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.

Targeting Trained Innate Immunity With Nanobiologics to Treat Cardiovascular Disease.

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An iterative sparse deconvolution method for simultaneous multicolor 19 F-MRI of multiple contrast agents.

PURPOSE: 19 F-MRI is gaining widespread interest for cell tracking and quantification of immune and inflammatory cells in vivo. Different fluorinated compounds can be discriminated based on their characteristic MR spectra, allowing in vivo imaging of multiple 19 F compounds simultaneously, so-called multicolor 19 F-MRI. We introduce a method for multicolor 19 F-MRI using an iterative sparse deconvolution method to separate different 19 F compounds and remove chemical shift artifacts arising from multiple resonances. METHODS: The method employs cycling of the readout gradient direction to alternate the spatial orientation of the off-resonance chemical shift artifacts, which are subsequently removed by iterative sparse deconvolution. Noise robustness and separation was investigated by numerical simulations. Mixtures of fluorinated oils (PFCE and PFOB) were measured on a 7T MR scanner to identify the relation between 19 F signal intensity and compound concentration. The method was validated in a mouse model after intramuscular injection of fluorine probes, as well as after intravascular injection. RESULTS: Numerical simulations show efficient separation of 19 F compounds, even at low signal-to-noise ratio. Reliable chemical shift artifact removal and separation of PFCE and PFOB signals was achieved in phantoms and in vivo. Signal intensities correlated excellently to the relative 19 F compound concentrations (r-2 = 0.966/0.990 for PFOB/PFCE). CONCLUSIONS: The method requires minimal sequence adaptation and is therefore easily implemented on different MRI systems. Simulations, phantom experiments, and in-vivo measurements in mice showed effective separation and removal of chemical shift artifacts below noise level. We foresee applicability for simultaneous in-vivo imaging of 19 F-containing fluorine probes or for detection of 19 F-labeled cell populations.

Model-driven engineering of supramolecular buffering by multivalency.

A supramolecular system in which the concentration of a molecule is buffered over several orders of magnitude is presented. Molecular buffering is achieved as a result of competition in a ring-chain equilibrium of multivalent ureidopyrimidinone monomers and a monovalent naphthyridine molecule which acts as an end-capper. While we previously only considered divalent ureidopyrimidinone monomers we now present a model-driven engineering approach to improve molecular buffering using multivalent ring-chain systems. Our theoretical models reveal an odd-even effect where even-valent molecules show superior buffering capabilities. Furthermore, we predict that supramolecular buffering can be significantly improved using a tetravalent instead of a divalent molecule, since the tetravalent molecule can form two intramolecular rings with different "stabilities" due to statistical effects. Our model predictions are validated against experimental 1H NMR data, demonstrating that model-driven engineering has considerable potential in supramolecular chemistry.

Investigating supramolecular systems using Förster resonance energy transfer.

Supramolecular systems have applications in areas as diverse as materials science, biochemistry, analytical chemistry, and nanomedicine. However, analyzing such systems can be challenging due to the wide range of time scales, binding strengths, distances, and concentrations at which non-covalent phenomena take place. Due to their versatility and sensitivity, Förster resonance energy transfer (FRET)-based techniques are excellently suited to meet such challenges. Here, we detail the ways in which FRET has been used to study non-covalent interactions in both synthetic and biological supramolecular systems. Among other topics, we examine methods to measure molecular forces, determine protein conformations, monitor assembly kinetics, and visualize in vivo drug release from nanoparticles. Furthermore, we highlight multiplex FRET techniques, discuss the field's limitations, and provide a perspective on new developments.

Directing the Self-Assembly Behaviour of Porphyrin-Based Supramolecular Systems.

The self-assembly behaviour of a library of tetra-amidated porphyrin molecules decorated with a variety of solubilizing wedges is investigated as dilute solutions in methylcyclohexane. Small changes in the solubilising wedge of the porphyrins resulted in different aggregated states, as evidenced by CD and UV/Vis absorption spectroscopy. The porphyrins form co-facially stacked H-aggregates, slip-stacked J-aggregates or a mixture of both. Detailed thermodynamic and kinetic analysis showed that in all cases the formation of J-aggregates proceeds via an isodesmic mechanism whereas H-aggregates are formed via a cooperative mechanism. It is shown that these aggregates assemble in a parallel pathway, in which both compete for the monomer, compared to a sequential pathway, in which one of the aggregates interconverts into the other. Interestingly, kinetic analysis of porphyrins that only form H-aggregates in thermodynamic equilibrium revealed that the competing pathway towards J-aggregates is operational in these systems as well. Our findings show that the balance between H- and J-aggregates depends on remarkably small changes in the architecture of the solubilising wedges.

End Groups of Functionalized Siloxane Oligomers Direct Block-Copolymeric or Liquid-Crystalline Self-Assembly Behavior.

Monodisperse oligodimethylsiloxanes end-functionalized with the hydrogen-bonding ureidopyrimidinone (UPy) motif undergo phase separation between their aromatic end groups and dimethylsiloxane midblocks to form ordered nanostructures with domain spacings of <5 nm. The self-assembly behavior of these well-defined oligomers resembles that of high degree of polymerization (N)-high block interaction parameter (χ) linear diblock copolymers despite their small size. Specifically, the phase morphology varies from lamellar to hexagonal to body-centered cubic with increasing asymmetry in molecular volume fraction. Mixing molecules with different molecular weights to give dispersity >1.13 results in disorder, showing importance of molecular monodispersity for ultrasmall ordered phase separation. In contrast, oligodimethylsiloxanes end-functionalized with an O-benzylated UPy derivative self-assemble into lamellar nanostructures regardless of volume fraction because of the strong preference of the end groups to aggregate in a planar geometry. Thus, these molecules display more classically liquid-crystalline self-assembly behavior where the lamellar bilayer thickness is determined by the siloxane midblock. Here the lamellar nanostructure is tolerant to molecular polydispersity. We show the importance of end groups in high χ-low N block molecules, where block-copolymer-like self-assembly in our UPy-functionalized oligodimethylsiloxanes relies upon the dominance of phase separation effects over directional end group aggregation.

Regulating Competing Supramolecular Interactions Using Ligand Concentration.

The complexity of biomolecular systems inevitably leads to a degree of competition between the noncovalent interactions involved. However, the outcome of biological processes is generally very well-defined often due to the competition of these interactions. In contrast, specificity in synthetic supramolecular systems is usually based on the presence of a minimum set of alternative assembly pathways. While the latter might simplify the system, it prevents the selection of specific structures and thereby limits the adaptivity of the system. Therefore, artificial systems containing competing interactions are vital to stimulate the development of more adaptive and lifelike synthetic systems. Here, we present a detailed study on the self-assembly behavior of a C2v-symmetrical tritopic molecule, functionalized with three self-complementary ureidopyrimidinone (UPy) motifs. Due to a shorter linker connecting one of these UPys, two types of cycles with different stabilities can be formed, which subsequently dimerize intermolecularly via the third UPy. The UPy complementary 2,7-diamido-1,8-naphthyridine (NaPy) motif was gradually added to this mixture in order to examine its effect on the cycle distribution. As a result of the C2v-symmetry of the tritopic UPy, together with small differences in binding strength, the cycle ratio can be regulated by altering the concentration of NaPy. We show that this ratio can be increased to an extent where one type of cycle is formed almost exclusively.

Protocol for the development of mRNA lipid nanoparticle vaccines and analysis of immunization efficiency in mice.

Here, we present a protocol for the development of mRNA-loaded lipid nanoparticle (LNP) vaccines for target antigen sequences of interest. We describe key steps required to design and synthesize mRNA constructs, their LNP encapsulation, and mouse immunization. We then detail quality control assays to determine RNA purity, guidelines to measure RNA immunogenicity using in vitro reporter systems, and a technique to evaluate antigen-specific T cell responses following immunization.

Uncovering atherosclerotic cardiovascular disease by PET imaging.

Assessing atherosclerosis severity is essential for precise patient stratification. Specifically, there is a need to identify patients with residual inflammation because these patients remain at high risk of cardiovascular events despite optimal management of cardiovascular risk factors. Molecular imaging techniques, such as PET, can have an essential role in this context. PET imaging can indicate tissue-based disease status, detect early molecular changes and provide whole-body information. Advances in molecular biology and bioinformatics continue to help to decipher the complex pathogenesis of atherosclerosis and inform the development of imaging tracers. Concomitant advances in tracer synthesis methods and PET imaging technology provide future possibilities for atherosclerosis imaging. In this Review, we summarize the latest developments in PET imaging techniques and technologies for assessment of atherosclerotic cardiovascular disease and discuss the relationship between imaging readouts and transcriptomics-based plaque phenotyping.

Macrophage PET imaging in mouse models of cardiovascular disease and cancer with an apolipoprotein-inspired radiotracer.

Macrophages are key inflammatory mediators in many pathological conditions, including cardiovascular disease (CVD) and cancer, the leading causes of morbidity and mortality worldwide. This makes macrophage burden a valuable diagnostic marker and several strategies to monitor these cells have been reported. However, such strategies are often high-priced, non-specific, invasive, and/or not quantitative. Here, we developed a positron emission tomography (PET) radiotracer based on apolipoprotein A1 (ApoA1), the main protein component of high-density lipoprotein (HDL), which has an inherent affinity for macrophages. We radiolabeled an ApoA1-mimetic peptide (mA1) with zirconium-89 (89Zr) to generate a lipoprotein-avid PET probe (89Zr-mA1). We first characterized 89Zr-mA1's affinity for lipoproteins in vitro by size exclusion chromatography. To study 89Zr-mA1's in vivo behavior and interaction with endogenous lipoproteins, we performed extensive studies in wildtype C57BL/6 and Apoe-/- hypercholesterolemic mice. Subsequently, we used in vivo PET imaging to study macrophages in melanoma and myocardial infarction using mouse models. The tracer's cell specificity was assessed by histology and mass cytometry (CyTOF). Our data show that 89Zr-mA1 associates with lipoproteins in vitro. This is in line with our in vivo experiments, in which we observed longer 89Zr-mA1 circulation times in hypercholesterolemic mice compared to C57BL/6 controls. 89Zr-mA1 displayed a tissue distribution profile similar to ApoA1 and HDL, with high kidney and liver uptake as well as substantial signal in the bone marrow and spleen. The tracer also accumulated in tumors of melanoma-bearing mice and in the ischemic myocardium of infarcted animals. In these sites, CyTOF analyses revealed that natZr-mA1 was predominantly taken up by macrophages. Our results demonstrate that 89Zr-mA1 associates with lipoproteins and hence accumulates in macrophages in vivo. 89Zr-mA1's high uptake in these cells makes it a promising radiotracer for non-invasively and quantitatively studying conditions characterized by marked changes in macrophage burden.

Polymersomes with splenic avidity target red pulp myeloid cells for cancer immunotherapy.

Regulating innate immunity is an emerging approach to improve cancer immunotherapy. Such regulation requires engaging myeloid cells by delivering immunomodulatory compounds to hematopoietic organs, including the spleen. Here we present a polymersome-based nanocarrier with splenic avidity and propensity for red pulp myeloid cell uptake. We characterized the in vivo behaviour of four chemically identical yet topologically different polymersomes by in vivo positron emission tomography imaging and innovative flow and mass cytometry techniques. Upon intravenous administration, relatively large and spherical polymersomes accumulated rapidly in the spleen and efficiently targeted myeloid cells in the splenic red pulp. When loaded with ß-glucan, intravenously administered polymersomes significantly reduced tumour growth in a mouse melanoma model. We initiated our nanotherapeutic's clinical translation with a biodistribution study in non-human primates, which revealed that the platform's splenic avidity is preserved across species.

Acid ceramidase regulates innate immune memory.

Innate immune memory, also called "trained immunity," is a functional state of myeloid cells enabling enhanced immune responses. This phenomenon is important for host defense, but also plays a role in various immune-mediated conditions. We show that exogenously administered sphingolipids and inhibition of sphingolipid metabolizing enzymes modulate trained immunity. In particular, we reveal that acid ceramidase, an enzyme that converts ceramide to sphingosine, is a potent regulator of trained immunity. We show that acid ceramidase regulates the transcription of histone-modifying enzymes, resulting in profound changes in histone 3 lysine 27 acetylation and histone 3 lysine 4 trimethylation. We confirm our findings by identifying single-nucleotide polymorphisms in the region of ASAH1, the gene encoding acid ceramidase, that are associated with the trained immunity cytokine response. Our findings reveal an immunomodulatory effect of sphingolipids and identify acid ceramidase as a relevant therapeutic target to modulate trained immunity responses in innate immune-driven disorders.

Multiparametric Immunoimaging Maps Inflammatory Signatures in Murine Myocardial Infarction Models.

In the past 2 decades, research on atherosclerotic cardiovascular disease has uncovered inflammation to be a key driver of the pathophysiological process. A pressing need therefore exists to quantitatively and longitudinally probe inflammation, in preclinical models and in cardiovascular disease patients, ideally using non-invasive methods and at multiple levels. Here, we developed and employed in vivo multiparametric imaging approaches to investigate the immune response following myocardial infarction. The myocardial infarction models encompassed either transient or permanent left anterior descending coronary artery occlusion in C57BL/6 and Apoe-/-mice. We performed nanotracer-based fluorine magnetic resonance imaging and positron emission tomography (PET) imaging using a CD11b-specific nanobody and a C-C motif chemokine receptor 2-binding probe. We found that immune cell influx in the infarct was more pronounced in the permanent occlusion model. Further, using 18F-fluorothymidine and 18F-fluorodeoxyglucose PET, we detected increased hematopoietic activity after myocardial infarction, with no difference between the models. Finally, we observed persistent systemic inflammation and exacerbated atherosclerosis in Apoe-/- mice, regardless of which infarction model was used. Taken together, we showed the strengths and capabilities of multiparametric imaging in detecting inflammatory activity in cardiovascular disease, which augments the development of clinical readouts.

Resolving sepsis-induced immunoparalysis via trained immunity by targeting interleukin-4 to myeloid cells.

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.

Embracing nanomaterials' interactions with the innate immune system.

Immunotherapy has firmly established itself as a compelling avenue for treating disease. Although many clinically approved immunotherapeutics engage the adaptive immune system, therapeutically targeting the innate immune system remains much less explored. Nanomedicine offers a compelling opportunity for innate immune system engagement, as many nanomaterials inherently interact with myeloid cells (e.g., monocytes, macrophages, neutrophils, and dendritic cells) or can be functionalized to target their cell-surface receptors. Here, we provide a perspective on exploiting nanomaterials for innate immune system regulation. We focus on specific nanomaterial design parameters, including size, form, rigidity, charge, and surface decoration. Furthermore, we examine the potential of high-throughput screening and machine learning, while also providing recommendations for advancing the field. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Employing nanobodies for immune landscape profiling by PET imaging in mice.

Noninvasive immunoimaging holds great potential for studying and stratifying disease as well as therapeutic efficacy. Radiolabeled single-domain antibody fragments (i.e., nanobodies) are appealing probes for immune landscape profiling, as they display high stability, rapid targeting, and excellent specificity, while allowing extremely sensitive nuclear readouts. Here, we present a protocol for radiolabeling an anti-CD11b nanobody and studying its uptake in mice by a combination of positron emission tomography imaging, ex vivo gamma counting, and autoradiography. Our protocol is applicable to nanobodies against other antigens. For complete details on the use and execution of this protocol, please see Priem et al. (2020), Senders et al. (2019), or Rashidian et al. (2017).

A modular approach toward producing nanotherapeutics targeting the innate immune system.

Immunotherapies controlling the adaptive immune system are firmly established, but regulating the innate immune system remains much less explored. The intrinsic interactions between nanoparticles and phagocytic myeloid cells make these materials especially suited for engaging the innate immune system. However, developing nanotherapeutics is an elaborate process. Here, we demonstrate a modular approach that facilitates efficiently incorporating a broad variety of drugs in a nanobiologic platform. Using a microfluidic formulation strategy, we produced apolipoprotein A1-based nanobiologics with favorable innate immune system-engaging properties as evaluated by in vivo screening. Subsequently, rapamycin and three small-molecule inhibitors were derivatized with lipophilic promoieties, ensuring their seamless incorporation and efficient retention in nanobiologics. A short regimen of intravenously administered rapamycin-loaded nanobiologics (mTORi-NBs) significantly prolonged allograft survival in a heart transplantation mouse model. Last, we studied mTORi-NB biodistribution in nonhuman primates by PET/MR imaging and evaluated its safety, paving the way for clinical translation.

Prosaposin mediates inflammation in atherosclerosis.

Macrophages play a central role in the pathogenesis of atherosclerosis. The inflammatory properties of these cells are dictated by their metabolism, of which the mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator. Using myeloid cell-specific nanobiologics in apolipoprotein E-deficient (Apoe -/-) mice, we found that targeting the mTOR and ribosomal protein S6 kinase-1 (S6K1) signaling pathways rapidly diminished plaque macrophages' inflammatory activity. By investigating transcriptome modifications, we identified Psap, a gene encoding the lysosomal protein prosaposin, as closely related with mTOR signaling. Subsequent in vitro experiments revealed that Psap inhibition suppressed both glycolysis and oxidative phosphorylation. Transplantation of Psap -/- bone marrow to low-density lipoprotein receptor knockout (Ldlr -/-) mice led to a reduction in atherosclerosis development and plaque inflammation. Last, we confirmed the relationship between PSAP expression and inflammation in human carotid atherosclerotic plaques. Our findings provide mechanistic insights into the development of atherosclerosis and identify prosaposin as a potential therapeutic target.

Nuclear imaging approaches facilitating nanomedicine translation.

Nanomedicine approaches can effectively modulate the biodistribution and bioavailability of therapeutic agents, improving their therapeutic index. However, despite the ever-increasing amount of literature reporting on preclinical nanomedicine, the number of nanotherapeutics receiving FDA approval remains relatively low. Several barriers exist that hamper the effective preclinical evaluation and clinical translation of nanotherapeutics. Key barriers include insufficient understanding of nanomedicines' in vivo behavior, inadequate translation from murine models to larger animals, and a lack of patient stratification strategies. Integrating quantitative non-invasive imaging techniques in nanomedicine development offers attractive possibilities to address these issues. Among the available imaging techniques, nuclear imaging by positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are highly attractive in this context owing to their quantitative nature and uncontested sensitivity. In basic and translational research, nuclear imaging techniques can provide critical quantitative information about pharmacokinetic parameters, biodistribution profiles or target site accumulation of nanocarriers and their associated payload. During clinical evaluation, nuclear imaging can be used to select patients amenable to nanomedicine treatment. Here, we review how nuclear imaging-based approaches are increasingly being integrated into nanomedicine development and discuss future developments that will accelerate their clinical translation.