Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T-cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. As expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8+ T-cell development was required to obtain a mature T-cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T-cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity.

The recognition and neutralization of tumour cells is one of the big challenges in immunity. The immune system has to recognize syngeneic tumour cells and has to be primed and respond in an adequate manner. Priming of a leukaemia‐specific immune response is a crucial step in tumour immunology that can mislead to tumour tolerance either by T cell ignorance, deletion or Treg induction. To resemble the situation of acute lymphoblastic leukaemia (ALL) in patients, we used the murine BALB/c model with syngeneic BM185 tumour cells. We established a tumour cell line that expresses the neo‐antigen ovalbumin (BM185‐OVA/GFP) to allow the application of T cell receptor transgenic, antigen‐specific CD4+ T cells. Here, we demonstrate that effective anti‐ALL immunity can be established by in vivo priming of CD4+ T cells that is sufficient to differentiate into effector cells. Yet they failed to control tumour alone, but initiated a Th1 response. An efficient tumour clearance was dependent on both antigen‐specific CD4+ T cells and CD8+ effector T cells from the endogenous repertoire. The tolerogeneic milieu was characterized by increased Tregs numbers and elevated IL‐10 level. Tregs hamper effective antitumour immune response, but their depletion did not result in reduced tumour growth. In contrast, neutralization of IL‐10 improved median mouse survival. Future therapies should focus on establishing a strong CD4+ T cells response, either by adjuvant or by adoptive transfer.

Co-transplantation of genetically engineered T cell precursors together with hematopoietic stem cells is a promising means of specifically target tumor-associated antigens but avoid non specific alloreactivity causing graft versus host disease complicating the adoptive transfer of allogeneic mature T cells. However, genetic modification of undifferentiated precursor cells bears the risk of insertional mutagenesis and off-target-toxicity. Novel safety mechanisms to control activity of genetically engineered cells would ease its transfer into clinical practice. Here, we have modified human cord blood-derived CD34+ cells with an inducible Caspase 9 suicide switch using alpharetroviral SIN vectors, which provide a favorable integration pattern lowering the risk of insertional mutagenesis. Additionally two different promotors and envelope genes were comparatively assessed. Transduction efficiency was twice as efficient when using the RD114/TR envelope as compared to VSVg. Transgene expression was three times higher with the MPSV promoter as compared to the EFS promotor. These differences are of highly relevant since iC9 was responsive to the respective dimerizer under MPSV control only. These results are important since they stress the necessity to carefully appropriate vector systems and lay the basis for the development of engineered off-the-shelf cellular products.

Adoptive transfer (AT) of TCR-gene modified T cells has been associated with a couple of draw-backs. 1. Rigorous in vitro stimulation required for TCR-gene transduction can profoundly impact in vivo function after AT, 2. TCR-chain mispairing can cause potentially life threatening autoreactivity, and 3. HLA-matched T cell donors are required. Here we present a system designed to transduce stem cells with an anti-leukemic TCR-gen whose expression is regulated by a tetracycline-response-element (TRE). The new vector was primarily evaluated on murine T 58 cells and human Jurkat cells. The addition of doxycycline resulted in prompt expression of the TCR and its respective reporter in vitro. Importantly, removal of the agent resulted in complete downregulation within two days. These switch on/off cycles could be repeated multiple times over several weeks showing the stability of the system. Consecutively we generated T-lineage committed lymphoid precursor cells (predominantly with a phenotype comparable to double CD4 and CD8 negative (DN)2 and DN3 thymocytes) using the bone-marrow-derived OP9 cells that express the Notch ligand DL1. T cell precursors were cultured from Lin-, Sca 1+, c-kithi stem cells using Flt3-ligand and IL-7. After transduction with our new plasmid T-cell precursors carrying the gene of interest were generated. This is relevant since it will allow co-transfer of third party precursors avoiding thymic negative selection of leukemia TCR-expressing immature precursors.