B-NDG hIL15 mice_Biocytogen Pharmaceuticals (Beijing) Co., Ltd._Biocytogen,drug development,antibody discovery

Strain Name	NOD.CB17-Prkdc^scidIl2rg^tm1Il15^tm1(IL15)/Bcgen	Common Name	B-NDG hIL15 mice
Background	B-NDG	Catalog number	110600
Related Genes	IL15(interleukin 15）
NCBI Gene ID	16168

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.

Protein expression analysis

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Strain specific IL15 expression analysis in wild-type B-NDG mice and heterozygous/homozygous B-NDG hIL15 mice by ELISA.Serum was collected from wildtype B-NDG mice (+/+), heterozygous B-NDG hIL15 mice (H/+) and homozygous B-NDG hIL15 mice (H/H) (male, 6-week-old) stimulated with or without poly(I:C) in vivo, and analyzed by ELISA with species-specific IL15 ELISA kit. Human IL15 was detectable in heterozygous and homozygous B-NDG hIL15 mice but not in B-NDG

mice.

HSC Human Immune System Engraftment

Human immune cell phenotyping in B-NDG hIL15 engrafted with human HSC. Human CD34+ cells (0.15 M) were intravenously injected into homozygote B-NDG hIL15 (female, 6 week-old, n=19) and B-NDG mice (female, 6 week-old, n=17). All mice were treated with 1.6 Gy irradiation. Representative flow cytometric analysis of peripheral blood lymphocyte from mice after engraftment with human CD34+ cells. B-NDG hIL15 show a higher percentage of human NK cells compared with B-NDG. Our results suggest that human NK, T, and B cells in reconstituted B-NDG IL15 mice were successfully propagated.

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 (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. (A-C) The mean ± SEM of tumor sizes. . K562 cells, MV-4-11, Panc-1 were successfully tumorigenic on a mouse model of human HSC immune system reconstitution, and tumor growth was delayed in B-NDG hIL15 mice compared to B-NDG mice.

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Human HSC immune system engraftment and CDX model

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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

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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

Raji-luc 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%. Raji-luc (5E5) were subcutaneously implanted into B-NDG hIL15 and B-NDG mice respectively. (A) fluorescence imager was used to monitor tumor fluorescence in mice. (B-C) Body weight and survival percentage changes during treatment. The results showed that Raji-luc were successfully tumorigenic on a mouse model of human HSC immune system reconstitution, and tumor growth was delayed in B-NDG hIL15 mice compared to B-NDG mice.

In vivo efficacy of anti-human CD20 antibody with human HSC engraftment

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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.

In vivo efficacy of anti-human Claudin18.2 antibody with human HSC engraftment

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Antitumor activity of anti-human Claudin18.2 antibodies in B-NDG hIL15 mice. Human CD34+ cells (0.15M) were intravenous implanted into homozygote B-NDG hIL15 mice. All mice were treated with 1.6gry-irradiation. A549-hCLDN18.2 were implanted into B-NDG IL15 mice. Mice were grouped when the tumor volume reached approximately 50 mm³ (n=5) at which time they was treated with CLN-zolbetuximab-IgG1 (in house) with dose and schedule indicated in panel. The results showed that CLN-zolbetuximab antibody had efficacies for tumor growth inhibition in B-NDG hIL15 mice with human HSC immune system reconstitution model.

In vivo efficacy of anti-human EGFR antibody with human HSC engraftment and PDX model

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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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Human PBMC immune system engraftment

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Human immune cell phenotyping in B-NDG hIL15 engrafted with human PBMC. Human PBMC cells (5E6) were intravenous implanted into homozygote B-NDG hIL15 (female, 5-week-old, n=15) and B-NDG mice (female, 5-week-old, n=10). Blood from B-NDG hIL15 and B-NDG mice was taken at different times after human PBMC engraftment for flow cytometric analysis. A. Human immune cell phenotyping. B. NK cell phenotyping. C. Survival. D. Body weight. Values were expressed mean ± SEM. Our results suggest that human NK and T cells in reconstituted B-NDG IL15 mice were successfully propagated.

Human PBMC immune system engraftment

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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.

Human PBMC immune system engraftment

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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

In vivo efficacy of anti-human Claudin18.2 antibody with human PBMC engraftment

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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.

In vivo efficacy of anti-human TIGIT antibody with human PBMC engraftment

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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.

Human immune reconstitution of NK cells purified from PBMC

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Analysis of CD56+ cells after NK cell reconstitution in B-NDG hIL15 mice. NK cells were purified from human PBMC using the sorting kit, and sorted NK cells (2.0 M) were intravenously injected into homozygote B-NDG hIL15 (female, 6 week-old, n=8) and B-NDG mice (female, 6 week-old, n=8). Representative flow cytometric analysis of peripheral blood lymphocyte from mice after engraftment with human NK cells. B-NDG hIL15 show a higher percentage of human NK cells compared with B-NDG, and it can maintain a high level of reconstitution 6 weeks after injection. Our results suggest that human NK cells in reconstituted B-NDG IL15 mice were successfully propagated.

NK cell function detection after NK cell reconstitution

Human NK cell function marker in B-NDG hIL15 engrafted with human NK cells. After the reconstitution of NK cells, the number of functional NK cell markers such as Granzyme B, NKG2A, NKG2D, NKp46 are significantly increased, and NK cells have a killing function, other functional NK markers such as perforin, CD107,NKp30, CD57 have similar trends (data not shown here).

In vivo efficacy of anti-human Claudin18.2 antibody with human NK cell engraftment

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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.

Antitumor effect of CLDN18.2 antibody in B-NDG hIL15 mice reconstituted with human NK cell

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.

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References

1.Ali, A. K., Nandagopal, N. & Lee, S. H. IL-15-PI3K-AKT-mTOR: A Critical Pathway in the Life Journey of Natural Killer Cells. Frontiers in immunology 6, 355, doi:10.3389/fimmu.2015.00355 (2015).

2.Fehniger, T. A. et al. Fatal leukemia in interleukin 15 transgenic mice follows early expansions in natural killer and memory phenotype CD8+ T cells. The Journal of experimental medicine 193, 219-231, doi:10.1084/jem.193.2.219 (2001).

3.Kennedy, M. K. et al. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. The Journal of experimental medicine 191, 771-780, doi:10.1084/jem.191.5.771 (2000).

4.Matsuda, M. et al. Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice. Life science alliance 2, doi:10.26508/lsa.201800195 (2019).