B-NDG hIL15 Mice: Where Enhanced Human Immune Reconstitution Fuels NK-Dependent Therapies
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B-NDG hIL15 Mice: Where Enhanced Human Immune Reconstitution Fuels NK-Dependent Therapies
Highly immunodeficient mouse models enable human immune system reconstitution, allowing the engraftment of human tissues or cells for research on immune function, therapeutic efficacy, and disease modeling. While these models effectively reconstitute human T cells, the lack of human IL15—an essential cytokine for NK cell maturation—hinders human NK cell reconstitution, limiting research on NK cell biology and the evaluation of NK-dependent immunotherapies (Gergues et al., 2021; Huang et al., 2022; Liu et al., 2023).
To address this challenge, Biocytogen developed B-NDG hIL15 mice, an IL15-humanized model based on our highly immunodeficient B-NDG mice. This advanced model expresses human IL15 but not mouse IL15, providing the necessary cytokine support for NK cell development. It significantly improves human NK cell reconstitution, making B-NDG hIL15 mice a powerful tool for studying NK cells and evaluating NK-dependent therapies.
Strain-specific IL15 expression in B-NDG and B-NDG hIL15 mice. Serum from wild-type B-NDG (+/+), heterozygous (H/+), and homozygous (H/H) B-NDG hIL15 mice (male, 6 weeks old) was analyzed by ELISA after in vivo poly(I:C) stimulation. Human IL15 was detectable in B-NDG hIL15 mice but not in B-NDG mice.
B-NDG hIL15 Mice Enhance Human NK Cell Reconstitution
Upon engrafting human CD34+ hematopoietic stem cells (HSCs), peripheral blood mononuclear cells (PBMCs), or NK cells, B-NDG hIL15 mice showed a significantincrease in both the frequency and absolute number of reconstituted human NK cells compared to B-NDG mice. Other immune cells like T cells, B cells, and myeloid cells also exhibited enhanced reconstitution.
Human CD34+ HSC-reconstituted model
When human HSCs were engrafted into B-NDG hIL15 mice (huHSC-B-NDG hIL15 mice), the reconstitution of human NK cells was significantly improved in both adult and newborn mice compared with B-NDG mice.
Human CD34+ HSC engraftment in B-NDG hIL15 mice (adult) enhances human NK cell reconstitution
Human immune cell phenotyping in B-NDG hIL15 mice engrafted with human HSCs. Human CD34+ cells were intravenously injected into irradiated (1.6 Gy) homozygous B-NDG hIL15 (n=19) and B-NDG mice (n=17), both female and 6 weeks old. Flow cytometry of peripheral blood lymphocytes showed a higher percentage of human NK cells in B-NDG hIL15 mice. Human NK, T, and B cells were successfully propagated.
Human CD34+ HSC engraftment in B-NDG hIL15 mice (neonatal) enhances the reconstitution of human NK cells and other immune cells like T cells, B cells, and myeloid cells
Engraftment of human CD34+ HSCs in neonatal B-NDG hIL15 mice successfully reconstituted human immune cells. Human CD34+ HSCs were engrafted via the facial vein into irradiated B-NDG hIL15 and B-NDG mice (24–48 hours old). Peripheral blood immune cell reconstitution was analyzed by flow cytometry. (A) Survival curve; (B) Body weight; (C) Percentages and numbers of human immune cells. huHSC-B-NDG hIL15 mice exhibited significantly higher percentages of human CD45+ and NK cells, along with greater numbers of CD45+, T, B, NK, and myeloid cells.
NK cells isolated from the spleen of huHSC-B-NDG hIL15 mice possess tumor-killing activity against Jurkat cells
NK cells isolated from huHSC-B-NDG hIL15 mice exhibit tumor-killing activity against Jurkat cells. Spleen-derived NK cells were collected 14 weeks after hCD34+ HSC engraftment (n=4) and assessed via LDH assay. Human PBMC-purified NK cells served as a positive control. (A) Reconstituted human CD45+ cells ranged from 56% to 81%. (B) Reconstituted human NK cell counts ranged from 22 to 44 cells/µL of blood. (C) The cytotoxicity of reconstituted human NK cells was comparable to that of PBMC-derived NK cells. (D-F) Activity markers of NK cells were analyzed by flow cytometry. NK cells expressing hCD69, hCD107a, and hGranzyme B were detected in reconstituted NK cells. After incubation with Jurkat cells, the proportion of NK cells expressing hCD69 and hCD107a increased, while hGranzyme B levels remained unchanged. Compared to PBMC-derived NK cells, the proportion of reconstituted NK cells expressing hCD69 and hCD107a was lower, whereas the proportion of cells expressing hGranzyme B was higher. These results indicate that reconstituted human NK cells in huHSC-B-NDG hIL15 mice exhibit cytotoxicity comparable to that of human NK cells purified from PBMCs.
B-NDG hIL15 mice exhibit a high and stable reconstitution rate of human CD34+ HSCs.
Reconstitution of Human CD34+ HSCs from Different Donors. (A) Human CD34+ HSCs engrafted in newborn B-NDG hIL15 mice stably reconstitute immune cells, with success rates varying by donor. (B) Mouse survival rate ranges from 80% to 90% at 24 weeks post-reconstitution. (C) Mice continue to gain weight after reconstitution.
Human PBMC-reconstituted model
Human immune cell phenotyping in B-NDG hIL15 mice engrafted with human PBMC. Human PBMCs were intravenously implanted into B-NDG hIL15 (n=15) and B-NDG (n=10) female mice (5 weeks old). Blood was collected at different time points for flow cytometric analysis. (A) Human immune cell phenotyping, (B) NK cell phenotyping, (C) Survival, (D) Body weight. Results indicate successful propagation of human NK and T cells in reconstituted B-NDG hIL15 mice.
Human NK cell-reconstituted model
Engraftment of human NK cells in B-NDG hIL15 mice enhanced human NK cell reconstitution. Irradiated B-NDG (n=7) and B-NDG hIL15 (n=8) mice (6 weeks old) were intravenously injected with purified human NK cells from PBMCs. Peripheral blood was analyzed weekly. NK cells purified from PBMCs reached 80%, and the number and proportion of reconstituted NK cells (CD3⁻CD56⁺) were significantly higher in B-NDG hIL15 mice. The NK cell proportion remained ~80% after 2 weeks and stayed higher after 6 weeks. Reconstituted NK cells were predominantly CD16⁺ NK cells with cytotoxic activity.
Applications in Preclinical Oncology Research
We have successfully established various cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models using HSC-, PBMC-, or NK cell-reconstituted B-NDG hIL15 mice, enabling in vivo evaluation of therapeutics in a humanized immune environment. These models have been leveraged to assess antibodies such as anti-human CLDN18.2 and anti-human CD3×HER2 bispecific antibodies, demonstrating their effectiveness in suppressing tumor growth.
In vivo efficacy of anti-human CLDN18.2 antibody in CDX model established with huHSC-B-NDG hIL15 mice
Antitumor activity of anti-human CLDN18.2 antibody (Zolbetuximab) in a lung cancer A549 CDX model established with huHSC-B-NDG hIL15 mice. Human CD34+ HSCs were intravenously engrafted into irradiated B-NDG hIL15 mice. When hCD45+ cells exceeded 25% in blood, B-hCLDN18.2 A549 cells were subcutaneously inoculated. Mice were grouped at 80-100 mm³ tumor volume and treated with zolbetuximab. Zolbetuximab effectively inhibited tumor growth, with high levels of human NK cells detected in blood and tumor tissues.
In vivo efficacy of anti-human CD3×HER2 BsAb in PDX model established with huHSC-B-NDG hIL15 mice
Antitumor activity of anti-human CD3×HER2 BsAb in a pancreatic cancer PDX model established with huHSC-B-NDG hIL15 mice. Human CD34+ HSCs were engrafted into irradiated B-NDG hIL15 mice. Pancreatic cancer PDX (BP0209) was inoculated, and mice were grouped at 100-150 mm³ tumor volume for intraperitoneal CD3×HER2 BsAb treatment. Treatment effectively inhibited tumor growth.
In vivo efficacy of anti-human CLDN18.2 antibody in CDX model established with huNK-B-NDG hIL15 mice
Antitumor activity of anti-human CLDN18.2 antibody in A549 CDX model established with huNK-B-NDG hIL15 mice. Human NK cells from PBMCs were intravenously engrafted into X-ray-irradiated B-NDG hIL15 mice. B-hCLDN18.2 A549 cells were subcutaneously inoculated. Mice were grouped when tumors reached ~100–150 mm³ and treated with zolbetuximab (n=6). Peripheral blood was collected weekly to assess human CD45⁺ and NK cell reconstitution. Results showed zolbetuximab effectively inhibited tumor growth, with high levels of human NK cells detected in peripheral blood.
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References
Gergues, Marina, et al. “Development of CD19 CAR Engineered Human Placental CD34+-Derived Natural Killer Cells (CAR19-CYNK) As an Allogeneic Cancer Immunotherapy.” Blood 138 (2021): 2779.
Huang, Dehao, et al. “Lateral plate mesoderm cell-based organoid system for NK cell regeneration from human pluripotent stem cells.” Cell Discovery 8.1 (2022): 121.
Liu, Xiaowei, et al. “Immune checkpoint HLA-E: CD94-NKG2A mediates evasion of circulating tumor cells from NK cell surveillance.” Cancer Cell 41.2 (2023): 272-287.
Fehniger, Todd A., and Michael A. Caligiuri. “Interleukin 15: biology and relevance to human disease.” Blood, The Journal of the American Society of Hematology 97.1 (2001): 14-32.