An optimized IL-12-Fc expands its therapeutic window, achieving strong activity against mouse tumors at tolerable drug doses.

BACKGROUND: Interleukin-12 (IL-12) has emerged as one of the most potent cytokines for tumor immunotherapy due to its ability to induce interferon γ (IFNγ) and polarize Th1 responses. Clinical use of IL-12 has been limited by a short half-life and narrow therapeutic index. METHODS: We generated a monovalent, half-life-extended IL-12-Fc fusion protein, mDF6006, engineered to retain the high potency of native IL-12 while significantly expanding its therapeutic window. In vitro and in vivo activity of mDF6006 was tested against murine tumors. To translate our findings, we developed a fully human version of IL-12-Fc, designated DF6002, which we characterized in vitro on human cells and in vivo in cynomolgus monkeys in preparation for clinical trials. FINDINGS: The extended half-life of mDF6006 modified the pharmacodynamic profile of IL-12 to one that was better tolerated systemically while vastly amplifying its efficacy. Mechanistically, mDF6006 led to greater and more sustained IFNγ production than recombinant IL-12 without inducing high, toxic peak serum concentrations of IFNγ. We showed that mDF6006's expanded therapeutic window allowed for potent anti-tumor activity as single agent against large immune checkpoint blockade-resistant tumors. Furthermore, the favorable benefit-risk profile of mDF6006 enabled effective combination with PD-1 blockade. Fully human DF6002, similarly, demonstrated an extended half-life and a protracted IFNγ profile in non-human primates. CONCLUSION: An optimized IL-12-Fc fusion protein increased the therapeutic window of IL-12, enhancing anti-tumor activity without concomitantly increasing toxicity. FUNDING: This research was funded by Dragonfly Therapeutics.

CNS imaging characteristics in fibromyalgia patients with and without peripheral nerve involvement.

We tested the hypothesis that reduced skin innervation in fibromyalgia syndrome is associated with specific CNS changes. This prospective case-control study included 43 women diagnosed with fibromyalgia syndrome and 40 healthy controls. We further compared the fibromyalgia subgroups with reduced (n = 21) and normal (n = 22) skin innervation. Brains were analysed for cortical volume, for white matter integrity, and for functional connectivity. Compared to controls, cortical thickness was decreased in regions of the frontal, temporal and parietal cortex in the fibromyalgia group as a whole, and decreased in the bilateral pericalcarine cortices in the fibromyalgia subgroup with reduced skin innervation. Diffusion tensor imaging revealed a significant increase in fractional anisotropy in the corona radiata, the corpus callosum, cingulum and fornix in patients with fibromyalgia compared to healthy controls and decreased FA in parts of the internal capsule and thalamic radiation in the subgroup with reduced skin innervation. Using resting-state fMRI, the fibromyalgia group as a whole showed functional hypoconnectivity between the right midfrontal gyrus and the posterior cerebellum and the right crus cerebellum, respectively. The subgroup with reduced skin innervation showed hyperconnectivity between the inferior frontal gyrus, the angular gyrus and the posterior parietal gyrus. Our results suggest that the subgroup of fibromyalgia patients with pronounced pathology in the peripheral nervous system shows alterations in morphology, structural and functional connectivity also at the level of the encephalon. We propose considering these subgroups when conducting clinical trials.

Relevance of Religiosity for Coping Strategies and Disability in Patients with Fibromyalgia Syndrome.

Coping strategies are essential for the outcome of chronic pain. This study evaluated religiosity in a cohort of patients with fibromyalgia syndrome (FMS), its effect on pain and other symptoms, on coping and FMS-related disability. A total of 102 FMS patients were recruited who filled in questionnaires, a subgroup of 42 patients participated in a face-to-face interview, and data were evaluated by correlation and regression analyses. Few patients were traditionally religious, but the majority believed in a higher existence and described their spirituality as "transcendence conviction". The coping strategy "praying-hoping" and the ASP dimension "religious orientation" (r = 0.5, P < 0.05) showed a significant relationship independent of the grade of religiosity (P < 0.05). A high grade of belief in a higher existence was negatively associated with the choice of ignoring as coping strategy (r = - 0.4, P < 0.05). Mood and affect-related variables had the highest impact on disability (b = 0.5, P < 0.05). In this cohort, the grade of religiosity played a role in the choice of coping strategies, but had no effects on health and mood outcome.

Iodine-Mediated Tryptathionine Formation Facilitates the Synthesis of Amanitins.

Synthetic methods on the macrocyclization of peptides are of high interest since they facilitate the synthesis of various types of potentially bioactive compounds, e.g. addressing targets like protein-protein-interactions. Herein, we report on an efficient method to construct tryptathionine-cross-links in peptides between the amino acids Trp and Cys. This reaction not only is the basis for the total synthesis of the death cap toxin α-amanitin but also provides rapid access to various new amanitin analogues. This study for the first time presents a systematic compilation of structure-activity relations (SAR) of amatoxins with regard to RNA polymerase II inhibition and cytotoxicity with one amanitin derivative of superior RNAP II inhibition. The present approach paves the way for the synthesis of structurally diverse amatoxins as future payloads for antibody-toxin conjugates in cancer therapy.

Design, Synthesis, and Biochemical Evaluation of Alpha-Amanitin Derivatives Containing Analogs of the trans-Hydroxyproline Residue for Potential Use in Antibody-Drug Conjugates.

Alpha-amanitin, an extremely toxic bicyclic octapeptide extracted from the death-cap mushroom, Amanita phalloides, is a highly selective allosteric inhibitor of RNA polymerase II. Following on growing interest in using this toxin as a payload in antibody-drug conjugates, herein we report the synthesis and biochemical evaluation of several new derivatives of this toxin to probe the role of the trans-hydroxyproline (Hyp), which is known to be critical for toxicity. This structure activity relationship (SAR) study represents the first of its kind to use various Hyp-analogs to alter the conformational and H-bonding properties of Hyp in amanitin.

Clustering fibromyalgia patients: A combination of psychosocial and somatic factors leads to resilient coping in a subgroup of fibromyalgia patients.

BACKGROUND: Coping strategies and their efficacy vary greatly in patients suffering from fibromyalgia syndrome (FMS). OBJECTIVE: We aimed to identify somatic and psychosocial factors that might contribute to different coping strategies and resilience levels in FMS. SUBJECTS AND METHODS: Standardized questionnaires were used to assess coping, pain, and psychological variables in a cohort of 156 FMS patients. Quantitative real-time polymerase chain reaction (qRT-PCR) determined gene expression of selected cytokines in white blood cells of 136 FMS patients and 25 healthy controls. Data of skin innervation, functional and structural sensory profiles of peripheral nociceptive nerve fibers of a previous study were included into the statistics. An exploratory factor analysis was used to define variance explaining factors, which were then included into cluster analysis. RESULTS: 54.9% of the variance was explained by four factors which we termed (1) affective load, (2) coping, (3) pain, and (4) pro-inflammatory cytokines (p < 0.05). Considering differences in the emerged factors, coping strategies, cytokine profiles, and disability levels, 118 FMS patients could be categorized into four clusters which we named "maladaptive", "adaptive", "vulnerable", and "resilient" (p < 0.05). The adaptive cluster had low scores in disability and in all symptom categories in contrast to the vulnerable cluster, which was characterized by high scores in catastrophizing and disability (p < 0.05). The resilient vs. the maladaptive cluster was characterized by better coping and a less pro-inflammatory cytokine pattern (p < 0.05). CONCLUSION: Our data suggest that problem- and emotion-focused coping strategies and an anti-inflammatory cytokine pattern are associated with reduced disability and might promote resilience. Additional personal factors such as low anxiety scores, ability of acceptance, and persistence further favor a resilient phenotype. Individualized therapy should take these factors into account.

MiR103a-3p and miR107 are related to adaptive coping in a cluster of fibromyalgia patients.

BACKGROUND: MicroRNA (miRNA) mainly inhibit post-transcriptional gene expression of specific targets and may modulate disease severity. OBJECTIVE: We aimed to identify miRNA signatures distinguishing patient clusters with fibromyalgia syndrome (FMS). SUBJECTS AND METHODS: We previously determined four FMS patient clusters labelled "maladaptive", "adaptive", "vulnerable", and "resilient". Here, we cluster-wise assessed relative gene expression of miR103a-3p, miR107, miR130a-3p, and miR125a-5p in white blood cell (WBC) RNA of 31 FMS patients and 16 healthy controls. Sum scores of pain-, stress-, and resilience-related questionnaires were correlated with miRNA relative gene expression. A cluster-specific speculative model of a miRNA-mediated regulatory cycle was proposed, and its potential targets verified by the online tool "target scan human". RESULTS: One-way ANOVA revealed lower gene expression of miR103a-3p, miR107, and miR130a-3p in FMS patients compared to controls (p < 0.05). Follow-up post-hoc tests indicated the highest peak of gene expression of miR103a-3p for the adaptive cluster (p < 0.05), i.e. in patients with low disability in all symptom categories. Gene expression of miR103a-3p correlated with FMS related disability and miR107 with the score "physical abuse" of the trauma questionnaire (p < 0.05). Target scan identified sucrose non-fermentable serine/threonine protein kinase, nuclear factor kappa-b, cyclin dependent kinase, and toll-like receptor 4 as genetic targets of the miR103a/107 miRNA family. CONCLUSION: We show an association between upregulated gene expression of miR103a, tendentially of miR107, and adaptive coping in FMS patients. Validation of this pair of miRNA may enable to identify a somatic resilience factor in FMS.

Harnessing single-cell genomics to improve the physiological fidelity of organoid-derived cell types.

BACKGROUND: Single-cell genomic methods now provide unprecedented resolution for characterizing the component cell types and states of tissues such as the epithelial subsets of the gastrointestinal tract. Nevertheless, functional studies of these subsets at scale require faithful in vitro models of identified in vivo biology. While intestinal organoids have been invaluable in providing mechanistic insights in vitro, the extent to which organoid-derived cell types recapitulate their in vivo counterparts remains formally untested, with no systematic approach for improving model fidelity. RESULTS: Here, we present a generally applicable framework that utilizes massively parallel single-cell RNA-seq to compare cell types and states found in vivo to those of in vitro models such as organoids. Furthermore, we leverage identified discrepancies to improve model fidelity. Using the Paneth cell (PC), which supports the stem cell niche and produces the largest diversity of antimicrobials in the small intestine, as an exemplar, we uncover fundamental gene expression differences in lineage-defining genes between in vivo PCs and those of the current in vitro organoid model. With this information, we nominate a molecular intervention to rationally improve the physiological fidelity of our in vitro PCs. We then perform transcriptomic, cytometric, morphologic and proteomic characterization, and demonstrate functional (antimicrobial activity, niche support) improvements in PC physiology. CONCLUSIONS: Our systematic approach provides a simple workflow for identifying the limitations of in vitro models and enhancing their physiological fidelity. Using adult stem cell-derived PCs within intestinal organoids as a model system, we successfully benchmark organoid representation, relative to that in vivo, of a specialized cell type and use this comparison to generate a functionally improved in vitro PC population. We predict that the generation of rationally improved cellular models will facilitate mechanistic exploration of specific disease-associated genes in their respective cell types.

Bioresponsive release of insulin-like growth factor-I from its PEGylated conjugate.

PEGylation of protein ligands, the attachment of polyethylene glycol (PEG) polymers to a therapeutic protein, increases therapeutics' half-life but frequently comes at the cost of reduced bioactivity. We are now presenting a bioinspired strategy leading out of this dilemma. To this end, we selected a position within insulin-like growth factor I (IGF-I) for decoration with a PEG30kDa-modified protease-sensitive peptide linker (PSL) using a combination of enzymatic and chemical bioorthogonal coupling strategies. The PSL sequence responded to matrix metalloproteinases (MMP) to provide a targeted release in diseased tissue. The IGF-PSL-PEG conjugate had different binding protein affinity, cell proliferation, and endocytosis patterns as compared to the wild type. Exposure of the conjugate to elevated levels of activated MMPs, as present in inflamed tissues, fully reestablished the wild type properties through effective PSL cleavage. In conclusion, this bioinspired approach provided a blueprint for PEGylated therapeutics combining the pharmacokinetic advantages of PEGylation, while locally restoring the full suite of biological potential of therapeutics.

Bioorthogonal strategies for site-directed decoration of biomaterials with therapeutic proteins.

Emerging strategies targeting site-specific protein modifications allow for unprecedented selectivity, fast kinetics and mild reaction conditions with high yield. These advances open exciting novel possibilities for the effective bioorthogonal decoration of biomaterials with therapeutic proteins. Site-specificity is particularly important to the therapeutics' end and translated by targeting specific functional groups or introducing new functional groups into the therapeutic at predefined positions. Biomimetic strategies are designed for modification of therapeutics emulating enzymatic strategies found in Nature. These strategies are suitable for a diverse range of applications - not only for protein-polymer conjugation, particle decoration and surface immobilization, but also for the decoration of complex biomaterials and the synthesis of bioresponsive drug delivery systems. This article reviews latest chemical and enzymatic strategies for the biorthogonal decoration of biomaterials with therapeutic proteins and inter-positioned linker structures. Finally, the numerous reports at the interface of biomaterials, linkers, and therapeutic protein decoration are integrated into practical advice for design considerations intended to support the selection of productive ligation strategies.

Bioorthogonal Modification of Cell Derived Matrices by Metabolic Glycoengineering.

Cell-derived matrices (CDMs) emerged as an attractive biomaterial in regenerative medicine. Here we present a strategy for site-specific decoration of CDMs with bioactive molecules deploying metabolic glycoengineering. NIH 3T3 fibroblasts were cultured in the presence of a tetra acetylated azide bearing monosaccharide to metabolically incorporate the synthetic sugar into the glycan structure of extracellular matrix proteins. Glycoengineered CDMs were isolated and analyzed for fibronectin as one of the most abundant ECM species by western blotting and fluorescence labeling of the azide-monosaccharides deploying bioorthogonal chemistries. Glycoengineered CDMs were biocompatible for incorporated and reseeded NIH 3T3 fibroblasts, respectively. Successful modification of glycoengineered CDMs was demonstrated by a therapeutic peptide inhibitor against myostatin. This study details a site-specific, effective, and biocompatible strategy for the decoration of glycan structures within CDMs for future tissue engineering application.

Site-Specific Conjugated Insulin-like Growth Factor-I for Anabolic Therapy.

Insulin-like growth factor-I (IGF-I) is an inducer of skeletal muscle hypertrophy and blocks skeletal muscle atrophy, rendering it a good therapeutic option for the treatment of severe burn injury for which localized treatment options are particularly interesting. For that, the therapeutic was redesigned via amber codon expression, leading to a propargyl-l-lysine (plk) modified IGF-I (plk-IGF-I Ea) with Ea peptide prolongation at the C-terminus, thereby becoming a substrate for copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) and other bio-orthogonal click chemistries. The plk-IGF-I Ea was site-specifically immobilized to agarose particles, resulting in homogeneous product outcome with retained potency while providing the necessary tools to maximize local and minimize systemic exposure. IGF-I decorated particles outperformed soluble IGF-I in C2C12 induced myotube formation, reflecting the impact of controlled multivalence on decorated materials.

Associação entre função endotelial e balanço autonômico em indivíduos saudáveis (y modulación autonómica en sujetos sanos) / Association between endothelial function and autonomic balance in healthy subjects

Objetivo: Avaliar a associação da função endotelial com o balanço autonômico, variáveis antropométricas e bioquímicas em indivíduos saudáveis (Evaluar la asociación de la función endotelial con el equilibrio autónomo, variables antropométricas y bioquímicas en sujetos sanos). Metodologia: Estudo transversal com 35 voluntários normotensos, ativos e muito ativos, de ambos os sexos (27.5 ± 5.8 anos de idade). O balanço autonômico foi estimado pela medida da variabilidade da frequência cardíaca, com o Polar RS800CX, por 20 minutos (10 minutos em ventilação espontânea/10 minutos com ventilação controlada). O sinal foi analisado pelo (La señal se analizó con el) software Kubios Heart Rate Variability. A frequência respiratória foi fixada em um (La frecuencia respiratoria se estableció con un) metrônomo ajustado a 12 irpm. A função endotelial foi avaliada por ultrassonografia da artéria braquial e estimada pela dilatação mediada pelo fluxo. Resultados: Houve correlação negativa entre a dilatação mediada pelo fluxo e a relação (Hubo una correlación negativa entre la dilatación mediada por el flujo y la relación) LF/HF (r = -0.43; p = 0.011), componente de baixa frequência (componente de baja frecuencia) (LF; r = -0.41; p = 0.016), índice de massa corporal (r = -0.43; p = 0.01) e peso (r = -0.39; p = 0.02). A dilatação mediada pelo fluxo esteve associada com o componente de alta frequência (HF; r = 0.41; p = 0.015). Não houve associação da dilatação mediada pelo fluxo ou de componentes do balanço autonômico (hubo una asociación de la dilatación mediada por flujo o de los componentes del equilibrio autonómico) com colesterol total, glicemia e triglicerídeos. Conclusão: A associação da função endotelial com o balanço autonômico em sujeitos saudáveis, ativos e muito ativos, demonstra a ação simultânea entre respostas biológicas das células endoteliais e estímulos neurohumorais do sistema nervoso autônomo, ambos visando o adequado controle cardiovascular (ambos con vistas al adecuado control cardiovascular).

Objective: To evaluate the association between endothelial function and autonomic balance, anthropometric and biochemical variables in healthy subjects. Methods: Cross-sectional study with 35 normotensive, active and very active volunteers of both sexes (27.5 ± 5.8 years). Autonomic balance was estimated by measuring heart rate variability, with the Polar RS800CX, for 20 minutes (10 minutes in spontaneous ventilation/10 minutes with controlled ventilation). The signal was analyzed by Kubios Heart Rate Variability software. Respiratory rate was fixed on a metronome set at 12 breaths/min. Endothelial function was assessed by ultrasound of the brachial artery and estimated by flow-mediated dilation. Results: There was a negative correlation between flow-mediated dilation and LF/HF ratio (r = -0.43; p = 0.011), low frequency component (LF; r = -0.41; p = 0.016), body mass index (r = -0.43; p = 0.01) and weight (r = -0.39; p = 0.02). Flow-mediated dilation was associated with the high-frequency component (HF, r = 0.41; p = 0.015). There was no association between flow-mediated dilation or components of the autonomic balance and total cholesterol, blood glucose and triglycerides. Conclusion: The association between endothelial function and autonomic balance in healthy, active and very active subjects demonstrates the simultaneous action between biological responses of endothelial cells and neuro-humoral stimuli of the autonomic nervous system, both with a view to proper cardiovascular control.

Matrix Metalloproteinase Responsive Delivery of Myostatin Inhibitors.

PURPOSE: The inhibition of myostatin - a member of the transforming growth factor (TGF-ß) family - drives regeneration of functional skeletal muscle tissue. We developed a bioresponsive drug delivery system (DDS) linking release of a myostatin inhibitor (MI) to inflammatory flares of myositis to provide self-regulated MI concentration gradients within tissues of need. METHODS: A protease cleavable linker (PCL) - responding to MMP upregulation - is attached to the MI and site-specifically immobilized on microparticle surfaces. RESULTS: The PCL disintegrated in a matrix metalloproteinase (MMP) 1, 8, and particularly MMP-9 concentration dependent manner, with MMP-9 being an effective surrogate biomarker correlating with the activity of myositis. The bioactivity of particle-surface bound as well as released MI was confirmed by luciferase suppression in stably transfected HEK293 cells responding to myostatin induced SMAD phosphorylation. CONCLUSIONS: We developed a MMP-responsive DDS for MI delivery responding to inflammatory flare of a diseased muscle matching the kinetics of MMP-9 upregulation, with MMP-9 kinetics matching (patho-) physiological myostatin levels. ᅟ: Graphical Abstract Schematic illustration of the matrix metalloproteinase responsive delivery system responding to inflammatory flares of muscle disease. The protease cleavable linker readily disintegrates upon entry into the diseased tissue, therby releasing the mystatin inhibitor.

Biocompatible Azide-Alkyne "Click" Reactions for Surface Decoration of Glyco-Engineered Cells.

Bio-orthogonal copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) has been widely used to modify azide- or alkyne-bearing monosaccharides on metabolic glyco-engineered mammalian cells. Here, we present a systematic study to elucidate the design space for the cytotoxic effects of the copper catalyst on NIH 3T3 fibroblasts and on HEK 293-F cells. Monitoring membrane integrity by flow cytometry and RT-PCR analysis with apoptotic and anti-apoptotic markers elucidated the general feasibility of CuAAC, with exposure time of the CuAAC reaction mixture having the major influence on biocompatibility. A high labeling efficiency of HEK 293-F cells with a fluorescent alkyne dye was rapidly achieved by CuAAC in comparison to copper free strain-promoted azide-alkyne cycloaddition (SPAAC). The study details effective and biocompatible conditions for CuAAC-based modification of glyco-engineered cells in comparison to its copper free alternative.

Fatty acid composition analysis in polysorbate 80 with high performance liquid chromatography coupled to charged aerosol detection.

The fatty acid (FA) composition of polysorbate 80 (PS80), a sorbitan oleic acid ester copolymerized with about 20mole of ethylene oxide, is typically characterized by gas chromatography. Here, an alternative method was developed. After saponification with potassium hydroxide the FA fraction was collected with liquid-liquid extraction using methyl-tert-butyl ether. HPLC in combination with a Corona® charged aerosol detector (CAD) was applied for the separation and detection. The method was fully validated in terms of specificity, repeatability, limits of quantification, linearity, range, accuracy and robustness. The characterization of 16 different PS80 batches demonstrated variability regarding their FA composition, with e.g. the amount of oleic acid ranging from 67.8±0.7% to 96.6±1.4%. Furthermore, we identified petroselinic acid, a double-bond positional isomer to oleic acid in all batches, an FA not known to pharmacopoeias at present. In addition, 11-hydroxy-9-octadecenoic acid, an oxidation product of oleic acid was identified. Structure elucidation was performed by means of HPLC-MS/MS. In addition, the method was expanded to the evaluation of the free FAs. Having determined the entire FA composition, the acid value according to EP and USP can be calculated.

Predicting critical micelle concentration and micelle molecular weight of polysorbate 80 using compendial methods.

This manuscript addresses the capability of compendial methods in controlling polysorbate 80 (PS80) functionality. Based on the analysis of sixteen batches, functionality related characteristics (FRC) including critical micelle concentration (CMC), cloud point, hydrophilic-lipophilic balance (HLB) value and micelle molecular weight were correlated to chemical composition including fatty acids before and after hydrolysis, content of non-esterified polyethylene glycols and sorbitan polyethoxylates, sorbitan- and isosorbide polyethoxylate fatty acid mono- and diesters, polyoxyethylene diesters, and peroxide values. Batches from some suppliers had a high variability in functionality related characteristic (FRC), questioning the ability of the current monograph in controlling these. Interestingly, the combined use of the input parameters oleic acid content and peroxide value - both of which being monographed methods - resulted in a model adequately predicting CMC. Confining the batches to those complying with specifications for peroxide value proved oleic acid content alone as being predictive for CMC. Similarly, a four parameter model based on chemical analyses alone was instrumental in predicting the molecular weight of PS80 micelles. Improved models based on analytical outcome from fingerprint analyses are also presented. A road map controlling PS80 batches with respect to FRC and based on chemical analyses alone is provided for the formulator.