• Human CD34+ HSCs (hematopoietic stem cells) engrafted humanized mice is a powerful model allows researchers to examine the human immune system and related primates' alien virus research and cellular immunotherapy preclinical evaluation. Although B-NDG humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. Such as frequencies of human NK cells did not reach physiological levels in B-NDG humanized mice.
• IL15 (Interleukin 15) encodes a pleiotropic cytokine of the interleukin family of proteins that plays important roles in the innate and adaptive cell homeostasis, as well as peripheral immune functions. It has been reported that IL15 supports innate lymphoid cell development^[1]. Studies using IL15 transgenic mice^[2] and IL15 knockout mice^[3] have shown that IL15 is essential for the development of NK cells, natural killer T (NKT) cells, and memory CD8+ T cells. The analysis of human HSCs engrafted mice showed that hIL15 is required for NK cell development ^[4].
• Biocytogen developed B-NDG hIL15 mice in which the coding sequence (CDS) of the human IL15 gene was inserted after the 5′ UTR of the mouse IL15 gene to allow the mouse to express human IL15, but not mouse IL15. When human HSCs were engrafted into B-NDG hIL15 mice, the reconstitution level of human NK cells was significantly improved in both adult and newborn mice compared with B-NDG mice. In addition to human HSCs, PBMC and NK cells purified from human PBMCs can also be used as a quick reconstitution of human NK cells in mice. B-NDG hIL15 mice can be used as a powerful tool to study the development and function of human NK cells, and to evaluate the efficacy of NK-dependent antibodies, especially antibodies with ADCC activity.

Expression analysis of functional markers and surface receptors in reconstituted human NK cells. NK cells were purified from human PBMC using the sorting kit, and sorted NK cells (2.0 M) were intravenously injected into B-NDG mice（female, 6-week-old, n=7）and B-NDG hIL15 mice（n=8）after treated with 1.2 Gy irradiation. Peripheral blood was collected weekly to detect cytotoxic markers Granzyme B and CD57, cell surface agonistic receptors NKG2D, NKp46 and NKp30, and the cell surface inhibitory receptor NKG2A in human NK cells. Results showed that compared with B-NDG mice, the proportions of hNKp46+ cells, hNKp30+ cells, Granzyme B+ cells and human NKG2A+ cells in B-NDG hIL15 mice after human NK cell reconstitution were significantly increased, while the proportions of hNKG2D+ cells and hCD57+ cells were slightly decreased. These results indicated that the reconstituted human NK cells have cytotoxic activity.

Engraftment of human NK cells in B-NDG hIL15 mice enhanced the reconstitution of human NK cells. Female 6-week-old of B-NDG mice (n = 7) and B-NDG hIL15 mice (n = 8) were irradiated with 1.2 Gy, and NK cells (2E6) obtained after purification of human PBMCs were intravenously injected. Peripheral blood was taken weekly to analyze the reconstitution level of human immune cells. The results showed that the proportion of NK cells obtained from PBMC purified reached 80%. Compared with B-NDG mice, the number and proportion of reconstituted human NK cells (CD3-CD56+) were significantly higher in B-NDG hIL15 mice. The proportion of NK cells remained around 80% after 2 weeks of reconstitution and a higher proportion of NK cells remained after 6 weeks of reconstitution. Reconstituted human NK cells were predominantly CD16+ NK cells with cytotoxic activity.

Antitumor activity of anti-human CLDN18.2 antibody in lung cancer cell line A549 CDX model established with huNK-B-NDG hIL15 mice. Human NK cells (2E6) purified from PBMCs were intravenously engrafted into B-NDG hIL15 mice irradiated with X-ray. B-hCLDN18.2 A549 cells (1E7) were subcutaneously inoculated into B-NDG hIL15 mice. The mice were grouped when the tumor volume reached approximately 100-150 mm³ at which time they was treated with anti-human CLDN18.2 antibody (zolbetuximab, in-house) with dose and schedule indicated in panel (n=6). Peripheral blood was collected weekly to analyze the reconstitution levels of human CD45+cells and NK cells. The results showed that zolbetuximab could effectively inhibit tumor growth in huNK-B-NDG hIL15 mice. High levels of human NK cells could be detected in peripheral blood.