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Background Patients with high-risk prostate cancer (PC) can experience biochemical relapse (BCR), despite surgery, and develop noncurative disease. The present study aimed to reduce the risk of BCR with a personalized dendritic cell (DC) vaccine, given as adjuvant therapy, after robot-assisted laparoscopic prostatectomy (RALP). Methods Twelve weeks after RALP, 20 patients with high-risk PC and undetectable PSA received DC vaccinations for 3 years or until BCR. The primary endpoint was the time to BCR. The immune response was assessed 7 weeks after surgery (baseline) and at one-time point during the vaccination period. Results Among 20 patients, 11 were BCR-free over a median of 96 months (range: 84-99). The median time from the end of vaccinations to the last follow-up was 57 months (range: 45-60). Nine patients developed BCR, either during (n = 4) or after (n = 5) the vaccination period. Among five patients diagnosed with intraductal carcinoma, three experienced early BCR during the vaccination period. All patients that developed BCR remained in stable disease within a median of 99 months (range: 74-99). The baseline immune response was significantly associated with the immune response during the vaccination period (p = 0.015). For patients diagnosed with extraprostatic extension (EPE), time to BCR was longer in vaccine responders than in non-responders (p = 0.09). Among 12 patients with the International Society of Urological Pathology (ISUP) grade 5 PC, five achieved remission after 84 months, and all mounted immune responses. Conclusion Patients diagnosed with EPE and ISUP grade 5 PC were at particularly high risk of developing postsurgical BCR. In this subgroup, the vaccine response was related to a reduced BCR incidence. The vaccine was safe, without side effects. This adjuvant first-in-man Phase I/II DC vaccine study showed promising results. DC vaccines after curative surgery should be investigated further in a larger cohort of patients with high-risk PC.

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.

Prostate cancer patients diagnosed with high Gleason score (≥ 8) and large tumors (≥T2c) are considered high-risk patients and >50% will develop an early biochemical relapse following radical surgery. Presently, there is no curative therapy available for patients once when biochemical relapse occur. Based on encouraging clinical results from 6 relapsed prostate cancer patients treated under hospital exemption with dendritic cell (DC) vaccines we started an adjuvant, first in man, phase I/II study using autologous, monocyte derived DCs targeting autologous tumor antigens from primary tumor, combined with hTERT and survivin. Twenty patients were included in the study. All patients had pathological stage pT2-pT3b and Gleason score 7b-10. Following surgery prostate specific antigen (PSA) was < 0,2 µg/L in all patients. Fifteen patients have received 3-days DCs generated according to our standard protocol with a maturation cocktail composed of GM-CSF, IL-4, TNFα, IL1β and PgE2. Five patients were treated with a TLR 7/8 ligand containing maturation cocktail, resulting in DCs with a polarized release of IL-12p70 and no or low IL-10. The patients received 4 weekly vaccinations, then DTH vaccine at week 8 and thereafter-monthly vaccinations the first year, and every third month the second and third year. All patients except of two have completed vaccination. Of the 15 first patients using the standard DCs, three patients developed PSA relapse during vaccination (PSA measured twice >0,5 μg/L with minimum 4 weeks interval) and two additional patients relapsed after completed 3 years of vaccination. From the last 5 patients given the new type of DCs none have experienced PSA relapse. Seventy-five percent of the patients remain without biochemical relapse with a mean observation time of 47,5 (range 29-82) months. We confirm that the study is feasible and safe. Immune responses in the patients are under investigation. Altogether, our clinical results are promising and the use of adjuvant DC vaccines might become a new approach to prevent biochemical relapse in high-risk prostate cancer patients following radical surgery. Citation Format: Anne Merete Tryggestad, Iris Bigalke, Karol Axcrona, Bjørn Brennhovd, T Kirsti Hønnåshagen, Lisbeth Skoge, Lena Tjeldhorn, Lene Mowinckel, Stein Sæbøe-Larsen, Jens A L Jørgensen, Grete S Andreassen, Gjertrud Skorstad, Dag Josefsen, Ulrika Axcrona, Steinar Aamadal, Wolfgang Lilleby, Svein Dueland, Gunnar Kvalheim. A first in man phase I/II adjuvant dendritic cell vaccine study in high-risk prostate cancer patients following radical surgery reduce the incidence of biochemical relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT016.

Although progress has been made in the treatment of AML in recent years, relapse rates following chemotherapy are still high resulting in only low long-term patient survival rates. Treatment options are often limited following relapse, emphasizing the continued need for new and more effective treatments options.We have developed an active immunotherapy using TLR7/8-polarized fast dendritic cells (DCs), which is currently under evaluation for treatment of patients with AML in morphological remission in a non-randomized phase I/II clinical trial (NCT02405338). The trial subjects receive repeated immunotherapy with autologous vaccine cells, transfected with RNA encoding two leukemia-associated antigens WT1 and PRAME. Patients are vaccinated for two years or until disease progression. Here we present data on the feasibility of clinical grade production of our TLR7/8-polarized fast DC vaccine for long-term vaccination of chemotherapeutically pretreated AML patients. A total of 20 patients with a median age of 59 years (range 24 - 73 years) were recruited to this ongoing trial. For vaccine production, autologous apheresis material was collected from each patient and fast DC generation was performed following isolation of DC precursor cells using a TLR7/8-agonist containing maturation cocktail. Final vaccine cells were cryopreserved in multiple aliquots prepared to deliver 5x106 - 10x106 cells per vaccine dose. Vaccine cells could be generated from all 20 AML patients. A second apheresis was performed during the trial for a second production in order to generate sufficient vaccine doses for the intended treatment period for 4 of 20 patients. The majority of production runs (17 out of 24) yielded 20 or more vaccine doses (8 batches: 20-29 doses, 6 batches: 30-39 doses, 2 batches: 40-49 doses and more than 60 doses from one batch). For seven patients less than 20 vaccine doses were cryopreserved from one production run. To evaluate vaccine cell purity, presence of autologous non-DC cells was determined and revealed a purity of more than 70% for 22 vaccine batches (17 batches 80% - 99%, 5 batches 71% - 77%). Only two productions yielded lower purities (range 64% - 69%). The average post-thawing vaccine cell viability was 83% (range 68%-95%) with the majority of cells showing a viability of >70%. Additionally, DCs generated from all patients showed a mature surface phenotype as demonstrated by high expression of typical DC surface markers such as CD80, CD86, CD83, CD40 and HLA-DR. Furthermore, high expression of the lymphocyte homing receptor CCR7 could be detected for all cell preparations. Taken together, these results clearly demonstrate the feasibility and robustness of our protocol for production of mature clinical grade TLR7/8-polarized fast DCs in high numbers from heavily pretreated post-remission AML patients allowing for long-term vaccination of trial subjects. Citation Format: Iris Bigalke, Lisbeth Johanne Skoge, Kirsti Hønnåshagen, Christiane Geiger, Gunnar Kvalheim, Dolores J. Schendel. Generation of clinical grade autologous TLR 7,8-polarized fast dendritic cell vaccines for active immunotherapy of patients with AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5644.

Patients diagnosed with acute myeloid leukemia (AML) may not be eligible for curative intensive treatment due to co-morbidity factors or age. Here we report results of 5 AML patients in morphological remission after incomplete induction/consolidation chemotherapy treated with dendritic cells (DCs) targeting WT-1 and PRAME. DCs were produced as described previously (Subklewe et al 2014), using a maturation cocktail containing the TLR 7/8 ligand R848. These DCs show a polarized release of IL-12p70 combined with low IL-10 upon stimulation. 2.5 or 5E+6 DCs per antigen were injected intradermally once weekly for 4 weeks (wks), in wk 6 and thereafter at monthly intervals. Blood and bone marrow (BM) samples were collected at regular intervals. Minimal residual disease (MRD) was measured in BM by quantitative PCR of WT-1 and PRAME expression and by morphology. Immune responses were assessed by analysis of intracellular interferon gamma expression or proliferation following stimulation with peptides spanning WT-1, PRAME, hTERT and survivin or after stimulation with autologous WT-1 and PRAME DCs. A 57 year old woman with intermediate risk M4 AML was vaccinated over 22 months after chemotherapy. Five weeks after start of vaccination she mounted strong CD8 responses against PRAME combined with an increase in hTERT response, suggesting epitope spreading. WT-1 signals in BM showed low positive levels throughout vaccination, but she remains in morphological remission 33 months after end of chemotherapy (EoC). A 50 year old man with M2 intermediate risk AML, initially not eligible for BM transplantation, showed specific immune responses against WT-1 during 10 months of vaccination. Due to Bell’s Palsy he was treated with cortisone which immediately reduced the vaccine effect, accompanied by increase of blasts in the bone marrow. Following new induction therapy he received BM transplantation and is currently in CR. A 68 year old woman with M1 intermediate risk AML is under vaccination for 24 months. WT-1 signals in BM continue to be slightly elevated without any sign of morphological relapse. CD4 responses and low CD8 responses are detected against WT-1, PRAME and hTERT. A 73 year old woman with M1 good risk AML relapsed after 6 months of DC vaccination without mounting specific immune responses. DC treatment was combined with 5-Azacytidine without any immunological and clinical effects. A 59 year old woman with good risk AML has been vaccinated for 14 months and is in remission for 21 months since EoC. WT-1 in BM remains slightly elevated whilst the initially positive PRAME signal is negative. Our results show that in 4 out of 5 AML patients fast TLR-polarized DC vaccination induced or supported specific T cell responses. 3 patients continue to be in CR after 21, 25 and 33 months respectively, following suboptimal primary chemotherapy. Immune responses are under investigation and will be presented. Citation Format: Iris Bigalke, Guri Solum, Dag Josefsen, Yngvar Fløisand, Kirsti Hønnåshagen, Lisbeth Skoge, Signe Spetalen, Stein Sæbøe-Larssen, Dolores J. Schendel, Gunnar Kvalheim. WT1 and PRAME mRNA transfected TLR 7/8-polarized fast DC vaccines in AML patients mount specific immune responses and impact progression free survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3659. doi:10.1158/1538-7445.AM2017-3659

We have previously used different protocols for GMP production of monocyte derived dendritic cells (DCs), loaded them with mRNA and conducted several clinical trials to investigate their therapeutic impact in treatment of different solid tumors like prostate carcinoma, glioblastoma or melanoma. These studies showed that only a group of patients mounted an immune response after DC vaccination within a timeframe of 3 to 6 months. A new generation of IL-12p70 producing DCs gave superior immunological responses in pre-clinical data when compared with DCs generated according to the standard protocol used for DC production so far. Aim one of our study is to show the feasibility of generating IL-12p70 producing monocyte derived DCs with starting material from patients with different types of malignancies in a large scale GMP setting and aim two is to investigate the clinical response of these new generation DCs in patients suffering from different types of cancer and acute myeloid leukemia (AML). Autologous mature DCs were loaded with mRNA (messenger Ribonucleic Acid) either encoding the tumor specific antigens hTERT and survivin, plus where available, autologous tumor mRNA, or the AML specific antigens WT-1 or PRAME. Patients received 4 initial vaccinations in weekly intervals, a delayed type hypersensitivity challenge in week 6 and further boost vaccinations every month. With each vaccination either 2,5 x10^6 or 5×10^6 DCs were injected intradermal. So far we have started treatment of one lung cancer patient, one prostate cancer patient, 4 glioblastoma patients and 3 AML-patients. The lung cancer patient suffering from stage IV disease with lung and brain metastases has now been vaccinated since 12/2011 and remained in stable disease. The glioblastoma patients, also with stage IV diseases, have been vaccinated for 16, 12, 12 and 11 months. All of them are in complete remission. One patient developed a pseudo relapse after 7 and again after 9 months of non-malignant origin. The Prostate cancer patient progressed before an immune response could be expected and immediately dropped out of the vaccination program after start of DC treatment. The first AML patient has started DC treatment in May 2014, the other two just recently and therefore are still too early to evaluate. Our investigations show that mRNA loaded new generation DCs can successfully be produced from patients with different types of cancer. All patients tolerated DC vaccination well. Some patients developed flu-like symptoms on the day of vaccination. Strong DTH responses at the injection sites appeared 2 to 4 days after DC injection from the second vaccination time point on indicating the presence of specific immune responses. Solid tumor patients suffering from advanced disease treated with our DC vaccines have a prolonged progression free survival, showing that this immunotherapeutic approach will be a promising alternative to current standard therapies. Citation Format: Iris Bigalke, Kirsti Honnashagen, Marianne Lundby, Guri Solum, Lisbeth Skoge, Else M. Suso Inderberg, Julitta Kasten, Stein Saboe-Larssen, Dolores J. Schendel, Gunnar Kvalheim. A new generation of dendritic cells to improve cancer therapy shows prolonged progression-free survival in patients with solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2516. doi:10.1158/1538-7445.AM2015-2516

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.