Mann-Whitney test, Wilcoxon signature ranking test, McNemar test, Friedman test, and Page's L trend test. These and other statistical tests are well known in the art and are described in detail in Hicks, CM, Research Methods for Clinical therapeutics: Applied Project Design and Analysis, Churchill Livingstone (publisher); 5 th edition, 2009; and Freund, RJ, et al, Statistical Methods, Academic Press; version 3, 2010.

The test substance used in the method of the invention may be any chemical entity, including, for example, synthetic or naturally occurring compounds or a combination of synthetic or naturally occurring compounds, organic or inorganic small molecules, antibodies (murine, chimeric or humanized), antibody fragments (Fab, f (ab)' 2), proteins, peptides, nucleic acids, carbohydrates, oligosaccharides, lipids or a combination of any of these.

According to aspects of the invention, the test agent is an immunotherapeutic agent, such as an antibody (murine, chimeric or humanized), an antibody fragment (Fab, f (ab)' 2), or a combination of any of these, or a non-immunotherapeutic agent such as a synthetic or naturally occurring compound, a combination of synthetic or naturally occurring compounds, an organic or inorganic small molecule, a protein or peptide that is not an antibody or antigen binding fragment, a nucleic acid, a carbohydrate, an oligosaccharide, a lipid, or a combination of any of these.

According to an aspect of the invention, the test substance is an anti-cancer agent. According to aspects of the invention, the anti-cancer agent is an anti-cancer immunotherapeutic agent, such as an anti-cancer antibody or antigen-binding fragment thereof. According to aspects of the invention, the anti-cancer agent is a non-immunotherapeutic agent, such as a synthetic or naturally occurring compound, a combination of synthetic or naturally occurring compounds, an organic or inorganic small molecule, a protein or peptide that is not an antibody or antigen-binding fragment, a nucleic acid, a carbohydrate, an oligosaccharide, a lipid, or a combination of any of these.

Anticancer agents are described, for example, in Brunton et al, (eds.), Goodman and Gilman's the pharmacological Basis of Therapeutics, 12 th edition, Macmillan Publishing Co., 2011.

Examples of the anticancer agent include acervocine (acivicin), aclarubicin (aclodazole), ecodazole (acodazole), ametrycin (oxycetacin), ciclopirox (capreomycin), ciclopirox (ciclopirox), ciclopirox-), ciclopirox-alone (ciclopirox), ciclopirox-alone (ciclopirox), ciclopirox-or (ciclopirox-), ciclovir (ciclopirox-or (ciclopirox), these, or the injection, these, the injection.

According to aspects of the invention, the anti-cancer agent is an anti-cancer immunotherapeutic agent, also known as an anti-cancer antibody. The anti-cancer immunotherapeutic agent used may be any antibody, or an effective portion of an antibody, which is effective in inhibiting at least one type of tumor, particularly a human tumor. The anti-cancer immunotherapeutic agent includes, but is not limited to, 3F8, 8H9, abafungumab (abagomab), abituzumab, adalimumab (adalimumab), adetuzumab, adacanazumab, adoctanumab, afuttuzumab (afutuzumab), alizeumab pegol, alemtuzumab (alemtuzumab), amatuximab, antatmomafenotox, anetuzumab ravtansine, adolizumab, arimomab, arcamumazumab, ascrinkumacumumab, alezumab (atezolizumab), bavituximab, belimumab (belimuzumab), bevacizumab (bevacizumab), bivatuzumab (betatuzumab), brituzumab (bravacetazumab), bravacetazumab (betatuzumab), bevacizumab (bevacizumab), bevacizumab (beauveteuzumab, abertuzumab (aberrab), bevacizumab (tartuzumab), bevacizumab, tartuzumab (tartuzumab, tartuzumab (tartuzumab), bevacizumab, tartuzumab (tartuzumab), bivattuzumab, tartuzumab (tartuzumab), bevacizumab, tartuzumab (tartuzumab), bevac, ganitumumab, gemtuzumab (gemtuzumab), girentiximab, glemtuzumab vedotin, ibratuzumab (ibritumomab), agonvomattuzumab (igomozetan), imab362, imaluumab, igatuzumab, indatuzumab ravatin, indatuzumab vedotin, inebrizumab, inotuzumab, indotuzumab, intetuzumab, ipilimumab, iratuzumab, isatuximab, lauzumab, lexatuzumab (lexatuzumab), lifastuzumab vedotatin, lintuzumab (lintuzumab), lirtuzumab, lortuzumab mertuzumab mertansine, wucatazumab (wurtuzumab), meltuzumab (miltuzumab), meltuzumab (veovatuzumab), rituximab (dolutab), rituximab (omamab), rituximab (orizumab, paratuzumab (omab), rituximab (omartamab), rituximab (omab, paratuzumab (omatuzumab), rituximab (omab), rituximab (paratuzumab (valtuzumab), rituximab (valtuzumab), rituximab (valtuzumab), valtuzumab (valtuzumab), valtuzumab (valtuzumab), valtuzumab (valtuzumab), valtuzumab (valtuzumab), valtuzumab (valtuzumab), valtuzumab (valtuzumab), valtuzumab (val, Prtuzumab, ractumomab, radiotuzumab, ramucirumab, rituximab (rilotumumab), rituximab (rituximab), robitumumab, sacitumumab govitetrane, samalizumab, seribant, sibutrumab, siltuximab, sofitumumab vedottin, tacitumumab tetraxetan, tarextitumumab, tenatemab, tepitumumab, tigatuzumab, tositumomab, tositumumab, trastuzumab (trastuzumab), trutelimumab, tuzumab beculmoleine, uklitiximab, unituzumab, vatuzumab, trastuzumab, truzumab, tuzumab, tuvelloub, uklituzumab, tuvelutumumab, netuzumab, trastuzumab, truveluttuzumab, tuveluttuvelutvazumab, radretazumab, radtuzumab, ritumumab, tuveluttuzumab, tuzumab, tuveluttuveluttuzumab, ritukutuveluttuveluttuvelutvazumab, tuveluttuvelutvatuvelab, tuveluttuveluttuvelab, etc.

According to an aspect of the invention, the test substance is a substance that specifically binds to one or more of: 1) cell surface proteins such as Clusters of Differentiated (CD) cell surface molecules; 2) intracellular proteins such as kinases; and 3) extracellular proteins, such as shed cell surface receptors or soluble ligands for cell surface receptors.

According to aspects of the present invention, the test substance is a substance that specifically binds to a protein expressed by leukocytes (e.g., lymphocytes or leukocytes of the myeloid lineage). In other options, the test substance is a substance that specifically binds to a ligand of a leukocyte. In other options, the test agent is an agent that specifically binds to a molecule expressed by the cancer cell.

According to aspects of the invention, the test agent can specifically bind to PD-1, PD-L1, or CTLA-4. According to aspects of the invention, the test agent may be an immune checkpoint inhibitor, such as a PD-1 inhibitor, a PD-L1 inhibitor or a CTLA-4 inhibitor. According to aspects of the invention, the immune checkpoint inhibitor is an antibody that specifically binds to PD-1, PD-L1 or CTLA-4, respectively, and is a PD-1 inhibitor, PD-L1 inhibitor or CTLA-4 inhibitor. According to an aspect of the invention, the test substance is an immune checkpoint inhibitor selected from the group consisting of alemtuzumab, avizumab, devoluumab, ipilimumab, nivolumab, pembrolizumab, and an antigen-binding fragment of any of the foregoing.

The test substance can be administered by any suitable route of administration, such as, but not limited to, oral, rectal, buccal, nasal, intramuscular, vaginal, ocular, otic, subcutaneous, transdermal, intratumoral, intravenous, and intraperitoneal.

Embodiments of the compositions and methods of the present invention are illustrated in the following examples. These examples are provided for illustrative purposes and are not to be construed as limiting the scope of the compositions and methods of the present invention.