MR Elastography-Based Assessment of Matrix Remodeling at Lesion Sites Associated With Clinical Severity in a Model of Multiple Sclerosis

Magnetic resonance imaging (MRI) with gadolinium based contrast agents (GBCA) is routinely used in the clinic to visualize lesions in multiple sclerosis (MS). Although GBCA reveal endothelial permeability, they fail to expose other aspects of lesion formation such as the magnitude of inflammation or tissue changes occurring at sites of blood-brain barrier (BBB) disruption. Moreover, evidence pointing to potential side effects of GBCA has been increasing. Thus, there is an urgent need to develop GBCA-independent imaging tools to monitor pathology in MS. Using MR-elastography (MRE), we previously demonstrated in both MS and the animal model experimental autoimmune encephalomyelitis (EAE) that inflammation was associated with a reduction of brain stiffness. Now, using the relapsing-remitting EAE model, we show that the cerebellum-a region with predominant inflammation in this model-is especially prone to loss of stiffness. We also demonstrate that, contrary to GBCA-MRI, reduction of brain stiffness correlates with clinical disability and is associated with enhanced expression of the extracellular matrix protein fibronectin (FN). Further, we show that FN is largely expressed by activated astrocytes at acute lesions, and reflects the magnitude of tissue remodeling at sites of BBB breakdown. Therefore, MRE could emerge as a safe tool suitable to monitor disease activity in MS

Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1

We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to preinhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties

Technical Developments and Ex Vivo Demonstration in a Mouse Model of Neuroinflammation

Neuroinflammation can be monitored using fluorine-19 (19F)-containing nanoparticles and 19F MRI. Previously we studied neuroinflammation in experimental autoimmune encephalomyelitis (EAE) using room temperature (RT) 19F radiofrequency (RF) coils and low spatial resolution 19F MRI to overcome constraints in signal-to-noise ratio (SNR). This yielded an approximate localization of inflammatory lesions. Here we used a new 19F transceive cryogenic quadrature RF probe (19F-CRP) that provides the SNR necessary to acquire superior spatially-resolved 19F MRI. First we characterized the signal-transmission profile of the 19F-CRP. The 19F-CRP was then benchmarked against a RT 19F/1H RF coil. For SNR comparison we used reference compounds including 19F-nanoparticles and ex vivo brains from EAE mice administered with 19F-nanoparticles. The transmit/receive profile of the 19F-CRP diminished with increasing distance from the surface. This was counterbalanced by a substantial SNR gain compared to the RT coil. Intraparenchymal inflammation in the ex vivo EAE brains was more sharply defined when using 150 μm isotropic resolution with the 19F-CRP, and reflected the known distribution of EAE histopathology. At this spatial resolution, most 19F signals were undetectable using the RT coil. The 19F-CRP is a valuable tool that will allow us to study neuroinflammation with greater detail in future in vivo studies

Iterative in situ click chemistry creates antibody-like protein-capture agents

Iterative in situ click chemistry (see scheme for the tertiary ligand screen) and the one-bead-one-compound method for the creation of a peptide library enable the fragment-based assembly of selective high-affinity protein-capture agents. The resulting ligands are water-soluble and stable chemically, biochemically, and thermally. They can be produced in gram quantities through copper (I)-catalyzed cycloaddition

A Phase I, First-in-Human Study of GSK2849330, an Anti-HER3 Monoclonal Antibody, in HER3-Expressing Solid Tumors

Background GSK2849330, an anti-HER3 monoclonal antibody that blocks HER3/Neuregulin 1 (NRG1) signaling in cancer cells, is engineered for enhanced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. This phase I, first-in-human, open-label study assessed the safety, pharmacokinetics (PK), pharmacodynamics, and preliminary activity of GSK2849330 in patients with HER3-expressing advanced solid tumors. Patients and Methods Patients with various tumor types were prospectively selected for HER3 expression by immunohistochemistry; a subset was also screened for NRG1 mRNA expression. In the dose-escalation phase, patients received GSK2849330 1.4-30 mg/kg every 2 weeks, or 3 mg/kg or 30 mg/kg weekly, intravenously (IV). In the dose-expansion phase, patients received 30 mg/kg GSK2849330 IV weekly. Results Twenty-nine patients with HER3-expressing cancers, of whom two expressed NRG1, received GSK2849330 (dose escalation: n = 18, dose expansion: n = 11). GSK2849330 was well tolerated. No dose-limiting toxicities were observed. The highest dose, of 30 mg/kg weekly, expected to provide full target engagement, was selected for dose expansion. Treatment-emergent adverse events (AEs) were mostly grade 1 or 2. The most common AEs were diarrhea (66%), fatigue (62%), and decreased appetite (31%). Dose-proportional plasma exposures were achieved, with evidence of HER3 inhibition in paired tissue biopsies. Of 29 patients, only 1 confirmed partial response, lasting 19 months, was noted in a patient with CD74-NRG1-rearranged non-small cell lung cancer (NSCLC). Conclusion GSK2849330 demonstrated a favorable safety profile, dose-proportional PK, and evidence of target engagement, but limited antitumor activity in HER3-expressing cancers. The exceptional response seen in a patient with CD74-NRG1-rearranged NSCLC suggests further exploration in NRG1-fusion-positive cancers. Implications for Practice This first-in-human study confirms that GSK2849330 is well tolerated. Importantly, across a variety of HER3-expressing advanced tumors, prospective selection by HER3/NRG1 expression alone was insufficient to identify patients who could benefit from treatment with this antibody-dependent cell-mediated cytotoxicity- and complement-dependent cytotoxicity-enhanced anti-HER3 antibody. The only confirmed durable response achieved was in a patient with CD74-NRG1-rearranged lung cancer. This highlights the potential utility of screening for NRG1 fusions prospectively across tumor types to enrich potential responders to anti-HER3 agents in ongoing trials

Neuroprotective Effect of Combination Therapy of Glatiramer Acetate and Epigallocatechin-3-Gallate in Neuroinflammation

Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system. However, studies of MS and the animal model, experimental autoimmune encephalomyelitis (EAE), indicate that neuronal pathology is the principle cause of clinical disability. Thus, there is need to develop new therapeutic strategies that not only address immunomodulation but also neuroprotection. Here we show that the combination therapy of Glatiramer acetate (GA), an immunomodulatory MS therapeutic, and the neuroprotectant epigallocatechin-3-gallate (EGCG), the main phenol in green tea, have synergistic protective effects in vitro and in the EAE model. EGCG and GA together led to increased protection from glutamate- and TRAIL-induced neuronal cell death in vitro. EGCG combined with GA induced regeneration of hippocampal axons in an outgrowth assay. The combined application of EGCG and GA did not result in unexpected adverse events in vivo. Neuroprotective and neuroregenerative effects could be translated in the in vivo model, where combination treatment with EGCG and GA significantly delayed disease onset, strongly reduced clinical severity, even after onset of symptoms and reduced inflammatory infiltrates. These results illustrate the promise of combining neuroprotective and anti-inflammatory treatments and strengthen the prospects of EGCG as an adjunct therapy for neuroinflammatory and neurodegenerative diseases

Mitoxantrone Induces Natural Killer Cell Maturation in Patients with Secondary Progressive Multiple Sclerosis

Mitoxantrone is one of the few drugs approved for the treatment of progressive multiple sclerosis (MS). However, the prolonged use of this potent immunosuppressive agent is limited by the appearance of severe side effects. Apart from its general cytotoxic effect, the mode of action of mitoxantrone on the immune system is poorly understood. Thus, to develop safe therapeutic approaches for patients with progressive MS, it is essential to elucidate how mitoxantrone exerts it benefits. Accordingly, we initiated a prospective single-arm open-label study with 19 secondary progressive MS patients. We investigated long-term effects of mitoxantrone on patient peripheral immune subsets using flow cytometry. While we corroborate that mitoxantrone persistently suppresses B cells in vivo, we show for the first time that treatment led to an enrichment of neutrophils and immunomodulatory CD8low T cells. Moreover, sustained mitoxantrone applications promoted not only persistent NK cell enrichment but also NK cell maturation. Importantly, this mitoxantrone-induced NK cell maturation was seen only in patients that showed a clinical response to treatment. Our data emphasize the complex immunomodulatory role of mitoxantrone, which may account for its benefit in MS. In particular, these results highlight the contribution of NK cells to mitoxantrone efficacy in progressive MS

Identification of Cellular Infiltrates during Early Stages of Brain Inflammation with Magnetic Resonance Microscopy

A comprehensive view of brain inflammation during the pathogenesis of autoimmune encephalomyelitis can be achieved with the aid of high resolution non-invasive imaging techniques such as microscopic magnetic resonance imaging (μMRI). In this study we demonstrate the benefits of cryogenically-cooled RF coils to produce μMRI in vivo, with sufficient detail to reveal brain pathology in the experimental autoimmune encephalomyelitis (EAE) model. We could visualize inflammatory infiltrates in detail within various regions of the brain, already at an early phase of EAE. Importantly, this pathology could be seen clearly even without the use of contrast agents, and showed excellent correspondence with conventional histology. The cryogenically-cooled coil enabled the acquisition of high resolution images within short scan times: an important practical consideration in conducting animal experiments. The detail of the cellular infiltrates visualized by in vivo μMRI allows the opportunity to follow neuroinflammatory processes even during the early stages of disease progression. Thus μMRI will not only complement conventional histological examination but will also enable longitudinal studies on the kinetics and dynamics of immune cell infiltration

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation

Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure

The Omicron, or Pango lineage B.1.1.529, variant of SARS-CoV-2 carries multiple spike mutations with high transmissibility and partial neutralizing antibody (nAb) escape. Vaccinated individuals show protection from severe disease, often attributed to primed cellular immunity. We investigated T and B cell immunity against B.1.1.529 in triple mRNA vaccinated healthcare workers (HCW) with different SARS-CoV-2 infection histories. B and T cell immunity against previous variants of concern was enhanced in triple vaccinated individuals, but magnitude of T and B cell responses against B.1.1.529 spike protein was reduced. Immune imprinting by infection with the earlier B.1.1.7 (Alpha) variant resulted in less durable binding antibody against B.1.1.529. Previously infection-naïve HCW who became infected during the B.1.1.529 wave showed enhanced immunity against earlier variants, but reduced nAb potency and T cell responses against B.1.1.529 itself. Previous Wuhan Hu-1 infection abrogated T cell recognition and any enhanced cross-reactive neutralizing immunity on infection with B.1.1.529