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author:("herman, Carlo")
1.  Targeting the tumor microenvironment to enhance antitumor immune responses 
Oncotarget  2014;6(3):1359-1381.
The identification of tumor-specific antigens and the immune responses directed against them has instigated the development of therapies to enhance antitumor immune responses. Most of these cancer immunotherapies are administered systemically rather than directly to tumors. Nonetheless, numerous studies have demonstrated that intratumoral therapy is an attractive approach, both for immunization and immunomodulation purposes. Injection, recruitment and/or activation of antigen-presenting cells in the tumor nest have been extensively studied as strategies to cross-prime immune responses. Moreover, delivery of stimulatory cytokines, blockade of inhibitory cytokines and immune checkpoint blockade have been explored to restore immunological fitness at the tumor site. These tumor-targeted therapies have the potential to induce systemic immunity without the toxicity that is often associated with systemic treatments. We review the most promising intratumoral immunotherapies, how these affect systemic antitumor immunity such that disseminated tumor cells are eliminated, and which approaches have been proven successful in animal models and patients.
PMCID: PMC4359300  PMID: 25682197
Intratumoral; Immunotherapy; Tumor microenvironment; Immunomodulation; Vaccination
3.  Intratumoral administration of mRNA encoding a fusokine consisting of IFN-β and the ectodomain of the TGF-β receptor II potentiates antitumor immunity 
Oncotarget  2014;5(20):10100-10113.
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 Fβ2, 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 Fβ2 to tumor cells resulted in a reduced proliferation and increased expression of MHC I but also PD-L1. Importantly, Fβ2 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 Fβ2, can modulate the tumor microenvironment, leading to effective antitumor T cell responses, which can be further potentiated through combination therapy.
PMCID: PMC4259408  PMID: 25338019
mRNA; IFN-β; TGF-β; cancer therapy; T cell
4.  Targeting of Human Antigen-Presenting Cell Subsets 
Journal of Virology  2013;87(20):11304-11308.
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.
PMCID: PMC3807283  PMID: 23864630
5.  Immunogenicity of targeted lentivectors 
Oncotarget  2014;5(3):704-715.
To increase the safety and possibly efficacy of HIV-1 derived lentivectors (LVs) as an anti-cancer vaccine, we recently developed the Nanobody (Nb) display technology to target LVs to antigen presenting cells (APCs). In this study, we extend these data with exclusive targeting of LVs to conventional dendritic cells (DCs), which are believed to be the main cross-presenting APCs for the induction of a TH1-conducted antitumor immune response. The immunogenicity of these DC-subtype targeted LVs was compared to that of broad tropism, general APC-targeted and non-infectious LVs. Intranodal immunization with ovalbumin encoding LVs induced proliferation of antigen specific CD4+ T cells, irrespective of the LVs' targeting ability. However, the cytokine secretion profile of the restimulated CD4+ T cells demonstrated that general APC targeting induced a similar TH1-profile as the broad tropism LVs while transduction of conventional DCs alone induced a similar and less potent TH1 profile as the non-infectious LVs. This observation contradicts the hypothesis that conventional DCs are the most important APCs and suggests that the activation of other APCs is also meaningful. Despite these differences, all targeted LVs were able to stimulate cytotoxic T lymphocytes, be it to a lesser extent than broad tropism LVs. Furthermore this induction was shown to be dependent on type I interferon for the targeted and non-infectious LVs, but not for broad tropism LVs. Finally we demonstrated that the APC-targeted LVs were as potent in therapy as broad tropism LVs and as such deliver on their promise as safer and efficacious LV-based vaccines.
PMCID: PMC3996667  PMID: 24519916
lentivector; targeting; antigen presenting cell; vaccine; antitumor immunotherapy
6.  Design of an Optimized Wilms' Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo 
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.
PMCID: PMC3889186  PMID: 24253259
7.  mRNA 
Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and “naked mRNA” for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters.
PMCID: PMC3859745  PMID: 23291946
mRNA; dendritic cell; immunotherapy; cancer
8.  Characterization of CD8+ T-Cell Responses in the Peripheral Blood and Skin Injection Sites of Melanoma Patients Treated with mRNA Electroporated Autologous Dendritic Cells (TriMixDC-MEL) 
BioMed Research International  2013;2013:976383.
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.
PMCID: PMC3581259  PMID: 23509826
9.  CD8α+ and CD8α− Subclasses of Dendritic Cells Direct the Development of Distinct T Helper Cells In Vivo  
Cells of the dendritic family display some unique properties that confer to them the capacity to sensitize naive T cells in vitro and in vivo. In the mouse, two subclasses of dendritic cells (DCs) have been described that differ by their CD8α expression and their localization in lymphoid organs. The physiologic function of both cell populations remains obscure. Studies conducted in vitro have suggested that CD8α+ DCs could play a role in the regulation of immune responses, whereas conventional CD8α− DCs would be more stimulatory. We report here that both subclasses of DCs efficiently prime antigen-specific T cells in vivo, and direct the development of distinct T helper (Th) populations. Antigen-pulsed CD8α+ and CD8α− DCs are separated after overnight culture in recombinant granulocyte/macrophage colony-stimulating factor and injected into the footpads of syngeneic mice. Administration of CD8α− DCs induces a Th2-type response, whereas injection of CD8α+ DCs leads to Th1 differentiation. We further show that interleukin 12 plays a critical role in Th1 development by CD8α+ DCs. These findings suggest that the nature of the DC that presents the antigen to naive T cells may dictate the class selection of the adaptative immune response.
PMCID: PMC2192907  PMID: 9927520
primary response; T helper cell type 1/type 2 balance; interleukin 12; tolerance; memory
10.  Effects of Granulocyte-Macrophage Colony-Stimulating Factor and Tumor Necrosis Factor Alpha on Trypanosoma cruzi Trypomastigotes 
Infection and Immunity  1998;66(6):2722-2727.
We have previously shown that the addition of exogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) to nonactivated mouse peritoneal macrophages (MPM) limits Trypanosoma cruzi infections in vitro (E. Olivares Fontt and B. Vray, Parasite Immunol. 17:135–141, 1995). Lower levels of infection were correlated with a higher level of production of tumor necrosis factor alpha (TNF-α) in the absence of nitric oxide (NO) release. These data suggested that GM-CSF and/or TNF-α might have a direct parasitocidal effect on T. cruzi trypomastigotes, independently of NO release. To address this question, T. cruzi trypomastigotes were treated with recombinant murine GM-CSF (rmGM-CSF), recombinant murine TNF-α (rmTNF-α), or both cytokines in a cell-free system. Treatment with rmGM-CSF but not rmTNF-α caused morphological changes in the parasites, and most became spherical after 7 h of incubation. Both cytokines exerted a cytolytic activity on the trypomastigotes, yet the trypanolytic activity of rmTNF-α was more effective than that of rmGM-CSF. Viable rmGM-CSF- and rmTNF-α-treated parasites were less able to infect MPM than untreated parasites, and this reduction in infectivity was greatest for rmGM-CSF. Treatments with both cytokines resulted in more lysis and almost complete inhibition of infection. The direct parasitocidal activity of rmTNF-α was inhibited by carbohydrates and monoclonal antibodies specific for the lectin-like domain of TNF-α. Collectively, these results suggest that cytokines such as GM-CSF and TNF-α may directly control the level of T. cruzi trypomastigotes at least in vitro and so could determine the outcome of infection in vivo.
PMCID: PMC108261  PMID: 9596739

Results 1-10 (10)