Mechanism Of Blockade Research Articles

Examining Downstream Effects of Concizumab in Hemophilia A with a Mathematical Modeling Approach

BackgroundTissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits FXa, the TF-FVIIa-FXa complex, and early forms of the prothrombinase complex. Concizumab is a monoclonal antibody that blocks FXa inhibition by TFPI and reduces bleeding in hemophilia. ObjectivesTo examine how concizumab impacts various reactions of TFPI to restore thrombin generation in hemophilia A using mathematical models. MethodsA compartment model was used to estimate plasma concentrations of free concizumab and its complexes with TFPIα and TFPIβ. Concizumab was integrated into a flow-mediated mathematical model of coagulation and a small injury was simulated under hemophilia A conditions. Simulations were then analyzed to determine how concizumab’s blockade of TFPI anticoagulant activities, specifically the inhibition of FXa in plasma and on platelets, inhibition of TF:VIIa at the subendothelium, and prior sequestration of plasma TFPIα to the endothelium via TFPIβ, altered thrombin generation. ResultsConcizumab improved simulated thrombin generation in hemophilia A by simultaneously altering all three mechanisms of TFPI anticoagulant blockade examined. Concizumab sequesters ∼75% of plasma TFPIα through formation of ternary TFPIα-concizumab-TFPIβ-complexes. For all TF levels, reducing the TFPIα plasma concentration had the largest impact on the lag time followed by blocking TFPIα inhibition of TF:VIIa:FXa and subsequently followed by blocking TFPIα inhibition of FXa in plasma and on the platelet surface. ConclusionsThe effectiveness of concizumab is mediated through blockade of TFPI anticoagulant activities in plasma and on multiple physiological surfaces. An important and previously unrecognized function of concizumab was sequestration of plasma TFPIα to the endothelium.

STAT3 blockade ameliorates LPS-induced kidney injury through macrophage-driven inflammation

BackgroundSignal transducer and activator of transcription 3 (STAT3), a multifaceted transcription factor, modulates host immune responses by activating cellular response to signaling ligands. STAT3 has a pivotal role in the pathophysiology of kidney injury by counterbalancing resident macrophage phenotypes under inflammation conditions. However, STAT3’s role in acute kidney injury (AKI), particularly in macrophage migration, and in chronic kidney disease (CKD) through fibrosis development, remains unclear.MethodsStattic (a JAK2/STAT3 inhibitor, 5 mg/kg or 10 mg/kg) was administered to evaluate the therapeutic effect on LPS-induced AKI (L-AKI) and LPS-induced CKD (L-CKD), with animals sacrificed 6–24 h and 14 days post-LPS induction, respectively. The immune mechanisms of STAT3 blockade were determined by comparing the macrophage phenotypes and correlated with renal function parameters. Also, the transcriptomic analysis was used to confirm the anti-inflammatory effect of L-AKI, and the anti-fibrotic role was further evaluated in the L-CKD model.ResultsIn the L-AKI model, sequential increases in BUN and blood creatinine levels were time-dependent, with a marked elevation of 0–6 h after LPS injection. Notably, two newly identified macrophage subpopulations (CD11bhighF4/80low and CD11blowF4/80high), exhibited population changes, with an increase in the CD11bhighF4/80low population and a decrease in the CD11blowF4/80high macrophages. Corresponding to the FACS results, the tubular injury score, NGAL, F4/80, and p-STAT3 expression in the tubular regions were elevated. STAT3 inhibitor injection in L-AKI and L-CKD mice reduced renal injury and fibrosis. M2-type subpopulation with CD206 in CD11blowF4/80high population increased in the Stattic-treated group compared with that in the LPS-alone group in the L-AKI model. Additionally, STAT3 inhibitor reduced inflammation driven by LPS-stimulated macrophages and epithelial cells injury in the co-culture system. Transcriptomic profiling identified 3 common genes in the JAK-STAT, TLR, and TNF signaling pathways and 11 common genes in the LPS with macrophage response. The PI3K-AKT (IL-6, Akt3, and Pik3r1) and JAK-STAT pathways were determined as potential Stattic targets. Further confirmation through mRNA and protein expressions analyses showed that Stattic treatment reduced inflammation in the L-AKI and fibrosis in the L-CKD mice.ConclusionsSTAT3 blockade effectively mitigated inflammation by retrieving the CD11blowF4/80high population, further emphasizing the role of STAT3-associated macrophage-driven inflammation in kidney injury.

Abstract 43: PAR1 Inhibition with Vorapaxar Enhances Renal Function and Modulates Inflammation Mediators in Diabetic Nephropathy

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure, and hypertension is a comorbidity in about 90% of all cases. Characteristics of pathological changes of DN include severe albuminuria, renal hyperfiltration, glomerular basement membrane thickening, and glomerulosclerosis. Identifying the molecular mechanisms underlying DN is critical for developing more effective therapeutic approaches. Thus, interventions targeting podocytes have become a major focus for diseases like DN due to their crucial role in regulating glomerular permeability and maintaining glomerular structures. Our previous studies have demonstrated that serine proteases and Protease-activated receptor 1 (PAR1) are involved in developing DN through TRPC6-mediated calcium signaling in podocytes. Utilizing a PAR1 heterozygous knockout rat model with a type 2 diabetic background, we showed that reducing PAR1 expression decreases microalbuminuria and glomerular damage. This study aims to define the effects and potential mechanisms of pharmacological blockade of PAR1 on the development of DN. To achieve this, 16-week-old T2DN rats were treated with a potent and selective PAR1 antagonist (vorapaxar (SCH 530348), 10 mg/kg of diet) for 16 weeks to determine the effect of PAR1 inhibition. We found improvements in glomerular filtration rate (GFR) in treated animals compared to control after 16 weeks of treatment (0.8 ± 0.2 vs 1.2 ± 0.2 mL/min/100g b.w., p=0.029, N≥4). We observed a trend toward differences in basal intracellular calcium concentration in podocytes between the control and treated groups, although these changes were not statistically significant (202 ± 95 vs 152 ± 75 nM, p=0.19, n≥10, N=3). Additionally, inhibition of PAR1 caused modulation in the inflammation-resolution mediators in the kidney with significant upregulation of Maresins Conjugates in Tissue Regeneration (MCTR1) and downregulation of hydroxy-docosahexaenoic acids (7-,14-,17-HDHA). Changes in GFR may be linked to reduced glomerular damage, indicating that the structural integrity of the glomeruli is better maintained in treated animals. Alterations in the lipidomic profile could indicate a reduction in the inflammatory processes typically associated with DN. Overall, our data indicates that vorapaxar improves glomerular filtration rate and modulates lipid mediators in a type 2 diabetic rat model. This suggests that targeting PAR1 could be a promising therapeutic strategy for diabetic nephropathy.

Abstract 2473: A novel anti-PD-1 and proIL-2 bifunctional fusion protein delivers potent anti-tumor activity via PD-1 checkpoint inhibition and tumor selective IL-2R agonism preferentially on antigen specific T cells

Abstract High-dose IL-2 (HD IL-2) induces complete and durable responses in RCC and melanoma but the treatment is associated with severe toxicities. PD-(L)1 blockade represents a recent breakthrough in immunotherapy with solid tumors, but only 20-30% of patients can benefit from the therapy and most responders eventually develop resistance. In more recent clinical studies with small number of subjects, HD IL-2 has shown promising responses in patients who progressed on anti-PD-1 therapy and combination therapy of HD-IL-2 and PD-1 blockade has resulted in high rate of objective response. In an attempt to maximize the clinical benefit of these immunotherapies to a broader patient population with deeper response and in the meantime to minimize the severe toxicity of HD IL-2, we have generated a novel bifunctional PD-1-proIL-2v fusion protein harnessing the mechanisms of PD-1 blockade and PD-1-cis directed tumor conditional IL-2R activation. PD-1-proIL-2v shows dose dependent inhibition of PD-1 immune checkpoint pathway measured by GM-CSF and IFNγ production in an MLR assay. Protease activated PD-1-proIL-2v, but not the intact drug, stimulates the phosphorylation of STAT5 of NK and T cells, the proliferation of CD8 T cells as well as the cytokine production in human whole blood. The potency of pSTAT5 by protease activated product is enhanced by 10-20 folds on PD-1+ T cells than T cells without PD-1. When tested in mouse tumor models resistant to PD-1 blockade, PD1-proIL-2v demonstrated high potency of tumor inhibition at as low as 0.1 mg/kg dose. In contrast, non-targeted proIL-2v at molar equivalent or higher dose showed minimal antitumor effect in either monotherapy or combination with anti-PD-1. Analysis of PD-1-proIL-2v treated mice showed dramatically increased tumor infiltrating CD4+ and CD8+ T cells, including PD-1+Tcf-1+ and PD-1+ IL-18Ra+TIM3− CD8+ T cells, referred to “stem-like” progenitor effector cells and “fresh” effector cells, respectively. Activation of the IL-2 procytokine in PD-1-proIL-2v was observed in mouse tumor tissues but not in the plasma. In monkey GLP-compliant 4-week repeat-dose toxicity study, PD-1-proIL-2v demonstrated excellent safety profiles without overt irAEs that are characteristic of IL-2. In conclusion, our preclinical data demonstrated that PD-1-proIL-2v effectively stimulated the expansion and reinvigoration of antigen specific T cells in the tumor tissue resulting in strong anti-tumor efficacy while it did not cause significant peripheral toxicity. Phase 1 clinical trial of PD-1-proIL-2v is planned to start in early 2024 to evaluate the safety, pharmacokinetics and anti-tumor activity in advanced solid tumors. Citation Format: Zijuan Li, Feifei Zhang, Ze Zhang, Harry Zhou. A novel anti-PD-1 and proIL-2 bifunctional fusion protein delivers potent anti-tumor activity via PD-1 checkpoint inhibition and tumor selective IL-2R agonism preferentially on antigen specific T cells [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 2473.

A Life-Threatening Neurotoxicity in Bungarus Candidus Snake Bite: A Case Report

Background: Bungarus Candidus, a snake inducing severe neurotoxic symptoms. Case: A 31-year-old female presented to the emergency department with complaints of pain in the whole body, limb weakness, shortness of breath after being bitten by a snake. Neurological examination, ptosis of both eyelids was found with normal movement of the eyeballs. Muscle strength in all extremities is 4 followed by decreased physiological reflexes. There are fang marks in the form of 2 slightly swollen and reddened black dots on the left foot. During observation, the patient had decreased consciousness, severe shortness of breath with desaturation without any signs of shock. Other clinical revealed bilateral dilated pupils, dysphonia, dysarthria, and muscle strength that was difficult to assess. The patient was intubated and subsequently admitted to the ICU. The patient regained consciousness but still had difficulty opening his eyes and weak muscle strength on the 2nd of ICU. The patient's condition improved on the 5th of ICU, extubating was performed and transferred to the non-ICU. The treatment obtained included 17 vials of SABU, analgesics, antibiotics, anticholinesterase, and anticholinergic. The patient was discharged after 11 days of hospitalization. Discussion: The neurotoxicity of snake venom is related to mechanisms of neuromuscular transmission blockade, pre-synaptic and post-synaptic. Bungarus Candidus has a peculiar type of toxin, candoxin that acts by inhibiting nicotinic acetylcholine receptors at the postsynaptic site. Recognition of symptoms and management interventions in snakebite cases are critical to patient outcomes and survival.

Photon blockade with a trapped Λ-type three-level atom in asymmetrical cavity.

We propose a scheme to manipulate strong and nonreciprocal photon blockades in asymmetrical Fabry-Perot cavity with a Λ-type three-level atom. Utilizing the mechanisms of both conventional and unconventional blockade, the strong photon blockade is achieved by the anharmonic eigenenergy spectrum brought by Λ-type atom and the destructive quantum interference effect induced by a microwave field. By optimizing the system parameters, the manipulation of strong photon blockade over a wide range of cavity detuning can be realized. Using spatial symmetry breaking introduced by the asymmetry of cavity, the direction-dependent nonreciprocal photon blockade can be achieved, and the nonreciprocity can reach the maximum at optimal cavity detuning. In particular, manipulating the occurring position of nonreciprocal photon blockade can be implemented by simply adjusting the cavity detuning. Our scheme provides feasible access for generating high-quality nonreciprocal single-photon sources.

ASRGL1 downregulation suppresses hepatocellular carcinoma tumorigenesis in a CDK1-dependent manner.

The asparaginase-like protein 1 (ASRGL1) catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Emerging evidences have shown a strong correlation between ASRGL1 expression and tumorigenesis. However, the expression and biological function of ASRGL1 in hepatocellular carcinoma (HCC) are still unclear. Here, we explored anti-tumor activity and fundamental mechanisms of ASRGL1 blockade in the HCC progression. Expression levels of ASRGL1 in patients with HCC were higher than those in the adjacent normal tissue. In addition, increased expression of ASRGL1 in HCC patients was correlated with poor overall survival. Knockdown of ASRGL1 gene in HepG2 and Li-7 cell lines inhibited cell proliferation, migration and invasion, but promoted apoptosis in vitro. ASRGL1 knockdown suppressed tumor growth in vivo. Conversely, ASRGL1 overexpression promoted cell proliferation, migration and invasion in HepG2 cells. Through bioinformatics analysis, we found that ASRGL1 might participate in the regulation of the cell cycle. Flow cytometry analysis conformed that ASRGL1 knockdown captured the cell cycle during the G2/M phase. ASRGL1 blockade promoted P53 protein expression and reduced expression of cyclin B and CDK1 proteins, as well as failed to binding. Moreover, CDK1 overexpression was able to reverse the decreased proliferation, migration and invasion of HepG2 cells induced by ASRGL1 knockdown. Collectively, our studies indicate that ASRGL1 blockade functions to inhibit cyclin B/CDK1-dependent cell cycle, leading to G2-to-M phase transition failure and tumor suppression in HCC.

Multimodal Integrative Genomics and Pathology Analyses in Neoadjuvant Nivolumab Treatment for Intermediate and Locally Advanced Hepatocellular Carcinoma

Introduction: Immunotherapy has resulted in pathologic responses in hepatocellular carcinoma (HCC), but the benefits and molecular mechanisms of neoadjuvant immune checkpoint blockade are largely unknown. Methods: In this study, we evaluated the efficacy and safety of preoperative nivolumab (anti-PD-1) in patients with intermediate and locally advanced HCC and determined the molecular markers for predicting treatment response. Results: Between July 2020 and November 2021, 20 treatment-naive HCC patients with intermediate and locally advanced tumors received preoperative nivolumab at 3 mg/kg for 3 cycles prior to surgical resection. Nineteen patients underwent surgical resection on trial. Seven (36.8%) of the 19 patients had major pathologic tumor necrosis (≥60%) in the post-nivolumab resection specimens, with 3 having almost complete (>90%) tumor necrosis. The tumor necrosis was hemorrhagic and often accompanied by increased or dense immune cell infiltrate at the border of the tumors. None of the patients developed major adverse reactions contradicting hepatectomy. RNA-sequencing analysis on both pre-nivolumab tumor biopsies and post-nivolumab resected specimens showed that, in cases with major pathologic necrosis, the proportion of CD8 T cells in the HCC tissues predominantly increased after treatment. Moreover, to investigate noninvasive biomarker for nivolumab response, we evaluated the copy number variation (CNV) using target-panel sequencing on plasma cell-free DNA of the patients and derived a CNV-based anti-PD-1 score. The score correlated with the extent of tumor necrosis and was validated in a Korean patient cohort with anti-PD-1 treatment. Conclusion: Neoadjuvant nivolumab demonstrated promising clinical activity in intermediate and locally advanced HCC patients. We also identified useful noninvasive biomarker predicting responsiveness.

The Antinociceptive Effect of Sympathetic Block is Mediated by Transforming Growth Factor β in a Mouse Model of Radiculopathy.

Although sympathetic blockade is clinically used to treat pain, the underlying mechanisms remain unclear. We developed a localized microsympathectomy (mSYMPX), by cutting the grey rami entering the spinal nerves near the rodent lumbar dorsal root ganglia (DRG). In a chemotherapy-induced peripheral neuropathy model, mSYMPX attenuated pain behaviors via DRG macrophages and the anti-inflammatory actions of transforming growth factor-β (TGF-β) and its receptor TGF-βR1. Here, we examined the role of TGF-β in sympathetic-mediated radiculopathy produced by local inflammation of the DRG (LID). Mice showed mechanical hypersensitivity and transcriptional and protein upregulation of TGF-β1 and TGF-βR1 three days after LID. Microsympathectomy prevented mechanical hypersensitivity and further upregulated Tgfb1 and Tgfbr1. Intrathecal delivery of TGF-β1 rapidly relieved the LID-induced mechanical hypersensitivity, and TGF-βR1 antagonists rapidly unmasked the mechanical hypersensitivity after LID+mSYMPX. In situ hybridization showed that Tgfb1 was largely expressed in DRG macrophages, and Tgfbr1 in neurons. We suggest that TGF-β signaling is a general underlying mechanism of local sympathetic blockade.

Lineage-Specific Induced Pluripotent Stem Cell-Derived Smooth Muscle Cell Modeling Predicts Integrin Alpha-V Antagonism Reduces Aortic Root Aneurysm Formation in Marfan Syndrome Mice.

The role of increased smooth muscle cell (SMC) integrin αv signaling in Marfan syndrome (MFS) aortic aneurysm remains unclear. Herein, we examine the mechanism and potential efficacy of integrin αv blockade as a therapeutic strategy to reduce aneurysm progression in MFS. Induced pluripotent stem cells (iPSCs) were differentiated into aortic SMCs of the second heart field (SHF) and neural crest (NC) lineages, enabling in vitro modeling of MFS thoracic aortic aneurysms. The pathological role of integrin αv during aneurysm formation was confirmed by blockade of integrin αv with GLPG0187 in Fbn1C1039G/+ MFS mice. iPSC-derived MFS SHF SMCs overexpress integrin αv relative to MFS NC and healthy control SHF cells. Furthermore, integrin αv downstream targets (FAK [focal adhesion kinase]/AktThr308/mTORC1 [mechanistic target of rapamycin complex 1]) were activated, especially in MFS SHF. Treatment of MFS SHF SMCs with GLPG0187 reduced p-FAK/p-AktThr308/mTORC1 activity back to control SHF levels. Functionally, MFS SHF SMCs had increased proliferation and migration compared to MFS NC SMCs and control SMCs, which normalized with GLPG0187 treatment. In the Fbn1C1039G/+ MFS mouse model, integrin αv, p-AktThr308, and downstream targets of mTORC1 proteins were elevated in the aortic root/ascending segment compared to littermate wild-type control. Mice treated with GLPG0187 (age 6-14 weeks) had reduced aneurysm growth, elastin fragmentation, and reduction of the FAK/AktThr308/mTORC1 pathway. GLPG0187 treatment reduced the amount and severity of SMC modulation assessed by single-cell RNA sequencing. The integrin αv-FAK-AktThr308 signaling pathway is activated in iPSC SMCs from MFS patients, specifically from the SHF lineage. Mechanistically, this signaling pathway promotes SMC proliferation and migration in vitro. As biological proof of concept, GLPG0187 treatment slowed aneurysm growth and p-AktThr308 signaling in Fbn1C1039G/+ mice. Integrin αv blockade via GLPG0187 may be a promising therapeutic approach to inhibit MFS aneurysmal growth.

Зміна «правил гри» в лікуванні пацієнтів із серцевою недостатністю: фокус на емпагліфлозин

Інгібітори натрійзалежних котранспортерів глюкози 2-го типу (іНЗКТГ2) є препаратами, що «змінили правила гри» в ендокринології, кардіології та нефрології. Емпагліфлозин, маючи виражені кардіоренальні органопротективні властивості незалежно від наявності ЦД 2-го типу, є, ймовірно, найуніверсальнішою молекулою серед іНЗКТГ2. У науковому огляді розглянуто механізми блокади емпагліфлозином одночасно НЗКТГ2-каналів та NHE3-каналів як причини натрійуретичного ефекту препарату. Блокада NHE-каналів у клітинах міокарда зменшує навантаження клітин натрієм та кальцієм. Наголошено на здатності емпагліфлозину знижувати артеріальний тиск, селективно зменшувати об’єм інтерстиційної рідини, впливати на геометрію лівого шлуночка, гальмуючи його ремоделювання. Здійснено огляд першого великого дослідження EMPA-REG OUTCOME в пацієнтів з атеросклеротичними серцево-судинними захворюваннями, у якому виявлено клінічно значущий вплив емпагліфлозину на великі серцево-судинні події, зниження ризику серцево-судинної смерті, смерті від будь-яких причин та госпіталізації з приводу серцевої недостатності. Проаналізовано результати досліджень емпагліфлозину в пацієнтів з хронічною серцевою недостатністю з різною фракцією викиду лівого шлуночка та в пацієнтів із гострою серцевою недостатністю (EMPEROR-REDUCED, EMPEROR-PRESERVED та EMPULSE). Зосереджено увагу на тому, що емпагліфлозин на сьогодні має достатню доказову базу, щоб лікувати усі категорії пацієнтів з хронічною та гострою серцевою недостатністю і починати терапію ще до моменту виписування зі стаціонару.

Sulfation at Glycopolymer Side Chains Switches Activity at the Macrophage Mannose Receptor (CD206) In Vitro and In Vivo.

The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight on the chemistry of CD206-ligand interactions and, importantly, effectively modulate its activity are currently lacking. Using novel SO4-3-Gal-glycopolymers targeting its cysteine-rich lectin ectodomain, this study uncovers and elucidates a previously unknown mechanism of CD206 blockade involving the formation of stable intracellular SO4-3-Gal-glycopolymer-CD206 complexes that prevents receptor recycling to the cell membrane. Further, we show that SO4-3-Gal glycopolymers inhibit CD206 both in vitro and in vivo, revealing hitherto unknown receptor function and demonstrating their potential as CD206 modulators within future immunotherapies.

Inhibition of the CD47-SIRPα axis for cancer therapy: A systematic review and meta-analysis of emerging clinical data

CD47-SIRPα interaction acts as a "don't eat me" signal and is exploited by cancer to downregulate innate and adaptive immune surveillance. There has been intense interest to develop a mechanism of blockade, and we aimed to analyze the emerging data from early clinical trials. We performed a systematic review and meta-analysis of relevant databases and conference abstracts including clinical trials using CD47 and/or SIRPα inhibitors in cancer treatment. Nonlinear mixed models were applied for comparison of response and toxicity. We retrieved 317 articles, 24 of which were eligible. These included 771 response-evaluable patients with hematologic (47.1%) and solid tumors (52.9%). Of these, 6.4% experienced complete response, 10.4% partial response, and 26.1% stable disease for a 16.7% objective response rate (ORR), 42.8% disease control rate, and 4.8-month median duration of response. ORR was significantly higher for hematologic cancers (25.3%) than solid cancers (9.1%, p=0.042). Comparing by mechanism, seven CD47 monoclonal antibodies (mAbs) and six selective SIRPα blockers were given alone or combined with checkpoint inhibitors, targeted therapy, and/or chemotherapy. In solid cancers, selective SIRPα blockade showed a higher ORR (16.2%) than anti-CD47 mAbs (2.8%, p=0.079), which was significant for combination therapies (ORR 28.3% vs 3.0%, respectively, p=0.010). Responses were seen in head and neck, colorectal, endometrial, ovarian, hepatocellular, non-small cell lung, and HER2+gastroesophageal cancers. Dose-limiting toxicity (DLT) was seen in 3.3% of patients (5.4% anti-CD47 mAbs, 1.4% selective SIRPα blockers; p=0.01). The frequency of treatment-related adverse events (TRAEs) ≥grade 3 was 18.0%, similar between the two groups (p=0.082), and mostly laboratory abnormalities. For anti-CD47 mAbs, the most common toxicities included grade 1-2 fatigue (27.2%), headache (21.0%), and anemia (20.5%). For selective SIRPα blockers, these included grade 1-2 infusion reaction (23.1%) and fatigue (15.8%). Anti-CD47 mAbs were significantly more likely than selective SIRPα blockers to cause grade 1-2 fever, chills, nausea/vomiting, headache, and anemia. In conclusion, combination therapies using selective SIRPα blockade had higher response rates in solid tumors than anti-CD47 mAb combinations. Hematologic changes were the main TRAEs, and selective SIRPα blockers seemed to have a better grade 1-2 toxicity profile. Treatment was well-tolerated with minimal DLTs.

147 (PB027) - Combination Therapy with Poly-gamma-glutamic Acid and Anti-PD-1 Results in Synergistic Therapeutic Effects against Cancers

Background: Combinatorial therapeutic strategies based on innate immunity have the potential to enhance the treatment efficacy of cancer therapies. We have been demonstrated that poly-γ-glutamic acid (γ-PGA) has a potent innate immune modulating activities including natural killer cell activation and interferon secretion through Toll-like receptor 4 and may be a useful mean for immunotherapeutic as reported previously. The programmed death protein 1 (PD-1) plays a critical part in downregulating immune responses and limiting immunogenic response in T cells. Here, we report the anti-cancer effects of γ-PGA combined with PD-1 blockade in murine colon adenocarcinoma and murine melanoma. Material and methods: It was performed to evaluate the in vivo combinatorial anti-cancer effects by oral administration of γ-PGA and intraperitoneal injection of anti-PD-1 in C57BL/6N mice transplanted with MC38 cell, a colorectal cancer cell. And the another study was carried out to examine the synergetic therapeutic efficacy by γ-PGA and anti-PD-1 against B16F10 melanoma tumor model in mice. Results: Combination therapy with γ-PGA and anti-PD-1 produced significantly longer tumor growth delay than other treatment and almost completely inhibited melanoma tumor growth, and also the improved longterm survival rate was shown compared to γ-PGA or anti-PD-1 alone. Similar experiment was performed in melanoma tumor and the result shows that co-treatment with γ-PGA and anti-PD-1 significantly prolonged survival rate compared with anti-PD-1 alone in MC38 tumor model and dramatically reduced MC38 tumor growth but had no synergistic effect. The results showed that the survival rate of the combinatorial treatment was superior to anti-PD-1 alone. Conclusions: Based on the resistance mechanism of PD-1 blockade with low response rates in many patients, γ-PGA treatment having an innate immune modulating activity combined with anti-PD-1 can induce stronger anti-tumor immunity and destroy the target cancer cells. In this study, oral administration of γ-PGA with intraperitoneal injection of anti-PD-1 dramatically inhibited tumor growth and enhanced the survival rate in MC38 and B16F10 tumor-bearing mice. The study demonstrates that the tumor microenvironment was controlled by administration of γ-PGA, thus results in exertion of immune checkpoint inhibitor. These data provide a strong preclinical evidence for a novel combinatorial therapeutic strategy that can be assessed in prospective clinical trials. No conflict of interest.

Electron tunneling energies of a quantum dot in a magnetic field

Theoretical analysis of the experimental energy data for an electron tunneling into a single electron state and to two interacting electrons state confined by a finite Gaussian potential in a two-dimensional quantum dot subjected to a uniform magnetic field perpendicular to the plane of the dot is presented. While a previously published analytic solution for the magnetic field at which the 2-electron ground state transitions from the spin-singlet to the spin-triplet state agreed well with the data, the calculated energy for an electron tunneling into the two-electron state at higher magnetic fields diverged from the experimental data. The discrepancy was attributed to the magnetic field dependent Fermi energy of the n + electrode of the tunnel capacitor in the experiment. It is shown that the one electron and the two-electron experimental data agree remarkably well with the theory when we use a mechanism in which as if the added electron tunnels from a fixed energy state and the confining potential is a finite Gaussian potential in contrast to the generally used infinite harmonic confining potential. That the tunneling of an electron into the quantum dot from a fixed intermediate state assumed in the analysis is equivalent to the true tunneling picture where a mechanism known as “Coulomb blockade” that regulates the tunneling of one electron at a time into the quantum dot and thus the measured electron tunneling energies are independent of the magnetic field dependent Fermi energy of the n + electrode is discussed. • First time theoretical analysis of experimental electron tunneling energy data of a quantum dot is presented. • Both 1-e and 2-e experimental data fit extremely well with the theoretical results obtained by using only two fit parameters for the Gaussian confining potential. • In addition to the electron tunneling data, the singlet-to-triplet 2-e state transition magnetic field value agrees remarkably well with the theory. • The effect of the finite confining potential compared to the infinite harmonic potential is clearly demonstrated by a significantly better fit with the experimental data. • Our theoretical analysis of the experimental data confirms the mechanism of Coulomb blockade for electron tunneling into a quantum dot.

Abstract P250: Targeting The Thrombospondin-CD47 Axis Attenuates Blood Pressure And Renal Injury In Female Hypertensive (mRen2)27 Rats

Alterations in the extracellular matrix (ECM) can influence vascular stiffness and compromise organ function contributing to hypertension. A key component of the ECM is the type I integral membrane receptor CD47 that regulates the ECM by binding to the matricellular protein thrombospondin-1(TSP1). Increased circulating levels of TSP1 and CD47 activation are associated with cardiac and renal pathologies. Thus, the objective of the current study determined the efficacy of targeting CD47 with an anti-sense morpholinos to reduce blood pressure and renal injury in 20-week old hypertensive (mRen2)27 female rats. The CD47 morpholino (Morph) or the missense probe (Control) were dissolved in sterile PBS and administered by tail vein injection twice weekly for 6 weeks. Systolic blood pressures (SBPs) were monitored in conscious rats by a tail cuff auscultation and expression of RAS components assessed by RT-qPCR; all data are means ± SEM with n=8-10 per group. Prior to treatment, the baseline SBPs did not differ between the Control and Morph groups [196 ± 5 vs.197 ± 4 mmHg; p=0.87]. However, as early as one week of treatment, the CD47 Morph group exhibited a significant reduction in SBPs compared to the Control transgenics [185 ± 5 vs. 204 ± 3 mmHg; p<0.01]. At the end of the 6 week treatment period, SBPs remained significantly lower in the CD47 Morph group compared to the Controls [185 ± 4 vs.199 ± 3 mmHg; p<0.02]. In addition, CD47 blockade was associated with reduced proteinuria in the CD47 Morph compared to Controls [7 ± 1 vs. 26 ± 7 mg protein/24 hr excretion; p<0.05]. Regarding the mechanism of CD47 blockade to reduce blood pressure and proteinuria, mRNA analysis revealed lower renal renin mRNA levels in the CD47 Morph versus the Control group [0.54 ± 0.11 vs.1.04 ± 0.11; p<0.01]. Moreover, CD47 Morph treatment significantly reduced the mRNA levels of renal ACE [0.78 ± 0.13 vs.1.05 ± 0.14; p<0.05] and the renal AT1A receptor [0.74 ± 0.07 vs.1.05 ± 0.12; p<0.05], however, renal angiotensinogen mRNA levels were not different among the CD47 Morph and Controls [1.04.± 0.10 vs. 1.16 ± 0.20, p=0.83]. We conclude that silencing CD47 attenuates hypertension and renal injury in the female (mRen2)27 potentially by reducing expression of the renin-ACE-AT1 receptor axis within the kidney.

Abstract 5534: NTX-1088, a first-in-class anti-PVR mAb mediates DNAM1-dependent antitumor immunity

Abstract Poliovirus receptor (PVR, CD155), expressed on the surface of cancer cells, represents a resistance mechanism to approved immune checkpoint inhibitors (ICIs). It is a key regulator of immune activation, that modifies function through multiple mechanisms. Increased PVR expression levels have been associated with resistance to anti-PD-(L)1 therapy, while loss of PVR led to reduced tumor growth. Targeting PVR using mAbs offers an intriguing therapeutic approach for patients with advanced cancers, who are not responding to other ICIs.NTX-1088 is a potent, first-in-class, anti-PVR mAb, developed for the treatment of patients with locally advanced and metastatic solid tumors. The antibody binds PVR with high affinity, blocks its interactions with TIGIT and CD96, and preventing their inhibitory signaling. Moreover, NTX-1088 block the critical interaction between PVR and DNAM1 (CD226). This blockade prevents internalization of DNAM1, restores its expression on the surface of immune cells and results in a robust antitumor activity.NTX-1088 was thoroughly investigated in vitro and in vivo. Various cancer cell lines were co-cultured with immune effector cells from healthy human donors, in the presence of NTX-1088, alone and in combination with anti-PD-1 mAb (pembrolizumab). NTX-1088 significantly increased immune cell activation, as measured by IFN-gamma release from activated polyclonal CD8+ T cells, induction of CD137 and killing of tumor cells. When tested in combination with pembrolizumab, NTX-1088 further improved all measured activation parameters. Synergistic effect was obtained when NTX-1088 was combined with the anti-CD112R mAb, NTX-2R13. The effect surpassed that of TIGIT and CD112R combined inhibition. When compared to anti-TIGIT mAb (tiragolumab), NTX-1088 demonstrated clear superiority in activating T and NK cells as a single agent. Furthermore, NTX-1088 in combination with pembrolizumab, was superior to the combination of pembrolizumab with tiragolumab. Importantly, NTX-1088 was the only intervention that significantly restored DNAM1 levels, leading to an enhanced antitumor immune surveillance. Numerous humanized murine models, including pembrolizumab-insensitive models, confirmed the above observations; NTX-1088 exhibited a robust tumor growth inhibition, accompanied by significantly higher prevalence of CD137+, DNAM1+, CD8+ T cells within the tumor bed, compared to mice treated by other ICIs. This is the first report of drug-induced DNAM1 restoration and immune activation. NTX-1088 shows, for the first time, exclusive triple mechanism of action, whereby simultaneous and effective blockade of TIGIT and CD96 is complemented by the efficient restoration of DNAM1. This is a step change in antitumor immune activation that provides a remarkable and differentiated addition to the armamentarium available to patients and their treating oncologists. An IND will be open during 2Q2022. Citation Format: Akram Obiedat, Anas Atieh, Guy Cinamon, Keren Paz, Paola Kucan Brilc, Lea Hirsl, Tihana Lenac Roviš, Ofer Mandelboim, Stipan Jonjic, Pini Tsukerman. NTX-1088, a first-in-class anti-PVR mAb mediates DNAM1-dependent 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 5534.

ENGOT-ov60/GOG-3052/RAMP 201: A phase 2 study of VS-6766 (RAF/MEK clamp) alone and in combination with defactinib (FAK inhibitor) in recurrent low-grade serous ovarian cancer (LGSOC).

TPS5615 Background: Low-grade serous ovarian cancer (LGSOC) constitutes up to 10% of all ovarian cancer and has clinical and molecular characteristics distinct from high-grade serous ovarian cancer. Approximately a third of patients (pts) with recurrent LGSOC harbor KRAS mutations (mt) and pts with KRAS wild-type (wt) LGSOC may have mutations in NRAS, BRAF, or other RAS pathway-associated genes. Prior clinical studies with single agent MEK inhibitors have shown response rates of 16-26% in recurrent LGSOC. VS-6766 is a unique small molecule RAF/MEK clamp that inhibits both RAF and MEK activities by trapping them in inactive complexes. This mechanism of blockade has been shown to limit compensatory MEK activation, thereby potentially enhancing efficacy of MEK inhibition. Focal adhesion kinase (FAK) activation is a putative resistance mechanism to RAF and MEK inhibition, and defactinib, a small molecule inhibitor of FAK, has shown synergistic anti-tumor activity with VS-6766 in preclinical models, including organoids from LGSOC pts. Furthermore, FAK inhibition combined with VS-6766 induces tumor regression in a KRAS mt ovarian cancer xenograft model. The combination of VS-6766 and defactinib is currently being evaluated in the ongoing Investigator Sponsored FRAME study (NCT03875820). In this proof-of-concept study, durable objective responses (ORR = 46%; 11/24) have been reported in recurrent LGSOC pts, including pts who have had a prior MEK inhibitor (Banerjee ESMO 2021) and the combination of VS-6766 + defactinib has received FDA Breakthrough Therapy Designation for recurrent LGSOC. These initial preclinical and clinical results support the ongoing phase 2 ENGOT-ov60/GOG-3052 in recurrent LGSOC. Methods: This is an international phase 2, adaptive, multicenter, randomized, open label study designed to evaluate the efficacy and safety of VS-6766 vs VS-6766 in combination with defactinib currently open to enrollment (NCT04625270). The study will be conducted in two parts. Part A will determine the optimal regimen based on confirmed overall response rate (independent radiology review) in KRAS mt and KRAS wt LGSOC. Part B will determine the efficacy of the optimal regimen identified in Part A in KRAS mt and KRAS wt LGSOC. The minimum expected enrollment is 104 pts, 52 pts with KRAS mt and 52 KRAS wt (64 pts in Part A and 40 pts in Part B). Pts will be randomized to receive VS-6766 (4.0 mg orally (PO), twice weekly 3 wks on, 1 wk off) or VS-6766 with defactinib (VS-6766 3.2 mg PO, twice weekly + defactinib 200 mg PO BID 3 wks on, 1 wk off) till progression. Key inclusion criteria include histologically confirmed LGSOC, known KRAS mutation status, prior systemic therapy including platinum for metastatic disease and up to 1 prior line of MEK inhibitor therapy permitted. Part A of this study has completed enrollment and Part B is currently enrolling pts. Clinical trial information: NCT04625270.

259-LB: Inhibition of the Polyamine-Hypusine Circuit Delimits Islet ß-Cell Stress in the Context of T1D via Suppression of IRE-1a Signaling

Recent data suggest that stress-induced molecular pathways in the β cell may trigger the display of neoantigens that initiate autoimmunity in type 1 diabetes (T1D) . Polyamines (putrescine, spermidine, and spermine) are biogenic amines that participate in stress-induced mRNA translation, in part, through the posttranslational hypusine modification of eIF5A. Rate-limiting enzymes in this circuit include the ODC, which converts Arg to polyamines, and DHPS, which controls eIF5A hypusination. Previously, we showed that inhibition of ODC or DHPS in non-obese diabetic mice using either DFMO or GC7, respectively, resulted in preserved β-cell function, enhanced β-cell mass, and significantly reduced T1D incidence. DFMO treatment subjects with recent-onset T1D was suggestive of reductions in β cell stress. We hypothesized that polyamine depletion with DFMO reduces inflammatory stress in β cells and the consequent accumulation of unhypusinated eIF5A limits stress-related protein production. To test the hypothesis, we treated the human islets with proinflammatory cytokine (PIC) cocktail to mimic T1D in presence or absence of 5 mM DFMO. We observed induction of ER stress as shown by increased expression of unfolded protein response proteins within 24 h of PIC treatment. Treatment with DFMO reduced protein levels of IRE1α and downstream XBP1. Gene expression analysis showed significant downregulation of genes in the IRE1α pathway (CHOP, TNXIP, and XBP1s) . To clarify a mechanism underlying suppression of stress protein production by unhypusinated eIF5A, we treated HEK cells with DFMO or GC7, both of which enhanced the association of unhypusinated eIF5A with GCN2, an eIF2α kinase, suggesting a non-ER stress mediated mechanism of mRNA translational blockade. Collectively, these data suggest that inhibition of the polyamines-hypusine circuit suppresses ER stress via IRE1α signaling and enhances non-ER stress mediated suppression of mRNA translation. Disclosure A. Anderson: None. A. Kulkarni: None. R. G. Mirmira: None.

PD33-03 IS ANGIOTENSIN SYSTEM INHIBITOR USE ASSOCIATED WITH IMPROVED OUTCOMES IN PATIENTS WITH LOCALIZED CLEAR CELL RCC?

You have accessJournal of UrologyCME1 May 2022PD33-03 IS ANGIOTENSIN SYSTEM INHIBITOR USE ASSOCIATED WITH IMPROVED OUTCOMES IN PATIENTS WITH LOCALIZED CLEAR CELL RCC? Paige Nichols, Andrew Zganjar, Christine Lohse, Sounak Gupta, John Cheville, Cameron Britton, Aaron Potretzke, Stephen Boorjian, Houston Thompson, Bradley Leibovich, and Vidit Sharma Paige NicholsPaige Nichols More articles by this author , Andrew ZganjarAndrew Zganjar More articles by this author , Christine LohseChristine Lohse More articles by this author , Sounak GuptaSounak Gupta More articles by this author , John ChevilleJohn Cheville More articles by this author , Cameron BrittonCameron Britton More articles by this author , Aaron PotretzkeAaron Potretzke More articles by this author , Stephen BoorjianStephen Boorjian More articles by this author , Houston ThompsonHouston Thompson More articles by this author , Bradley LeibovichBradley Leibovich More articles by this author , and Vidit SharmaVidit Sharma More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002584.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Preclinical studies demonstrate VEGF expression is regulated by angiotensin II. Increased angiotensin II receptor expression in clear cell renal cell carcinoma (ccRCC) may be associated with higher tumor grade and poorer survival. It is unknown if angiotensin system inhibitors, in the form of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs), are associated with improved outcomes for hypertensive patients with ccRCC. METHODS: A large nephrectomy registry was queried to identify adults treated with radical or partial nephrectomy for localized ccRCC from 2005-2012. Patients were divided into three groups: (1) patients without HTN, (2) patients with HTN but not treated with ACEIs or ARBs, and (3) patients with HTN treated with ACEIs or ARBs. The primary outcome of disease progression-free survival (PFS) and secondary outcomes of cancer-specific survival and CKD stage PFS were estimated using the Kaplan-Meier method. Associations with disease progression, death from RCC, and CKD stage progression were evaluated using multivariable Cox models. We adjusted for a disease progression score and a death from RCC score in the multivariable models for disease progression and death from RCC, respectively. RESULTS: Among 1,203 patients, there were 432 (36%), 359 (30%), and 412 (34%) in groups 1, 2, and 3, respectively, with a median follow-up of 10.1 (IQR 7.9-12.5) years. Disease PFS for the three groups is shown in Figure 1. On multivariable analysis, group 3 was associated with improved disease PFS compared to group 2 (HR 0.76, 95% CI 0.57-1.00, p=0.05) but not group 1 (HR 0.89, 95% CI 0.68-1.17, p=0.40). A subset multivariable analysis of group 3 treated with ACEIs alone relative to group 2 demonstrated improved disease PFS (HR 0.68, 95% CI 0.49-0.93, p=0.02). There were no significant differences between groups for either cancer specific survival or CKD stage PFS on multivariable analysis. CONCLUSIONS: The use of ACEIs or ARBs is associated with improved disease PFS in patients with HTN and localized ccRCC after radical or partial nephrectomy. No appreciable differences in cancer specific survival or CKD stage PFS were identified. Further studies are warranted to validate this finding and elucidate underlying mechanisms of angiotensin blockade in ccRCC patients. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e553 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Paige Nichols More articles by this author Andrew Zganjar More articles by this author Christine Lohse More articles by this author Sounak Gupta More articles by this author John Cheville More articles by this author Cameron Britton More articles by this author Aaron Potretzke More articles by this author Stephen Boorjian More articles by this author Houston Thompson More articles by this author Bradley Leibovich More articles by this author Vidit Sharma More articles by this author Expand All Advertisement PDF DownloadLoading ...