异种移植模型|BioMice百奥动物

The Immune-deficient B-NDG mouse model (NOD.CB17-Prkdc^scidIl2rg^tm1/Bcgen) was independently designed and generated by Biocytogen. B-NDG mice are generated by deleting the IL2rg gene from NOD-scid mice with severe immunodeficiency phenotype. Lacking mature T cells, B cells or functional NK cells, and displaying cytokine signaling deficiencies , this mouse model has the highest degree of immunodeficiency and thus is most suitable for engraft and growth of human hematopoietic stem cells (HSCs), peripheral blood mononuclear cells (PBMCs) and human tumor cells or tissues.

• NOD-scid (non-obese diabetes, severe combined immunodeficiency) genetic background: mice of NOD genetic background and with Prkdc (protein kinase DNA-activated catalytic) knockout. Functional T cells, B cells and complement system in these mice are lost, and the activity of NK cells is greatly weakened.

• IL2rg null: the gamma chain of Interleukin-2 receptor (IL-2R γc, also called CD132) is on the mouse X chromosome, and is the common receptor subunit of cytokines IL2, IL-4, IL-7, IL-9, IL-15 and IL-21 with important immune functions. After IL2r is knocked out, mouse immunity function is greatly weakened, activities of NK cells, which are almost completely lost.

• Prkdc null (DNAPK, scid）: Prkdc (protein kinase DNA-activated catalytic) null mutation is characterized by significantly deficient of functional T cells and B cells, and an absence of lymphocytes, recapitulating severe combined immunodeficiency (scid) in human patients.

Advantages of B-NDG Mice

• Model currently holds the highest degree of immunodeficiency among all immunodeficiency models

• Longer lifespan than NOD-scid mice; 1.5 years on average

• Minimal to absent rejection of human-derived cells and tissues

• More efficient for CDX and PDX model generation

• No B lymphocyte leakage

Major Applications

• Human-derived cell or tissue engraftment
• Tumor and tumor stem cell research
• ES and iPS cell research
• Hematopoiesis and immunology studies
• Human infectious disease studies
• Development of humanized models

Application for B-NDG Trademark Registration

Beijing Biocytogen Co., Ltd. has applied for the B-NDG trademark registration for its use in different classes of goods/services (Class 5, Class 31, Class 42 and Class 44). Each letter in the name stands for: B-Biocytogen; N-NOD background; D-DNAPK (Prkdc) null; G-IL2rg knockout.

Detailed information of each class (until the date of application):

Class 5：medicines for human purposes; leeches for medical purposes; diagnostic biomarker reagents for medical purposes; preparations of microorganisms for medical or veterinary use; dietetic foods adapted for medical purposes；reagents for veterinary purposes；insecticides；wadding for medical purposes; stem cells for veterinary purposes

Class 31：live animals; poultry, live; fish, live; crustaceans, live; trees; grains [cereals]; plants; seeds for planting / plant seeds; hay; animal foodstuffs

Class 42： quality control; chemical research; biological research; clinical trials; material testing

Class 44：pharmacy advice; hospital services; artificial insemination services; diet and nutrition guidance; animal breeding; veterinary assistance; artificial insemination services (for animals); in vitro fertilization services/in vitro fertilization services (for animals); rental of sanitation facilities; in vitro fertilization services/in vitro fertilization services

Animal breeding and maintenance

Health status of housing

B-NDG mice are housed in isolators instead of IVCs in our facility. Based on our experience, the mice can live up to 2 months in SPF standard IVCs. This time frame matches the requirements of most experiments performed with B-NDG mice. To improve facility standards, strict sanitation procedures are recommended: cages and bedding need to be sterilized by autoclaving or Co60 irradiation before use, and cages need to be changed in laminar flow hoods weekly. Keeping a clean, high standard housing environment helps to improve the life span of B-NDG mice.

Animal husbandry

Food

5CJL from Labdiet (USA) is recommended to use for breeding B-NDG mice (19.3% protein, 6.2% fat, 20 ppm Vitamin K). Co60 radiation is recommended to sterilizethe food before use.

Water

B-NDG mice are housed in pathogen-free isolators in our facility. Autoclaved purified water is used.

For SPF standard facilities, we recommend following the Jackson Lab standard for water supply: acidified water (adjust pH to 2.5-3.0 using HCl), autoclaved to prevent Pseudomonas and Staphylococcus aureus infection. Autoclaved purified water can also be used with more frequent water changes. Bottle must be changed every 3 days regardless if there is still water left in the bottle.

Bedding

Shavings are the recommended bedding material for B-NDG mice. The bedding material needs to be sterile, soft, dust-free, odor-free and have high moisture absorbance. Sterilizing by autoclaving or irradiation is required before use.

Bedding needs to be changed weekly in laminar flow hoods if the mice are not housed in isolators. Mice need to be transferred into new cages with fresh bedding using sterile tweezers or forceps.

Housing environment

Enough light time and appropriate light intensity are necessary for breeding. We use a standard light cycle, which is 12-hours of light followed by 12-hours of dark.

Housing temperature is strictly 20-26℃. The temperature difference between day and night should not be more than 4℃.

Cages need to be made from non-toxic material and must be easy to clean and disinfect. Thorough cleaning and disinfection is required every week at least.

Animal breeding and maintenance

Transportation

Biocytogen’s B-NDG mouse can be shipped using land and/or air. Although the courier is notified to handle the crate with care, stress response of mice during shipment is still inevitable. Although enough supply of water jelly and food will be provided in cages, increased metabolism and fecal excretion caused by the stress may result in dehydration and loss of body weight. General percentage weight loss due to shipment is ~10%. The percentage can be as high as 15% if the shipment procedure is longer and the cage is populated. Usually, the most of the lost body weight is regained (although cannot reach 100%) after 5-7 days of adaptive feeding (Labdiet food is recommended)).

Adaptive feeding

Importance of adaptive feeding

Before performing experiments, at least 5-7 days of feeding in the receiving facility are required so that the animals can adapt to their new environment, and the stress response caused by transportation can be eliminated or alleviated.

Brief procedure description of adaptive feeding

Perform animal husbandry following 1.10.1.2. Monitor the health status of animals by observing their appearance (e.g. hair), feces and activity. Separate the animals from other animals in the facility as the sound and smell (e.g. Ammonia smelling feces) from other animals may be stimuli. Adaptive feeding is a critical prerequisite for successful experiments.

Generation of gene editing mouse model

Targeting strategy

Phenotypic analysis

Body weight growth

Figure 1. Body weight growth curve of B-NDG mice after birth

Newborn pups (50 males and 50 females, respectively) were obtained at weaning (Week 3; birthdate +/- 3 days). Body weight was measured once every week (on the same day each week) for 8 weeks.

Serum Antibody Response

Figure 2. IgG, IgM and IgG subclasses response in the sera of BALB/c, B-NDG and Blank

(A) Value of OD450 in the sample from BALB/c mice is significantly higher than that from Blank (PBS) and B-NDG mice (~0.04), indicating little or no IgG or IgM in the sera of B-NDG mice.(B) Value of OD450 in the sample from BSA is ~ 0.06 (baseline is 0.1), indicating there is no cross-reaction among antibody capture, linking of enzyme to the antibody and BSA. Compared with the value from BALB/c mice, the value from B-NDG mice is below the baseline. This result suggests that there is no IgG subclasses in the sera of B-NDG mice, confirming it is an ideal mouse model with severe immunodeficiency.

Flow-cytometric Analysis Using Specific Markers for T, B and NK Cells

Figure 3. Analysis of spleen leukocyte subpopulations by FACS

Splenocytes were isolated from female BALB/c, NOD-scid and B-NDG mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for mCD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells was detectable in BALB/c mice, NK cells was detectable in NOD-scid, but not B-NDG mice.

Histology of spleen from B-NDG mice

Figure 4. Histological sections of spleen from 9-week-old C57BL/6, NOD-scid and B-NDG mice.

Spleen from C57BL/6 mouse has normal structure with well-defined follicles. Spleen from NOD-scid mouse shows hypoplasia of white pulp. Spleen from B-NDG mouse shows complete loss of follicular structure.

Immunohistochemistry of spleen from B-NDG mice

Figure 5. Histological sections of spleen from 9-week-old C57BL/6, NOD-scid, and B-NDG mice (n=3).

Spleen from C57BL/6 mouse has normal CD3ε and CD19 expression (brown), No CD3ε and CD19 expression in mice B-NDG mice.

Hematology test results

Figure 6. Complete blood count (CBC) test results for B-NDG mice Blood from B-NDG mice (n=6, 6 week-old) was collected and analyzed for CBC. Values are expressed as mean ± SEM.

Biochemical test results for Blood

Figure 7. Blood biochemical test results of B-NDG mice(n=6).

Serum from the B-NDG mice (n=6, 6 week-old) was collected and analyzed for levels of ALT, AST, CHOL, CR, GLU, TRIG and UREA. Values are expressed as mean ± SEM.

Instrument：Thermo Fisher scientific # Indiko

CDX tumor models

CDX lymph cancer metastasis model in B-NDG mouse

Figure 8. Raji B cells (5X10⁶) were injected in to each B-NDG, NOD-scid and BALB/C Nude mice.

We recorded and analyzed the following parameters in the mice at different time points: (A) Mouse survival after cell inoculation, constructed Kaplan-Merier survival curves. (B) Body weight (g) change each week after inoculation, calculated weights relative to weights the day animals were inoculated. (C) The percentage change of human cells in mouse peripheral blood. Inoculation of Raji cells, we took 100 μl of whole blood via retro-orbital venous plexus each week, extracted DNA, and determined the ratio of human cells in peripheral blood of mice by q-PCR. (D) Comparison of liver from mice after Raji B cell injection.Once the weight of the mice was reduced by more than 20% after inoculation, we euthanized the mice, dissected the viscera and took pictures. (E) Immunohistochemical staining of mice livers and spleens after Raji cell inoculation. Once the weight of the mice was reduced by more than 20% after inoculation, we embedded mouse livers and spleens into paraffin for immunohistochemical assays.

Drug in vivo efficacy in CDX tumor models

Drug in vivo efficacy study using Raji lymphoma CDX Tumor metastasis model in B-NDG mice.

Figure 9. Raji-Fluc cells (5×10⁵) were injected into B-NDG mice and the same dose of antibody X was given at day 3 and day 10. (A) In vivo imaging recorded at different time points to observe disease progression in mice. (B) Tumor curve for tumor cell fluorescence curves in different groups of mice. The effect of early treatment (at day 3, day 10) is remarkable, and this effect is significantly reduced for the late treatment (at day 10).

Human CD47 antibody in vivo efficacy study using Raji lymphoma CDX Tumor model in B-NDG mice.

Figure 10. Antitumor activity of anti-human CD47 antibodies in B-NDG mice.
Human B-luciferase-GFP Raji cells (B lymphocytes) were caudal vein implanted into B-NDG. Mice were grouped when the fluorescence intensity reached approximately 1.0E+06 (n=6) at which time they were treated with anti-human CD47 antibodies with doses and schedules indicated in panel A. (A) fluorescence imager was used to monitor tumor fluorescence in mice. (B) Body weight changes during treatment. The results showed that 3 antibodies had efficacies for tumor growth inhibition in B-NDG mice. B-NDG is a powerful model for human CD47 antibody efficacy study. Values are expressed as mean ± SEM.

CAR-T in vivo efficacy study using Raji lymphoma CDX Tumor model in B-NDG mice.

Figure 11. Antitumor activity of CAR-T therapy in B-NDG mice.

Human B-luciferase-GFP Raji cells (B lymphocytes) were caudal vein implanted into B-NDG. Mice were grouped when the fluorescence intensity reached approximately 1E+06 (n=6) at which time they were treated with CAR-T cells (1E+07) with schedules indicated in panel A. . (A) Signal intensity of Raji-Luciferase cell line treated with different CAR-T cells; (B) Body weight changes during treatment. The results showed that 4 CAR-T cells differently inhibited tumor growth in in B-NDG mice. B-NDG is a powerful model for human CAR-T cells efficacy study. Values are expressed as mean ± SEM.

Human Immune System Reconstituted Models and Efficacy Evaluation

Immune Humanized B-NDG Mice via Human PBMCs Engraftment

Figure 18. Analysis of peripheral blood lymphocyte subpopulations by FACS. Human PBMC cells(5E6) were intravenous implanted into homozygote B-NDG mice(female, 6 week-old, n=6) . Blood from B-NDG mice was taken at different times after implantation of huamn PBMC for flow cytometric analysis. B-NDG mice showed a high percentage of human CD45+ cells, T cells. B-NDG mice exhibit reduced body weight and shortened survival likely due to GVHD effects caused by a high proportion of human immune cell reconstitution.

Bispecific antibody in vivo efficacy evaluation in B-NDG mice reconstituted with human PBMCs

Figure 19. . B-NDG mice reconstituted with PBMCs cells were used for bispecific antibody efficacy studies

Human B-luciferase-GFP Raji cells (5E5), PBMC (5E6) and antibodies mixture were intravenously injected into B-NDG mice (n=4). (A) fluorescence imager was used to monitor tumor fluorescence in mice. (B) Body weight changes during treatment. Bispecific antibody shows significant inhibitory effects. The results indicate that establishing a CDX tumor model in B-NDG mice with reconstituted PBMCs provide a powerful preclinical model for in vivo evaluation of antibodies. Values are expressed as mean ± SEM.

Human Immune System Reconstituted Models and Efficacy Evaluation

Bispecific antibody in vivo efficacy evaluation in B-NDG mice reconstituted with human PBMCs

Figure 20. B-NDG mice reconstituted with PBMCs cells were used for CD3×Claudin18.2 bispecific antibody efficacy studies
NUGC4 cells (5E6) were subcutaneously implanted after human PBMCs (5E6) were intravenous implanted into B-NDG mice (female, 7 week-old, n=6). The animals were grouped into control and treatment when the tumor size was approximately 50-80 mm³ and the percentage of human blood hCD45% were above 10%, at which time they were treated with drugs. (A) Anti human CD3×Claudin18.2 bispecific antibody (AMG 910 analog) inhibited NUGC4 tumor growth in human PBMC reconstituted B-NDG mice. (B) Body weight changes during treatment. CD3×Claudin18.2 bispecific antibody shows significant tumor inhibitory effects. The results indicate that establishing a CDX tumor model in B-NDG mice with reconstituted PBMCs provide a powerful preclinical model for in vivo evaluation of antibodies. Values are expressed as mean ± SEM.

High engraftment of human CD34+ cells in B-NDG mice.

Figure 21. B-NDG mice are well suited for HSC engraftment

(A) Reconstitution of human CD34+ cells prolongs the life-span of irradiated B-NDG mice. (B) Body weight of B-NDG mice and CD34+ reconstituted B-NDG mice after radiation transplantation.(C) The percentage of human CD45+ cells was measured sequentially at the time points shown after engraftment of 2x10⁵ CD34+ cells into B-NDG mice. (D) The percentage of human CD19+ cells among the human CD45+ cells. (E) The percentage of human CD3+ cells among the human CD45+ cells.

B-NDG免疫缺陷鼠+人免疫细胞重建

近年来，免疫检查点抑制剂在肿瘤治疗中取得突破性进展，在多种实体瘤中均取得了较好的治疗效果，免疫治疗因而备受关注。百奥赛图B-NDG小鼠表现为缺乏成熟的T、B、NK细胞，是目前国际公认的免疫缺陷程度高、适合人源细胞或组织移植的工具小鼠。但由于模型本身免疫系统的缺陷，无法进行免疫治疗的药效评价。对免疫缺陷鼠进行人免疫系统重建，能够很好的解决这一问题：将人的免疫细胞、造血干细胞移植到B-NDG及B-NDG衍生小鼠中构建的免疫系统重建小鼠，能够更好的模拟人的免疫系统，进行免疫学研究和免疫药物评价。

根据人免疫系统重建移植的细胞类型，百奥赛图目前可提供三类免疫重建模型：人外周血单核细胞（PBMC）重建，人造血干细胞（CD34+ HSC）重建和NK细胞重建。利用免疫重建小鼠模型，能够进行肿瘤免疫治疗药物的药效评价，为新药研发及临床前评估提供有力支持。

B-NDG mice 免疫重建模型+CDX

在人PBMC重建的B-NDG小鼠中进行单抗和双特异性抗体抗CDX肿瘤药效研究

将人 B-luc-GFP Raji(5E5)、PBMC (5E6) 和抗体混合物静脉注射到 B-NDG 小鼠 (n = 4) 中。(A) 小动物成像仪监测小鼠肿瘤荧光。(B) 治疗期间的体重变化。单抗表现出明显的抑瘤效果，双特异性抗体的抑制作用更加显著。结果表明，使用 PBMC重建的B-NDG小鼠建立CDX肿瘤模型为体内评价抗体提供了有力的临床前模型。数值表示为平均值±SEM。

原位肿瘤模型

原位瘤和转移瘤模型汇总

Pancreatic cancer：B-luc MIA Paca-2 #4+ B-NDG mice（orthotopic）

Drug evaluation in Pancreatic orthotopic model

Liver cancer：B-luc Hep 3B plus + B-NDG hIL15 mice（orthotopic）

Colorectal cancer：B-Tg(Luc) HCT 116（mix）+ B-NDG mice （orthotopic）

Lung cancer：B-Tg(Luc) A549（mix）+ B-NDG mice（ i.v.）

Colorectal cancer：B-Tg(Luc) RKO（mix）+ B-NDG mice（orthotopic）

Breast cancer：4T1+ Balb/c mice（orthotopic）

PDX模型服务

在B-NDG小鼠中成功建立PDX模型

PDX模型中供试品的疗效评估

1、实体瘤PDX

案例：胰腺癌—BP0062

不同代次B-NDG小鼠的生长曲线

将BP0062植入B-NDG小鼠皮下。体积以mm³表示，公式V=0.5a X b²，其中a、b分别为肿瘤长径和短径。肿瘤平均体积±SEM。

结果:胰腺癌BP0062 PDX模型能够在B-NDG小鼠中稳定建立肿瘤模型，并能有效跟踪肿瘤体积。

模型验证

Patient-derived xenografts were found to recapitulate the structures in original patient samples and maintain similar heterogeneity in different generations. 患者来源的异种移植物被发现重现了原始患者样本中的结构，并在不同代中保持了相似的异质性。

对CLDN18.2和EGFR的全面免疫组化评估显示，不同代的个体肿瘤之间没有明显的异质性。

靶向EGFR的药物在B-NDG小鼠中的抗肿瘤活性。(A)分子靶向小分子抗癌药物轻度抑制BP0062在B-NDG小鼠体内的肿瘤生长。将BP0062的PDX模型植入B-NDG小鼠(雌性，6周龄，n=6)皮下。当肿瘤体积达到约100 mm3时对小鼠进行分组，在这个时间点，小鼠接受不同的靶向药物治疗，治疗期间的体重变化见图(B)。如图A所示，分子靶向小分子抗癌药物疗效显著，表明BP0062的PDX模型可用于建立肿瘤模型，为EGFR阳性细胞提供了一种强大的临床前胰腺肿瘤模型。值表示为平均值±SEM。

不同品系小鼠在相同PDX模型中的反应

体内药效:HER2 X TROP2 BsADC在HER2低的PDX模型中显示出强大的抗肿瘤活性

2、血液瘤PDX

案例：白血病 PDX—BP2010

WES(Whole Exome Sequencing) Examples

不同品系小鼠的生长曲线

将BP2010经尾静脉接种于CB-17 SCID小鼠。每周2次测量肿瘤生长和小鼠体重，流式细胞术分析肿瘤(n=10)。(A)肿瘤平均生长速度±SEM，(B)小鼠平均体重±SEM。

结果:白血病PDX模型PDX BP2010在B- ndg中能够稳定地建立肿瘤模型，而在CB-17 SCID中不能生长。

化药体内疗效

当hCD45>10%时，给药组治疗没有显著效果；当10%>hCD45>1%时，给药组抑瘤效果显著。结论：给药过程中的建模程度对小分子药物的疗效影响较大。

抗体药体内疗效

抗体在B-NDG小鼠中的抗肿瘤活性。（A）人CD47在BP2010的肿瘤细胞表面高度表达。（B）抗体CD47-Hu5F9可抑制BP2010在B-NDG小鼠中的肿瘤生长。通过尾静脉将BP2010的PDX模型接种到B-NDG小鼠（雌性，n=7）中。当小鼠外周血中的hCD45阳性细胞达到约1%时，对小鼠进行分组，此时用抗体药物和表（C）所示的方案对它们进行治疗。治疗期间体重变化。数值表示为平均值±SEM。