Abstract
Abstract 3010
Current therapeutic regimens for Acute Myeloid Leukemia (AML) are still associated with high rates of relapse. In the last years, great interest has been focused on the identification of surface molecules that are preferentially expressed by AML cells and leukemic stem cells (LSCs), in order to selectively target the tumor population, whilst sparing the normal counterpart of hematopoietic stem/progenitor cells (HSPC), and possibly impeding disease recurrence. Immunotherapy with T-cells genetically modified to express chimeric antigen receptors (CARs) represents a valid and innovative cell therapy approach for hematological malignancies. In this study we developed a new CAR molecule specific for the IL-3Rα (CD123) target antigen, which is overexpressed on AML blasts, CD34+ leukemic progenitors, and leukemic stem cells (AML-LSCs) compared to normal hematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine Induced Killer (CIK) cells, ex-vivo expanded T cells with spontaneous antitumoral activity, were transduced with an SFG-retroviral vector encoding an anti-CD123.CAR and CAR functionality has been evaluated by short-term cytotoxicity assay. Transduced CIK cells strongly killed CD123+ THP-1 cell line (60%±5.4%, Effector:Target –E:T- ratio of 5:1, n=3), as well as primary AML blasts (59%±5.4%, E:T ratio of 3:1, n=4). With the aim to better characterize the ability of anti-CD123.CAR+CIK cells to kill leukaemia cells over time we performed long-term cytotoxicity assay, observing a leukemic cell recovery for THP-1 of 3.5%±1.5% (n=5) and for primary AML cells of 2.4%±1.4% (n=3) when co-cultured with CIK cells expressing anti-CD123.CAR, compared to an average target survival of up to 80%, when co-cultured with unmanipulated (NT) CIK cells. Interestingly, secondary colonies experiments after co-culture of healthy donor cord blood-derived HSPCs (Lin-) with anti-CD123.CAR+CIK cells demonstrated that this newly generated CAR molecule better preserved the normal haematopoietic reconstitution in contrast to a previously generated anti-CD33.CAR (total number of colonies of 146.8±6.6, 66.4±5.1, 117.6±4.6, for Lin- cells co-cultured with NT CIK cells, anti-CD33.CAR+CIK cells, anti-CD123.CAR+CIK cells respectively, n=4), while keeping identical cytotoxicity profile towards AML. Furthermore, a limited killing of normal CD123 expressing monocytes and CD123-low expressing endothelial cells was measured, accompanied by a lesser release of stimulatory cytokines such as IFN-gamma, TNF-alfa and TNF-beta when compared to the levels released after stimulation with CD123+ leukemic cells (THP-1 and AML), thus indicating a low toxicity profile of the anti-CD123.CAR. Taken together, our results indicate that CD123-specific CAR strongly enhances anti-leukemic CIK functions towards AML, while sparing HSPCs and normal CD123-expressing tissues, paving the way for the development of novel immunotherapy approaches for the treatment of resistant forms of AML, particularly for a precocious intervention in presence of minimal residual disease, in the context of early relapse after HSCT.
Disclosures:
No relevant conflicts of interest to declare.
CD33
‐directed immunotherapy with third‐generation chimeric antigen receptor T cells and gemtuzumab ozogamicin in intact and
CD33
‐edited acute myeloid leukemia and hematopoietic stem and progenitor cells
