Current strategies in immunotherapy for acute myeloid leukemia.

The prognosis of acute myeloid leukemia, particularly when associated with adverse chromosomal or molecular aberrations, is poor due to a high relapse rate after induction chemotherapy. Postremission therapy for elimination of minimal residual disease remains a major challenge. Allogeneic hematopoietic stem cell transplantation has proven to provide a potent antileukemic effect. Novel strategies are needed for patients ineligible for this treatment. Here current immunotherapeutic concepts in acute myeloid leukemia in a nonallogeneic hematopoietic stem cell transplantation setting are reviewed. Data gathered with different monoclonal antibodies are discussed. Adoptive transfer of NK and T cells is reviewed, including evolving data on T-cell engineering. Results of systemic cytokine administration and of therapeutic vaccinations with peptides, modified leukemic cells and dendritic cells are presented. One particular focus of this review is the integration of currently running clinical trials. Recent immunotherapeutic studies have been encouraging and further interesting results are to be expected

Immunotherapy for acute myeloid leukemia.

Despite longstanding efforts in basic research and clinical studies, the prognosis for patients with acute myeloid leukemia (AML) remains poor. About half of the patients are not medically fit for intensive induction therapy to induce a complete remission and are treated with palliative treatment concepts. The patients medically fit for intensive induction therapy have a high complete remission rate but the majority suffers from relapse due to chemo-refractory leukemic cells. Allogeneic stem cell transplantation as post-remission therapy can significantly reduce the likelihood of relapse, but it is associated with a high rate of morbidity and mortality. Novel therapeutic concepts are therefore urgently sought after. During recent years, the focus has shifted towards the development of novel immunotherapeutic strategies. Some of the most promising are drug-conjugated monoclonal antibodies, T-cell engaging antibody constructs, adoptive transfer with chimeric antigen receptor (CAR) T cells, and dendritic cell vaccination. Here, we review recent progress in these four fields and speculate about the optimal time points during the course of AML treatment for their application

Impaired NK cells and increased T regulatory cell numbers during cytotoxic maintenance therapy in AML.

Cyclic cytotoxic maintenance therapy can be applied to patients with AML in post-remission. We studied the immune status of AML patients in complete remission and the effect of maintenance therapy on different immune cell populations. Patients in complete remission had reduced NK, TH and Treg counts and a reduced NK activation capacity. In the course of cytotoxic maintenance therapy, NK counts further declined, while TH and Treg cells increased, with lower proliferative potential of TH cells. We conclude that immunotherapeutic approaches in post-remission have to consider reduced NK cell function and further impairment of cellular immune responses during cytotoxic therapy

New generation dendritic cell vaccine for immunotherapy of acute myeloid leukemia.

Dendritic cell (DC)-based immunotherapy is a promising strategy for the elimination of minimal residual disease in patients with acute myeloid leukemia (AML). Particularly, patients with a high risk of relapse who are not eligible for hematopoietic stem cell transplantation could benefit from such a therapeutic approach. Here, we review our extensive studies on the development of a protocol for the generation of DCs with improved immunogenicity and optimized for the use in cell-based immunotherapy. This new generation DC vaccine combines the production of DCs in only 3 days with Toll-like receptor-signaling-induced cell maturation. These mature DCs are then loaded with RNA encoding the leukemia-associated antigens Wilm's tumor protein 1 and preferentially expressed antigen in melanoma in order to stimulate an AML-specific T-cell-based immune response. In vitro as well as in vivo studies demonstrated the enhanced capacity of these improved DCs for the induction of tumor-specific immune responses. Finally, a proof-of-concept Phase I/II clinical trial is discussed for post-remission AML patients with high risk for disease relapse

Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission.

Active dendritic cell (DC) immunization protocols are rapidly gaining interest as therapeutic options in patients with acute myeloid leukemia (AML). Here we present for the first time a GMP-compliant 3-day protocol for generation of monocyte-derived DCs using different synthetic Toll-like receptor (TLR) agonists in intensively pretreated patients with AML. Four different maturation cocktails were compared for their impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation in 20 AML patients and 25 healthy controls. Maturation cocktails containing the TLR7/8 agonists R848 or CL075, with and without the addition of the TLR3 agonist poly(I:C), induced DCs that had a positive costimulatory profile, secreted high levels of IL-12(p70), showed chemotaxis to CCR7 ligands, had the ability to activate NK cells, and efficiently stimulated antigen-specific CD8+ T cells. Our results demonstrate that this approach translates into biologically improved DCs, not only in healthy controls but also in AML patients. This data supports the clinical application of TLR-matured DCs in patients with AML for activation of innate and adaptive immune responses

T cells are functionally not impaired in AML: Increased PD-1 expression is only seen at time of relapse and correlates with a shift towards the memory T cell compartment.

BACKGROUND: T cell function is crucial for the success of several novel immunotherapeutic strategies for the treatment of acute myeloid leukemia (AML). However, changes in phenotype and function of T cells have been described in various hematologic malignancies, mimicking T cell exhaustion known from chronic viral infections. Detailed knowledge about phenotype and function of T cells in AML patients at different stages of the disease is indispensable for optimal development and application of immunotherapeutic strategies for this disease. METHODS: We used flow cytometry-based assays to characterize T cell phenotype and function in peripheral blood and bone marrow of AML patients at diagnosis, at relapse after intensive chemotherapy, and at relapse after allogeneic stem cell transplantation (SCT). Surface expression of CD244, PD-1, CD160, and TIM-3 was determined, and proliferation and production of IFN-γ, TNF-α, and IL-2 were measured. RESULTS: We detected similar expression of inhibitory molecules on T cells from patients at diagnosis and from age-matched healthy controls. At relapse after SCT, however, PD-1 expression was significantly increased compared to diagnosis, both on CD4(+) and CD8(+) T cells. This pattern was not associated with age and cytomegalovirus (CMV) status but with a shift towards effector memory cells in relapsed AML patients. Proliferation and cytokine production assays did not reveal functional defects in T cells of AML patients, neither at diagnosis nor at relapse. CONCLUSION: We thus conclude that T cell exhaustion does not play a major role in AML. Immunotherapeutic strategies targeting autologous T cells thus have particularly good prospects in the setting of AML. &nbsp

Identifying immune resistance mechanisms to CD33/CD3 BiTE® antibody construct (AMG 330) mediated cytotoxicity.

In our previous work, we showed elimination of primary acute myeloid leukemia (AML) cells by CD33/CD3 BiTE® antibody construct (AMG 330) mediated cytotoxicity (Krupka et al, Blood 2014). The goal of the present study was to identify innate and adaptive resistance mechanisms to AMG 330 mediated lysis of AML cells. Immune checkpoint molecules have been shown to be a highly relevant escape mechanism of malignant cells to evade innate and adaptive immunity. Previously, it was shown that AML cells upregulate the expression of inhibitory ligands in response to proinflammatory cytokines (Krönig et al, 2014). As AMG 330 mediated T-cell activation induces high levels of the proinflammatory cytokines IFNγ and TNFα, we assessed the constitutive and inducible expression profile of different immune checkpoint molecules on AML cell lines and primary AML cells, including PD-L1, HVEM, ILT3 and SLAMF7 by flow cytometry. No constitutive expression was observed for PD-L1 at time of primary diagnosis in 83.7% of the cases (103/123). In contrast, constitutive expression of HVEM and ILT3 was detected in 73.7% (42/57) and 91.9% (68/74) of patient samples, respectively. Adaptive resistance was evaluated by incubating AML cell lines and primary AML samples with IFNγ and TNFα. We observed an upregulation of PD-L1 and SLAMF7 on AML cell lines and on primary AML patient samples whereas HVEM and ILT3 did not show a significant change in expression level. To test the functional relevance of the immune checkpoint molecules upon AMG 330 mediated lysis, we used an ex vivo long term culture system that enabled us to analyse the dynamic process of receptor-ligand interaction over time. Blockade of the PD-1/PD-L1 interaction resulted in a significantly increase in AMG 330 mediated lysis of primary AML cells (n=9, p=0.03). Currently, blockade of the inducible molecule SLAMF7 in AMG 330 mediated cytotoxicity is being tested. Blocking of HVEM or ILT3 did not result in a significant increase in T cell activation and concomitant lysis of AML cells suggesting a less relevant role of HVEM and ILT3 in resistance to AMG 330 mediated cytotoxicity. The latter might also be influenced by the cytokine microenvironment which favours immune resistance of AML cells.  Using a bead based multiplex assay we screened the bone marrow (BM) plasma from 16 AML patients and 3 healthy donors (HD) for the presence of 33 cytokines. The cytokine profile differed between AML patients and healthy donors (HDs). The plasma levels of IL-8, IP-10 and CXCL-16 were higher in the AML samples compared to those of HDs (p=0.0041, 0.0248 and 0.0289, respectively). In contrast, EGF, FLT3-ligand, RANTES and IL-4 were significantly lower in AML samples compared to HDs (p=0.0227, 0.0145, 0.0041 and 0.0041, respectively). However, we did observe a high inter-patient variability of cytokine composition in AML. To explore the functional relevance of the BM plasma on AMG 330 mediated cytotoxicity, cocultures of AML cell lines and HD T cells were set up using different sources of plasma including fetal calf serum (FCS) and patient derived BM plasma. Interestingly, AMG 330 mediated cytotoxicity was significantly reduced using patient derived BM plasma (n=5) compared to cultures containing FCS (n=4) (mean % lysis FCS 97.4 vs PT 70.6). This was accompanied by a considerable impairment in T-cell proliferation (mean % proliferation FKS 44.7% vs PT 26.6%). Currently, we are investigating which soluble factors are responsible for the immunosuppressive effects and if we can increase lysis efficacy and T-cell proliferation through specific blocking of them. In summary we have identified possible resistance mechanisms of AML cells to AMG 330 mediated cytotoxicity. Dynamic receptor-ligand interactions between target and effector cells as well as soluble factors contribute to AMG 330 mediated lysis of primary AML cells. We hypothesize that AMG 330 mediated cytotoxicity can be augmented through combinatorial approaches including PD-1 blockade. The significance of our findings will first be validated in an in vivo mouse model and prospectively translated into human studies