Intratumoral (IT) myDC play a pivotal role in initiating antitumor immune responses within the tumor microenvironment. IT injection of the oncolytic virus T-VEC may lead to the release of tumor antigens and maturation signals that can be captured and processed by CD1c (BDCA-1)+/CD141 (BDCA-3)+ myDC, thereby reinvigorating the cancer immunity cycle.

Intratumoral (IT) myDC play a pivotal role in initiating antitumor immune responses within the tumor microenvironment. IT injection of the oncolytic virus T-VEC may lead to the release of tumor antigens and maturation signals that can be captured and processed by CD1c (BDCA-1)+/CD141 (BDCA-3)+ myDC, thereby reinvigorating the cancer immunity cycle.

Abstract Abstract 1894 Mesenchymal stem cells (MSCs) give rise to bone marrow (BM) stromal cells and play an essential role in the formation and function of the MM microenvironment. Some recent studies revealed that MSCs from myeloma patients (MM-hMSCs) show an enhanced spontaneous and myeloma cell-induced production of cytokines and a distinctive gene expression profile, when compared to MSCs from normal donors (ND-hMSCs). However, regarding the osteogenic differentiation ability of MM-hMSCs conflicting observations were reported. In this study, we observed that MM-hMSCs, especially for those from MM patients with bone lesions, exhibited in the presence of osteogenic differentiation (OD) medium, significantly decreased alkaline phosphatase (ALP) activity, reduced expression of specific osteogenic markers (OPN, BMP2, OTX and BSP) and impaired matrix mineralization, compared to ND-hMSCs. However, MGUS-hMSCs, did not show a significantly impaired osteogenesis ability. Primary CFU-ALP assay from BM samples of diseased mice in the 5T33MM model also confirmed that the osteogenic differentiation ability of MSCs was impaired. Previous reports indicated that MM cells can suppress MSCs osteogenesis by HGF and DKK1 as observed in vitro (Giuliani et al, Cancer Res. 2007; Standal et al, Blood. 2007). Since MM-hMSCs have been cultured in vitro for several weeks and without any stimulation of MM cells, we believe that the impaired osteogenic differentiation of MM-hMSCs was due to an intrinsic abnormality. Several reports suggested that NOTCH signalling can maintain bone marrow mesenchymal progenitors in a more undifferentiated state by suppressing osteoblast differentiation (Hilton et al, Nat Med. 2008; Zanotti et al, Endocrinology. 2008). Therefore, we postulate that impaired osteogenic ability of MM-hMSCs might be (at least partly) related to abnormal NOTCH activity during osteogenesis. We found by quantitative real time PCR that NOTCH1, NOTCH2, Dll-1, Jagged-1, and NOTCH pathway downstream genes hes1, hey1, hey2, heyL were considerably decreased in ND-hMSCs after shifting them from normal culture medium to OD medium, indicating that NOTCH signalling was gradually suppressed during MSC osteogenesis. However, it was observed that the expression of NOTCH1, Jagged-1, Hes1 and Hes5 in MM-hMSCs did not decrease to the level of ND-hMSC with statistical difference. This implicates that the NOTCH signaling pathway remains in MM-hMSCs over-activated even in the presence of osteogenesis inducing signals. When the NOTCH signalling inhibitor DAPT was added to MM-hMSCs in OD medium, we found that hes1 expression was suppressed while, RUNX2 expression, a key transcription factor for osteoblastogenesis, as well as ALP activity, osteogenic genes expression and mineralization deposition were all increased. In conclusion our data indicate that MM-hMSCs exhibit in vitro lower osteogenic differentiation ability compared to ND-hMSCs, and that this impairement is associated with an inappropriate NOTCH pathway deactivation during the osteogenesis process. Targeting hMSCs in vivo by NOTCH inhibitors might have therapeutical potential to control bone disease in MM patients. Disclosures: No relevant conflicts of interest to declare.

The final quality control of cryopreserved progenitor cells is a successful and persistent three lineage engraftment after transplantation. Of course, the stem cell providing institution is obliged to have a program for controlling and monitoring the manufacturing of cellular therapy products before the patients’ conditioning therapy is started. The FACT-JACIE Standards [1] and the Netcord ⁄ FACT Stan- dards prescribe that the director of the institute shall define tests and procedures for measuring and assaying cellular therapy products to ensure their safety, viability and integ- rity and shall also ensure that products meet predetermined release specifications. This requires specifications of assays and the definition of thresholds to allow release.

2519 Background: Autologous monocyte derived dendritic cells (DC) electroporated with synthetic messenger RNA (smRNA) encoding CD40 ligand, a constitutively active TLR4, and CD70 (TriMixDC), together with smRNA encoding fusion-proteins of a HLA-class II targeting signal and a melanoma associated antigen are immunogenic when administered ID (Wilgenhof et al. ASCO AM 2009). The TriMixIDIV clinical trial investigates the safety, immunogenicity, and activity of combined ID and IV administration in patients with pretreated melanoma. METHODS Following leukapheresis, immature DC (derived from adherent PBMC cultured for 6 days in IL-4/GM-CSF supplemented medium) are electroporated with smRNA encoding MAGE-A3, MAGE-C2, Tyrosinase and gp100 linked to DC-LAMP, and TriMix smRNA. TriMix-DC are cryopreserved and 24.106 viable DC are administered by 4 ID/IV-injections q2w, and a 5th-injection on w16. The number of IV administered DC was escalated from 4.106, over 12.106, to 20.106 in cohort 1, 2 and 3 respectively. Tumor response assessments are performed by 18F-PET/CT at baseline and in w8, 16 and 24. Immunomonitoring is performed by analysis of DTH infiltrating lymphocytes (DIL) at an ID injection site. RESULTS 11 pts (9M/2F; med age 54, range 40-77) with stage IV pretreated melanoma, have initiated study treatment. TriMix-DC related AE's: gr2 local skin injection site reactions (all pts); fever & lethargy (gr1, 1 pt in cohort 1); chills (gr2, 2 pts in cohort 3). Vaccine-specific DIL were documented in 6/7 pts (5/7 pts had a CD137+CD8+ and 4/7 pts a CD4+ T-cell response). As of Jan 2011, 8 pts were evaluated for response (RECISTv1.1): 1 CR, 1 PR, 3 SD, and 3 PD; regression of metastases occurred in lung- (2 pts) and lymph node metastases (3 pts); 4/8 pts remain progression-free after respectively 3.1, 3.6, 7.7 and 8.2 mths of follow-up. CONCLUSIONS TriMix-DC therapy by combined IV/ID administration is feasible, safe, and immunogenic. Durable anti-melanoma activity is observed across the investigated IV-dose levels and compares favorably with our prior observations with TriMix-DC administered ID-only.

9024 Background: Electroporation of dendritic cells (DC) with mRNA encoding fusion-proteins of a HLA-class II targeting signal and a melanoma associated antigen (MAA) together with mRNA encoding CD40 ligand, a constitutively active TLR4 and CD70 (TriMix) improves the immunostimulatory capacity of autologous DC. Methods: Following leukapheresis, immature DCs (derived from adherent PBMC cultured for 6 days in IL-4 / GM-CSF supplemented medium) are electroporated with mRNA encoding MAGE-A3, MAGE-C2, Tyrosinase and gp100 linked to DC-LAMP, and TriMix mRNA. TriMix-DC (12.5 106/antigen) are cryopreserved and administered by 4 ID-injections q2w, and q8w thereafter. After the 4th vaccination, interferon alfa-2b (IFN- a2b, 5 MIU TIW) is initiated. Immune monitoring is performed by skin biopsy of a vaccine injection site. Biopsies are investigated by IHC and by analyzing the activation (CD137+), cytolytic capacity (CD107a+), and cytokine release (IFN-γ and TNF-α) of DTH infiltrating T-cells in response to autologous EBV-B cells expressing MAA. Results: 29 pts (17M/12F; med age 49, range 28–75) with stage III/IV melanoma, nl LDH, and no CNS metastases were recruited. Vaccine related AE's (first 24 pts): gr2 local injection site reactions (all pts); fever & lethargy (gr2, 1 pt). Pts (20) who initiated IFN-a2b experienced constitutional side effects (gr3, 1 pt). Vaccine-specific DTH infiltrating T cells were documented post-vaccination in 13/17 pts (10/13 pts had a CD137+CD8+ and 2/13 pts a CD4+ response). Out of the 11 pts without evaluable disease, 2 had a local recurrence (salvaged by surgery). After a mFU of 7.8 mths (range 4.3–13.7) all pts remain disease-free. Out of the 13 pts with measurable disease, BOR (RECIST) was 8 SD and 5 PD; 1 pt with initial PD subsequently obtained a PR. Regression of metastases occurred in lung- (2 pts), orbita- (1 pt) and lymph node metastases (3 pts). After a mFU of 7 mths (range 1–14), the mPFS is 3,1 mths (95% CI 2,29–4,08); 4 pts remain progression-free after respectively 5, 8, 10 and 11 mths of follow-up. Conclusions: Therapeutic vaccination with TriMix-DC combined with sequential IFN-a2b is feasible, safe, immunogenic and associated with anti-tumor activity in patients with advanced melanoma. [Table: see text]

BACKGROUND Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure or with 1 vial of CD34 reagent is limited. METHODS In this retrospective evaluation of 643 CliniMACS CD34-selection procedures, we validated the capacity of CliniMACS tubing sets and CD34 reagent. Endpoints of this study were the recovery and purity of CD34+ cells, T-cell depletion efficiency and recovery of colony-forming units-granulocyte-macrophage (CFU-GM). RESULTS Overloading normal or large-scale tubing sets with excess numbers of total nucleated cells, without exceeding the maximum number of CD34+ cells, had no significant effect on the recovery and purity of CD34+ cells. In contrast, overloading normal or large-scale tubing sets with excess numbers of CD34+ cells resulted in a significantly lower recovery of CD34+ cells. Furthermore, the separation capacity of 1 vial of CD34 reagent could be increased safely from 600 x 10(6) CD34+ cells to 1000 x 10(6) CD34+ cells with similar recovery of CD34(+) cells. Finally, T-cell depletion efficiency and the fraction of CD34+ cells that formed CFU-GM colonies were not affected by out-of-specification procedures. DISCUSSION Our validated increase of the capacity of CliniMACS tubing sets and CD34 reagent will reduce the number of selection procedures and thereby processing time for large HPC products. In addition, it results in a significant cost reduction for these procedures.

AIM Stem cell homing to injured tissue is necessary for local tissue repair. But homing of stem cells in chronic ischemic heart disease (CIHD) is poorly understood. This study investigated homing of peripheral blood stem cells (PBSC) expressing the CD133 antigen. After intracoronary injection. The cells were (111)In labeled for in vivo visualization. METHODS PBSC were mobilized with granulocyte-colony stimulating factor and collected by apheresis on d-1. On d0, CD133+ cells were enriched up to a median purity of 89% (range: 79-97%) with an immunomagnetic separation device (CliniMACS, Miltenyi). A fraction of the cells was radiolabeled with [(111)In]oxine in 0.1 M TRIS at pH 7.4 for 45-60 min. Cell viability after labeling was assessed using trypan-blue. The cells were injected at a radioactive concentration of 0.9 MBq/10(6) cells into the target open coronary vessel through a balloon catheter. During balloon inflation [(99m)Tc]sestamibi was injected intravenously to identify the myocardium and the target vascular territory. Eight patients (mean age: 53 years; range: 50-72 years) with stable CIHD and reduced left ventricular function (NYHA class I-II) after acute myocardial infarction (>12 months) were studied. After a first cohort of 3 patients received an injectate of 5-10 x 10(6) cells, our final protocol was applied in 5 patients in whom an average of 34.4 x 10(6) (range: 18.6-49.4) CD133+ cells was injected. Whole body and single photon emission computed tomography (SPECT) scans were acquired at different time points after injection (energy windows set at 140, 171 and 245 keV). Residual activity in the heart was assessed by drawing a region of interest around the heart on the anterior whole body views. RESULTS Mean labeling efficiency of [111In]oxine labeling was 51.2% and cell viability after labeling averaged 88%. In the 5 patients receiving the higher amount of labeled cells, a clear (111)In-signal was observed in the heart region up to 3 days after administration. Fused [(99m)Tc]sestamibi/(111)In SPECT images demonstrated that the regional distribution of the transplanted cells within the target zone, as delineated by the flow tracer, remained unchanged over time. A biodistribution study in 2 patients showed a residual activity in the heart, liver and spleen of 6.9-8%, 23.1-26.8%, 3.1-3.7%, respectively, after 1-2 h and 2.3-3.2% 23.8-28.3%, 3.5-3.8%, respectively, after 12 h (decay corrected and expressed as a percentage of total body initial activity). No adverse events were observed during the procedure and up to 3 months follow-up. CONCLUSIONS Radiolabeling with [(111)In]oxine is a suitable method for follow-up of cell distribution during the first days after transplantation. A significant amount of CD133+ PBSC home to the heart after intracoronary injection in patients with CIHD. The results of this study are useful for the design of trials that evaluate the tissue repair potential of CD133+ PBSC in the setting of CIHD.

Neighboring adipocytes participate in the bone marrow microenvironment of multiple myeloma cells