High-affinity Receptor Research Articles

Hyaluronic Acid-Stabilized Fe3O4 Nanoparticles for Promoting In Vivo Magnetic Resonance Imaging of Tumors.

The use of iron oxide (Fe3O4) nanoparticles as novel contrast agents for magnetic resonance imaging (MRI) has attracted great interest due to their high r 2 relaxivity. However, both poor colloidal stability and lack of effective targeting ability have impeded their further expansion in the clinics. Here, we reported the creation of hyaluronic acid (HA)-stabilized Fe3O4 nanoparticles prepared by a hydrothermal co-precipitation method and followed by electrostatic adsorption of HA onto the nanoparticle surface. The water-soluble HA functions not only as a stabilizer but also as a targeting ligand with high affinity for the CD44 receptor overexpressed in many tumors. The resulting HA-stabilized Fe3O4 nanoparticles have an estimated size of sub-20 nm as observed by transmission electron microscopy (TEM) imaging and exhibited long-term colloidal stability in aqueous solution. We found that the nanoparticles are hemocompatible and cytocompatible under certain concentrations. As verified by quantifying the cellular uptake, the Fe3O4@HA nanoparticles were able to target a model cell line (HeLa cells) overexpressing the CD44 receptor through an active pathway. In addition, we showed that the nanoparticles can be used as effective contrast agents for MRI both in vitro in HeLa cells and in vivo in a xenografted HeLa tumor model in rodents. We believe that our findings shed important light on the use of active targeting ligands to improve the contrast of lesion for tumor-specific MRI in the nano-based diagnosis systems.

The Role of Neurotrophin Signaling in Age-Related Cognitive Decline and Cognitive Diseases.

Neurotrophins are a family of secreted proteins expressed in the peripheral nervous system and the central nervous system that support neuronal survival, synaptic plasticity, and neurogenesis. Brain-derived neurotrophic factor (BDNF) and its high affinity receptor TrkB are highly expressed in the cortical and hippocampal areas and play an essential role in learning and memory. The decline of cognitive function with aging is a major risk factor for cognitive diseases such as Alzheimer’s disease. Therefore, an alteration of BDNF/TrkB signaling with aging and/or pathological conditions has been indicated as a potential mechanism of cognitive decline. In this review, we summarize the cellular function of neurotrophin signaling and review the current evidence indicating a pathological role of neurotrophin signaling, especially of BDNF/TrkB signaling, in the cognitive decline in aging and age-related cognitive diseases. We also review the therapeutic approach for cognitive decline by the upregulation of the endogenous BDNF/TrkB-system.

Allergen-Specific IgA Antibodies Block IgE-Mediated Activation of Mast Cells and Basophils.

Mast cells and basophils have long been implicated in the pathogenesis of IgE-mediated hypersensitivity reactions. They express the high-affinity IgE receptor, FcϵRI, on their surface. Antigen-induced crosslinking of IgE antibodies bound to that receptor triggers a signaling cascade that results in activation, leading to the release of an array of preformed vasoactive mediators and rapidly synthesized lipids, as well as the de novo production of inflammatory cytokines. In addition to bearing activating receptors like FcεRI, these effector cells of allergy express inhibitory ones including FcγR2b, an IgG Fc receptor with a cytosolic inhibitory motif that activates protein tyrosine phosphatases that suppress IgE-mediated activation. We and others have shown that food allergen-specific IgG antibodies strongly induced during the course of oral immunotherapy (OIT), signal via FcγR2b to suppress IgE-mediated mast cell and basophil activation triggered by food allergen challenge. However, the potential inhibitory effects of IgA antibodies, which are also produced in response to OIT and are present at high levels at mucosal sites, including the intestine where food allergens are encountered, have not been well studied. Here we uncover an inhibitory function for IgA. We observe that IgA binds mouse bone marrow-derived mast cells (BMMCs) and peritoneal mast cells. Binding to BMMCs is dependent on calcium and sialic acid. We also found that IgA antibodies inhibit IgE-mediated mast cell degranulation in an allergen-specific fashion. Antigen-specific IgA inhibits IgE-mediated mast cell activation early in the signaling cascade, suppressing the phosphorylation of Syk, the proximal protein kinase mediating FcεRI signaling, and suppresses mast cell production of cytokines. Furthermore, using basophils from a peanut allergic donor we found that IgA binds to basophils and that activation by exposure to peanuts is effectively suppressed by IgA. We conclude that IgA serves as a regulator of mast cell and basophil degranulation, suggesting a physiologic role for IgA in the maintenance of immune homeostasis at mucosal sites.

Cariprazine

Cariprazine is a partial agonist of dopamine and serotonin with high affinity for D3 receptor. There is excess endogenous dopamine both in schizophrenia and bipolar disorder. Cariprazine binds to D3/D2 receptor as antagonist and agonist in schizophrenia and bipolar depression, respectively. Thus, the dopamine pathway inhibited and stimulated in the respective cases. The United States Food and Drug Administration (USFDA) approval now covers the full-spectrum Bipolar I disorder, yet to hit the Indian market.

Non-steroidal mineralocorticoid receptor antagonists in cardiorenal disease.

Despite existing treatments, patients with heart failure and chronic kidney disease (CKD) remain at high risk for adverse outcomes and progression to end-stage disease. Steroidal mineralocorticoid receptor antagonists (MRAs) such as spironolactone and eplerenone reduce mortality but remain under-prescribed due to the perceived risk of hyperkalaemia and hormonal side effects. The discovery of non-steroidal MRAs represents a major new dimension in cardiorenal disease therapy. Non-steroidal MRAs have high affinity and specificity for the mineralocorticoid receptor (MR) and differ from both steroidal agents and each other with respect to important physiochemical, pharmacodynamic, and pharmacokinetic parameters. Similar to their steroidal counterparts, they have beneficial anti-inflammatory, anti-remodelling, and anti-fibrotic properties in the kidneys, heart, and vasculature. There are several non-steroidal MRAs under development and clinical assessment; of these, only esaxerenone and finerenone are approved for treatment globally. In Japan, esaxerenone is approved for essential hypertension and has been studied in diabetic nephropathy. Compared with steroidal MRAs, finerenone more potently inhibits MR co-regulator recruitment and fibrosis and distributes more evenly between the heart and kidneys. The landmark Phase III trials FIGARO-DKD and FIDELIO-DKD demonstrated that finerenone-reduced major kidney and cardiovascular events on top of maximally tolerated renin-angiotensin-aldosterone system inhibition in patients with CKD associated with Type 2 diabetes. Non-steroidal MRAs are currently under evaluation in heart failure and for synergistic treatment with sodium-glucose contransporter 2 inhibitors. These ground-breaking agents could become an important therapy across the spectrum of cardiorenal disease.

Abstract 4224: BPT-143: a fully synthetic alpha-dead IL-2 with a best-in-class preclinical pharmacodynamic and efficacy profile supporting first-in-human clinical development

Abstract High-dose recombinant IL-2 (aldesleukin) is approved for treatment of advanced melanoma and renal cell carcinoma, however, major limitations restrict its therapeutic use. Wild-type IL-2 acts via binding to the medium-affinity IL-2 receptor βγ (IL2Rβγ) expressed in CD8+ T effector cells and NK cells. At the same time, its efficacy is dampened due to strong activation of regulatory T cells (Treg) expressing the high-affinity IL-2 receptor αβγ (IL2Rαβγ). Furthermore, binding to CD25/IL2Rα on endothelial cells and type 2 innate lymphoid cells is thought to be involved in the induction of severe toxicity including vascular leak syndrome (VLS). In addition, aldesleukin exhibits a very short half-life that, combined with its safety risks, requires a burdensome inpatient treatment schedule. We set out to rationally design a variant of human IL-2 that addresses and overcomes the major limitations of aldesleukin. Using our chemical protein synthesis technology, we introduced select modified amino acids including site-specific chemical conjugation handles to optimize the properties of IL-2 for cancer therapy while maintaining high homology to the wild-type IL-2 sequence. Bright Peak’s enhanced cytokine shows increased binding to CD122/IL2Rβ and does not interact with CD25/IL2Rα to improve safety and prevent the preferential activation of Tregs compared to CD8+ T effector cells. Site-specific conjugation to a 30 kDa PEG for half-life extension resulted in the generation of BPT-143, which is equipotent to aldesleukin in activating CD8+ T cells in vitro. In mice, BPT-143 induces a strong expansion of CD8+ T cells with only transient and minor effects on Tregs in vivo and exhibits improved PK properties allowing for a convenient dosing schedule. In the syngeneic CT26 tumor model, BPT-143 showed strong anti-tumor efficacy as a single agent as well as enhanced efficacy in combination with an anti-PD-1 antibody. BPT-143 induced a 55% complete response rate and, upon tumor re-challenge, all cured animals rejected CT26 tumor cells indicating the development of immunologic memory. In multiple-dose PK/PD studies in non-human primates (NHP), BPT-143 was well tolerated and induced robust and repeated expansion of CD8+ T cells, CD4+ conventional T cells and NK cells while showing only negligible effects on Tregs and eosinophils. Both in vivo efficacy studies in murine tumor models as well as PD effects observed in NHP indicate that BPT-143 has a best-in-class profile among “not-alpha” IL-2 compounds currently in development. IND-enabling studies are ongoing and a first in human trial is planned to start in 2022. Citation Format: Jean-Philippe Carralot, Rubén Alvarez Sanchez, Matilde Arévalo Ruiz, Magali Muller, Vijaya R. Pattabiraman, Bertolt Kreft. BPT-143: a fully synthetic alpha-dead IL-2 with a best-in-class preclinical pharmacodynamic and efficacy profile supporting first-in-human clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4224.

Abstract 3185: Treatment with an engineered decoy receptor targeting the CLCF1-CNTFR signaling axis prevents oncogenesis in Kras mutant lung adenocarcinoma via both cell autonomous and non-cell autonomous mechanisms

Abstract Proinflammatory cytokines in the tumor microenvironment can promote tumor growth, yet their value as therapeutic targets remain underexploited. Previously, we validated the functional significance of the cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling axis in lung adenocarcinoma (LUAD) and generated a high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1, thereby inhibiting its oncogenic effects. eCNTFR-Fc inhibits tumor growth in multiple xenograft models and in an autochthonous, highly aggressive genetically engineered mouse model of LUAD, driven by activation of oncogenic Kras and loss of Trp53. Abrogation of CLCF1 through eCNTFR-Fc appears most effective in tumors driven by oncogenic KRAS. From our genetically engineered mouse and our syngeneic modelling of LUAD we have observed that eCNTFR seems to work via two mechanisms, firstly in a tumor intrinsic fashion where it may modulate altered guanosine triphosphate loading, as we see differential effectiveness of eCNTFR-Fc, depending on the presence of KRAS mutations that retain the intrinsic capacity to hydrolyze guanosine triphosphate. Alternatively, eCNTFR also effects the tumor microenvironment and alters the composition of the tumor immune infiltrate, where using single cell genomics we see vast changes to the myeloid compartment, and alteration of the CAF populations, which we have previously shown to be the primary location of CLCF1 expression in mouse LUAD. To further enhance the efficacy of eCNTFR-Fc we have identified potential targets for combination therapy from both the autonomous and non-cell autonomous mechanisms of eCNTFR-Fc. We conclude that eCNTFR-Fc acts through two distinct mechanisms to be a highly effective treatment for Kras mutant LUAD, and combining eCNTER with targets, identified by a rational combination approach, could further enhance its therapeutic potential. Citation Format: Kieren D. Marini, Emma C. Champion, Jennifer R. Cochrane, E. Alejandro Sweet-Cordero. Treatment with an engineered decoy receptor targeting the CLCF1-CNTFR signaling axis prevents oncogenesis in Kras mutant lung adenocarcinoma via both cell autonomous and non-cell autonomous mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3185.

Abstract LB032: CXCR7 drives prostate cancer growth through AURKA activation

Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer among males in the United States. The mainstay treatment for metastatic PCa patients is androgen-deprivation therapies, including high-affinity androgen receptor antagonists, such as enzalutamide. Unfortunately, most patients develop resistance and relapse with castration-resistant PCa (CRPC). We have previously reported up-regulation of CXCR7, an atypical chemokine receptor, in CRPC. CXCR7 interacts with beta-arrestin (ARRB2), a cytoplasmic scaffold protein, and translocates to the cytoplasm to induce signal transduction and PCa progression. However, much remains to be known regarding CXCR7-driven downstream signaling and its therapeutic targeting in advanced PCa. To unveil CXCR7-downstream signaling, we utilized RNA-seq to profile gene expression. Gene ontology analyses revealed remarkable enrichment of CXCR7-regulated genes in G2/M cell cycle progression. Comprehensive phospho-proteomics analysis of CXCR7-knockdown PCa cells showed markedly reduced phosphorylation of AURKA substrates. AURKA, a serine/threonine-protein kinase, is a key regulator of mitosis and G2/M progression and is frequently upregulated in CRPC and the late-stage neuroendocrine PCa (NEPC). Activated AURKA experiences autophosphorylation and phosphorylates downstream substrates, e.g., TACC3, that mediate proper cell division. Immunoblotting confirmed that CXCR7 depletion reduces phosphorylation of AURKA and TACC3. Co-immunoprecipitation revealed protein-protein interaction between CXCR7 and AURKA, which is mediated by ARRB2. Accordingly, ARRB2-knockdown similarly inhibits AURKA phosphorylation and arrests cell proliferation. Immunofluorescence coupled with confocal imaging demonstrated that CXCR7 is packed into clathrin-coated vesicles and locates in the plasma membrane, cytoplasm, and perinuclear Golgi complex, which surrounds the centrosomal AURKA and ARRB2. We found that CXCR7 translocates to the Golgi complex along microtubule-dependent trafficking. Finally, we found that pharmacological inhibitors of AURKA abolish CXCR7-driven PCa cell proliferation in vitro and tumor growth in vivo. Conclusions: Our results describe for the first time that the CXCR7-ARRB2 complex interacts with AURKA and promotes its activation. Our data suggest that AURKA-targeting is a promising therapeutic strategy for CRPC and NEPC with CXCR7 up-regulation. Citation Format: Galina Gritsina, Zhuoyuan Lin, Ka Wing Fong, Xiaodong Lu, Changsheng Zhao, Jindan Yu. CXCR7 drives prostate cancer growth through AURKA activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB032.

Abstract 366: Combinatorial effect of fisetin and paclitaxel encapsulated PBM nanoparticles in ovarian cancer therapy

Abstract High-grade Serous Ovarian Cancer (HGSOC) accounts for 68% of Ovarian Cancers (OvCas). It is a clinically aggressive neoplasm that even after treatment, a substantial proportion of advanced OvCas will develop resistance within 18 months. Thus, it is imperative to formulate anti-cancer agents that target cancer cells without affecting normal cells. The proposed study evaluated a novel method for targeted delivery of fisetin alone or combined with paclitaxel (PTX) at desired rates. We have used a natural polysaccharide (starch; FDA approved) as a core for planetary ball milled (PBM) nanoparticles which binds high-affinity folate receptors on OvCa cells via folate conjugated poly (ε-caprolactone)/poly (ethylene glycol) copolymer coating of the PBM nanoparticles. Treatment of OvCa cells (CAOV-3 and OVCAR-3) with PBM-NPs has shown its cytotoxic effect. The cell viability (MTT) assay was performed to determine the optimal IC50 values of fisetin, PTX alone, and in combination. These particles are rapidly internalized and induce significant cancer cell death at lower doses than unformulated fisetin or PTX. Importantly, negative controls showed that PBM nanoparticles without fisetin or paclitaxel have no toxic effects on OvCa cells. The immunofluorescence study confirmed the effectiveness of the combined drug; dead nuclei were found more within the cells treated by the combination than fisetin or PTX alone. The combined drug treatment was most effective in inhibiting proliferation and inducing apoptosis; accordingly, it showed noticeable increases in pro-apoptotic (BAX, BID), apoptotic (PARP, Caspase-3), and decreases in anti-apoptotic genes (BCL-XL and MCL-1) gene in a western blot and RT-PCR analysis. Taken together, we observed that the combination of PTX and fisetin resulted in significant cytotoxicity and apoptosis as compared to alone. Our results suggest that fisetin can be a potential agent in enhancing the efficacy of PTX in OvCa treatment. These finding highlights the promising role of natural bioactive compounds and provide the rationale for further transitional research. Citation Format: Melayshia McFadden, Santosh Kumar Singh, Rajesh Singh. Combinatorial effect of fisetin and paclitaxel encapsulated PBM nanoparticles in ovarian cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 366.

Abstract 5308: Therapeutical antibody against CD25 mediating depletion of regulatory T cells without interruption of IL-2 signaling enhances antitumor immunity

Abstract Regulatory T cell or suppressor T cell in tumor diminishes immune response to immunotherapy. CD25, the high-affinity receptor subunit for interleukin (IL)-2, is highly expressed on Treg and considered a selective target for Treg depletion. However, CD25 is also expressed on activated effector T cells (Teff), and antibodies against CD25 with blocking IL-2 signaling have limited antitumor activity. Here we developed a humanized antibody against CD25 (H3F1-4V2) without blocking IL-2 signaling. This antibody was derived from hybridoma of mice immunized with CD25-mFc. Two CD25 antibodies Daclizumab and RG6292 were applied as reference antibodies, and Daclizumab blocked IL-2 signaling, and RG6292 did not block IL-2 signaling. H3F1-4V2 had higher affinity to 293T cells overexpressing human CD25 than Daclizumab (EC50=0.4nM vs 0.45nM), with lower Kd (0.88nM vs 1.5nM). Because of high affinities, H3F1-4V2 and Daclizumab both efficiently depleted Treg from PBMC in vitro, while RG6292 depleted Treg with weaker ability because of low affinity to CD25. In the Teff, downstream signaling of IL-2 was analyzed, and Daclizumab significantly decreased pSTAT5, while RG6292 and H3F1-4V2 kept the pSTAT5 level intact. PBMC was cultured in the presence of CD25 antibodies, and Daclizumab inhibited T cell proliferation (ki67+%) and activation (Granzyme B+%), but not for RG6292 and H3F1-4V2. At last, these CD25 antibodies were tested in humanized mice whose CD25 was replaced with human CD25, and MC38 tumor cells were transplanted into these mice.H3F1-4V2 completely suppressed tumor growth, while RG6292 also suppressed tumor growth with weaker efficiency and Daclizumab had little effect on tumor growth. These results suggested H3F1-4V2 was the best candidate targeting CD25 for cancer therapy. Citation Format: Hao Peng, Shuangshuang Hu, Yuli Li, li Ding, Jinying Ning, Guojin Wu. Therapeutical antibody against CD25 mediating depletion of regulatory T cells without interruption of IL-2 signaling enhances antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5308.

Application of Three-Dimensional Culture Method in the Cardiac Conduction System Research.

Congenital heart defects (CHD) are the most common type of birth defects. Several human case studies and genetically altered animal models have identified abnormalities in the development of ventricular conduction system (VCS) in the heart. While cell-based therapies hold promise for treating CHDs, translational efforts are limited by the lack of suitable in vitro models for feasibility and safety studies. A better understanding of cell differentiation pathways can lead to development of cell-based therapies for individuals living with CHD/VCS disorders. Here, we describe a new and reproducible 3-D cell culture method for studying cardiac cell lineage differentiation in vitro. We used primary ventricular cells isolated from embryonic day 11.5 (E11.5) mouse embryos, which can differentiate into multiple cardiac cell types including VCS cells. We compared 3-D cultures with three types of basement membrane extracts (BME) for their abilities to support E11.5 ventricular cell differentiation. In addition, the effects of atrial natriuretic peptide (ANP) and an inhibitor for its high affinity receptor were tested on cell differentiation in 3-D cultures. Following the cell culture, protocols for immunofluorescence imaging, cell extraction and protein isolation from the 3-D culture matrix and in-cell western methods are described. Further, these approaches can be used to study the effects of various ligands and genetic interventions on VCS cell development. We propose that these methodologies may also be extended for differentiation studies using other sources of stem cells such as induced pluripotent stem cells.

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

In vivo HSC transduction in rhesus macaques with an HDAd5/3+ vector targeting desmoglein 2 and transiently overexpressing cxcr4

AbstractWe developed a new in vivo hematopoietic stem cell (HSC) gene therapy approach that involves only IV injections and does not require myeloablation/conditioning and HSC transplantation. In this approach, HSCs are mobilized from the bone marrow into the peripheral bloodstream and transduced with IV injected helper-dependent adenovirus (HDAd) vectors. A fraction of transduced HSCs returns to the bone marrow and persists there long term. Here, we report desmoglein 2 (DSG2) as a new receptor that can be used for in vivo HSC transduction. HDAd5/3+ vectors were developed that use DSG2 as a high-affinity attachment receptor, and in vivo HSC transduction and safety after IV injection of an HDAd5/3+ vector expressing green fluorescent protein (GFP) in granulocyte colony-stimulating factor/AMD3100 (plerixafor)-mobilized rhesus macaques were studied. Unlike previously used CD46-targeting HDAd5/35++ vectors, HDAd5/3+ virions were not sequestered by rhesus erythrocytes and therefore mediated ∼10-fold higher GFP marking rates in primitive HSCs (CD34+/CD45RA–/CD90+ cells) in the bone marrow at day 7 after vector injection. To further increase the return of in vivo transduced, mobilized HSCs to the bone marrow, we transiently expressed cxcr4 in mobilized HSCs from the HDAd5/3+ vector. In vivo transduction with an HDAd5/3+GFP/cxcr4 vector at a low dose of 0.4 × 1012 viral particles/kg resulted in up to 7% of GFP-positive CD34+/CD45RA–/CD90+ cells in the bone marrow. This transduction rate is a solid basis for in vivo base or prime editing in combination with natural or drug-induced expansion of edited HSCs. Furthermore, our study provides new insights into HSC biology and trafficking after mobilization in nonhuman primates.

Tyrosine 146 of the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) Is Essential for Its Hepatitis B Virus (HBV) Receptor Function and HBV Entry into Hepatocytes.

Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1) is a hepatic bile acid uptake carrier participating in the enterohepatic circulation of bile acids. Apart from its transporter function, NTCP acts as the high-affinity liver-specific receptor for the hepatitis B virus (HBV), which attaches via its preS1-peptide domain of the large surface protein to NTCP, subsequently leading to endocytosis of the virus/NTCP-receptor complex. Although the process of NTCP-dependent HBV infection of hepatocytes has received much attention over the last decade, the precise molecular sites of the virus/NTCP interaction have not been fully identified. Inspection of the primary protein sequence of human NTCP revealed 139YIYSRGIY146 as a highly conserved tyrosine-rich motif. To study the role of Y139, Y141 and Y146 amino acids in NTCP biology, the aforementioned residues were substituted with alanine, phenylalanine or glutamate (mimicking phosphorylation) using site-directed mutagenesis. Similar to wt NTCP, the Y139A, Y141A, Y146A, Y141F, Y146F, and Y146E mutants were expressed at the plasma membrane of HEK293 cells and exhibited intact bile acid transport function. Y146A, Y146E, and Y146F demonstrated transport kinetics comparable to wild-type NTCP with Km values of 57.3–112.4 µM and Vmax values of 6683–7579 pmol/mg protein/min. Only Y141E was transport deficient, most likely due to an intracellular accumulation of the mutant protein. Most importantly, Y146A and Y146E mutation completely abrogated binding of the viral preS1-peptide to NTCP, while the Y146F mutant of NTCP showed some residual binding competence for preS1. Consequently, the NTCP mutants Y146A and Y146E, when expressed in HepG2 hepatoma cells, showed complete loss of susceptibility for in vitro HBV infection. In conclusion, tyrosine 146, and to some extent tyrosine 141, both belonging to the tyrosine-rich motif 139YIYSRGIY146 of human NTCP, are newly identified amino acid residues that play an essential role in the interaction of HBV with its receptor NTCP and, thus, in the process of virus entry into hepatocytes.

Endothelial ACKR3 drives atherosclerosis by promoting immune cell adhesion to vascular endothelium

Atherosclerosis is the foundation of potentially fatal cardiovascular diseases and it is characterized by plaque formation in large arteries. Current treatments aimed at reducing atherosclerotic risk factors still allow room for a large residual risk; therefore, novel therapeutic candidates targeting inflammation are needed. The endothelium is the starting point of vascular inflammation underlying atherosclerosis and we could previously demonstrate that the chemokine axis CXCL12–CXCR4 plays an important role in disease development. However, the role of ACKR3, the alternative and higher affinity receptor for CXCL12 remained to be elucidated. We studied the role of arterial ACKR3 in atherosclerosis using western diet-fed Apoe−/− mice lacking Ackr3 in arterial endothelial as well as smooth muscle cells. We show for the first time that arterial endothelial deficiency of ACKR3 attenuates atherosclerosis as a result of diminished arterial adhesion as well as invasion of immune cells. ACKR3 silencing in inflamed human coronary artery endothelial cells decreased adhesion molecule expression, establishing an initial human validation of ACKR3’s role in endothelial adhesion. Concomitantly, ACKR3 silencing downregulated key mediators in the MAPK pathway, such as ERK1/2, as well as the phosphorylation of the NF-kB p65 subunit. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in ACKR3-deficient mice. Lack of smooth muscle cell-specific as well as hematopoietic ACKR3 did not impact atherosclerosis in mice. Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways.

Looking for novel interactors of IL-13 receptor α2 (IL13Rα2) and tyrosine-protein phosphatase non-receptor type 1 (PTPN1/ PTP1B) by using proximity-dependent biotinylation (BioID)

Introduction: IL13Rα2 is a high affinity receptor for IL-13 that is re-expressed in late neoplastic stages. The IL- 13/IL- 13Rα2 axis triggers invasion in colorectal cancer (CRC) and other cancers in a process mediated, among others, by the phosphatase PTPN1 (aka PTP1B), involved in the dephosphorylation of the autoinhibitory Src Tyr416, and further Src activation. PTPN1 takes part in IL13Rα2 internalization and associates with poor outcome in glioblastoma and CRC patients. Inhibition of IL13Rα2 pathway decreases invasion of multiple cancer cell lines (i.e., SKOV-3, PC-3 and KM12SM). To better define the oncogenic properties of IL13Rα2 and PTPN1, we aim to identify novel interactors by using proximity-dependent biotinylation coupled to mass spectrometry. Materials and methods: PTPN1 and IL13Rα2 were cloned into pcDNA.5-pDEST-BirA-Flag-Ct by using LR clonase enzyme, obtaining the desired plasmids containing fusion genes PTPN1-BirA and IL13Rα2-BirA. Flp-In™ T-REx™ cells were co-transfected with pOG44 and pcDNA.5 vectors and selected with hygromycin. Then doxycycline and biotin were added to induce the expression of the fusion gene and proximity labelling. Biotinylated proteins were purified by affinity using streptavidin-sepharose, trypsin-digested and analysed in a Q-Exactive mass spectrometer. Raw data were analysed with MaxQuant, followed by Perseus for statistical analysis and SAINT for probabilistic scoring of protein-protein interaction data, which were subjected to gene ontology (GO) analysis. Results: For IL13Rα2, 735 proteins were identified and 94 were scored as significant interactors by SAINT. GO enrichment analysis showed association with cadherin binding and response to reticulum stress, among others. 1009 proteins were identified and 217 were scored as significant PTPN1 interactors. GO enrichment analysis showed important association with mitotic cell cycle regulation, such as CDK1 (G2/M transition) or PCTN (centrosome component). Among the 19 IL13Rα2 and PTPN1 common interactors stand out those related to SNARE complex (YKT6, SNAP29 and VAMP3), which is involved in regulation of cancer invasion, drug resistance and kinase phosphorylation. Another interesting finding was the presence of VRK2, a kinase involved in MAPK pathway. Conclusions: This study reveals novel individual interactors of IL13Rα2 and PTPN1, but also identifies common interactors of both proteins such as members of the SNARE complex and VRK2, opening new strategies for the inhibition of IL13Rα2 and PTPN1 in cancer metastasis.

Human IgE monoclonal antibody recognition of mite allergen Der p 2 defines structural basis of an epitope for IgE cross-linking and anaphylaxis in vivo.

Immunoglobulin E (IgE) antibody is a critical effector molecule for adaptive allergen-induced immune responses, which affect up to 40% of the population worldwide. Allergens are usually innocuous molecules but induce IgE antibody production in allergic subjects. Allergen cross-linking of IgE bound to its high affinity receptor (FcεRI) on mast cells and basophils triggers release of histamine and other mediators that cause allergic symptoms. Little is known about the direct allergen–IgE antibody interaction due to the polyclonal nature of serum IgE and the low frequency of IgE-producing B cells in blood. Here, we report the X-ray crystal structure of a house dust mite allergen, Der p 2, in complex with Fab of a human IgE monoclonal antibody (mAb) isolated by hybridoma technology using human B cells from an allergic subject. This IgE mAb, 2F10, has the correct pairing of heavy and light chains as it occurs in vivo. Key amino acids forming the IgE epitope on Der p 2 were identified. Mutation of these residues ablated their functional ability to cross-link IgE in a mouse model of passive systemic anaphylaxis. These analyses revealed an important conformational epitope associated with the IgE antibody repertoire to a major mite allergen.

Treatment of Cognitive Impairment in Schizophrenia

Cognitive function in schizophrenia is one of the main elements significantly related to functional outcomes, accounting for approximately 25–50% of the variance in real-world functioning. Treatment approaches to cognitive dysfunctions include both pharmacological and psychosocial interventions. Second Generation Antipsychotics (SGAs) may partially improve cognitive dysfunction, due to their relatively high affinity for serotonin 5HT2A receptors. The effects of glutamatergic agents indicated benefits on cognition of a group of amino acids that act as glutamate agonists by binding to the glycine site on NMDA receptors. The administration of muscarinic antagonists potentiates cognitive impairments, while the α7 nicotinic acetylcholine receptors have been shown to play an important role in cognition with potential therapeutic applications in schizophrenia. Studies on drugs targeting neuroinflammation and oxidative stress emerged. Cognitive remediation has proved to be effective in improving cognitive dysfunctions and psychosocial functioning in people with schizophrenia, however there is still a limited understanding of how the putative active therapy ingredients contribute to changes in the brain and translate into improved real-world functioning. Cogrem exerts its maximal benefit when delivered in the context of other psychiatric rehabilitative interventions, but it is not yet clear what are the mechanisms of effectiveness of integrated treatment. It is important that future research shift from studies of group efficacy to individual efficacy of treatments, in the perspective of precision medicine. Issues related to individual predictors of effectiveness and of interactions between specific pharmacologic, specific cognitive remediation technique and individual patients’ characteristics should be furtherly addressed.DisclosureIn the last two years, Prof Vita received, directly or indirectly, support for clinical studies or trials, conferences, consultancies, Congress presentations, advisory boards from: Angelini, Boehringer Ingelheim, Innovapharma, Janssen-Cilag, Lundbeck,

Artificial sweeteners sucralose and acesulfame potassium: A bittersweet story for the cardiovascular system?

Artificial sweeteners (AS) are well known to activate two categories of receptors; sweet taste receptor with high affinity and bitter taste receptors with low affinity. Interestingly, recent data from our and other groups suggested that these two receptors families are expressed throughout the arterial tree. Therefore, it could be interesting to explore if AS may have an impact on vascular function, and to decipher the role of these taste receptors. The aims of this ongoing work is to investigate whether AS, like sucralose and acesulfame potassium (AceK) (1) exhibit vasomotor effect and (2) in that case determine the underlying mechanisms. Biomolecular and functional investigations have been performed on isolated rodent aortas, human dermal micro-arteries and omental arteries. Ex vivo vasomotor function was assessed using isometric tension measurements in organ bath systems. We first confirmed that both endothelial and smooth muscle cells from rodent and human arteries express the transcripts encoding for TAS1R2 and TAS1R3; the heterodimer receptor known to be responsible for the sweet taste. Our ex vivo data showed that only high concentrations (about 10 mM) of AceK and sucralose induce vasomotor responses. While AceK exhibited a vasoconstrictive effect, sucralose induced vasorelaxation. Both responses were found to be independent of the endothelium. Pharmacological inhibition (gurmarin and lactisol) and the use of a TAS1R3 KO mice model demonstrated that AS vasomotor effects do not rely on the sweet taste receptors. In contrast, inhibition of TAS2Rs abolished all responses suggesting that bitter taste receptors are more likely responsible for the AS-induced vascular effects. Additionally, our last observations show that the vasoconstrictive effect of AceK is mediated by RhoA/ROCK pathway. These evidences support the idea that AS can alter vasomotor signaling in the smooth muscle cells. These acute effects are probably mediated by the bitter taste receptor family. Their activation at the vascular level as well as the underlying pathways remains poorly understood, and the physiopathological consequences need to be further investigated.

A phase 1/2 study of REGN5093-M114, a METxMET antibody-drug conjugate, in patients with mesenchymal epithelial transition factor (MET)-overexpressing NSCLC.

TPS8593 Background: MET, also called hepatocyte growth factor receptor (HGFR), is a high-affinity transmembrane protein receptor for HGF. MET is overexpressed in various malignancies, including non-small cell lung cancer (NSCLC). MET overexpression can accompany MET exon 14 alteration or de novo/acquired MET amplification. REGN5093-M114 is an antibody drug conjugate composed of a novel linker-payload (M114, carrying the maytansine derivative M24, a potent inhibitor of microtubule assembly) covalently bound to lysine residues on a MET-targeting human IgG4p bispecific antibody, REGN5093. In preclinical models of MET overexpressing cancers, REGN5093-M114 demonstrated significant dose-dependent antitumor activity. Methods: This is an open label, phase 1/2, first-in-human, multicenter dose-escalation study with cohort expansion evaluating REGN5093-M114 in patients with MET-overexpressing NSCLC (NCT04982224). Patients must have advanced stage NSCLC for which there are no approved therapies available expected to confer clinical benefit, with tumor overexpressing MET (≥75% tumor cell staining at 2+) as centrally confirmed by immunohistochemistry. For the expansion phase, patients must have at least one lesion that is measurable by RECIST 1.1. REGN5093-M114 will be administered intravenously once every 3 weeks over 30 minutes until disease progression, intolerable adverse events, withdrawal of consent, or study withdrawal. The primary objectives in dose escalation are to evaluate safety, tolerability, PK, and maximum tolerated dose and/or recommended phase 2 dosing regimen of REGN5093-M114. PKs will include the assessment of REGN5093-M114, total antibody, and payload M24 concentrations. The primary objective in dose expansion is to assess preliminary anti-tumor activity of REGN5093-M114 in MET-overexpressing NSCLC as measured by the objective response rate. The secondary objectives of both phases of the study include an evaluation of treatment durability, and the immunogenicity of REGN5093-M114. This study is currently open to enrollment. Clinical trial information: NCT04982224.