Background

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.

Major Applications 

Animal breeding and maintenance

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 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)).

Phenotypic analysis

Body weight growth 

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

Histology 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).

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

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. 

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

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.

Figure 21. B-NDG mice reconstituted with CD34+ cells were used for drug efficacy studies.

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。

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。

在人CD34+重建的B-NDG小鼠中进行抗体的抗CDX肿瘤药效研究

利用人CD34+细胞重建的人源化 B-NDG 小鼠模型静脉注射人B-luc-GFP Raji细胞 (5E5)。肿瘤细胞植入后5天，用人抗体 X 处理小鼠。在第7天观察到人抗体X对肿瘤细胞生长的显著抑制作用。结果表明，基于B-NDG的HSC人源化小鼠模型为体内抗体评价提供了有力的临床前模型。数值表示为平均值±SEM。
RC：Raji-Fluc Control.  XH: Antibody X/humanized mice. naïve B-NDG: B-NDG mice without Raji-Fluc and antibody treatment

在人NK细胞重建的B-NDG小鼠中进行抗体的抗CDX肿瘤药效研究

使用NK细胞重建的B-NDG hIL15小鼠进行IMAB362抗体药效研究

NK细胞(2E6)重建的人源化B-NDG hIL15小鼠模型皮下移植A549-hCLDN18.2细胞 (1E6)。肿瘤细胞植入后11天分组，用IMAB362处理小鼠，表现出明显的抑瘤效果。结果表明，基于B-NDG hIL15小鼠进行NK细胞重建的小鼠模型为体内抗体评价提供了有力的临床前模型。数值表示为平均值±SEM。

Summary of orthotopic tumor models (Services available) 

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

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

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

PDX Models are Successfully Established in B-NDG Mice

Efficacy evaluation of test articles in PDX models

1、实体瘤PDX

案例：Pancreatic Cancer—BP0062 

• Growth Curve in different passages of B-NDG mice

Results: The PDX model of Pancreatic cancer BP0062 is able to establish tumor model steadily in B-NDG mice and can effectively track tumor volume.

• Model Validation

Patient-derived xenografts were found to recapitulate the structures in original patient samples and maintain similar heterogeneity in different generations. 

A thorough IHC assessment of CLDN18.2 and EGFR revealed no observable heterogeneity among individual turmous in different generations. 

Antitumor activity of Drugs target EGFR in B-NDG mice . (A) Molecular targeted small-molecule anti-cancer drugs slightly inhibited tumor growth of BP0062 in B-NDG mice. PDX model of BP0062 was subcutaneously implanted into B-NDG mice (female, 6 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with different targeted drugs and schedules indicated in panel (B) Body weight changes during treatment. As shown in panel A, Molecular targeted small-molecule anti-cancer drugs were efficacious, demonstrating that PDX model of BP0062 can be used to establish tumor model and provide a powerful preclinical pancreatic tumor model with EGFR positive cells. Values are expressed as mean ± SEM.

• Response of different mice strains in the same PDX models

• In Vivo Efficacy: HER2 X TROP2 BsADC Showed Potent Anti-Tumor Activity in HER2-low PDX Models

2、血液瘤PDX

案例：Leukemia PDX—BP2010 

• WES(Whole Exome Sequencing) Examples 

• Growth Curve in different strains of mice

BP2010 was inoculated into CB-17 SCID mice via caudal vein. Tumor growth and mice body weight were measured twice a week. Tumor was analyzed by flow cytometry (n=10). (A) Tumor average growth ± SEM, (B) Mice average weight ± SEM
Results: The PDX model of leukemia PDX BP2010 is able to establish tumor model steadily in B-NDG while can not grow in CB-17 SCID.

When hCD45 > 10%, the group was administered, and the treatment had no significant effect; when 10% > hCD45 > 1%, the group was administered, and the tumor inhibition effect was significant. 
Conclusion: The degree of modeling during administration has a greater impact on the efficacy of small molecule drugs.

• In vivo Efficacy of Antibody

Antitumor activity of antibody in B-NDG mice . (A) Human CD47 was highly expressed on the surface of tumor cells of BP2010.(B) Antibody CD47-Hu5F9 could inhibite tumor growth of BP2010 in B-NDG mice. PDX model of BP2010 was inoculated into B-NDG mice via caudal vein (female, n=7). Mice were grouped when hCD45 positive cells of PB reached approximately 1%, at which time they were treated with antibody drug and schedules indicated in panel (C) Body weight changes during treatment. Values are expressed as mean ± SEM.