Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

Intravital visualization of interactions of murine Peyer's patch-resident dendritic cells with M cells.

The small intestine hosts specialized lymphoid structures, the Peyer's patches, that face the gut lumen and are overlaid with unique epithelial cells, called microfold (M) cells. M cells are considered to constitute an important route for antigen uptake in the mucosal immune system. Here, we used intravital microscopy to define immune cell populations, which are in close contact with M cells and potentially sample antigen. We present live evidence that DCs enter M cell pockets and highlight the abundance of mononuclear phagocytes in these structures. Taking advantage of the respective reporter animals, we focused on classical DCs that express Zbtb46 and analyzed how these cells interact with M cells in steady state and sample antigen for T cell activation in the Peyer's patches following challenge.

Data Trustworthiness Evaluation in Mobile Crowdsensing Systems with Users' Trust Dispositions' Consideration.

Mobile crowdsensing is a powerful paradigm that exploits the advanced sensing capabilities and ubiquity of smartphones in order to collect and analyze data on a scale that is impossible with fixed sensor networks. Mobile crowdsensing systems incorporate people and rely on their participation and willingness to contribute up-to-date and accurate information, meaning that such systems are prone to malicious and erroneous data. Therefore, trust and reputation are key factors that need to be addressed in order to ensure sustainability of mobile crowdsensing systems. The objective of this work is to define the conceptual trust framework that considers human involvement in mobile crowdsensing systems and takes into account that users contribute their opinions and other subjective data besides the raw sensing data generated by their smart devices. We propose a novel method to evaluate the trustworthiness of data contributed by users that also considers the subjectivity in the contributed data. The method is based on a comparison of users' trust attitudes and applies nonparametric statistic methods. We have evaluated the performance of our method with extensive simulations and compared it to the method proposed by Huang that adopts Gompertz function for rating the contributions. The simulation results showed that our method outperforms Huang's method by 28.6% on average and the method without data trustworthiness calculation by 33.6% on average in different simulation settings.

Nanoparticulate vaccine inhibits tumor growth via improved T cell recruitment into melanoma and huHER2 breast cancer.

Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle-immune cell interactions and treatments regime is required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-γ cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction.

Regulatory aspects on nanomedicines.

Nanomedicines have been in the forefront of pharmaceutical research in the last decades, creating new challenges for research community, industry, and regulators. There is a strong demand for the fast development of scientific and technological tools to address unmet medical needs, thus improving human health care and life quality. Tremendous advances in the biomaterials and nanotechnology fields have prompted their use as promising tools to overcome important drawbacks, mostly associated to the non-specific effects of conventional therapeutic approaches. However, the wide range of application of nanomedicines demands a profound knowledge and characterization of these complex products. Their properties need to be extensively understood to avoid unpredicted effects on patients, such as potential immune reactivity. Research policy and alliances have been bringing together scientists, regulators, industry, and, more frequently in recent years, patient representatives and patient advocacy institutions. In order to successfully enhance the development of new technologies, improved strategies for research-based corporate organizations, more integrated research tools dealing with appropriate translational requirements aiming at clinical development, and proactive regulatory policies are essential in the near future. This review focuses on the most important aspects currently recognized as key factors for the regulation of nanomedicines, discussing the efforts under development by industry and regulatory agencies to promote their translation into the market. Regulatory Science aspects driving a faster and safer development of nanomedicines will be a central issue for the next years.

DC Respond to Cognate T Cell Interaction in the Antigen-Challenged Lymph Node.

Dendritic cells (DC) are unrivaled in their potential to prime naive T cells by presenting antigen and providing costimulation. DC are furthermore believed to decode antigen context by virtue of pattern recognition receptors and to polarize T cells through cytokine secretion toward distinct effector functions. Diverse polarized T helper (TH) cells have been explored in great detail. In contrast, studies of instructing DC have to date largely been restricted to in vitro settings or adoptively transferred DC. Here we report efforts to unravel the DC response to cognate T cell encounter in antigen-challenged lymph nodes (LN). Mice engrafted with antigen-specific T cells were immunized with nanoparticles (NP) entrapping adjuvants and absorbed with antigen to study the immediate DC response to T cell encounter using bulk and single cell RNA-seq profiling. NP induced robust antigen-specific TH1 cell responses with minimal bystander activation. Fluorescent-labeled NP allowed identification of antigen-carrying DC and focus on transcriptional changes in DC that encounter T cells. Our results support the existence of a bi-directional crosstalk between DC and T cells that promotes TH1 responses, including involvement of the ubiquitin-like molecule Isg15 that merits further study.

Immunization with mannosylated nanovaccines and inhibition of the immune-suppressing microenvironment sensitizes melanoma to immune checkpoint modulators.

A low response rate, acquired resistance and severe side effects have limited the clinical outcomes of immune checkpoint therapy. Here, we show that combining cancer nanovaccines with an anti-PD-1 antibody (αPD-1) for immunosuppression blockade and an anti-OX40 antibody (αOX40) for effector T-cell stimulation, expansion and survival can potentiate the efficacy of melanoma therapy. Prophylactic and therapeutic combination regimens of dendritic cell-targeted mannosylated nanovaccines with αPD-1/αOX40 demonstrate a synergism that stimulates T-cell infiltration into tumours at early treatment stages. However, this treatment at the therapeutic regimen does not result in an enhanced inhibition of tumour growth compared to αPD-1/αOX40 alone and is accompanied by an increased infiltration of myeloid-derived suppressor cells in tumours. Combining the double therapy with ibrutinib, a myeloid-derived suppressor cell inhibitor, leads to a remarkable tumour remission and prolonged survival in melanoma-bearing mice. The synergy between the mannosylated nanovaccines, ibrutinib and αPD-1/αOX40 provides essential insights to devise alternative regimens to improve the efficacy of immune checkpoint modulators in solid tumours by regulating the endogenous immune response.

Poly(lactic acid)-based particulate systems are promising tools for immune modulation.

Poly(lactic acid) (PLA) is one of the most successful and versatile polymers explored for controlled delivery of bioactive molecules. Its attractive properties of biodegradability and biocompatibility in vivo have contributed in a meaningful way to the approval of different products by the FDA and EMA for a wide range of biomedical and pharmaceutical applications, in the past two decades. This polymer has been widely used for the preparation of particles as delivery systems of several therapeutic molecules, including vaccines. These PLA vaccine carriers have shown to induce a sustained and targeted release of different bacterial, viral and tumor-associated antigens and adjuvants in vivo, triggering distinct immune responses. The present review intends to highlight and discuss the major advantages of PLA as a promising polymer for the development of potent vaccine delivery systems against pathogens and cancer. It aims to provide a critical discussion based on preclinical data to better understand the major effect of PLA-based carrier properties on their interaction with immune cells and thus their role in the modulation of host immunity. STATEMENT OF SIGNIFICANCE: During the last decades, vaccination has had a great impact on global health with the control of many severe diseases. Polymeric nanosystems have emerged as promising strategies to stabilize vaccine antigens, promoting their controlled release to phagocytic cells, thus avoiding the need for multiple administrations. One of the most promising polymers are the aliphatic polyesters, which include the poly(lactic acid). This is a highly versatile biodegradable and biocompatible polymer. Products containing this polymer have already been approved for all food and some biomedical applications. Despite all favorable characteristics presented above, PLA has been less intensively discussed than other polymers, such as its copolymer PLGA, including regarding its application in vaccination and particularly in tumor immunotherapy. The present review discusses the major advantages of poly(lactic acid) for the development of potent vaccine delivery systems, providing a critical view on the main properties that determine their effect on the modulation of immune cells.

Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming.

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4+ and CD8+ T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4+ and CD8+ lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30-40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll-like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4+ and CD8+ T cells, in vivo. Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects.

Translational Peptide-associated Nanosystems: Promising Role as Cancer Vaccines.

Cancer is a heterogeneous disease that results from a multi-step process, being characterized by uncontrolled proliferation, invasion and metastasis. The understanding that tumor cells can be recognized by host immune cells has highlighted the potential advantages of using vaccination purposes to eliminate cancer cells, while avoiding severe side effects associated to conventional cancer treatments. Interesting outcomes have been obtained with the new identified tumor associated antigens (TAAs), including recombinant proteins and peptides. However, these molecules are weakly immunogenic, demanding the concomitant use of adjuvants to boost and achieve a strong tumor-specific immune response. Different classes of nanosystems have been used to protect and deliver several vaccine components. In vitro and preclinical studies have emphasized their promising role to attain a prolonged eradication of cancer cells, including metastasis. However, some studies support the co-entrapment of multiple adjuvants and TAAs within a single particulate carrier, while others indicate that stronger immune responses were obtained using a mixture of nanocarriers entrapping different combinations of TAAs and adjuvants. These apparently contradictory results may be related to nanocarrier physicochemical properties, which have a profound impact on their interaction with targeted cells and consequent biological effects. This review discusses the application of nanoscale systems as cancer vaccines, highlighting the particular characteristics of tumor biology and immunology that have been used to guide the design of these nanodelivery tools. We also aim to explore the major weaknesses that have prevented their wide application in the clinic to overcome the delivery, efficacy and safety issues associated to biological entities.

In vivo delivery of peptides and Toll-like receptor ligands by mannose-functionalized polymeric nanoparticles induces prophylactic and therapeutic anti-tumor immune responses in a melanoma model.

We hypothesized that the co-entrapment of melanoma-associated antigens and the Toll-like receptor (TLR) ligands Poly(I:C) and CpG, known to be Th1-immunopotentiators, in mannose-functionalized aliphatic polyester-based nanoparticles (NPs) could be targeted to mannose receptors on antigen-presenting cells and induce anti-tumor immune responses. High entrapment efficiencies of antigens and immunopotentiators in 150nm NPs were obtained. The co-entrapment of the model antigen ovalbumin and the TLR ligands was crucial to induce high IgG2c/IgG1 ratios and high levels of IFN-γ and IL-2. Mannose-functionalization of NPs potentiated the Th1 immune response. The nanoparticulate vaccines decreased the growth rate of murine B16F10 melanoma tumors in therapeutic and prophylatic settings. The combination of mannose-functionalized NPs containing MHC class I- or class II-restricted melanoma antigens and the TLR ligands induced the highest tumor growth delay. Overall, we demonstrate that the multifunctional properties of NPs in terms of targeting and antigen/adjuvant delivery have high cancer immunotherapeutic potential.

Biophysical implications of sphingosine accumulation in membrane properties at neutral and acidic pH.

Sphingosine (Sph) is a simple lipid involved in the regulation of several biological processes. When accumulated in the late endosomal/lysosomal compartments, Sph causes changes in ion signaling and membrane trafficking, leading to the development of Niemann-Pick disease type C. Little is known about Sph interaction with other lipids in biological membranes; however, understanding the effect of Sph in the physical state of membranes might provide insights into its mode of action. Using complementary established fluorescence approaches, we show that Sph accumulation leads to the formation of Sph-enriched gel domains in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/sphingomyelin (SM)/cholesterol (Chol) model membranes. These domains are more easily formed in membrane models mimicking the neutral pH plasma membrane environment (PM) as compared to the acidic lysosomal membrane environment (LM), where higher Sph concentrations (or lower temperatures) are required. Electrophoretic light scattering measurements further revealed that in PM-raft models (POPC/SM/Chol), Sph is mainly neutral, whereas in LM models, the positive charge of Sph leads to electrostatic repulsion, reducing the Sph ability to form gel domains. Thus, formation of Sph-enriched domains in cellular membranes might be strongly regulated by Sph charge.