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The cancer-testis antigen PRAME (PReferentially expressed Antigen in MElanoma) is expressed at high levels in several oncological indications, including leukemias and solid tumors, as confirmed by mRNA or protein expression studies. Its low to absent expression on normal tissues allows suitable discrimination of tumors for targeted immunotherapy. As an intracellular protein, PRAME-derived peptides can be presented by several HLA class I allotypes, including HLA-A*02:01, and specific T cell recognition has been reported for different PRAME peptide-HLA (pHLA) ligands. Thus, developing a small set of TCRs covering different HLAs could increase the number of PRAME-positive patients eligible for treatment with T cell receptor (TCR)-based immunotherapy approaches. We determined PRAME expression in a group of Caucasian patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS). In addition, we analyzed the HLA-A genotype distribution in a set of healthy blood donors to determine how many and which different TCRs would be needed to optimally cover a Caucasian population in Germany. For the analysis of PRAME expression, bone marrow (BM) and peripheral blood (PB) samples from 165 patients (AML=133, MDS=32), obtained from multiple clinical sites across Germany, were studied. Ninety-six samples were from newly diagnosed patients (58%) while 68 samples were from relapsed/refractory patients (41%), irrespective of percentage of leukemic blasts. Disease status was not known for one patient. PRAME mRNA expression was measured by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). A threshold for PRAME-negativity was derived by measuring average PRAME mRNA expression in 22 healthy individuals. Detection of 99 or more copies of PRAME, as measured in 12.5 ng RNA, was considered positive. Of the 165 AML/MDS patients, 79 (48%; AML= 69, MDS=10) were positive for PRAME mRNA. Among relapsed/refractory patients, 52% (35/68) were classified as PRAME-positive, versus 45% (43/96) of first-diagnosis AML/MDS patients. NGS high-resolution HLA-A genotyping showed that 55% of all patients had an HLA-A*02:01 allotype, which is higher than the approximately 45% expected for a random Caucasian population and accounted for by the fact that some patients were included with known HLA-A2 status due to prior assessment for transplant eligibility. In combination, 43 (26%) AML/MDS patients were positive for PRAME above the threshold level as well as for HLA-A*02:01, thereby being potential candidates for treatment with PRAME-specific, HLA-A*02:01-restricted TCR immunotherapy. In a group of 141 healthy local blood donors, NGS high-resolution HLA-A genotyping showed an expected distribution of HLA-A*02:01, HLA-A*01:01, HLA-A*03:01, HLA-A*24:02 and HLA-A*11:01 of 43%, 26%, 25%, 19% and 8% respectively, with some donors heterozygous for two of these particular allotypes. To determine the smallest number of TCRs needed to address the largest number of patients, we assessed how the non-overlapping distribution for each potential HLA-A allotype would contribute to patient coverage. Starting with the 43% of patients who would be covered by an HLA-A*02:01-restricted TCR immunotherapy product, additional coverage of 21%, 14%, 9%, and 1% of patients could be achieved with TCR products using HLA-A*01:01-, HLA-A*03:01-, HLA-A*24:02- and HLA-A*11:01-restricted TCRs, respectively. In conclusion, approximately half of AML/MDS patients are PRAME-positive and could be addressed with PRAME-specific TCR immunotherapy approaches. An HLA-A*02:01-restricted TCR addresses the most patients in a population in Germany at 43%, while coverage could be increased to 87% with addition of three TCRs restricted by the next most common HLA-A allotypes, contingent upon suitable TCR discovery. Disclosures Addo: Medigene Immunotherapies GmbH: Current Employment. Davari:Medigene Immunotherapies: Current Employment. Raffegerst:Medigene Immunotherapies GmbH: Current Employment, Current equity holder in publicly-traded company. Tippmer:Medigene Immunotherapies: Current Employment. Schendel:Medigene AG: Current Employment, Current equity holder in publicly-traded company. Pinkernell:Medigene Immunotherapies GmbH: Current Employment, Current equity holder in publicly-traded company.

Patients with acute myeloid leukemia (AML), not eligible for allogeneic hematopoietic stem cell transplantation (AHSCT), have a significant risk of disease relapse and death due to a lack of long-term disease control. To investigate the possibility of relapse prevention after initial chemotherapy, a safety and feasibility, single-center, open label, first in human phase I/II study using dendritic cells (DC) targeting PRAME and WT-1 was recently completed. Mature autologous DCs were rapidly (3-4 days) generated using full-length PRAME and WT-1 antigens and a maturation cocktail with a TLR 7/8 agonist. Intradermal vaccination of 2.5-5x106 WT-1 and 2.5-5x106 PRAME RNA-loaded DCs was performed at week 1, 2, 3, 4, 6 and then monthly for the remainder of a 2-year study period. Patients with WT1-positive AML, with or without PRAME positivity, had to be in morphological remission (+/- hematological recovery) after induction chemotherapy without being eligible for AHSCT. The study consisted of a phase I part (n=6) to assess early safety followed by a phase II part (n=14) which assessed further safety as well as clinical and immunological effects of vaccination. A total of 20 eligible patients (5 female and 15 male, ECOG score of 0) with a median age of 59 years (range 24 to 73) were included into the study. Risk groups based on HOVON/SAKK 102 criteria at screening identified 13 patients as good, 5 as intermediate and 2 as poor risk. All patients were positive for WT-1 and 17 for PRAME prior to chemotherapy. The mean time from first diagnosis to first vaccination was 10.1±3.7 months. Vaccinations were well tolerated, without any withdrawals from the study due to toxicity. No related serious or unexpected adverse events (AEs) were reported and the most common AEs were injection site related, accounting for 28% of all AEs, which were mild and transient in nature (Grade I; CTCAE v4.03). Grade I/II toxicities were experienced by 18/20 patients, whilst 5 (25%) experienced Grade III toxicity unlikely or not related to treatment. The 2-year survival probability from time of first vaccination was 80% (95% CI: 55-92). Importantly, patients at or above the age of 60 years (n=10) also had an 80% survival rate at 2 years (risk groups: 4 good, 4 intermediate, 2 poor). Nine patients relapsed under vaccination, of whom 4 died thereafter (three due to the underlying disease, one due to GvHD after transplantation) within the 2-year study duration, equating to a probability of progression free survival (PFS) of 55% (95% CI: 31-74). The PFS rate for the elderly patients (≥ 60 years) was 50%, while it was 60% for the younger patient group. Most relapses (5/9) appeared early, within 80 days after first vaccination. Out of the 9 relapsed patients, 6 could be successfully transplanted (104 to 380 days after first vaccination; risk groups: 4 good and 2 poor risk) and 4 were still alive at the end of the 2-year observation period. The DC manufacturing process reproducibly yielded a high number of DCs expressing the transfected tumor antigens, WT-1 or PRAME, as well as high surface levels of co-stimulatory molecules. After freeze-thawing, DCs were capable of CCL19-directed migration and, upon stimulation, secreted IL-12, but not IL-10. These phenotypic and functional characteristics indicate efficient DC maturation and activation despite the patients´ previous therapeutic regimens. DC properties were comparable irrespective of the patients' clinical outcome. Immune-monitoring of bulk T cells from vaccinated patients revealed that patients in stable remission had higher levels of HLA-DR-expressing T cells when compared to relapsing patients, both in peripheral blood and bone marrow. Such difference was observed prior to vaccination and throughout the entire treatment period, suggesting that T cell activation may play a role in maintaining remission. Analysis of 23 common AML mutations revealed that 6/9 relapsed patients had at least 1 mutation, with 4 patients having 3-6 mutations. In contrast, only 1 mutation already present at baseline was found in 4/11 patients in remission. Administration of autologous DC transfected with PRAME- and WT1-RNA is feasible, safe and well tolerated. The 2-year 80% survival rate, particularly in the elderly, and the 55% progression free survival warrant further studies to assess the efficacy of this vaccine approach in improving outcomes in patients with AML. Disclosures Raffegerst: Medigene Immunotherapies GmbH: Current Employment, Current equity holder in publicly-traded company. Schnorfeil:Medigene Immunotherapies GmbH: Current Employment. Addo:Medigene Immunotherapies GmbH: Current Employment. Schendel:Medigene AG: Current Employment, Current equity holder in publicly-traded company. Pinkernell:Medigene Immunotherapies GmbH: Current Employment, Current equity holder in publicly-traded company.

Acute myeloid leukemia (AML) is a lethal hematological malignancy characterized by high rates of relapse (50%). Studies of immune responses in AML patients led to identification of two leukemia-associated antigens (Wilms' tumor protein 1 (WT1) and preferentially expressed antigen of melanoma (PRAME) for which cellular and humoral immune responses were detected. Based on expression profiles in AML and normal tissues, WT1 and PRAME were selected as target antigens for a DC vaccine, aiming to elicit adaptive and innate immune responses in AML patients. The primary objective of the trial (EudraCT No.: 2014-003520-44) is to evaluate safety and feasibility of active immunotherapy with antigen-loaded autologous DCs. The secondary objectives are to assess DC vaccination-induced T cell responses against WT1 and PRAME and to evaluate the clinical outcome over a period of 2 years or until disease progression/relapse. WT1-positive AML patients, with or without PRAME positivity were included, if they had a morphological remission with or without hematological recovery (CRi) after induction chemotherapy and were not eligible for allogeneic hematopoietic stem cell transplantation. DCs were administered intradermally (2.5x106 WT1 plus 2.5x106 PRAME DCs) on a weekly basis for 4 consecutive weeks, followed by administration on week 6 and then every 4 weeks until 2 years, tumor progression or patient withdrawal. This report focusses on the immune-monitoring data collected during the first year of treatment. Multiple parameters were evaluated, including WT1- and PRAME specific T cell responses in peripheral blood (IFN-g ELISPOT), the recurrence of WT1- and/or PRAME-positive AML blasts in bone marrow (qRT-PCR) and peripheral blood (flow cytometry), and occurrence of AML-specific mutations in bone marrow (NGS). DCs were assessed for expression of WT1 and PRAME and costimulatory molecules by flow cytometry, while secretion of IL10 and IL12 was evaluated by double-color ELISPOT. Twenty CR/CRi AML patients were vaccinated, and vaccination was well tolerated, and no signs of autoimmunity were observed. 12/20 patients remained stable throughout the first year of treatment, while 8/20 relapsed. 2/8 relapsed patients died due to progression of disease during the first year. In patients in stable remission, 7/12 patients had no detectable IFN-γ ELISPOT response in peripheral blood, 3/12 mounted a response and 2/12 were not evaluable due to high background production of IFN-γ. Positive IFN-γ ELISPOT responses were observed within 1-30 weeks of treatment and associated with a decrease over time in levels of WT1 (3/3) and/or PRAME mRNA (1/3) expression. Except for 2 patients who remained MRD-negative throughout the first year of treatment, ELISPOT-negative patients in remission (5/7) exhibited low, relatively stable levels of target antigens. Stable remissions were associated with one (3/12) or no (8/12) AML-related mutations while one patient was not evaluable. AML relapses were observed within 50 days in 4 patients, within 51-100 days in 1 patient, and after more than 150 days in 3 patients. Overall, 6/8 relapsing patients exhibited positive IFN-γ ELISPOT responses, while 2 patients relapsing in In conclusion, IFN-γ responses to WT1 and/or PRAME were detected in peripheral blood of 75% (6/8) of relapsing patients, but only 25% (3/12) of the patients in remission. It is tempting to speculate that detection of IFN-γ response in peripheral blood is linked to the concomitant presence of AML blasts in the periphery of relapsing patients. Stable or decreasing levels of WT1 and/or PRAME mRNA in bone marrow of most patients in remission is compatible with the hypothesis that a local response against AML antigens may be ongoing, despite a negative IFN-γ response in peripheral blood. Monitoring of the patients currently in remission may shed some light on the role of DC vaccination on the prevention of AML recurrence. Disclosures Eckl: Medigene Immunotherapies GmbH: Employment. Raffegerst:Medigene Immunotherapies GmbH: Employment. Schnorfeil:Medigene Immunotherapies GmbH: Employment. Prinz-Schulz:Medigene Immunotherapies GmbH: Employment. Fingerhut:Medigene Immunotherapies GmbH: Employment. Bigalke:BioNTech Innovative Manufacturing Services GmbH: Employment. Pinkernell:Medigene AG: Employment. Schendel:Medigene AG: Employment, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Tafuri:Medigene Immunotherapies GmbH: Employment.

High rates of early disease relapse are observed in patients with acute myeloid leukemia (AML). Due to poor health status, many patients cannot tolerate intensive chemotherapy and/or stem cell transplantation, resulting in a high unmet medical need for new therapy options. Clinical benefit of SCT is associated with immune responses that can control residual leukemia. To replace SCT in non-eligible patients, we designed an autologous dendritic cell (DC) vaccine approach that is given 4 times weekly at the beginning (immunization phase) followed by booster vaccines at week six and then monthly, with the intention to induce immune responses that delay or prevent relapse. The DCs secrete bioactive IL-12, but not IL-10, allowing activation of innate and adaptive responses. T cell responses are directed to two target antigens expressed in AML: Wilm's tumor-1 (WT-1) and preferentially expressed in melanoma (PRAME). Prior to initiation of a Phase I/II study implementing this vaccine approach (EudraCT No.: 2014-003520-44; clinicaltrials.gov No.: NCT02405338), five patients were treated under compassionate use (CU) using DC vaccines prepared according to an approved GMP manufacturing protocol. Here we report immune monitoring studies, exploring quality of vaccines and specificity and persistence of T cell responses. DC vaccines produced in batch lots were cryopreserved in multiple aliquots for thaw prior to application. Extensive characterization showed DCs to be of mature phenotype, with high levels of positive co-stimulatory molecules and fewer negative regulatory molecules. Antigen-loading by electroporation of in vitro transcribed RNA, led to full length intracellular protein expression in most cells. Chemokine-directed migration was measured for all DC preparations. A novel dual-color ELISpot assay showed that DCs of each patient secreted IL-12 but not IL-10 after CD40 ligand stimulation. Despite extensive chemotherapy and impaired hematopoiesis, high quality DC vaccines with essential functions were easily generated from monocytes of these patients. T cell responses to target antigens were assessed with two assays. Intracellular interferon-gamma production was measured in PBL acquired at various time points during treatment. Standard ELISpot assays independently confirmed T cell responses to both antigens. In addition, responses to non-immunizing antigens (hTERT and survivin) were detected in some cases, demonstrating antigen-spreading during treatment. T cell responses to one or both targets were found in 4/5 patients; thus, both self-antigens were immunogenic when presented by polarized mDCs. Persistent vaccination allowed maintenance of T cell responses over extended periods of time in 4/5 patients. Interestingly, antigen-specific responses to over-lapping peptides were only found in two patients when anti-PD-1 blocking antibody was present in the stimulation cultures, thereby enabling T cells to produce cytokine after antigen-specific stimulation. To date 3/5 patients are alive without current signs of relapse. Two patients received DC vaccines for 22 and 30 months and have been followed for 53 and 41 months respectively, since completion of chemotherapy. The third patient had signs of relapse and vaccination was halted after 10 months. This individual was then given allogeneic SCT and remains in CR after 35 months. The two remaining patients are deceased. One patient who showed no T cell activity relapsed after 12 months of vaccination and died at month 20 during chemotherapy. The fifth patient was vaccinated for 24 months without signs of relapse but died due to unrelated heart disease. The ongoing clinical study using extended DC vaccination will provide more information whether persistent vaccination against these self-antigens contributes to immune responses that prevent disease relapse in AML patients. Disclosures Eckl: Medigene Immunotherapies GmbH: Employment. Geiger:Medigene Immunotherapies GmbH: Employment. Schendel:Medigene Immunotherapies GmbH: Employment, Patents & Royalties: DC Vaccines.

The hyaluronan-mediated motility receptor (HMMR/Rhamm) is overexpressed in numerous tumor types, including acute lymphoid leukemia and acute myeloid leukemia (AML). Several studies have reported the existence of T-cell responses directed against HMMR in AML patients that are linked to better clinical outcome. Therefore, we explored the use of HMMR-specific TCRs for transgenic expression in lymphocytes and their in vivo impact on HMMR+ solid tumors and disseminated leukemia. We obtained TCRs via an in vitro priming approach in combination with CD137-mediated enrichment. Recipient lymphocytes expressing transgenic TCR revealed the specific tumor recognition pattern seen with the original T cells. Adoptive transfer experiments using a humanized xenograft mouse model resulted in significantly retarded solid tumor outgrowth, which was enhanced using IL-15–conditioned, TCR-transgenic effector memory cells. These cells also showed an increased potency to retard the outgrowth of disseminated AML, and this was further improved using CD8-enriched effector memory cells. To define a safe clinical setting for HMMR-TCR gene therapy, we analyzed transgenic T-cell recognition of hematopoietic stem cells (HSCs) and found on-target killing of HLA-A2+ HSCs. Our findings clearly limit the use of HMMR-TCR therapy to MHC- mismatched HSC transplantation, in which HLA-A2 differences can be used to restrict recognition to patient HSCs and leukemia.

AML is frequently diagnosed in elderly patients, with a median age of 69. Many older patients cannot tolerate intensive chemotherapy and/or stem cell transplantation, making curative treatment difficult and rates of early relapse high. Immunotherapy with dendritic cell (DC) vaccines after chemotherapy was shown by others to provide clinical benefit to some AML patients (van Tendeloo et al. 2010). Here we report results in four AML patients receiving DC vaccines targeting the antigens Wilm's tumor-1 (WT-1) and preferentially expressed antigen in melanoma (PRAME), applied in compassionate use, employing new generation monocyte-derived fast DCs, matured with a cocktail containing the TLR7/8 ligand R848. The mature DCs show high expression of CD83, strong up-regulation of HLA-DR and co-stimulatory molecules, down-regulation of CD14 and polarized release of IL-12p70, with no or low IL-10 secretion, upon T cell encounter. After informed consent and hematopoietic recovery from chemotherapy, mononuclear cells were collected by apheresis and mature DC vaccines were prepared to separately express full length mRNA encoding the two target antigens (Subklewe et al. Cancer Immunol. Immunother. 2014). DCs were administered intradermally, once weekly for 4 wks, at wk6 and then on a monthly basis. Blood and bone marrow (BM) samples were collected throughout treatment. Minimal residual disease (MRD) was measured in BM and blood by quantitative PCR of WT-1 expression and BM was monitored by morphology. Table 1 summarizes the salient features of the patients, treatment parameters, MRD monitoring and initial immune response assessment. DTH reactions were detected in all patients challenged with DCs at wk6. Immune responses of CD4 and CD8 T cells demonstrating intracellular interferon gamma (IFNg) expression were assessed by flow cytometry of PBL stimulated overnight with peptides spanning WT-1, PRAME, and hTERT and survivin as vaccine-unrelated antigens. Responses were scored positive when two-fold or greater frequencies of IFNg-expressing T cells were found compared to unstimulated controls. Patient (Pt.)CU030 and Pt.CU031 showed CD4 and CD8 responses to different test antigens. Pt.CU030 displayed strong and persistent CD8 responses to PRAME and a surprising increase in hTERT reactivity, potentially representing epitope spreading. The pt. continues to receive monthly vaccination and displays a low fluctuating WT-1 PCR signal in BM but no signal is seen in blood at wk61 after start of vaccination. Pt.CU031 displayed WT-1-specific immune responses until wk37 when responses decreased and WT-1 PCR signals increased in BM. The pt. developed Bell's palsy and immune responses were no longer detected after cortisone therapy. WT-1 signals then increased strongly in BM, accompanied by an increase of blasts. Pt. CU033 had no significant T cell response during 9 months (m) of vaccination. WT-1 signals now increase slowly in BM but relapse cannot be confirmed by morphology and WT-1 PCR remains negative in blood. Pt.CU040 has only received DC vaccines for 5 m, remains in morphological remission and immune response and MRD monitoring are ongoing. These results show that fast, TLR-polarized DCs induce or enhance specific T cell responses in elderly and undertreated AML patients, with individual strengths and specificities. Preliminary assessments suggest that changes in MRD are related to increase or loss of vaccine-associated immune responses. Table 1. Characteristics of AML patients receiving DC vaccines Patient CU030 CU031 CU033 CU040 Age 57 50 68 73 Sex f m f f AML Classification M4 M2 M1 M1 Risk Classification intermed intermed intermed good Chemotherapy cycles Induction/Consolidation 2/0 2/4 2/0 2/0 Time between chemo-therapy and vaccination 5 m 8 m 3 m 7 m Months of vaccination as of (08/2015) 16 m 10 m 9 m 5 m DTH responses at w6 toWT-1/PRAME DC challenge pos/pos pos/pos pos/pos pos/pos IFNg-positive T cell responses to overlapping peptides of WT-1, PRAME, hTERT, and Survivin Strong and persistent CD8 responses to PRAME and hTERT Early CD4 & CD8 responses to WT-1; decrease at wk37; full loss after cortisone therapy No significant responses detected up to wk33 To be done after acquisition of further samples MRD (WT-1 PCR) in BM/blood fluctuating low /neg rapid increase after cortisone /pos slow increase /neg ongoing BM morphology (most recent test) neg pos neg neg Time since completion of chemotherapy 21 m 18 m 12 m 12 m Disclosures Eckl: Medigene Immunotherapies GmbH: Employment. Schendel:Medigene Immunotherapies GmbH: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: for DC maturation cocktail. Kvalheim:Medigene Immunotherapies GmbH: Other: Scientific collaboration.

Adoptive transfer of T cells expressing transgenic T-cell receptors (TCRs) with antitumor function is a hopeful new therapy for patients with advanced tumors; however, there is a critical bottleneck in identifying high-affinity TCR specificities needed to treat different malignancies. We have developed a strategy using autologous dendritic cells cotransfected with RNA encoding an allogeneic major histocompatibility complex molecule and a tumor-associated antigen to obtain allo-restricted peptide-specific T cells having superior capacity to recognize tumor cells and higher functional avidity. This approach provides maximum flexibility because any major histocompatibility complex molecule and any tumor-associated antigen can be combined in the dendritic cells used for priming of autologous T cells. TCRs of allo-restricted T cells, when expressed as transgenes in activated peripheral blood lymphocytes, transferred superior function compared with self-restricted TCR. This approach allows high-avidity T cells and TCR specific for tumor-associated self-peptides to be easily obtained for direct adoptive T-cell therapy or for isolation of therapeutic transgenic TCR sequences.

We have previously shown in preclinical models that a new generation of fast dendritic cells (DCs), using a maturation cocktail containing IL-1β, TNFα, IFNγ, PGE2 and the TLR7/8 ligand R848, is more efficient than the standard 7 day DCs matured with a cytokine cocktail as described by Jonuleit (standard DCs). These DCs show a high up-regulation of HLA-DR and co-stimulatory molecules like CD80, CD83, CD86 and CD40 combined with a down regulation of CD14. They have a good migratory capacity towards CCL19 and their special characteristic is IL-12p70 production when stimulated with CD40L whilst IL-10 production is low. We investigated if the new generation DCs maintain their properties when produced by GMP standards and if they are able to mount specific immune responses in patients. Monocytes were enriched using the Elutra cell separator and cultivated either fresh or after cryopreservation. Monocytes were cultured in Teflon bags in the presence of IL-4 and GM-CSF and the maturation cocktail was added on day 2 or 3. After 24 hours DCs were harvested and electroporated with mRNA. After 2-4 hours recovery, cells were frozen with either 2.5E+6 or 5E+6 transfected DCs per vial. DC productions from one lung cancer, one prostate cancer, four glioblastoma and one AML patient showed the same characteristics with only some variations in the amounts of IL-12p70 released after co-culture with CD40L transfected mouse fibroblasts. The first patient treated with the new generation DCs transfected with hTERT and survivin mRNA suffered from stage IV lung cancer with brain metastases. Following diagnosis in June 2011 she was treated with chemotherapy and radiotherapy. Since December 2011 she has been vaccinated and has obtained a status of stable disease. DC vaccination was interrupted in 2/2013 when an attempt was made to re-open an occluded bronchus with radiotherapy. During irradiation the patient developed an inflammation of the pleura, which was treated with high dose cortisone. While on cortisone therapy the patient developed 2 new brain metastases, which were treated with local radiotherapy using the Cyberknife. DC vaccination was continued in 6/2013 and health conditions gradually improved bringing the patient again into a stable disease with no further development of brain metastasis. The second patient receiving DC vaccination had a hormone resistant prostate cancer in a very advanced stage and dropped out immediately after start of treatment. Additionally, four stage IV glioblastoma patients have been treated with hTERT and survivin transfected DCs plus either with autologous tumor mRNA or, where no tumor RNA was available, with hCMVpp65 mRNA DCs. The observation time of these four patients is currently 14, 11, 11 and 10 months and all patients remained in complete remission. All patients treated with the new generation DCs show strong local DTH responses and flu-like symptoms after vaccination at a significantly earlier time point then previously observed by us in patients treated with standard DCs. Altogether our results show that the new generation DCs can successfully be produced from patients with different kinds of cancer. Clinical results, strong DTH reactions and flu like symptoms following DC vaccinations suggest that specific immune responses have occurred. To further confirm these observations immune monitoring of individual patients is under investigation and results from these studies will be presented. Disclosures Schendel: Medigene: Employment, Equity Ownership, Patents & Royalties.

Dendritic cell (DC) vaccination is considered a promising immunotherapeutic strategy for AML patients with minimal residual disease (MRD). We have developed a three-day manufacturing protocol containing a synthetic TLR7/8 agonist to generate DCs with a positive costimulatory profile and high IL-12p70 secretion (TLR-DCs). These proved to be superior with respect to type 1 polarization of T cells and activation of NK cells compared to conventional monocyte-derived DCs. In an attempt to further optimize the maturation protocol, we compared the use of standard pooled human serum (PHS) with autologous human serum (AHS) from healthy donors (HDs, n=16) and AML patients (n=6) within the generation process of TLR-DCs. Surprisingly, we found that AHS-DCs from HDs were significantly inferior to PHS-DCs concerning viability (81% vs. 91%, p=0.002) and recovery (3.8% vs. 7.1%, p=0.037) after the maturation process. A similar effect was seen in AHS-DCs from AML patients. In a functional analysis, IL-12p70 secretion was reduced and migratory capacity of the DCs to CCL19 was impaired. We are currently working on the identification of immunomodulatory components within the AHS that are responsible for these detrimental effects. In a preliminary cytokine analysis, we found that pro-inflammatory (TNF-α) as well as anti-inflammatory (IL-10) cytokines were significantly increased in the AHS samples. PHS is therefore highly recommendable for the maturation process and was used in all subsequent experiments. With this protocol, we were able to show that TLR-DCs can also be generated from monocytes of AML patients. No differences in phenotype and function were found compared to samples from healthy blood donors. Efficient and controllable expression of leukemia-associated antigens was induced by RNA electroporation. These cells are capable of both priming naïve T cells and reactivating antigen-specific pre-primed effector cells in vitroand are therefore highly suitable for DC vaccination strategies in the setting of postremission AML. In order to further enhance the immunostimulatory capacity of TLR-DCs, we are currently evaluating combinatorial approaches with inhibitors of receptor-ligand immune checkpoints. Their high therapeutic potential has recently been demonstrated for various tumor entities. In a comprehensive analysis of immune checkpoint molecules, it was revealed that TLR-DCs, albeit showing a preferentially positive costimulatory profile, express significant amounts of immunoinhibitory molecules from different families (PD-L1, B7-H3, HVEM, ILT3, ILT4, 4-1BBL). We have set up an ex vivo coculture system for activation and expansion of T cells by autologous DCs. Using blocking antibodies against PD-1, we could demonstrate a significant increase of IFN-γ secretion by T cells in samples of healthy donors and HIV patients as well as AML patients. The ultimate goal is to figure out which combination of immune checkpoint inhibitors is the most appropriate to increase the effect of a DC-based immunotherapy in the clinical setting of AML immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 358FN2 Dendritic cells (DCs) are important regulators of the human immune response. By means of direct intercellular interactions and secretion of cytokines, they can induce a stimulatory or a regulatory response, depending on the environment in which they developed. In vitro, it is possible to imitate this process by addition of various cytokines. The aim of this study was to evaluate DCs matured by different cytokine cocktails for expression of immunostimulatory and -inhibitory molecules and correspondent activation of T helper 1 (Th1) and natural killer (NK) cells. The selection of these cocktails was guided by potential clinical application and usage in a GMP setting. We compared three different ways of DC generation from peripheral blood monocytes of healthy donors: 1) maturation by a cocktail including the TLR7/8 agonist R848 (TLR-mDCs), 2) DC generation by the standard combination of proinflammatory cytokines (IL-4, GM-CSF, IL-1β, PGE2, TNF-α, and IL-6) applied in many clinical studies so far (cc-mDCs), and 3) addition of IL-10 in order to induce a more regulatory phenotype (IL10-mDCs). The expression of a broad range of costimulatory and coinhibitory molecules (CD80, CD86, CD273, CD274, CD275, CD276, B7-H4, HVEM, CD30L, CD70, CD134L = OX40L, CD137L = 4-1BBL) on the surface of these DC populations was analyzed by FACS. Secretion of various cytokines crucial for interaction with other immune cells (IL-12p70, IL-10, TNF-α, IFN-γ, IL-2, and TGF-β) was measured by cytometric bead array after stimulation with CD40 ligand. In order to assess the functional importance of these signals, we performed in vitro polarization assays for T helper cells after co-culture with DCs and measured the in vitro stimulatory potential of the DCs for natural killer (NK) cells by CD69 upregulation and intracellular IFN-γ staining. We could show that TLR-mDCs were characterized by a predominance of costimulatory (e.g. CD80, CD86) relative to coinhibitory molecules (e.g. CD273, CD274, HVEM). When stimulated by CD40L, they displayed a cytokine profile with very high IL-12p70 and TNF-α, but little if any IL-10 production. In a co-culture with autologous T cells, the combination of these signals resulted in a strong polarization toward IFN-γ secreting Th1 cells, with little or no stimulation of Th2 and Th17 cells. The costimulatory profile of cc-mDCs, in comparison, was shifted toward a lower expression of costimulatory molecules and similar or higher expression of coinhibitory molecules (ratio of CD86 to CD273 expression around 40 compared to > 60 for TLR-mDCs, p < 0.005). No IL-12p70 and low levels of IL-10 were secreted. These signals were reflected in a less pronounced type 1 polarization of T helper cells. IL10-mDCs expressed very low levels of CD80 and CD86 and displayed a coinhibitory molecule pattern similar to cc-mDCs. Additionally, they secreted the immunoregulatory molecule IL-10 in higher amounts and did not activate T helper cells at all. As IL-12p70 is an important factor for NK cell activation, only TLR-mDCs were capable of upregulating the activation marker CD69 on NK cells and inducing significant secretion of IFN-γ. Both Th1 and NK cells play an important role in tumor defense. With this set of data, we clearly showed that TLR-mDCs, in consequence of their positive costimulatory profile and their high IL-12p70 secretion, are superior with respect to type 1 polarization of T cells and activation of NK cells. They are therefore highly suitable for application in cancer immunotherapy. This DC type will be used in a phase I/II trial for postremission therapy in patients with non-favorable AML, which will start in our clinic in 2012. Disclosures: No relevant conflicts of interest to declare.

Purified recombinant human interleukin-11 (rhuIL-11) was assessed for its in vivo effects on the proliferation and differentiation of hematopoietic progenitors as well as its capacity to accelerate the recovery of a drug-suppressed hematopoietic system. Dosage and time sequence studies demonstrated that administration of IL-11 to normal mice resulted in increases in absolute numbers of femoral marrow and splenic myeloid (granulocyte-macrophage colony-forming unit [CFU-GM], burst-forming unit-erythroid [BFU-E], CFU-granulocyte, erythroid, macrophage, megakaryocyte) progenitor cells and in stimulation of these progenitors to a higher cell cycling rate. This was associated with increased numbers of circulating neutrophils. Administration of IL-11 to mice pretreated with cyclophosphamide decreased the time required to regain normal levels of neutrophil and platelet counts in peripheral blood. In addition, IL-11 accelerated reconstitution to normal range of myeloid progenitors from bone marrow and spleen of myelosuppressed mice. These data suggest that IL-11 may play an important role in the regulation of hematopoiesis, and the application of this novel cytokine may have clinical therapeutic benefits.

Abstract 2195 Introduction: Therapeutic vaccination with dendritic cells (DC) is currently considered as an investigational therapy in acute myeloid leukemia (AML) for eradication of minimal residual disease (MRD). Dendritic cells derived from autologous peripheral blood monocytes have been tested as cellular adjuvants for therapeutic vaccination of malignancies and proven feasibility and safety, but overall clinical response rates remain very low. The vast majority of DCs used for clinical trials were differentiated with a standard maturation cocktail composed of the cytokines TNF-a, IL-1b, IL-6 and PGE2 leading to DCs unable to secrete biologically active IL-12. This cytokine is fervently desired because of its leading role in promoting T helper 1 cell polarization and therefore fostering the appropriate adaptive immune responses needed to combat minimal residual disease. Cocktails containing synthetic Toll-like receptors (TLR) agonists emerged as an attractive alternative for the induction of DC maturation with T helper type 1 polarizing capacity. Our present investigation was designed to study the feasability of a clinical grade DC 3-day mDC generation protocol from nonleukemic monocytes of intensively pretreated AML patients with novel maturation cocktails containing different TLR-agonists in vitro and assessment of their potency to induce adaptive and innate immune responses. Material & Methods: Monocytes isolated from peripheral blood of AML patients in CR and healthy donors were differentiated into immature DC with GM-CSF and IL-4. After 48 hours DC were additionally cultured with TNF-a, IL1-b, INF-g, PGE2 and corresponding to the defined cocktail with the TLR7/8 agonists R848 (R) or CL075 (C) with or without the TLR3 agonist poly(I:C) (P) for 24 hours. mDCs were analyzed for expression of maturation surface markers, costimulatory profile, IL-12(p70)/IL-10 ratio, migratory capacity, NK cell activation and polarization of T cells. Results: No significant difference in absolute monocyte counts and percentage of DC recovery between healthy controls and AML patients in CR was found using different maturation cocktails (C, CP, R, and RP). Phenotype analysis of surface marker expression revealed no substantial differences between the different DC generation protocols used in healthy donors and AML patients in CR. The costimulatory profile assesed by the expression of two members of the B7 family, CD80 (B7.1) and CD274 (B7-H1 or PD-L1), was in healthy donors superior to AML patients, but these differences were not statistically significant. Variations were noted in the capacity of DCs derived from different donors to produce IL-12(p70) and IL-10, but importantly no significant differences between AML patients in CR and healthy controls could be observed. Interestingly, both healthy donor and AML derived DCs secrete a significantly higher proportion of IL-12(p70) with R848 containing cocktails compared to CL075. Treatment with the CP cocktails even leads to a inverse Il12/IL-10 ratio in AML patients. The high CCR7 expression was paralleled by a strong migratory capacity as well as positive chemotactic reponses to CCL19 chemokine signals. DCs matured with these novel cocktails induced potent alloresponses and strongly activated NK cells measured by upregulation of CD69 expression and IFN-g secretion. No differences beetween R848 and CL075 could be observed. Conclusion: Here we report for the first time a clinically applicable, time- and resource saving 3-day TLR-agonist containing maturation protocol for the generation of IL-12(p70) secreting DCs from AML patients in remission validated with healthy controls which allowed efficient generation, easy harvesting, stable maturation and substantial recoveries of mature DCs. Comparison of different TLR7/8 agonists showed superiority of R848 in IL-12(p70) production to CL075. We believe that these studies point the way to improved DCs that will induce better and long lasting immune responses in the vaccination against acute myelo Disclosures: No relevant conflicts of interest to declare.

Introduction. Mesenchymal stem cells (MSC) show immunosuppressive capacity in an allogeneic host. Severe progressive systemic sclerosis (SSc) is an autoimmune disease in which prognosis is worsened in individuals presenting with diffuse cutaneous, lung and renal involvement. Immunosuppression can improve the course of the disease. Methods. We transplanted a 41 year old female patient suffering from diffuse cutaneous systemic sclerosis with MSC from her father after in vitro expansion of the MSC. Results. The patient presented with a remarkable clinical response. Her ulcer surface decreased, the 17 Site Modified Rodnan Skin Score, vascular ultrasound, mouth aperture, the Visual Analogue Scale for pain (VAS), the Valentini Activity Score and the Hannover Functional Questionnaire showed encouraging improvements. The patient did not experience any adverse events during the first 300 days after transplantation. Conclusions. The transplantation of MSC from a haploidentical donor into a patient with systemic sclerosis is feasible, safe and effective. Furthermore is the transplantation of MSC from a haploidentical donor into an adult without severe pharmacological immunosuppression at the time of transplantation feasible and safe.