Currently there is no knowledge about the relevance of MDSC in ovarian cancer patients. However, it is known that both Tregs and suppressive macrophages are increased in this malignancy and have been suggested to correlate with worse prognosis. In order to obtain data, we are collecting blood and ascitic fluid of ovarian cancer patients to study the presence of immature myeloid populations as compared to healthy donor blood. We have identified two candidate populations that will be tested for the ability to suppress T cells. We also aim to characterize these cel...

Data has been obtained on the phenotype and function of myeloid derived suppressor cells in melanoma in patients. Accumulation of myeloid derived suppressor cells (MDSC) with the ability to suppress T cells has been observed in serveral types of cancer, including melanoma. Using blood from melanoma patients we are investigating phenotypic markers that can be used to further characterize MDSC, focusing on molecules that are related to their suppressive function. As it is believed that recruitment from the bone marrow by tumor derived factors leads to increased pre...

BruCells is collaborating with Professor De Witte from ULB for the preclinical study in which circulating tumour-specific CD8+ T-cells will be detected in the blood of newly diagnosed glioblastoma patients and from which data will be obtained. In order to obtain the blood samples and tumour biopsies from the glioblastoma patients, a file has been submitted to the Ethics Committee of the Erasme Hospital and has been recently approved.

This test, described for glioblastoma, performed with PBMC from newly diagnosed glioblastoma patients will verify the ability of tumour-specific T-cells to recognize the fusion vaccine. Data will be made available. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

This test will verify whether the fusion vaccine possesses the properties of both parental cell types for inducing primary CD8+ T-cell responses (data will become available). Detection of circulating tumour-specific CD8+ T-cells. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

Data will be available from in-vitro pre-clinical tests on the pharmacodynamic activity of the fusion vaccines. This test will verify the capacity of the fusion vaccine to present certain tumour-antigen derived peptides corresponding to antigens present in the fused cancer cells. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

We explored for the first time transfection of CD4(+) and CD8(+) T cells with mRNA encoding recombinant immunoreceptors for use in the adoptive immunotherapy of cancer and obtained data from this study. CD4(+) and CD8(+) T cells were efficiently transfected with immunoreceptors specific for ErbB2 and CEA. The immunoreceptor expression was transient with half-maximal expression at day 2 and no detectable immunoreceptor expression at day 9 after electroporation. Immunoreceptor-transfected T cells were specifically activated upon coincubation with ErbB2(+) and CEA(+...

We have data available from a comparison of the use of fresh peripheral blood mononuclear cells (PBMC) versus fresh heparinised whole blood. Only when peptide pools as an antigen source are used PBMC exhibited a slightly higher stimulation capacity compared to whole blood. However, slightly higher background was observed when using PBMC to stimulate with antigen. Lowest background was observed when either peptides or protein were used for stimulation enabling assessment also of rare antigen-specific Th-cells below frequencies of 0.05% of peripheral CD4+ Th-cell...

Data were obtained from an ex-vivo analysis of different peptide specific T cell functions like degranulation and production of cytokines such as IFN? and TNF? by 8-color flow cytometry. In general, pre-existing immune-responses were rather mono-functional with dominating “IFN?-only”, “TNF?-only” and “degranulation-only” cells in the CD8+ compartment. Vaccination not only increased some of these monofunctional subpopulations but also induced occurrence of tumorantigen specific polyfunctional subsets. The further course of our melanoma patients will sho...

We obtained data on the T-cell stimulatory capacity of human monocyte-derived DCs co-electroporated with different combinations of CD40L, CD70, and constitutively active toll-like receptor 4 (caTLR4) encoding mRNA. The effectiveness of the dendritic cell (DC) vaccination protocols that are currently in use could be improved by providing the DCs with a more potent maturation signal. We therefore investigated whether the T-cell stimulatory capacity of human monocyte-derived DCs could be increased by co-electroporation with different combinations of CD40L, CD70, and...

Data has been obtained on TriMix DCs co-electroporated with whole tumorantigen encoding mRNA. A critical factor determining the effectiveness of currently used dendritic cell (DC)-based vaccines, is the DC’s activation or maturation status. We have shown recently that the T cell stimulatory capacity of DCs pulsed with tumorantigen-derived peptides can be considerably increased by activating the DCs through electroporation with CD40L, CD70 and constitutively active TLR4 encoding mRNA (TriMix DCs). Here, we investigate whether TriMix DCs can be co-electroporate...

We have used agonists of the dectin-1/Syk pathway as adjuvants in vivo to induce CD8+ T cell responses and have data available on this. In summary, we have shown that this can result in CTL capable of destroying tumours.

Data is available from an evaluation of different stimulation conditions for the cytometric assessment of antigen-specific Th-cells based on CD154 expression induced during short-term in vitro activation, a method established by us during previous DC-THERA activities. This is important and essential for implementation of the technology into clinical immune monitoring in e.g. vaccination trials. Moreover, the affect of stimulation length was determined. Differences with respect to the induced frequencies of antigen-specific, activated CD154+ Th-cells were minor. I...

We have obtained data on the immunogenicity induced by chemotherapeutics.

Data has been generated in HHD2 transgenic mice on dendritic cell derived-exosomes (DEX), nanomeric vesicles harboring MHC/peptide complexes capable of promoting primary T cell responses and tumor rejection in the presence of adjuvants. We showed that, in the absence of adjuvants, DEX mediate potent antigen dependent-antitumor effects against preestablished tumors in mice pretreated with immunopotentiating dosing of cyclophosphamide (CTX). CTX could i) abolish the suppressive function of CD4+CD25+Foxp3+ regulatory T cells (Treg), ii) markedly enhance the mag...

We further our understanding of different DC populations and their soluble products in adults but in particular in neonatal mice. We have concentrated on the evaluation of the interplay between the hematopoetic factor Flt3L and IFNalpha,beta and have data available from our studies. We found that the cooperation between IFNalpha,beta and Flt3L (FL) plays an important role in the defense against Herpes simplex virus type 1 (HSV-1) in neonates. Treatment of neonatal mice with recombinant IFNalpha has a short-term, FL-independent and a long-term, FL-dependent protect...

We have data available on dendritic cell development from hematopoietic stem and progenitor cells in steady-state and in inflammatory conditions, and, in particular, gene-expression data of Lin–c-KitintFlt3+M-CSFR+ cells in mouse bone marrow. Lymphoid tissue plasmacytoid and conventional dendritic cells (DCs) are continuously regenerated from hematopoietic stem cells. The cytokine dependence and biology of plasmacytoid and conventional DCs suggest that regeneration might proceed through common DC-restricted developmental intermediates. By selecting for cytokin...

Protein expression data is available for our pre-clinical work aiming at developing DC based vaccines expressing the tumor antigens PSA or Her2/neu. Monocyte rich fractions were produced by elutriation under GMP conditions and cultured for 5 days with GM-CSF and IL-4. Conditions for transfection with electroporation (Amaxa) or with Adenovirus constructs have been established. Protein expression (Her2/neu, PSA) following transfection with a panel of different full length or truncated Her2/neu or PSA constructs was assessed using Facs analysis (Her2/neu) or assessme...

We have obtained data addressing the effectiveness of TLR ligand-long peptide conjugates in vivo by either direct vaccination or ex vivo DC loading prior to vaccination. Both approaches show similar effectiveness in CTL priming suggesting that TLRL-peptide conjugates efficiently target DC in vivo.

Data has been obtained on DCs loaded with Ag-IgG immune complexes (ICs) as a means of prophylactic immunization in mice. Therapeutic vaccinations strongly delay tumor growth or even prevent tumors from growing out. By depleting CD4+ and CD8+ cell populations prior to tumor challenge, CD8+ cells were identified as the main effector cells involved. Importantly, DCs pre-loaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of non-complexed protein. FcgammaRs on DCs were required for effici...