ALBERT

Description: Abstract 3904 Agaricus bM (AbM) is an edible Basidiomycetes mushroom from Brazilian rain forest that is used in traditional medicine against cancer and other diseases. It is rich in immunomodulating substances like β-glucans and is shown to inhibit tumors and adjuvate hepatitis B virus DNA vaccine in mouse models. Previously, we have found that an AbM-based (82%) extract, AndoSan™, additionally containing Basidiomycetes mushrooms Hericium erinaceum and Grifola frondosa, also protected against gram positive and gram-negative sepsis and allergy in mice. Now, to examine the mechanisms behind AbM's biological effects and to see whether it is beneficial as adjuvant in DC cancer vaccines, cellular surface markers on stimulated MDDC were studied by flow cytometry and cytokine production by a 17-plex Luminex system. Transcription factor NF-kB activation, which is linked to cytokine production, cell cyclus regulation and more, was then determined by a luciferase assay system and western blotting as translocation of NF-kB from cytosol to nucleus in AbM stimulated promonocytic THP-1 cells and in HEK293 cells, transfected with CD14/MD2 and TLR2 or TLR4. In both cell lines, AbM induced NF-kB activation via TLR2, but inhibited such activation induced via TLR4. After 24h incubation of MDDC with AndoSan™, there was down-regulated CD11c, de novo CD69 and enhanced CD86 expression on the cells, as well as increased levels in the culture supernatants of cytokines TNFα, IL-1β, IL-2, IL-6, IL-8, MIP-1β, IFNγ and G-SCF. Moreover, when this mixed mushroom extract was added as adjuvant to a DC melanoma vaccine, there was increased proliferation (H3-thymidine incorporation) of cancer-peptide-specific T cells. We conclude that in monocytes and probably also MDDC, AbM activates transcription factor NF-kB via TLR2 stimulation but inhibits such activation induced via the LPS receptor, TLR4. In MDDC the AbM-based extract AndoSan™ induced differential expression of cell surface markers for cell adhesion, activation and antigen presentation, and increased production of a leukocyte growth factor and proinflammatory, chemotactic and some Th1-type but not Th2-type cytokines in vitro. This may explain some of the known antitumor properties of AbM and its effect against infection and allergy. Since preliminary data suggest that the AbM-based extract, AndoSan™, may also have positive adjuvant effect in MDDC-based cancer vaccine, it will be tested further ex vivo and in clinical trials with cancer patients subjected to such DC vaccines. Disclosures: Hetland: Immunpharma AS: Patents & Royalties, Research Funding. Tangen:Immunopharma AS: Research Funding. Johnson:Immunopharma AS: Patents & Royalties.

Description: Background The popular concept that human cancers might be driven by rare self-renewing cancer stem cells (CSCs) has extensive implications for cancer biology and modelling, as well for development of more efficient and targeted therapies. However, experimental support for the existence of distinct and rare CSCs in human malignancies remain contentious, particularly in light of compelling evidence that cancer-propagating cells frequently fail to read out in existing human stem cell assays. Therefore, to unequivocally establish the existence and identity of human CSCs, the challenge is first to identify candidate CSCs, and to establish their unique ability to self-renew and replenish molecularly and functionally distinct non-tumorigenic progeny followed by functional in situ validation within the patients themselves. Methods We have in the hematological malignancy myelodysplastic syndromes (MDS) characterize candidate hematopoietic stem and progenitor stages in the bone marrow of low-intermediate risk MDS patients by flow cytometry. Distinct cell populations were functionally characterised for lineage commitment in standard colony forming cell (CFC) assays, and for self-renewal potential in long-term culture initiating cell (LTC-IC) assays and in immune-deficient (NSG) mice. Moreover, we tracked the cellular origin of all identified somatic genetic lesions identified in each patient by targeted next-generation sequencing of genomic DNA isolated from each purified MDS stem and progenitor cell population. Results In low-intermediate risk MDS patients, regardless whether they were del(5q) (n=19) or non-del(5q) (n=11), we could identify rare but distinct Lin-CD34+CD38-CD90+CD45RA- candidate stem cells, granuclocyte-monocyte progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs) with frequencies within total BM similar to that of normal age-matched controls. Global gene expression analysis by RNA sequencing of MDS stem cells, GMPs and MEPs suggested that these are molecularly distinct populations. Myeloid and erythroid gene expression signatures were restricted to the GMPs and MEPs, respectively, whereas a transcriptional stem cell signature was restricted to the MDS stem cells. GMPs and MEPs isolated from del(5q) (n=12) and non-del(5q) (n=8) MDS patients displayed lineage-restricted myeloid and erythroid differentiation potentials, respectively. Self-renewal in LTC-IC assay was restricted exclusively to MDS Lin-CD34+CD38-CD90+CD45RA- stem cells in del(5q) (n=11) and non-del(5q) (n=8) MDS patients. Xenotransplantation into NSG mice also confirmed that only Lin-CD34+CD38-CD90+CD45RA- MDS stem cells have in vivo self-renewal potential, and these experiments also demonstrated their ability to replenish downstream CMPs, GMPs and MEPs, establishing the hierarchical relationship of MDS stem and progenitor cells. Targeted DNA sequencing of 88 genes recurrently mutated in MDS and other myeloid malignancies was pursued to identify somatic genetic lesions within the bulk bone marrow of MDS patients (n=13). In total we identified 30 presumed genetic driver lesions, including del(5q) and mutations in key transcription factors (RUNX1), signalling pathways (JAK2, CSF3R), epigenetic regulators (TET2, ASXL1), apoptosis regulators (TP53), and spliceosome components (SF3B1, SRSF2, U2AF2, SRSF6). Importantly, in support of their unique ability to self-renew and replenish lineage-restricted MDS progenitors, all stable somatic genetic lesions identified could in each MDS patient be backtracked to the rare stem cell population as defined phenotypically by flow cytometry and functionally by LTC-IC or xenograft potential, unequivocally establishing their unique stem cell identity within the malignant clone. Conclusions These findings provide definitive evidence for the existence of rare and distinct stem cells in MDS, a finding with extensive implications for therapeutic strategies in MDS and other cancers whose existence might also strictly depend on the persistence of rare CSCs. MDS stem cells typically acquire multiple driver mutations, together conferring a competitive advantage over normal stem cells, while even in combination failing to inflict self-renewal ability on MDS myelo-erythroid progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Description: Postremission therapy for acute myeloid leukemia (AML) is critical for elimination of minimal residual disease (MRD). In patients not eligible for allogeneic stem cell transplantation, alternative treatment options are needed. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with leukemia-associated antigens (LAAs) is a promising treatment strategy to induce anti-leukemic immune responses and to eradicate chemorefractory cells. We have developed a GMP-compliant 3-day protocol including a TLR7/8 agonist to differentiate monocytes of intensively pretreated AML patients into next-generation DCs. A phase I/II proof-of-concept study has been initiated using next-generation DCs as postremission therapy of AML patients with a non-favorable genetic risk profile in CR after intensive induction therapy (NCT01734304). DCs are loaded with in vitro transcribed RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant and surrogate antigen. Patients are vaccinated intradermally with 5x106 DCs of each antigen species up to 10 times within 26 weeks. The primary endpoint of the phase I/II trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control. Based on the safety and toxicity profile of the phase I trial (n=6), phase II has been initiated. In total, 10 patients have been enrolled into the study. DCs of sufficient number and quality were generated from leukapheresis in 8/9 cases. DCs exhibited an immune-stimulatory profile based on high surface expression of positive costimulatory molecules, the capacity to secrete IL-12p70, the migration towards a chemokine gradient and processing and presentation of antigen. 5 patients have completed the vaccination schedule; the 6th and 7th patient have received 7/10 and 4/10 vaccinations, respectively. We observed delayed-type hypersensitivity (DTH) responses at the vaccination site in 6/6 patients, accompanied by slight erythema and indurations at the injection site, but no grade III/IV toxicities. TCR repertoire analysis by next-generation sequencing revealed an enrichment of particular clonotypes at DTH sites. Limited by HLA restriction, we have so far analyzed 4 patients by multimer staining. All of them mounted DC vaccination-specific T cell responses: We detected an increase of WT1-specific T cells in one patient and strong expansion/induction of CMVpp65-specific T cells in one CMV-seropositive and two CMV-seronegative patients. In an individual treatment attempt, an enrolled patient with impending relapse was treated with a combination of DC vaccination and 5-azacytidine, resulting in MRD conversion. Long-term disease control and immunological responses are studied in the ongoing phase II trial. We conclude that vaccination with next-generation LAA-expressing DCs in AML is feasible, safe and induces anti-leukemia-specific immune responses in vivo. Disclosures Subklewe: AMGEN Research (Munich): Research Funding.

Description: High dose therapy followed by autologous stem cell support (HDT) is a curative treatment in selected lymphoma patients. Moreover, HDT has been shown to significantly increase progression-free survival in multiple myeloma (MM) patients. To obtain a quick and sustained engraftment after high dose therapy, a minimal number of 2x106 CD34+ cells/kg are desirable. However, in 15-20% of the lymphoma and MM patients insufficient numbers of CD34+ cells are harvested. These patients, characterized as poor mobilizers, will either be considered for remobilization, or can not be offered high dose therapy. Furthermore, previous findings have shown that poorly mobilizing lymphoma patients given HDT have a less favorable prognosis than good mobilizers. Recently, new mobilizing agents including plerixafor, a CXCR4 antagonist, have been developed. We, like others, have demonstrated that with a low concentration of CD34+ cells (5-10 cells/µL) and a total white cell count 〉10x109/L, addition of plerixafor resulted in successful stem cell harvesting in these poor mobilizing patients. However, little is known about the clinical outcome following high dose therapy of hard to mobilize patients given plerixafor. In the present study, we have mobilized and harvested 29 poor-mobilizers from January 2010 to December 2012, including 17 lymphoma patients and 12 MM patients. Ten patients were remobilized with G-CSF and plerixafor (8 MM patients and 2 lymphoma patients), whereas 19 patients were given plerixafor upfront in the primary mobilization attempt in addition to chemotherapy and G-CSF. The 17 lymphoma patients were followed up after reinfusion of autologous stem cells with regard to short-term and long-term engraftment as well as relapse and death. The day prior to harvest of the 29 patients, the level of leukocytes was 11.0x109 cells/L (median; range 3.0-40.5), and the CD34+ concentration was 5.5x106/L (median; range 1.9-20.8). Following plerixafor injection, the concentration of CD34+ cells increased to 25.5 x106/L (median; range 5.5-65.6). The patients were then successfully harvested (median: 3.9x106 CD34+cells/kg; range: 1.8-7.2) with 1-2 days of apheresis, although one of the patients only obtained 1.8x106CD34+cells/kg. In the 17 lymphoma patients receiving high dose therapy followed by autologous stem cell support we observed that time to short-term engraftment, defined by neutrophils 〉0.5x109/L and thrombocytes 〉20x109/L were 11 days (median; range 8-19) and 14 days (median; range 10-100) respectively. As an estimate of long-term engraftment we also examined the thrombocyte levels at day 100 after reinfusion. The median level was 135x109/L (range 27-398). Moreover, in contrast to previous findings we have observed durable responses with event free survival of 77% and overall survival of 93% (Median observation time: 24 months). In the MM patients the overall survival observed is 66%. In conclusion, our findings show that addition of plerixafor is successful in mobilization of hematopoietic stem cells in poorly mobilizing patients resulting in a fast and sustained engraftment as well as equal prognosis for both good mobilizing lymphoma and MM patients after high dose therapy with autologous stem cell support. Our clinical findings therefore justify the extra costs of plerixafor in hard-to-mobilize patients. Disclosures: Josefsen: Genzyme: Travel fund 2011 Other.

Description: Abstract 1804 Introduction: Advanced stage follicular lymphoma (FL) cannot be cured by current conventional therapies. Treatment with idiotype cancer vaccines has thus far been disappointing, and is expensive and labor intensive. Hence, simpler principles for vaccinations that do not require production of patient-specific tumor antigens are warranted. We designed a novel strategy involving intratumoral injections of low-dose anti-CD20 antibody (Rituximab) and autologous dendritic cells (DC) in irradiated lymph nodes. The aim was to facilitate antigen up-take and presentation by DCs to T cells to achieve systemic anti-tumor responses in patients with FL. Method: Ten untreated patients with stage III/IV follicular lymphoma not in need of conventional therapy underwent standard work-up, including CT-scans, FDG PET/CT and bone marrow samples. Single tumor cells were frozen for later immunoassays. Apheresis was performed for isolation of monocytes subsequently cultured with IL-4 and GM-CSF for 5 days to generate immature dendritic cells (DC). The first 6 patients received a single 8 Gy dose of radiotherapy against an enlarged node on day 1, followed by intratumoral injections of DCs (1 × 108) and GM-CSF (50 μg) on days 3 and 4. The treatment was repeated against a different enlarged node after 6 weeks. Thereafter, the protocol was changed, and patients received intratumoral injections of a small dose of rituximab (5 mg) on days 1 and 3 in order to enhance ADCC, radiotherapy (8 Gy) on day 2 and intratumoral injections of DCs and GM-CSF sc on days 4 and 5. This therapy was then repeated after 2 and 4 weeks, targeting other lymph nodes. Follow-up included repeated PET/CT-scans, bone marrow sampling and preparation of peripheral blood mononuclear cells (PBMC) for later immune studies. Tumor-specific helper and cytotoxic T cell responses were monitored by flow cytometry after a 5-day co-culture of single tumor cells with autologous PBMC sampled before, and 2 and 4 months after, treatment. Proliferation was studied by incorporation of CFSE, whereas degranulation (CD107a/b) and interferon gamma release was measured following re-stimulation with tumor cells for 5h. Result: Ten patients with FL of a total of 20 planned for this study were treated so far. No adverse events were observed. Among the first 6 patients treated by the original strategy, two showed a systemic metabolic response by PET/CT and one of these had a good response by regular CT not qualifying for PR. The protocol was then modified as described above. The first patient treated by the new strategy that included intratumoral rituximab, had a systemic metabolic response by PET/CT after 2 and 4 months and was PET negative with a normal CT by 8 months (Figure 1). Another patient showed a systemic PET response at 2 and 4 months. Analysis of T cell responses against autologous tumor cells has been initiated and we have so far demonstrated a vigorous CD8 dominated T cell response, as assayed by proliferation, degranulation and cytokine production, in the patient who achieved a negative PET-scan at 8 months (Figure 2). In parallel with the metabolic response, the immune response became stronger during follow-up after treatment. One patient without clinical response was also analysed, and no immune response was demonstrated. Further analysis of immune responses in the other patients is on-going. Conclusion: The present strategy is a novel principle to generate T cell responses and clinical anti-tumor responses detected by PET/CT in FL. Disclosures: No relevant conflicts of interest to declare.

Description: 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.