Abstract
BackgroundAcute Myeloid Leukemia (AML) is still associated with high relapse rates when treated with conventional chemotherapeutic and hematopoietic transplantation regimens. Thus, new treatment options are urgently needed.Immunotherapy adopting T cells engineered to express tumor-directed Chimeric Antigen Receptors (CARs) has shown striking results particularly in the context of B-cell malignancies, sparking a keen interest in extending this approach also to other hematological malignancies such as AML.Among the surface molecules identified, the CD33 molecule represents so far one of the main validated target in AML and, being broadly expressed on AML blasts, represents a suitable antigen to be targeted with CAR-T cells.ObjectivesThe aim of the present study is to preclinically evaluate the efficacy and safety profiles of CD33.CAR redirected Cytokine Induced Killer (CIK) cells alone and in combination with standard chemotherapeutic agents.MethodsDonor derived- and autologous-CIK cells were stably or transiently transduced with a third generation anti-CD33.CAR by Sleeping Beauty transposon- or mRNA-mediated engineering.In vitro anti-AML activity has been assessed by means of Flow cytometry-based cytotoxicity (AnnV-7AAD staining), proliferation (Ki67 staining and CFSE dilution) and cytokine production (intracellular IFNg and IL2 detection) assays, upon challenge with AML samples.In vivo efficacy has been evaluated in NSG mice transplanted with MA9-NRas AML cell line or primary AML samples. Moreover, an already established xenograft chemotherapy model has been exploited to examine the potential benefit of combining CD33.CAR-CIK cells with standard AML induction therapy (Ara-C and doxorubicin).ResultsCD33.CAR stably expressing CIK cells were able to induce a potent anti-leukemic activity in vitro, in terms of specific killing either in short term (>70% at 4h, E:T ratio 5:1) and long term cytotoxic assays (>90% at 1 week, E:T ratio 1:10), with statistically significant differences as compared to the unmanipulated condition. Moreover, CD33.CAR-CIK cells were able to retain a significant cytotoxic activity when re-challenged with the CD33+ target following a previous stimulation (up to 65%). The proliferative response to AML target cells was also considerable and CAR-specific (up to 60% of Ki67+CAR-CIK cells and up to 70% of CFSE diluted CAR-CIK cells), as well as the cytokine production (up to 35% of IFN-γ producing CAR-CIK cells and up to 25% of IL-2 producing CAR-CIK cells). CIK cells transiently expressing the CD33.CAR were also effective towards the AML target.In vivo results showed that CD33.CAR-CIK cells were able to control the disease in MA9 grafted mice in all the districts analyzed (peripheral blood, bone marrow, spleen, liver and kidney), as compared to untreated mice.To evaluate the effect of CD33.CAR-CIK cell immunotherapy particularly on Leukemia Initiating Cells (LICs), CD33.CAR-CIK cells were administered as an early treatment approach, treating mice 5 days after i.v. injection of a secondary transplanted PDX sample. We observed a clear engraftment reduction in the treated cohort, nearly undetectable in 2 out 5 mice, while a high leukemic burden has been detected in untreated mice (up to 70% of engraftment in bone marrow).Furthermore, by exploiting CD33.CAR-CIK cell treatment in mice experiencing disease recurrence after the “5+3” chemotherapy-induction protocol, preliminary data showed that CD33.CAR-CIK cells were also capable to target chemotherapy resistant/residual AML cells.ConclusionsConsidering our in vivo preliminary results, we aim to further evaluate CD33.CAR-CIK cell immunotherapy efficacy, particularly against chemotherapy resistant/residual AML cells.Concerning the safety aspect, since the CD33 targeting raises concerns for a potential myelotoxicity, we will assess the potential long-term off-target effects of CD33.CAR-CIK cells (comparing stably with transiently expressed CD33.CARs) on normal hematopoietic stem/myeloid progenitor cells. DisclosuresNo relevant conflicts of interest to declare.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have