Cleo Goyvaerts

Preclinical Evaluation of TriMix and Antigen mRNA-Based Antitumor Therapy

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.

Development of the Nanobody display technology to target lentiviral vectors to antigen-presenting cells

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.

Proinflammatory characteristics of SMAC/DIABLO-induced cell death in antitumor therapy

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.

Anti-melanoma vaccines engineered to simultaneously modulate cytokine priming and silence PD-L1 characterized using ex vivo myeloid-derived suppressor cells as a readout of therapeutic efficacy

Efficacious antitumor vaccines strongly stimulate cancer-specific effector T cells and counteract the activity of tumor-infiltrating immunosuppressive cells. We hypothesised that combining cytokine expression with silencing programmed cell death ligand 1 (PD-L1) could potentiate anticancer immune responses of lentivector vaccines. Thus, we engineered a collection of lentivectors that simultaneously co-expressed an antigen, a PD-L1-silencing shRNA, and various T cell-polarising cytokines, including interferon γ (IFNγ), transforming growth factor β (TGFβ) or interleukins (IL12, IL15, IL23, IL17A, IL6, IL10, IL4). In a syngeneic B16F0 melanoma model and using tyrosinase related protein 1 (TRP1) as a vaccine antigen, we found that simultaneous delivery of IL12 and a PD-L1-silencing shRNA was the only combination that exhibited therapeutically relevant anti-melanoma activities. Mechanistically, we found that delivery of the PD-L1 silencing construct boosted T cell numbers, inhibited in vivo tumor growth and strongly cooperated with IL12 cytokine priming and antitumor activities. Finally, we tested the capacities of our vaccines to counteract tumor-infiltrating myeloid-derived suppressor cell (MDSC) activities ex vivo. Interestingly, the lentivector co-expressing IL12 and the PD-L1 silencing shRNA was the only one that counteracted MDSC suppressive activities, potentially underlying the observed anti-melanoma therapeutic benefit. We conclude that (1) evaluation of vaccines in healthy mice has no significant predictive value for the selection of anticancer treatments; (2) B16 cells expressing xenoantigens as a tumor model are of limited value; and (3) vaccines which inhibit the suppressive effect of MDSC on T cells in our ex vivo assay show promising and relevant antitumor activities.

Targeting of human antigen-presenting cell subsets

ABSTRACT 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.

Assessing T-cell responses in anticancer immunotherapy: Dendritic cells or myeloid-derived suppressor cells?

Since dendritic cells operate as professional antigen-presenting cells (APCs) and hence are capable of jumpstarting the immune system, they have been exploited to develop a variety of immunotherapeutic regimens against cancer. In the few past years, myeloid-derived suppressor cells (MDSCs) have been shown to mediate robust immunosuppressive functions, thereby inhibiting tumor-targeting immune responses. Thus, we propose that the immunomodulatory activity of MDSCs should be carefully considered for the development of efficient anticancer immunotherapies.

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.

Cancer-Associated Myeloid Regulatory Cells

Myeloid cells are critically involved in the pathophysiology of cancers. In the tumor microenvironment (TME), they comprise tumor-associated macrophages (TAMs), neutrophils (TANs), dendritic cells, and myeloid-derived suppressor cells, which are further subdivided into a monocytic subset and a granulocytic subset. Some of these myeloid cells, in particular TAMs and TANs, are divided into type 1 or type 2 cells, according to the paradigm of T helper type 1 or type 2 cells. Type 1-activated cells are generally characterized as cells that aid tumor rejection, while all other myeloid cells are shown to favor tumor progression. Moreover, these cells are often at the basis of resistance to various therapies. Much research has been devoted to study the biology of myeloid cells. This endeavor has proven to be challenging, as the markers used to categorize myeloid cells in the TME are not restricted to particular subsets. Also from a functional and metabolic point of view, myeloid cells share many features. Finally, myeloid cells are endowed with a certain level of plasticity, which further complicates studying them outside their environment. In this article, we challenge the exclusive use of cell markers to unambiguously identify myeloid cell subsets in the TME. We further propose to divide myeloid cells into myeloid regulatory or stimulatory cells according to their pro- or antitumor function, because we contend that for therapeutic purposes it is not targeting the cell subsets but rather targeting their protumor traits; hence, myeloid regulatory cells will push antitumor immunotherapy to the next level.

Intratumoral delivery of TriMix mRNA results in T-cell activation by cross-presenting dendritic cells

Intratumoral injection of CTL-stimulatory agents could provide another avenue for immunotherapy. TriMix mRNA, comprising three DC-oriented stimulatory mRNAs, was examined in mouse models and provides a rationale for clinical testing in solid and accessible tumors. Modulating the activity of tumor-infiltrating dendritic cells (TiDC) provides opportunities for novel cancer interventions. In this article, we report on our study of the uptake of mRNA by CD8α+ cross-presenting TiDCs upon its intratumoral (i.t.) delivery. We exploited this property to deliver mRNA encoding the costimulatory molecule CD70, the activation stimuli CD40 ligand, and constitutively active Toll-like receptor 4, referred to as TriMix mRNA. We show that TiDCs are reprogrammed to mature antigen-presenting cells that migrate to tumor-draining lymph nodes (TDLN). TriMix stimulated antitumor T-cell responses to spontaneously engulfed cancer antigens, including a neoepitope. We show in various mouse cancer models that i.t. delivery of TriMix mRNA results in systemic therapeutic antitumor immunity. Finally, we show that the induction of antitumor responses critically depends on TiDCs, whereas it only partially depends on TDLNs. As such, we provide a platform and a mechanistic rationale for the clinical testing of i.t. administration of TriMix mRNA. Cancer Immunol Res; 4(2); 146–56. ©2015 AACR.

Immune modulation by genetic modification of dendritic cells with lentiviral vectors

Our work over the past eight years has focused on the use of HIV-1 lentiviral vectors (lentivectors) for the genetic modification of dendritic cells (DCs) to control their functions in immune modulation. DCs are key professional antigen presenting cells which regulate the activity of most effector immune cells, including T, B and NK cells. Their genetic modification provides the means for the development of targeted therapies towards cancer and autoimmune disease. We have been modulating with lentivectors the activity of intracellular signalling pathways and co-stimulation during antigen presentation to T cells, to fine-tune the type and strength of the immune response. In the course of our research, we have found unexpected results such as the surprising immunosuppressive role of anti-viral signalling pathways, and the close link between negative co-stimulation in the immunological synapse and T cell receptor trafficking. Here we review our major findings and put them into context with other published work.