Leukemia-derived Dendritic Cells Research Articles

Anti-Leukemic Effects Induced by Dendritic Cells of Leukemic Origin from Leukemic Blood Samples Are Comparable under Hypoxic vs. Normoxic Conditions.

Hypoxia can modulate the immune system by affecting the function and activity of immune cells, potentially leading to altered immune responses. This study investigated the generation of leukemia-derived dendritic cells (DCleu) from leukemic blasts and their impact on immune cell activation under hypoxic (5-10% O2) compared to normoxic (21% O2) conditions using various immunomodulatory Kits. The results revealed that DC/DCleu-generation was similar under hypoxic and normoxic conditions, with no significant differences observed in frequencies of generated DC/DCleu. Furthermore, the study showed that the activation of immune cells and their anti-leukemic activity improved when T cell-enriched immunoreactive cells were co-cultured with DC/DCleu which were generated with Kit I and M compared to the control after mixed lymphocyte cultures. The anti-leukemic activity was improved under hypoxic compared to normoxic conditions after MLCWB-DC Kit M. These findings suggest that DC/DCleu-cultures of leukemic whole blood with Kits under hypoxic conditions yield comparable frequencies of DC/DCleu and can even increase the anti-leukemic activity compared to normoxic conditions. Overall, this research highlights the potential of utilizing DC/DCleu (potentially induced in vivo with Kits) as a promising approach to enhance immune response in patients with acute myeloid leukemia.

Induction of Cellular and Humoral Immune Responses Is Associated with Durable Remissions in MRD+ AML-Patients after Maintenance Treatment with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine

Background. Maintaining remission in AML after intensive chemotherapy continues to be a challenge, specifically in patients with measurable residual disease (MRD+). Immunotherapy could be an effective way to induce durable disease control for patients in remission. This phase 2 study used an allogenic leukemia-derived dendritic cell vaccine to induce an effective anti-leukemic response to control or eradicated MRD, leading to durable disease control. (ADVANCE-II, Clintrials.gov: NCT03697707). Methods. A total of 20 evaluable AML patients, in complete remission (CR1/CRi), MRD + and ineligible for allo-HSCT at inclusion, were scheduled to receive 4 biweekly doses of vididencel, followed by 2 booster doses at week 14 and 18. Patients aged 34-79 (median 60 years) had an ELN2017 risk score assessed of 13 favorable, 6 intermediate and 1 adverse, with mutations in NPM1 (n=10), CBFB-MYH11 (n=4), IDH2 (n=2), RUNX1-RUNX1T1 (n=2), TP53 (n=1) and CEBPA (n=1). Patients were vaccinated after a median of 8.9 months (range 3 to 60.8 Mo) after reaching CR1/CRi. MRD was assessed at baseline, week 14, 20 and 32 by flow cytometry and/or molecular analyses on bone marrow samples. Immune responses were evaluated on peripheral blood samples taken before and during treatment by a) IFNγ ELISPOT using three antigens (WT1, PRAME and RHAMM), known to be expressed by vididencel, by b) flow cytometry, using a single 40-marker panel, and c) skin biopsies taken 2 days after the first, fourth and sixth dose to assess immune responses in the skin. Evaluation of relapse-free and overall survival was done at data cut-off (3 rd July 2023). Results. All 20 patients have received 4 initial vaccinations, 17 patients received all booster vaccinations and 1 patient received only one booster vaccination. No serious adverse events (AE) or severe AE (grade 3 or higher) related to the treatment have been reported. Related AEs are mainly injection site reactions, such as redness, warmth and swelling, occurring within 48 hours after intradermal administration. To date, after a median follow-up of 24.8 Mo (range 6.6- 54.8) (N=20) 14 patients are still alive with 12 being relapse-free for 24.4 to 54.7 months. The median RFS and OS (not shown) have not yet been reached (Figure 1A). Estimated RFS and OS at 12 months is 73% (range 51-86%) and 87% (range 67-95%), respectively, whereas the 24 months RFS is 59% (range 35-77%) and OS is 73% (range 50-87%). Induction of at least one vaccine induced response (VIR), as measured by IFNγ ELISPOT, was observed in 17 out of 20 patients, with a sustained vaccine induced response (sVIR) in 9 patients. There is a clear correlation (LogRank p=0.03) between number of VIRs and overall survival (Figure 1B), with best survival in patients with at least 3 VIRs. Of these patients, 4 out of 7 had shown MRD conversion (MRD -). Analysis of the immune cell composition at baseline and after vaccination revealed that higher baseline levels of B-cells (p <0.05) , cDC1, cDC2 were observed in patients remaining in CR. Vaccination also improved immune cell frequencies of dendritic cells with highest levels correlating with longer RFS (p<0.05) and OS (p<0.05). Immune histochemistry on skin biopsies confirmed the large influx of CD4+ and CD8+ T-cells as well as antigen presenting cells, in all patients explaining the clinical observation of swelling, warmth and redness in almost all patients. Conclusion/discussion. Vaccination with vididencel resulted in durable disease control, reflected by 2-year RFS and OS of 59% and 73%, respectively and the emerging plateau in the corresponding Kaplan Meier curves. Immune response assays indicate the induction of a strong immune response, with induction of specific T-cell responses towards tumor associated antigens, increased frequencies of B-cells and dendritic cells, all correlating with longer RFS and OS. The locally skin induced immune response leading to influx of a plethora of immune cells, confirmed the mode of action of immune priming through intradermal administration of the cancer vaccine. These results warrant further studies combining vididencel with SOC such as hypomethylating agents in AML maintenance.

Vaccination Using an Allogeneic Leukemia-Derived Dendritic Cell Vaccine, Maintains and Improves Frequencies of Circulating Antigen Presenting Dendritic Cells Correlating with Relapse Free and Overall Survival in AML Patients

Background. Active immunotherapy relies on the patient's immune system for induction of an effective immune response. Not only the frequency, number and exhaustion or activation status of CD8 T cells is important for an efficacious tumor cell lysis, but the quality, number and distribution of different antigen presenting cells is of equal importance. Vididencel is an allogeneic leukemia-derived dendritic cell vaccine used in AML patients in first complete remission (CR1/CRi), but with MRD positivity, to mount an immune response through intradermal vaccination (ADVANCE-II, Clintrials.gov: NCT03697707). Intradermal vaccination is known to induce a specific immune response, by recruitment of skin and blood derived dendritic cells, which process antigen and present them to the immune system (Zuo et al., 2021). In this study circulating dendritic cells and monocytes have been investigated before and during vaccination to investigate the effect of vididencel treatment on the composition of antigen presenting cells in relation to clinical responses. Methods. A total of 20 evaluable AML patients, in CR1/CRi, with measurable residual disease (MRD +) and ineligible for HSCT at inclusion, received 4 biweekly doses of vididencel, followed by 2 booster doses at week 14 and 18. Peripheral blood mononuclear cells were taken at baseline, week 6, 11, 14, 18, 20 and 32, ficoll isolated and cryopreserved in liquid nitrogen. Analysis of immune cell composition was performed by spectral flow cytometry, using a single 40-marker panel. Immune subset analysis of dendritic (live, single, CD45+, Lin-(CD3, 56, 19), CD16-, CD15-) cells was performed and correlated to clinical response, relapse free and overall survival, by high dimensional analysis using FlowSOM. Results. Dendritic cell subsets increased upon vaccination, with levels before and during vaccination being the highest in patients remaining in CR. Gating of the dendritic cell subsets by spectral flow cytometry analyses could be hampered by presence of leukemic blasts in the peripheral blood, expressing for example CD123, a marker required for gating of pDCs. Using gating on the lineage negative, CD15-, CD16- and HLA-DR+ cells, a deep immunophenotyping was performed on dendritic cells in peripheral blood. Baseline analysis showed a significantly higher frequency of CD45RA+, HLA-DR+ cells in patients who remain in CR. Using tSNE and UMAP plots generated to analyse changes in the Lin-, CD14-, CD15-, CD16- and CD11b-, HLA-DR+ population; changes and increases in several subsets were observed, most notably in subsets with cDC1, cDC2 and pDC characteristics, like expression of CD141, CLEC9A, CD1c and CD123. Higher frequencies at baseline of dendritic cells (cDC1 and cDC2) correlated positively with both relapse-free and overall survival. In patients remaining in CR, vaccination further increased or maintained dendritic cell subset frequencies, of which classical cDC1 and cDC2 correlated with longer overall survival (p<0.05). Conclusion: Patients who remain in CR after vididencel treatment had highest baseline levels of HLA-DR+ CD45RA+ cells, which could be myeloid cells able to differentiate into dendritic cell subsets, as in general CD45RA+ is lost during maturation from precursor to matured dendritic cells. Vaccination might improve and induce maturation of dendritic cell subsets, such as cDC1, cDC2 and pDC, which enhance antigen capturing, processing and presentation to tumor-reactive T cells, ultimately leading to improved survival.

Intradermal Vaccination with Vididencel in MRD+ AML-Patients Leads to Increase in Antigen Presenting Cells and T-Cells to the Injection Site, Visualized Using Imaging Mass Cytometry, Showing Local Immune Cell Interactions Leading to Systemic Immune Responses

Background. Vididencel is an allogeneic leukemia-derived dendritic cell vaccine being developed as maintenance treatment in AML patients. Intradermal vaccination using vididencel is known to induce a local skin reaction, with local antigen processing by skin dendritic cells and direct presentation to T cells as well as dendritic cells, crawling out of the skin and presentation of the antigen in the draining lymph nodes for a full-blown immune response. It was previously shown that vididencel is able to induce an influx of immune cells in the skin using standard immunohistochemistry (van de Loosdrecht, 2018). In a phase 2 study vididencel was investigated as AML maintenance treatment after induction of CR1 with chemotherapy (ADVANCE-II, Clintrials.gov: NCT03697707). Using imaging mass cytometry up to 43 markers can be assessed in the same tissue section, to allow in depth analysis of the immune cells on a single cell level infiltrating in the dermis at the injection site. Methods. AML patients, in first complete remission with measurable residual disease (MRD +) and not planned for HSCT at inclusion, received 4 biweekly doses of vididencel followed by 2 booster doses at week 14 and 18. Skin biopsies of the injection site were taken 2 days after the 1 st, 4 th and 6 th vaccination. Control samples were collected from unvaccinated/unaffected skin in two patients and from healthy controls. Samples were fixed and embedded in paraffin, and stained by standard immunohistochemistry for immune cell markers like CD4, CD8 and CD68 in the central lab. For patients treated at Haukeland University Hospital (N=6), tissue sections were prepared, mounted and stained with the panel of 29 antibodies labelled with heavy metal isotopes for use in the Hyperion mass cytometry imaging. For each sample a region of interest (ROI) of 2.8mm 2 was scanned based on the localization of the immune infiltrates, guided by standard H&E staining from neighboring slides. Results In 88.5% of patients injection site reactions were reported in this phase 2 study, characterized by redness, swelling and warmth at the site of intradermal injection, which were mild (maximum grade 1 or 2), and were of short duration (generally resolved within 12 days). Skin biopsies of a subset of 6 patients were analyzed using Hyperion. In standard single stain immunohistochemistry a large influx of CD4, CD8 and CD68 cells was observed in contrast to normal skin. Imaging mass cytometry confirmed these dense immune infiltrations. Using additional markers to characterize monocytes, dendritic cells and T-cells revealed that these cells are in close proximity and suggestive for cell-to-cell interaction (Figure 1). At sites of dense immune infiltration, influx of GranzymeB expressing CD8 T-cells and CD38 positive CD4 T-cells were observed as well as CD1c+ or CD141+ dendritic cells with high HLA-DR expression. These T-cells and dendritic cells are in close contact suggestive for patients' dendritic cells migrating from blood to skin, inducing a local immune response. Conclusion/discussion. Vaccination with vididencel triggered an inflammatory skin reaction in the majority of patients, with redness, warmth and swelling, of short duration, indicative for an active immune response at the site of injection. Skin biopsies using standard immunohistochemistry showed influx of activated CD4 and CD8 T cells as well as antigen presenting cell, including dendritic cells.Extended analysis with multiparameter imaging mass cytometry showed specific immune cell subsets infiltrating in these site of injection sites. The observed co-localization of the different immune subsets following vaccination supports the hypothesis of local antigen capturing by blood derived and local antigen presenting cells (Zuo et al, 2021). The local immune response may ultimately leads to a systemic immune response and disease control.

Durable Responses and Survival in High-risk Myelodysplastic Syndrome and Acute Myeloid Leukemia Patients Receiving the Allogeneic Leukemia-derived Dendritic Cell Vaccine DCP-001.

Durable Responses and Survival in High-risk Myelodysplastic Syndrome and Acute Myeloid Leukemia Patients Receiving the Allogeneic Leukemia-derived Dendritic Cell Vaccine DCP-001.

Volatile Phases Derived from Serum, DC, or MLC Culture Supernatants to Deduce a VOC-Based Diagnostic Profiling Strategy for Leukemic Diseases.

Volatile organic compounds (VOCs) reflect the metabolism in healthy and pathological conditions, and can be collected easily in a noninvasive manner. They are directly measured using electronical nose (eNose), and may qualify as a systemic tool to monitor biomarkers related to disease. Myeloid leukemic blasts can be transformed into leukemia-derived dendritic cells (DCleu) able to improve (anti-leukemic) immune responses. To profile immunological changes in healthy and acute myeloid leukemic (AML) patients' ex vivo cell cultures, we correlated the cell biological data with the profiles of cell culture supernatant-derived VOCs. DC/DCleu from leukemic or healthy whole blood (WB) were generated without (Control) or with immunomodulatory Kit M (Granulocyte macrophage-colony-stimulating-factor (GM-CSF) + prostaglandin E1 (PGE1)) in dendritic cell cultures (DC culture). Kit-pretreated/not pretreated WB was used to stimulate T cell-enriched immunoreactive cells in mixed lymphocyte cultures (MLC culture). Leukemia-specific adaptive and innate immune cells were detected with a degranulation assay (Deg) and an intracellular cytokine assay (InCyt). Anti-leukemic cytotoxicity was explored with a cytotoxicity fluorolysis assay (CTX). VOCs collected from serum or DC- and MLC culture supernatants (with vs. without Kit M pretreatment and before vs. after culture) were measured using eNose. Compared to the Control (without treatment), Kit M-pretreated leukemic and healthy WB gave rise to higher frequencies of mature (leukemia-derived) DC subtypes of activated and (memory) T cells after MLC. Moreover, antigen (leukemia)-specific cells of several lines (innate and adaptive immunity cells) were induced, giving rise to blast-lysing cells. The eNose could significantly distinguish between healthy and leukemic patients' serum, DC and MLC culture supernatant-derived volatile phases and could significantly separate several supernatant (with vs. without Kit M treatment, cultured vs. uncultured)-derived VOCs within subgroups (healthy DC or leukemic DC, or healthy MLC or leukemic MLC supernatants). Interestingly, the eNose could indicate a Kit M- and culture-associated effect. The eNose may be a prospective option for the deduction of a VOC-based profiling strategy using serum or cell culture supernatants and could be a useful diagnostic tool to recognize or qualify AML disease.

Induction of a Systemic Immune Response during Use of an Allogenic Leukemia-Derived Dendritic Cell Vaccine in MRD+ AML Patients Correlates with Clinical Response and MRD Conversion

Induction of a Systemic Immune Response during Use of an Allogenic Leukemia-Derived Dendritic Cell Vaccine in MRD+ AML Patients Correlates with Clinical Response and MRD Conversion

Use of an Allogeneic Leukemia-Derived Dendritic Cell Vaccine in MRD+ AML-Patients Results in MRD Conversion, Improved Relapse-Free Survival and Vaccine Induced Immune Responses to Tumor Antigens

Background. Maintenance therapy in AML patients with measurable residual disease (MRD) remains challenging. Immunotherapy could be an effective way to induce durable disease control. This phase II study aims to show efficacy of priming the immune system, using an allogenic leukemia-derived dendritic cell vaccine (DCP-001), to induce an effective anti-leukemic response to control or kill remaining malignant cells (ADVANCE-II, Clintrials.gov: NCT03697707). Methods. A total of 20 evaluable AML patients in first complete remission (CR1) with MRD positivity and ineligible for HSCT received 4 biweekly doses of 25e6 or 50e6 cells per intradermal vaccination (cells/vc) and 2 doses as boosts at week 14 and 18 of 10e6 cells/vc. MRD, as primary endpoint, was assessed at baseline, week 14, 20 and 32 by flow cytometry and/or molecular analyses. Evaluation of secondary endpoints relapse-free (RFS) and overall survival (OS) was done at data cut-off (18th July 2022). Peripheral blood samples were taken before and during treatment for analysis of vaccine induced responses as measured by IFNγ ELISPOT and general immune profiles by flow cytometry using a 40-marker panel. Results. Patients aged 34-79 (median 60y) had a cytogenetic risk status assessed of 4 better, 15 intermediate and 1 poor-risk, with mutations in NPM1 (n=10), CBFB-MYH11 (n=4), IDH2 (n=2), RUNX1-RUNX1T1 (n=2), TP53 (n=1) and CEBPA (n=1). All 20 patients have received 4 initial vaccinations, 17 patients received all booster vaccinations and 1 patient received one booster. No serious adverse events (AE) or severe AE (grade 3 or higher) related to the treatment have been reported. Related AEs are mainly injection site reactions, such as redness, warmth and swelling, occurring within 48 hours after intradermal administration. To date, median RFS and OS have not yet been reached (Figure 1), with a median follow up period of 15.4 (range 6.6- 16.8) months for the entire study population. Estimated RFS and OS at 6 months is 83.9% (range 64-94%) and 97% (range 79-99%), respectively. Estimated RFS and OS at 12 months is 70% (range 48-85%) and 87% (67-95%), respectively. MRD response (conversion or >10-fold decrease of MRD) was observed in 7 patients: 5 patients converted to negative and 2 showed a MRD decrease. Responses were observed within 14-20 weeks and durable in 4 out of 5 patients, remaining MRD negative up to week 32. Seven patients remained in CR with stable MRD levels and 6 patients relapsed before week 32. Three patients with stable MRD became eligible for HSCT during study and were transplanted successfully. Immune responses were observed to tumor associated antigens expressed by DCP-001; WT1, RHAMM and PRAME, with specific vaccine induced responses (VIR) in the majority of patients (17/20) and highest number of VIR per antigen observed in patients with MRD responses (Figure 2). Differences in immune profiles at baseline were observed; relapsed patients had a high number of CD8RO+ cells, in particular CD8 CM. On the contrary, MRD responders had the highest level of CD8RA+ cells at baseline, with CD8 EMRA cells at the highest level. Patients with stable MRD had comparable, but slightly lower CD8RO+ levels than MRD responders. Conclusion/discussion. Vaccination with DCP-001 resulted in durable MRD responses with five patients converting to MRD negativity, remaining relapse-free and alive during follow up. This translates in promising survival data with the median RFS for the full patient population not reached to date, at a median FU time of 15.4 months. Highest level of vaccine-induced T-cell responses to leukemia specific antigens were detected in MRD responders. Baseline levels of CD8 memory profiles were markedly different in MRD responders, which might be indicative that lower number of memory cells, and thus especially higher number of CD8 EMRA could reflect the ability to induce a tumor specific cytotoxic T-cell response after vaccination. Relapse on study was limited, with an estimated RFS at 6 months of 83.7%, which is higher than expected for this MRD+ AML patient population, which would be around 45% as shown for placebo-treated patients from a recent phase III study in a similar patient population (Quazar AML001). Immunotherapy with DCP-001 as AML maintenance therapy is safe and effective in triggering immune responses and converting patients from MRD positive to MRD negative, leading to promising relapse-free survival. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1309: DYSREGULATED EXPRESSIONS OF INHIBITORY CHECKPOINT MOLECULES AND THEIR LIGANDS ON T-CELLS AND BLASTS IN AML RELAPSES AFTER STEM CELL TRANSPLANTATION (SCT)

Background: Upregulation of inhibitory checkpoint molecules (ICM) on T-cells and their ligands on leukemic blasts may be a mechanism of acute myeloid leukaemia (AML) relapse after allogeneic stem cell transplantation (SCT). Aims: Better understanding of relapse biology could improve treatment efficacy. Methods: We examined peripheral blood (PB) and bone marrow (BM) samples of 5 AML patients (PTs) relapsing after SCT, and PB of 5 healthy individuals (H), including 2 stem cells donors. ICM (PD-1, CTLA-4) expressions on T-cells and their ligands (CD86, PD-L1, PD-L2) on leukemic blasts were assessed through flow cytometry. PTs’ PB was cultivated with and without “KitM” (GM-CSF+PGE-1) to generate leukemia-derived dendritic cells (DCleu), followed by MLC, enriched by PTs’ or donors’ T-cells. After MLC, immune activation and functionality (degranulation, intracellular cytokine production, blast lysis) was assessed. Results: 1. Whilst all patients showed high expressions of PD-1 on their T-Cells, additional overexpression of CTLA-4 was negatively correlated with responses to relapse treatment. Expression of ICM was low on T-cells from 4/5 healthy individuals (Figure 1). 2. Influence of KitM on ICM/ligand expressions on T-cells/blasts - ICM/ligand expressions on uncultured T-cells/blasts: In contrast to H, PTs presented high co-expressions of CTLA-4 and PD-1 on PB T-cells (Figure 1). In addition, PTs showed high frequencies of PB/BM blasts co-expressing CD86. - DC/DCleu in PB: Generation of DCleu in AML as well as generation of DC in H was successful with KitM pretreated PB vs. Control. - ICM co-expression on T-cells after MLC with KitM pretreated PB: MLC of KitM treated PB enriched with unstimulated PTs T-cells resulted in reduced frequencies of ICM-positive T-cells in 3/5 PTs, and increased frequencies of activated (leukemia specific) T-cells in 3/5 PTs. Beyond, blast lysis was improved in 4/5 samples treated with KitM. Both findings were not observed when PB cells were cultivated in the absence of KitM. 3. Possible impact of ICM profiles on clinical outcome in a particular casePT1 suffered from early relapse both after 1st and 2nd SCT from her healthy father. A role of ICM in relapse mechanisms was suggested by CTLA-4/PD-1 expression on her T-cells and CD86 expression on AML blasts. In addition, >90% of the healthy father’s T-cells expressed CTLA-4/PD-1, which might have contributed to treatment failure. In contrast, T-cells from PT1’s mother presented with low ICM levels (Figure 1), suggesting that the mother might have been a better donor. Stimulation of PT1’s PB cells with KitM generated DCleu, decreased ICM-expressions and increased T-cell activity. KitM pretreated samples also revealed improved blast lysis after MLC. Image:Summary/Conclusion: T-cells and blasts of AML PTs relapsing after SCT uniformly co-expressed ICM and their ligands, which could be a reason for inferior immune responses. High aberrant ICM-expressions, in particular CTLA-4, on donor T-cells could be responsible for relapse after SCT by inactivating antileukemic immune reactions. Hence, checkpoint-inhibitory antibodies might represent a specific therapeutic option in these patients. Further, interactions between ICM-expressions on healthy (fresh) donor T-cells could have an impact in therapy responses and should be evaluated in donor selection. Finally, generation of DCleu by treatment with KitM triggers immune responses in MLC alongside with reduced ICM-expressions on T-cells, possibly reducing their inhibitory effects and therefore improving antileukemic responses.

In Vitro Generated Dendritic Cells of Leukemic Origin Predict Response to Allogeneic Stem Cell Transplantation in Patients With AML and MDS.

Allogeneic stem cell transplantation (alloSCT) is the treatment of choice for many patients with acute myeloid leukemia (AML) and myelodysplastic syndrome. The presentation of leukemic or allospecific antigens by malignant blasts is regarded as a crucial trigger for an effective allogeneic immune response. Conversely, insufficient stimulatory capacity by the leukemic blasts is thought to be a relevant escape mechanism from cellular immunotherapy (alloSCT). Our purpose was to test, whether the ability of malignant blasts to differentiate in vitro toward dendritic cells of leukemic origin (DCleu) is associated with clinical outcome. We isolated leukemic blasts from peripheral blood or bone marrow of AML and myelodysplastic syndrome patients before alloSCT (n=47) or at relapse after alloSCT (n=22). A panel of 6 different assays was used to generate DCleu in vitro. Results were correlated with clinical outcome. DCleu could be generated from all 69 samples. Significantly higher mean frequencies of DCleu were found in clinical long-term responders versus nonresponders to SCT (76.8% vs. 58.8%, P=0.006). Vice versa, the chance for response to SCT was significantly higher, if a DCleu+/dendritic cells (DC) ratio of >50% could be reached in vitro (P=0.004). Those patients were characterized by a longer time to relapse (P=0.04) and by a higher probability for leukemia-free survival (P=0.005). In vitro generation of DC and DCleu from leukemic blasts correlated with the clinical outcome. This observation may support a role of leukemic antigen presentation by "leukemia-derived DC" for the stimulation of an allogeneic immune response in AML.

Treatment with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine in AML Patients Shows MRD Conversion and Improved Survival

Treatment with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine in AML Patients Shows MRD Conversion and Improved Survival

Conversion of AML-blasts to leukemia-derived dendritic cells (DCleu) in ‘DC-culture-media’ shifts correlations of released chemokines with antileukemic T-cell reactions

Dendritic cells (DC) and T-cells are mediators of CTL-responses. Autologous (from patients with acute myeloid leukaemia (AML) or myelodysplasia (MDS)) or allogeneic (donor)-T-cells stimulated by DCleu, gain an efficient lysis of naive blasts, although not in every case. CXCL8, -9, -10, CCL2, -5 and Interleukin (IL-12) were quantified by Cytometric Bead Array (CBA) in supernatants from 5 DC-generating methods and correlated with AML-/MDS-patients’ serum-values, DC-/T-cell-interactions/antileukemic T-cell-reactions after mixed lymphocyte culture (MLC) and patients’ clinical course. The blast-lytic activity of T-cells stimulated with DC or mononuclear cells (MNC) was quantified in a cytotoxicity assay. Despite great variations of chemokine-levels, correlations with post-stimulation (after stimulating T-cells with DC in MLC) improved antileukemic T-cell activity were seen: higher released chemokine-values correlated with improved T-cells’ antileukemic activity (compared to stimulation with blast-containing MNC) - whereas with respect to the corresponding serum values higher CXCL8-, -9-, and -10- but lower CCL5- and -2-release correlated with improved antileukemic activity of DC-stimulated (vs. blast-stimulated) T-cells. In DC-culture supernatants higher chemokine-values correlated with post-stimulation improved antileukemic T-cell reactivity, whereas higher serum-values of CXCL8, -9, and -10 but lower serum-values of CCL5 and -2 correlated with post-stimulation improved antileukemic T-cell-reactivity. In a context of ‘DC’-stimulation (vs serum) this might point to a change of (CCL5 and -2-associated) functionality from a more ‘inflammatory’ or ‘tumor-promoting’ to a more ‘antitumor’-reactive functionality. This knowledge could contribute to develop immune-modifying strategies that promote antileukemic (adaptive) immune-responses.

Conversion from MRD Positive to Negative Status in AML Patients in CR1 after Treatment with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine

Background. Persistence of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) remains a poor prognostic factor and unmet medical need. The allogeneic leukemia-derived dendritic cell vaccine, DCP-001, has shown in a phase I study to generate both humoral and cellular immune responses and is safe for clinical practice (van de Loosdrecht et al., Cancer Immunol. Immunother. 2018). In the current phase II study (Clintrials.gov: NCT03697707) we show the capability of DCP-001 to convert MRD positive patients to negative, leading to deeper remissions. Additionally, it is shown that DCP-001 induces immune responses, also specifically against tumor associated antigens known to be present in DCP-001 and relevant for AML. Methods. The current trial aims to enroll up to 20 AML -patients, ineligible for HSCT, who are in in first complete remission (CR1) but who are still MRD positive. MRD is assessed in the bone marrow through flow cytometry and/or qPCR (eg NPM1). Patients receive a primary vaccination regimen of 4 times 25.106or 50.106cells per vaccination, biweekly, followed by two booster vaccinations (10.106cells/vaccination) at week 14 and 18 after start of treatment. Primary endpoints of this trial are the safety and tolerability of the two vaccination schedules and the effect of vaccination on the MRD status. Additionally, cellular and humoral immune response induced by DCP-001 are evaluated in peripheral blood using several assay methods, including flow cytometry and IFN-ϒ ELISpot against known TAA's such as WT-1, RHAMM and PRAME. Results. Up to 15 July 2020, ten patients (age 41-76; 5 male, 5 female) have been enrolled and dosed within the study, completing the first dose cohort of 25.106cells/vaccination. All vaccinations were well tolerated and adverse events to the vaccine were limited to local injection site reactions such as redness, swelling and warmth (maximum grade 2). Three of the ten patient relapsed before the vaccination schedule was completed. Four patients could be evaluated for MRD; Two patients became MRD negative at the first timepoint after the initial vaccinations (week 14), and remained negative until end of active FU (week 32), two other patients remained in CR, but with MRD positivity. For the remaining three patients, to date, no MRD outcome is available yet but patients remain in CR (see swimmers plot for overview, Figure 1). Evaluation of the immune response before, during and after DCP-001 vaccination has only be performed in a small subset of three patients thus far. Of these patients one showed a clinical response becoming MRD negative after vaccination. In this patient we observed an increase in tumor specific functional T-cells assessed by IFNγ ELISPOT, either as a recall or primary response, as shown by the response observed to WT-1 (see for example Figure 2). These tumor associated antigens are known to be expressed by DCP-001 and included WT1, PRAME and RHAMM. Induction of specific memory CD8 and CD4 cells could be observed upon vaccination which might be related to the clinical response. Conclusion/discussion.Preliminary data from this ongoing study confirms that vaccination with DCP-001 is able to generate a tumor-specific immune response and may lead to potential tumor control. Two patients were actually converted from being MRD positive at start of vaccination to MRD negative during the study. These patients continue to be in complete remission for at least a year after vaccination. This study continues to enroll patients at a higher vaccine dose of 50.106cells per vaccination and additional data on induced immune responses and MRD status will be shown. Disclosures van de Loosdrecht: novartis: Honoraria; celgene: Honoraria. Wagner:Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Other: Travel grand; MSD: Membership on an entity's Board of Directors or advisory committees; Medac: Other: Travel grand; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Amgen: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Geron: Consultancy, Honoraria. Rovers:DCprime: Current Employment.

Potential of immunotherapies in the mediation of antileukemic responses for patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) - With a focus on Dendritic cells of leukemic origin (DCleu)

New (non-immunotherapeutic) treatment-strategies for AML/MDS-patients are under development. Dendritic cells (DCs) and ‘leukemia-derived DC’ (DCleu) connect the innate and the adaptive immunesystem and (re-)activate it, in their capacity as professional antigen-presenting cells (APCs). They can be generated ex vivo from peripheral blood mononuclear cells (PBMNCs) or whole blood (WB), containing the –physiological-cellular/soluble microenvironment of individual patients using various DC/DCleu-generating methods or (for WB) minimalized ‘Kits’, containing granulocyte-macrophage-colony-stimulating-factor (GM-CSF) and a second response-modifier. Proof for DC/DCleu-mediated activation of the immune-system after T-cell-enriched mixed lymphocyte culture (MLC) is done by flowcytometry, demonstrating increased fractions of certain activated, leukemia-specific or antileukemic cell-subsets of the innate and the adaptive immune-system.Generation of DC/DCleu is possible independent of patients' age, MHC-, mutation- or transplantation-status. In vivo-treatment of AML-/MDS-patients with blast-modulating, DC/DCleu- inducing ‘Kits’ could contribute to create migratory DCs, as well as antileukemically reactivated and memory-mediating immune-cells, which patrol tissue and blood and could contribute to stabilizing disease or remissions.

Durable Responses and Survival in High Risk AML and MDS Patients Treated with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine

Background. Maintenance of induced responses remains a significant unmet medical need in patients with acute myeloid leukemia (AML) or high risk myelodysplastic syndrome (MDS) not eligible for allogeneic hematopoietic stem cell transplantation (HSCT). The allogeneic leukemia-derived dendritic cell vaccine, DCP-001, has shown to generate both humoral and cellular immune responses and is safe for clinical practice (van de Loosdrecht et al., Cancer Immunol. Immunother. 2018). In this study we show additional data on long-term follow up and patient-related risk factors from the phase 1 study. Methods. AML- or MDS-patients (n=12), ineligible for HSCT, were included in a post-hoc clinical analysis of the DCP-001 phase I trial, receiving DCP-001 vaccination at the Amsterdam University Medical Center, VU University Medical Center, Amsterdam, the Netherlands (NCT01373515). All clinical data, including baseline characteristics as cytogenetics, pre-and post-vaccination treatment and morphologic blast counts in follow up, were retrospectively collected from corresponding patient files. Targeted next generation sequencing (NGS) gene panel data and cytogenetics were utilized to determine (cyto-)genetic risks following the European Leukemia Net (ELN) 2017 criteria. We defined 'response' as decreased or stable morphologic blast counts in bone marrow at day 126 (last day of official study evaluation) compared to study entry. Results. Seven out of twelve patients responded to treatment, the other patients showed progressive disease (data are summarized in table 1). The median relapse free survival (RFS) for the responding patients was 420 days (range 90-1849) and overall survival (OS) was 1090 days (90-2160); two patients died early in complete remission (CR) due to infection (respectively at 90 and 184 days). Notably, three of these patients were intermediate risk and four were poor risk patients based on the ELN risk classification for AML or Revised International Prognostic Scoring System (IPSS-R) for MDS. The median OS for the non-responders was 144 days (range 59-209). These five patients had relapsed or refractory disease at start of vaccination with detectable circulating peripheral blasts; four of them received additional azacitidine (AZA) therapy prior to vaccination. Moreover, they were vaccinated after induction and consolidation therapy with a median time between diagnosis and first vaccination of 611 days (219-2310) compared to 240 days (105-1398) for the seven responding patients. Of the latter, all were in complete remission (CR) (n=5) or had morphologic marrow blast counts <10% (n=2) at the start of vaccination. Two responders that progressed and two that relapsed after vaccination were treated with AZA, resulting in one complete remission and two partial responses (one non-evaluable due to sample failure), see figure 1. Conclusion/discussion. Clinical data of the phase 1 trial show that DCP-001 may prolong duration of remission or smoldering disease in intermediate and high risk AML or MDS patients. Response was associated with treatment by vaccination shortly after achieving CR. This should be taken into account in selection criteria of future vaccination studies. Hence, an international multi-center phase 2 study (ADVANCE II; NCT03697707) is currently being conducted to determine the effect of DCP-001 on measurable residual disease (MRD) in AML patients, aiming to convert them to a MRD negative state. Furthermore, we demonstrated that AZA can be applied as rescue therapy upon progression after vaccination. Disclosures Rovers: DCprime: Employment. Cloos:Daiicchi Sankyo: Speakers Bureau; Glycomimetics: Research Funding; Takeda: Research Funding; Novartis: Other: MRD assessments of clinical trials, Research Funding; Merus: Other: MRD assessment of a clinical trial , Research Funding; Genentech: Consultancy, Research Funding; Helsinn: Other: MRD assessment of a clinical trial; DCPrime: Other: MRD assessment of a clinical trial. de Gruijl:DCprime: Consultancy, Membership on an entity's Board of Directors or advisory committees.

PGE1-Containing Protocols Generate Mature (Leukemia-Derived) Dendritic Cells Directly from Leukemic Whole Blood.

Dendritic cells (DCs) and leukemia-derived DC (DCleu) are potent stimulators of various immunoreactive cells and they play a pivotal role in the (re-) activation of the immune system. As a potential treatment tool for patients with acute myeloid leukemia, we developed and analyzed two new PGE1-containing protocols (Pici-PGE1, Kit M) to generate DC/DCleu ex vivo from leukemic peripheral blood mononuclear cells (PBMCs) or directly from leukemic whole blood (WB) to simulate physiological conditions. Pici-PGE1 generated significantly higher amounts of DCs from leukemic and healthy PBMCs when compared to control and comparable amounts as the already established protocol Pici-PGE2. The proportions of sufficient DC-generation were even higher after DC/DCleu-generation with Pici-PGE1. With Kits, it was possible to generate DCs and DCleu directly from leukemic and healthy WB without induction of blast proliferation. The average amounts of generated DCs and DCleu-subgroups were comparable with all Kits. The PGE1 containing Kit M generated significantly higher amounts of mature DCs when compared to the PGE2-containing Kit K and increased the anti-leukemic-activity. In summary PGE1-containing protocols were suitable for generating DC/DCleu from PBMCs as well as from WB, which reliably (re-) activated immunoreactive cells, improved the overall ex vivo anti-leukemic activity, and influenced cytokine-release-profiles.

Role of Interferon (IFN)α in "Cocktails" for the Generation of (Leukemia-derived) Dendritic Cells (DCleu) From Blasts in Blood From Patients (pts) With Acute Myeloid Leukemia (AML) and the Induction of Antileukemic Reactions.

Strategies to stabilize remissions by specific elimination of residual acute myeloid leukemia (AML) blasts are needed. Leukemia-derived dendritic cell (DCleu/DC) generated from myeloid blasts improve antileukemic T-cell reactivity and install T-cell memory. Interferon (IFN)α-DC methods produce DCleu from chronic myeloid leukemia-patients (pts') blood. Various INFα-containing versus other DC methods were studied to produce DCleu (evaluated by flowcytometry) from AML-pts' blast-containing mononuclear (MNC) or whole blood (WB). After DCleu/DC stimulation in mixed lymphocyte cultures, T cells' potential to gain antileukemic cytotoxicity was studied and correlated with different DC methods and DCleu/DC counts. (1) Generation of DCleu/DC: (a) "IFN-GIT" [containing granulocyte macrophage-colony stimulating factor (GM-CSF)+IFNα+ tumor necrosis factor (TNF)-α] produced DC successfully (≥10% DC, ≥5% DCleu/cells) from AML-MNC (WB) in 54 (56%), "MCM-Mimic" in 76 (75%), "Picibanil" in 83 (64%), and "Calcium-ionophore" in 42 (67%) of cases. Proportions of DC subtypes in MNC (WB) were comparable with all DC methods, (b) IFNα combinations containing only GM-CSF+IFNα or only IFNα showed low efficiency to produce DCleu/DC from MNC (WB) compared with "IFN-GIT." (2) Antileukemic functionality: DCleu/DC-stimulated T cells showed improved leukemia cytotoxicity compared with blast cells or unstimulated T cells. The highest blast proliferation (=insufficient T cells) was seen with "IFN-GIT" DC-stimulated T cells. Probability to respond to immunotherapy or to obtain blast lysis of DC-stimulated T cells correlated with high proportions of DCleu/DC after DC culture, independent of DC-generating methods. (3) Cytokine release profiles: levels of interleukin-6, IFN-γ, and interleukin-2 were significantly lower in DC culture supernatants (from MNC/WB) with "IFN-GIT" compared with "MCM," "Pici," and "Ca" DC supernatants. Our data show that (1) WB culture simulates AML-pts' in vivo situation, (2) DC generation is possible from AML-MNC (WB) with IFNα-containing and other DC methods, (3) successful IFNα-DC generation needs GM-CSF+IFNα+TNF-α (IFN-GIT); however, "IFN-GIT" produces less DCleu/DC compared with other (non-IFNα) DC methods, (4) T cells stimulated with "IFN-GIT"-produced DCleu/DC yielded comparable antileukemic cytotoxicity; however, in cases without achieved blast lysis, an increased blast proliferation was observed.

Modulation of AML-blasts with clinically approved response modifiers to leukemia-derived dendritic cells (DCleu) ex vivo: DC-, T-cell- and cytokine profiles are predictive for antileukemic T-cell reactivity

Modulation of AML-blasts with clinically approved response modifiers to leukemia-derived dendritic cells (DCleu) ex vivo: DC-, T-cell- and cytokine profiles are predictive for antileukemic T-cell reactivity

Conversion of AML-blasts to leukemia-derived dendritic cells (DCleu) in ‘DC-culture-media’ shifts the (serum) chemokine-release profile to a more ‘inflammatory’ (in culture) going along with improved antileukemic T-cell-reactivity

Conversion of AML-blasts to leukemia-derived dendritic cells (DCleu) in ‘DC-culture-media’ shifts the (serum) chemokine-release profile to a more ‘inflammatory’ (in culture) going along with improved antileukemic T-cell-reactivity

Significance of Frequencies, Compositions, and/or Antileukemic Activity of (DC-stimulated) Invariant NKT, NK and CIK Cells on the Outcome of Patients With AML, ALL and CLL

Invariant natural killer T (iNKT)/natural killer (NK)/cytokine-induced killer (CIK) cells are important for immune surveillance. (I) Novel combinations of antibody 6B11 (targeting the Vα24-Jα18-invariant T-cell receptor) with CD4/CD8/CD1d/Vα24 for iNKT subset detection and "T/NK cell-like"-iNKT subsets were defined. Compared with healthy peripheral blood mononuclear cells (MNC) (significantly) lower proportions of iNKT cells (6B11/6B11CD3/6B11CD161), NK cells (CD3CD56/CD3CD161), and CIK cells (CD3CD56/CD3CD161) were found in peripheral blood MNC from acute myeloid (AML)/acute myeloid, lymphoid (ALL)/chronic lymphoid leukemia (CLL) patients in acute disease stages. Subtyping of iNKT cells revealed (significantly) higher proportions of CD3 T cells and CD161 NK cells in AML/ALL/CLL expressing 6B11 compared with healthy MNC. Prognostic evaluations showed higher proportions of iNKT/NK/CIK cells in favorable AML subgroups (younger age, primary, no extramedullary disease, achievement/maintenance of complete remission) or adult ALL and CLL patients. (II) iNKT/NK/CIK cell frequencies increased after (vs. before) mixed lymphocyte cultures of T-cell-enriched immune reactive cells stimulated with MNC/whole blood with or without pretreatment with "cocktails" (dendritic cells generating methods/kits inducing blasts' conversion to leukemia-derived dendritic cells from AML patients). Individual "cocktails" leading to "highest" iNKT cell frequencies could be defined. Antileukemic blast lytic activity correlated significantly with frequencies of iNKT/NK/CIK cells. In summary healthy MNC show significantly more iNKT/NK/CIK cells compared with AML/ALL/CLL MNC, a shift in the iNKT cell composition is seen in healthy versus leukemic samples and iNKT/NK/CIK cell-proportions in AML/ALL/CLL MNC samples correlate with prognosis. "Cocktail"-treated AML blasts lead to higher iNKT/NK/CIK cell frequencies and samples with antileukemic activity show significantly higher frequencies of iNKT/NK/CIK cells. Proportions of iNKT/NK/CIK cells should regularly be evaluated in AML/ALL/CLL diagnosis panels for quantitative/prognostic estimation of individual patients' antileukemic potential and their role in dendritic cells/leukemia-derived dendritic cells triggered immune surveillance.