Mice description

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. Studies using IL15 transgenic mice and IL15 knockout mice 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.

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, 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.

mRNA expression analysis

Strain specific analysis of IL15 gene expression in wild-type B-NDG and B-NDG hIL15 mice by RT-PCR. Mouse Il15 mRNA was detectable only in splenocytes of wild-type B-NDG (+/+) mice. Human IL15 mRNA was detectable only in homozygous B-NDG hIL15 (H/H), but not in B-NDG mice. 

Engraftment of human CD34+ HSCs in B-NDG hIL15 mice enhances reconstruction of human NK cells. Female 6-week-old of B-NDG mice (n = 17) and B-NDG hIL15 mice (n = 19) were irradiated with 1.6 Gy, respectively, and human CD34+ HSCs (1.5E5) were injected into the tail vein. The peripheral blood was taken at different time points to analyze the reconstitution levels of human immune cells. The results showed that the proportion of reconstituted human NK cells in B-NDG hIL15 mice was significantly higher compared with B-NDG mice, and the proportion could be stably maintained from 2 weeks to 14 weeks of reconstitution.

Engraftment of human CD34+ HSCs in B-NDG hIL15 mice enhances reconstitution of human NK cells (neonatal mice)

CDX model were well established on human immune system engraftment mice. Human CD34+ cells(0.15M) were intravenous implanted into homozygote B-NDG hIL15 (female, 7 week-old, n=30) and B-NDG mice (female, 7 week-old, n=30). All mice were treated with 1.6gry-irradiation. Mice were grouped as hCD45% ≥ 10%. K562 cells(1E6), MV-4-11(1E7), Panc-1(5E6) were subcutaneously implanted into B-NDG hIL15 and B-NDG mice respectively. During the establishment of CDX model using K562 cells (A), MV-4-11 (B), and Panc-1 (C) cells, the proportion of immune cells in the blood of mice were measured. The results showed that during the establishment of tumor models, the proportion of leukocytes, T cells and NK cells in the blood of B-NDG hIL15 mice was higher than  B-NDG mice.

Human HSC immune system engraftment and CDX model

CDX model were well established on human immune system engraftment mice Human CD34+ cells(0.15M) were intravenous implanted into homozygote B-NDG hIL15 and B-NDG mice. All mice were treated with 1.6gry-irradiation. Mice were grouped as hCD45% ≥ 10%. MV-4-11(1E7) and Panc-1(5E6) were subcutaneously implanted into B-NDG hIL15 and B-NDG mice respectively. The proportion and function of NK cells in mouse blood and tumor tissues were measured at the end of the experiment. The results showed that B-NDG hIL15 show a higher percentage of human NK cells compared with B-NDG, and NK cell express functional proteins.

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Human HSC immune system engraftment and Raji-luc CDX model

Antitumor activity of anti-human CD20 antibodies in B-NDG hIL15 mice. Human CD34+ cells(0.15M) were intravenous implanted into homozygote B-NDG hIL15 and B-NDG mice. All mice were treated with 1.6gry-irradiation. Raji-luc(5E5) were implanted into B-NDG IL15 mice. Mice were grouped when the tumor volume reached approximately 100-150mm³ (n=5) at which time they were treated with rituximab (in house) with doses and schedules indicated in panel . The proportion of leukocyte and NK cells in mouse blood were measured at the different time of the experiment. The results showed that CD20 antibodies had efficacies for tumor growth inhibition in B-NDG hIL15 mice with human HSC  immune system reconstitution model.

Human CD34+ HSCs reconstituted B-NDG hIL15 mice can effectively evaluate efficacy of anti-human CLDN18.2 antibody

Female 6-week-old B-NDG hIL15 mice (n = 6) were irradiated with 1.2 Gy and injected with human HSCs (1.5E5) via the tail vein. CLDN18.2-overexpressed human lung cancer B-hCLDN18.2 A549 cells (1E7) were innoculated subcutaneously after 6 weeks. Anti-human CLDN18.2 antibody (zolbetuximab, in house) was injected intraperitoneally 7 days after tumor innoculation. Peripheral blood was taken weekly to analyze the reconstitution levels of human NK cells and T cells. Tumor tissues were taken at the end of the experiment to detect the level of infiltrating human NK cells and T cells. The results showed that anti-human CLDN18.2 antibody could effectively inhibit tumor growth in B-NDG hIL15 mice with a tumor inhibition rate of 36.2%. High levels of human NK cells could be detected in both peripheral blood and tumor tissues. Human T cells could also be detected in peripheral blood and tumor tissues, but their reconstitution is slower than that of human NK cells. Nevertheless, the level of human T cells in peripheral blood maintained a gradually increasing trend.

Antitumor activity of anti-human EGFR antibodies in B-NDG hIL15 mice. Human CD34+ cells (0.15M) were intravenous implanted into homozygote B-NDG hIL15 and B-NDG mice. All mice were treated with 1.6gry-irradiation. Pancreatic cancer PDX(BP0160-R4P7) were implanted into B-NDG IL15 mice. Mice were grouped when the tumor volume reached approximately 100 mm3 (n=5) at which time they were treated with cetuximab (in house) with dose and schedule indicated in panel. The proportion of leukocyte and NK cells in mouse blood were measured at the different time of the experiment. The results showed that cetuximab antibody had efficacies for tumor growth inhibition in B-NDG hIL15 mice with human HSC immune system reconstitution model.

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Analysis of CD45+ and NK cells after PBMC engraftment and K562 CDX model generation in B-NDG hIL15 mice . Human PBMC cells(5E6) were intravenous implanted into homozygote B-NDG hIL15 (female, 6 week-old, n=5) and B-NDG mice (female, 6 week-old, n=5). K562 cells(1E6)  were subcutaneously implanted into B-NDG hIL15 and B-NDG mice on the same day as PBMCs. (A) The mean ± SEM of tumor sizes (B) The mean ± SEM of Body weight. (C) Blood from B-NDG hIL15 and B-NDG mice was taken at different times for flow cytometric analysis. K562 cells were successfully tumorigenic on a mouse model of human PBMC  immune system reconstitution, and B-NDG hIL15 showed a higher percentage of human CD45+ cells and cell number of human NK cells  compared to B-NDG.

Analysis of CD45+ and NK cells after PBMC engraftment and PANC-1 CDX model generation in B-NDG hIL15 mice. Human PBMC cells(5E6) were intravenous implanted into homozygote B-NDG hIL15 (female, 6 week-old, n=5) and B-NDG mice (female, 6 week-old, n=5). PANC-1 cells(5E6)  were subcutaneously implanted into B-NDG hIL15 and B-NDG mice on the same day as PBMCs. (A) The mean ± SEM of tumor sizes (B) The mean ± SEM of Body weight. (C)Blood from B-NDG hIL15 and B-NDG mice was taken at different times for flow cytometric analysis. PANC-1 cells were successfully tumorigenic on a mouse model of human PBMC  immune system reconstitution, and tumor growth was significantly delayed in B-NDG hIL15 mice compared to B-NDG mice. B-NDG hIL15 showed a higher percentage of human CD45+ cells and cell number of human NK cells  compared to B-NDG

Antitumor activity of anti-human Claudin18.2 antibodies in B-NDG hIL15 mice. Human PBMC cells(1E6) were intravenous implanted into homozygote B-NDG hIL15 mice. NUGC4-CLDN18.2(6E6) were implanted into B-NDG IL15 mice. Mice were grouped when the tumor volume reached approximately 70-100mm³ (n=5) at which time they were treated with IMAB362 (in house) with doses and schedules indicated in panel. (A) Tumor volume changes during treatment, (B) Body weight changes during treatment. The results showed that CLDN18.2 antibodies had efficacies for tumor growth inhibition in B-NDG hIL15 mice with human PBMC immune system reconstitution model.

Antitumor activity of anti-human TIGIT antibodies in B-NDG hIL15 mice. Human PBMC cells(1E6) were intravenous implanted into homozygote B-NDG hIL15 mice. A375(4E6) were implanted into B-NDG hIL15 mice. Mice were grouped the day after tumor inoculation (n=5) and treated with Tiragolumab (in house) with doses and schedules indicated in panel. (A) Tumor volume changes during treatment, (B) Body weight changes during treatment. The results showed that TIGIT antibodies had efficacies for tumor growth inhibition in B-NDG hIL15 mice with human PBMC immune system reconstitution model.

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.

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. Result showed that compared with B-NDG mice, the proportions of hNKp46+ cells, hNKp30+ cells, hGranzyme 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 indicate that the reconstituted human NK cells have cytotoxic activity.

Antitumor activity of anti-human Claudin18.2 antibodies in B-NDG hIL15 mice. Human NK cells(2E6) purified from PBMC were intravenous implanted into homozygote B-NDG hIL15 mice. A549-hCLDN18.2 (1E7) were implanted into B-NDG IL15 mice. Mice were grouped when the tumor volume reached approximately 100-150mm³ (n=6) at which time they were treated with IMAB362 (in house) with doses and schedules indicated in panel.  (A) Tumor volume changes during treatment, (B) Body weight changes during treatment. The results showed that CLDN18.2 antibodies had efficacies for tumor growth inhibition in B-NDG hIL15 mice with human NK immune system reconstitution model.

Human immune cell phenotyping in B-NDG hIL15 engrafted with human NK cells during the pharmaceutical assay. The change of the proportion of immune cells in the blood was observed, and the proportion of human CD45, CD56 and CD16 in the administration group was higher than that of the control group. This indicates that NK cells also maintained high levels of ration during the experiment.

References