The Immune-deficient B-NDG mouse model (NOD.CB17-PrkdcscidIl2rgtm1/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.
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
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)).
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.
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。
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
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）