Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of beta-arrestin 2 coupling

Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc’s epitope which may partially overlap with the known PTH (1–34) binding site. However, PTH (1–34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits β-arrestin-2 recruitment after PTH (1–34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias

Laser‐facilitated epicutaneous immunotherapy with hypoallergenic beta‐glucan neoglycoconjugates suppresses lung inflammation and avoids local side effects in a mouse model of allergic asthma

Background Allergen-specific immunotherapy via the skin targets a tissue rich in antigen-presenting cells, but can be associated with local and systemic side effects. Allergen-polysaccharide neoglycogonjugates increase immunization efficacy by targeting and activating dendritic cells via C-type lectin receptors and reduce side effects. Objective We investigated the immunogenicity, allergenicity, and therapeutic efficacy of laminarin-ovalbumin neoglycoconjugates (LamOVA). Methods The biological activity of LamOVA was characterized in vitro using bone marrow-derived dendritic cells. Immunogenicity and therapeutic efficacy were analyzed in BALB/c mice. Epicutaneous immunotherapy (EPIT) was performed using fractional infrared laser ablation to generate micropores in the skin, and the effects of LamOVA on blocking IgG, IgE, cellular composition of BAL, lung, and spleen, lung function, and T-cell polarization were assessed. Results Conjugation of laminarin to ovalbumin reduced its IgE binding capacity fivefold and increased its immunogenicity threefold in terms of IgG generation. EPIT with LamOVA induced significantly higher IgG levels than OVA, matching the levels induced by s.c. injection of OVA/alum (SCIT). EPIT was equally effective as SCIT in terms of blocking IgG induction and suppression of lung inflammation and airway hyperresponsiveness, but SCIT was associated with higher levels of therapy-induced IgE and TH2 cytokines. EPIT with LamOVA induced significantly lower local skin reactions during therapy compared to unconjugated OVA. Conclusion Conjugation of ovalbumin to laminarin increased its immunogenicity while at the same time reducing local side effects. LamOVA EPIT via laser-generated micropores is safe and equally effective compared to SCIT with alum, without the need for adjuvant

EBAG9-silencing exerts an immune checkpoint function without aggravating adverse effects

Chimeric antigen receptor (CAR) T cells have revolutionized treatment of B-cell malignancies. However, enhancing the efficacy of engineered T cells without compromising their safety is warranted. The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) inhibits release of cytolytic enzymes from cytotoxic T lymphocytes. Here, we examined the potency of EBAG9-silencing for the improvement of adoptive T cell therapy. Micro-RNA-mediated EBAG9 downregulation in transplanted CTLs from immunized mice improved their cytolytic competence in a tumor model. In tolerant female recipient mice that received organ transplants, a minor histocompatibility antigen was turned into a rejection antigen by Ebag9 deletion, indicating an immune checkpoint function for EBAG9. Considerably less EBAG9-silenced human CAR T cells were needed for tumor growth control in a xenotransplantation model. Transcriptome profiling did not reveal additional risks regarding genotoxicity or aberrant differentiation. A single-step retrovirus transduction process links CAR or TCR expression with miRNA-mediated EBAG9 downregulation. Despite higher cytolytic efficacy, release of cytokines associated with cytokine release syndrome remains unaffected. Collectively, EBAG9-silencing enhances effector capacity of TCR- and CAR-engineered T cells, results in improved tumor eradication, facilitates efficient manufacturing, and decreases the therapeutic dose

Comparison of FACS and PCR for detection of BCMA-CAR-T cells

Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02-0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred

Desaminierungen von #alpha#-Aminonitrilen

SIGLEAvailable from TIB Hannover: MA 7067 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

Biomarker responses in Lumbricus terrestris exposed to model drugs

2nd International Conference on Risk Assessment of Pharmaceuticals in the Environment, Barcelona, 28-29 November 2019.-- 1 pageThe earthworm Lumbricus terrestris, is a common anecic key species in natural soils used also as bioindicator in soil pollution assessment. Specimens of L. terrestris were exposed in Petri dishes to filter paper embedded with 1 mg/mL to different model compounds for 48 h. The chemicals were: Lamotrigine, Cocaine, Fipronil (as Regent¿800WG) and the organophosphorus pesticide bis-4-nitrophenyl phosphate (BNPP). At the end of this period, earthworms were immediately frozen in liquid N2 and their tissues, homogenized by cryogrinding, targeted to determine chemical exposure and metabolite formation as well as to biomarker determinations. The biomarkers analyzed were the activities of the enzymes Acetylcholinesterase (AChE), Glutathione-S-transferase (GST) and Carboxylesterase (CES) using different substrates. The results obtained revealed differences in enzymatic activity among substrates, being CE activities with the substrates 4-nitrophenyl butyrate (4-NPB) and 1-naphthyl butyrate (1-NB) the most responsive to cocaine and BNPP. These results for BNPP, corroborate data obtained after in vitro exposure. This study highlights the utility of long chain butyrate esters in CE measures in invertebrate and the agreement between in vivo and in vitro responses in L. terrestris. Chemical analysis and metabolite formation should confirm the extent of the exposure and other range of concentrations should be used to assess any dose-response effectsTo the WaterJPI-2015 AWARE project (PCIN-2017-067) and the Spanish national project (RTI2018-097158-B-C33