Therapeutic Blockade Research Articles

Candida infections in patients with psoriasis and psoriatic arthritis treated with interleukin-17 inhibitors and their practical management.

Interleukin 17A (IL-17A) is a pro-inflammatory cytokine produced by helper T cells (Th17) and other cells of the immune system and exerts pleiotropic effects on multiple cell lines. The role of IL-17 in the pathogenesis of numerous inflammatory disorders is well-documented. IL-17 activates signaling through the IL-17 receptor, which induces other proinflammatory cytokines, antimicrobial peptides, and neutrophil chemokines that are important for antifungal activity. Healthy levels of IL-17 can protect the host against extracellular bacterial and fungal infections in mucous membranes and epithelia. IL-17 deficiency reduces control of certain infections, while excessive IL-17 can produce unwanted inflammatory effects. Although the efficacy of the therapeutic blockade of this cytokine has been proven in several autoimmune diseases such as psoriasis and psoriatic arthritis, this strategy could also exacerbate fungal infections in such patients. Therefore, a better understanding of IL-17-mediated immunity to Candida is necessary for the development of autoimmune therapeutics that maintain antifungal immunity. In this review, we include a study of the new anti-IL-17 biological agents (secukinumab, ixekizumab, and bromalizumab) used for moderate-to-severe psoriasis and psoriatic arthritis treatment in clinical practice, as well as pivotal trials with bimekizumab. We study the relationship of these biological agents and the appearance of candidiasis in its various clinical forms.

Covid-19: Anti-TNF drug adalimumab to be trialled for patients in the community.

<h3>Background</h3> The pancreatic cancer vaccine, GVAX, induces novel lymphoid aggregates in the otherwise immune quiescent pancreatic ductal adenocarcinoma (PDAC). GVAX also upregulates the PD-1/PD-L1 pathway, and a pre-clinical model demonstrated the anti-tumor effects of combination GVAX and anti-PD-1 antibody therapy (GVAX/αPD-1). Resistance to GVAX was associated with an immune-suppressive myeloid cell infiltration, which may limit further therapeutic gains of GVAX/αPD-1 therapy. The expression of CSF-1R, a receptor important for myeloid cell migration, differentiation and survival, and the effect of its therapeutic blockade in the context of GVAX in PDAC has not been investigated. <h3>Methods</h3> Lymphoid aggregates appreciated in 24 surgically resected PDAC from patients who received one dose of neoadjuvant GVAX were analyzed with multiplex immunohistochemistry. Flow cytometry analysis of tumor infiltrating T-cells in a murine model of PDAC was performed to investigate the therapeutic effects and mechanism of anti-CSF-1R/anti-PD-1/GVAX combination immunotherapy. <h3>Results</h3> High CSF-1R expression in resected PDAC from patients who received neoadjuvant GVAX was associated with a higher myeloid to lymphoid cell ratio (<i>p</i> &lt; 0.05), which has been associated with poorer survival. This higher CSF-1R expression was associated with a higher intra-tumoral infiltration of immature dendritic cells (<i>p</i> &lt; 0.05), but not mature dendritic cells (<i>p</i> = 0.132). In the pre-clinical murine model, administering anti-CSF-1R antibody prior to and after GVAX/αPD-1 (“pre/post-αCSF-1R + αPD-1 + GVAX”) enhanced the survival rate compared to GVAX/αPD-1 dual therapy (<i>p</i> = 0.005), but administering anti-CSF-1R only before GVAX/αPD-1 did not (<i>p</i> = 0.41). The “pre/post-αCSF-1R + αPD-1 + GVAX” group also had higher intra-tumoral infiltration of PD-1 + CD8+ and PD-1 + CD4+ T-cells compared to αPD-1/GVAX (<i>p</i> &lt; 0.001). Furthermore, this regimen increased the intra-tumoral infiltration of PD-1 + CD137 + CD8+, PD-1 + CD137 + CD4+ and PD-1 + OX40 + CD4+ T-cells (<i>p</i> &lt; 0.001). These PD-1 + CD137 + CD8+ T-cells expressed high levels of interferon-γ (median 80–90%) in response to stimulation with CD3/CD28 activation beads, and this expression was higher than that of PD-1 + CD137-CD8+ T-cells (<i>p</i> &lt; 0.001). <h3>Conclusions</h3> The conversion of exhausted PD-1+ T-cells to CD137+ activated effector T-cells may contribute to the anti-tumor effects of the anti-CSF-1R/anti-PD-1/GVAX combination therapy. Anti-CSF-1R antibody with anti-PD-1 antibody and GVAX have the potential be an effective therapeutic strategy for treatment of PDAC.

Dominant-negative NFKBIA mutation promotes IL-1β production causing hepatic disease with severe immunodeficiency.

Although IKK-β has previously been shown as a negative regulator of IL-1β secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1β expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1β secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1β correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1β release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1β secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

Oxysophocarpine suppresses hepatocellular carcinoma growth and sensitizes the therapeutic blockade of anti-Lag-3 via reducing FGL1 expression.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with limited effective treatments and ranks as the second most lethal tumor. Immunotherapy has brought great hope for HCC treatment. Oxysophocarpine is a bioactive alkaloid which poses various pharmacological functions including neuroprotective, anti‐virus, anti‐convulsant, and anti‐nociception. However, there is little systematic study of Oxysophocarpine against HCC and its underlying potential and mechanism combined with immunotherapy in HCC treatment remain poorly unknown. This study was aimed to investigate whether Oxysophocarpine can distinctly suppress HCC cells and sensitize the immunotherapy of CD8+ T cells against HCC. We used HepG2, Hepa1‐6, and primary CD8+ T cells to perform in vitro assays and Hepa1‐6 subcutaneous tumor to conduct in vivo assay. Oxysophocarpine inhibited the proliferation and increased the apoptosis of HepG2 and Hepa1‐6 cells, meanwhile suppressed the migration of HepG2 and Hepa1‐6 cells. Oxysophocarpine sensitized the Lag‐3 immunotherapy effect of CD8+ T cells against HCC in vivo and in vitro by decreasing Fibrinogen‐like protein 1 (FGL1) expression through downregulating IL‐6‐mediated JAK2/STAT3 signaling, whereas Oxysophocarpine treatment had a little effect of CD8+ T cells cytotoxicity function against HCC with PD‐1, Tim‐3, or TIGIT blockade. Our studies provided preclinical basis for clinical application of Oxysophocarpine.

Abstract 917: Functional blocking of CD5 on T cells to enhance the efficacy of therapeutic PD-1 blockade in treatment of mouse 4T1 breast tumors

Abstract Blockade of programmed cell death protein 1 (PD-1) is approved for treatment of multiple human cancers and is the focus of multiple studies due to its key role in T cell function. However, only patients with ‘'hot'' tumors (i.e. those with increased numbers of tumor-infiltrating CD8+ T cells) respond well to the blockade. This suggests that new strategies to increase infiltration of CD8+ T cells into tumors will increase the number of tumor types and patients benefiting from anti-PD-1 therapy. CD5, a member of the scavenger receptor cysteine-rich (SRCR) superfamily, is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signaling and impair cytotoxic T lymphocyte (CTL) activation. CD5 knockout mice have increased anti-tumor immunity and reduced homograft tumor growth: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumor response and increase tumor-infiltrating CD8+ T cells. We report that in vivo administration of anti-CD5 blocking MAb treatment increased primary T cell activation in response to 4T1 tumor cell homografts and ex vivo activation as measured by increased CD69, Fas, Fas ligand, IFNγ, PD-1, and apoptosis. Further, anti-CD5 treatment enhanced the capacity of primary T cells to kill 4T1 tumor cells ex vivo. Mice receiving anti-CD5 and anti-PD-1 in combination exhibited increased overall survival compared to mice treated with either agent alone. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumor immunity and PD-1 blockade treatment. Citation Format: Faizah Alotaibi, Mateusz Rytelewski, Rene Figueredo, Ronak Zareardalan1, Saman Maleki Vareki, Xiufen Zheng, Meng Zhang, Peter Ferguson, Mikal El-Hajjar, Yousef Najajreh, Wei-ping Min, James Koropatnick. Functional blocking of CD5 on T cells to enhance the efficacy of therapeutic PD-1 blockade in treatment of mouse 4T1 breast tumors [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 917.

Abstract 3052: Targeting AXL in PIK3R2-amplified ovarian cancer

Abstract The p85 regulatory subunit and its associated p110 catalytic subunit are components of the class IA PI3K that mediates the downstream signal of receptor tyrosine kinases. PIK3R2 (encoding p85β, one of the p85 isoforms) is commonly amplified in ovarian cancer patients. Importantly, high PIK3R2 expression significantly correlated with worse patient survival. Here, we showed that p85β promoted tumorigenic properties of ovarian cancer cells. The protein expression of a receptor tyrosine kinase AXL was positively regulated by p85β. AXL is a member of the TAM receptor family. The regulation was specific to AXL because the other two members (Tyro3 and MERTK) were not altered by p85β. Drug sensitivity assays suggested that inhibition of AXL reduced oncogenic activities of p85β in vitro and in vivo. Together, we propose p85β as a potential therapeutic target in ovarian cancer and therapeutic blockade of AXL signaling may be an effective strategy for ovarian cancer with PIK3R2 amplification. Citation Format: Ling Rao, Victor C. Mak, Yuan Zhou, Dong Zhang, Xinran Li, Yiling Lu, Gordon B. Mills, Lydia W. Cheung. Targeting AXL in PIK3R2-amplified ovarian cancer [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 3052.

PD-L1 and CD8-Tumor-Infiltrating Lymphocytes Expression in Laryngeal Squamous Cell Carcinoma

Abstract Studies in recent years have shown that the immune system plays an important role in the prognosis of patients with different types of malignancies. The tumor immune microenvironment has been studied in various cancers in an attempt to find new antitumoral therapies. The therapeutic blockade of PD-1/PD-L1 has revolutionized the treatment of oncological pacients, with anti-PD-L1 monoclonal antibodies having remarkable results in malignant melanoma, cervical, renal and lung cancer. In laryngeal cancer, few reports are available on the immune microenvironment and PD-L1 expression. Considering these, we retrospectively review 23 laryngeal cancer patients with laryngectomy performed in the Otorhinolaryngology Department - Emergency County Clinical Hospital St Andrew Constanta. The aim of our research was to evaluate the immunohistochemical expression of PD-L1, using combined positive score which includes both viable tumoral cells and intratumoral lymphocytes, as well as the level of CD8 intratumoral lymphocytes and to analise the corelation between these markers and the clinicopathological characteristics of the pacients, in order to set these two parameters, in the near future, as new biomarkers. PD-L1 expression was shown to be a prognostic factor for disease-free survival in laryngeal cancer patients (p = 0.006) and was higher in tumors with elevated CD8 lymphocyte levels, with a significant correlation correlation between the two parameters (p = 0.019). The level of CD8 lymphocytes was also a predictive factor for free-disease survival, correlated with patient smoker status (p = 0.04), being higher in non-smoking patients.

Therapeutic blockade of inflammation in severe COVID-19 infection with intravenous N-acetylcysteine

Glucose 6-phosphate dehydrogenase (G6PD) deficiency facilitates human coronavirus infection due to glutathione depletion. G6PD deficiency may especially predispose to hemolysis upon coronavirus disease-2019 (COVID-19) infection when employing pro-oxidant therapy. However, glutathione depletion is reversible by N-acetylcysteine (NAC) administration. We describe a severe case of COVID-19 infection in a G6PD-deficient patient treated with hydroxychloroquine who benefited from intravenous (IV) NAC beyond reversal of hemolysis. NAC blocked hemolysis and elevation of liver enzymes, C-reactive protein (CRP), and ferritin and allowed removal from respirator and veno-venous extracorporeal membrane oxygenator and full recovery of the G6PD-deficient patient. NAC was also administered to 9 additional respirator-dependent COVID-19-infected patients without G6PD deficiency. NAC elicited clinical improvement and markedly reduced CRP in all patients and ferritin in 9/10 patients. NAC mechanism of action may involve the blockade of viral infection and the ensuing cytokine storm that warrant follow-up confirmatory studies in the setting of controlled clinical trials.

Issues of therapy of chronic non-specific lumbodynia

Low back pain or lumbalgia is one of the most common reasons for going to the doctor. Non-specific (musculoskeletal) pain is the most common (90%) cause of chronic lumbalgia. The chronic course of lumbalgia is determined not only by the anatomical sources of pain, but also by psychological and social factors that should be considered when managing patients. In the treatment of chronic nonspecific lumbalgia, a comprehensive multidisciplinary approach is effective, including optimization of drug therapy and motor activity, psychological methods (cognitive-behavioral therapy), an educational program, and manual therapy. When identifying common emotional disorders and insomnia, their therapy is required. Kinesitherapy (therapeutic gymnastics) is the most effective direction in the treatment of chronic nonspecific lumbalgia; оf primary importance are the regularity of physical exercises under the supervision of a specialist, the exclusion of sudden and excessive movements. In combination with activities, regular walking is highly effective. Cognitive-behavioral therapy is the most effective psychological direction in the management of patients with chronic nonspecific lumbalgia. It should be aimed not only at pain, but also at those often found in chronic non-specific lumbalgia insomnia, depression, and anxiety disorders. In some patients with damage to the facet joints, the sacroiliac joint, radiofrequency denervation or therapeutic blockades with anesthetics and corticosteroids can be used. Nonsteroidal anti-inflammatory drugs are used as medicines for chronic nonspecific lumbalgia, when prescribing them, it is necessary to take into account the presence and nature of risk factors for adverse events, concomitant diseases, interaction with other drugs The use of etoricoxib in chronic non-specific lumbalgia is discussed.

Diagnostic blockade under sonographic control in patients with lumbosacral ligamentoses

Objective: to develop standardized methods for performing diagnostic blockades increasing their effectiveness in lumbosacral ligamentoses. Material and methods. The study included the results of sonographically controlled blockades of the iliolumbar (66 patients), posterior long sacroiliac (126 patients), and sacrotuberal ligaments (25 patients). All of the patients had manifestations of low back pain syndrome. Results. The use of the standardized method of therapeutic and diagnostic blockades in all the patients with ligament-induced low back pain syndrome led to a marked decrease in the intensity of the pain syndrome and allowed verification of its cause. Conclusion. Sonographically controlled blockade of the iliolumbar, posterior long sacroiliac, and sacrotuberal ligaments is an effective way to verify and treat low back pain syndrome in lumbosacral ligamentoses.

372-OR: Ablation of the Hepatokine TSK Protects Mice against Obesity and Metabolic Disorders Caused by MC4R Deficiency

Endocrine hormones released by peripheral tissues, such as adipose tissue, skeletal muscle and the liver, play an important role in metabolic crosstalk and homeostasis and contribute to disease pathogenesis. We recently identified Tsukushi (TSK) as a liver-enriched secreted factor that is highly inducible in response to increased energy expenditure. TSK null mice were resistant to diet-induced obesity, insulin resistance, and diet-induced NASH as a result of enhanced sympathetic activation and brown fat thermogenesis. However, the mechanisms underlying TSK action remain incompletely elucidated. Melanocortin 4 receptor (MC4R) is a key regulator of energy balance and its mutations represent the most common monogenic cause of obesity in human. In this study, we generated TSK/MC4R double knockout mice to explore potential crosstalk between the TSK and MC4R signaling pathways. While MC4R-null mice developed severe obesity when fed standard rodent chow, TSK inactivation nearly completely abolished the defects of energy balance caused by MC4R deficiency. Remarkably, ablation of TSK normalized food intake and corrected hepatic steatosis, adipose tissue inflammation, and insulin resistance in MC4R null mice. TSK deficiency also rescued the impairments of thermogenic gene expression caused by MC4R deficiency. Those results suggest that TSK likely acts downstream or in parallel to MC4R signaling to regulate feeding behavior and energy expenditure. Our work supports a rationale for therapeutic blockade of TSK for the treatment of obesity and metabolic disease caused by MC4R mutations. Disclosure Q. Wang: None. J. Lin: None. Funding National Institutes of Health (DK102456, AG055379, DK114220)

Macrolets: Outsized Extracellular Vesicles Released from Lipopolysaccharide-Stimulated Macrophages that Trap and Kill Escherichia coli.

SummaryMacrophages release a variety of extracellular vesicles (EVs). Here we describe a previously unreported class of EVs that are released from macrophages in response to Escherichia coli endotoxin, lipopolysaccharide (LPS), that we have named "macrolets" since they are extruded as large "droplets" released from macrophages. Morphologically, macrolets are anuclear, bounded by a single lipid membrane and structurally dependent on an actin cytoskeleton. Macrolets are enriched in tetraspanins and separable on this basis from their parent macrophages. Macrolets are distinguished from classic exosomes by their larger size (10–30 μm), discoid shape, and the presence of organelles. Macrolets are rich in both interleukin 6 (IL-6) and interleukin 6 receptor (IL-6R),and are capable of trapping and killing E. coli in association with production of reactive oxygen species. Our observations offer insights into the mechanisms by which macrophage activities may be amplified in sites of infection, inflammation, and healing.

STAT3 induces the expression of fatty acid transport proteins (FATP2 and FATP4) to promote myeloid-derived suppressor cells function in tumor

Abstract Myeloid-derived suppressor cells are critical to regulation of immune responses; their accumulation in cancer patients inhibit antitumor immunity and suppress T-cells activity thereby promoting cancer progression and metastases. Metabolic reprogramming has been identified as a crucial factor in modulating MDSCs function; however, how lipid metabolism promotes MDSCs activity during tumor remains unknown. Here we report the upregulation of fatty acid transport proteins (FATP2 and FATP4) enhanced the exogenous uptake of fatty acids resulting in hyperlipidemic MDSCs during tumor. Accumulation of unsaturated fatty acids in GM-CSF-induced MDSCs in-vitro released IL-6, ARG1 and iNOS thereby inhibiting T-cells function. Increased fatty acid uptake in MDSCs induces the expression of FATP2 and FATP4 via activation of STAT3. Interestingly, therapeutic blockade of STAT3 decreased lipid accumulation and repressed FATP2 and FATP4 expression in MDSCs. Also, FATP2 specific inhibitor (Lipofermata) abrogated tumor progression in immunocompetent B16F10-tumor bearing mice in MDSCs dependent manner. Therefore, our data suggests therapeutic manipulation of lipid metabolic signaling pathways in MDSCs could be a promising approach to effective cancer immunotherapy.

Endothelial E-selectin inhibition improves acute myeloid leukaemia therapy by disrupting vascular niche-mediated chemoresistance

The endothelial cell adhesion molecule E-selectin is a key component of the bone marrow hematopoietic stem cell (HSC) vascular niche regulating balance between HSC self-renewal and commitment. We now report in contrast, E-selectin directly triggers signaling pathways that promote malignant cell survival and regeneration. Using acute myeloid leukemia (AML) mouse models, we show AML blasts release inflammatory mediators that upregulate endothelial niche E-selectin expression. Alterations in cell-surface glycosylation associated with oncogenesis enhances AML blast binding to E-selectin and enable promotion of pro-survival signaling through AKT/NF-κB pathways. In vivo AML blasts with highest E-selectin binding potential are 12-fold more likely to survive chemotherapy and main contributors to disease relapse. Absence (in Sele−/− hosts) or therapeutic blockade of E-selectin using small molecule mimetic GMI-1271/Uproleselan effectively inhibits this niche-mediated pro-survival signaling, dampens AML blast regeneration, and strongly synergizes with chemotherapy, doubling the duration of mouse survival over chemotherapy alone, whilst protecting endogenous HSC.

Blocking the death checkpoint protein TRAIL improves cardiac function after myocardial infarction in monkeys, pigs, and rats.

Myocardial infarction (MI) is a leading cause of death worldwide for which there is no cure. Although cardiac cell death is a well-recognized pathological mechanism of MI, therapeutic blockade of cell death to treat MI is not straightforward. Death receptor 5 (DR5) and its ligand TRAIL [tumor necrosis factor (TNF)-related apoptosis-inducing ligand] are up-regulated in MI, but their roles in pathological remodeling are unknown. Here, we report that blocking TRAIL with a soluble DR5 immunoglobulin fusion protein diminished MI by preventing cardiac cell death and inflammation in rats, pigs, and monkeys. Mechanistically, TRAIL induced the death of cardiomyocytes and recruited and activated leukocytes, directly and indirectly causing cardiac injury. Transcriptome profiling revealed increased expression of inflammatory cytokines in infarcted heart tissue, which was markedly reduced by TRAIL blockade. Together, our findings indicate that TRAIL mediates MI directly by targeting cardiomyocytes and indirectly by affecting myeloid cells, supporting TRAIL blockade as a potential therapeutic strategy for treating MI.

Pembrolizumab in patients with non-small-cell lung cancer of performance status 2 (PePS2): a single arm, phase 2 trial

Therapeutic blockade of the axis of programmed cell death 1 (PD-1) and its ligand (PD-L1) has transformed the management of non-small-cell lung cancer (NSCLC). Clinical trials with pembrolizumab have enrolled patients with performance status (PS) 0-1. However, around 18% of patients with NSCLC are PS2, and the activity and safety of pembrolizumab in these patients is unclear. We aimed to evaluate the safety and efficacy of pembrolizumab in these patients. We did a multicentre, single-arm, open-label, phase 2 trial (PePS2) in ten hospitals in the UK, in which patients with NSCLC and a rigorous ascription of PS2 were given pembrolizumab 200 mg every 3 weeks. No masking was used in this trial. We stratified the treatment evaluation by tumour proportion score (TPS) and line of therapy. Co-primary outcomes were: (1) durable clinical benefit (DCB), defined as the occurrence of complete response, partial response, or stable disease that continues until at least the second CT scan scheduled at 18 weeks; and (2) toxicity, defined as the occurrence at any time of treatment-related dose delay or treatment discontinuation due to an adverse event. Analysis included all patients who received any pembrolizumab. As well as reporting simple observed incidence for the co-primary outcomes, DCB and toxicity, we also estimated incidence using a model-based method for correlated binary outcomes. This study is registered with ClinicalTrials.gov, NCT02733159; EudraCT, 2015-002241-55; and ISRCTN, 10047797. Between Jan 4, 2017, and Feb 13, 2018, of 112 patients assessed for eligibility, we recruited 62 patients. 60 patients were evaluable for the co-primary outcomes. Median age was 72 years (IQR 65-75); 33 (55%) of participants were male and 27 (45%) were female. The observed incidence for DCB was 38% (95% CI 21-57) in first-line patients (n=24) and 36% (22-52) in subsequent-line patients (n=36); DCB was 22% (11-41) in patients with a TPS less than 1% (n=27), 47% (25-70) in patients with a TPS of 1-49% (n=15), and 53% (30-75) in patients with a TPS of 50% or greater (n=15). An increase in DCB incidences with TPS was also shown in model-based estimates. Toxicity was observed in 28% (95% CI 19-41) of patients, 11 (18%) of 60 due to dose delay and 6 (10%) of 60 due to drug discontinuation. No grade 5 treatment-related adverse events were observed and no early deaths were attributed to hyperprogression. The most common grade 3-4 adverse events were dyspnoea (n=9), hyponatraemia (n=5), and anorexia (n=4). There were ten serious adverse events considered to be related to treatment, comprising diarrhoea (n=3) and acute kidney injury, adrenal insufficiency, hyperbilirubinaemia, oral mucositis, rash, urinary tract infection, and vomiting (n=1 each). Patients with NSCLC of PS2 are a group of patients of unmet therapeutic need. The PePS2 trial shows that pembrolizumab can be safely administered to these patients, with no increase in the risk of immune-related or other toxicities. Efficacy outcomes are at least as good as those in patients with PS0-1 and the data provides clinicians with the confidence to incorporate pembrolizumab into the treatment pathway of patients with NSCLC of PS2. Merck, Sharp & Dohme.

Dual MicroRNA Blockade Increases Expression of Antioxidant Protective Proteins: Implications for Ischemia-Reperfusion Injury.

MicroRNAs (miRNAs) are short noncoding RNAs which each cause repression of many target genes. Previous work has demonstrated that therapeutic blockade of single miRNAs is possible. miR-24-3p and miR-145-5p are reported to have a detrimental role in ischemia-reperfusion injury. As the action of miRNAs is inhibitory, we hypothesized that dual blockade of both miRNAs could synergistically upregulate shared target genes. Quantification of miRNA expression in donated kidneys was performed using polymerase chain reaction (PCR) panels. Ischemia-reperfusion injury was modeled in vitro by placing human umbilical vein endothelial cells into a hypoxic incubator (1% O2) for 24 hours, with reoxygenation for 6 hours. RNA expression was quantified with reverse transcription quantitative polymerase chain reaction and protein expression assessed with Western blot. Antisense oligonucleotides were used to inhibit miRNAs. miR-24-3p and miR-145-5p were highly expressed in human kidneys following extended cold ischemia. In vitro, hypoxia caused significant upregulation of miR-24-3p (P ≤ 0.001) and miR-145-5p (P ≤ 0.001) and significant downregulation in messenger RNA of shared targets superoxide dismutase 2 (P ≤ 0.001) and heme oxygenase 1 (P ≤ 0.001). These changes were mirrored at the protein level. Dual inhibition of both miR-24-3p and miR-145-5p caused significant upregulation of superoxide dismutase 2 and heme oxygenase 1 protein following hypoxia-reoxygenation; fold change of 3.17 (P ≤ 0.05) and 6.97 (P ≤ 0.05) respectively. Dual inhibition resulted in reduced cellular reactive oxygen species production compared with negative control (P ≤ 0.05) and single blockade of miR-24-3p (P ≤ 0.01) or miR-145-5p (P ≤ 0.05). Dual blockade of 2 miRNAs can act synergistically to increase the expression of shared gene targets. Dual blockade of miR-24-3p and miR-145-5p represents a novel therapeutic option worthy of further research.

B cell-Derived IL35 Drives STAT3-Dependent CD8+ T-cell Exclusion in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy characterized by a paucity of tumor-proximal CD8+ T cells and resistance to immunotherapeutic interventions. Cancer-associated mechanisms that elicit CD8+ T-cell exclusion and resistance to immunotherapy are not well-known. Here, using a Kras- and p53-driven model of PDA, we describe a mechanism of action for the protumorigenic cytokine IL35 through STAT3 activation in CD8+ T cells. Distinct from its action on CD4+ T cells, IL35 signaling in gp130+CD8+ T cells activated the transcription factor STAT3, which antagonized intratumoral infiltration and effector function of CD8+ T cells via suppression of CXCR3, CCR5, and IFNγ expression. Inhibition of STAT3 signaling in tumor-educated CD8+ T cells improved PDA growth control upon adoptive transfer to tumor-bearing mice. We showed that activation of STAT3 in CD8+ T cells was driven by B cell- but not regulatory T cell-specific production of IL35. We also demonstrated that B cell-specific deletion of IL35 facilitated CD8+ T-cell activation independently of effector or regulatory CD4+ T cells and was sufficient to phenocopy therapeutic anti-IL35 blockade in overcoming resistance to anti-PD-1 immunotherapy. Finally, we identified a circulating IL35+ B-cell subset in patients with PDA and demonstrated that the presence of IL35+ cells predicted increased occurrence of phosphorylated (p)Stat3+CXCR3-CD8+ T cells in tumors and inversely correlated with a cytotoxic T-cell signature in patients. Together, these data identified B cell-mediated IL35/gp130/STAT3 signaling as an important direct link to CD8+ T-cell exclusion and immunotherapy resistance in PDA.

Pilot study: deficiency of mannose-binding lectin-dependent lectin pathway, a novel modulator in outcome from pancreatic islet auto-transplantation.

BackgroundNumerous factors influence pancreatic islet survival following auto-transplantation. Of these, the host immune response in the early peri-operative period is one of the most important. In this study we investigated the role of the mannose-binding lectin (MBL)-dependent pathway in a group of total pancreatectomy (TP) islet auto-transplantation (TPIAT) patients and classified them as competent or deficient in MBL activity. Complement pathway activities, MBL protein and inflammatory cytokine concentrations were evaluated from eleven pancreatic islet auto-transplant patients from two institutions.MethodsEleven patients from two institutions were prospectively recruited. Serum was screened at different time points for 29 different cytokines and compared according to their MBL deficient or competent status. Twelve patients from previous TPIAT patients also underwent screening of MBL pathway activity.ResultsA total nine of twenty three patients (39%) were MBL pathway deficient. MCP-1, IL-7 and IL-1a concentrations were significantly lower in the MBL deficient cohort compared to the normal MBL group (P=0.0237, 0.0001 and 0.0051 respectively). IL-6 and IL-8 concentrations were significantly raised in the normal MBL group. MBL functional activity was lower in insulin-independent group compared to the insulin-dependent group.ConclusionsComplement activation is an important, possibly damaging response during intra-portal islet infusion. MBL pathway deficiency appears common in this population and the cytokine response was attenuated in MBL pathway deficient patients. Therapeutic MBL pathway blockade during and following islet auto-transplantation (IAT) may improve islet survival and function and thereby clinical outcome.

Downregulation of PD-L1 via amide analogues of brefelamide: Alternatives to antibody-based cancer immunotherapy.

The therapeutic blockade of immune checkpoint has emerged as an effective treatment option for a broad range of tumors. However, the objective tumor response is still limited to a small number of cases and tumor types. The full utility of monoclonal antibody (mAb)-based treatment is hindered by several inherent limitations. Thus, there is an urgent requirement to explore alternative modalities targeting the same pathways. In the present study, two amide analogues of brefelamide, TPFS-201 and TPFS-202, were identified as small molecular immune checkpoint inhibitors, as they downregulated PD-L1 expression in tumor cells. PD-L1 was suppressed in cancer cells treated with TPFD compounds at both mRNA and protein levels, as detected by reverse transcription quantitative PCR and flow cytometric analysis, respectively. Reporter assays using a PD-L1 promoter luciferase construct confirmed the transcriptional inhibition of PD-L1 by TPFS compunds. TPFS compound-mediated PD-L1 downregulation in cancer cells consequently restored T cell activity, as identified by the reduction of apoptosis and an increase in interleukin-2 promoter activity in Jurkat T cells, which were co-cultured with TPFS compound-treated A549 cells. TPFS compound-mediated PD-L1 inhibition was partially abolished by the disruption of the putative transcriptional co-activator with PDZ (TAZ)/TEA domain (TEAD)-binding motif in the PD-L1 promoter. The inhibitory effect of TPFS compounds on PD-L1 was markedly inhibited in mouse cell lines, which is consistent with previous research demonstrating that PD-L1 regulation by TAZ is not conserved in mice due to distinct promoter sequences flanking the TAZ/TEAD-binding motif. Together, the data of the current study indicated the potential utility of the brefelamide amide analogues as small molecule immune checkpoint inhibitors, thereby providing therapeutic alternatives, which could be used as monotherapy or in combination with mAbs-based treatment.