Oral PD-L1 inhibitor GS-4224 selectively engages PD-L1 high cells and elicits pharmacodynamic responses in patients with advanced solid tumors.

BACKGROUND: Checkpoint inhibitors targeting the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) pathway are effective therapies in a range of immunogenic cancer types. Blocking this pathway with an oral therapy could benefit patients through greater convenience, particularly in combination regimens, and allow flexible management of immune-mediated toxicities. METHODS: PD-L1 binding activity was assessed in engineered dimerization and primary cell target occupancy assays. Preclinical antitumor activity was evaluated in ex vivo and in vivo human PD-L1-expressing tumor models. Human safety, tolerability, pharmacokinetics, and biomarker activity were evaluated in an open-label, multicenter, sequential dose-escalation study in patients with advanced solid tumors. Biomarkers evaluated included target occupancy, flow cytometric immunophenotyping, plasma cytokine measurements, and T-cell receptor sequencing. RESULTS: GS-4224 binding caused dimerization of PD-L1, blocking its interaction with PD-1 and leading to reversal of T-cell inhibition and increased tumor killing in vitro and in vivo. The potency of GS-4224 was dependent on the density of cell surface PD-L1, with binding being most potent on PD-L1-high cells. In a phase 1 dose-escalation study in patients with advanced solid tumors, treatment was well tolerated at doses of 400-1,500 mg once daily. Administration of GS-4224 was associated with a dose-dependent increase in plasma GS-4224 exposure and reduction in free PD-L1 on peripheral blood T cells, an increase in Ki67 among the PD-1-positive T-cell subsets, and elevated plasma cytokines and chemokines. CONCLUSIONS: GS-4224 is a novel, orally bioavailable small molecule inhibitor of PD-L1. GS-4224 showed evidence of expected on-target biomarker activity, including engagement of PD-L1 and induction of immune-related pharmacodynamic responses consistent with PD-L1 blockade. TRIAL REGISTRATION NUMBER: NCT04049617.

Safety, pharmacodynamics, and antiviral activity of selgantolimod in viremic patients with chronic hepatitis B virus infection.

Background & Aims: Novel finite therapies for chronic hepatitis B (CHB) are needed, since lifelong treatment is usually required with current available oral antivirals. This phase II study (NCT03615066) evaluated the safety, pharmacodynamics, and antiviral activity of selgantolimod (a Toll-like receptor 8 agonist [TLR8]) with tenofovir alafenamide (TAF). Methods: Viremic patients with CHB not receiving treatment were stratified by HBeAg status and randomized 2:2:1 to TAF 25 mg/day with selgantolimod 3 mg orally once weekly (QW), selgantolimod 1.5 mg QW, or placebo. Combination therapy continued until week (W)24, followed by TAF monotherapy until W48; patients then discontinued TAF and were followed until W96 (treatment-free follow-up [TFFU] period). The primary efficacy endpoint was the proportion with ≥1 log10 IU/ml HBsAg decline at W24. Results: Sixty-seven patients received study drug; 27 were followed during TFFU. Nausea, headache, vomiting, fatigue, and dizziness were the most common adverse events. Most adverse events were grade 1. Alanine aminotransferase flares were not observed up to W48. Four patients experienced alanine aminotransferase and hepatitis flares during TFFU; all had HBV DNA increases. Selgantolimod increased serum cytokines and chemokines and redistributed several circulating immune cell subsets. No patients achieved the primary efficacy endpoint. Mean HBsAg changes were -0.12, -0.16, and -0.12 log10 IU/ml in the selgantolimod 3 mg, selgantolimod 1.5 mg, and placebo groups, respectively, at W48; HBV DNA declined in all groups by ≥2 log10 IU/ml as early as W2, with all groups rebounding to baseline during TFFU. No HBsAg or HBeAg loss or seroconversion was observed throughout TFFU. Conclusions: Selgantolimod up to 3 mg was safe and well tolerated. Pharmacodynamics and antiviral activity in viremic patients support continued study of selgantolimod in combination CHB therapies. Impact and implications: Novel therapeutics for chronic HBV infection are needed to achieve a functional cure. In this study, we confirmed the safety and tolerability of selgantolimod (formerly GS-9688, a TLR8) when administered with tenofovir alafenamide over 24 weeks in viremic patients with chronic HBV infection. Overall, declines in HBsAg levels with selgantolimod treatment were modest; subgroup analysis indicated that patients with alanine aminotransferase levels greater than the upper limit of normal had significantly greater declines compared to those with normal alanine aminotransferase levels (-0.20 vs. -0.03 log10 IU/ml; p <0.001). These findings suggest a potential differential response to selgantolimod based on patients' baseline HBV-specific immune response, which should be considered in future investigations characterizing the underlying mechanisms of selgantolimod treatment and in HBV cure studies using similar immunomodulatory pathways. Clinical trial number: NCT03615066 be found at https://www.gileadclinicaltrials.com/transparency-policy/.

Safety and efficacy of the oral TLR8 agonist selgantolimod in individuals with chronic hepatitis B under viral suppression.

BACKGROUND & AIMS: Selgantolimod (GS-9688) is a Toll-like receptor 8 (TLR8) agonist that suppresses HBV in vitro. In a phase II study, we evaluated the safety and efficacy of weekly selgantolimod treatment in virally suppressed individuals with chronic HBV taking oral antiviral treatment. METHODS: Forty-eight patients were randomized into two cohorts (hepatitis B e antigen [HBeAg]-positive and -negative [n = 24 each]) to receive oral selgantolimod 3 mg, 1.5 mg, or placebo (2:2:1) once weekly for 24 weeks while maintaining oral antivirals. The primary efficacy endpoint was the percentage of patients with a ≥1 log10 IU/ml decline in hepatitis B surface antigen (HBsAg) from baseline to week 24. Post-treatment, patients continued on oral antivirals for 24 weeks. RESULTS: The primary endpoint was reached by one participant, who was HBeAg-negative and received selgantolimod 1.5 mg. In contrast with placebo-treated patients (n = 9), only selgantolimod-treated patients (n = 39 total) had HBsAg declines greater than 0.1 log10 IU/ml at weeks 24 (18%, 7/39) and 48 (26%, 10/39), HBsAg loss (5%, 2/39 through 48 weeks), or HBeAg loss (16%, 3/19 through 48 weeks). The most common adverse events in selgantolimod-treated groups were nausea (46%), upper respiratory tract infection (23%), and vomiting (23%). Gastrointestinal disorders were mostly mild and transient. Selgantolimod induced transient dose-dependent increases in serum cytokines, including IL-12p40, IFN-γ, and IL-1RA, as well as rapid redistribution of some circulating immune cell subsets. CONCLUSION: Oral selgantolimod up to 3 mg once weekly for 24 weeks was generally safe and well tolerated and led to serologic changes associated with progression to durable cure in two individuals by week 48. GOV IDENTIFIER: NCT03491553. IMPACT AND IMPLICATIONS: The only robust criterion for stopping treatment in chronic hepatitis B is loss of hepatitis B surface antigen (known as functional cure), which is rare during nucleos(t)ide analogue therapy. It is likely that novel antiviral and immunomodulatory therapies will be needed to achieve finite functional cure. Selgantolimod is an oral Toll-like receptor 8 agonist that has shown antiviral activity in vitro as well as safety in a phase I clinical trial with weekly dosing. In this phase II study, selgantolimod therapy was associated with transient increases in serum cytokines, rapid redistribution of circulating immune cell subsets, modest reductions in HBsAg and HBeAg levels, and occasional loss of HBsAg (5%) and HBeAg (16%) among participants with chronic hepatitis B on nucleos(t)ide analogue therapy with viral suppression. Our results support continued development of selgantolimod as a component of a future hepatitis B cure regimen.

Seven-colour multiplex immunochemistry/immunofluorescence and whole slide imaging of frozen sections.

Multiplex Immunochemistry/Immunofluorescence (mIHC/IF) aims to visualise multiple biomarkers in a single tissue section and is especially powerful when used on slide scanners coupled with digital analysis tools. mIHC/IF is commonly employed in immuno-oncology to characterise features of the tumour microenvironment (TME) and correlate them with clinical parameters to guide prognostication and therapy. However, mIHC/IF can be applied to a wide range of organisms in any physiological or disease context. Recent innovation has extended the number of markers that can be detected using slide scanners well beyond the 3-4 markers typically reported in traditional fluorescence microscopy. However, these methods often require sequential antibody staining and stripping, and are not compatible with frozen tissue sections. Using fluorophore-conjugated antibodies, we have established a simple mIHC/IF imaging workflow that enables simultaneous staining and detection of seven markers in a single section of frozen tissue. Coupled with automated whole slide imaging and digital quantification, our data efficiently revealed the tumour-immune complexity in metastatic melanoma. Computational image analysis quantified the immune and stromal cell populations present in the TME as well as their spatial interactions. This imaging workflow can also be performed with an indirect labelling panel consisting of primary and secondary antibodies. Our new methods, combined with digital quantification, will provide a valuable tool for high-quality mIHC/IF assays in immuno-oncology research and other translational studies, especially in circumstances where frozen sections are required for detection of particular markers, or for applications where frozen sections may be preferred, such as spatial transcriptomics.

A randomised controlled trial of long NY-ESO-1 peptide-pulsed autologous dendritic cells with or without alpha-galactosylceramide in high-risk melanoma.

AIM: We have previously reported that polyfunctional T cell responses can be induced to the cancer testis antigen NY-ESO-1 in melanoma patients injected with mature autologous monocyte-derived dendritic cells (DCs) loaded with long NY-ESO-1-derived peptides together with α-galactosylceramide (α-GalCer), an agonist for type 1 Natural Killer T (NKT) cells. OBJECTIVE: To assess whether inclusion of α-GalCer in autologous NY-ESO-1 long peptide-pulsed DC vaccines (DCV + α-GalCer) improves T cell responses when compared to peptide-pulsed DC vaccines without α-GalCer (DCV). DESIGN, SETTING AND PARTICIPANTS: Single-centre blinded randomised controlled trial in patients ≥ 18 years old with histologically confirmed, fully resected stage II-IV malignant cutaneous melanoma, conducted between July 2015 and June 2018 at the Wellington Blood and Cancer Centre of the Capital and Coast District Health Board. INTERVENTIONS: Stage I. Patients were randomised to two cycles of DCV or DCV + α-GalCer (intravenous dose of 10 × 106 cells, interval of 28 days). Stage II. Patients assigned to DCV + α-GalCer were randomised to two further cycles of DCV + α-GalCer or observation, while patients initially assigned to DCV crossed over to two cycles of DCV + α-GalCer. OUTCOME MEASURES: Primary: Area under the curve (AUC) of mean NY-ESO-1-specific T cell count detected by ex vivo IFN-γ ELISpot in pre- and post-treatment blood samples, compared between treatment arms at Stage I. Secondary: Proportion of responders in each arm at Stage I; NKT cell count in each arm at Stage I; serum cytokine levels at Stage I; adverse events Stage I; T cell count for DCV + α-GalCer versus observation at Stage II, T cell count before versus after cross-over. RESULTS: Thirty-eight patients gave written informed consent; 5 were excluded before randomisation due to progressive disease or incomplete leukapheresis, 17 were assigned to DCV, and 16 to DCV + α-GalCer. The vaccines were well tolerated and associated with increases in mean total T cell count, predominantly CD4+ T cells, but the difference between the treatment arms was not statistically significant (difference - 6.85, 95% confidence interval, - 21.65 to 7.92; P = 0.36). No significant improvements in T cell response were associated with DCV + α-GalCer with increased dosing, or in the cross-over. However, the NKT cell response to α-GalCer-loaded vaccines was limited compared to previous studies, with mean circulating NKT cell levels not significantly increased in the DCV + α-GalCer arm and no significant differences in cytokine response between the treatment arms. CONCLUSIONS: A high population coverage of NY-ESO-1-specific T cell responses was achieved with a good safety profile, but we failed to demonstrate that loading with α-GalCer provided an additional advantage to the T cell response with this cellular vaccine design. CLINICAL TRIAL REGISTRATION: ACTRN12612001101875. Funded by the Health Research Council of New Zealand.

Rapid culture of human keratinocytes in an autologous, feeder-free system with a novel growth medium.

BACKGROUND AIMS: The ability to culture human keratinocytes is beneficial in the treatment of skin injury and disease, as well as for testing chemicals in vitro as a substitute for animal testing. RESULTS: We have identified a novel culture medium for the rapid growth of keratinocytes from human skin. "Kelch's medium" supports keratinocyte growth that is as rapid as in the classical Rheinwald and Green method, but without the need for cholera toxin or xenogeneic feeder cells. It enables keratinocytes to out-compete co-cultured autologous fibroblasts so that separation of the epidermis from the dermis is no longer required before keratinocyte culture. Enzymatic digests of whole human skin can therefore be used to generate parallel cultures of autologous keratinocytes, fibroblasts and melanocytes simply by using different cell culture media. CONCLUSIONS: This new keratinocyte medium and the simplified manufacturing procedures it enables are likely to be beneficial in skin engineering, especially for clinical applications.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses.

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell-bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

Live-Cell Microscopy Reveals That Human T Cells Primarily Respond Chemokinetically Within a CCL19 Gradient That Induces Chemotaxis in Dendritic Cells.

The ability to study migratory behavior of immune cells is crucial to understanding the dynamic control of the immune system. Migration induced by chemokines is often assumed to be directional (chemotaxis), yet commonly used end-point migration assays are confounded by detecting increased cell migration that lacks directionality (chemokinesis). To distinguish between chemotaxis and chemokinesis we used the classic "under-agarose assay" in combination with video-microscopy to monitor migration of CCR7+ human monocyte-derived dendritic cells and T cells in response to a concentration gradient of CCL19. Formation of the gradients was visualized with a fluorescent marker and lasted several hours. Monocyte-derived dendritic cells migrated chemotactically towards the CCL19 gradient. In contrast, T cells exhibited a biased random walk that was largely driven by increased exploratory chemokinesis towards CCL19. This dominance of chemokinesis over chemotaxis in T cells is consistent with CCR7 ligation optimizing T cell scanning of antigen-presenting cells in lymphoid tissues.

Migratory cues controlling B-lymphocyte trafficking in human lymph nodes.

B-cell migration within lymph nodes (LNs) is crucial to adaptive immune responses. Chemotactic gradients are proposed to drive migration of B cells into follicles, followed by their relocation to specific zones of the follicle during activation, and ultimately egress. However, the molecular drivers of these processes and the cells generating chemotactic signals that affect B cells in human LNs are not well understood. We used immunofluorescence microscopy, flow cytometry and functional assays to study molecular mechanisms of B-cell migration within human LNs, and found subtle but important differences to previous murine models. In human LNs we find CXCL13 is prominently expressed at the follicular edge, often associated with fibroblastic reticular cells located in these areas, whereas follicular dendritic cells show minimal contribution to CXCL13 expression. Human B cells rapidly downregulate CXCR5 on encountering CXCL13, but recover CXCR5 expression in the CXCL13-low environment. These data suggest that the CXCL13 gradient in human LNs is likely to be different from that proposed in mice. We also identify CD68+ CD11c+ PU.1+ tingible body macrophages within both primary and secondary follicles as likely drivers of the sphingosine-1-phosphate (S1P) gradient that mediates B-cell egress from LNs, through their expression of the S1P-degrading enzyme, S1P lyase. Based on our findings, we present a model of B-cell migration within human LNs, which has both similarities and interesting differences to that proposed for mice.

Distinctive Subpopulations of Stromal Cells Are Present in Human Lymph Nodes Infiltrated with Melanoma.

Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases modulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90+ SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first population (CD90+ podoplanin+ CD105+ CD146+ CD271+ VCAM-1+ ICAM-1+ α-SMA+) corresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The second (CD90+ CD34+ CD105+ CD271+) represents a novel population of CD34+ SCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demonstrated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90+ CD146+ CD36+ NG2- pericytes in the walls of high endothelial venules and other small vessels, and CD90+ CD146+ NG2+ CD36- pericytes in the walls of larger vessels. Distinguishing between these CD90+ SC subpopulations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes.

Results of a randomized, double-blind phase II clinical trial of NY-ESO-1 vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in participants with high-risk resected melanoma.

BACKGROUND: To compare the clinical efficacy of New York Esophageal squamous cell carcinoma-1 (NY-ESO-1) vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in a randomized, double-blind phase II study in participants with fully resected melanoma at high risk of recurrence. METHODS: Participants with resected stage IIc, IIIb, IIIc and IV melanoma expressing NY-ESO-1 were randomized to treatment with three doses of NY-ESO-1/ISCOMATRIX or ISCOMATRIX adjuvant administered intramuscularly at 4-week intervals, followed by a further dose at 6 months. Primary endpoint was the proportion free of relapse at 18 months in the intention-to-treat (ITT) population and two per-protocol populations. Secondary endpoints included relapse-free survival (RFS) and overall survival (OS), safety and NY-ESO-1 immunity. RESULTS: The ITT population comprised 110 participants, with 56 randomized to NY-ESO-1/ISCOMATRIX and 54 to ISCOMATRIX alone. No significant toxicities were observed. There were no differences between the study arms in relapses at 18 months or for median time to relapse; 139 vs 176 days (p=0.296), or relapse rate, 27 (48.2%) vs 26 (48.1%) (HR 0.913; 95% CI 0.402 to 2.231), respectively. RFS and OS were similar between the study arms. Vaccine recipients developed strong positive antibody responses to NY-ESO-1 (p≤0.0001) and NY-ESO-1-specific CD4+ and CD8+ responses. Biopsies following relapse did not demonstrate differences in NY-ESO-1 expression between the study populations although an exploratory study demonstrated reduced (NY-ESO-1)+/Human Leukocyte Antigen (HLA) class I+ double-positive cells in biopsies from vaccine recipients performed on relapse in 19 participants. CONCLUSIONS: The vaccine was well tolerated, however, despite inducing antigen-specific immunity, it did not affect survival endpoints. Immune escape through the downregulation of NY-ESO-1 and/or HLA class I molecules on tumor may have contributed to relapse.

Neuroserpin regulates human T cell-T cell interactions and proliferation through inhibition of tissue plasminogen activator.

T cells play a key role in mounting an adaptive immune response. T cells are activated upon recognition of cognate Ag presented by an APC. Subsequently, T cells adhere to other activated T cells to form activation clusters, which lead to directed secretion of cytokines between communicating cells. T cell activation clusters have been implicated in regulating activation, proliferation, and memory formation in T cells. We previously reported the expression of the protease inhibitor neuroserpin by human T cells and showed that expression and intracellular localization is regulated following T cell activation. To gain a better understanding of neuroserpin in the proteolytic environment postactivation we assessed its role in human T cell clustering and proliferation. Neuroserpin knockdown increased T cell proliferation and cluster formation following T cell activation. This increased cluster formation was dependent on the proteases tissue plasminogen activator (tPA) and plasmin. Furthermore, neuroserpin knockdown or plasmin treatment of T cells increased the cleavage of annexin A2, a known plasmin target that regulates the actin cytoskeleton. Live cell imaging of activated T cells further indicated a role of the actin cytoskeleton in T cell clustering. The inhibition of actin regulators myosin ATPase and Rho-associated protein kinase signaling completely reversed the neuroserpin knockdown-induced effects. The results presented in this study reveal a novel role for neuroserpin and the proteolytic environment in the regulation of T cell activation biology.

Synthesis and Evaluation of Novel TLR2 Agonists as Potential Adjuvants for Cancer Vaccines.

Cancer immunotherapy has gained increasing attention due to its potential specificity and lack of adverse side effects when compared to more traditional modes of treatment. Toll-like receptor 2 (TLR2) agonists are lipopeptides possessing the S-[2,3-bis(palmitoyloxy)propyl]-l-cysteine (Pam2Cys) motif and exhibit potent immunostimulatory effects. These agonists offer a means of providing "danger signals" in order to activate the immune system toward tumor antigens. Thus, the development of TLR2 agonists is attractive in the search of potential immunostimulants for cancer. Existing SAR studies of Pam2Cys with TLR2 indicate that the structural requirements for activity are, for the most part, very intolerable. We have investigated the importance of stereochemistry, the effect of N-terminal acylation, and homologation between the two ester functionalities in Pam2Cys-conjugated lipopeptides on TLR2 activity. The R diastereomer is significantly more potent than the S diastereomer and N-terminal modification generally lowers TLR2 activity. Most notably, homologation gives rise to analogues which are comparatively active to the native Pam2Cys containing constructs.

Synthesis and SAR Analysis of Lipovelutibols B and D and Their Lipid Analogues.

The first syntheses of the cytotoxic peptides lipovelutibols B and D are described. While lipovelutibol D was prepared using solid-phase peptide synthesis followed by an O-N acyl migration to install the C-terminal amino alcohol, a different strategy was required to access lipovelutibol B and a series of N-terminal lipid analogues of the natural products. A cytotoxicity structure-activity relationship study revealed that the lipovelutibol D framework, whereby serine is substituted for alanine in the fifth position, provided the most potent analogues. Modification of the lipid tail was generally well tolerated, with longer alkyl chains enhancing analogue cytotoxicity.

High-resolution 3D imaging and topological mapping of the lymph node conduit system.

The conduit network is a hallmark of lymph node microanatomy, but lack of suitable imaging technology has prevented comprehensive investigation of its topology. We employed an extended-volume imaging system to capture the conduit network of an entire murine lymph node (comprising over 280,000 segments). The extensive 3D images provide a comprehensive overview of the regions supplied by conduits, including perivascular sleeves and distinctive "follicular reservoirs" within B cell follicles, surrounding follicular dendritic cells. A 3D topology map of conduits within the T-cell zone showed homogeneous branching, but conduit density was significantly higher in the superficial T-cell zone compared with the deep zone, where distances between segments are sufficient for T cells to lose contact with fibroblastic reticular cells. This topological mapping of the conduit anatomy can now aid modeling of its roles in lymph node function, as we demonstrate by simulating T-cell motility in the different T-cell zones.

Anti-PD-1 blockade with nivolumab with and without therapeutic vaccination for virally suppressed chronic hepatitis B: A pilot study.

BACKGROUND & AIMS: To evaluate the hypothesis that increasing T cell frequency and activity may provide durable control of hepatitis B virus (HBV), we administered nivolumab, a programmed death receptor 1 (PD-1) inhibitor, with or without GS-4774, an HBV therapeutic vaccine, in virally suppressed patients with HBV e antigen (HBeAg)-negative chronic HBV. METHODS: In a phase Ib study, patients received either a single dose of nivolumab at 0.1 mg/kg (n = 2) or 0.3 mg/kg (n = 12), or 40 yeast units of GS-4774 at baseline and week 4 and 0.3 mg/kg of nivolumab at week 4 (n = 10). The primary efficacy endpoint was mean change in HBV surface antigen (HBsAg) 12 weeks after nivolumab. Safety and immunologic changes were assessed through week 24. RESULTS: There were no grade 3 or 4 adverse events or serious adverse events. All assessed patients retained T cell PD-1 receptor occupancy 6-12 weeks post-infusion, with a mean total across 0.1 and 0.3 mg/kg cohorts of 76% (95% CI 75-77), and no significant differences were observed between cohorts (p = 0.839). Patients receiving 0.3 mg/kg nivolumab without and with GS-4774 had mean declines of -0.30 (95% CI -0.46 to -0.14) and -0.16 (95% CI -0.33 to 0.01) log10 IU/ml, respectively. Patients showed significant HBsAg declines from baseline (p = 0.035) with 3 patients experiencing declines of >0.5 log10 by the end of study. One patient, whose HBsAg went from baseline 1,173 IU/ml to undetectable at week 20, experienced an alanine aminotransferase flare (grade 3) at week 4 that resolved by week 8 and was accompanied by a significant increase in peripheral HBsAg-specific T cells at week 24. CONCLUSIONS: In virally suppressed HBeAg-negative patients, checkpoint blockade was well-tolerated and led to HBsAg decline in most patients and sustained HBsAg loss in 1 patient. LAY SUMMARY: Chronic hepatitis B virus infection (CHB) is characterized by a dysfunctional immune response. In patients with CHB, inhibitory receptors, such as programmed death receptor 1 (PD-1) are overexpressed on T cells, leading to an ineffective immune response in the liver. Herein, we show that the PD-1 inhibitor, nivolumab, is safe and effective for the treatment of virally suppressed patients with CHB. Australian New Zealand Clinical Trials Registry (http://www.anzctr.org.au/) number: ACTRN12615001133527.

Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Are a Heterogeneous Population That Demonstrate Rapid Culture-Induced Changes.

Human adipose-derived mesenchymal stromal cells (ASC) are showing clinical promise for the treatment of a range of inflammatory and degenerative conditions. These lipoaspirate-derived cells are part of the abundant and accessible source of heterogeneous stromal vascular fraction (SVF). They are typically isolated and expanded from the SVF via adherent cell culture for at least 2 weeks and as such represent a relatively undefined population of cells. We isolated ex vivo ASC directly from lipoaspirate using a cocktail of antibodies combined with immunomagnetic bead sorting. This method allowed for the rapid enrichment of a defined and untouched ex vivo ASC population (referred to as MACS-derived ASC) that were then compared to culture-derived ASC. This comparison found that MACS-derived ASC contain a greater proportion of cells with activity in in vitro differentiation assays. There were also significant differences in the secretion levels of some key paracrine molecules. Moreover, when the MACS-derived ASC were subjected to adherent tissue culture, rapid changes in gene expression were observed. This indicates that culturing cells may alter the clinical utility of these cells. Although MACS-derived ASC are more defined compared to culture-derived ASC, further investigations using a comprehensive multicolor flow cytometry panel revealed that this cell population is more heterogeneous than previously appreciated. Additional studies are therefore required to more precisely delineate phenotypically distinct ASC subsets with the most therapeutic potential. This research highlights the disparity between ex vivo MACS-derived and culture-derived ASC and the need for further characterization.

Safety, Pharmacokinetics, and Pharmacodynamics of Multiple Rising Doses of BI 655064, an Antagonistic Anti-CD40 Antibody, in Healthy Subjects: A Potential Novel Treatment for Autoimmune Diseases.

BI 655064 is a humanized antagonistic anti-cluster of differentiation (CD) 40 monoclonal antibody that selectively blocks the CD40-CD40L interaction. The CD40-CD40L pathway is a promising treatment target for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis. The safety, tolerability, pharmacokinetics, and pharmacodynamics of repeated once-weekly BI 655064 subcutaneous dosing over 4 weeks were evaluated in a multiple-dose study in healthy subjects. Subjects (N = 40) were randomized 4:1 to four sequential BI 655064 dose groups (80, 120, 180, 240 mg) or to placebo. Safety and tolerability, plasma exposure, CD40 receptor occupancy, and CD40L-induced CD54 upregulation were assessed over 64 and 78 days for the 80- to 180-mg and 240-mg dose groups, respectively. BI 655064 exposure increased in a supraproportional manner, due to target-mediated drug clearance, for doses between 80 mg and 120 mg, but was near proportional for doses greater than 120 mg. Terminal half-life ranged between 6 and 8 days. Dose-dependent accumulation of BI 655064 supports the use of a loading dose in future clinical studies. Following 4 weeks of dosing, >90% CD40 receptor occupancy and inhibition of CD54 upregulation were observed at all dose levels, lasting for 17 days after the last dose. BI 655064 was generally well tolerated. There were no serious adverse events and the frequency and intensity of adverse events were similar for BI 655064 and placebo; no dose relationship or relevant signs of an acute immune reaction were observed. These findings support further investigation of BI 655064 as a potential treatment for autoimmune diseases.

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

The natural compound, formononetin, extracted from Astragalus membranaceus increases adipocyte thermogenesis by modulating PPARγ activity.

BACKGROUND AND PURPOSE: Increasing energy expenditure through adipocyte thermogenesis is generally accepted as a promising strategy to mitigate obesity and its related diseases. However, few clinically effective and safe agents are known to promote adipocyte thermogenesis. In this study, 20 traditional Chinese herbal medicines were screened to examine whether they induced adipocyte thermogenesis. EXPERIMENTAL APPROACH: The effects of Chinese herbal medicines or components isolated from extracts of A. membranaceus, on adipocyte thermogenesis were analysed by assessing expression of uncoupling protein 1 (UCP1) by qPCR. Eight-week-old C57BL6/J male mice were fed a high-fat diet for 8 weeks and then randomized to two groups treated with vehicle or formononetin for another 8 weeks. Glucose tolerance tests and staining of adipose tissue with haematoxylin and eosin were carried out. Whole-body oxygen consumption was measured with an open-circuit indirect calorimetry system. KEY RESULTS: Extracts of A. membranaceus increased expression of Ucp1 in primary cultures of mouse adipocytes. Formononetin was the only known component of A. membranaceus extracts to increase adipocyte Ucp1 expression. Diet-induced obese mice treated with formononetin gained less weight and showed higher energy expenditure than untreated mice. In addition, formononetin binds directly with PPARγ. CONCLUSIONS AND IMPLICATION: Taken together, our study demonstrates that the Chinese herbal medicine from A. membranaceus and its constituent formononetin have the potential to reduce obesity and associated metabolic disorders. Our results suggest that formononetin regulates adipocyte thermogenesis as a non-classical PPARγ agonist.