Project description:Chimeric Antigen Receptor T cells express an extracellular domain consisting of a single-chain fragment variable (scFv) targeting a surface tumor-associated antigen (TAA), it is important to select scFv regarding safety profile through evaluation of the efficacy/toxicity balance, especially in cases where the target antigen is also expressed on healthy cells. Here, we developed five CAR T cells targeting CD123 (CD123 CAR-T) by substituting only the scFv, looking for differences in terms of efficacy and on-target/off-tumor effects. Using in vitro models, we found that three of the five CD123 CAR-T were more cytotoxic against leukemic cells, without increasing lysis of monocytes or endothelial cells. Using the Incucyte system, we confirmed the low cytotoxicity of CD123 CAR-T on endothelial cells. Hematotoxicity evaluated by progenitor culture and CD34 cell lysis showed that two of the five CD123 CAR-T were less cytotoxic against HSC, and we confirmed in a humanized mouse model that CD123- cells were not eliminated by the CD123 CAR-T. These two CD123 CAR-T reduced tumor infiltration and increased overall survival of mice in three in vivo models of BPDCN. Moreover, in BPDCN aggressive model, bulk RNA-sequencing analysis showed that these CD123 CAR-T upregulated genes associated with cytotoxicity and activation/exhaustion a few days after the injection. Finally, we were able to produce CD123 CAR-T selected previously from patient T cells. Together, these results emphasize the importance of screening different scFv for the development of CAR-T constructs, to select the one with the optimal risk-benefit ratio for clinical development.
Project description:To reveal which pathways linked to CD33 and CD123 overexpression could be involved in leukemic proliferation, we performed unbiased bulk RNA sequencing comparison between OCI-AML3 wt and CD123 and or CD33 KO clones
Project description:The comparative characterization of hematopoietic stem cells from healthy stem cell donors and patients with acute myeloid leukemia on a proteome level has the potential to reveal differentially regulated proteins which might be candidates for specific immunotherapy target molecules. Exemplarily, we analyzed the proteome of the cytosolic and the membrane fraction of CD34 and CD123 co-expressing FACS-sorted leukemic progenitors from five patients with acute myeloid leukemia employing mass spectrometry. As a reference, CD34+CD123+ normal hematopoietic progenitor cells from five healthy stem cell donors were analyzed. In this TMT 10-plex labeling based approach 2068 proteins were identified with 256 proteins differentially regulated in one or both cellular compartments. This study demonstrates the feasibility of a mass spectrometry based proteomic approach to detect differentially expressed proteins in two compartment fractions of leukemic stem cells as compared to their healthy stem cell counterparts.
Project description:The NUP98::NSD1 fusion gene is associated with extremely poor prognosis in patients with acute myeloid leukemia (AML). NUP98::NSD1 induces self-renewal and blocks differentiation of hematopoietic stem cells, leading to leukemia development. Despite its association with poor prognosis, targeted therapy for NUP98::NSD1-positive AML is lacking, as the details of NUP98::NSD1 function are unknown. Here, we generated 32D cells (a murine interleukin-3 (IL-3)-dependent myeloid progenitor cell line) expressing mouse Nup98::Nsd1 to explore the function of NUP98::NSD1 in AML, including by comprehensive gene expression analysis. We identified two properties of Nup98::Nsd1+ 32D cells in vitro: first, Nup98::Nsd1 promoted blocking of AML cell differentiation, consistent with a previous report; second, Nup98::Nsd1 increased dependence on IL-3 for cell proliferation, due to overexpression of the alpha subunit of the IL-3 receptor (IL3-RA, also known as CD123). Consistent with our in vitro data, IL3-RA was also up-regulated in samples from patients with NUP98::NSD1-positive AML. These results highlight CD123 as a potential new therapeutic target in NUP98::NSD1-positive AML.
Project description:In our study, MegaClust - an unsupervised, data-driven algorithm - barely described mDCs and pDC subsets were identified. To confirm these findings we performed RNA sequencing Facs sorted of mDCs (CD123-CD11c+CD4+HLA-DR+), pDCs (CD123+CD11c-CD4+HLA-DR+) and monocytes (CD14+) from healthy donors and compared these with publicly available data.
Project description:Single cell gene expression profiling of splenic cells in mice treated with gated CD33-CD123 CARs compared to those with conventional CD123 CAR expression
