First-in-Human Phase I/II ICONIC Trial of the ICOS Agonist Vopratelimab Alone and with Nivolumab: ICOS-High CD4 T-Cell Populations and Predictors of Response.

PURPOSE: The first-in-human phase I/II ICONIC trial evaluated an investigational inducible costimulator (ICOS) agonist, vopratelimab, alone and in combination with nivolumab in patients with advanced solid tumors. PATIENTS AND METHODS: In phase I, patients were treated with escalating doses of intravenous vopratelimab alone or with nivolumab. Primary objectives were safety, tolerability, MTD, and recommended phase II dose (RP2D). Phase II enriched for ICOS-positive (ICOS+) tumors; patients were treated with vopratelimab at the monotherapy RP2D alone or with nivolumab. Pharmacokinetics, pharmacodynamics, and predictive biomarkers of response to vopratelimab were assessed. RESULTS: ICONIC enrolled 201 patients. Vopratelimab alone and with nivolumab was well tolerated; phase I established 0.3 mg/kg every 3 weeks as the vopratelimab RP2D. Vopratelimab resulted in modest objective response rates of 1.4% and with nivolumab of 2.3%. The prospective selection for ICOS+ tumors did not enrich for responses. A vopratelimab-specific peripheral blood pharmacodynamic biomarker, ICOS-high (ICOS-hi) CD4 T cells, was identified in a subset of patients who demonstrated greater clinical benefit versus those with no emergence of these cells [overall survival (OS), P = 0.0025]. A potential genomic predictive biomarker of ICOS-hi CD4 T-cell emergence was identified that demonstrated improvement in clinical outcomes, including OS (P = 0.0062). CONCLUSIONS: Vopratelimab demonstrated a favorable safety profile alone and in combination with nivolumab. Efficacy was observed only in a subset of patients with a vopratelimab-specific pharmacodynamic biomarker. A potential predictive biomarker of response was identified, which is being prospectively evaluated in a randomized phase II non-small cell lung cancer trial. See related commentary by Lee and Fong, p. 3633.

ICOS agonism by JTX-2011 (vopratelimab) requires initial T cell priming and Fc cross-linking for optimal T cell activation and anti-tumor immunity in preclinical models.

Immunotherapy checkpoint inhibitors, such as antibodies targeting PD-1 and CTLA-4, have demonstrated the potential of harnessing the immune system to treat cancer. However, despite encouraging results particularly with respect to survival, only a minority of patients benefit from these therapies. In clinical studies aimed at understanding changes in the immune system following immunotherapy treatment, ICOS (Inducible T cell CO-Stimulator) was shown to be significantly up-regulated on CD4+ T cells and this was associated with clinical activity, indicating that ICOS stimulatory activity may be beneficial in the treatment of solid tumors. In this report, we describe the generation of specific, species cross-reactive, agonist antibodies to ICOS, including the humanized clinical candidate, JTX-2011 (vopratelimab). Preclinical studies suggest that the ICOS stimulating antibodies require Fc receptor cross-linking for optimal agonistic activity. Notably, the ICOS antibodies do not exhibit superagonist properties but rather require T cell receptor (TCR)-mediated upregulation of ICOS for agonist activity. Treatment with the ICOS antibodies results in robust anti-tumor benefit and long-term protection in preclinical syngeneic mouse tumor models. Additional benefit is observed when the ICOS antibodies are administered in combination with anti-PD-1 and anti-CTLA-4 therapies. Based on the preclinical data, JTX-2011 is currently being developed in the clinical setting for the treatment of solid tumors.

Differentially expressed mRNAs and lncRNAs shared between activated human hepatic stellate cells and nash fibrosis.

We previously reported dysregulated expression of liver-derived messenger RNA (mRNA) and long noncoding RNA (lncRNA) in patients with advanced fibrosis resulting from nonalcoholic fatty liver disease (NAFLD). Here we sought to identify changes in mRNA and lncRNA levels associated with activation of hepatic stellate cells (HSCs), the predominant source of extracellular matrix production in the liver and key to NAFLD-related fibrogenesis. We performed expression profiling of mRNA and lncRNA from LX-2 cells, an immortalized human HSC cell line, treated to induce phenotypes resembling quiescent and myofibroblastic states. We identified 1964 mRNAs (1377 upregulated and 587 downregulated) and 1460 lncRNAs (665 upregulated and 795 downregulated) showing statistically significant evidence (FDR ≤0.05) for differential expression (fold change ≥|2|) between quiescent and activated states. Pathway analysis of differentially expressed genes showed enrichment for hepatic fibrosis (FDR = 1.35E-16), osteoarthritis (FDR = 1.47E-14), and axonal guidance signaling (FDR = 1.09E-09). We observed 127 lncRNAs/nearby mRNA pairs showing differential expression, the majority of which were dysregulated in the same direction. A comparison of differentially expressed transcripts in LX-2 cells with RNA-sequencing results from NAFLD patients with or without liver fibrosis revealed 1047 mRNAs and 91 lncRNAs shared between the two datasets, suggesting that some of the expression changes occurring during HSC activation can be observed in biopsied human tissue. These results identify lncRNA and mRNA expression patterns associated with activated human HSCs that appear to recapitulate human NAFLD fibrosis.

AEBP1 expression increases with severity of fibrosis in NASH and is regulated by glucose, palmitate, and miR-372-3p.

Factors governing the development of liver fibrosis in nonalcoholic steatohepatitis (NASH) are only partially understood. We recently identified adipocyte enhancer binding protein 1 (AEBP1) as a member of a core set of dysregulated fibrosis-specific genes in human NASH. Here we sought to investigate the relationship between AEBP1 and hepatic fibrosis. We confirmed that hepatic AEBP1 expression is elevated in fibrosis compared to lobular inflammation, steatosis, and normal liver, and increases with worsening fibrosis in NASH patients. AEBP1 expression was upregulated 5.8-fold in activated hepatic stellate cells and downregulated during chemical and contact induction of biological quiescence. In LX-2 and HepG2 cells treated with high glucose (25 mM), AEBP1 expression increased over 7-fold compared to normal glucose conditions. In response to treatment with either fructose or palmitate, AEBP1 expression in primary human hepatocytes increased 2.4-fold or 9.6-fold, but was upregulated 55.8-fold in the presence of fructose and palmitate together. AEBP1 knockdown resulted in decreased expression of nine genes previously identified to be part of a predicted AEBP1-associated NASH co-regulatory network and confirmed to be upregulated in fibrotic tissue. We identified binding sites for two miRNAs known to be downregulated in NASH fibrosis, miR-372-3p and miR-373-3p in the AEBP1 3' untranslated region. Both miRNAs functionally interacted with AEBP1 to regulate its expression. These findings indicate a novel AEBP1-mediated pathway in the pathogenesis of hepatic fibrosis in NASH.

Chemokine ligand 20 (CCL20) expression increases with NAFLD stage and hepatic stellate cell activation and is regulated by miR-590-5p.

CCL20 (CC chemokine ligand 20) is emerging as an important regulatory molecule in a pathway common to virus infection, alcoholic hepatitis, and non-alcoholic fatty liver disease (NAFLD) leading to the development of hepatic fibrosis. We previously observed upregulation of CCL20 in patients with NAFLD fibrosis and human hepatic stellate cells (LX-2 cells) in response to lipid loading. To date, the mechanisms mediating the relationship between CCL20 and hepatic fibrogenesis remain unknown. In this study, we sought to characterize the molecular mechanisms by which CCL20 may contribute to fibrogenesis in NAFLD. We observed that CCL20 levels increased with worsening severity of liver histology in NAFLD patients (normal < steatosis < inflammation < fibrosis) and during LX-2 cell activation in a time-dependent manner. We found that treatment of LX-2 cells with CCL20 corresponded with increased levels of CCL20 and ACTA2, and decreased levels of PLAU and SERPINE1, effects mitigated by CCL20 knockdown. We identified a putative binding site for miR-590-5p, which we previously reported to be downregulated in NAFLD fibrosis, in the CCL20 3' untranslated region (3'UTR), and found that exogenous miR-590-5p functionally interacted with the CCL20 3'UTR to downregulate its expression. Transfection of LX-2 hepatic stellate cells with miR-590-5p mimic or silencing RNA resulted in decreased or increased CCL20 levels, respectively. Our results indicate an association between CCL20 and hepatic stellate cell activation that includes modulation of key ECM components and functional interactions with a miRNA previously implicated in NAFLD fibrosis. Together, these findings support a novel mechanism by which CCL20 may promote fibrogenesis in NAFLD.

The Role of Long Non-Coding RNAs (lncRNAs) in the Development and Progression of Fibrosis Associated with Nonalcoholic Fatty Liver Disease (NAFLD).

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of conditions ranging from hepatic steatosis to inflammation (nonalcoholic steatohepatitis or NASH) with or without fibrosis, in the absence of significant alcohol consumption. The presence of fibrosis in NASH patients is associated with greater liver-related morbidity and mortality; however, the molecular mechanisms underlying the development of fibrosis and cirrhosis in NAFLD patients remain poorly understood. Long non-coding RNAs (lncRNAs) are emerging as key contributors to biological processes that are underpinning the initiation and progression of NAFLD fibrosis. This review summarizes the experimental findings that have been obtained to date in animal models of liver fibrosis and NAFLD patients with fibrosis. We also discuss the potential applicability of circulating lncRNAs to serve as biomarkers for the diagnosis and prognosis of NAFLD fibrosis. A better understanding of the role played by lncRNAs in NAFLD fibrosis is critical for the identification of novel therapeutic targets for drug development and improved, noninvasive methods for disease diagnosis.

Interleukins 7 and 15 Maintain Human T Cell Proliferative Capacity through STAT5 Signaling.

T lymphocytes require signals from self-peptides and cytokines, most notably interleukins 7 and 15 (IL-7, IL-15), for survival. While mouse T cells die rapidly if IL-7 or IL-15 is withdrawn, human T cells can survive prolonged withdrawal of IL-7 and IL-15. Here we show that IL-7 and IL-15 are required to maintain human T cell proliferative capacity through the STAT5 signaling pathway. T cells from humanized mice proliferate better if stimulated in the presence of human IL-7 or IL-15 or if T cells are exposed to human IL-7 or IL-15 in mice. Freshly isolated T cells from human peripheral blood lose proliferative capacity if cultured for 24 hours in the absence of IL-7 or IL-15. We further show that phosphorylation of STAT5 correlates with proliferation and inhibition of STAT5 reduces proliferation. These results reveal a novel role of IL-7 and IL-15 in maintaining human T cell function, provide an explanation for T cell dysfunction in humanized mice, and have significant implications for in vitro studies with human T cells.

Ultrasound or near-infrared vascular imaging to guide peripheral intravenous catheterization in children: a pragmatic randomized controlled trial.

BACKGROUND: Peripheral intravenous catheterization in children is challenging, and success rates vary greatly. We conducted a pragmatic randomized controlled trial to determine whether the use of ultrasound or near-infrared vascular imaging to guide catheterization would be more effective than the standard approach in achieving successful catheter placement on the first attempt. METHODS: We enrolled a convenience sample of 418 children in a pediatric emergency department who required peripheral intravenous catheterization between June 2010 to August 2012. We stratified them by age (≤ 3 yr and > 3 yr) and randomly assigned them to undergo the procedure with the standard approach, or with the help of either ultrasound or near-infrared vascular imaging. The primary outcome was the proportion of patients who had successful placement of a catheter on the first attempt. RESULTS: The rate of successful first attempts did not differ significantly between either of the 2 intervention groups and the standard approach group (differences in proportions -3.9%, 95% confidence interval [CI] -14.2% to 6.5%, for ultrasound imaging; -8.7%, 95% CI -19.4% to 1.9%, for near-infrared imaging). Among children 3 years and younger, the difference in success rates relative to standard care was also not significant for ultrasound imaging (-9.6%, 95% CI -29.8% to 10.6%), but it was significantly worse for near-infrared imaging (-20.1%, 95% CI -40.1% to -0.2%). Among children older than 3 years, the differences in success rates relative to standard care were smaller but not significant (-2.3%, 95% CI -13.6% to 9.0%, for ultrasound imaging; -4.1%, 95% CI -15.7% to 7.5%, for near-infrared imaging). None of the pairwise comparisons were statistically significant in any of the outcomes. INTERPRETATION: Neither technology improved first-attempt success rates of peripheral intravenous catheterization in children, even in the younger group. These findings do not support investment in these technologies for routine peripheral intravenous catheterization in children. TRIAL REGISTRATION: ClinicalTrials.gov, no. NCT01133652.

9-cis-retinoic Acid and troglitazone impacts cellular adhesion, proliferation, and integrin expression in K562 cells.

Retinoids are established pleiotropic regulators of both adaptive and innate immune responses. Recently, troglitazone, a PPAR gamma agonist, has been demonstrated to have anti-inflammatory effects. Separately, retinoids and troglitazone are implicated in immune related processes; however, their combinatory role in cellular adhesion and proliferation has not been well established. In this study, the effect of 9-cis-retinoic acid (9-cis-RA) and troglitazone on K562 cellular adhesion and proliferation was investigated. Troglitazone exposure decreased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin, FN-120, and vitronectin in a concentration and time-dependent manner. In the presence of troglitazone, 9-cis-retinoic acid restores cellular adhesion to levels comparable to vehicle treatment alone on fibronectin, FN-120, and vitronectin substrates within 72 hours. Due to the prominent role of integrins in attachment to extracellular matrix proteins, we evaluated the level of integrin α5 subunit expression. Troglitazone treatment results in decrease in α5 subunit expression on the cell surface. In the presence of both agonists, cell surface α5 subunit expression was restored to levels comparable to vehicle treatment alone. Additionally, troglitazone and 9-cis-RA mediated cell adhesion was decreased in the presence of a function blocking integrin alpha 5 inhibitor. Further, through retinoid metabolic profiling and HPLC analysis, our study demonstrates that troglitazone augments retinoid availability in K562 cells. Finally, we demonstrate that troglitazone and 9-cis-retinoic acid synergistically dampen cellular proliferation in K562 cells. Our study is the first to report that the combination of troglitazone and 9-cis-retinoic acid restores cellular adhesion, alters retinoid availability, impacts integrin expression, and dampens cellular proliferation in K562 cells.