Receptor-mediated Clearance Research Articles

Embelin Elevates Endoplasmic Reticulum Calcium Levels and Blocks the Sterol Regulatory Element-Binding Protein 2 Mediated Proprotein Convertase Subtilisin/Kexin Type 9 Expression and Improves the Low-Density Lipoprotein Receptor Mediated Lipid Clearance on Hepatocytes.

Cardiovascular diseases (CVDs) continue to be one of the leading causes of morbidity and mortality worldwide, with a significant increase in recent years. Atherosclerosis, the pathological basis and prime reason for CVDs is primarily driven by dysregulated lipid metabolism and inflammation. Recently, proprotein convertase subtilisin kexin9 (PCSK9) has been evolved to be highly implicated in the circulatory low-density lipoprotein cholesterol levels by its modulatory effects on the low-density lipoprotein receptor (LDLR) mediated clearance. Even though not economical, the therapies targeting PCSK9 demonstrated appreciable levels of efficiency in managing hyperlipidaemic conditions. Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) is a naturally occurring para-benzoquinone isolated from dried berries of Embelia ribes, which possess several effects in maintaining the cholesterol homeostasis. In this study, we have analysed the role of embelin in sterol regulatory element-binding protein 2 (SREBP2) mediated PCSK9 expression in cultured hepatocytes. The study showed that the embelin treatment attenuates the endoplasmic reticulum (ER) stress-induced reactive oxygen species levels and ER stress markers on cultured hepatocytes. The treatment of embelin modulates the mRNA and protein level expression of SREBP2 and its downstream targets like PCSK9, LDLR, and HMG-CoA reductase (HMGCR). Interestingly the Ca2+ levels and the calcium binding protein of ER were significantly increased with embelin treatment. The work revealed a putative mechanism of embelin in lowering PCSK9 levels by boosting ER Ca2+ levels, thereby blocking SREBP2 nuclear translocation. Further, this reduces LDLR degradation and increases receptor-mediated circulatory lipid clearance. The study summarized the potential clinical applications of embelin in addressing the cardio vascular diseases.

Kinetics of endothelin-1 and effect selective ETA antagonism on ETB activation: a mathematical modeling analysis.

Endothelin-1 (ET-1) regulates renal and vascular function, but the clinical utility of selective ETA receptor antagonists has been limited due to associated fluid retention. The mechanisms underlying fluid retention remain poorly understood but could be a consequence of changes in ET-1 binding to the unantagonized ETB receptor, either through increased ET-1 or non-selective ETB. A mathematical model of ET-1 kinetics was developed to quantify effects of ETA antagonist exposure and selectivity on concentrations of ET-1 and its complexes with ETA and ETB receptors. The model describes ET-1 production, tissue and plasma distribution, ETA and ETB receptor binding, and receptor-mediated clearance, and was calibrated and validated with human ET-1 infusion studies. The model confirmed the significant role of ETB in ET-1 clearance. By varying both drug ETA selectivity (Kib/Kia) and concentration over a wide range, simulations predicted that while selective ETA antagonist (selectivity >1) always decreased [ET1-ETA], the change in [ET1-ETB] was more complex. It increased up to 45% as drug concentrations approached and exceeded Kia, but the increase was diminished as drug concentration increased further and fell below baseline at high concentrations. The drug concentration required to cause a decrease in [ET1-ETB] was lower as ETA selectivity decreased. This is the first mechanistic mathematical model of ET-1 kinetics that describes receptor-mediated clearance, and the consequence of ETB blockade on ET-1 concentrations. It provides a useful tool that can coupled with experimental studies to quantitively understand and investigate this complex and dynamic system.

Uncovering the interleukin-12 pharmacokinetic desensitization mechanism and its consequences with mathematical modeling.

The cytokine interleukin-12 (IL-12) is a potential immunotherapy because of its ability to induce a Th1 immune response. However, success in the clinic has been limited due to a phenomenon called IL-12 desensitization - the trend where repeated exposure to IL-12 leads to reduced IL-12 concentrations (pharmacokinetics) and biological effects (pharmacodynamics). Here, we investigated IL-12 pharmacokinetic desensitization via a modeling approach to (i) validate proposed mechanisms in literature and (ii) develop a mathematical model capable of predicting IL-12 pharmacokinetic desensitization. Two potential causes of IL-12 pharmacokinetic desensitization were identified: increased clearance or reduced bioavailability of IL-12 following repeated doses. Increased IL-12 clearance was previously proposed to occur due to the upregulation of IL-12 receptor on T cells that causes increased receptor-mediated clearance in the serum. However, our model with this mechanism, the accelerated-clearance model, failed to capture trends in clinical trial data. Alternatively, our novel reduced-bioavailability model assumed that upregulation of IL-12 receptor on T cells in the lymphatic system leads to IL-12 sequestration, inhibiting the transport to the blood. This model accurately fits IL-12 pharmacokinetic data from three clinical trials, supporting its biological relevance. Using this model, we analyzed the model parameter space to illustrate that IL-12 desensitization occurs over a robust range of parameter values and to identify the conditions required for desensitization. We next simulated local, continuous IL-12 delivery and identified several methods to mitigate systemic IL-12 exposure. Ultimately, our results provide quantitative validation of our proposed mechanism and allow for accurate prediction of IL-12 pharmacokinetics over repeated doses.

Abstract 719: A tumor-activated PD1/IL2 bispecific molecule, designed to overcome IL-2 receptor-mediated clearance, improve tolerability and stimulate antigen-experienced CD8+ T cells in the tumor microenvironment of murine models

Abstract PD-1/PD-L1 therapies have shown significant activity across a range of tumor types, however, 70-90% of patients do not derive durable benefit. In preclinical settings, delivery of PD-1 blockade and IL-2 agonism in cis via a bispecific molecule, comprising a PD-1 antibody fused to IL-2, has demonstrated superior activity compared to delivery of individual components or their combination. Targeting of IL-2 to PD-1+ cells in cis has been shown to drive a unique differentiation path endowing CD8+ T cells with enhanced functionality. However, we show that the IL-2 component in an unmasked PD1/IL2 bispecific molecule dominates its pharmacology, driving marked peripheral activation of immune cells, rapid IL-2 receptor-mediated clearance and systemic toxicity. To address these challenges and enable tumor-specific activity, we designed a PD1/IL2 bispecific molecule that incorporates a single domain antibody-based mask to bind to the IL-2 component and maintain it in a quiescent state until activated by prevalent and dysregulated proteases in the tumor microenvironment (TME). In preclinical models, the affinity-optimized mask prevented peripheral IL-2 receptor binding, associated toxicity and receptor-mediated clearance. Improved pharmacokinetics of our tumor-activated PD1/IL2 enabled PD1 antibody-like exposures and is projected to achieve complete blockade of PD1 signaling. Tumor-activated PD1/IL2 significantly inhibited tumor growth compared to anti-PD-1 alone or vehicle and demonstrated improved tolerability compared to an unmasked PD1/IL2 bispecific in mouse models. In addition, pharmacodynamic activity of tumor-activated PD1/IL2 was restricted to the TME. Consistent with the masked design, we observed no significant change in CD8+ T cells in the periphery and significant increases in antigen specific CD8+ T cells, memory CD8+ T cells and TCF1+ stem-like CD8+ T cells in the TME. Ex vivo protease cleavage assays performed in human samples demonstrated efficient activation of tumor-activated PD1/IL2 by multiple tumor types and minimal activation in human plasma. In non-human primates, tumor-activated PD1/IL2 achieved tolerable exposures that were comparable to those of existing PD-1 blocking immunotherapies. Taken together, these preclinical data suggest that tumor-activated PD1/IL2 has the potential to improve upon existing PD-1/PD-L1 immunotherapies by inhibiting PD-1/PD-L1 axis and simultaneously directing immunostimulatory IL-2 to antigen-experienced effector cell populations in the TME. Citation Format: Ertan Eryilmaz, Wilson Guzman, Stephanie Hsiao, Parker Johnson, Lisa Quinn, Jimit Lakhani, Brendan Whalen, Rosa Quiroz, Kurt Jenkins, Bernard Lanter, Oleg Yerov, Will Scott, Justin Greene, Zhen Liu, Jason Hedges, Megan McLaughlin, Sallyann Vu, Chelsea Turcotte, Jacob Taylor, Caitlin O'Toole, Magali Pederzoli-Ribeil, Haley Duprey, Natalia Malkova, Damiano Fantini, David Crowe, Kyle Smith, Joseph Card, Janice Lee, Kerri Smith, Benjamin Nicholson, Jennifer O'Neil, Carl U. Bialucha. A tumor-activated PD1/IL2 bispecific molecule, designed to overcome IL-2 receptor-mediated clearance, improve tolerability and stimulate antigen-experienced CD8+ T cells in the tumor microenvironment of murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 719.

Myristica fragrans Extract Inhibits Platelet Desialylation and Activation to Ameliorate Sepsis-Associated Thrombocytopenia in a Murine CLP-Induced Sepsis Model.

Sepsis, characterized by an uncontrolled host inflammatory response to infections, remains a leading cause of death in critically ill patients worldwide. Sepsis-associated thrombocytopenia (SAT), a common disease in patients with sepsis, is an indicator of disease severity. Therefore, alleviating SAT is an important aspect of sepsis treatment; however, platelet transfusion is the only available treatment strategy for SAT. The pathogenesis of SAT involves increased platelet desialylation and activation. In this study, we investigated the effects of Myristica fragrans ethanol extract (MF) on sepsis and SAT. Desialylation and activation of platelets treated with sialidase and adenosine diphosphate (platelet agonist) were assessed using flow cytometry. The extract inhibited platelet desialylation and activation via inhibiting bacterial sialidase activity in washed platelets. Moreover, MF improved survival and reduced organ damage and inflammation in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. It also prevented platelet desialylation and activation via inhibiting circulating sialidase activity, while maintaining platelet count. Inhibition of platelet desialylation reduces hepatic Ashwell-Morell receptor-mediated platelet clearance, thereby reducing hepatic JAK2/STAT3 phosphorylation and thrombopoietin mRNA expression. This study lays a foundation for the development of plant-derived therapeutics for sepsis and SAT and provides insights into sialidase-inhibition-based sepsis treatment strategies.

Insulin Clearance in Health and Disease.

Insulin action is impaired in type 2 diabetes. The functions of the hormone are an integrated product of insulin secretion from pancreatic β-cells and insulin clearance by receptor-mediated endocytosis and degradation, mostly in liver (hepatocytes) and, to a lower extent, in extrahepatic peripheral tissues. Substantial evidence indicates that genetic or acquired abnormalities of insulin secretion or action predispose to type 2 diabetes. In recent years, along with the discovery of the molecular foundation of receptor-mediated insulin clearance, such as through the membrane glycoprotein CEACAM1, a consensus has begun to emerge that reduction of insulin clearance contributes to the disease process. In this review, we consider the evidence suggesting a pathogenic role for reduced insulin clearance in insulin resistance, obesity, hepatic steatosis, and type 2 diabetes.

Abstract 3515: Engineered IL18 heterodimeric Fc-fusions featuring improved stability, reduced potency, and insensitivity to IL18BP

Abstract Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. After processing of the inactive precursor within monocytes and macrophages, mature IL18 can promote the expansion, survival, and cytotoxicity of T and NK cells expressing its receptor subunits IL-18Rα (IL18R1) and IL18Rβ (IL18RAP). Preclinical studies with recombinant IL18 have demonstrated anti-tumor activity in animal models, including impressive synergy with both immune checkpoint inhibitors and CAR-T therapy. However, clinical development exhibited poor pharmacokinetics and an overall lack of efficacy despite heavy dosing. IL18 participates in a negative feedback loop with a very high affinity natural inhibitor, IL18 binding protein (IL18BP), whose expression is induced by IFNγ. As IL18BP upregulation was observed in early phase clinical trials, it likely limited the efficacy of recombinant IL18. Here, we have engineered next generation IL18 cytokines that aim to improve on the poor PK of WT and contend with IL18BP induction. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Following that principle, we generated monovalent IL18-Fc fusions upon our XmAb® heterodimeric Fc platform and introduced substitutions that could modulate IL18 stability, affinity toward the IL18 heterodimeric receptor, and affinity toward IL18BP. Initially, IL18-Fc was difficult to produce but yields improved to typical bispecific antibody levels after stabilization. An engineered disulfide bridge increased the WT thermal denaturation temperature from 45 °C to 65 °C, and demonstrated a significant improvement in serum clearance in mouse PK experiments. Variants at IL18 positions along the receptor and IL18BP interfaces were explored in vitro by measuring PD-L1 induction on KG-1 cells, with and without a high concentration of IL18BP. Recombining a select number of hits generated a potency series with variants exhibiting over a 2,000-fold reduction in PD-L1 induction potency as compared to WT IL18-Fc. Importantly, we identified variants that no longer detectably bound IL18BP, relieving natural inhibition of our engineered IL18-Fc. In vivo immune-mediated inflammation was explored in human PBMC engrafted mouse models of graft versus host disease (GvHD). We observed potency-dependent exacerbation of GvHD, with corresponding dramatic increases in the numbers and activation of T and NK cells as compared to a human PBMC only control. Analysis of serum from the study revealed up to 100-fold increases in IFNγ levels. Our engineered, reduced-potency IL18-Fc cytokines demonstrate robust inflammation activity in vivo, improved pharmacokinetics in mice, and insensitivity to IL18BP inhibition. Citation Format: Alex Nisthal, Sung-Hyung Lee, Christine Bonzon, Ruschelle Love, Kendra N. Avery, Rumana Rashid, Panida Lertkiatmongkol, Nicole Rodriguez, Hanh Nguyen, Araz Eivazi, Sher Karki, Norm Barlow, Seung Y. Chu, Greg L. Moore, John R. Desjarlais. Engineered IL18 heterodimeric Fc-fusions featuring improved stability, reduced potency, and insensitivity to IL18BP [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 3515.

Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α2-macroglobulin

Human α2-macroglobulin (hα2M) is a multidomain protein with a plethora of essential functions, including transport of signaling molecules and endopeptidase inhibition in innate immunity. Here, we dissected the molecular mechanism of the inhibitory function of the ∼720-kDa hα2M tetramer through eight cryo–electron microscopy (cryo-EM) structures of complexes from human plasma. In the native complex, the hα2M subunits are organized in two flexible modules in expanded conformation, which enclose a highly porous cavity in which the proteolytic activity of circulating plasma proteins is tested. Cleavage of bait regions exposed inside the cavity triggers rearrangement to a compact conformation, which closes openings and entraps the prey proteinase. After the expanded-to-compact transition, which occurs independently in the four subunits, the reactive thioester bond triggers covalent linking of the proteinase, and the receptor-binding domain is exposed on the tetramer surface for receptor-mediated clearance from circulation. These results depict the molecular mechanism of a unique suicidal inhibitory trap.

Molecular Engineering of Efficacious Mono-Valent Ultra-Long Acting Two-Chain Insulin-Fc Conjugates.

Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element. Fc conjugation leads to recycling by the neonatal Fc receptor and increase in the molecular size, both contributing to the ultra-long pharmacokinetic and pharmacodynamic profiles.

Abstract 1743: IL12 heterodimeric Fc-fusions engineered for reduced potency exhibit strong anti-tumor activity and improved therapeutic index compared to native IL12 agents

Abstract Interleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 may help to establish an inflammatory tumor microenvironment critical for anti-tumor responses. Preclinical studies in mice revealed that native IL12 can dramatically shrink syngeneic tumors, however clinical studies in humans resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and extend half-life. IL12 is a heterodimeric protein consisting of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity of human IL12-Fc were assessed in a human PBMC engrafted mouse MCF7 tumor model. Surrogate mouse IL12-Fc were evaluated in additional murine tumor models. Tolerability and pharmacodynamic activity were assessed in non-human primates. IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to native IL12-Fc. Anti-tumor activity was achieved with potency-reduced IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Combined, these data indicate that potency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to narrow therapeutic index associated with recombinant native IL12 or IL12-Fc agents. Citation Format: Matthew J. Bernett, Ke Liu, Christine Bonzon, Rumana Rashid, Nicole Rodriguez, Nargess Hassanzadeh-Kiabi, Connie Ardila, Katrina Bykova, Michael Hackett, Norm Barlow, Irene Leung, Duc-Hanh Nguyen, Araz Eivazi, Seung Y. Chu, Rajat Varma, Umesh S. Muchhal, John R. Desjarlais. IL12 heterodimeric Fc-fusions engineered for reduced potency exhibit strong anti-tumor activity and improved therapeutic index compared to native IL12 agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1743.

Vincristine-Loaded Platelets Coated with Anti-CD41 MAbs: A New Proposal for the Treatment of Immune Thrombocytopenia

Objective: Immune thrombocytopenia (ITP) is an autoimmune disorder in which platelet-reactive autoantibodies accelerate the destruction of platelets. Macrophages play a crucial role in ITP through IgG-Fcγ receptor-mediated platelet clearance. Patients responding to initial therapies often troubled with frequent relapses, severe reverse reactions and poor life quantity. Therefore, searching new effective strategies for ITP treatment is in urgent need.Methods: In this study, platelets coated with anti-CD41 antibodies, which can mimic autoantibody-binding platelets in ITP patients, were used as a smart drug delivery system to load vincristine (VCR). Vincristine-loaded platelets coated with anti-CD41 mAbs (CD41-VLP) can selective release VCR in macrophages through IgG-Fcγ receptor-mediated phagocytosis. Scanning electron microscopy (SEM), dynamic light scattering (DLS), and platelet aggregation assays were performed to evaluate the morphological and functional changes of CD41-VLP. The encapsulation efficiency, drug loading and intracellular VCR concentration were determined using high performance liquid chromatography (HPLC). Macrophages were derived from THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The expression of cell differentiation antigen (CD11b and CD14) and was detected by flow cytometry (FCM). The growth inhibition of CD41-VLP on macrophages was detected by Cell Counting Kit-8 (CCK-8) assay and the macrophage apoptosis was quantified by FCM. The possible mechanism of CD41-VLP on macrophages was further investigated by western blotting.Results: platelets coated with anti-CD41 antibodies could load VCR with high drug loading and encapsulation efficiency. There is no significant difference in morphology and function between platelets and CD41-VLP. Meanwhile, VCR was released from CD41-VLP in a pH-triggered behavior and VCR would be rapidly released in an acidic condition. The THP-1 derived macrophages was detected with a strong ability of phagocytosis and a high expression of CD11b and CD14. In the group of macrophages treated with CD41-VLP, intracellular concentration of VCR was significant increased compared with other groups, which demonstrates that Cd41-VLP can selectively deliver VCR to macrophages. In the CD41-VLP group, the growth inhibition rate was also much higher than other groups and the apoptosis rate was significant increased compared with other groups. These results suggest that CD41-VLP can enhance the cytotoxicity of VCR against macrophages. Furthermore, In the CD41-VLP group, the expressions of pro-apoptotic proteins,including Bax, caspase-3 and CyclinB1, were significantly increased and the expression of Bcl-2, anti-apoptotic protein, was decreased. This finding indicates that CD41-VLP might improve the effects of VCR on macrophages through regulating the expression of apoptosis-related proteins.Conclusion: CD41-VLP can improve the cytotoxicity of VCR against macrophages, thus establishing a new targeted therapy for ITP treatment. DisclosuresNo relevant conflicts of interest to declare.

PAC1 receptor-mediated clearance of tau in postsynaptic compartments attenuates tau pathology in mouse brain.

Accumulation of pathological tau in synapses has been identified as an early event in Alzheimer's disease (AD) and correlates with cognitive decline in patients with AD. Tau is a cytosolic axonal protein, but under disease conditions, tau accumulates in postsynaptic compartments and presynaptic terminals, due to missorting within neurons, transsynaptic transfer between neurons, or a failure of clearance pathways. Using subcellular fractionation of brain tissue from rTg4510 tau transgenic mice with tauopathy and human postmortem brain tissue from patients with AD, we found accumulation of seed-competent tau predominantly in postsynaptic compartments. Tau-mediated toxicity in postsynaptic compartments was exacerbated by impaired proteasome activity detected by measuring lysine-48 polyubiquitination of proteins targeted for proteasomal degradation. To combat the accumulation of tau and proteasome impairment in the postsynaptic compartments of rTg4510 mouse brain, we stimulated the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) with its ligand PACAP administered intracerebroventricularly to rTg4510 mice. We observed enhanced synaptic proteasome activity and reduced total tau in postsynaptic compartments in mouse brain after PACAP treatment. The clearance of tau from postsynaptic compartments correlated with attenuated tauopathy and improved cognitive performance of rTg4510 transgenic mice on two behavioral tests. These results suggest that activating PAC1R could prevent accumulation of aggregate-prone tau and indicate a potential therapeutic approach for AD and other tauopathies.

Design of the ForesiGHt Trial: A Multicenter, Randomized, Placebo- and Active-Controlled Trial to Compare Once-Weekly TransCon hGH (lonapegsomatropin) to Placebo and Daily Somatropin in Adults With Growth Hormone Deficiency (GHD)

BackgroundAdult GHD results from insufficient growth hormone (GH) secretion from the anterior pituitary gland and may represent either a continuation of childhood-onset GHD or GHD acquired during adulthood. Clinically, adult GHD is associated with central adiposity, decreased lean muscle mass, increased fat mass, decreased bone mineral density, and reduced quality of life. Current standard of care consists of GH replacement via daily injections.Lonapegsomatropin is a long-acting prodrug of somatropin (hGH), designed to deliver unmodified hGH with a weekly exposure profile. Lonapegsomatropin consists of somatropin that is transiently bound to a carrier via a proprietary TransCon Linker. The carrier extends the duration of somatropin in the circulation through a shielding effect that minimizes renal excretion and receptor-mediated clearance of the prodrug. At physiologic conditions, lonapegsomatropin releases fully active, unmodified somatropin via autocleavage of the linker in a controlled manner.The safety and efficacy of lonapegsomatropin have previously been evaluated in two phase 3 trials and one long-term extension trial in pediatric GHD. Changes in body composition are an objective and sensitive endpoint that reflects biologic activity of GH and clinical efficacy of GH replacement. Methods: The primary objective of the foresiGHt trial is to evaluate the efficacy of once-weekly lonapegsomatropin compared to placebo at 38 weeks in adults with GHD. The trial will be conducted at approximately 120 sites in North America, Europe, Asia, and Oceania. Approximately 240 subjects will be randomized 1:1:1 to once-weekly lonapegsomatropin, once-weekly placebo, or daily somatropin (Norditropin®). Subjects will be treatment-naïve or without GH therapy for at least 12 months. Subjects with well-controlled non-insulin dependent diabetes mellitus (HbA1c ≤ 7.5%) will be eligible. Following screening, the 38-week treatment period will consist of a 12-week gradual dose titration period and 26 weeks of fixed dose maintenance. Fixed dosing will be used to ensure maximal comparability across the treatment arms in the trial. Three dosing groups per arm will be established to allow for differences in age and oral estrogen intake in women.The primary endpoint is change from baseline in trunk percent fat at week 38, as measured by dual energy X-ray absorptiometry (DXA). Secondary efficacy endpoints are change from baseline in trunk fat mass and change in total body lean mass at week 38. Exploratory efficacy endpoints include total body fat mass, trunk lean mass, visceral adipose tissue, and patient-reported outcomes. Conclusion: The ongoing global phase 3 foresiGHt trial is designed to assess the efficacy, safety, and tolerability of lonapegsomatropin by weekly administration, compared to weekly placebo and daily hGH replacement therapy in adults with GHD.

Modulation of the tumor micro-environment by CD8+ T cell-derived cytokines.

Upon their activation, CD8+ T cells in the tumor micro-environment (TME) secrete cytokines such as IFNγ, TNFα, and IL-2. While over the past years a major interest has developed in the antigenic signals that induce such cytokine release, our understanding of the cells that subsequently sense these CD8+ T-cell secreted cytokines is modest. Here, we review the current insights into the spreading behavior of CD8+ T-cell-secreted cytokines in the TME. We argue for a model in which variation in the mode of cytokine secretion, cytokine half-life, receptor-mediated clearance, cytokine binding to extracellular components, and feedback or forward loops, between different cytokines or between individual tumors, sculpts the local tissue response to natural and therapy-induced T-cell activation in human cancer.

Phosphatidylethanolamine and Phosphatidylserine Synergize To Enhance GAS6/AXL-Mediated Virus Infection and Efferocytosis.

Phosphatidylserine (PS) receptors mediate clearance of apoptotic cells-efferocytosis-by recognizing the PS exposed on those cells. They also mediate the entry of enveloped viruses by binding PS in the virion membrane. Here, we show that phosphatidylethanolamine (PE) synergizes with PS to enhance PS receptor-mediated efferocytosis and virus entry. The presence of PE on the same surface as PS dramatically enhances recognition of PS by PS-binding proteins such as GAS6, PROS, and TIM1. Liposomes containing both PE and PS bound to GAS6 and were engulfed by AXL-expressing cells much more efficiently than those containing PS alone. Further, infection of AXL-expressing cells by infectious Zika virus or Ebola, Chikungunya, or eastern equine encephalitis pseudoviruses was inhibited with greater efficiency by the liposomes containing both PS and PE compared to a mixture of liposomes separately composed of PS and PE. These data demonstrate that simultaneous recognition of PE and PS maximizes PS receptor-mediated virus entry and efferocytosis and underscore the important contribution of PE in these major biological processes.IMPORTANCE Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are usually sequestered to the inner leaflet of the plasma membrane of the healthy eukaryotic cells. During apoptosis, these phospholipids move to the cell's outer leaflet where they are recognized by so-called PS receptors on surveilling phagocytes. Several pathogenic families of enveloped viruses hijack these PS receptors to gain entry into their target cells. Here, we show that efficiency of these processes is enhanced, namely, PE synergizes with PS to promote PS receptor-mediated virus infection and clearance of apoptotic cells. These findings deepen our understanding of how these fundamental biological processes are executed.

An IL-2 mutein engineered to promote expansion of regulatory T cells arrests ongoing autoimmunity in mice.

Interleukin-2 (IL-2) controls the homeostasis and function of regulatory T (Treg) cells, and defects in the IL-2 pathway contribute to multiple autoimmune diseases. Although recombinant IL-2 therapy has been efficacious in certain inflammatory conditions, the capacity for IL-2 to also activate inflammatory effector responses highlights the need for IL-2-based therapeutics with improved Treg cell specificity. From a panel of rationally designed murine IL-2 variants, we identified IL-2 muteins with reduced potency and enhanced Treg cell selectivity due to increased dependence on the IL-2 receptor component CD25. As an Fc-fused homodimer, the optimal Fc.IL-2 mutein induced selective Treg cell enrichment and reduced agonism of effector cells across a wide dose range. Furthermore, despite being a weaker agonist, overall Treg cell growth was greater and more sustained due to reduced receptor-mediated clearance of the Fc.IL-2 mutein compared with Fc-fused wild-type IL-2. Preferential Treg cell enrichment was also observed in the presence of activated pathogenic T cells in the pancreas of nonobese diabetic (NOD) mice, despite a loss of Treg cell selectivity in an IL-2R proximal response. These properties facilitated potent and extended resolution of NOD diabetes with infrequent dosing schedules.

Abstract 5549: Potency-reduced IL12 heterodimeric Fc-fusions exhibit strong anti-tumor activity

Abstract Interleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 are promising for cancer treatment and may help to convert immunologically suppressed “cold” tumors into inflamed “hot” tumors. Preclinical studies in mice revealed that IL12 can have a dramatic effect on shrinking syngeneic tumors, however clinical studies in humans have resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created various IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency in order to improve tolerability, slow receptor-mediated clearance, and prolong half-life. IL12 is a heterodimeric protein consisting of two subunits, and thus we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on normal human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity was assessed by engrafting pp65-MCF-7 cells into human PBMC engrafted NSG MHC class I and II double-knockout mice and by measuring tumor volume, lymphocyte activation/proliferation, and IFNγ production over time. IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and weaker variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to a wild-type IL12-Fc. In vivo anti-tumor activity in the huPBMC-pp65-MCF7 model was achieved with reduced-potency IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells, as evidenced by upregulation of CD25. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum cytokine indicated up to 200-fold increases in IFNγ levels. Combined, these data indicate that reduced-potency IL12-Fc retain strong anti-tumor activity, with potential for improvement of therapeutic index. Citation Format: Rajat Varma, Ke Liu, Christine Bonzon, Rumana Rashid, Nicole Rodriguez, Nargess Hassanzadeh-Kiabi, Connie Ardila, Seung Y. Chu, Umesh S. Muchhal, John R. Desjarlais, Matthew J. Bernett. Potency-reduced IL12 heterodimeric Fc-fusions exhibit strong anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5549.

Hsa-miR-4271 downregulates the expression of constitutive androstane receptor and enhances in vivo the sensitivity of non-small cell lung cancer to gefitinib

The efficacy of molecular targeting agents is dependent on the metabolism or nuclear receptor-mediated clearance of chemotherapy resistance-related factors such as cytochrome P450 (CYP) or ATP binding cassette subfamily B member 1 (ABCB1). In this study, we revealed the roles of the microRNA-4271/CAR (constitutive androstane receptor) axis in the regulation of the resistance to molecular anticancer targeting agents in non-small cell lung cancer (NSCLC) cells including two main categories of NSCLC: lung adenocarcinoma (AC) and large cell lung cancer (LCC). The expression of miR-4271 was negatively correlated with CAR expression in NSCLC tissues. MiR-4271 targeted CAR and inhibited the activation of the CAR signaling pathway. Overexpression of CAR in NSCLC enhanced the resistance of NSCLC cells to molecular targeting agents and miR-4271-infected NSCLC cells enhanced their sensitivity to molecular targeting agents such as Gefitinib. The mechanism-data showed that overexpression of miR-4271 decelerated the mechanism or the clearance of molecular targeting agents by targeting the 3′UTR (3′ un-translation region). These results suggest that miR-4271 may contribute to the development of more effective strategies for the treatment of advanced NSCLC.

Dissection of Clinical and Gene Expression Signatures of Familial versus Multifactorial Chylomicronemia.

Familial chylomicronemia syndrome (FCS) is a rare disorder associated with chylomicronemia (CM) and an increased risk of pancreatitis. Most individuals with CM do not have FCS but exhibit multifactorial CM (MCM), which differs from FCS in terms of risk and disease management. This study aimed to investigate clinical and gene expression profiles of FCS and MCM patients. Anthropometrics, clinical, and biochemical variables were analyzed in 57 FCS and 353 MCM patients. Gene expression analyses were performed in a subsample of 19 FCS, 28 MCM, and 15 normolipidemic controls. Receiver operating characteristic (ROC) curve analyses were performed to analyze the capacity of variables to discriminate FCS from MCM. Sustained fasting triglycerides ≥20 mmol/L (>15 mmol/L with eruptive xanthomas), history of pancreatitis, poor response to fibrates, diagnosis of CM at childhood, body mass index <22 kg/m2, and delipidated apolipoprotein B or glycerol levels <0.9 g/L and <0.05 mmol/L, respectively, had an area under the ROC curve ≥0.7. Gene expression analyses identified 142 probes differentially expressed in FCS and 32 in MCM compared with controls. Among them, 13 probes are shared between FCS and MCM; 63 are specific to FCS and 2 to MCM. Most FCS-specific or shared biomarkers are involved in inflammatory, immune, circadian, postprandial metabolism, signaling, docking systems, or receptor-mediated clearance mechanisms. This study reveals differential signatures of FCS and MCM. It opens the door to the identification of key mechanisms of CM expression and potential targets for the development of new treatments.

Endosomal Acid-Base Homeostasis in Neurodegenerative Diseases.

Neurodegenerative disorders are debilitating and largely untreatable conditions that pose a significant burden to affected individuals and caregivers. Overwhelming evidence supports a crucial preclinical role for endosomal dysfunction as an upstream pathogenic hub and driver in Alzheimer's disease (AD) and related neurodegenerative disorders. We present recent advances on the role of endosomal acid-base homeostasis in neurodegeneration and discuss evidence for converging mechanisms. The strongest genetic risk factor in sporadic AD is the ε4 allele of Apolipoprotein E (ApoE4), which potentiates pre-symptomatic endosomal dysfunction and prominent amyloid beta (Aβ) pathology, although how these pathways are linked mechanistically has remained unclear. There is emerging evidence that the Christianson syndrome protein NHE6 is a prominent ApoE4 effector linking endosomal function to Aβ pathologies. By functioning as a dominant leak pathway for protons, the Na+/H+ exchanger activity of NHE6 limits endosomal acidification and regulates β-secretase (BACE)-mediated Aβ production and LRP1 receptor-mediated Aβ clearance. Pathological endosomal acidification may impact both Aβ generation and clearance mechanisms and emerges as a promising therapeutic target in AD. We also offer our perspective on the complex role of endosomal acid-base homeostasis in the pathogenesis of neurodegeneration and its therapeutic implications for neuronal rescue and repair strategies.