The antitumor effect of adoptively transferred tumor-specific cytotoxic T lymphocytes (CTLs) is impaired by the limited capacity of these cells to expand within the tumor microenvironment. Administration of interleukin 2 (IL-2) has been used to overcome this limitation, but the systemic toxicity and the expansion of unwanted cells, including regulatory T cells, limit the clinical value of this strategy. To discover whether transgenic expression of lymphokines by the CTLs themselves might overcome these limitations, we evaluated the effects of transgenic expression of IL-2 and IL-15 in our model of Epstein Barr Virus-specific CTLs (EBVCTLs). We found that transgenic expression of IL-2 or IL-15 increased the expansion of EBV-CTLs both in vitro and in vivo in a severe combined immunodeficiency disease (SCID) mouse model and enhanced antitumor activity. Although the proliferation of these cytokine genes transduced CTLs remained strictly antigen dependent, clinical application of this approach likely requires the inclusion of a suicide gene to deal with the potential development of T-cell mutants with autonomous growth. We found that the incorporation of an inducible caspase-9 suicide gene allowed efficient elimination of transgenic CTLs after exposure to a chemical inducer of dimerization, thereby increasing the safety and feasibility of the approach.
Summary 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.
The online version of this article has a Supplementary Appendix. BackgroundCytokine-induced killer cells are ex vivo-expanded cells with potent antitumor activity. The infusion of cytokine-induced killer cells in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses have been 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 MethodsSFG-retroviral vectors coding for anti-CD33-ζ and anti-CD33-CD28-OX40-ζ chimeric receptors were used to transduce cytokine-induced killer cells. Transduced cells were characterized in vitro for their ability to lyse leukemic targets (4-hour 51 chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate ( 3 H-thymidine-incorporation assay) and to secrete cytokines (flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34 + hematopoietic progenitor cells was evaluated by analyzing the colony-forming unit capacity after co-incubation. ResultsCytokine-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 prominent CD33-specific proliferative activity, with the release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of the anti-leukemic activity of cytokine-induced killer cells. Importantly, even though the cytokine-induced killer cells transduced with anti-CD33 chimeric receptors showed toxicity against normal hematopoietic CD34 + progenitor cells, residual clonogenic activity was preserved. ConclusionsOur results indicate that anti-CD33 chimeric receptors strongly enhance anti-leukemic cytokine-induced killer cell functions, suggesting that cytokine-induced killer cells transduced with these molecules might represent a promising optimized tool for acute myeloid leukemia immunotherapy.Key words: chimeric T-cell receptor, acute myeloid leukemia, CD33, cell therapy, gene therapy. Haematologica 2010;95(12):2144-2152. doi:10.3324/haematol.2010 This is an open-access paper. © F e r r a t a S t o r t i F o u n d a t i o n
Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) can induce objective clinical responses in patients with malignant diseases. The option of providing a proliferative and survival advantage to adoptively transferred CTLs remains a challenge to improve their efficacy. Host lymphodepletion and administration of recombinant interleukin-2 (IL-2) are currently used to improve CTL survival and expansion after adoptive transfer, but these approaches are frequently associated with significant side effects and may increase proliferation of T regulatory cells. IL-7 is a crucial homeostatic cytokine that has been safely administered as a recombinant protein. However, while IL-7 induces robust expansion of naive and memory T lymphocytes, the lack of expression of the IL-7 receptor alpha chain (IL-7Ralpha) by CTLs precludes their response to this cytokine. We found that CTLs can be genetically modified to re-express IL-7Ralpha, and that this manipulation restores the response of these cells to IL-7 without apparent modification of their antigen specificity or dependency, and without changing their response to other common gamma (gammac) chain cytokines. This approach may allow selective expansion of CTLs without the unwanted effects associated with IL-2.
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