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Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells.

ABSTRACT: Discrimination between hematopoietic stem cells and leukemic stem cells remains a major challenge for acute myeloid leukemia immunotherapy. CAR T cells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells before consolidation with allogeneic hematopoietic stem cell transplantation in absence of cytotoxic conditioning. Here we exploit non-viral technology to achieve early termination of CAR T cell activity to prevent incoming graft rejection. Transient expression of an anti-CD117 CAR by mRNA conferred T cells the ability to eliminate CD117+ targets in vitro and in vivo. As an alternative approach, we used a Sleeping Beauty transposon vector for the generation of CAR T cells incorporating an inducible Caspase 9 safety switch. Stable CAR expression was associated with high proportion of T memory stem cells, low levels of exhaustion markers, and potent cellular cytotoxicity. Anti-CD117 CAR T cells mediated depletion of leukemic cells and healthy hematopoietic stem cells in NSG mice reconstituted with human leukemia or CD34+ cord blood cells, respectively, and could be terminated in vivo. The use of a non-viral technology to control CAR T cell pharmacokinetic properties is attractive for a first-in-human study in patients with acute myeloid leukemia prior to hematopoietic stem cell transplantation.

SUBMITTER: Magnani CF

PROVIDER: S-EPMC10424000 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells.

Magnani Chiara F CF Myburgh Renier R Brunn Silvan S Chambovey Morgane M Ponzo Marianna M Volta Laura L Manfredi Francesco F Pellegrino Christian C Pascolo Steve S Miskey Csaba C Ivics Zoltán Z Shizuru Judith A JA Neri Dario D Manz Markus G MG

Molecular therapy oncolytics 20230719

Discrimination between hematopoietic stem cells and leukemic stem cells remains a major challenge for acute myeloid leukemia immunotherapy. CAR T cells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells before consolidation with allogeneic hematopoietic stem cell transplantation in absence of cytotoxic conditioning. Here we exploit non-viral technology to achieve early termination of CAR T cell activity to prevent incoming graft rejection. Transient express ...[more]

PMID: 37583386

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Project description:Hematopoietic recovery is considered to be associated with the number of multipotent hematopoietic stem cells in the bone marrow, as observed in functional assays involving stem cell transplantation. However, there is little evidence related to hematopoietic recovery in non-transplantation settings, which is accomplished by endogenous hematopoietic cells. A recent study suggested that progenitors are the main contributors during this steady-state hematopoiesis, which differs from exogenous transplantation. We hypothesized that endogenous progenitor support post-chemotherapy hematopoietic recovery. To investigate the potential impact of these progenitor cell percentage on hematopoietic recovery, we retrospectively analyzed the percentage of CD34+CD38+CD117+HLA-DR+CD13+CD33+ cells (P cells) and hematopoietic recovery in 223 newly diagnosed acute myeloid leukemia patients during two courses of consolidation chemotherapy after complete remission. We found that a lower P cell percentage was significantly associated with prolonged neutropenia recovery time after the first and second courses of consolidation chemotherapy (p = 0.001; p = 0.045, respectively). We also observed similar results with regard to platelet recovery time after the first course of consolidation chemotherapy (p = 0.000). Univariate analysis showed that P cell percentage and consolidation chemotherapy regimens, and not gender, age, induction chemotherapy regimens, infection grade, WHO classification and NCCN risk category, were associated with neutrophil recovery after chemotherapy. Multivariate analysis demonstrated that P cell percentage is an independent factor affecting neutrophil recovery capacity for both the first and second courses (p = 0.008; p = 0.032, respectively). Our results indicate that CD34+CD38+CD117+HLA-DR+CD13+CD33+ cells before each course of chemotherapy is independently associated with chemotherapy-related hematopoietic reconstitution capacity. These findings may help modify future chemotherapy regimens based on progenitor cell percentages.

Project description:BackgroundChronic myeloid leukemia stem cells (CML-LSCs), which exhibit resistance to tyrosine kinase inhibitors (TKIs), are the leading cause of treatment failure and recurrence in chronic myeloid leukemia (CML). This highlights the urgent need for novel therapies aimed at eliminating these CML-LSCs. Chimeric antigen receptor macrophages (CAR-M) not only perform phagocytosis on target cells but also function as antigen-presenting cells, thereby activating the anti-tumor immune response.CD26 (dipeptidyl peptidase 4, DPP IV) is abundantly expressed in CML-LSCs and functions as a tumor-specific antigen (TSA) in CAR-M treatment. The purpose of this study is to evaluate CAR-M's efficacy in targeting CD26-positive CML cells and to develop a novel strategy for CML treatment.MethodsCD26 CAR-M was constructed using mouse-derived macrophage Raw264.7 cells. CD26 was overexpressed in CML cell lines BP210 and BP210-T315I. The targeting phagocytosis of CAR-M was verified using confocal microscopy and flow cytometry. X-ray was used to eliminate the tumorigenicity of CAR-M, and the safety of CAR-M was verified through CCK-8, clone formation assays, and animal experiments. To assess the anti-leukemia ability of CAR-M in the CML mouse model, the survival, peripheral blood white blood cell counts, and CML cell infiltration in the liver, spleen, and bone marrow (BM) were measured. Additionally, CD26 CAR-THP1 was constructed, and its phagocytic ability against CD26-positive cells NCI-H2452 was confirmed by confocal microscopy.ResultsWe successfully constructed CD26 CAR-M and validated its targeted phagocytosis of CD26-positive CML cells both in vitro and in vivo. The data indicate that CAR-M has higher phagocytic efficiency in CD26-positive CML cells than in CD26-negative cells. CAR-M-treated CML mice demonstrated extended survival and reduced CML invasion. In addition, CAR-THP1 demonstrated targeted phagocytosis of NCI-H2452 cells that normally express CD26.ConclusionThis study demonstrates that CD26 CAR-M effectively targets and phagocytizes CD26-positive CML cells, implying that targeting CD26 with CAR-M could be a viable method for eradicating CML-LSCs. Furthermore, our discoveries illuminate the potential application of CAR-M in treating hematological malignancies.

Dataset Information

Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells.

Publications

Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells.

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