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 with restricted tumor specificity provides a promising approach to immunotherapy of cancers. However, the isolation of autologous cytotoxic T cells that recognize tumor-associated antigens is time consuming and fails in many instances. Alternatively, gene modification with tumor antigen-specific T-cell receptors (TCR) or chimeric antigen receptors (CARs) can be used to redirect the specificity of large numbers of immune cells toward the malignant cells. Chimeric antigen receptors are composed of the single-chain variable fragment (scFv) of a tumor-recognizing antibody cloned in frame with human T-cell signaling domains (e.g., CD3zeta, CD28, OX40, 4-1BB), thus combining the specificity of antibodies with the effector functions of cytotoxic T cells. Upon antigen binding, the intracellular signaling domains of the CAR initiate cellular activation mechanisms including cytokine secretion and cytolysis of the antigen-positive target cell.In this chapter, we provide detailed protocols for large-scale ex vivo expansion of T cells and manufacturing of medium-scale batches of CAR-expressing T cells for translational research by mRNA electroporation. An anti-CD19 chimeric receptor for the targeting of leukemias and lymphomas was used as a model system. We are currently scaling up the protocols to adapt them to cGMP production of a large number of redirected T cells for clinical applications.

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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

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