ALBERT

Description: It has been demonstrated that in a proportion of patients with Acute Myeloid Leukaemia (AML) their leukaemic blasts can be induced to differentiate into Dendritic Like Leukaemic Cells (DLLC) in vitro under the influence of GMCSF, IL-4 and TNF alpha. DLLC hold promise as cellular vaccines aimed at eradicating Minimal Residual Disease following successful induction of remission by chemotherapy. In A Phase I/II Multi-Centre Study we assessed leukaemic blasts of 21 patients with AML for their cytokine driven potential to differentiate into DLLC in vitro. 7 out of 21 (33%) patients showed DLLC conversion of their AML blasts based on morphological appearances and immunophenotype. 5 patients have completed chemotherapy and were then eligible to receive 4 escalating doses of subcutaneous DLLC vaccine. Immune responses to the administration of the vaccine are monitored by a combination of methods. The emergence of leukaemia specific T-cells following vaccination is demonstrated using Elispot assaying of Interferon gamma release in an in vitro re-stimulation assay. In WT1 expressing patients HLA-Tetramers allow to determine the frequency of WT1 specific T-cells. Regulatory T-cells (CD4/CD25 positive) are monitored by flow-cytometry. Monitoring of Minimal Residual Disease (MRD) is undertaken by means of real-time quantitative RT-PCR for leukaemia specific fusion transcripts or WT1 gene expression. 4 patients have so far completed the DLLC vaccination course. Vaccination was well tolerated with minimal side effects. A more than two-fold increase of leukaemia specific cytotoxicity was demonstrated following DLLC vaccination in one patient, while reduction of MRD was seen in several patients during the vaccination. The increase in CD4 /CD25 positive regulatory T-cells observed in several patients post vaccination may serve to dampen the induced anti-leukaemic immunity.

Description: Dendritic cell (DC) vaccines in leukemia show promise as a novel treatment modality however to date clinical evidence of efficacy has been limited. This is likely to be as a consequence of a combination of factors, which include insufficient immunogenicity of the DC vaccine and vaccination taking place in an environment adverse for generation of effective immune responses i.e. in patients with active disease. Our study aims to generate more efficient cytotoxic T cell (CTL) responses by improving DC uptake and presentation of leukemia cells in the remission state and will be applicable to both acute and chronic leukemias. Monoclonal antibodies (MoAbs) have been used to treat malignant cells prior to co-culture with DCs to enhance cross-presentation and generation of specific CTLs. We investigated whether this approach could improve DC induction of CTL responses in comparison to DCs loaded with UVB irradiated apoptotic leukemia cells. In this in vitro study we generated dendritic cells from adherent mononuclear cells (differentiation with GM-CSF and IL-4) of patients in remission following chemotherapy for acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). The immature DCs were loaded with autologous leukemia cells from the patients’ presentation samples. The presentation leukemia cells were treated with either UVB irradiation or appropriate monoclonal antibodies (the anti-CD33 MoAb Mylotarg in AML and CML; the anti CD20 MoAb Rituximab or the anti-CD 52 MoAb Alemtuzumab in CLL). Apoptosis was assessed by Annexin/Propidium iodide labelling. Treatment of the leukemia cells by different MoAbs induced varying degrees of apoptosis. DC uptake of antibody treated or apoptotic leukemia cells was assessed by dual colour staining. Leukemia cells were stained with PKH and DCs labelled with FITC-CD80 or CD86. DC uptake was more efficient with MoAb treated cells irrespective of the degree of apoptosis induced by the MoAb. DCs were matured with TNFa for two days then co-cultured with autologous T cells for one week. T cell subsets and Regulatory T cells were assessed on the presentation and remission samples.The T cells were harvested and their cytoxicity assessed in an Interferon Gamma (IFNg) ELISPOT assay where the unmodified blasts were used as stimulators. Initial results show enhanced anti-leukemia activity in the MoAb treated group as compared to the irradiated group. A similar set up using allogeneic DCs and T cells confirmed the augmentation of CTL responses with MoAb treatment of leukemia cells.The use of MoAb in this setting shows promise for improvement in the success and applicability of DC vaccine strategies in leukemia.