Heleen Dewitte

Combinatorial strategies for the induction of immunogenic cell death.

1 Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France, 2 U1138, INSERM, Paris, France, Metabolomics and Cell Biology Platforms, Gustave Roussy Campus Cancer, Villejuif, France, 4 Faculté de Medecine, Université Paris-Sud, Le Kremlin-Bicêtre, France, 5 Department of Chemistry, University of Coimbra, Coimbra, Portugal, 6 Laboratory for General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent, Belgium, 7 Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Jette, Belgium, 8 Sotio a.c., Prague, Czech Republic, 9 Department of Immunology, 2nd Faculty of Medicine, University Hospital Motol, Charles University, Prague, Czech Republic, Gustave Roussy Campus Cancer, Villejuif, France, 11 Université Paris Descartes, Paris, France, 12 Université Pierre et Marie Curie, Paris, France, 13 Pôle de Biologie, Hopitâl Européen George Pompidou, AP-HP, Paris, FranceThe term “immunogenic cell death” (ICD) is commonly employed to indicate a peculiar instance of regulated cell death (RCD) that engages the adaptive arm of the immune system. The inoculation of cancer cells undergoing ICD into immunocompetent animals elicits a specific immune response associated with the establishment of immunological memory. Only a few agents are intrinsically endowed with the ability to trigger ICD. These include a few chemotherapeutics that are routinely employed in the clinic, like doxorubicin, mitoxantrone, oxaliplatin, and cyclophosphamide, as well as some agents that have not yet been approved for use in humans. Accumulating clinical data indicate that the activation of adaptive immune responses against dying cancer cells is associated with improved disease outcome in patients affected by various neoplasms. Thus, novel therapeutic regimens that trigger ICD are urgently awaited. Here, we discuss current combinatorial approaches to convert otherwise non-immunogenic instances of RCD into bona fide ICD.

mRNA-Lipoplex loaded microbubble contrast agents for ultrasound-assisted transfection of dendritic cells.

In cancer immunotherapy the immune system should be triggered to specifically recognize and eliminate tumor cells in the patients body. This could be achieved by loading dendritic cells (DCs) with tumor-associated antigens (TAAs). This can be achieved by transfecting DCs with messenger RNA encoding a tumor-associated antigen. Here we demonstrate transient transfection of dendritic cells by means of mRNA-lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-mediated delivery of mRNA-complexes led to efficient transfection of DCs. When mRNA encoding luciferase was used, maximal levels of the enzyme activity were detected 8 h after ultrasound application. Upon longer incubation protein expression gradually declined. This treatment did not affect viability of the cells. Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labeled lipoplexes. Over 50% of DCs contained fluorescently labeled mRNA-complexes. In the absence of additional maturation signals, transfection of immature DCs with mRNA-lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the expression level of maturation markers (CD40 and CD86). However, in the presence of the activation stimulus (LPS), cells were able to further mature as shown by a significant up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-transfection of cultured DCs.

The ReNAissanCe of mRNA-based cancer therapy

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.

The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy.

Dendritic cell (DC)-based cancer vaccines, where the patients 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.

Particle-mediated Intravenous Delivery of Antigen mRNA Results in Strong Antigen-specific T-cell Responses Despite the Induction of Type I Interferon.

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.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.

Theranostic mRNA-loaded Microbubbles in the Lymphatics of Dogs: Implications for Drug Delivery

Microbubbles have shown potential as intralymphatic ultrasound contrast agents while nanoparticle-loaded microbubbles are increasingly investigated for ultrasound-triggered drug and gene delivery. To explore whether mRNA-nanoparticle loaded microbubbles could serve as theranostics for detection of and mRNA transfer to the lymph nodes, we investigate the behavior of unloaded and mRNA-loaded microbubbles using contrast-enhanced ultrasound imaging after subcutaneous injection in dogs. Our results indicate that both types of microbubbles are equally capable of rapidly entering the lymph vessels and nodes upon injection, and novel, valuable and detailed information on the lymphatic structure in the animals could be obtained. Furthermore, additional observations were made regarding the dynamics of microbubble lymph node uptake. Importantly, neither the microbubble migration distance within the lymphatics, nor the observed contrast signal intensity was influenced by mRNA-loading. Although further optimization of acoustic parameters will be needed, this could represent a first step towards ultrasound-guided, ultrasound-triggered intranodal mRNA delivery using these theranostic microbubbles.

Evading innate immunity in nonviral mRNA delivery : don't shoot the messenger

In the field of nonviral gene therapy, in vitro transcribed (IVT) mRNA has emerged as a promising tool for the delivery of genetic information. Over the past few years it has become widely known that the introduction of IVT mRNA into mammalian cells elicits an innate immune response that has favored mRNA use toward immunotherapeutic vaccination strategies. However, for non-immunotherapy-related applications this intrinsic immune-stimulatory activity directly interferes with the aimed therapeutic outcome, because it can seriously compromise the expression of the desired protein. This review presents an overview of the immune-related obstacles that limit mRNA advance for non-immunotherapy-related applications.

Hitchhiking nanoparticles: Reversible coupling of lipid-based nanoparticles to cytotoxic T lymphocytes

Following intravenous injection of anti-cancer nanomedicines, many barriers need to be overcome en route to the tumor. Cell-mediated delivery of nanoparticles (NPs) is promising in terms of overcoming several of these barriers based on the tumoritropic migratory properties of particular cell types. This guided transport aims to enhance the NP accumulation in the tumor and moreover enhance the infiltration of regions that are typically inaccessible for free NPs. Within this study, cytotoxic CD8(+) T cells were selected as carriers based on both their ability to migrate to the tumor and their intrinsic cytolytic activity against tumor cells. Many anti-cancer nanomedicines require tumor cell internalization to mediate cytosolic drug delivery and enhance the anti-cancer effect. This proof-of-concept therefore reports on the reversible attachment of liposomes to the surface of cytotoxic T lymphocytes via a reduction sensitive coupling. The activation status of the T cells and the liposome composition are shown to strongly influence the loading efficiency. Loading the cells with liposomes does not compromise T cell functionalities like proliferation and cytolytic function. Additionally, the triggered liposome release is demonstrated upon the addition of glutathione. Based on this optimization using liposomes as model NPs, a small interfering RNA (siRNA)-loaded NP was developed that can be coupled to the surface of CD8(+) T cells.

Design and evaluation of theranostic perfluorocarbon particles for simultaneous antigen-loading and 19F-MRI tracking of dendritic cells

Perfluorocarbon (PFC) particles are currently on the rise as cell labeling agents for ¹⁹F-MRI tracking of dendritic cell (DC)-based vaccines. In this work, we design theranostic PFC particles for single-step loading of DCs with both antigenic protein and with a liquid PFC for ¹⁹F-MRI detection of the antigen-loaded cells. Upon addition to DCs in vitro, the antigen-loaded PFC particles are efficiently internalized, resulting in intracellular presence of up to 40 pmol ¹⁹F atoms per cell. At the same time, the DCs become loaded with antigenic proteins, that can be efficiently processed, without important effects on cell viability or altering the DCs phenotype and the cells capacity to respond to danger signals. In addition, antigen-loaded PFC particle containing DCs are capable of inducing extensive proliferation of antigen-specific CD8⁺ T cells in vitro. Importantly, the antigen-coated PFC particles allow in vitro ¹⁹F-MRI-based detection of the antigen-containing DCs with detection limits as low as 10³ cells μl⁻¹. The dual-modality characteristics of the designed particles could assure that only those DCs that have taken up the antigen, and hence are responsible for an immune response, are traceable via ¹⁹F-MRI. Taken together, these novel dual-modality particles represent an interesting strategy in the development of a traceable DC vaccine.

Choose your models wisely: How different murine bone marrow-derived dendritic cell protocols influence the success of nanoparticulate vaccines in vitro

Dendritic cell (DC)-based cancer vaccination has shown great potential in cancer immunotherapy. As a result, novel nanoparticles aiming to load DCs with tumor antigens are being developed and evaluated in vitro. For this, murine bone marrow-derived DCs (BM-DCs) are most commonly used as model DCs. However, many different protocols exist to generate these cells. Therefore, we investigated to what extent different BM-DC culture protocols impact on the immunobiology of the cells, as well as their response to particulate antigens. We evaluated 4 different BM-DC protocols with 2 main variables: bovine serum and cytokine combinations. Our results show distinct differences in yield, phenotypical maturation status and the production of immune stimulatory and immune suppressive cytokines by the different BM-DCs. Importantly, we demonstrate that the antigen-loading of these different BM-DCs via transfection with mRNA lipoplexes results in large differences in transfection efficiency as well as in the capacity of mRNA-transfected BM-DCs to stimulate antigen-specific T cells. Thus, it is clear that the BM-DC model can have significant confounding effects on the evaluation of novel nanoparticulate vaccines. To take this into account when testing novel particulate antigen-delivery systems in BM-DC models, we propose to (1) perform a thorough immunological characterization of the BM-DCs and to (2) not only judge a particles potential for cancer vaccination based on transfection efficiency, but also to include an evaluation of functional end-points such as T cell activation.