Asia-Inclusive Clinical Research and Development Enabled by Translational Science and Quantitative Clinical Pharmacology: Toward a Culture That Challenges the Status Quo.

Access lag to innovative therapies in Asian populations continues to present a challenge to global health. Recent progressive changes in the global regulatory landscape, including newer guidelines, are enabling simultaneous global drug development and near-simultaneous global drug registration. The International Conference on Harmonization (ICH) E17 guideline outlines general principles for the design and analysis of multiregional clinical trials (MRCTs). We posit that translational research and quantitative clinical pharmacology tools are core enablers for Asia-inclusive global drug development aligned with ICH E17 principles. Assessment of ethnic sensitivity should be initiated early in the development lifecycle to inform the need for, and extent of, Asian phase I ethno-bridging data. Relevant ethno-bridging data may be generated as standalone Asian phase I trials, as part of Western First-In-Human trials, or under accelerated development settings as a lead-in phase in an MRCT. Quantitative understanding of human clearance mechanisms and pharmacogenetic factors is vital to forecasting ethnic sensitivity in drug exposure using physiologically-based pharmacokinetic models. Stratification factors to control heterogeneity in MRCTs can be identified by reverse translational research incorporating pharmacometric disease models and model-based meta-analyses. Because epidemiological variations can extend to the molecular level, quantitative systems pharmacology models may be useful in forecasting how molecular variation in therapeutic targets or pathway proteins across populations might impact treatment outcomes. Through prospective evaluation of conservation in drug- and disease-related intrinsic and extrinsic factors, a pooled East Asian region can be implemented in Asia-inclusive MRCTs to maximize efficiency in substantiating evidence of benefit-risk for the region at-large with a Totality of Evidence approach.

Activation of Tumor-Cell STING Primes NK-Cell Therapy.

Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist-induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.

Survival Outcomes and Treatment Patterns in Patients With NFE2L2 and/or KEAP1 Mutation-Positive Advanced Squamous Cell NSCLC Using a Real-World Clinico-Genomic Database.

BACKGROUND: NFE2L2 and/or KEAP1 mutations are associated with worse prognosis in all non-small cell lung cancer (NSCLC). We determined real-world survival outcomes and treatment patterns among patients with advanced squamous cell NSCLC by NFE2L2 and KEAP1 mutation status. PATIENTS AND METHODS: A retrospective study (January 2011-December 2018) was conducted using a de-identified US-based clinico-genomic database. Adult patients with advanced squamous cell NSCLC with ≥ 2 in-network visits and comprehensive genomic profiling during the study period were included. Outcomes included real-world progression free survival (rwPFS) by line of therapy and overall survival (OS). The real-world effectiveness of anti-PD-1/PD-L1 first-line therapy was also evaluated in patients with a NFE2L2 and/or KEAP1 mutation. RESULTS: Of 703 patients included (median age: 70.0 years), 31.6% had a NFE2L2 and/or KEAP1 mutation. The most common first- and second-line treatments regardless of mutation status were platinum-based chemotherapies and anti-PD-1/PD-L1 therapies. The most common third-line treatment was anti-PD-1/PD-L1 therapy in patients with a NFE2L2 and/or KEAP1 mutation and single-agent chemotherapy in patients with wild-type disease. Patients with a NFE2L2 and/or KEAP1 mutation versus wild-type disease had significantly shorter rwPFS (4.54 vs. 6.25 months; P = .003) following first- but not second- or third-line therapy and shorter median OS (13.59 vs. 17.37 months; P  =  .4105). No survival differences were observed in patients with a NFE2L2 and/or KEAP1 mutation receiving first-line anti-PD-1/PD-L1 therapies versus other therapies. CONCLUSIONS: Patients with advanced squamous cell NSCLC with a NFE2L2 and/or KEAP1 mutation have poor real-world survival, highlighting the need for a genotype-directed therapeutic strategy in this population.

Challenges in IBD Research: Novel Technologies.

Novel technologies is part of five focus areas of the Challenges in IBD research document, which also includes preclinical human IBD mechanisms, environmental triggers, precision medicine and pragmatic clinical research. The Challenges in IBD research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of a multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the novel technologies section is focused on prioritizing unmet clinical needs in IBD that will benefit from novel technologies applied to: 1) non-invasive detection and monitoring of active inflammation and assessment of treatment response; 2) mucosal targeted drug delivery systems; and 3) prevention of post-operative septic complications and treatment of fistulizing complications. Proposed approaches include development of multiparametric imaging modalities and biosensors, to enable non invasive or minimally invasive detection of pro-inflammatory signals to monitor disease activity and treatment responses. Additionally, technologies for local drug delivery to control unremitting disease and increase treatment efficacy while decreasing systemic exposure are also proposed. Finally, research on biopolymers and other sealant technologies to promote post-surgical healing; and devices to control anastomotic leakage and prevent post-surgical complications and recurrences are also needed.

Five-step authorship framework to improve transparency in disclosing contributors to industry-sponsored clinical trial publications.

Authorship guidelines have established criteria to guide author selection based on significance of contribution and helped to define associated responsibilities and accountabilities for the published findings. However, low awareness, variable interpretation, and inconsistent application of these guidelines can lead to confusion and a lack of transparency when recognizing those who merit authorship. This article describes a research project led by the Medical Publishing Insights and Practices (MPIP) Initiative to identify current challenges when determining authorship for industry-sponsored clinical trials and develop an improved approach to facilitate decision-making when recognizing authors from related publications. A total of 498 clinical investigators, journal editors, publication professionals and medical writers were surveyed to understand better how they would adjudicate challenging, real-world authorship case scenarios, determine the perceived frequency of each scenario and rate their confidence in the responses provided. Multiple rounds of discussions about these results with journal editors, clinical investigators and industry representatives led to the development of key recommendations intended to enhance transparency when determining authorship. These included forming a representative group to establish authorship criteria early in a trial, having all trial contributors agree to these criteria and documenting trial contributions to objectively determine who warrants an invitation to participate in the manuscript development process. The resulting Five-step Authorship Framework is designed to create a more standardized approach when determining authorship for clinical trial publications. Overall, these recommendations aim to facilitate more transparent authorship decisions and help readers better assess the credibility of results and perspectives of the authors for medical research more broadly. Please see related article: http://www.biomedcentral.com/1741-7015/12/214.

Noninvasive optical imaging of apoptosis by caspase-targeted activity-based probes.

Imaging agents that enable direct visualization and quantification of apoptosis in vivo have great potential value for monitoring chemotherapeutic response as well as for early diagnosis and disease monitoring. We describe here the development of fluorescently labeled activity-based probes (ABPs) that covalently label active caspases in vivo. We used these probes to monitor apoptosis in the thymi of mice treated with dexamethasone as well as in tumor-bearing mice treated with the apoptosis-inducing monoclonal antibody Apomab (Genentech). Caspase ABPs provided direct readouts of the kinetics of apoptosis in live mice, whole organs and tissue extracts. The probes produced a maximum fluorescent signal that could be monitored noninvasively and that coincided with the peak in caspase activity, as measured by gel analysis. Overall, these studies demonstrate that caspase-specific ABPs have the potential to be used for noninvasive imaging of apoptosis in both preclinical and clinical settings.

Naive CD4 t cell proliferation is controlled by mammalian target of rapamycin regulation of GRAIL expression.

In this study, we demonstrate that the E3 ubiquitin ligase gene related to anergy in lymphocytes (GRAIL) is expressed in quiescent naive mouse and human CD4 T cells and has a functional role in inhibiting naive T cell proliferation. Following TCR engagement, CD28 costimulation results in the expression of IL-2 whose signaling through its receptor activates the Akt-mammalian target of rapamycin (mTOR) pathway. Activation of mTOR allows selective mRNA translation, including the epistatic regulator of GRAIL, Otubain-1 (Otub1), whose expression results in the degradation of GRAIL and allows T cell proliferation. The activation of mTOR appears to be the critical component of IL-2R signaling regulating GRAIL expression. CTLA4-Ig treatment blocks CD28 costimulation and resultant IL-2 expression, whereas rapamycin and anti-IL-2 treatment block mTOR activation downstream of IL-2R signaling. Thus, all three of these biotherapeutics inhibit mTOR-dependent translation of mRNA transcripts, resulting in blockade of Otub1 expression, maintenance of GRAIL, and inhibition of CD4 T cell proliferation. These observations provide a mechanistic pathway sequentially linking CD28 costimulation, IL-2R signaling, and mTOR activation as important requirements for naive CD4 T cell proliferation through the regulation of Otub1 and GRAIL expression. Our findings also extend the role of GRAIL beyond anergy induction and maintenance, suggesting that endogenous GRAIL regulates general cell cycle and proliferation of primary naive CD4 T cells.

The single subunit transmembrane E3 ligase gene related to anergy in lymphocytes (GRAIL) captures and then ubiquitinates transmembrane proteins across the cell membrane.

The ubiquitin E3 ligase gene related to anergy in lymphocytes (GRAIL) (Rnf128) is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger. GRAIL also contains an equally large luminal region consisting primarily of an uncharacterized protease-associated (PA) domain. Using two-hybrid technology to screen for proteins that bound the PA domain we identified CD151, a member of the tetraspanin family of membrane proteins. GRAIL bound to the luminal/extracellular portion of both CD151 and the related tetraspanin CD81 using its PA domain, which promoted ubiquitination of cytosolic lysine residues. GRAIL exhibited specificity for lysines only within the tetraspanin amino terminus even in the presence of other cytosolic lysine residues in the substrate. GRAIL-mediated ubiquitination promoted proteasomal degradation and cell surface down-regulation of tetraspanins via Lys-48 linkages. As a result, the juxtaposition of PA and RING finger domains across a lipid bilayer facilitates the capture of transmembrane substrates for subsequent ubiquitination. These findings identify for the first time a single subunit E3 ligase containing a substrate-binding domain spatially restricted by a membrane from its E2 recruitment domain as well as an E3 ligase for members of the tetraspanin family.

Cutting edge: The transmembrane E3 ligase GRAIL ubiquitinates the costimulatory molecule CD40 ligand during the induction of T cell anergy.

Activation of naive T lymphocytes is regulated through a series of discrete checkpoints that maintain unresponsiveness to self. During this multistep process, costimulatory interactions act as inducible signals that allow APCs to selectively mobilize T cells against foreign Ags. In this study, we provide evidence that the anergy-associated E3 ubiquitin ligase GRAIL (gene related to anergy in lymphocytes) regulates expression of the costimulatory molecule CD40L on CD4 T cells. Using its luminal protease-associated domain, GRAIL binds to the luminal/extracellular portion of CD40L and facilitates transfer of ubiquitin molecules from the intracellular GRAIL RING (really interesting new gene) finger to the small cytosolic portion of CD40L. Down-regulation of CD40L occurred following ectopic expression of GRAIL in naive T cells from CD40(-/-) mice, and expression of GRAIL in bone marrow chimeric mice was associated with diminished lymphoid follicle formation. These data provide a model for intrinsic T cell regulation of costimulatory molecules and a molecular framework for the initiation of clonal T cell anergy.

Molecular mechanisms of CD4+ T-cell anergy.

Directing both innate and adaptive immune responses against foreign pathogens with correct timing, location and specificity is a fundamental objective for the immune system. Full activation of CD4+ T cells requires the binding of peptide-MHC complexes coupled with accessory signals provided by the antigen-presenting cell. However, aberrant activation of the T-cell receptor alone in mature T cells can produce a long-lived state of functional unresponsiveness, known as anergy. Recent studies probing both immune signalling pathways and the ubiquitin-proteasome system have helped to refine and elaborate current models for the molecular mechanisms underlying T-cell anergy. Controlling anergy induction and maintenance will be a key component in the future to mitigate unwanted T-cell activation that leads to autoimmune disease.

A novel E3 ubiquitin ligase substrate screen identifies Rho guanine dissociation inhibitor as a substrate of gene related to anergy in lymphocytes.

Ubiquitination of eukaryotic proteins regulates a broad range of cellular processes, including regulation of T cell activation and tolerance. We have previously demonstrated that gene related to anergy in lymphocytes (GRAIL), a ring finger ubiquitin E3 ligase, is required for the induction of T cell anergy; however, the substrate(s) for GRAIL E3 ligase activity is/are unknown. In this study, we report a novel prokaryotic system developed to screen for substrates of E3 ligases. Using this screen, Rho guanine dissociation inhibitor (RhoGDI) was identified as a potential substrate of GRAIL. GRAIL was subsequently demonstrated to bind and ubiquitinate RhoGDI, although GRAIL-mediated ubiquitination of RhoGDI did not result in proteosomal degradation. Expression of GRAIL in T cells resulted in specific inhibition of RhoA GTPase activation; activation of Rac1, cdc42, and Ras GTPases were not affected. Interestingly, stable T cell lines expressing dominant-negative RhoA mimicked the GRAIL-mediated IL-2 inhibition phenotype, and T cells expressing constitutively active RhoA were able to overcome GRAIL-mediated inhibition of IL-2 expression. These findings validate our prokaryotic screen as a method of identifying substrates for ubiquitin E3 ligases and suggest a role for Rho effector molecules in T cell anergy.

T cell anergy: where it's LAT.

T cell receptor engagement activates selective signaling pathways in T lymphocytes under different conditions. In this issue of Immunity, demonstrate that anergic T cells are selectively defective in LAT activation.