Search

As significant numbers of acute myeloid leukemia (AML) patients are still refractory to conventional therapies or experience relapse, immunotherapy using T-cells expressing chimeric antigen receptors (CARs) might represent a valid treatment option. AML cells frequently overexpress the myeloid antigens CD33 and CD123, for which specific CARs can be generated. However, CD33 is also expressed on normal hematopoietic stem/progenitors cells (HSPCs), and its targeting could potentially impair normal hematopoiesis. In contrast, CD123 is widely expressed by AML, while low expression is detected on HSPCs, making it a much more attractive target. In this study we describe the in vivo efficacy and safety of using cytokine-induced-killer (CIK) cells genetically modified to express anti-CD33 or anti-CD123 CAR to target AML. We show that both these modified T-cells are very efficient in reducing leukemia burden in vivo, but only the anti-CD123 CAR has limited killing on normal HSPCs, thus making it a very attractive immunotherapeutic tool for AML treatment.Leukemia accepted article preview online, 7 February 2014; doi:10.1038/leu.2014.62.

Despite the progress in the treatment of acute myeloid leukemia (AML) achieved in the last decades, a significant number of patients are still refractory to or relapse after standard cures. Hence, to improve cure rates of AML, it is crucial to develop novel therapeutic strategies. Immunotherapy with T cells genetically modified to express chimeric antigen receptors (CARs) represent a valid option in this sense. CARs are artificial molecules constituted by an extracellular-antigen-binding domain derived from a monoclonal antibody and an intracellular-signalling region that is immediately triggered after antigen recognition. Therefore, CARs combine the antigen binding properties of mononoclonal antibodies to T cell mediated effector functions, including the killing mechanism -that might be active against antibody resistant targets-, cytokine secretion- that might boost the anti-tumoral immune response- and capacity to efficiently home and infiltrate tumor sites. Different CARs have been generated so far, against a wide range of surface molecules expressed by many tumors and, currently, several phase I clinical trials are undergoing and the results obtained so far are very encouraging. The CARs approach can be employed to selectively target AML cells due to the overexpression of myeloid antigens, like CD33 and CD123. We recently demonstrated that expression of CD33-specific CARs in a population of ex-vivo activated T cells, called “cytokine induced killer” (CIK) cells, confers them potent in vitro anti-leukemic functions. However, since CD33 antigen is also expressed on normal haematopoietic stem/progenitors cells (HSPCs) resulting in a potential severe impairment of normal myelopoiesis, CD123 has recently been proposed as a new potential attractive molecule based on its differential expression pattern, being widely overexpressed by AML population and at the same time less expressed on HSPCs. In order to improve the safety profile against these cells we develop and tes

Current therapeutic regimens for acute myeloid leukaemia (AML) are still associated with high rates of relapse. Immunotherapy with T-cells genetically modified to express chimeric antigen receptors (CARs) represents an innovative approach. Here we investigated the targeting of the interleukin three receptor alpha (IL3RA; CD123) molecule, which is overexpressed on AML bulk population, CD34(+) leukaemia progenitors, and leukaemia stem cells (LSC) compared to normal haematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine-induced killer (CIK) cells were transduced with SFG-retroviral-vector encoding an anti-CD123 CAR. Transduced cells were able to strongly kill CD123(+) cell lines, as well as primary AML blasts. Interestingly, secondary colony experiments demonstrated that anti-CD123.CAR preserved in vitro HSPCs, in contrast to a previously generated anti-CD33.CAR, while keeping an identical cytotoxicity profile towards AML. Furthermore, limited killing of normal monocytes and CD123-low-expressing endothelial cells was noted, thus indicating a low toxicity profile of the anti-CD123.CAR. Taken together, our results indicate that CD123-specific CARs strongly enhance anti-AML CIK functions, while sparing HSPCs and normal low-expressing antigen cells, paving the way to develop novel immunotherapy approaches for AML treatment.

Use of adoptive T-cell therapy (ACT) is increasing; however, T-cell therapy can result in severe toxicity. Consequently, several suicide-gene strategies that allow selective destruction of the infused T cells have been described. We compared effectiveness of four such strategies in vitro in Epstein Barr virus (EBV)-cytotoxic T lymphocytes (CTLs). Herpes simplex virus thymidine kinase (HSV-TK), human inducible caspase 9 (iCasp9), mutant human thymidylate kinase (mTMPK), and human CD20 codon optimized genes were cloned in frame with 2A-truncated codon optimized CD34 (dCD34) in a retroviral vector. Codon-optimization considerably improved CD20 expression. EBV-CTLs could be efficiently transduced in all constructs, with transgene expression similar to the control vector containing dCD34 alone. Expression was maintained for prolonged cultures. Expression of the suicide genes was not associated with alterations in immunophenotype, proliferation, or function of CTLs. Activation of HSV-TK, iCasp9, and CD20 ultimately resulted in equally effective destruction of transduced T cells. However, while iCasp9 and CD20 effected immediate cell-death induction, HSV-TK-expressing T cells required 3 days of exposure to ganciclovir to reach full effect. mTMPK-transduced cells showed lower T-cell killing all time points. Our results suggest that the faster activity of iCasp9 might be advantageous in treating certain types of acutely life-threatening toxicity. Codon-optimized CD20 has potential as a suicide gene

Adoptive transfer of specific cytotoxic T lymphocytes (CTL) and Cytokine Induced Killer Cells (CIK) following genetic engineering of T-cell receptor zeta hold promising perspective in immunotherapy. In the present work we focused on the mechanisms of anti-tumor action of effectors transduced with an anti-CD19 chimaeric receptor in the context of B-lineage acute lymphoblastic leukemia (B-ALL). Primary B-ALL blasts were efficiently killed by both z-CD19 CTL and z-CD19 CIK effectors. The use of death receptor mediated apoptosis of target cells was excluded since agonists molecules of Fas and TRAIL-receptors failed to induce cell death. Perforin/granzyme pathway was found to be the mechanism of chimaeric effectors mediated killing. Indeed, cytolytic effector molecules perforin as well as granzymes were highly expressed by CTL and CIK. CD19 specific stimulation of transduced effectors was associated with degranulation as attested by CD107 membrane expression and high IFN- and TNF- release. Moreover inhibitors of the perforin-based cytotoxic pathway, Ca 2+-chelating agent EGTA and Concanamycin A, almost completely abrogated B-ALL blast killing. In conclusion we show that the cytolysis response of z-CD19 chimaeric effectors is predominantly mediated via perforin/granzyme pathway and is independent of death receptors signaling in primary B-ALL. Copyright © 2010 David Laurin et al.

Background. Cytokine induced killer cells are ex-vivo expanded cells with potent antitumoral activity. Cytokine induced killer cells infusion in acute myeloid leukemia patients relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses were observed. To improve their effector functions against acute myeloid leukemia, we genetically modified cytokine induced killer cells with chimeric receptors specific for the CD33 myeloid antigen. Design and Methods. SFG-retroviral vectors coding for anti-CD33-zeta and anti-CD33-CD28-OX40-zeta chimeric receptors were used to transduce cytokine induced killer cells. Transduced cells were in vitro characterized for their ability to lyse leukemic targets (4-hour-51Chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate (3H-thymidine-incorporation assay) and to secrete cytokines (Flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34+ hematopoietic progenitors was evaluated by analyzing the colony forming unit capacity after co-incubation. Results. Cytokine induced killer cells were efficiently transduced with the anti-CD33 chimeric receptors, maintaining their native phenotype and functions and acquiring potent cytotoxicity (up to 80% lysis after 4-hour incubation) against different acute myeloid leukemia targets, as also confirmed in long-term killing experiments. Moreover, introduction of the anti-CD33 chimeric receptors was accompanied by a prominent CD33-specific proliferative activity, with a release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of cytokine induced killer cells anti-leukemic activity. Importantly, even though anti-CD33 chimeric receptors-transduced cytokine induced killer cells showed toxicity against normal hematopoietic CD34+ progenitors, a residual clonogenic activ