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Dendritic cells (DCs) are an attractive tool for immunomodulation, targeting mature DCs (mDCs) for immunization or immature/semimature DCs (iDCs) for tolerization. Therefore, introducing antigens into DCs has become a prime topic in various immunological disciplines. Numerous studies have shown that lentiviruses are an efficient vehicle for this purpose. This study evaluates the effects of lentiviral transduction on iDC activation. Immature DCs are efficiently transduced with increasing doses of lentivirus without affecting cell viability. Transduction at low multiplicities of infection (MOIs) did not result in phenotypical or functional maturation. Higher doses of lentivirus, however, resulted in upregulation of adhesion, costimulatory, and HLA molecules, as well as in increased allostimulatory capacity and secretion of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha. Production of IL-12 p70, IL-10, and interferon-alpha was observed only at extremely high doses. Protein kinase R phosphorylation on transduction at an MOI of 150 was demonstrated by Western blotting. A Toll-like receptor (TLR)-driven luciferase reporter assay showed dose-dependent activation of TLR2, TLR3, and TLR8, which was independent of the pseudotype, production, or transduction protocol and was abrogated on heat inactivation. These data show that lentiviral vectors provide not only the antigen but also appropriate activation signals to iDCs, favoring their use for immunotherapy and vaccine development.

In situ modification of antigen-presenting cells garnered interest in cancer immunotherapy. Therefore, we developed APC-targeted lentiviral vectors (LVs). Unexpectedly, these LVs were inferior vaccines to broad tropism LVs. Since IL-12 is a potent mediator of antitumor immunity, we evaluated whether this proinflammatory cytokine could enhance antitumor immunity of an APC-targeted LV-based vaccine. Therefore, we compared subcutaneous administration of broad tropism LVs (VSV-G-LV) with APC-targeted LVs (DC2.1-LV)-encoding enhanced GFP and ovalbumin, or IL-12 and ovalbumin in mice. We show that codelivery of IL-12 by VSV-G-LVs or DC2.1-LVs augments CD4(+) or CD8(+) T-cell proliferation, respectively. Furthermore, we demonstrate that codelivery of IL-12 enhances the CD4(+) TH 1 profile irrespective of its delivery mode, while an increase in cytotoxic and therapeutic CD8(+) T cells was only induced upon VSV-G-LV injection. While codelivery of IL-12 by DC2.1-LVs did not enhance CD8(+) T-cell performance, it increased expression of inhibitory checkpoint markers Lag3, Tim3, and PD-1. Finally, the discrepancy between CD4(+) T-cell stimulation with and without functional CD8(+) T-cell stimulation by VSV-G- and DC2.1-LVs is partly explained by the observation that IL-12 relieves CD8(+) T cells from CD4(+) T-cell help, implying that a T(H)1 profile is of minor importance for antitumor immunotherapy if IL-12 is exogenously delivered.

Cancer immunotherapy has been proposed as a powerful treatment modality. Active immunotherapy aspires to stimulate the patient's immune system, particularly T cells. These cells can recognize and kill cancer cells and can form an immunological memory. Dendritic cells (DCs) are the professional antigen-presenting cells of our immune system. They take up and process antigens to present them to T cells. Consequently, DCs have been investigated as a means to stimulate cancer-specific T-cell responses. An efficient strategy to program DCs is the use of mRNA, a well-defined and safe molecule that can be easily generated at high purity. Importantly, vaccines consisting of mRNA-modified DCs showed promising results in clinical trials. Therefore, we will introduce cancer immunotherapy and DCs and give a detailed overview on the application of mRNA to generate cancer-fighting DC vaccines.

About 25 years ago, mRNA became a tool of interest in anticancer vaccination approaches. However, due to its rapid degradation in situ, direct application of mRNA was confronted with considerable skepticism during its early use. Consequently, mRNA was for a long time mainly used for the ex vivo transfection of dendritic cells, professional antigen-presenting cells known to stimulate immunity. The interest in direct application of mRNA experienced a revival, as researchers became aware of the many advantages mRNA offers. Today, mRNA is considered to be an ideal vehicle for the induction of strong immune responses against cancer. The growing numbers of preclinical trials and as a consequence the increasing clinical application of mRNA as an off-the-shelf anticancer vaccine signifies a renaissance for transcript-based antitumor therapy. In this review, we highlight this renaissance using a timeline providing all milestones in the application of mRNA for anticancer vaccination.

Dendritic cells (DC) are professional antigen-presenting cells that are used in vaccine approaches to cancer. Classically, mature monocyte-derived DC are generated in vitro in the presence of interleukin (IL)-4, granulocyte macrophage-colony stimulating factor, and inflammatory cytokines (G4-DC). Recently, it has been described that DC can also be generated in the presence of IL-3 and interferon (IFN)-β and that these DC are efficiently matured using polyriboinosinic polyribocytidylic acid (I3-DC). In this study, a series of in vitro experiments was performed to compare side-by-side I3-DC and G4-DC as vaccine adjuvants. Phenotypic characterization of the DC revealed differences in the expression of the monocyte marker CD14 and the maturation marker CD83. Low expression of CD14 and high expression of CD83 characterized G4-DC, whereas I3-DC displayed intermediate expression of CD14 and CD83. Both types of DC were as potent in the induction of allogeneic T cell proliferation. Upon CD40 ligation, G4-DC produced lower amounts of IFN-α and pulmonary and activation-regulated chemokine, similar amounts of IL-6, macrophage-inflammatory protein (MIP)-1α, and MIP-1β, and higher amounts of IL-12 p70, tumor necrosis factor α, and MIP-3β than I3-DC. We further evaluated whether the DC could be frozen/thawed without loss of cell number, viability, phenotype, and function. After freezing/thawing, 56.0% ± 9.0% of I3-DC and 77.0% ± 3.0% of G4-DC (n=9) were recovered as viable cells, displaying the same phenotype as their fresh counterparts. Finally, in vitro stimulations showed that fresh and frozen peptide-loaded I3-DC are more potent inducers of Melan-A-specific CD8+ T cell responses than G4-DC. The antigen-specific T cells were functional as shown in cytotoxicity and IFN-γ secretion assay.

An optimal anticancer vaccine probably requires the cooperation of both CD4+ Th cells and CD8+ CTLs. A promising tool in cancer immunotherapy is, therefore, the genetic modification of dendritic cells (DCs) by introducing the coding region of a tumor Ag, of which the antigenic peptides will be presented in both HLA class I and class II molecules. This can be achieved by linking the tumor Ag to the HLA class II-targeting sequence of an endosomal or lysosomal protein. In this study we compared the efficiency of the targeting signals of invariant chain, lysosome-associated membrane protein-1 (LAMP1) and DC-LAMP. Human DCs were electroporated before or after maturation with mRNA encoding unmodified enhanced green fluorescent protein (eGFP) or eGFP linked to various targeting signals. The lysosomal degradation inhibitor chloroquine was added, and eGFP expression was evaluated at different time points after electroporation. DCs were also electroporated with unmodified MAGE-A3 or MAGE-A3 linked to the targeting signals, and the presentation of MAGE-A3-derived epitopes in the context of HLA class I and class II molecules was investigated. Our data suggest that proteins linked to the different targeting signals are targeted to the lysosomes and are indeed presented in the context of HLA class I and class II molecules, but with different efficiencies. Proteins linked to the LAMP1 or DC-LAMP signal are more efficiently presented than proteins linked to the invariant chain-targeting signal. Furthermore, DCs electroporated after maturation are more efficient in Ag presentation than DCs electroporated before maturation.

BACKGROUND & AIMS: The peroxisome proliferator-activated nuclear receptors (PPAR-alpha, PPAR-beta, and PPAR-gamma), which modulate the expression of genes involved in energy homeostasis, cell cycle, and immune function, may play a role in hepatic stellate cell activation. Previous studies focused on the decreased expression of PPAR-gamma in hepatic stellate cell activation but did not investigate the expression and role of the PPAR-alpha and -beta isotypes. The aim of this study was to evaluate the expression of the different PPARs during hepatic stellate cell activation in vitro and in situ and to analyze possible factors that might contribute to their expression. In a second part of the study, the effect of a PPAR-beta agonist on acute liver injury was evaluated. METHODS: The effects of PPAR isotype-specific ligands on hepatic stellate cell transition were evaluated by bromodeoxyuridine incorporation, gel shifts, immunoprecipitation, and use of antisense PPAR-beta RNA-expressing adenoviruses. Tumor necrosisfactor alpha-induced PPAR-beta phosphorylation and expression was evaluated by metabolic labeling and by using specific P38 inhibitors. RESULTS: Hepatic stellate cells constitutively express high levels of PPAR-beta, which become further induced during culture activation and in vivo fibrogenesis. No significant expression of PPAR-alpha or -gamma was found. Stimulation of the P38 mitogen-activated protein kinase pathway modulated the expression of PPAR-beta. Transcriptional activation of PPAR-beta by L165041 enhanced hepatic stellate cell proliferation. Treatment of rats with a single bolus of CCl(4) in combination with L165041 further enhanced the expression of fibrotic markers. CONCLUSIONS: PPAR-beta is an important signal-transducing factor contributing to hepatic stellate cell proliferation during acute and chronic liver inflammation.

Recently, human dendritic cells (DCs) pulsed with mRNA encoding a broad range of tumor antigens have proven to be potent activators of a primary anti-tumor-specific T-cell response in vitro. The aim of this study was to improve the mRNA pulsing of murine DC. Compared to a standard lipofection protocol and passive pulsing, electroporation was, in our hands, the most efficient method. The optimal conditions to electroporate murine bone marrow-derived DCs with mRNA were determined using enhanced green fluorescent protein and a truncated form of the nerve growth factor receptor. We could obtain high transfection efficiencies around 70-80% with a mean fluorescence intensity of 100-200. A maximal expression level was reached 3 hours after electroporation. A clear dose-response effect was seen depending on the amount of mRNA used. Importantly, the electroporation process did not affect the viability nor the allostimulatory capacity or phenotype of the DC. To study the capacity of mRNA-electroporated DCs to present antigen in the context of MHC classes I and II, we made use of chimeric constructs of ovalbumin. The dose-dependent response effect and the duration of presentation were also determined. Together, these results demonstrate that mRNA electroporation is a useful method to generate genetically modified murine DC, which can be used for preclinical studies testing immunotherapeutic approaches.

// Kevin Van der Jeught 1 , Patrick Tjok Joe 1 , Lukasz Bialkowski 1 , Carlo Heirman 1 , Lidia Daszkiewicz 1 , Therese Liechtenstein 2 , David Escors 2, 3 , Kris Thielemans 1 , Karine Breckpot 1 1 Laboratory of Molecular and Cellular Therapy, Department of Biomedical Sciences, Vrije Universiteit Brussel, Brussels, Belgium 2 Rayne Institute, University College London, London, UK 3 Biomedical Research Centre NavarraBiomed-Fundacion Miguel Servet, National Health Service of Navarre, Pamplona, Navarre, Spain Correspondence to: Dr. Karine Breckpot, e-mail: kbreckpo@vub.ac.be Keywords: mRNA, IFN-β, TGF-β, cancer therapy, T cell Received: July 04, 2014 Accepted: September 06, 2014 Published: September 19, 2014 ABSTRACT It is generally accepted that the success of immunotherapy depends on the presence of tumor-specific CD8 + cytotoxic T cells and the modulation of the tumor environment. In this study, we validated mRNA encoding soluble factors as a tool to modulate the tumor microenvironment to potentiate infiltration of tumor-specific T cells. Intratumoral delivery of mRNA encoding a fusion protein consisting of interferon-β and the ectodomain of the transforming growth factor-β receptor II, referred to as Fβ 2 , showed therapeutic potential. The treatment efficacy was dependent on CD8 + T cells and could be improved through blockade of PD-1/PD-L1 interactions. In vitro studies revealed that administration of Fβ 2 to tumor cells resulted in a reduced proliferation and increased expression of MHC I but also PD-L1. Importantly, Fβ 2 enhanced the antigen presenting capacity of dendritic cells, whilst reducing the suppressive activity of myeloid-derived suppressor cells. In conclusion, these data suggest that intratumoral delivery of mRNA encoding soluble proteins, such as Fβ 2 , can modulate the tumor microenvironment, leading to effective antitumor T cell responses, which can be further potentiated through combination therapy.

Peripheral blood T cells transduced with a tumor-specific T-cell receptor (TCR) face problems of auto-reactivity and lack of efficacy caused by cross-pairing of exogenous and endogenous TCR chains, as well as short term in vivo survival due to activation and growth factor-induced differentiation. We here studied an alternative strategy for the efficient generation of naive CD8(+) T cells with a single TCR. TCR-transduced human postnatal thymus-derived and adult mobilized blood-derived hematopoietic progenitor cells (HPCs) were differentiated to CD4(+)CD8(+) double-positive T cells using OP9-Delta-like 1 (OP9-DL1) cultures. Addition of the agonist peptide induced double positive cells to cross-present the peptide, leading, in the absence of co-stimulation, to cell cycle arrest and differentiation into mature CD8(+) T cells. Comprehensive phenotypic, molecular and functional analysis revealed the generation of naive and resting CD8(+) T cells through a process similar to thymic positive selection. These mature T cells show a near complete inhibition of endogenous TCRA and TCRB rearrangements and express high levels of the introduced multimer-reactive TCR. Upon activation, specific cytokine production and efficient killing of tumor cells were induced. Using this strategy, large numbers of high-avidity tumor-specific naive T cells can be generated from readily available HPCs without TCR chain cross-pairing.

Corrigendum to ‘A phase I/IIa immunotherapy trial of HIV-1-infected patients with Tat, Rev and Nef expressing dendritic cells followed by treatment interruption’ [Clin. Immunol. 142 (2012) 252–268] Sabine D. Allard⁎, Brenda De Keersmaecker, Anna L. de Goede, Esther J. Verschuren, Jeanette Koetsveld , Mariska L. Reedijk , Carolien Wylock, Annelies V. De Bel , Judith Vandeloo, Frank Pistoor , Carlo Heirman, Walter E.P. Beyer , Paul H.C. Eilers , Jurgen Corthals , Iman Padmos, Kris Thielemans, Albert D.M.E. Osterhaus , Patrick Lacor, Marchina E. van der Ende, Joeri L. Aerts , Carel A. van Baalen, Rob A. Gruters c,2

Dendritic cell (DC)-based cancer vaccines, where the patient's own immune system is harnessed to target and destroy tumor tissue, have emerged as a potent therapeutic strategy. In the development of such DC vaccines, it is crucial to load the DCs with tumor antigens, and to simultaneously activate them to become more potent antigen-presenting cells. For this, we report on microbubbles, loaded with both antigen mRNA as well as immunomodulating TriMix mRNA, which can be used for the ultrasound-triggered transfection of DCs. In vivo experiments with in vitro sonoporated DCs show the effective induction of antigen-specific T cells, resulting in specific lysis of antigen-expressing cells. Especially in a therapeutic setting, sonoporation with TriMix has an important added value, resulting in a significant reduction of tumor outgrowth and a marked increase in overall survival. What is more, complete tumor regression was observed in 30% of the antigen+TriMix DC vaccinated animals, which also displayed long-term antigen-specific immunological memory. As a result, DC sonoporation using microbubbles loaded with a combination of antigen and TriMix mRNA can elicit powerful immune responses in vivo, and might serve as a potential tool for further in vivo DC vaccination applications.

The micro-environment in multiple myeloma (MM) is highly immunosuppressive with increased numbers of regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs) favoring tumorcell survival and hampering immunotherapeutic strategies such as dendritic cell vaccination. Immunomodulatory drugs (IMiDs) are known to enhance T- and NK-cell function. In this study we evaluated the effects of low dose (0.5 microM) lenalidomide (Len) and pomalidomide (Pom) on the functionality of CD8+ and CD4+ T cells, MDSCs, Tregs and ex-vivo generated mononuclear derived dendritic cells (moDCs) obtained from MM patients after first autologous stem cell transplantation (ASCT). Peripheral blood mononuclear cell fractions were obtained by leukapheresis from 9 MM patients (age 29-62 years), in very good partial response (4/9) or complete response (5/9) after ASCT. The magnitude of cytokine release (mean +/- standard error of the mean, in ng/ml) by purified CD8+ T cells after 144 hours stimulation with anti-CD3/anti-CD28 coated microbeads was significantly increased after addition of Len and Pom to the culture medium, respectively : IFN-gamma (217.5 +/- 62.1 and 437.1 +/- 137.1** vs 66.4 +/- 21.0) , TNF-alpha (21.4 +/- 5.4 and 44.9 +/- 9.4*** vs 4.9 +/- 1.7) and IL-2 (5.3 +/- 2.7 and 12.7 +/- 6.6 vs 1.9 +/- 1.7 ng/ml) (** p< 0.01, *** p< 0.001). We also evaluated the number of different types of cytokines/chemokines on a per cell basis by intracellular flow cytometry staining for IFN-gamma, TNF-alpha, IL-2 and MIP-1beta and observed increased polyfunctionality of CD8+ and CD4+ T cells. After 72 h of stimulation with anti-CD3/CD28 microbeads the number of single, double, triple or quadruple functional CD8+ T cells increased from 5.96 %, 2.82 %, 0.1 %, 0 % (culture medium alone) to 9.68 %, 7.57 %, 0.41%, 0.03 % (Len) and 12.57 %, 8.96 %, 0.81 %, 0.03 % (Pom), respectively. A similar observation was made for CD4+ T cells. A significant percentage, median 5.7 % (4.0-7.2 %) of CD4+ CD25high CD127low (Tregs) was found in the CD4+ T cell population in 8 out of 9 patients, demonstrating the highly suppressive immune environment in myeloma patients even with low disease burden. Effector T cells (Teffs) were stimulated with anti-CD3/CD28 microbeads and cocultured at varying ratios with purified Tregs. After 144 h of coculture, Len and Pom reduced the suppressive effects of Tregs on Teffs proliferation and IFN-gamma and TNF-alpha production (see figure). A similar effect was observed for MDSC but did not reach statistical significance (data not shown). TriMix DCs (moDCs matured by electroporation with mRNA encoding TLR4, CD40L and CD70) and cytokine matured moDCs were cocultured with autologous CD4+ and CD8+ T cells and anti-CD3 microbeads. Adding IMiDs resulted in more polyfunctional CD4+ and CD8+ T cells with both types of DCs but effects were most pronounced with the TriMix variant. Our study shows that Len and Pom restore effector cell functions in myeloma patients with low tumor burden after ASCT. These findings provide a immunomechanistic explanation for IMiD-based maintenance therapy. They also offer a rationale to combine IMiD-based maintenance with immunotherapeutic approaches such as dendritic cell vaccination in this particular setting. Disclosures: No relevant conflicts of interest to declare.

Follicular lymphoma (FL) cells have inherited an activated hypermutation mechanism from their origin of germinal centre B cells. Based on today's knowledge of the intrinsic properties related to this mechanism and the VH base composition, reconsideration of previous reports should be made on a broader range of samples. The present study examined the mutation pattern of the VH genes expressed by 55 cases of FL. FL VH genes showed evidence of antigenic selection in 30% of cases with 88% carrying a functional sIg and 78.2% showing intraclonal variation. VH family and gene segment utilization was found to be roughly similar to that of normal B lymphocytes. FL VH genes revealed extensive variations. 17% of the VH exons harboured a total of five deletions, three duplications and two insertions as compared to the most homologous germline counterpart. The VH genes of one tumour displayed three populations with varying CDR3 length at diagnosis. At relapse, emergence of a differently mutated gene, additional mutations reminiscent of ongoing mutations or no variation was prominent. From this study the heterogeneity of FLs is well established and ongoing mutations are seen in the scope of the activated status of the hypermutation mechanism rather than antigen-stimulated tumour growth.

Recently it was reported that Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) infects bone marrow (BM) dendritic cells (DC) in multiple myeloma (MM) patients and therefore might play a role in MM development. Because of the use of myeloid growth factors like GM-CSF and G-CSF for the mobilization of peripheral blood progenitor cells (PBPC), the subsequent increase of DC precursors might imply a risk for KSHV contamination in PBPC grafts. Therefore, in this study leukapheresis products and ex vivo cultured CD34+ cell suspensions were analysed. KSHV DNA could not be amplified in any of them.

As demonstrated in several preclinical models, bispecific Abs are attractive immunotherapeutic agents for tumor treatment. We have previously reported that a bacterially produced anti-CD3 × antitumor bispecific single chain variable fragment of Ab fragment (BsscFv), which is capable of retargeting CTLs toward BCL1 tumor cells, exhibits antitumor activity in vitro. To further facilitate BsscFv production, the coding sequence was subcloned in a eukaryotic expression vector and introduced into Chinese hamster ovary cells for large-scale production. In this report, we have determined the serum stability and the clearance rate from the circulation of BsscFv. Most important, we prove here the therapeutic value of BsscFv in the treatment of BCL1 lymphoma, a murine model for human non-Hodgkin’s lymphoma. Tumor-bearing mice that were treated with rscFv in combination with staphylococcal enterotoxin B superantigen, human rIL-2, or murine rIL-12 showed long-term survival, whereas untreated mice all died. This is the first report of the successful in vivo use of BsscFv as an immunotherapeutic agent. Furthermore, long-term survival was the result of complete tumor removal and was not due to the induction of dormancy.

This report summarizes our experimental data concerning the use of bispecific antibodies (bsAb) for the treatment of the murine BCL1 B cell lymphoma model. Initially we used a hybrid-hybridoma-derived bsAb with specificity for the TcR/CD3 complex on T cells and the idiotype of the membrane-bound IgM on the tumor cells. The bsAb used as a single agent could cure animals with a low tumor load (resembling minimal residual disease). However, in experiments aimed at increasing the therapeutic effect in animals with a higher tumor burden, we could demonstrate the importance of additional T-cell-costimulatory signals and the careful timing of the bsAb administration. Recently we have generated a bispecific single-chain Fv (bsscFv) fusion protein with the same dual specificity as the hybrid-hybridoma-derived bsAb. Immunotherapy with this smaller molecule also resulted in tumor elimination in BCL1-bearing mice. A second bsscFv (alpha-CD19 x alpha-CD3) with a broader applicability is now being characterized and tested in vivo.

Regulatory T cells (Tregs) counteract anticancer immune responses through a number of mechanisms, limiting dendritic cell (DC)–based anticancer immunotherapy. In this study, we investigated the influence of various DC activation stimuli on the Treg functionality. We compared DCs activated by electroporation with mRNA encoding constitutively active TLR4 (caTLR4) and CD40 ligand (DiMix-DCs), or these factors together with mRNA encoding the costimulatory molecule CD70 (TriMix-DCs) with DCs maturated in the presence of a mixture of inflammatory cytokines (DCs maturated with a combination of the cytokines IL-1β, IL-6, TNF-α, and PGE2) for their ability to counteract Tregs on different levels. We first demonstrated that there was no difference in the extent of Treg induction starting from CD4+CD25− T cells under the influence of the different DC maturation stimuli. Second, we showed that both DiMix- and TriMix-DCs could partly alleviate Treg inhibition of CD8+ T cells. Third, we observed that CD8+ T cells that had been precultured with DiMix-DCs or TriMix-DCs were partially protected against subsequent Treg suppression. Finally, we showed that Tregs cocultured in the presence of TriMix-DCs, but not DiMix-DCs, partially lost their suppressive capacity. This was accompanied by a decrease in CD27 and CD25 expression on Tregs, as well as an increase in the expression of T-bet and secretion of IFN-γ, TNF-α, and IL-10, suggesting a shift of the Treg phenotype toward a Th1 phenotype. In conclusion, these data suggest that TriMix-DCs are not only able to suppress Treg functions, but moreover could be able to reprogram Tregs to Th1 cells under certain circumstances.

Background Autologous monocyte-derived dendritic cells (DCs) electroporated with synthetic messenger RNA (mRNA) encoding a CD40 ligand, a constitutively active Toll-like receptor 4 and CD70, together with mRNA encoding fusion proteins of a human leukocyte antigen (HLA)-class II targeting signal (DC-LAMP) and a melanoma-associated antigen (MAA); either MAGE-A3, MAGE-C2, tyrosinase or gp100) (TriMixDC-MEL) are superiorly immunogenic. Patients and methods In this phase IB clinical trial, 24 million viable DCs were administered by four biweekly combined intradermal (id) and intravenous (iv) administrations, and a fifth administration on week 16. The number of iv-administered DCs was escalated in four sequentially treated cohorts. Immune responses were assessed by analysis of antigen specificity of blood-derived T-cells and skin infiltrating lymphocytes (SKILs). Results Fifteen patients with pretreated advanced melanoma tolerated administration of TriMixDC-MEL well. Two patients achieved a complete response and two patients a partial response. All objective responders are progression-free after a follow-up of, respectively, 24+, 28+, 33+, and 34+ months. Post-therapy antigen-specific SKILs were documented in 6 of 12 patients, and antigen-specific CD8+ T-cells were detected in the blood of 4 of 5 patients. Conclusions Cellular immunotherapy with TriMixDC-MEL is safe and immunogenic. Antitumor activity with durable disease control is observed across the investigated iv-dose levels. ClinicalTrials.gov Identifier NCT01066390.

Antigen-presenting cells are a heterogeneous group of cells that are characterized by their functional specialization. Consequently, targeting specific antigen-presenting cell subsets offers opportunities to induce distinct T cell responses. Here we report on the generation and use of nanobodies (Nbs) to target lentivectors specifically to human lymph node-resident myeloid dendritic cells, demonstrating that Nbs represent a powerful tool to redirect lentivectors to human antigen-presenting cell subsets.

Treatment of melanoma patients with mRNA electroporated dendritic cells (TriMixDC-MEL) stimulates T-cell responses against the presented tumor-associated antigens (TAAs). In the current clinical trials, melanoma patients with systemic metastases are treated, requiring priming and/or expansion of preexisting TAA-specific T cells that are able to migrate to both the skin and internal organs. We monitored the presence of TAA-specific CD8+T cells infiltrating the skin at sites of intradermal TriMixDC-MEL injection (SKILs) and within the circulation of melanoma patients treated in two clinical trials. In 10 out of fourteen (71%) patients screened, CD8+T cells recognizing any of the four TAA presented by TriMixDC-MEL cellular vaccine were found in both compartments. In total, 30 TAA-specific T-cell responses were detected among the SKILs and 29 among peripheral blood T cells, of which 24 in common. A detailed characterization of the antigen specificity of CD8+T-cell populations in four patients indicates that the majority of the epitopes detected were only recognized by CD8+T cells derived from either skin biopsies or peripheral blood, indicating that some compartmentalization occurs after TriMix-DC therapy. To conclude, functional TAA-specific CD8+T cells distribute both to the skin and peripheral blood of patients after TriMixDC-MEL therapy.

Tumor antigen-encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.Molecular Therapy-Nucleic Acids (2013) 2, e134; doi:10.1038/mtna.2013.54; published online 19 November 2013.

Autologous monocyte-derived synthetic mRNA electroporated dendritic cells (TriMixDC-MEL) are immunogenic and can induce anti-tumor activity in pretreated advanced melanoma patients when administered by the intradermal (id) and intravenous (iv) route. We investigated the safety and activity of TriMixDC-MEL in stage III/IV melanoma patients who are disease free following the local treatment of macrometastases. We performed a randomized, controlled, non-comparative phase II clinical trial where TriMixDC-MEL was administered iv (20.106 DCs) and id (4.106 DCs) at 2 separate sites of the body every 2 weeks for a total of 4 administrations and a fifth administration after 16 weeks in patients randomized to the treatment arm. Patients on the control arm did not receive treatment but were allowed to “cross-over” and be treated with TriMixDC-MEL at the time of non-salvageable recurrence. Tumor evaluations on both arms were performed by total-body 18-FDG-PET/CT every 12 weeks. The primary endpoint was the percentage of patients who were alive and free from melanoma macrometastases at 1 year following randomization. Patients were allowed to undergo local salvage treatments for loco-regional melanoma recurrences during the study (week 0-52). If local salvage treatment during the study resulted in a disease-free status that was confirmed at any of the planned assessment, but not earlier than 16 weeks after the salvage treatment, these patients were considered not to have reached the primary endpoint of the study. Between November 2012 and November 2014, 41 eligible patients were randomized between the TriMixDC-MEL treatment arm (n = 21) and control-arm (n = 20). Baseline characteristics (22M/19F; median age 57.5y (range 24-81); AJCC stage III/IV-M1a/-M1b/-M1c: 33/1/6/1 patients, ulcerated primary melanoma: 12 patients) were well balanced between both groups. After a median follow-up of 28 months (range 15 to 40 months) 21 patients experienced a non-salvageable melanoma recurrence (7 on the DC- and 14 on the control-arm). The rate of patients who were disease-free at 1 year (evaluable population = 41 patients) was higher in the TriMixDC-MEL treated group (68% [95%CI 46-86] vs. 35% [14-55]). The time-to-non-salvageable melanoma recurrence was significantly lower in the TriMixDC-MEL treated group (log-rank p = .021). Seven patients in the TriMixDC-MEL group and 3 patients in the control arm were offered local salvage therapy at first recurrence (surgery: 9 patients; RT: 1pt). TriMixDC-MEL was well tolerated (there were no grade ≥3 AE related to TriMixDC-MEL). AE were limited to: local skin injection reactions (grade 1-2; 17/21 patients), flu-like symptoms (grade 1-2; 4/21 patients), and post-infusion chills (grade 1-2; 4 [19%] patients). The results of this non-comparative randomized controlled phase II clinical trial of TriMixDC-MEL id/iv versus observation support the further evaluation of TriMixDC-MEL as a well-tolerated adjuvant therapy for melanoma patients following the resection of macrometastases. Citation Format: Bart Neyns, Yanina Jansen, Jurgen Corthals, Sofie Wilgenhof, Max Schreuer, Carlo Heirman, Kris Thielemans. A randomized controlled phase II clinical trial on mRNA electroporated autologous dendritic cells for stage III/IV melanoma patients who are disease-free following the local treatment of macrometastases [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B003.

Cancer vaccines based on mRNA are extensively studied. The fragile nature of mRNA has instigated research into carriers that can protect it from ribonucleases and as such enable its systemic use. However, carrier-mediated delivery of mRNA has been linked to production of type I interferon (IFN) that was reported to compromise the effectiveness of mRNA vaccines. In this study, we evaluated a cationic lipid for encapsulation of mRNA. The nanometer-sized, negatively charged lipid mRNA particles (LMPs) efficiently transfected dendritic cells and macrophages in vitro. Furthermore, i.v. delivery of LMPs resulted in rapid expression of the mRNA-encoded protein in spleen and liver, predominantly in CD11c(+) cells and to a minor extent in CD11b(+) cells. Intravenous immunization of mice with LMPs containing ovalbumin, human papilloma virus E7, and tyrosinase-related protein-2 mRNA, either combined or separately, elicited strong antigen-specific T-cell responses. We further showed the production of type I IFNs upon i.v. LMP delivery. Although this decreased the expression of the mRNA-encoded protein, it supported the induction of antigen-specific T-cell responses. These data question the current notion that type I IFNs hamper particle-mediated mRNA vaccines.

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient's human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8(+) and CD4(+) T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8(+) response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4(+) response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8(+) and CD4(+) T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.

The use of tumor-associated antigen (TAA) mRNA for therapeutic purposes is under active investigation. To be effective, mRNA vaccines need to deliver activation stimuli in addition to TAAs to dendritic cells (DC). In this study, we evaluated whether intranodal delivery of TAA mRNA together with TriMix, a mix of mRNA encoding CD40 ligand, constitutive active Toll-like receptor 4 and CD70, results in the in situ modification and maturation of DCs, hence, priming of TAA-specific T cells. We showed selective uptake and translation of mRNA in vivo by lymph node resident CD11c+ cells. This process was hampered by codelivery of classical maturation stimuli but not by TriMix mRNA. Importantly, TriMix mRNA induced a T-cell–attracting and stimulatory environment, including recruitment of antigen-specific CD4+ and CD8+ T cells and CTLs against various TAAs. In several mouse tumor models, mRNA vaccination was as efficient in CTL induction and therapy response as vaccination with mRNA-electroporated DCs. Together, our findings suggest that intranodal administration of TAA mRNA together with mRNA encoding immunomodulating molecules is a promising vaccination strategy. Cancer Res; 72(7); 1661–71. ©2012 AACR.

Molecular mimetics of the caspase activator second mitochondria-derived activator of caspase (SMAC) are being investigated for use in cancer therapy, but an understanding of in vivo effects remains incomplete. In this study, we offer evidence that SMAC mimetics elicit a proinflammatory cell death in cancer cells that engages an adaptive antitumor immune response. Cancer cells of different histologic origin underwent apoptosis when transduced with lentiviral vectors encoding a cytosolic form of the SMAC mimetic LV-tSMAC. Strikingly, treatment of tumor-bearing mice with LV-tSMAC resulted in the induction of apoptosis, activation of antitumor immunity, and enhanced survival. Antitumor immunity was accompanied by an increase of tumor-infiltrating lymphocytes displaying low PD-1 expression, high lytic capacity, and high levels of IFN-γ when stimulated. We also noted in vivo a decrease in regulatory T cells along with in vitro activation of tumor-specific CD8+ T cells by dendritic cells (DC) isolated from tumor draining lymph nodes. Last, tumor-specific cytotoxic T cells were also found to be activated in vivo. Mechanistic analyses showed that transduction of cancer cells with LV-tSMAC resulted in exposure of calreticulin but not release of HMGB1 or ATP. Nevertheless, DCs were activated upon engulfment of dying cancer cells. Further validation of these findings was obtained by their extension in a model of human melanoma using transcriptionally targeted LV-tSMAC. Together, our findings suggest that SMAC mimetics can elicit a proinflammatory cell death that is sufficient to activate adaptive antitumor immune responses in cancer. Cancer Res; 72(6); 1342–52. ©2012 AACR.

Antigen-specific immunity is crucially important for containing viral replication in human immunodeficiency virus (HIV)-1-infected hosts. Several epitopes have been predicted for the early expressed HIV-1 proteins Tat and Rev, but few have been studied in detail. We characterized the human leukocyte antigen (HLA)-B44-restricted Rev epitope EELLKTVRL (EL9) in an HIV-1-infected subject treated with antiretroviral therapy. Interestingly, a high sequence similarity was found between the EL9 epitope and the human nucleolar protein 6 (NOL6). However, this similarity does not seem to impede immunogenicity as CD8(+) T-cells, previously stimulated with EL9-pulsed dendritic cells, were able to specifically recognize the HIV-1 Rev epitope without cross-recognizing the human self-antigen NOL6. After the subject interrupted antiretroviral therapy and virus rebounded, mutations within the EL9 epitope were identified. Although the emerging mutations resulted in decreased or abolished T-cell recognition, they did not impair Rev protein function. Mutations leading to escape from T-cell recognition persisted for up to 124 weeks following treatment interruption. This study shows that the HLA-B44-restricted Rev CD8(+) T-cell epitope EL9 is immunogenic notwithstanding its close resemblance to a human peptide. The epitope mutates as a consequence of dynamic interaction between T-cells and HIV-1. Clinical status, CD4(+) T-cell count and viral load remained stable despite escape from T-cell recognition.

Antigen-specific immunity is crucially important for containing viral replication in human immunodeficiency virus (HIV)-1-infected hosts. Several epitopes have been predicted for the early expressed HIV-1 proteins Tat and Rev, but few have been studied in detail. We characterized the human leukocyte antigen (HLA)-B44-restricted Rev epitope EELLKTVRL (EL9) in an HIV-1-infected subject treated with antiretroviral therapy. Interestingly, a high sequence similarity was found between the EL9 epitope and the human nucleolar protein 6 (NOL6). However, this similarity does not seem to impede immunogenicity as CD8(+) T-cells, previously stimulated with EL9-pulsed dendritic cells, were able to specifically recognize the HIV-1 Rev epitope without cross-recognizing the human self-antigen NOL6. After the subject interrupted antiretroviral therapy and virus rebounded, mutations within the EL9 epitope were identified. Although the emerging mutations resulted in decreased or abolished T-cell recognition, they did not impair Rev protein function. Mutations leading to escape from T-cell recognition persisted for up to 124 weeks following treatment interruption. This study shows that the HLA-B44-restricted Rev CD8(+) T-cell epitope EL9 is immunogenic notwithstanding its close resemblance to a human peptide. The epitope mutates as a consequence of dynamic interaction between T-cells and HIV-1. Clinical status, CD4(+) T-cell count and viral load remained stable despite escape from T-cell recognition.

Lentiviral vectors (LVs) provide unique opportunities for the development of immunotherapeutic strategies, as they transduce a variety of cells in situ, including antigen-presenting cells (APCs). Engineering LVs to specifically transduce APCs is required to promote their translation towards the clinic. We report on the Nanobody (Nb) display technology to target LVs to dendritic cells (DCs) and macrophages. This innovative approach exploits the budding mechanism of LVs to incorporate an APC-specific Nb and a binding-defective, fusion-competent form of VSV. G in the viral envelope. In addition to production of high titer LVs, we demonstrated selective, Nb-dependent transduction of mouse DCs and macrophages both in vitro and in situ. Moreover, this strategy was translated to a human model in which selective transduction of in vitro generated or lymph node (LN)-derived DCs and macrophages, was demonstrated. In conclusion, the Nb display technology is an attractive approach to generate LVs targeted to specific cell types.

In a phase I/IIa clinical trial, 17 HIV-1 infected patients, stable on cART, received 4 vaccinations with autologous dendritic cells etectroporated with mRNA encoding Tat, Rev and Nef, after which cART was interrupted. Vaccination was safe and feasible. During the analytical treatment interruption (All), no serious adverse events were observed. Ninety-six weeks following ATI, 6/17 patients remained off therapy. Although induced and/or enhanced CD4(+) and CD8(+) T-cell responses specific for the immunogens were observed in most of the patients, we found no correlation with the number of weeks off cART. Moreover, CD4(+) T-cell counts, plasma viral load and the time remaining off cART following ATI did not differ from historical control data. To conclude, the vaccine was safe, well tolerated and resulted in vaccine-specific immune responses. Since no correlation with clinical parameters could be found, these results warrant further research in order to optimize the efficacy of vaccine-induced T-cell responses. (C) 2011 Elsevier Inc. All rights reserved.

Dendritic cells (DCs) electroporated with mRNA encoding CD70, CD40L and a constitutively active toll-like receptor 4 (TriMix-DC) have an increased T-cell stimulatory capacity. In a prospective phase IB clinical trial, we treated melanoma patients with intradermal and intravenous injections of autologous TriMix-DC co-electroporated with mRNA encoding full-length MAGE-A3, MAGE-C2, tyrosinase and gp100. We report here the immunological and clinical results obtained in one patient with a particularly favorable outcome. This patient had stage IV-M1c melanoma with documented progression during dacarbazine chemotherapy and received 5 TriMix-DC injections. Following DC therapy, a broad CD8(+) T-cell response against multiple epitopes derived from all four treatment antigens was found in the blood and among T cells derived from DTH biopsy. In addition, CD4(+) T cells recognizing different MAGE-A3-derived epitopes were detected in DTH-derived cells. A spontaneous anti-MAGE-C2 CD8(+) T-cell response was present prior to TriMix-DC therapy and increased during treatment. The tumor response was assessed with 18-fluorodeoxyglucose-positron emission/computed tomography. We documented a partial tumor response according to RECIST criteria with a marked reduction in (18)F-FDG-uptake by lung, lymph node and bone metastases. The patient remains free from progression after 12 months of follow-up. This case report indicates that administration of autologous TriMix-DC by the combined intradermal and intravenous route can mediate a durable objective tumor response accompanied by a broad T-cell response in a chemorefractory stage IV-M1c melanoma patient.

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.

Previous studies showed that stimulation of T cells derived from HIV-1-infected patients with autologous dendritic cells electroporated with mRNA encoding HIV antigens can induce antigen-specific T cell responses in vitro. Linking the antigen to an MHC class II-targeting sequence, such as dendritic cell lysosome-associated membrane protein (DC-LAMP), in the mRNA construct results in presentation of antigenic peptides in both MHC class I and class II molecules and therefore enhances the induced T cell responses. To analyze whether the lumenal domain of DC-LAMP is required for optimal induction of cellular immunity against HIV antigens, we compared fusion constructs with or without the lumenal domain of the DC-LAMP protein. A human codon-optimized consensus Gag sequence and a chimeric cDNA sequence encompassing Tat, Rev, and Nef codons (TaReNef ) were cloned into a vector containing the DC-LAMP sequence with or without its lumenal domain. The Gag protein lacking the DC-LAMP-derived sequence altogether elicited only weak T cell responses. DCs electroporated with Gag or TaReNef linked to DC-LAMP were able to elicit similar levels of antigen-specific CD4(+) and CD8(+) T cell responses for both Gag and TaReNef, irrespective of the addition of the DC-LAMP lumenal domain. These data show that DC-LAMP-mediated antigen targeting is absolutely required for optimal T cell stimulation, but that in our experimental setup, the lumenal part of DC-LAMP does not improve the overall induction of antigen-specific T cell responses.

Since decades, the main goal of tumor immunologists has been to increase the capacity of the immune system to mediate tumor regression. Considerable progress has been made in enhancing the efficacy of therapeutic anticancer vaccines. First, dendritic cells (DCs) have been identified as the key players in orchestrating primary immune responses. A better understanding of their biology and the development of procedures to generate vast amounts of DCs in vitro have accelerated the development of potent immunotherapeutic strategies for cancer. Second, tumor-associated antigens have been identified which are either selectively or preferentially expressed by tumor cells and can be recognized by the immune system. Finally, several studies have been performed on the genetic modification of DCs with tumor antigens. In this regard, loading the DCs with mRNA, which enables them to produce/process and present the tumor antigens themselves, has emerged as a promising strategy. Here, we will first overview the different aspects that must be taken into account when generating an mRNA-based DC vaccine and the published clinical studies exploiting mRNA-loaded DCs. Second, we will give a detailed description of a novel procedure to generate a vaccine consisting of tumor antigen-expressing dendritic cells with an in vitro superior capacity to induce anti-tumor immune responses. Here, immature DCs are electroporated with mRNAs encoding a tumor antigen, CD40 ligand (CD40L), CD70, and constitutively active (caTLR4) to generate mature antigen-presenting DCs.

Since decades, the main goal of tumor immunologists has been to increase the capacity of the immune system to mediate tumor regression. Considerable progress has been made in enhancing the efficacy of therapeutic anticancer vaccines. First, dendritic cells (DCs) have been identified as the key players in orchestrating primary immune responses. A better understanding of their biology and the development of procedures to generate vast amounts of DCs in vitro have accelerated the development of potent immunotherapeutic strategies for cancer. Second, tumor-associated antigens have been identified which are either selectively or preferentially expressed by tumor cells and can be recognized by the immune system. Finally, several studies have been performed on the genetic modification of DCs with tumor antigens. In this regard, loading the DCs with mRNA, which enables them to produce/process and present the tumor antigens themselves, has emerged as a promising strategy. Here, we will first overview the different aspects that must be taken into account when generating an mRNA-based DC vaccine and the published clinical studies exploiting mRNA-loaded DCs. Second, we will give a detailed description of a novel procedure to generate a vaccine consisting of tumor antigen-expressing dendritic cells with an in vitro superior capacity to induce anti-tumor immune responses. Here, immature DCs are electroporated with mRNAs encoding a tumor antigen, CD40 ligand (CD40L), CD70, and constitutively active (caTLR4) to generate mature antigen-presenting DCs.

A decade after its discovery, RNA interference has proven to be an instant success both in fundamental research and clinical applications. Lentiviral delivery of shRNAs is one of the most popular approaches to study gene functionalities in both developmental biology and disorders. During the past 10 years, several adaptations and novel techniques have emerged to improve (conditional) transgene expression and to meet researchers' needs. However, due to this magnitude of diversity, it is sometimes difficult to select the most suitable approach for a specific experimental setup. Here, we summarize the different systems and techniques available for every step in the generation of shRNA-bearing lentiviruses. The most crucial point is inevitably the selection of the target sequence itself. A good shRNA design is indispensable and determines almost completely the success of the experiments. In addition, an adequate promoter that drives the shRNA expression has to be chosen depending on its strength, inducibility, tissue-specificity, At this point, the researcher has also to decide whether the expression of the shRNA should be inducible or not. Another point one has to keep in mind is the choice of lentiviral vector in which the silencing cassette will be incorporated; single- or double-copy vectors are available. The last 2 years, shRNA multiplex approaches in which several targets are silenced with one vector have emerged and have shown a lot of potential in complex studies (like HIV-1). Finally, in the last section, we will discuss the possible induction of an immune response by short dsRNA molecules.

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]

Richter's syndrome (RS) can be defined as the emergence of an aggressive lymphoma in patients suffering from chronic lymphocytic leukemia (CLL). The authors performed immunophenotypic and Southern blot analysis of the peripheral blood and tissue specimen of a patient with RS. Using immunoperoxidase and immunogold-silver staining techniques and a panel of monoclonal antibodies, the authors found that the large cells characteristic of RS showed an altered immunophenotype as compared with the CLL cells and did not express mu heavy chain. Southern blot analysis revealed identical kappa light chain rearrangements in both tumoral cell populations consistent with a common clonal origin. Using the JH probe and several restriction enzymes, the authors also found evidence for a postrearrangement deletion of the heavy chain mu gene. These findings suggest that in this case of RS, a deletion of the heavy chain mu gene resulted in loss of mu expression by the larger cells that were characteristic of RS and was associated with their altered phenotype.

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 constitutively active toll-like receptor 4 (caTLR4) encoding mRNA. We show that immature DCs electroporated with CD40L and/or caTLR4 mRNA, but not those electroporated with CD70 mRNA, acquire a mature phenotype along with an enhanced secretion of several cytokines/chemokines. Moreover, these DCs are very potent in inducing naive CD4(+) T cells to differentiate into interferon-gamma (IFN-gamma)-secreting type 1 T helper (Th1) cells. Further, we assessed the capacity of the electroporated DCs to activate naive HLA-A2-restricted MelanA-specific CD8(+) T cells without the addition of any exogenous cytokines. When all three molecules were combined, a >500-fold increase in MelanA-specific CD8(+) T cells was observed when compared with immature DCs, and a >200-fold increase when compared with cytokine cocktail-matured DCs. In correlation, we found a marked increase in cytolytic and IFN-gamma/tumor necrosis factor-alpha (TNF-alpha) secreting CD8(+) T cells. Our data indicate that immature DCs genetically modified to express stimulating molecules can induce tumor antigen-specific T cells in vitro and could prove to be a significant improvement over DCs matured with the methods currently in use.

Antigen-loaded dendritic cells (DCs) have been intensively investigated as potential cellular antitumor vaccines. Several recent reports have indicated that loading DCs with whole tumor derived mRNA or defined tumor-antigen-encoding mRNA represents an effective nonviral strategy to stimulate T cell responses both for in vitro and in vivo models. Here, we describe the electroporation method as a tool for introducing in vitro transcribed capped mRNA into human DCs for tumor vaccination. We use MART-1/Melan-A as a model tumor-associated antigen for the generation of a DC-based vaccine against melanoma cancer. In addition to efficient antigen loading, it is important to obtain a maximal number of potent antigen-presenting cells. Another prerequisite for the development of a DC-based cancer vaccine is to obtain mature DCs. In this chapter, we describe the basic techniques required for the successful genetic modification of DCs by using the mRNA electroporation method.

Antigen-loaded dendritic cells (DCs) have been intensively investigated as potential cellular antitumor vaccines. Several recent reports have indicated that loading DCs with whole tumor derived mRNA or defined tumor-antigen-encoding mRNA represents an effective nonviral strategy to stimulate T cell responses both for in vitro and in vivo models. Here, we describe the electroporation method as a tool for introducing in vitro transcribed capped mRNA into human DCs for tumor vaccination. We use MART-1/Melan-A as a model tumor-associated antigen for the generation of a DC-based vaccine against melanoma cancer. In addition to efficient antigen loading, it is important to obtain a maximal number of potent antigen-presenting cells. Another prerequisite for the development of a DC-based cancer vaccine is to obtain mature DCs. In this chapter, we describe the basic techniques required for the successful genetic modification of DCs by using the mRNA electroporation method.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4(+) and CD8(+) cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection. (C) 2008 Elsevier Ltd. All rights reserved.

CD4+CD25+ regulatory T cells (Treg) have been described as an important hurdle for immunotherapy. Engagement of glucocorticoid-induced TNF receptor-related protein (GITR) has emerged recently as an important mechanism to control the suppression of CD4+CD25+ Treg. Furthermore, it has been documented extensively that GITR ligation is costimulatory for naive and activated T cells in the murine setting. However, little is known about the role of the human GITR ligand (huGITRL). We wanted to explore whether huGITRL could enhance antigen-specific T cell priming by dendritic cells (DC). First, we confirmed the endogenous expression of GITRL on HUVEC. We also detected GITRL expression on EBV-B cell lines, whereas no GITRL expression was observed on human monocyte-derived DC. Electroporation of GITRL mRNA in monocyte-derived DC resulted in a strong and long-lasting surface expression of GITRL. In contrast to data obtained in mice, no significant abrogation of Treg suppression by GITRL-expressing human DC was observed. Consistent with our mouse data, we showed that huGITRL is costimulatory for responder T cells. Furthermore, we found that GITRL-expressing DC primed increased numbers of Melan-A-specific CD8+ T cells. We conclude that although huGITRL is not capable of alleviating Treg suppression of responder T cells, huGITRL overexpression on monocyte-derived DC enhances their capacity to induce antigen-specific T cell responses. Thus, GITRL incorporation in DC might improve the antitumor immune response after vaccination.

Despite the enormous advances that have been made in the treatment of the plasma cell malignancy multiple myeloma (MM) this disease remains most often fatal. RAS, a family of GTPase genes, is frequently mutated in MM patients (20-45%). It has been shown that RAS oncoproteins, next to their classical roles in stimulating growth and survival, also have tumor suppressive effects. These effects are partially mediated through the Ras-Association Domain Family (RASSF), a group of 10 proteins and numerous isoforms. These tumor suppressors are often silenced in cancer cells due to methylation of the promotor. So far, however, no data about RASSF proteins is available in MM. Here, we investigated the expression and role of RASSF4 in MM. Using gene expression profiling data of 2 independent patient cohorts, we found that RASSF4 expression is significantly downregulated in MM cells compared to healthy bone marrow plasma cells and that a low expression at time of diagnosis associates with a bad prognosis. In addition, treatment with the histone deacetylase inhibitor quisinostat and/or the DNA methyltransferase inhibitor decitabine significantly increased RASSF4 expression both in vitro and in vivo, thus indicating that epigenetic modifications are involved in this silencing. Next, to elucidate the biological role of RASSF4 in MM we used a conditional lentiviral delivery system. Tree human myeloma cell lines (HMCL), namely AMO1, JJN3 and the IL-6 dependent cell line XG7, were transduced with either the empty pLenti 6.3 vector (control) or pLenti6.3 huRASSF4. Overexpression of RASSF4 in the HMCL revealed that RASSF4 has a strong negative effect on MM cell viability and potently induces apoptosis. This induction of apoptosis was found to be caspase-3 mediated. In addition, RASSF4 was found to inhibit the clonogenic capacity of the HMCL. Mechanistically, immunofluorescence stainingfor RASSF4 showed that RASSF4 is co-localized with the centrosome. In addition, RASSF4 overexpression was found to downregulate the expression of the anti-apoptotic protein Bcl-2. In conclusion, we have identified RASSF4 as a target for epigenetic silencing in MM and demonstrated its tumor suppressive function in MM cells. Further insights into the molecular mechanisms of RASSF4 in MM will continue to be explored both in vitro and in vivo.

Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.