Posttransplantation lymphoproliferative disease (PTLD) associated with EpsteinBarr virus (EBV) is

Background: For optimal T cell activation it is desirable that dendritic cells (DCs) display peptides within MHC molecules as signal 1, costimulatory molecules as signal 2 and, in addition, produce IL12p70 as signal 3. IL-12p70 polarizes T cell responses towards CD4 + T helper 1 cells, which then support the development of CD8 + cytotoxic T lymphocytes. We therefore developed new maturation cocktails allowing DCs to produce biologically active IL-12p70 for large-scale cancer vaccine development.

Material

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

Objectives Innovative post‐remission therapies are needed to eliminate residual AML cells. DC vaccination is a promising strategy to induce anti‐leukaemic immune responses. Methods We conducted a first‐in‐human phase I study using TLR7/8‐matured DCs transfected with RNA encoding the two AML‐associated antigens WT1 and PRAME as well as CMVpp65. AML patients in CR at high risk of relapse were vaccinated 10× over 26 weeks. Results Despite heavy pretreatment, DCs of sufficient number and quality were generated from a single leukapheresis in 11/12 cases, and 10 patients were vaccinated. Administration was safe and resulted in local inflammatory responses with dense T‐cell infiltration. In peripheral blood, increased antigen‐specific CD8+ T cells were seen for WT1 (2/10), PRAME (4/10) and CMVpp65 (9/10). For CMVpp65, increased CD4+ T cells were detected in 4/7 patients, and an antibody response was induced in 3/7 initially seronegative patients. Median OS was not reached after 1057 days; median RFS was 1084 days. A positive correlation was observed between clinical benefit and younger age as well as mounting of antigen‐specific immune responses. Conclusions Administration of TLR7/8‐matured DCs to AML patients in CR at high risk of relapse was feasible and safe and resulted in induction of antigen‐specific immune responses. Clinical benefit appeared to occur more likely in patients <65 and in patients mounting an immune response. Our observations need to be validated in a larger patient cohort. We hypothesise that TLR7/8 DC vaccination strategies should be combined with hypomethylating agents or checkpoint inhibition to augment immune responses. Trial registration The study was registered at https://clinicaltrials.gov on 17 October 2012 (NCT01734304) and at https://www.clinicaltrialsregister.eu (EudraCT‐Number 2010‐022446‐24) on 10 October 2013.

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.

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.

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