NSG mice lacking MHC class I and class II

Abstract

A NOD.Cg-Prkdcscid NSG) mouse which is genetically modified such that the NSG mouse lacks functional major histocompatibility complex I (MHC I) and lacks functional major histocompatibility complex II (MHC II) is provided according to aspects of the present invention. According to specific aspects the genetically modified NSG mouse is a NOD.Cg- Prkdcscid H2-K1tml Bpe H2-Ab1em Mvw H2-D1tml H2rg Wjl / SzJ NSG- RIP-DTR (IAnull) mouse, or a NOD.Cg-B2m'm" Une Prkdcscid H2dlAbl-Ea H2rg!"1 Wjl / SzJ (NSG- B2M ull (IA mouse. Human immune cells and / or human tumor cells are administered to a genetically modified immunodeficient mouse according to aspects described herein and assays of one or more test substances can be performed using the provided mice. 20 24 20 47 25 09 J ul 2 02 4 A B S T R A C T 2 0 2 4 2 0 4 7 2 5 0 9 J u l 2 0 2 4

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of Australian Patent Application No. 2018265887, filed on 8 November 2019, and is related to International Patent Application No. PCT / US2018 / 032548, filed on 14 May 2018, and claims priority from U.S. Provisional Patent Application Serial Nos. 62 / 505,264, filed May 12, 2017 and 62 / 649,099, filed March 28, 2018; each of which are incorporated herein by reference in their entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. 1R24 OD018259 and Grant No. OD011190, both awarded by the National Institutes of Health. The Government has certain rights in the application. FIELD OF THE INVENTION

[0003] Generally described are mouse models of functional human cells and tissues. According to specific aspects, genetically modified immunodeficient mice are provided that are deficient in MHC class I and MHC class II. According to further specific aspects, genetically modified immunodeficient mice are provided that are deficient in MHC class I and MHC class II and which include 1) engrafted functional human T cells and 2) allogeneic or xenogeneic cells, such as human patient-derived tumor cells. BACKGROUND OF THE INVENTION

[0004] Humanized mice, e.g. immunodeficient mice engrafted with functional human cells and tissues, have been widely used to model human immune cell function in vivo. A major limitation for studying human T cell function in such mouse models has been the rapid development of graft versus host disease (GVHD) that not only shortens the experimental time window, but also confounds the analysis of human T cell function due to the underlying ongoing acute GVHD that eventually kills the mice. These issues have hindered studies of human T cell function.

[0005] Some attempts were made to generate humanized mouse models lacking the major histocompatibility complex (MHC) class I or class II. For example, Vugmeyster et al. disclose a mouse model deficient in MHC molecules encoded by the H-2K and H-2D genes (KbDb - / mice) (Vugmeyster et al., Proc. Natl. Acad. Sci. USA 95: 12492-12497, 1998). Ashizawa et al. describe a humanized immunodeficient NOG mouse -2 - 2024204725   23 Jun 2026 (NOD / Shi-scid-IL2rYnull) [NOD / Shi-Prkdcscd-IL2rYnull] knockout of the MHC Class I / II (Ashizawa et al., Clin Cancer Res; 23(1), 149-158, 2017).

[0006] There is a continuing need for mouse models of functional human cells and tissues. [0006a]      Reference to any prior art in the specification is not an acknowledgement or 5 suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be combined with any other piece of prior art by a skilled person in the art. SUMMARY OF THE INVENTION [0006b]       In a first aspect of the invention, there is provided a method, comprising: 10         engrafting a humanized immunodeficient non-obese diabetic (NOD) mouse with xenogeneic cells, wherein the genome of the humanized immunodeficient mouse comprises (i) a mutation in an IL2rg gene, (ii) a scid mutation in a Prkdc gene, and (iii) a mutation in each of a H2-K1 gene, a H2-Ab1 gene, and a H2-D1 gene; administering a therapeutic agent to the immunodeficient mouse; and 15          assaying for an effect of the therapeutic agent on the xenogeneic cells and / or the immunodeficient mouse, wherein the immunodeficient mouse has an IL2RG deficiency, a severe combined immune deficiency, a major histocompatibility complex (MHC) class I deficiency, and an MHC class II deficiency. 20   [0006c] In a second aspect of the invention, there is provided a selective breeding method, comprising breeding a first parental strain of mouse with a second parental strain of mouse to produce offspring, wherein the first parental strain of mouse is a non-obese diabetic (NOD) mouse, and the genome of the NOD mouse comprises (i) a mutation in an IL2rg gene, (ii) a scid mutation in a Prkdc gene, and (iii) a mutation in each of a H2-K1 gene, a H2-Ab1 gene, 25 and a H2-D1 gene. [0006d]       In a third aspect of the invention, there is provided a genetic modification method, comprising: introducing, into a non-obese diabetic (NOD) mouse preimplantation embryo or fertilized oocyte, RNA molecules encoding a Clustered Regularly Interspaced Short 30 Palindromic Repeats (CRISPR)-Cas and at least one oligonucleotide targeting a predetermined target site, wherein the genome of the NOD mouse preimplantation embryo or fertilized oocyte -2a - 2024204725   23 Jun 2026 comprises (i) a mutation in an IL2rg gene, (ii) a scid mutation in a Prkdc gene, and (iii) a mutation in each of a H2-K1 gene, a H2-Ab1 gene, and a H2-D1 gene; selecting the NOD mouse preimplantation embryo or fertilized oocyte with a desired genetic modification; and 5          transferring the NOD mouse preimplantation embryo or fertilized oocyte with a desired genetic modification to a pseudopregnant female. [0006e]       In a fourth aspect of the invention, there is provided a genetic modification method, comprising: introducing, into a non-obese diabetic (NOD) mouse stem cell, RNA molecules 10 encoding a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas and at least one oligonucleotide targeting a predetermined target site, wherein the genome of the NOD mouse stem cell comprises (i) a mutation in an IL2rg gene, (ii) a scid mutation in a Prkdc gene, and (iii) a mutation in each of a H2-K1 gene, a H2-Ab1 gene, and a H2-D1 gene; selecting the NOD mouse stem cell with a desired genetic modification; 15          transferring the NOD mouse stem cell with a desired genetic modification to a mouse preimplantation embryo or fertilized oocyte; and transferring the mouse preimplantation embryo or fertilized oocyte to a pseudopregnant female. [0006f]       In a fifth aspect of the invention, there is provided an immunodeficient non- 20 obese diabetic (NOD) mouse whose genome comprises (i) a mutation in an IL2rg gene, (ii) a scid mutation in a Prkdc gene, and (iii) a mutation in each of a H2-K1 gene, a H2-Ab1 gene, and a H2-D1 gene, wherein the immunodeficient mouse has an IL2RG deficiency, a severe combined immune deficiency, a major histocompatibility complex (MHC) class I deficiency, and an MHC class II deficiency. 25

[0007] A NOD.Cg-Prkdcscid Il2rgtmiWrSzJ (NOD-scid-IL2rYnull, NSG) mouse which is genetically modified such that the NSG mouse lacks functional major histocompatibility complex I (MHC I) and lacks functional major histocompatibility complex II (MHC II) is provided according to aspects of the present invention. According to specific aspects the genetically modified NSG mouse is a NOD.Cg-Prkdcscid H2-K1tm1Bpe H2-Ab1em1Mvw H2- 30 D1tm1Bpe Il2rgtm1Wjl / SzJ (NSG-(Kb Db)null (IAnull)) mouse, NSG-RIP-DTR (Kb Db)null (IAnull) mouse, or a NOD.Cg-B2mtm1Unc Prkdcscid H2dlAb1-Ea Il2rgtm1Wjl / SzJ (NSG-B2Mnull (IA IEnull)) mouse.

[0008] A NOD.Cg-Prkdcscid Il2rgtnlWjj / SzJ (NOD-scid-IL2rynuU, NSG) mouse which is genetically modified such that the NSG mouse lacks functional major histocompatibility - 2b - 2024204725   23 Jun 2026 complex I (MHC I) and lacks functional major histocompatibility complex II (MHC II) is provided according to aspects of the present invention which includes human immune cells. According to specific aspects the genetically modified NSG mouse is a NOD.Cg-Prkdcscid H2-K1tm1Bpe H2-Ab1em1Mvw H2-D1tm1Bpe Il2rgtm1Wjl / SzJ (NSG-(Kb Db)null (IAnull)) mouse which 5 includes human immune cells, NSG-RIP-DTR (Kb Db)null (IAnull) mouse which includes human immune cells, or a NOD.Cg-B2mtm1Unc Prkdcscid H2dlAb1-Ea Il2rgtm1Wjl / SzJ (NSG-B2Mnull (IA IEnull)) mouse which includes human immune cells.

[0009] A NOD.Cg-Prkdcscid Il2rgtmlWjj / SzJ (NOD-scid-IL2rYnull, NSG) mouse which is genetically modified such that the NSG mouse lacks functional major histocompatibility 10 complex I (MHC I) and lacks functional major histocompatibility complex II (MHC II) is provided according to aspects of the present invention which includes human peripheral blood mononuclear cells. According to specific aspects the genetically modified NSG mouse is a NOD.Cg-Prkdcscid H2-KltmlBpe H2-AblemlMvw H2-DltmlBpe Il2rgtmlWjl / SzJ (NSG-(Kb Db)null (IAnull)) mouse which includes human peripheral blood mononuclear cells, NSG-RIP-DTR (Kb 15   Db)null (IAnull) mouse which includes human peripheral blood mononuclear cells, or a NOD.Cg- B2mtmlUnc Prkdcscid H2dlAbl-Ea 2024204725   09 Jul 2024 30 -3 - ll2rgm- W-IS'zJ Q^^G-B2Mwb (lA I^liy) mouse which includes-human peripheral blood mononuclear cells. [00101   A NOD.CgGVfcfc^ ll2r^*1 ^ / SzJ -(N0D-s«cZ4L2^, NSG) mouse which is genetically modified such that the NSG mouse lacks functional major histocompatibility complex I (MHC I) and lacks functional .major histocompatibility complex II (MHC II) is provided according to: aspects of the present invention which includes human T cells. According to specific aspects the genetically modified NSG mouse is a NOD,Cg-Prfe / c^ H2-Kltm11^ H2,Ablet*tM™' H2AAl™JBpe 112^1^1^ mouse which includes human T ceils, NSG-RIP-DTR. (X® (IAmi>) mouse which includes human T cells, or a NOD,CguS2Wm'PrkdcSC!ci B2r^mlWjll^ QAPA2~B2Mwil (1A      mouse which includes human T cells.

[0011] A NOD.Cg-PrM^ 7K^    (NOD-^^II^rY™”, NSG) mouse which is genetically modified such that the NSG mouse lacks functional major histocompatibility complex I (MHC- I) and kicks functional major histocompatibility complex II (MHC II) is provided according to aspects of the present invention which: includes human immune cells and human tumor cells. According to specific aspects the genetically modified NSG mouse is a NOD.Cg-Prfc / / ^ H2~KJlmlSpl! lf2-Abl*m'lMv* -H2-ji2r^ms ^ / SzJ (NSG-(K*            mouse which includes human immune cells and human tumor cells, NSG-RIP-DTR l^^1-1 {JA™11) mouse which includes human immune cells and human tumor cells, or a NOD-Cg-jOrmlb,1c Prkdc*"1' R2r^m" ZSzJ (JASG-B2APul‘ (1A 11^)) mouse which includes human immune cells and human tumor cells.

[0012] A NODGg-Prh^^          (^OD~scidAL2^ NSG) mouse which is genetically modified such, that the NSG mouse lacks functional major histocompatibility complex I (MHC I) and lacks functional major histocompatibility complex II (MHC II) is provided according to aspects of the present invention which includes human peripheral blood mononuclear cells and human tumor cells. According to specific aspects the genetically modified NSG mouse is a NOD^Cg-JV-Wc^ H2-Klfni^ H2-AbIemlM™         H2r^lf3i2 (NSG-(^ {IA^ mouse which includes human peripheral blood mononuclear cells and human tumor cells, NSG-RIP-DTR         (JA”*1) mouse which includes human peripheral blood mononuclear cells and human, tumor cells, or a NOD.Cg-jBWw / ^' PrkdiP^          Il2r^m^ / SzJ 2024204725   09 Jul 2024 (NSG-.^A / *^ (LA mouse which includes human peripheral blood mononuclear cells and human tumor cells.

[0013] A NOD.Cg-Prfcfc^' E2r^mt ^ / SzJ (NQD-scidAL2^, NSG> mouse which is genetically modified such that the NSG mouse lacks functional major histocompatibility complex I (MHC I) and lacks functional major histocompatibility complex II (MHC II) is provided according to aspects of the present invention which includes human T cells and human tumor cells. According to specific aspects the genetically modified NSG mouse is a NOD.CgrPrWe^'H2-AbEm .H2-H2rgmli^f^ (NSG-<* {Unvli) mouse which includes human T cells and human tumor cells, NSG-RIP-DTR.         ([A”*11) mouse which includes human T cells and human tumor cells, or a NQD.Cg-^ / M^'’'7 Prkdc^ (NSG-^jt / *^ (IA IE™11)') mouse which includes human T cells and human tumor cells.

[0014] An NSG-fA* (LA™11)) mouse of the present invention is characterized by clearance of no more than 60%, such as clearance of no more than 70%, 80%, or 90%, of administered human IgG in a time period of 2 days following administration of the human IgG.

[0015] 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 a NOD / Shi-mz^ mouse characterized by p2m (component of MHC I) knockout and LAP (light chain of MHC II) knockout According to particular aspects, the mouse further includes human immune cells such as human peripheral blood mononuclear cells and such as human T cells. According to particular aspects, the mouse further includes human immune cells such as human peripheral blood mononuclear cells and such as human T cells and further includes human tumor cells.

[0016] A method for modeling an effect of a human immune system, or one or more components thereof, in a genetically modified immunodeficient mouse is provided which includes administering a test substance to genetically modified immunodeficient mouse of the present invention; and assaying the effect of the human immune system, or one or more components thereof in the genetically modified immunodeficient mouse. The test substance 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. to V, th 2024204725   09 Jul 2024 modified immunodeficient mouse is provided wherein the genetically modified immunodeficient mouse is a NOD.Cg-PrWc4^ H2~Klifnl^pe            H2~Dlm^ 1121^ ^SzJ (NSG-f^ #F11      mouse, NSG-RIKDTR Dbrn mouse. g O o & so e a <x ■S ¢3 components thereof, in a genetically modified immunodeficient mouse is provided wherein the genetically modified immunodeficient mouse is a                  H2~ KJ1^^6 ffl-Abl™1**™ H2-DltmiBl*           .(NSG-f^ &T" mouse, NSG- RIP-DTR (t              mouse, or a                   Prkdecili 20                  (^G-B2&ftl>ti (IA 1^^}) mouse, wherein, the method includes PD-L1 inhibitor, or a CTLA-4 inhibitor. The test substance can be an immune checkpoint inhibitor selected from .atezolizum-ab, avelumab, duryalumab, ipilimumab, nivolumab, or pembrolizumab, or an antigen-binding fragment of any one of the foregoing. The test substance can be an anti-cancer agent.

[0018] A method for modeling an effect of a human immune system, or one or more 2024204725   09 Jul 2024 ft g IK’ s CT S' S o ft R ft o’ immunodeficient mouse; and assaying the effect of the human PMBC in the genetically modified immunodeficient mouse. The test substance can be, but is not limited to an i^mmunodeficient mouse is provided wherein the genetically modified immunodefident mouse is a NOD.Cg-Prhfc^ H2-K2tmiSpe H2-AbrMlMw H2-Dl!m}Spe Il2r^mimi / SzJ (NSG-^Z^1^       NSG-RIP-DTR           mouse, or a NOD^g- Pr^                           (U mouse, wherein the method includes administering a test substance to the genetically modified > i .£>• 2024204725   09 Jul 2024 -7- including, but not limited to, a PD-1 inhibitor PD-L1 inhibitor, or a CTLA-4 inhibitor. The test substance can be an inmiune checkpoint inhibitor selected from atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, or pembrolizumab, or an antigenbinding fragment of any one of the foregoing. The test substance can be: an anti-cancer 5 agent, BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figures 1A-1C show representative flow cytometry of MHC class I and class II expression in NSG-^D')™iS (lA^1) and NSG-R^A / ™^^ IErl‘ mice. Spleens from 10 NSG, NSG-«& D^nai! and NSG-&W- (IA knockout mice were disaggregated by enzymatic and mechanical digestion,.

[0023] Figure 1A is a graph showing that monocyte derived dendritic cells were identified in viable cells as CD 11 bT, LyOGdim, CD 11 c+ and Ly6C-.

[0024] Figure IB is a graph showing results of evaluation of monocyte derived 15 dendritic cells recovered from each strain for expression of mouse H2Kd and H2Kb. Representative staining is shown for all stains 0^=2).

[0025] Figure IC is a graph showing results of evaluation of monocyte derived dendritic cells recovered from each strain for expression of mouse H2 IAg7 and H2 IAb. Representative staining is shown for all stains (N~2). 20

[0026] Figure 2 is a graph showing human IgG half-life in the serum, of NSG-fX* and MSG-SAW™47 (IA mice, Mice were injected IV with 200 pg of human IgG and bled at the indicated time points to recover serum. Serum was used for ELISA analysis of circulating human IgG. The first bleed at 2 minutes post-injection was considered as 100% serum IgG. Each point represents the mean ± standard error of IgG 25 in 5 males who were 2-3 months of age.

[0027] Figures 3 A and 3B show survival of NSG mice lacking the expression of both mouse MHC class I and II following injection of Human Peripheral Blood Mononuclear Cells (PBMC):. Recipient mice were injected intravenously (IV) with 10 x 10s PBMC, and mice were monitored for overall health and survival. 30

[0028] Figure 3A is a graph showing % survival when NSG, MSG-fM™8), NSG-(K° and NSG-(K° D(Mw) mice were used as recipients of PBMC. The data are representative of 3 independent experiments. Survival distributions between groups were tested using the log. rank statistic. 2024204725   09 Jul 2024 - s - (0()29] Figure: 3B is a graph showing % survival when NSG, NSG-(A4 IE)1™11, NSG-322^^, and NSG- (IA 13)^ mice were used as: recipients of PBMC. The data are representative of 3 independent experiments, -Survival' distributions between groups were tested using the log rank statistic. 5

[0030] Figures 4A-4D show human CD45+ cell chimerism levels in NSG mice lacking the expression of both mouse MHC class I and. n following injection of PBMC. Recipient mice were injected IV 'with 10 x 106 PBMC, and mice were monitored for levels of human cell chimerism by determining the proportion of human CD45+ cells in the peripheral blood (Figure 4A and Figure 4G) and spleen (Figure 4B and Figure 4D). 10   [0031 ]    Figure 4 A is a graph showing human cell chimerism levels as monitored in the blood of NSG, NSG-CZr^ NSG-f^ and NSG-(K* ^ / uH mice injected with PBMC over a 10 week time period. The data are representative of 3 independent experiments. A 2-way ANOVA was used to determine significant differences between groups at each time point. Week 6; NSG vs NSG-;(X*p < 0,01 15 and NSG vs NSG-^W“" (lA^-p < 0.001; NSG-(j^) vs NSG-(X*Dhf p < 0.01, and NSG 4IA™il} vs NSG-^^T^ (TA^p < 0.001.

[0032] Figure 4B is a graph showing human cell chimerism levels as monitored in the spleens of NSG, NSG^M^)* NSG-:^ I^p11, and NSG-^           mice injected with. PBMC when mice were euthanized. A. one-way ANOVA was used to 20 determine- significant differences between groups. * represents p <0.05, ** represents p <0.01.

[0033] Figure 40 is a graph showing human cell chimerism levels as monitored in the blood of NSG, NSG-(M 1^, ^SG-B2Mtua, and NSG-K?^ (IA lE)^1 mice injected with PBMC over a 10 week time period.. The data are representative of 3 25 independent experiments. A 2-way ANOVA was used to determine significant. differences between groups at each time point. Week 4; NSG vs NSG-J&M"^ (IA IE^"ua' P < 0.01, NSG-(M IEf‘M vs. NSG-BdA / ^ (IA lEf^p < 0.01, and NSG-B2^ vs NSG-32M”,a (IA I^p < 0.05, Week 6; NSG vs NSG-52^ (IA lEf^1 p < 0,05, NSG~(M IEf^ vs NSG-^2^ p < 0.05, NSG-fM IEfn vs NSG-^^' (IA IE)nuli p < 0.01. 30 Week 8; NSG vs NSG-52^ p < 0.001, NSG vs NSG-52^ (U lEf1^ p < OM, NSG<£4 IEflil vs N^G-^AC* p < 0.001, NSG-(M IEf‘n vs NSG-52M^ (IA lE)^ p < 0.01. Week 10; NSG vs           p < 0.01, NSG vs NSG-i^Ar" (IA lEf*11 p < 2024204725   09 Jul 2024 -9 0.01, and NSG~(M IEfu!l vs KSG-B2M*11 p < 0.01, NSG-fM lEf* vs NSG-^^ (M IErUp<Q£G

[0034] Figure 4D is a graph showing human cell chimerism levels as monitored in the spleens of NSG, NSG-(Z4 NSG-Olrt and NSG^Af^ (M mice injected with PBMC when mice were euthanized. A one-way ANOVA was used to determine significant differences between groups. ** representsp <0.01,

[0035] Figures 5A-5D show engraftment of human T cells and B cells in NSG mice: lacking the expression of both mouse MHC class I and II following injection of PBMC. Recipient mice were injected IV with 10 xlO PBMC, and mice were monitored for levels of human CD3+ T ceils (Figure 5A and Figure 5C) and CD20+ B cells (Figure 5B and Figure 5D) in peripheral blood. {0036] Figure 5A is a graph showing human CD3+ cells (% of CD45) when NSG (N-7). NSG-(«W / ) (N-5), NSG-(^ (N=7), and NSG-fA6            (N=8) mice were used as recipients of PBMC. The data are representative of 3 independent experiments. A 2-way ANOVA was used to determine significant differences between groups at each time point * represents p < 0.05,

[0037] Figure 5B Is a graph showing, human. CD20+ cells (% of CD45) when NSG (N-7), NSG-CZr^): (N=5), NSG-^ 1^)^ (N-7), and NSG-^ (1^: (N=8) mice were- used as recipients of PBMC. The data are representative of 3 independent experiments. A 2-way ANOVA was used to determine significant differences between groups at each time point. * represents p < 0.05, (0038] Figure 5C is a graph showing, human CD3+ cells (% of CD45) when NSG (N=6), NSG-(Z4 IE)™11 (N=6), NSG-^Af"" (N=5)s and NSG-BW^' (JA lEf^1 (N=7) mice were used as recipients of PBMC. The data are representative of 3 independent experiments. A 2-way ANOVA was used to determine significant differences between groups at each time point, * represents p < 0.05.

[0039] Figure 5D is a graph showing human CD20+ cells (% of CD45) when NSG (N=6)5 NSG-fM W“ / Z NSG-^AT^ (N=5), and NSG-^JAf^ (M lEf* (N-7) mice were used as recipients of PBMC, The data are representative of 3 independent experiments. A 2-way ANOVA was used to determine significant differences between groups at each time point * representsp < 0,05.

[0040] Figures: 6A-6H show phenotypic analysis of human T cells; engrafting in NSG, NSG-(MWZ), NSG- / X* and NSG-fK6             mice injected, with b-> Vi NJ O 2024204725   09 Jul 2024 © q x © Cu 3 c? &J t-o JX 25 C» o to by E? cP Q © B' o o’ g ft O I kJ o 4^ ^5 p z; ->3 ? rd

[0045] Figures 6G and 6H are graphs showing CD4 and CD8 T cells, respectively, that were evaluated for expression of CD45RA and CCR7 by flow cytometry. Percentages of T cell subsets are shown with CD45RA± / CCR7^ cells labeled as naive, CD45RA- / CCR7^ cells labeled as central memory, CD45RA- / CCR7- cells labeled as injected with PBMC,

[0043] Figure 6C is a graph showing PD-1 expression by CDS T cells determined by flow cytometry for NSG,           NSG-(X*D^”4Pand NSG-^          mice 15 injected with PBMC,

[0044] Figures 6D-6F are graphs showing representative CD4, CD8, and PD1 g 'X- & £3 ,n Er o "01 - 2024204725   09 Jul 2024 o S' 2024204725   09 Jul 2024

[0058] Figures 9A-9H show expression of human IL2 in PBMC engrafted NSG mice and NSG-fX* (lA^1) mice enhances survival of human CD4+ Treg. Recipient NSG and NSG-          (lA^) mice were injected IP with 2.5 x 10!! particles of AAV-IL2 or injected with PBS. Two weeks later mice were injected intraperitoneally 5 (IP) with 1 x 106 PBMC.

[0059] Figures 9A-9C are graphs showing levels of human CD45+ cells (Figure 9A), CD3+ T cells (Figure 9B) and CD4+ / CD25+ / CD127-ZFOXP3+ Treg (Figure 9C) as determined by flow cytometry. A 2-way ANOVA was used to determine significant differences between groups. *** represents p < 0.005, and **** represents p < 0.001. 10

[0060] Figure 9D shows representative staining of CD4-H- T cells for CD25, CD127 and FOXP3 for the indicated groups.

[0061] Figure 9E is a graph showing. % survival of recipient mice was monitored, and survival distributions between the indicated groups was tested using the log rank statistic. 15

[0062] Figure 9F is a graph showing levels of CD4 and CD8 T cell determined by flow cytometry and expressed as a ratio of CD4 to CDS T cells. Closed black triangles represent NSG mice, open black triangles represent NSG mice injected with AAV-IL2, closed circles represent NSG- (if ifmice and open circles represent NSG-if{L4™a) mice injected with AAV-IL2. 20

[0063] Figure 9G is a graph showing results of evaluation of CD8 T cells for expression of CD45RA and CCR7 by flow cytometry. Percentages of T cell subsets are shown with CD45RA:NCCR7+ cells labeled as naive, CD45RA- / CCR7+ cells labeled as central memory, CD45RA- / CCR7- cells labeled as effector / effector memory, and CD45RA+ / CCR7- cells labeled as TEMRA. Closed black, triangles represent NSG mice, 25 open black triangles represent NSG mice injected with AAV-IL2, closed circles represent NSG- (if Dn fM (lA^1) mice and open circles represent NSG- (^ Iff^ (lA”1^ mice injected with AAV-IL2.

[0064] Figure 9H Is a graph showing Granzyme B expression by CD8 T cells as determined by flow cytometry and representative staining is shown. A t-test was used to 30 determine significant differences between mice treated with AAV-IL2 and controls. **♦ represents p < 0.005, **** represents p < 0.001. The data are representative of 3 independent experiments. 2024204725   09 Jul 2024

[0065] Figure I0A is a graph showing percent survival of a group of NSG mice coinjected with PBMC and human patient-derived tumor cells and a group of NSG-fK* £^nuii mice co-injected with PBMC and human patient-derived tumor cells.

[0066] Figure 10B is a graph showing tumor growth in 1) NSG mice injected with human patient-derived tumor cells: 2) NSG mice co-injected with PBMC and human patient-derived-tumor cells;               CM™1) mice injected with PBMC; and NSG- (Kb            mice co-injected with PBMC and human patient-derived tumor cells. DETAILED DESCRIPTION OF THE INVENTION

[0067] 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 defined and used in context in various standard references illustratively including J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. .Alberts & al., Molecular Biology of the Cell, 4th Ed., Garland,. 2002; D.L. Nelson and M.M, Cox, Lehninger .Principles of Biochemistry, 4th Ed., W.H. Freeman & Company, 2004; A. Nagy, M. Gertsenstein, K. Vintersten, R, Behringer, Manipulating: the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press; December 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,, Physicians’ Cancer Chemotherapy Drug Manual, Jones & Bartlett Publishers, 2005; LM, Kirkwood el al, Eds., Current Cancer Therapeutics, 4th Ed., Cun-ent Medicine Group, 2001; Remington; The Science and Practice of Pharm acy , Lippincott Williams & Wilkins, 21st Ed., 2005; L.V. Allen, Jr. el al, Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed,, Philadelphia, PA: Lippincott, Williams & Wilkins, 2004;and L, Brunton el at, Goodman & Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill Professional, 12th Ed., 2011,

[0068] The singular terms "a," "an," and "the" are not intended to be limiting and include plural referents unless explicitly stated otherwise or the context .clearly indicates otherwise. 2024204725   09 Jul 2024

[0069] The term "functional" as used generally herein refers to a protein, complex, cell, or other substance that retains the biological function of the corresponding nativeprotein, complex, cell, or other substance

[0070] By contrast, the term "norMunctionai" as used generally herein refers to a 5 protein, complex, cell, or other substance that does not retain the biological function of the corresponding native protein, complex, cell, or other substance,

[0071] Genetically modified immunodeficient mice that are deficient in. MHC class I and MHC class II are provided by the present invention.

[0072] According to aspects, a genetically modified immunodeficient mouse is 10 provided which includes in its genome at least one mutation effective to reduce or eliminate expression of functional MHC I a protein and / or reduce or eliminate expression of functional pSmficroglobulm such that MHC I is not present or is nonfunctional in the mouse; and which includes in its genome at least one mutation effective to reduce or eliminate expression of functional MHC II a protein and / or expression of 15 functional MHC II p protein such that MHC II is not present or is non-functional in the mouse.

[0073] According to aspects, the genetically modified immunodeficient mouse is a genetically modified NSG mouse. NSG MHC I / II knockout mice according to aspects of the present invention are useful in various applications, including study of human 20 immunity in the absence of GVHD and evaluation of antibody-based therapeutics.

[0074] MHC I

[0075] The terms "MHC F and “MHC class F are used interchangeably to refer to a complex formed by MHC I a protein and P2~mieroglobulin protein.

[0076] MHC I a protein includes an extracellular domain (which has three 25 subdomains: al, a2, and a3), a transmembrane domain, and a cytoplasmic tail. The al and «2 subdomains form the peptide-binding cleft,, while the «3 subdomain interacts with p2-m.icroglobulin. The terms “H2-K”, “H2-D” and “112-17”, refer to mouse MHC I a protein subclasses, all of which are encoded on mouse Chromosome-17.

[0077] p2-microglobulin associates noncovalently with the «3 subdomain of MHC I 30 a protein. The gene encoding: mouse p2-mieroglobulin is encoded on Chromosome- 2 (Chr2:122147686-122153083bp, + strand, GRCm38).

[0078] MHC II 2024204725   09 Jul 2024

[0079] The terms "MHC II" and “MHC class II” are used interchangeably to refer to a complex formed by two non-covalently associated proteins: an MHC II a protein and .an MHC II p protein. The terms "H-2A" and "H-2E" (often abbreviated as I-A and I-E, respectively) refer to subclasses of MHC II. The MHC II a: protein and MHC II p 5 proteins span the plasma membrane and each contains an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The extracellular portion of the MHC II a protein includes MHC II al and MHC II a2 domains, and the extracellular portion of the MHC II p protein includes MHC 11. pl and MHC II [32 domains. {0080] The term * functional” as used herein in reference to a functional MHC I a 10 protein, a functional p2mncroglobulin protein, a functional MHC II ft protein, a functional MHC II P protein, functional MHC I or functional MHC II, refers to MHC I a protein, p2.-microglobulin protein, MHC II a protein, MHC II p protein, MHC T or MHC II that retains the biological function of the corresponding native MHC I a protein, p:2-microglobulin protein, MHC II a protein, MHC II p protein, MHC I or MHC II. 15

[0081] By contrast, the term "non-functionar as used herein in reference to a non functional MHC I a protein, P2~microglobulin protein, MHC II a protein, MHC II p 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 a protein, [32-microglobulin protein, MHC II a protein, MHC II p protein, MHC I or MHC IL 20

[0082] The term, “native” as used herein refers to an unmutated protein or nucleic acid.

[0083] As used herein, the term “genetically modified” refers to modification of genomic DNA in a mouse that disrupts expression of at least one of: functional MHC I a protein, and functional: p2-microglobulin; and at least one of: functional MHC II a 25 protein and functional MHC II P protein such that the mouse that lacks functional MHC I and functional MHC IL

[0084] The term “expression” refers to transcription of a nucleic acid sequence to produce a corresponding mRNA and / or translation of the mRNA to produce the corresponding protein. 30

[0085] As used herein, the term “target gene” refers to a nucleic acid sequence that defines a mouse MHC I a gene, mouse p2-microglobulin gene, mouse MHC II a gene or mouse MHC II (3 gene. 2024204725   09 Jul 2024 ui sx (jo rt> * so Ui © to to .© to J# o o g o p << xs tQ D " -O Q s xs 2024204725   09 Jul 2024 o re n B © ca ag s tn re< w fa N, O B-re re S S’ re ¢5 re £5 O B re B fa re P re c» re B re re' oo o © 3- B re as ,® tn re » «c re {3 re 2024204725   09 Jul 2024 10 15 20 25 30 not limited to, CRISPR methodology, TAL (transcription activator-like Effector methodology, Zinc Finger-Mediated Genome Editing or DRAP to produce a genetically modified mouse provided according to embodiments of the present invention,

[0096] As used herein, the terms “target site” and “target sequence” in the context of a nuclease genetic editing technique refer to 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, provided sufficient, conditions for binding exist.

[0097] CRISHACg&Sysfom

[0098] CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea and confer resistance to foreign DNA elements, see Horvath, 2010, Science, 327: 167-170; Barrangou et al., 2007, Science, 315: 1709-1712; and Makarova et al, 2011, Nature Reviews Microbiology. 9:.467-477..

[0099] CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic. CRISPR repeats are separated by spacers of similar length.

[0100] The CRISPR-associated (eas) genes are often associated with CRISPR repeatspacer arrays. More than forty different Cas protein families have been described (Haft et al. 2005, PLoS Comput Biol. 1. (6): e60). Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes, some of which are associated with an additional gene module encoding repeat-associated mysterious proteins (RAMPs).

[0101] There are diverse CRISPR'systems in different organisms, and one of the simplest is the type II CRISPR system, .from. Streptococcus pyogenes* only a single gene encoding the Cas9 protein and two RNAs, a mature CRISPR RNA (crRNA) and a partially complementary trans-actmg- RNA (tracrRNA),. are necessary and sufficient for RNA-guided silencing of foreign. DNAs (Gasiunas et at, 2012, PNAS 109: E2579-E2586; Jinek et al, 2012, Science 337: 816-821). Maturation of crRNA requires tracrRNA and RNase III (Defteheva et al, 2011, Nature 471: 602-607), However, this requirement can be bypassed by using an engineered small guide RNA (sgRNA) containing a designed hairpin that mimics the tracrRNA-crRNA complex (Jinek et al, 2012, Science 337: 816-821). Base pairing between the sgRNA and. target DNA causes 2024204725   09 Jul 2024 20 25 30 double-strand breaks (DSBs) due to the endonuclease activity of Cas9.. Binding specificity is determined by both sgRNA-DNA base pairing and a short DNA motif (protospacer adjacent motif [PAM] sequence: NGG) juxtaposed to the DNA complementary region (Marraffini & Sontheimer, 2010, Nature Reviews Genetics, 11: 181-190). For example, the CRISPR system requires a minimal set of two molecules, the Cas9 protein and the sgRNA, and therefore can be used as a host-independent genetargeting platform. The Cas9 / CRISPR can be harnessed for site-selective RNA-guided genome editing, such as targeting insertion see for example. Carroll, 2012, Molecular Therapy 20:1658-1660; Chang et al, 2013, Cell Research 23: 465-472; Cho et al, 2013, Nature Biotephno'l 31: 230-232; Cong elal,, 2013, Science 339: 819-823; Hwang et al„ 2013, Nature Biotechnol 31: 227-229; Jiang el al., 2013, Nature Biotechnol 31: 233-239; Mali et al,, 2013, Science 339: 823-826; Qi et al,, 2013, Cell 152: 1173-1183; Shen et aL, 2013, Cell Research 23: 720-723; and Wang et al, 2013, Cell 153: 910-918). In particular, Wang et al. 2013, Cell 153: 910-918 describe targeted insertion using, the CRJSPR / Cas9 system combined with oligonucleotides,

[0102] Generation of a genetically modified immunodeficient mouse according to aspects of the present invention may include injection or transfection of appropriate nucleic acids, such as an expression construct encoding ,cas9 and an expression construct encoding a guide RNA specific for the gene to be targeted, for use in CRISPR, into a preimplantation embryo or stem cells, such' as embryonic stem (ES) cells or induced pluripotent stem. (IPS) cells. Optionally, cas9 and the guide RNA are encoding in a single expression construct.

[0103] TAL (transcription actb mor-hke) [ Hectors

[0104] Transcription activator-like (TAL) effectors or TALE (transcription activatorlike effector) are derived from a plant pathogenic bacteria genus, Xanthomonas, and these proteins mimic plant transcriptional activators and manipulate the plant transcript, see Kay et al, 2007, Science, 318:648-651.

[0105] TAL effectors contain a centralized domain of tandem repeats, each repeat containing approximately 34 amino acids, which are key to the: DNA binding specificity of these proteins.. In addition, they contain a nuclear localization, sequence and an acidic transcriptional activation domain, for a review see Schornack et al., 2006, J, Plant Physiol., 163(3): 256-272; Scholze and Boch, 2011, Cun* Opin' Microbiol, 14:47-53, 2024204725   09 Jul 2024

[0106] Specificity of TAL effectors depends on the sequences found in the tandem repeats, The repeated sequence includes approximately 102 bp and the repeats are typically 91-100% homologous with each other (Bonas et al., 1989, Mol Gen Genet 218: 127-136). Polymorphism of the repeats is usually located at positions 12 and 13 and 5 there appears to be a one-to-one correspondence between the identity of the bypervariable diresidues at positions 12 and 13 with the identity of the contiguous nucleotides in the TAL-effector's target sequence, see Moscou and Bogdanove 2009, Science 326: 1501: and Boch et al, 2009. Science 326:1509-1512. The two hypervariable residues are known as repeat variable diresidues (RVDs), whereby one 10 RVD recognizes one nucleotide of DNA sequence and ensures that the DNA binding domain of each TAL-effector can target large recognition sites with high precision (15 -30nt). Experimentally, the code for DNA recognition of these TAL-effectors has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, C, G or T, NN binds to A or G, and IG binds to T. 15 These DNA binding repeats have been assembled into proteins with new combinations and numbers of repeats, to make artificial transcription factors that are able to interact with new sequences and activate tire expression of a reporter gene in plant cells (Boch et al., 2009, Science 326:1509-1512). These DNA binding domains have been shown to have general applicability in the field of targeted genomic editing or targeted gene 20 regulation in all ceil types, see Gaj et al., Trends in Bioteehnol, 2013, 31(7):397-405. Moreover, engineered TAL effectors have been shown to function in association with exogenous functional protein effector domains such as a nuclease, not naturally found in natural Xanthomonas TAL-effect or proteins in mammalian cells. TAL nucleases (TALNs or TALENs) can be constructed by combining TALs with a nuclease, e.g, FokI 25 nuclease domain at the N-terminus or C-terminus, Kim et al. 1996, PNAS 93:1156-1160; Christian et al., 2010, Genetics 186:757-761; Li et al., 2011, Nucleic Acids Res 39: 6315-6325; and Miller et al., 2011, Nat Bioteehnol 29: 143-148. The functionality of TALENs to cause deletions by NHEJ has been shown in rat, mouse, zebrafish, Xenopus, medaka, rat and human cells, Ansai et al„ 2013, Genetics, 193: 739-749; Carlson et al., 30   2012, PNAS, 109: 17382-17387; Hockemeyer et al., 2011, Nature Bioteehnol., 29: 731 734; Lei et al., 2012, PNAS, 109: 17484-17489; Moore et al., 2012, PLoS ONE, 7: e37877; Stroud et al., 2()13, J. Biol. Chern., 288: 1685-1690; Sung et al., 2013, Nature Bioteehnol 31: 23-24; Wefers et al., 2013, PNAS 110: 3782-3787. 2024204725   09 Jul 2024 [0107) For TALEN, methods of making such are further- described in U.S. latent Nos. 8,420,782; 8,450,471; 8,450,107; 8,440,432; 8,440,431 and U.S, Patent Publication Nos. US20130137161 and 14820130137174.

[0108] Other useful endonucleases may include, for example, HhaL Hindlll, Notl, 5 BbvCI, EcoRI, Bg / I, and AlwL The fact that some endonucleases (e.g, FokI) only function as dimers can be capitalized upon to enhance the target specificity of the TAL effector. 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 do the inactive monomers come together to create 10 a. functional, enzyme. By requiring DNA binding to activate the nuclease, a highly sitespecific restriction enzyme can be created.

[0109] In some embodiments, the TALEN may farther include a nuclear localization signal or sequence (NLS). A NTS is an amino acid sequence that facilitates targeting the TALEN nuclease protein into the nucleus to introduce a double stranded break at the 15 target sequence in the chromosome.

[0110] Nuclear localization signals are known in the art, see, for example, Makkerh et rd. 1996, Curr Biol. 6:1025-1027. NLS include the sequence from SV40 Large T-antigen, Kalderon 1984, Cell, 39: 499-509; the NLS from nucleoplasmim described in detail in Dingwall et al, 1988, J Cell Biol., 107, 841-9. Further examples are described 20 in McLane and Corbett. 2009,1UBMB Life, 61, 697-70; Duple et al 2012, PNAS, 109, E544-E552. ]0111] The cleavage domain may be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which a cleavage domain may be derived include, but are not limited to, restriction endonucleases and homing 25 endonucleases. See, for example, 2002-2003 Catalog, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional 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.) Nucleases, Cold Spring: Harbor Laboratory Press, 1993, One or more of these enzymes, or 30 functional fragments thereof, may be used as a source of cleavage domains.

[0112] Fax 1 s tge»-Xkd med Genome I dm ng |0113] The use of zinc finger nucleases (ZEN) for gene editing, such as for targeted insertion via a homology-directed repair process, has been well established. For 2024204725   09 Jul 2024 example, see Carbery et al... 2010, Genetics, 186: 451-459; Cui et aL, 2011, Nature BiotechnoL, 29: 64-68; Hauschild etaL, 2011,PNAS, 108: 12013-12017; Orlando et al., 2010, Nucleic Acids Res., 38:. e!52-e:152; and Porteus & Carroll, 2005, Nature Biotechnology, 23: 967-973, 5

[0114] Components of the ZFN-mediated process include a zinc finger nuclease with a DNA binding domain and a cleavage domain. Such are described for example in Beerli el 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) Cun’ Opin. Biotechnol., 12:632-637; and Choo et al. (2000) Cwt Opin. Struct Biol., 10:41110: 416; and U.S. Pat. Nos. 6,453,242 and 6,534,261. Methods to design and select a zinc finger binding domain to a target sequence are known in the art, see for example Sera, et aL Biochemistry 2002,41,7074-7081; U.S. Pat. Nos. 6,607,882; 6,534,261 and 6,453,242.

[0115] In some embodiments, the zinc finger nuclease may further include a nuclear 15 localization signal or sequence (NLS). A NLS is aa amino acid sequence that facilitates targeting the zinc finger nuclease protein into the nucleus to introduce a double stranded break at the target sequence in the chromosome. Nuclear localization signals are known in the art. See, for example, Makkerh et al. (1996) Current Biology 6:1025-1027 and others described herein. 20

[0116] The cleavage domain may be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which a cleavage domain may be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalog, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes 25 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.) Nucleases, Cold Spring Harbor Laboratory Press, 1993. One or more of these enzymes (or functional fragments thereof) may be used as a source of cleavage domains. A cleavage domain also may be derived, from: an enzyme or portion thereof, as described above, that 30 requires dimerization for cleavage activity.

[0117] Two zinc finger nucleases may be required for cleavage, as each nuclease includes a monomer of the active enzyme dimer. Alternatively, a single zinc finger nuelease may include both monomers to create an active enzyme dimer. Restriction 2024204725   09 Jul 2024 endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and d caving DNA at or near the site of binding. 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 FokI catalyzes double stranded cleavage of .DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example. U.S, Pat. Nos. 5,356,802; 5,436250 and 5,487,994; as well as Li et al. (1992) PNAS 89:4275-4279; Li er al. (1993) PNAS 90:2764-2768; Kim et al. (1994) PNAS 91:883-887; Kim et at (1994) J. Biol. Chem. 269:31, 978-31, 982. Thus, a zinc finger nuclease may include the 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 disclosure of which is incorporated by reference herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and these also are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res, 31: 418-420. An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is FokI. This particular enzyme is active as a dimer (Bitinaite et al. 1998, PNAS 95: 10,570-10,575). Accordingly, for the purposes of th© present disclosure, the portion of the FokI enzyme used in a zinc finger nuclease is considered a cleavage monomer. Thus, for targeted double stranded cleavage using a Fold cleavage domain, two zinc finger nucleases, each: including a FokI cleavage monomer, may be used to reconstitute an active enzyme dimer. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage monomers may also be used. In certain embodiments, the cleavage domain may include 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, each of which is incorporated by reference herein in its entirety. By way of 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 ofFokl are all targets for influencing dimerization of the FokI cleavage half-domains. Exemplary engineered cleavage monomers of FokI that form obligate heterodimers include a pair in which a first cleavage monomer includes mutations at amino acid residue positions 490 and 538 TO rs oc Ch ©e --4 OS g CL, O', ce UJ o sp o kj c* Ch w 4a. n> 09 VO ¢5 C1 <r o S ci o, IK-' S' S' V! UJ ■TO o O> a TO 5' o S w fss 2024204725   09 Jul 2024

[0122] For targeted gene disruption, e,g. mutation, a gene targeting vector is made using recombinant DNA techniques and includes 5' and 3' sequences which are homologous to the stem eel! endogenous target gene. The gene targeting vector optionally and preferably further includes a selectable marker such as neomycin phosphotransferase, hygromycin or puromycin. Those of ordinary skill in the art are capable of selecting: sequences for inclusion in a gene targeting vector and using' these with no more than routine experimentation. Gene targeting vectors can be generated, recombinantly or synthetically using, well-known methodology.

[0123] For methods of DNA injection of a gene targeting vector into a preirn.pl an tati on embryo, the gene targeting vector is linearized before injection into nonhuman preimplantation embryos. Preferably, the gene targeting vector is injected into fertilized oocytes. Fertilized oocytes are collected from superovulated females the day-after mating (0.5 dpc) and injected with the expression construct. The injected oocytes are either cultured overnight or transferred directly into oviducts of 0.5-day p.c. pseudopregnant females. Methods for superovuiaiion, harvesting of oocytes, gene targeting vector injection and embryo transfer .are known in the art and described in Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press; December 15, 2002, ISBN-10: 0879695919. Offspring can be tested for the presence of target gene disruption, e.g. mutation, by DNA analysis, such as PGR, Southern blot or sequencing. Mice having a disrupted, e.g. mutated, target gene can be tested for expression of the target protein such as by using ELISA or Western blot analysis and / or mRNA expression such as by RT-PCR,

[0124] Alternatively the gene targeting vector may be transfected into stem cells (ES cells or IPS cells) using well-known methods, such as electroporation, calciumphosphate precipitation and lipofection.

[0125] .Mouse ES cells are grown in media optimized for the particular line. Typically ES media contains 15% fetal bovine serum (FBS) or synthetic or semi-sypihctic- equivalents, 2 mMglutamine, 1 mM Na Pyruvate, 0.1 mM non-essential amino acids, 50 U / ml penicillin and streptomycin, 0.1 mM 2-mercaptoethanol and 1000 U / ml LIF (plus, for some cell lines chemical inhibitors of differentiation) in Dulbecco's Modified Eagle Media (DMEM). A detailed description is known in the art (Tremml et al, 2008, Current Protocols in Stem Cell Biology, Chapter l:Unit IC A For review of inhibitors of ES cell differentiation, see Buehr, M.,e / al, (2003). Genesis of embryonic 2024204725   09 Jul 2024 io 15 20 25 30 stem: cells. Philosophical Transactions of the Royal Society B: Biological Sciences 358, 1397-1402.

[0126] The cells .are screened for target gene disruption, e.g. mutation, by DNA analysis, such as PCR, Southern blot or sequencing. Cells: with the correct homologous recombination event disrupting the target gene can be tested for expression of the target protein such as by using ELISA or Western blot analysis and / or mRNA expression such as by RT-PCR. If desired, the selectable marker can be removed by treating the stem cells with Cre recombinase. After Cre recombinase- treatment the cells are analyzed for the presen ce of the nucleic acid encoding the target protein.

[0127] Selected stem cells with the correct genomic event disrupting the target gene can be injected into preimplantation embryos. For microinjection, ES or iPS cell are rendered to single cells using a mixture of trypsin and ED TA, followed by resuspension in ES media. Groups of single cells are selected using a finely drawn-out glass needle (20-25 micrometer inside diameter) and introduced through the embryo's zona pellucida and into: the blastocysts cavity (blastocoel) using an inverted microscope fitted with micromampulators. As an alternative to blastocyst injection, stem, cells can be injected into early stage embryos (e.g. 2-cell, 4-cell, 8-cell, premorula or morula). Injection may be assisted with a laser or piezo pulses drilled opening the zona pellucida. Approximately 9-10 selected stem cells (ES or IPS cells) are injected per blastocysts, or 8-eell stage embryo, 6-9 stem cells per 4-cell stage embryo, and about 6 stem cells per 2cell stage embryo. Following stem cell introduction, embryos are allowed to recover for a few hours at 37°C in 5% CO2, 5% O2 in nitrogen or cultured overnight before transfer into pseudopregnant recipient females. In a further alternative to stem cell injection, stem cells can be aggregated with morula stage embryos. All 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, 3rd edition (A. Nagy, M. Gertsenstein, K, Vinterstem R, Behringer, Cold Spring Harbor Laboratory Press; December 15, 2002, ISBN-10: 0879695919, Nagy et at, 1990, Development 110, 815-821; US7576259: Method for making genetic modifications, US7659442, US 7,294,754, Kraus et al. 2010, Genesis 48, 394-399). [012§| Pseudopregnant embryo recipients are prepared using methods known in the art. Briefly, fertile female mice- between 6-8 weeks of age are mated with vasectomized or sterile males to induce a hormonal state conductive to supporting surgically 2024204725   09 Jul 2024 to w s.o Q Ct «5 ¢3 O' w <1 as p p 3' S' o g cL £3 00 S» Cl. 53 co c> S 2024204725   09 Jul 2024 io mouse which has a target gene disruption such that expression of the target gene is absent or reduced to produce offspring which are immunodeficient and have a target gene disruption such that expression of the target protein is: absent or reduced.

[0133] Aspects of the invention provide a genetically modified immunodeficient mouse that includes a target gene disruption in substantially all of their cells, as well as a genetically modified mouse that include a target gene disruption in some, but not all their cells.

[0134] Imm unodefi ciency

[0135] The term ‘‘immunodeficient non-human animal” refers to a non-human animal characterized by one or more of: a lack of functional immune cells, such as T cells and B cells; a DNA repair defect; a defect in the rearrangement of genes encoding antigen-specific receptors on lymphocytes; and a lack of immune functional molecules such as IgM, IgGI, IgG2a, IgG2b, IgG3 and IgA.

[0136] According to aspects of the present invention, a genetically modified immunodeficient non-human animal whose genome includes a genetic modification, wherein the genetic modification renders the non-human animal deficient in MHO I and MHO II activity, provided according to aspects of the present invention is a mouse. While description herein refers primarily to aspects of the present 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 non-human primate.

[0137] The term “immunodeficient mouse” refers to a mouse characterized by one or more of: a lack of functional immune cells, such as T cells and. B cells; a DNA repair defect; a defect in the rearrangement of genes encoding antigen-specific- receptors on lymphocytes; and a lack of immune functional molecules such as IgM, IgGI, IgG2a, IgG2b, IgG3 and IgA. Immunodeficient mice can be characterized by one or more deficiencies in a gene involved in immune function, such as Rag? and Rag2 (Oettinger, M.A et al.f Science, 248:1517-1523, 1990; and Schatz, D. G. etal, Cell, 59:1035-1048,: 1989) Immunodeficient mice may have any of these or other defects which result in abnormal immune function in the mice,

[0138] A particularly useful immunodeficient mouse strain is NOD.Cg- / MricAW commonly referred to as NOD scid gamma (NSG) mice, described in detail in Shultz LD et al.. 2005, J. Immunol, 174:6477-89. NSG is representative of the JO Ui & s o’ 3 O So <t> O o & «s a P e GQ & Q 2024204725   09 Jul 2024 Artemis gene mutations and the lymphocyte phenotype T(-) B(-) NK(+), CD45 gene mutations and the lymphocyte phenotype T(-) B(+) NK(m-

[0141] In further aspects, a genetically modified immunodeficient mouse has a. defect in its endogenous gene encoding DNA-dependent protein kinase, catalytic subunit (Prkdc) which causes the mouse- to express a defective endogenous I)NA~dependent B(A) NK(-); and autosomal recessive SCID characterized by Jaki gene mutations and the lymphocyte phenotype T(-) B(+): NK(-), ADA gene: .mutations- and the lymphocyte phenotype T(-) 80 NK(~), IL-7R. alpha-chain mutations and the lymphocyte phenotype T(-) B(+) NK(+), CD3 delta or epsilon mutations and the lymphocyte phenotype T(-) B(+) NK(+X RAG 1 / RAG2 mutations and the lymphocyte phenotype T(-) B(~): 5' p- “O ft o $ a 3 KJ ■O o -65 $2 13 o 2 B’ «> n vx z» S* Er a S' » ri o modification, wherein the genetic modification renders the immunodeficient mouse deficient in MHC I and MHC II, such that the genetically modified immunodeficient NRG mouse lacks functional MHC I and lacks functional MHC II. g o OS o as 2024204725   09 Jul 2024 o iri ri S3. TO rs 2024204725   09 Jul 2024 - .31 - NSG- / X*             mouse lacks fimctional MHC II due to a homozygous null mutation of H-2A subclass of MHC II (abbreviated as Z4”“'").

[0149] Although bath N^G-^            and            (1A mice lack functional MHC I and MHC II, unexpectedly, human IgG clearance in NSG-f^ D6 / ”51 5          mice differs significantly from that of NSG~B2Afltl‘ (14. lE)^1 mice. While NSG- (X*              mice exhibit a slow human IgG clearance pattern (similar to that observed in NSG mice; note that NSG mice have functional MHC 1 and MHC II), the NSG“B2M"" / (M IE)mlt mice exhibits a rapid IgG clearance (see Figure 2) such that it renders this mouse model not suitable for use in antibody testing, An NSG~(K* D0)^ 10   CM"*")) mouse of the present invention is characterized by clearance of no more than 60%, such as clearance o f no more than 70%, 80%, or 90%, of administered human IgG in a time period of 2 days following administration of the human IgG. About 90% of human IgG was cleared in NSG-fX*            mice after about 2 weeks. The term “clearance” used in reference to human IgG administered to a mouse refers to a process 15 of removal of functional human IgG from the mouse. 10150]

[0151] According to aspects of the present invention, a genetically modified immunodeficient mouse deficient in MHC class I and MHC class II which lacks functional MHC I and lacks functional MHC II is                             H2- 20 Abr!j^v 122-1) / ^^ n2r^m1^ Tg(Ins2-HBEGF)6832Ugfm / Sz (abbreviated as NSG-62^11 (M lE)^'} mouse. The             (lA lE)^1'' mouse lacks functional MHC I due: to a homozygous null mutation of £2 microglobulin (abbreviated B2Af u^ The NSG-&2A / "'6 (lA IEyw,b mouse lacks functional MHC II due to a homozygous null mutation of H-2A and H-2E subclasses of MHC II (abbreviated as (IT IE)™11). 25

[0152] Rapid clearance of human IgG in NSG-BIM”'” (IA mice was observed. About 90% of human IgG was cleared in            (M l^1}- mice after about 2 days, see Fig. 2. [01531    NSG-R1M>-| R / M           mice

[0154] According to aspects of the present invention, a genetically modified 30 immunodeficient mouse deficient in MHC class I and MHC class II which lacks functional MHC I and lacks functional MHC II is a                             H2~ H2~DllmiBpe E2r^iWjf Tg(Ins2-HBEGF)6832Ugfm / Sz transgenic mouse, abbreviated as NSG-RIP-DTR (Kh Dby''in (JA / !U":), which expresses the diphtheria toxin LA LA more types of cells or tissues.

[0158] Allogeneic and / or xenogeneic cells or tissues administered include, but are not limited to, non-human pancreatic cells; non-human pancreatic islets; non-human Ci c: co § o' «5 same type which do not lack functional MHCI and functional MHC IL

[0157] The allogeneic and / or xenogeneic cells or tissues administered to a genetically modified immunodeficient mouse lacking functional MHC I and functional MHC II are not limited with respect to source or type. Administration of the allogeneic and / or xenogeneic cells or tissues to a genetically modified immunodeficient mouse is observed due to reduction or absence of graft versus host disease (GVHD) since the mice lack functional MHC I and functional MHC IL For examples the genetically modified immunodeficient mice lacking functional MHC I and functional MHC II survive longer following: administration of allogeneic and / or xenogeneic cells or tissues to the genetically modified immunodeficient mice than in immunodeficient mice of the g S 3 3 2024204725   09 Jul 2024 B a & G Bx B B’ S’ W cL g te B ft P B ps CT d> « g. a a administered to an imnumodeficient genetically modified mouse lacking functional MHC I and functional MHC IL The human T cells can 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 the mouse, or as a mixed population of cells of which human T cells are a subset. to Ch V B* § g: r; g B: ! B B g S 5 S3 B o o ?p :O 4S B a & o © (10,00()) to about 1 x 10s (100,000,000), about 1 x 104 (10,000) to about 1 x IO7 (10,000,000), about 1 x 10s (100,000) to about 1 x 10' (10,000,000), about 1 x 103 (1,000) to about 1 x 104 (10,000), about 5 x 10J (5,000) to about 5 x 104 (50,000), about 1 X 104 (10,000) to about 1 x 105 (100,000), about 5 x 104 (50,000), to about 5 x 105 (500,000), about 1 x 10° (1,000,000) to about 1 x 10s (100,000,000), about 5 x 106 considered limiting. Thus, the number of administered allogeneic and / or xenogeneic cells is generally in the range of 1 x 10J to 1 x 108 (1,000 to 100,000,000), although more or fewer can be used.

[0167] Thus, a method aecording to aspects of the present invention can include administering about 1 x 10J (1000) to about 1 x 108 (100,000,000), about I x 104 Ct. 3 s X fWTOi sr rJQ 3 B 0 a >v>»< 0 s s 0 G to to< dS CXs n © Ct1 J- o 0 cr 0* C- 0 0 2-. to R "d &> 0; o 0 o Cz k—J 0 to q S” B* GO o 2- G 0L & 0. 3 Q p Cl to >-(- to 2 :B *p Cl 0 ■0 s a fx ET .0 K* Ci-' B cr 0 B o 0 5' to 0 to S' 0 “"4 o to' to' 0 S’ p .0 S" B 2 Q 0 g to to "e to* ,g S' rig 2 & ST 0 o 0,. -¾. io'* :Cj 1 IP 0 S o r 0 0 O S' p 0 ■4*1 ?r S' <—> |T 0 0 o’ to S 05 Cl cr g CL O o ¢5 ©. 0 to 0 2 o' 0’ CT p-. s M S~ to c 0 CS © 0 0 g 5* 2 aS to 0- O f5 R cr 2' §" 2 23. 2< X* 3' B 3 0' 09 d5 & g o’ 2 a ts 3 SB. o cL irwM & 0 3 0 0 0 0 Wy cr S / ■“3 0 §' z*; 0 8' x. s' n' g GC 2 3 o' £3 uq 3 g S d5 S o *** * aa Bl 0 cl B S o 3 0 £ 0 n A" s to 0 S" 0 to o' Sg o’ o' as ©" to 0 0^ s 0 2 dT ’■<! 2 Q to* £X B 3 3 2. 3* to O ™» 0 2- © ■Q o 3 Cl 0 CL to to a o to* 0 CL 0 C. 2 ©■ 0 JW to 0 to 0 2* 2024204725   09 Jul 2024 - 35 - about 5 x 10s (500,000), about 6 x 105 (600,000), about 7 x 105 (700,000), about 8 x 105 (800,000),  about 9 x 10' (900,000), about 1 x 10* (1,000,000), about 2 x 106 (2,000,000), about 3 x 106 (3,000,000), about 4 x 106 (4,000,000), about 5 x 106 (5,000,000), about 6 x 106 (000,000), about 7 x 106 (7,000,000), about 8 x 10s 5   (8,000,000),. about 9 x 10* (9,000,000), about 1 x 107 (10,000,000), about 2 x 107 (2(),00(),000), about 3 x 107 (30,000,000), about 4 x 107 (40,000,000), about 5 x IO7 (50,000,000), about 6 x 10' (60^000,000), about 7 x 107 (70,000,000), about 8 x 10' (80,000,000), about 9 x 10' (90,000,000), or about 1 x 108 (100,000,000), allogeneic and / or xenogeneic cells to the immunodeficient genetically modified mouse. Those of 10 ordinary skill will be able to determine a number of .allogeneic and / or xenogeneic cells to be administered to a specific mouse using no more than routine experimentation.

[0168] Administering allogeneic and / or xenogeneic cells to a mouse can include administering a composition comprising allogeneic and / or xenogeneic cells to the mouse. The composition can further include, for example, water, a tonicity-adjusting agent (&g., 15 a salt such as sodium chloride), a pH buffer (e.g., citrate), and / or a sugar (e.g., glucose),

[0169] Engraftment of allogeneic and / or xenogeneic hematopoietic stem, cells in genetically modified immunodeficient animals is characterized by the presence of differentiated allogeneic and / or xenogeneic cells, such as hematopoietic cells in the genetically modified immunodeficient mice of the present invention. Engraftment of 20 allogeneic and / or xenogeneic cells can be assessed by any of various methods, such as, but not limited to, flow cytometric analysis of cells: in the animals to which the allogeneic and / or xenogeneic are administered at one or more time points following the administration of the cells.

[0170] Tumor Xenograft 25

[0171] Various aspects of the invention relate to administering xenogeneic tumor cells to a genetically modified immunodeficient mouse of the present invention.

[0172] Xenogeneic tumor cells administered to a genetically modified immunodeficient, mouse of the present invention can be any of various tumor cells, including but not limited to, cells of a tumor cell line and primary tumor cells. The 30 xenogeneic tumor cells may be derived from any of various organisms, preferably mammalian, including human, non-human primate, rat, guinea pig, rabbit, cat, dog, horse, cow, goat, pig. and sheep. S3 w CT 2 Q Ci ct Ci 5' ct 2. & to Ws CT CT caused by cancer include, but are not limited to, lymphoma, leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma: of the lung, squamous carcinoma of the lung, cancer of the peritoneum, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, brain cancer, breast cancer, triple negative breast cancer, central or peripheral nervous system cancers, cervical cancer, colon, cancer, g T SI 03 x' 6c CT 2024204725   09 Jul 2024 tji © <8 OS CT vi. g CT «5 b' Ui © S’ s» CT I* P CT 2024204725   09 Jul 2024 © sg Os o g. to is & z SB, g OS X 00 a Of o s &> <3" o C! 8 JX tumor cell can expand into a detectable tumor in the genetically modified immunodeficient animals described herein. The number of administered tumor cells is generally in the range of 10J (1,000) - 1x10* (100,000,000), tumor cells, although more or fewer can be administered. s» B s i-i n 2024204725   09 Jul 2024 the same time or at different times. S ¢3 o o £ 2 S <o w £ a & o 3 § £ J*r c n> rt> p g cc s rs n* <T 3 1 $£ 103 (8,000), about 9 x 103 (9,000), about 1 x 10-(10,()00), about 2 x 104(20,000), about 3 x 1()4 (30,000), about 4 x 104 (40,000), about 5 x 104 (50,000), about 6 X 104 (60,000), about 7 x 104 (70,000), about 8 x 104 (80,000), about 9 x 104 (90,000), about 1 x 105 (10(),()00), about 2 x 105 (200,000), about 3 x 105 (300,000), about 4 x 10s (400,000), about 5 x 10 s (500,000), about. 6 x 105 (600,000), about 7 x 105 (700,000), about 8 x 10’ cells, to the immunodeficient QUAD mouse. The method can include administeriag about I x 10^ (100), about 2 x 102 (200), about 3 x 102 (300), about 4 X 102 (400), about 5 x 102 (500), about 6 x W2 (600), about 7 x 102 (700), about 8 x 102 (800), about 9 x 102 (900), about 1 x 103(l,000), about 2 x 103 (2,000), about 3 x 107(3,000), about 4 x 10’ (4,000), about 5 x 10^(5,000), about 6 x 10’ (6,000), about. 7 x 10^(7,000), about 8 x 5® o o o CT VI o g GO O X X LA N? Ui oc M g EL o’ w ¢6

[0187] According to aspects of the present invention, the tumor cells are derived from the same species as the administered PBMC. According to aspects, both the tumor cells and the PBMC administered to a genetically modified immunodeficient mouse of the present invention are human ceils. [ 01881     Cond i t i on i n a g EL o the present invention are human cells.

[0186] According to aspects of the present invention, xenogeneic tumor cells and xenogeneic PBMC are administered to a genetically modified immunodeficient mouse. The xenogeneic tumor cells and xenogeneic PBMC can be administered at the same time or at different times. n p vT er o> o cr o Er 2024204725   09 Jul 2024 - 40 - 15 2:0

[0190] Methods are provided according to aspects of the present invention which include administration of xenogeneic immune cells, such as, but not limited to, leukocytes, T cells, PBMC or other cells, to an immunodeficient genetically modified mouse without “conditioning” the immunodeficient genetically modified mouse prior to administration of the xenogeneic immune cells, such as. but not limited to, leukocytes, T cells, PBMC, or other cells. Methods are provided according to aspects of the present invention which include administration of xenogeneic immune cells, such as, but not limited to, leukocytes, T cells, PBMC, or other cells, to an immunodeficient genetically modified mouse without “conditioning” by radiation or radiomimetic drags of the immunodeficient genetically modified mouse prior to administration of the xenogeneic xenogeneic immune cells.

[0191] Assays [01921 Methods of assaying an effect of a putative therapeutic agent are provided according to aspects of the present invention which include administering an amount of the putative therapeutic agent to a genetically modified immunodeficient mouse including allogeneic and / or xenogeneic cells or tissues; and measuring the effect of the putative therapeutic agent.

[0193] A putative therapeutic agent used in a method of the present invention can be any chemical entity, illustratively including a synthetic or naturally occurring compound or a combination of a synthetic or naturally occurring compound, a small organic or inorganic molecule, a protein, a peptide, a nucleic acid, a carbohydrate, an oligosaccharide, a lipid or a combination of any of these.

[0194] Standards suitable for assays are well-known in the art and the standard used can be any appropriate standard.

[0195] Assay results can be analyzed using statistical analysis by any of various: methods, exemplified by parametric or mtn-parametric tests, analysis of variance, analysis of covariance, logistic regression for multivariate analysis, Fisher's exact test, the chi-square test, Student’s T-test, the Mann-Whitney test, Wilcoxon signed ranks test, McNemar test, Friedman test and Page’s L trend test. 'These and other statistical tests are well-known in the art as detailed in Hicks, CM, Research Methods for Clinical Therapists: Applied Project Design and Analysis, Churchill Livingstone (publisher);: 5 th Ed,, 2009; and Freund, RJ el al, Statistical Methods, Academic Press; 3rd Ed,, 2010. »3 e> E3 2024204725   09 Jul 2024 modified immunodeficient mouse, wherein the xenogeneic tumor cells form a solid or iron-solid tumor In the genetically modified immunodeficient mouse; administering a test substance to the genetically modified immunodeficient mouse; assaying a response of the xenogeneic tumor .and / or tumor cells to the test substance, wherein an inhibitory effect of the test substance on the tumor and / or tumor cells identifies the test substance- as having anti-tumor activity. (0198] Methods for identifying anti-tumor activity of a test substance according to aspects of the present invention include providing a genetically modified immunodeficient mouse, wherein the genetically modified immunodeficient mouse has engrafted xenogeneic PMBG; administering xenogeneic tumor cells to the genetically E s* 'jy s s o a o' g S3 2024204725   09 Jul 2024 OS W OS o’ c SS B. o well-known in the art and the standard used can be any appropriate standard. In one example, a standard is: a compound known to have an anti-tumor effect. In a further example, non-treatnient of a comparable xenogeneic tumor provides a base level indication of the tumor growth without treatment for comparison of the effect of a test substance. A standard may be a reference level of expected tumor growth previously B 5 o B" o S ts g cL 2024204725   09 Jul 2024 ©' co S>’ B. & ft B' (K g » & p ■P <ns Ka ft O & ¢3 OS « *3 © dS R‘ £L’ O g r& B o (X S3 2024204725   09 Jul 2024 i, 44 - 10 15 20 25 recombinant interleukin II or rIL2), interferon alfa-2a, interferon alfa-2b, -interferon aifa-n.I, interferon alfa-n.3, interferon beta-la, interferon gamma-lb, iproplatin, irinotecan, lanreoti.de, letrozole> leuprolide, liarozole, lometrexol, lomustine, losoxantrone, masoprocol, maytansine, mechlorethamine hydrochiride, megestrol, melengestrol acetate, melphalan, menogaril, merc-aptopurine, methotrexate, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitoginin, mitomycin, mitomycin, mitosper, mitotane, mitoxarstrone, mycophenolic acid, pelarabine, nocodazole, nogalamycin, ormnaplatin, oxisuran, paclitaxel, pega&pargase, peliomycin, pentamustine, peplomycin, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plieamycin, ploiiiestane, porfimer, por&omycin, prednimustine, procarbazine, -puromydn, pyTazoforin, riboprine, rogletimide, safingol, semustine, simtrazene, sparfosate, sparsomycin, spirogermamum, spiromustine, spiroplatin, streptomgrin, streptozocin, sulofenur, talisomycin, tamoxifen, tecogalan, tegafur, teloxantrone, temoporfm, teniposide, teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, topotecan, toremifene, trestolone, tritiribine, trimetrexate, triptorelin, tubulozole, uracil mustard, uredepa, vapreotide, veninrafenib, verteporfin, vinblastine, vincristine sulfate, vindesine, vinepidine, vinglycinate, vinleurosine, vinorelbine, vinrosidine, vinzolidine, vorozoie, zeniplatin, zinostatin, zoledronate, zorubicin, and the like, {0209] According to aspects of the present invention,: an anti-cancer agent is an anti-cancer immuriotherapeutic agent, also called an anti-cancer antibody. An anti-caneer immunotherapeutic agent used can be any antibody, or effective portion of an antibody, effective to inhibit at least one type of tumor, particularly a human tumor. Anti-cancer immunotherapeutic agents include, but are not limited to, 3F8, 8H9, abagovomab, abituzumab, adalimumab, .adecatumumab, aducanumab, afutuzumab, alacizumab pegol, alemtuzmnab, amatuximab, anatumomab mafenatox, anetumab ravtansine, apoLizumab, arcitumomab, ascrinvacumab, atezolizumab, bavituximab, beliniumab, bevaeizumab, bivatuzumab mertansme, brentuximab vedotin, brontictuzumab, cantuzumab mertansine, eantuzumab ravtansine, capromab pendetide, catumaxomab, cetuximab, citatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan,, coltuximab ravtansine, conaiumumab, daeetuzumab, dalotuzumab, demeizumab, deuintuzumab mafodotin, depatuxizumab mafodotin, durvalumab, dusighnmab, edrecoiomab, elotuzumab, emactuzumab, emibetuzumab, enoblituznmab, enfortumab vedotin, enavatuzumab, epratuzumab, 2024204725 09 Jul 2024 g EL Ci- iff Ci ks> o O g § a •s G $ <0 fa' o S Ef* 8 ST &. 6 pankomab, parsatuzumab, patritumab, pembrolizamab, pemtumamab, pectozumab, pidilizumab, pmatuzumab vedotin, polatuzumab vedotin, pritiimumab, racotumomab, radretumab, ramucirumab, rilotomumab, rituximab, robatumumab, sacituzumab govitecan, samalizumab, seribantumab, sibrotuzumab, -siltuximab,. soiituzumab vedotin, taeatuzamab tetraxetan, tarextumab, tenatumomab, teprotumumab, tetuiomab. - 45 - 2024204725   09 Jul 2024 s © © g s' B B n S G C TS « £2L w X 2 M O O B

[0217] All mice used in these studies were raised in breeding colonies at the Jackson Laboratory.                               (NOD-scid IL2rdni‘l, NSG) mice have been described previously in Shultz LD, et al., 2005, J Immunol 174:6477-6489. 20

[0218] NSG mice were maintained through sib matings. NOD.Cg-Prfefc*^ H2- mddd!,!ls^         (abbreviated as NS'G^ Won(Zrt Us

[0214] Embodiments of inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.

[0215] Examples

[0216] Mice ’d 2 § B’ 46- 3 A e. GO 00 Ch 2g ¢, g 8' i-J GO g & g 8 o R to Ch 4-©0 xO ft cages, given autoclaved food and maintained on acidified autoclaved water at The Jackson Laboratory or alternated weekly between acidified autoclaved water and sulfamethoxazole-trimethoprim medicated water (Goldlme Laboratories, Ft. Lauderdale, FL) at the University of Massachusetts Medical School. (0222] Antibodies and Flow Cytometry & Q oJ !Z2 US                M O                 Ql treated with BD FACS lysing solution for whole blood. At least 50,000 events were acquired on LSRII or FACSCalibur instruments (BD Biosciences); For human cell phenotyping, mouse cells were identified and excluded from analysis by staining with a mAh specific for murine CD45 (clone 30-FH, BD Biosciences). Data analysis was performed with FlowJo (Tree Star, Inc,, Ashland, OR) software. whole blood were washed with FACS buffer (PBS supplemented with 2% fetal bovine serum (FBS) and ().02% sodium azide) and then pre-ineubated with rat anti-mouse FcRllb mAh (clone 2.4G2, BD Biosciences) to block binding to mouse Fc receptors. 15 Specific: mAbs were then added to the samples and incubated for 30 min at 4°C. Stained samples were washed and fixed with 2% parafoimaldehyde for cell suspensions or ©                 co 48 -

[0232] dsAAV Vectors

[0233] The- dsAAV vectors were engineered ami packaged as previously described He Y,.stal., 2013, Huw. Gene Then, 24:545-553). 15

[0234] Briefly, full-length cDNA encoding human IL2 or EGFP was subeloned into a dsAAV plasmid (McCarty DM, et al, 2001, Gene Ther 8:1248-1254) containing the o -6F 2024204725   09 Jul 2024 -50- The absence of MHC class I and class II in both strains was confirmed by flow cytometry (Figure 1). Due to absence of immune cells that express readily detectable levels of mouse MHC II, spleens were enzymatically disaggregated and gated to analyze the dendritic cell population. Figure 1A demonstrates the gating strategy of excluding doublets and dead cells and proceeds to gate on monocyte derived dendritic cells (GDI lb+ Ly6cdim CDnc+). The NSG mouse demonstrates the expected staining pattern of H2Kd positive. H2K° negative for MHC class I (Figure IB), and I-Ag7 positive, I-A9 negative for MHC class II (Figure IC). The NSG-(^          and NSG-W*^ IE™") knockout mice both lack MHC class I and II molecules, normally expressed by NOD and C57BL / 6 mice,

[0240] Due to the requirement of B2M for appropriate expression of murine FcRm the receptor responsible for prolonging the half-life- of IgG in the circulation, the clearance of human. IgG in both stocks of mice was compared. Mice were injected IV with 200 pg of human IgG and bled at intervals for ELISA analysis of circulating human IgG. The first bleed at 2 minutes post-injection was considered as 100% serum IgG. Rapid clearance of human IgG in NSG-^A / 8^ (IA IE™'11) mice was observed whereas IgG clearance in NSG-(K&            mice was similar to that observed in NSG mice (Figure 2).

[0241] Survival of PBMC-eugrafted NSG and NSG-MHC class I kuockout, NSG-MHC class II knockout, and NSG-MHC IZH knockout mice

[0242] NSGteKl^         In determine whether the absence of mouse MHC class I and II altered the incidence and kinetics of xenogeneic GVHD following human PBMC engraftment in NSG MHC I / II knockout mice, NSG strains deficient in MHC class I, MHC class. II or the two NSG double .knockout strains: were- engrafted with 10 x 10'"' PBMC and their survival was compared to that of NSG mice. As previously reported, NSG and NSG-(ZXW? / ) showed relatively similar short survival, similar to that observed in NSG mice. Tn. contrast, as expected, NSG-fX* mice had an extended period of survival as compared to NSG mice. However, when both MHC class I and class II were knocked-out in NSG-(K" j / / - mice, survival was past 100 days, and 13 of 15 of these MHC I / II knockout mice still alive at the end of the observation period (125 days) with no symptoms of GVHD, (Figure 3AJ.

[0243] XSGriW" ( / J     >: S:milar extended survival results were observed in PBMC-engrafted             (M IE1™11) mice. For this MHC I / II knockout strain, the 2024204725   09 Jul 2024 knockout and MHC class II knockout mice. o s di •—2 i j tA 5 era' s <? n g' if sr £6 n o I™4 a 5' c

[0251] Phenotypic analysis of human T cells, engrafted in NSG, NSG-^M*^ NSG-^D^, and NSG«*D4 / “" (£4^) mice injected with PBMC

[0252] The CD4:CD8 ratio in NSG mice at 4 weeks post PfiMC-eftgraftment was approximately 4:1 .(Figure 6A), In contrast, very few CD4+ T cells engrafted in NSG-mice, while high levels of CD4+ T cells engrafted in NSG-fK* mice. !Zi o «5 'O' Q § % m. £ is s» OS fc S3 Ct S3 s. o Q Er ft Ci % Ci ft o V, fti S' Ci s <6 ft S' ?r o ft g n ft G® > o Ws S' S' © \ J s 01 1 o + |0253] Phenotypic analysis &f human T cells engrafting in 1NSG, NSG-(£4 / E) , NSG-J5>r^ NSG-jB^^ (lA lET11, .and NSG-jmr*® (IA. lE)^ mice injected with PBMC received human PBMCs; the one mouse that did not revert to hyperglycemia had levels of C~peptide similar to that observed in islet recipients that were not transplanted with .allogeneic PBMCs (Figure 8E). However, in all 4 mice that were given the allogeneic PBMCs, the quantity of human insulin observed in the islet grafts was significantly lower as compared islet transplant recipients that were not given human PBMCs. (Figure S’ s o o S g SL S' CO c tZJ n 2 <? pg - 2024204725   09 Jul 2024 MS I > r * i'-j jg, 8 i ft s B o' G » b> H~ V? 3 t increase in the proportion of CD4+ cells that expressed a I" regulatory (Treg) phenotype (CD44'CD25'«D127-FOXP3'+) at 2, 4 and 6 weeks in NSG mice and at 2 and 4 weeks in NSG-tX^              mice post PBMC injection (Figure 9Cj. Representative staining of CD4+ T cells with antibodies to CD25 and CD 127 is shown in the upper row and the expression of F0XP3 in the putative CD4+CD25+CD127- T cells in NSG and Sr engraftment of human fetal liver and thymus, Le.. the BLT model. Injection of AAV~ huIL2 led to a transient expansion of human CD45+ cells in the blood of NSG and NSG-(Kbmice that had been engrafted with lOxlO” PBMC for 2 weeks (Figure 9A). AA.V-IL2 did not alter the proportion of human CD45+ cells that were CD3+ over the 8 week course of the experiment (Figure 9B). However, there was a significant 2024204725   09 Jul 2024 IgG clearance was comparable to observed in NSG mice whereas IgG clearance in the NSG-821^4 (IA      strain was extremely rapid. 'Si g R S g 2024204725   09 Jul 2024

[0268] Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.

[0269] The compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.

[0270] Other embodiments of the invention as described herein are defined in the following paragraphs: 1.        A NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NOD-scid-IL2rYnull, NSG) mouse genetically modified such that the 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 paragraph 1, wherein the mouse is a NOD.Cg-Prkdcscid H2-K1tm1Bpe H2-Ab1em1Mvw H2-D1tm1Bpe Il2rgtm1Wjl / SzJ (NSG-(Kb Db)null (IAnull)) mouse. 3.        The genetically modified NSG mouse of paragraph 1, wherein the mouse is a NSG- RIP-DTR (Kb Db)null (IAnull) mouse. 4.        The genetically modified NSG mouse of paragraph 1, wherein the mouse is a NOD.Cg-B2mtm1Unc Prkdcscid H2dlAb1-Ea Il2rgtm1Wjl / SzJ (NSG-B2Mnull (IA IEnull)) mouse. 5.         The genetically modified NSG mouse of any of paragraphs 1, 2, 3 or 4, further comprising human immune cells. 6.        The genetically modified NSG mouse of paragraph 5, wherein the human immune cells are human peripheral blood mononuclear cells. 7.        The genetically modified NSG mouse of paragraph 5, wherein the human immune cells are human T cells. 2024204725   09 Jul 2024 8.        The genetically modified NSG mouse of any of paragraphs 1 to 7, further comprising human tumor cells. 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 a NOD / Shi-scid-IL2rYnull mouse characterized by 02m (component of MHC I) knockout and IA0 (light chain of MHC II) knockout. 10. The genetically modified immunodeficient mouse of paragraph 9, further comprising human immune cells. 11. The genetically modified immunodeficient mouse of paragraph 10, wherein the human immune cells are human peripheral blood mononuclear cells. 12. The genetically modified immunodeficient mouse of paragraph 10, wherein the human immune cells are human T cells. 13. The genetically modified NSG mouse of any of paragraphs 9 to 12, further comprising human tumor cells. 14. A method for modeling an effect of a human immune system in a genetically modified immunodeficient mouse, comprising: administering a test substance to the genetically modified immunodeficient mouse according to any of paragraphs 5 to 8 or 10 to 13; and assaying the effect of the human immune system in the genetically modified immunodeficient mouse. 15.       The method of paragraph 14 wherein the test substance is an anti-cancer agent. 16.       The method of paragraph 14 or 15, wherein the test substance is an immunotherapeutic agent. 2024204725   09 Jul 2024 17. The method of paragraph 14, 15, or 16 wherein the test substance is an anti-cancer immunotherapeutic agent. 18. The method of any one of paragraphs 14 to 17, wherein the test substance is an immune checkpoint inhibitor. 19. The method of paragraph 18, wherein the immune checkpoint inhibitor is a PD-1 inhibitor, PD-L1 inhibitor, or CTLA-4 inhibitor. 20. The method of paragraph 18 or 19, wherein the immune checkpoint inhibitor is atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, or pembrolizumab, or the immune checkpoint inhibitor comprises an antigen-binding fragment of any one of the foregoing. 21.       A genetically modified mouse substantially as described or shown herein. 22.       A method of use of a genetically modified mouse substantially as described or shown herein. Still further embodiments are in the scope of the following claims.

Claims

1. A method, comprising:engrafting a humanized immunodeficient non-obese diabetic (NOD) mouse with5 xenogeneic cells, wherein the genome of the humanized immunodeficient mouse comprises (i) a mutation in an IL2rg gene, (ii) a scid mutation in a Prkdc gene, and (iii) a mutation in each of a H2-K1 gene, a H2-Ab1 gene, and a H2-D1 gene;administering a therapeutic agent to the immunodeficient mouse; andassaying for an effect of the therapeutic agent on the xenogeneic cells and / or the10 immunodeficient mouse,wherein the immunodeficient mouse has an IL2RG deficiency, a severe combined immune deficiency, a major histocompatibility complex (MHC) class I deficiency, and an MHC class II deficiency.15   2.     The method of claim 1, wherein the humanized NOD immunodeficient mouse isengrafted with human stem cells and / or human peripheral blood mononuclear cells.

3. The method of claim 1 or 2, wherein the humanized NOD immunodeficient mouse isengrafted with human leukocytes, red blood cells, lymphocytes, monocytes, neutrophils,20 eosinophils, basophils, platelets, and / or NK cells.

4. The method of any one of claims 1-3, wherein the humanized NOD immunodeficientmouse is engrafted with human hepatocytes or human pancreatic beta cells.25   5.     The method of any one of claims 1-4, wherein the xenogeneic cells comprise humantumor cells.

6. The method of any one of the preceding claims wherein the therapeutic agent is animmunotherapeutic agent.

307. The method of claim 6, wherein the immunotherapeutic agent is an antibody or antibodyfragment.2024204725   23 Jun 20268.     The method of claim 6 or 7, wherein the immunotherapeutic agent is an immunecheckpoint inhibitor.

9. The method of any one of claims 1-8, wherein the therapeutic agent specifically binds a5 protein that is expressed by leukocytes.

10. The method of any one of claims 1-8, wherein the therapeutic agent specifically binds a protein that is expressed by cancer cells.10   11. The method of any one of the preceding claims, wherein the therapeutic agent isadministered to the immunodeficient mouse intravenously, subcutaneously, or intraperitoneally.

12. The method of any one of the preceding claims, wherein assaying for an effect of the therapeutic agent on the xenogeneic cells and / or the immunodeficient mouse comprises15 assaying for an anti-cancer effect of the therapeutic agent on the xenogeneic cells and / or the immunodeficient mouse.

13. A selective breeding method, comprising breeding a first parental strain of mouse with a second parental strain of mouse to produce offspring, wherein the first parental strain of mouse20 is a non-obese diabetic (NOD) mouse, and the genome of the NOD mouse comprises (i) a mutation in an IL2rg gene, (ii) a scid mutation in a Prkdc gene, and (iii) a mutation in each of a H2-K1 gene, a H2-Ab1 gene, and a H2-D1 gene.

14. A genetic modification method, comprising:25          introducing, into a non-obese diabetic (NOD) mouse preimplantation embryo orfertilized oocyte, RNA molecules encoding a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas and at least one oligonucleotide targeting a predetermined target site, wherein the genome of the NOD mouse preimplantation embryo or fertilized oocyte comprises (i) a mutation in an IL2rg gene, (ii) a scid mutation in a Prkdc gene, and (iii) a30 mutation in each of a H2-K1 gene, a H2-Ab1 gene, and a H2-D1 gene;selecting the NOD mouse preimplantation embryo or fertilized oocyte with a desired genetic modification; andtransferring the NOD mouse preimplantation embryo or fertilized oocyte with a desired genetic modification to a pseudopregnant female.2024204725   23 Jun 202615. A genetic modification method, comprising:introducing, into a non-obese diabetic (NOD) mouse stem cell, RNA molecules encoding a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas and at5 least one oligonucleotide targeting a predetermined target site, wherein the genome of the NOD mouse stem cell comprises (i) a mutation in an IL2rg gene, (ii) a scid mutation in a Prkdc gene, and (iii) a mutation in each of a H2-K1 gene, a H2-Ab1 gene, and a H2-D1 gene;selecting the NOD mouse stem cell with a desired genetic modification;transferring the NOD mouse stem cell with a desired genetic modification to a mouse10 preimplantation embryo or fertilized oocyte; andtransferring the mouse preimplantation embryo or fertilized oocyte to a pseudopregnant female.

16. An immunodeficient non-obese diabetic (NOD) mouse whose genome comprises (i) a15 mutation in an IL2rg gene, (ii) a scid mutation in a Prkdc gene, and (iii) a mutation in each of a H2-K1 gene, a H2-Ab1 gene, and a H2-D1 gene, wherein the immunodeficient mouse has an IL2RG deficiency, a severe combined immune deficiency, a major histocompatibility complex (MHC) class I deficiency, and an MHC class II deficiency.20   17. The immunodeficient mouse of claim 16, wherein the mouse is humanized.

18. The immunodeficient mouse of claim 16 or 17 engrafted with xenogeneic cells.

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