Development and characterization of the ultra‐immunodeficient NOD.CB17‐PrkdcscidIL2rgtm1/Bcgen (B‐NDG) mouse model

Abstract

The “B‐NDG” (NOD.CB17‐PrkdcscidIL2rgtm1/Bcgen) mouse is the latest advancement to provide an alternative option in the highly immunodeficient mouse model category for the oncology, immunology and other biomedical research communities. This model was designed and generated by Biocytogen. IL2rg (common gamma) gene was deleted in NOD‐scid mice resulting in a mouse that lacks mature B cells, T cells, NK cells and has a deficiency in cytokine signaling. This level of immunodeficiency makes this model ideal for engraftment with human immune cells and human tumor cells or tissues. Herein we describe the development and characterization of the B‐NDG model, which includes growth curve, complete blood count, serum chemistry, flow cytometry, tumor growth and humanization. A cohort of B‐NDG mice (N=50 males and 50 females) were weighed weekly from 3 to 9 weeks of age. At 9 weeks of age males range from 26–31 grams and females range from 19–24 grams. A cohort of six male B‐NDG were analyzed to determine baseline levels for complete blood cell count and serum chemistry. Splenocytes of C57BL/6, NOD‐scid and B‐NDG mice were isolated and examined for total T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and regulatory T cells using flow cytometry. Data showed complete lack of all T cells, B cells and NK cells in the B‐NDG model. Raji cells were injected into BALB/c Nude, NOD‐scid and B‐NDG mice at 5×106 and survival, body weight and percentage of human cells in whole blood was measured over the next 30 days. Survival and body weight was lowest in the B‐NDG model with 0% survival by day 19 post injection and 20% body weight loss by day 18. The number of human cells in the blood of the animals was highest in the B‐NDG at over 60% by day 18. At day 18 the B‐NDG mice showed a significantly higher amount of liver metastasis than the NOD‐scid mice. A gastric cancer PDX model was also examined in B‐NDG mice compared to C.B17 scid mice, with 67% and 0% take rate, resepctively. Two different methods of humanization were examined in the B‐NDG model. Human peripheral blood mononuclear cells (hPBMCs) were injected at 5×106 into the caudal vein of three B‐NDG mice. After 24 days, the hCD45+ and mCD45+ cells were examined and mice had 30–80% hCD45+ cells. Of those hCD45+ cells, over 99% were identified as T cells. The second humanization method used was injection of 1×105 human CD34+ hematopoietic stem cells after irradiation. The percentage of human CD45+ cell in the peripheral blood was over 20% by eight weeks post injection in over 70% engrafted mice. At 16 weeks post injection, of the total number of human lymphocytes in the humanized mice, 60% were B cells, 2% were NK cells and over 10% were T cells. The percentage of human T cells increases steadily with the extended observation window. We have demonstrated that B‐NDG mice have several features that translate many benefits as compared to other immunodeficient models. This model has demonstrated better tumor growth than the NOD‐scid and C.B17 scid mice for cell lines as well as PDX lines. We have also shown that this model is extremely useful for studies that require humanization of an immunodeficient mouse model.