33DC54DCm33DC54DCmRBC33DC54DCmRBCWe aim to understand genetic and biological heterogeneity and com-58Normal WBChCD45T cell enrichmentMNCsEnrichment of T cellsby negative selectionIn vitro cultureCAR-T therapy (4w)CD3LLeeuukkeemmiiaaNormal WBCFew THuman:96.7%hCD45CXCR4 expression potentiates CD25-targeted CAR-T cell therapyCD25-targetedCAR-T injection intoxenograftof patientleukemic cells(24hr)Lentiviral transduction(48hr)CXCR4CAR-T therapy (4w)CD30.0%Normal WBCCCAARR--TTHuman:16.3%CXCR4CAR-T therapy (5m)hCD45CD3Human:5.3%To strengthen the therapeutic effect of small molecules targeting the intra-cellular vulnerability proteins, we set out to engineer T cells binding particular cell surface antigens and exhibiting potent cytotoxic activity against patient leukemic cells. In AML, considering the lack of or only low-level expression of putative targets in normal hematopoietic stem/progenitor cells and non-hematopoietic tissues, as well as the prognostic impact of the target’s expression on patient survival, we chose CD25/IL2RA as one of the therapeutic cell surface molecules. Though CD25-targeted CAR-T cell therapy resulted in variable ef-ficacy in the AML xenograft model, additional engineering resulting in expres-sion of CXCR4 in the CAR-T cells led to complete elimination of human leuke-mic cells in bone marrow, spleen, liver, and peripheral blood. In vivo treatment of normal, non-leukemic humanized mice with the CXCR4-expressing CD25-targeted CAR-T cells did not result in a severe reduction of normal human immune subsets including regulatory T cells. We are currently performing and analyzing single-cell TCR and gene expression profiling as well as mass cytom-etry, which thus far is indicating that maintenance of central memory or stem cell memory CD4+ T cells and over-represented expression of TCF7, IL-7R and CD226/DNAM1 are crucial cellular and molecular signatures of prevention of cellular exhaustion and supporting successful treatment efficacy of the CAR-T cells.plexity of poor prognosis leukemia. Through a collaboration with Dr. Shuichi Taniguchi’s group at Toranomon Hospital, we have collected hundreds of leukemia patient samples. By extracting DNA and RNA from those samples as well as setting up xenogeneic transplantation using NOD/SCID/Il2rgKO newborns, we identified mutations and chromosomal aberrations critical in leukemia initiation. Furthermore, we discovered non-mutated key survival molecules in mitochondria and in the cytoplasm in a patient-specific manner.Figure: Engineering of chemokine receptor-expressing CAR-T cells for AML treatment(Upper) A schematic showing how to prepare CD25-targeted CAR-T cells for injecting into mice bearing an AML patient-derived xenograft. (Lower left) In an AML xenograft mouse injected with CD25-targeted CAR-T cells, we did not observe good therapeutic efficacy with the remaining CD33+ AML cells. (Lower middle) How-ever, additional engineering to cause CXCR4 expression resulted in the complete elimination of AML cells and good recovery of normal hematopoiesis. (Lower right) The potent therapeutic effect against AML was main-tained up until 5 months post-injection.Recent Major PublicationsHashimoto M, Saito Y, Nakagawa R, Ogahara I, Takagi S, Takata S, Amitani H, Endo M, Yuki H, Manabe R, Watanabe T, Ozaki K, Kaneko A, Kajita H, Fujiki S, Sato K, Honma T, Uchida N, Okazaki Y, Ohara O, Shultz LD, Yamada M, Taniguchi S, Vyas P, de Hoon M, Momozawa Y, Ishikawa F. Maximal vulnerability converges to XIAP and BCL2 in leukemia with diverse genetic aberrations. Nat Cancer 2: 340-356 (2021)De Groot A, Saito Y, Kawakami E, Hashimoto M, Aoki Y, Ono R, Ogahara I, Fujiki S, Kaneko A, Watanabe T, Takagi M, Tomizawa D, Koh K, Eguchi M, Ishii E, Ohara O, Shultz LD, Mizutani S, Ishikawa F. Targeting critical kinases and anti-apoptotic molecules overcomes steroid resistance in MLL-rearranged leukemia. EBioMedicine 64, 103235 (2021)Saito Y, Shultz L, Ishikawa F. Understanding normal and malignant human hematopoiesis using next-generation humanized mice. Trends Immunol 41, 706-720 (2020)Invited presentationsAri Itoh-Nakadai. “Targeting poor prognosis leukemia with CD25-targeted chemokine receptor expressing CAR T cell therapy” Annual Meeting of Japan Biochemistry Society (online) November 2021Ishikawa F. “Finding vulnerabilities in genetically-complex hematologic tumors” The 80th Annual Meeting for Japan Cancer Association (Yokohama, Japan) October 2021Ishikawa F. “Humanized Mice for studying normal and diseased blood and immune systems” Lecture for Gradu-ate School at Yokohama City University (Yokohama, Japan) October 2021Ishikawa F. “Humanized Mice for Normal and Malignant Human Hematopoiesis” China-Japan High-level Expert Symposium on Animal Models (Beijing, China/Online) August 2021Ishikawa F. “Identification of vulnerabilities in genetical-ly-complex AML” Annual Meeting of European Hematol-ogy Association 2021(Online) June 2021Laboratory for Human Disease ModelsTeam Leader: Fumihiko Ishikawa
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