We describe successful treatment of 3 cases of human herpesvirus 6 (HHV-6) encephalitis/myelitis following cord blood transplantation (CBT). Ganciclovir (GCV) (10 mg/kg/day) reduced HHV-6 load to undetectable levels in cerebrospinal fluid (CSF). Early dose reduction in the presence of HHV-6 detectable in CSF resulted in an increased HHV-6 load. GCV was capably shifted to valganciclovir (VGCV) with an almost equivalent concentration. GCV/VGCV may be effective for HHV-6 encephalitis/myelitis after CBT, although HHV-6 load in CSF should be monitored.
Objectives As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)‐T cells for AML are more challenging than those targeting CD19 in B‐cell malignancies. We recently developed piggyBac ‐modified ligand‐based CAR‐T cells that target CD116/CD131 complexes, also known as the GM‐CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR‐T cells. Methods To further improve the efficacy of the original GMR CAR‐T cells, we have developed novel GMR CAR vectors incorporating a mutated GM‐CSF for the antigen‐binding domain and G4S spacer. All GMR CAR‐T cells were generated using a piggyBac ‐based gene transfer system. The anti‐tumor effect of GMR CAR‐T cells was tested in mouse AML xenograft models. Results Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR‐T cells exhibited potent cytotoxic activities against CD116 + AML cells in vitro . Furthermore, GMR CAR‐T cells incorporating a G4S spacer significantly improved long‐term in vitro and in vivo anti‐tumor effects. By employing a mutated GM‐CSF at residue 21 (E21K), the anti‐tumor effects of GMR CAR‐T cells were also improved especially in long‐term in vitro settings. Although GMR CAR‐T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. Conclusions GMR CAR‐T cell therapy represents a promising strategy for CD116 + R/R AML.
Background: The prognosis of relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor; therefore, novel treatment strategies are required urgently. Meanwhile, recent clinical trials have demonstrated that CAR-T cells for AML have been less successful than those targeting CD19 for B cell malignancies. Recently, we developed piggyBac-modified ligand-based CAR-T cells that target CD116, also called granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) α chain, for treating juvenile myelomonocytic leukemia (Nakazawa, et al. J Hematol Oncol. 2016). Since CD116 is overexpressed in 60%-80% of AML cases, the present study aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. Methods: CD116 expression in AML cell lines or primary leukemia cells were examined using flow cytometry. The original piggyBac transposon plasmid for GMR CAR comprises GM-CSF as an antigen recognition site, IgG1 CH2CH3 hinge region, CD28 costimulatory domain, and CD3ζ chain. To improve the in vivo persistency and anti-tumor effects, two types of spacer (∆CH2H3 and G4S) that lack CH2CH3 lesion were newly constructed. In order to modulate the antigen recognition ability, mutated ligand-based GMR CAR vectors were constructed with a mutation at residue 21 of GM-CSF that is reported to play a critical role in its biological activity (Lopez, et al. Embo j. 1992). All the GMR CAR-T cells were generated with piggyBac gene modification. To investigate the in vitro anti-tumor activity, GMR CAR-T cells were co-cultured with AML cell lines. In order to evaluate the in vivo anti-tumor effects, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were intravenously injected with THP-1, THP1-ffLuc, or MV4-11 and then treated with GMR CAR-T cells. To characterize the safety profile of GMR CAR-T cells, peripheral blood mononuclear cells or polymorphonuclear cells were co-cultured with GMR CAR-T cells at an effector:target ratio of 1:1 for 3 days. Thereafter, B cells, NK cells, neutrophils, and monocytes were quantified using flow cytometry using counting beads. Results: Approximately 80% of the AML cells predominant in myelomonocytic leukemia expressed CD116. PiggyBac-modified GMR CAR-T cells displayed a favorable CD45RA+CCR7+-dominant phenotype, consistent with our previous findings. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. GMR CAR-T cells incorporating a G4S spacer significantly improved the long-term in vitro and in vivo anti-tumor effects as compared to those incorporating a ∆CH2CH3 spacer. Furthermore, by employing a mutated GM-CSF at residue 21 (E21K and E21R) as an antigen recognition site, the in vivo anti-tumor effects were also substantially improved along with prolonged survival (Figure 1) over controls (PBS or CD19.CAR-T cells) (all, p < 0.01) as well as over GMR CAR-T cells with a wild-type GM-CSF ligand (E21R: p < 0.01; E21K: p = 0.02), with 4 out of 5 mice surviving for > 150 days. Safety tests revealed that the toxicity of GMR CAR-T cells was restricted to normal monocytes. It is noteworthy that the cytotoxic effects of GMR CAR-T cells on normal neutrophils, T cells, B cells, and NK cells were minimal. Conclusions: GMR CAR-T cell therapy appears to be a potentially useful strategy for CD116+ R/R AML. Based on the promising results, we plan to perform the first-in-human clinical trial of GMR CAR-T cells. Disclosures Saito: Toshiba Corporation: Research Funding. Hasegawa:Toshiba Corporation: Research Funding. Inada:Kissei Pharmaceuticals: Ended employment in the past 24 months. Nakashima:Toshiba Corporation: Research Funding. Yagyu:Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.
Objectives Chimeric antigen receptor (CAR)‐T cell therapy redirected to specific antigens on tumor cells is a promising immunotherapy strategy for various cancers. Most target antigens are also expressed on normal tissues at varying levels, and therefore, a considerable challenge in the field is determining safety profiles, including life‐threatening off‐tumor and off‐target toxicities. The granulocyte–macrophage colony‐stimulating factor receptor (hGMR) is a promising target for CAR T‐cell therapy for a subset of acute myelocytic leukaemia, although it is also expressed on normal cells including monocytes, macrophages, CD34‐positive haematopoietic cells and vascular endothelial cells. hGMR and other immune‐related proteins are highly conserved between humans and cynomolgus macaques (Macaca fascicularis). Therefore, in this study, we engineered cynomolgus T cells to express CAR molecules redirected to hGMR by piggyBac (PB) transposon‐based gene transfer and adoptively transferred autologous hGMR‐CAR T cells into cynomolgus macaques. Methods We established PB‐mediated human GMR (hGMR)‐specific CAR T cells using cynomolgus peripheral blood mononuclear cells and transferred them into autologous individuals, and evaluated the potential toxicity related to hGMR‐CAR T cells. Results hGMR‐CAR T cells did not exert overt organ toxicities such as bone marrow suppression, monocytopenia and vasculitis, although they recognised and killed cynomolgus monocytes and macrophages in vitro. Conclusion Although our model did not simulate a tumor‐bearing model, it supports the safety of hGMR‐CAR T cells and demonstrates the usefulness of a non‐human primate model to evaluate the safety of T‐cell products by assessing off‐tumor/off‐target toxicity before clinical trials.
Background: The prognosis of relapsed/refractory (R/R) myeloid malignancies remains poor, and the development of novel treatment strategies is crucial. Although chimeric antigen receptor (CAR)-modified T cell therapy for B cell malignancies has shown excellent clinical efficacy, the use of CAR-T cells for myeloid malignancies has been more challenging, partly due to the heterogeneous expression of candidate target antigens in leukemia cells and shared expression of those antigens in normal myeloid cells or progenitor cells. We previously developed the piggyBac-modified chimeric antigen receptor (CAR)-T cells targeting CD116, also known as GM-CSF receptor alpha chain (GMR) (Nakazawa Y, et al. J Hematol Oncol. 2016 and Hasegawa A, et al. Clin Transl Immunology. 2021). GMR CAR-T cells showed substantial antitumor effects against both acute myeloid leukemia and juvenile myelomonocytic leukemia. Moreover, modulation of the spacer and antigen recognition site of the CAR vector further enhanced the anti-tumor effects of GMR CAR-T cells. GMR CAR-T cells showed an acceptable safety profile with limited cytotoxicity on normal hematopoietic cells except monocytes. Based on these results, we have initiated a first-in-human clinical trial of GMR CAR-T cell therapy in March 2021 in Japan. Study Design and Methods: The study is a phase I/II, single-center, dose-escalation study with a traditional 3+3 dose-escalation design (Table 1). Maximum of 18 patients will be recruited. Primary objectives of this study are to determine the safety and severe adverse events of piggyBac-modified GMR CAR-T cells for CD116 + relapsed/refractory myeloid malignancies by assessing the dose-limiting toxicity within 28 days from the single infusion of GMR CAR-T cells. The patients with CD116 + myeloid malignancies aged more than 1 year with myeloid malignancies who experienced an induction failure or a relapse after hematopoietic stem cell transplantation (HSCT) will be recruited. CD116 is defined as positive when a CD116 relative mean fluorescence (RFI) in leukemia cells ≥ 2. RFI was calculated by dividing the MFI of samples with that of the isotype control. Major exclusion criteria are as follows, acute promyelocytic leukemia, acute graft-versus-host disease (GVHD) (Grade ≥ 2), extensive chronic GVHD, and concurrent treatment with corticosteroid (≥ 6 mg/m2). Statistical analysis will be performed when the data will be fixed. Peripheral blood mononuclear cells will be harvested from the patient by leukapheresis and then will be transduced with GMR CAR vector by piggyBac transposon system. All manufacturing process of GMR CAR-T cells is performed in Cell Processing Center in Shinshu University Hospital under good manufacturing practice conditions. The patient will be treated with lymphodepleting chemotherapy consisting of fludarabine and cyclophosphamide, followed by CAR-T cell infusion. The dose of CAR-T cells will be 3 x 10 5 and 1 x 10 6 in cohorts 1 & 2 and cohort 3, respectively (Table 1). Kinetics of GMR CAR-T cells will be determined by quantifying the GMR CAR gene in the peripheral blood using real-time PCR after the CAR-T cell infusion. All the patients will be required to receive HSCT by day 56 following CAR-T cell infusion. The primary endpoint of the study is the safety, pharmacokinetics, and efficacy of GMR CAR-T therapy (Trial registration: jRCT2033210029). Conclusion: We herein described the protocol of first-in-human GMR CAR-T cells for relapsed/refractory myeloid malignancies. By employing the optimized CAR vector and production protocol, the safety and efficacy of GMR CAR-T cells will be evaluated in this study. Figure 1 Figure 1. Disclosures Saito: Toshiba corporation: Research Funding. Yagyu: AGC Inc.: Research Funding. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.