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.

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.

Engineering cytokine therapeutics.

Cytokines have pivotal roles in immunity, making them attractive as therapeutics for a variety of immune-related disorders. However, the widespread clinical use of cytokines has been limited by their short blood half-lives and severe side effects caused by low specificity and unfavourable biodistribution. Innovations in bioengineering have aided in advancing our knowledge of cytokine biology and yielded new technologies for cytokine engineering. In this Review, we discuss how the development of bioanalytical methods, such as sequencing and high-resolution imaging combined with genetic techniques, have facilitated a better understanding of cytokine biology. We then present an overview of therapeutics arising from cytokine re-engineering, targeting and delivery, mRNA therapeutics and cell therapy. We also highlight the application of these strategies to adjust the immunological imbalance in different immune-mediated disorders, including cancer, infection and autoimmune diseases. Finally, we look ahead to the hurdles that must be overcome before cytokine therapeutics can live up to their full potential.

In vivo imaging of cerebral glucose metabolism informs on subacute to chronic post-stroke tissue status - A pilot study combining PET and deuterium metabolic imaging.

Recanalization therapy after acute ischemic stroke enables restoration of cerebral perfusion. However, a significant subset of patients has poor outcome, which may be caused by disruption of cerebral energy metabolism. To assess changes in glucose metabolism subacutely and chronically after recanalization, we applied two complementary imaging techniques, fluorodeoxyglucose (FDG) positron emission tomography (PET) and deuterium (2H) metabolic imaging (DMI), after 60-minute transient middle cerebral artery occlusion (tMCAO) in C57BL/6 mice. Glucose uptake, measured with FDG PET, was reduced at 48 hours after tMCAO and returned to baseline value after 11 days. DMI revealed effective glucose supply as well as elevated lactate production and reduced glutamate/glutamine synthesis in the lesion area at 48 hours post-tMCAO, of which the extent was dependent on stroke severity. A further decrease in oxidative metabolism was evident after 11 days. Immunohistochemistry revealed significant glial activation in and around the lesion, which may play a role in the observed metabolic profiles. Our findings indicate that imaging (altered) active glucose metabolism in and around reperfused stroke lesions can provide substantial information on (secondary) pathophysiological changes in post-ischemic brain tissue.

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.

Systematically evaluating DOTATATE and FDG as PET immuno-imaging tracers of cardiovascular inflammation.

In recent years, cardiovascular immuno-imaging by positron emission tomography (PET) has undergone tremendous progress in preclinical settings. Clinically, two approved PET tracers hold great potential for inflammation imaging in cardiovascular patients, namely FDG and DOTATATE. While the former is a widely applied metabolic tracer, DOTATATE is a relatively new PET tracer targeting the somatostatin receptor 2 (SST2). In the current study, we performed a detailed, head-to-head comparison of DOTATATE-based radiotracers and [18F]F-FDG in mouse and rabbit models of cardiovascular inflammation. For mouse experiments, we labeled DOTATATE with the long-lived isotope [64Cu]Cu to enable studying the tracer's mode of action by complementing in vivo PET/CT experiments with thorough ex vivo immunological analyses. For translational PET/MRI rabbit studies, we employed the more widely clinically used [68Ga]Ga-labeled DOTATATE, which was approved by the FDA in 2016. DOTATATE's pharmacokinetics and timed biodistribution were determined in control and atherosclerotic mice and rabbits by ex vivo gamma counting of blood and organs. Additionally, we performed in vivo PET/CT experiments in mice with atherosclerosis, mice subjected to myocardial infarction and control animals, using both [64Cu]Cu-DOTATATE and [18F]F-FDG. To evaluate differences in the tracers' cellular specificity, we performed ensuing ex vivo flow cytometry and gamma counting. In mice subjected to myocardial infarction, in vivo [64Cu]Cu-DOTATATE PET showed higher differential uptake between infarcted (SUVmax 1.3, IQR, 1.2-1.4, N = 4) and remote myocardium (SUVmax 0.7, IQR, 0.5-0.8, N = 4, p = 0.0286), and with respect to controls (SUVmax 0.6, IQR, 0.5-0.7, N = 4, p = 0.0286), than [18F]F-FDG PET. In atherosclerotic mice, [64Cu]Cu-DOTATATE PET aortic signal, but not [18F]F-FDG PET, was higher compared to controls (SUVmax 1.1, IQR, 0.9-1.3 and 0.5, IQR, 0.5-0.6, respectively, N = 4, p = 0.0286). In both models, [64Cu]Cu-DOTATATE demonstrated preferential accumulation in macrophages with respect to other myeloid cells, while [18F]F-FDG was taken up by macrophages and other leukocytes. In a translational PET/MRI study in atherosclerotic rabbits, we then compared [68Ga]Ga-DOTATATE and [18F]F-FDG for the assessment of aortic inflammation, combined with ex vivo radiometric assays and near-infrared imaging of macrophage burden. Rabbit experiments showed significantly higher aortic accumulation of both [68Ga]Ga-DOTATATE and [18F]F-FDG in atherosclerotic (SUVmax 0.415, IQR, 0.338-0.499, N = 32 and 0.446, IQR, 0.387-0.536, N = 27, respectively) compared to control animals (SUVmax 0.253, IQR, 0.197-0.285, p = 0.0002, N = 10 and 0.349, IQR, 0.299-0.423, p = 0.0159, N = 11, respectively). In conclusion, we present a detailed, head-to-head comparison of the novel SST2-specific tracer DOTATATE and the validated metabolic tracer [18F]F-FDG for the evaluation of inflammation in small animal models of cardiovascular disease. Our results support further investigations on the use of DOTATATE to assess cardiovascular inflammation as a complementary readout to the widely used [18F]F-FDG.

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.

Immune Checkpoint Inhibitor Therapy Aggravates T Cell-Driven Plaque Inflammation in Atherosclerosis.

BACKGROUND: Immunotherapy has revolutionized cancer treatment. However, immune checkpoint inhibitors (ICIs) that target PD-1 (programmed cell death protein-1) and/or CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) are commonly associated with acute immune-related adverse events. Accumulating evidence also suggests that ICIs aggravate existing inflammatory diseases. OBJECTIVES: As inflammation drives atherosclerotic cardiovascular disease, we studied the propensity of short-term ICI therapy to aggravate atherosclerosis. METHODS: We used 18F-FDG (2-deoxy-2-[fluorine-18]fluoro-D-glucose) positron emission tomography-computed tomography to detect macrophage-driven vascular and systemic inflammation in pembrolizumab and nivolumab/ipilimumab-treated melanoma patients. In parallel, atherosclerotic Ldlr -/- mice were treated with CTLA-4 and PD-1 inhibition to study the proinflammatory consequences of immune checkpoint inhibition. RESULTS: ICI treatment did not affect 18F-FDG uptake in the large arteries, spleen, and bone marrow of melanoma patients, nor myeloid cell activation in blood and lymphoid organs in hyperlipidemic mice. In contrast, we found marked changes in the adaptive immune response (i.e., increased CD4+ effector T cell and CD8+ cytotoxic T cell numbers in lymphoid organs and the arterial wall of our hyperlipidemic mice). Although plaque size was unaffected, plaques had progressed toward a lymphoid-based inflammatory phenotype, characterized by a 2.7-fold increase of CD8+ T cells and a 3.9-fold increase in necrotic core size. Increased endothelial activation was observed with a 2.2-fold and 1.6-fold increase in vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, respectively. CONCLUSIONS: This study demonstrates that combination therapy with anti-CTLA-4 and anti-PD-1 antibodies does not affect myeloid-driven vascular and systemic inflammation in melanoma patients and hyperlipidemic mice. However, short-term ICI therapy in mice induces T cell-mediated plaque inflammation and drives plaque progression.

Tumor Targeting by αvß3-Integrin-Specific Lipid Nanoparticles Occurs via Phagocyte Hitchhiking.

Although the first nanomedicine was clinically approved more than two decades ago, nanoparticles' (NP) in vivo behavior is complex and the immune system's role in their application remains elusive. At present, only passive-targeting nanoformulations have been clinically approved, while more complicated active-targeting strategies typically fail to advance from the early clinical phase stage. This absence of clinical translation is, among others, due to the very limited understanding for in vivo targeting mechanisms. Dynamic in vivo phenomena such as NPs' real-time targeting kinetics and phagocytes' contribution to active NP targeting remain largely unexplored. To better understand in vivo targeting, monitoring NP accumulation and distribution at complementary levels of spatial and temporal resolution is imperative. Here, we integrate in vivo positron emission tomography/computed tomography imaging with intravital microscopy and flow cytometric analyses to study αvß3-integrin-targeted cyclic arginine-glycine-aspartate decorated liposomes and oil-in-water nanoemulsions in tumor mouse models. We observed that ligand-mediated accumulation in cancerous lesions is multifaceted and identified "NP hitchhiking" with phagocytes to contribute considerably to this intricate process. We anticipate that this understanding can facilitate rational improvement of nanomedicine applications and that immune cell-NP interactions can be harnessed to develop clinically viable nanomedicine-based immunotherapies.

Imaging Cardiovascular and Lung Macrophages With the Positron Emission Tomography Sensor 64Cu-Macrin in Mice, Rabbits, and Pigs.

BACKGROUND: Macrophages, innate immune cells that reside in all organs, defend the host against infection and injury. In the heart and vasculature, inflammatory macrophages also enhance tissue damage and propel cardiovascular diseases. METHODS: We here use in vivo positron emission tomography (PET) imaging, flow cytometry, and confocal microscopy to evaluate quantitative noninvasive assessment of cardiac, arterial, and pulmonary macrophages using the nanotracer 64Cu-Macrin-a 20-nm spherical dextran nanoparticle assembled from nontoxic polyglucose. RESULTS: PET imaging using 64Cu-Macrin faithfully reported accumulation of macrophages in the heart and lung of mice with myocardial infarction, sepsis, or pneumonia. Flow cytometry and confocal microscopy detected the near-infrared fluorescent version of the nanoparticle (VT680Macrin) primarily in tissue macrophages. In 5-day-old mice, 64Cu-Macrin PET imaging quantified physiologically more numerous cardiac macrophages. Upon intravenous administration of 64Cu-Macrin in rabbits and pigs, we detected heightened macrophage numbers in the infarcted myocardium, inflamed lung regions, and atherosclerotic plaques using a clinical PET/magnetic resonance imaging scanner. Toxicity studies in rats and human dosimetry estimates suggest that 64Cu-Macrin is safe for use in humans. CONCLUSIONS: Taken together, these results indicate 64Cu-Macrin could serve as a facile PET nanotracer to survey spatiotemporal macrophage dynamics during various physiological and pathological conditions. 64Cu-Macrin PET imaging could stage inflammatory cardiovascular disease activity, assist disease management, and serve as an imaging biomarker for emerging macrophage-targeted therapeutics.

Multimodal imaging of bacterial-host interface in mice and piglets with Staphylococcus aureus endocarditis.

Acute bacterial endocarditis is a rapid, difficult to manage, and frequently lethal disease. Potent antibiotics often cannot efficiently kill Staphylococcus aureus that colonizes the heart's valves. S. aureus relies on virulence factors to evade therapeutics and the host's immune response, usurping the host's clotting system by activating circulating prothrombin with staphylocoagulase and von Willebrand factor-binding protein. An insoluble fibrin barrier then forms around the bacterial colony, shielding the pathogen from immune cell clearance. Targeting virulence factors may provide previously unidentified avenues to better diagnose and treat endocarditis. To tap into this unused therapeutic opportunity, we codeveloped therapeutics and multimodal molecular imaging to probe the host-pathogen interface. We introduced and validated a family of small-molecule optical and positron emission tomography (PET) reporters targeting active thrombin in the fibrin-rich environment of bacterial colonies. The imaging agents, based on the clinical thrombin inhibitor dabigatran, are bound to heart valve vegetations in mice. Using optical imaging, we monitored therapy with antibodies neutralizing staphylocoagulase and von Willebrand factor-binding protein in mice with S. aureus endocarditis. This treatment deactivated bacterial defenses against innate immune cells, decreased in vivo imaging signal, and improved survival. Aortic or tricuspid S. aureus endocarditis in piglets was also successfully imaged with clinical PET/magnetic resonance imaging. Our data map a route toward adjuvant immunotherapy for endocarditis and provide efficient tools to monitor this drug class for infectious diseases.

Probing myeloid cell dynamics in ischaemic heart disease by nanotracer hot-spot imaging.

Ischaemic heart disease evokes a complex immune response. However, tools to track the systemic behaviour and dynamics of leukocytes non-invasively in vivo are lacking. Here, we present a multimodal hot-spot imaging approach using an innovative high-density lipoprotein-derived nanotracer with a perfluoro-crown ether payload (19F-HDL) to allow myeloid cell tracking by 19F magnetic resonance imaging. The 19F-HDL nanotracer can additionally be labelled with zirconium-89 and fluorophores to detect myeloid cells by in vivo positron emission tomography imaging and optical modalities, respectively. Using our nanotracer in atherosclerotic mice with myocardial infarction, we observed rapid myeloid cell egress from the spleen and bone marrow by in vivo 19F-HDL magnetic resonance imaging. Concurrently, using ex vivo techniques, we showed that circulating pro-inflammatory myeloid cells accumulated in atherosclerotic plaques and at the myocardial infarct site. Our multimodality imaging approach is a valuable addition to the immunology toolbox, enabling the study of complex myeloid cell behaviour dynamically.