)l( ytimgp( γNFxototyC)%ci0I/0with DC/Galwith DC/GaliPS-NKT alonewith NALM-6iPS-NKT alonewith NALM-6CCBB5000AA72100806040200.5:10.25:1ET ratioCD19CARwild-type40003000200010000.1:1Day 0 NALM6 2x10^7 s.c.Day 14, 17, 19 iPS‐NKT 3x10^6peutic potential in many areas, including regenerative medi-cine and immune therapy. On a collaborative basis with individ-ual IMS research laboratories, the core facility for iPS research is aiming to put cancer immunotherapy with iPS-derived NKT cells into clinical use.The facility has operated an IMS Cell Manufacturing Unit to produce iPS-derived human invariant NKT (Vα24+iNKT) cells under Good Manufacturing Practice/Good Gene, Cellular, and Tissue-based Products Manufacturing Practice guidelines. The facility completed the first clinical trial of iPS-Vα24+iNKT cells for head and neck cancer and determined that it will be safe. The next clinical trial is being planned by a licensee. We have also been conducting another clinical trial using a combination therapy with iPS-Vα24+iNKT cells and activated dendritic cells One of the merits of the Humanized Mice is functional defi-nition of normal and malignant stem cells. By injecting heterog-enous blood cells of human samples into immune-compromised NOD/SCID/Il2rgKO newborns, we can identify which cell popu-lations are able to give rise to multiple blood and immune cells and which ones initiate leukemia. Based on transcriptomics and proteomics using normal and malignant stem cells, we develop therapies specifically targeting tumor cells and sparing normal cells. Another merit of the Humanized Mouse system is to evalu-ate therapeutic efficacy and safety of new treatment modalities. We have created a xenograft model for CAR-T cell treatment in which engineered T cells were injected into NSG mice engrafted for head and neck cancer.To maximize the efficacy of iPS-Vα24+iNKT cells, the facil-ity has focused on introduction of Chimeric Antigen Receptors (CARs) into these cells. Among several CARs, the facility chose CD19 and CD25 CARs to treat leukemia. In 2022, we showed a proof of concept. CD19-CAR iPS-Vα24+iNKT cells suppressed leukemia in mice (Figure). Currently, we are generating GMP grade CD19-CAR iPS cells for a future clinical trial.The facility also has been developing a protocol with shorter culture times and higher induction efficiency. We have found that iPS-derived early progenitors are multipotential for cell fate determination prior to lymphoid-lineage commitment in our current protocol, which possibly causes low induction efficiency. Thus we are planning to improve this situation by using chemical compounds and/or cytokines.with patient leukemic cells. After the in vivo treatment studies, both T cells and leukemia cells are recovered from bone marrow and spleen and then subjected to single cell RNA sequencing to identify which genes are associated with treatment efficacy or treatment resistance. We are also comparing how human T cells behave in the xenogeneic environment and in patients. We hope to connect the merits of the xeno-transplant/xenograft system and those of direct examination of patient specimens. In any case, immune-compromised mice engrafted with patient tumors or healthy immune system play critical roles in testing newly-developed therapeutics. We hope to bring our research findings into the clinic in the future.Figure: Anti-tumor effects of CD19CAR iPS-NKTA) Direct cytotoxicity of CD19CAR iPS-NKT and wild-type iPS-NKT toward the CD19+ NALM-6 human acute lymphoblastic leukemia cell line. B) IFN-γ production by CD19CAR iPS-NKT and wild-type iPS-NKT cocul-tured with NALM-6 ± α-galactosylceramide-pulsed dendritic cells (DC/Gal). C) Antitumor effect of iPS-NKT in humanized mouse NALM-6 lymphoma model.Figure: Integrative studies using patient-de-rived xenografts, in vivo CAR-T cell treatment and scRNAseq/mass cytometryTo develop CAR-T cells, we incorporated the sequence of the Fab portion of a human CD25 antibody into a CAR construct with the 4-1BB signaling domain. Acute myeloid leukemia (AML) patient-derived xenograft (PDX) models were created by transplantation of sorted patient-derived AML-initiating cells into newborn NOD/SCID/Il2rgKO mice. CAR-T cells were created using cord blood (CB)-derived T cells which were HLA-matched to the patient from whom the PDX was created. In AML-engrafted PDX mice, a one-time CAR-T cell injection was performed, followed by analysis of treatment ef-ficacy and in vivo T cell populations by flow cytometry, histology, scRNAseq, and mass cytometry.iPS ProjectInduced pluripotent stem (iPS) cells possess tremendous thera-Humanized Mouse ProjectThe Humanized Mouse has been used for diverse purposes.
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