P477: combination of different immunotherapeutic drugs enhances specific t cell immune responses against leukemic cells and leukemic progenitor/stem cells in acute myeloid leukemia

Background: Immunotherapeutic approaches have become increasingly important in cancer treatment for various solid tumors, malignant lymphatic diseases, but also for acute myeloid leukemia (AML). Some of the immunotherapies in AML, such as allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion, have long been part of routine clinical practice, whereas other immunotherapeutic approaches have only recently entered clinical practice or need to be further developed. Aims: For immunotherapies, the mechanisms of the underlying immune responses and the responsible antigen structures still need to be further investigated. Combination treatments seem to be very promising and are therefore further investigated in this study using primary leukemic cells and leukemic progenitor and stem cells (LPC/LSC) from AML patients. Methods: We used functional colony forming immunoassays and ELISpot assays. We investigated the influence of several drugs on its own or in combination on the antigen-specific immune responses by specific cytotoxic T cells (CTL) stimulated with Leukemia-associated antigens (LAA). These CTL were targeted against leukemic cells and also LPC/LSC of AML patients. The drugs used were anti-PD-1 (Nivolumab), anti-CTLA-4 (Ipilimumab), All-trans Retinoic Acid (ATRA), 5-Azacitidine (5-Aza) and Lenalidomide. Each drug was tested on its own as well as in combination with anti-PD-1. Results: Without additional drugs, we were able to detect a relevant frequency of immune reactions against different LAAs (NPM-1, WT-1, RHAMM, PRAME). 16 AML patients were tested who showed an immune response against at least one of these antigens. The samples were then stimulated with the LAA that showed a strong response to test the drugs mentioned above alone or in combination with the anti-PD-1 antibody. We tested immune responses against leukemic cells, focusing on LPC/LSC. Immune effects increased adding anti-PD-1 to the stimulated specific T cells, 9/16 patients showed a significant reduction of colonies (range 11-75%) with a mean reduction of 40%. In 8/16 patients a reduction of colonies was found when T cells were incubated with anti-CTLA-4. The combination of anti-PD-1 and anti-CTLA-4 showed no additional effect on immune responses compared to anti-PD-1 or anti-CTLA-4 alone. Using 5-Aza alone had an effect on 8/16 patients, the mean reduction was 54% (range 14-100% in responders), the effect of anti-PD1 was enhanced by adding 5-Aza in 13 of 16 patients. In fact, employing LAA in combination with anti-PD-1 and with 5-Aza had a particularly strong effect with a mean reduction of 62% (range 22-100% in responders). Lenalidomide also showed an increased effect in 8/16 patients and the combination with anti-PD-1 augmented this effect (Lena alone mean 49% versus 53% in combination with anti-PD-1 in responders), but the effects were not as strong compared with the combination of 5-Aza/anti-PD-1. ATRA showed just a slightly augmented effect in a few patients (4/16) and in the combination with anti-PD1 the effect increased. Summary/Conclusion: Taken together, combinations of immunotherapeutic approaches increase antigen-specific immune responses against leukemic cells but also LPC/LSC, especially the combination of LAA-Peptides with the anti-PD-1 antibody and one further immunomodulating drug, like 5-Aza, could be an interesting option for further clinical studies. Keywords: Immunotherapy, Acute myeloid leukemia, Leukemic stem cell, Leukemia-associated antigen