Explore the vast range of titles available.

Immune clearance of Hepatitis B virus (HBV) is characterized by broad and robust antiviral T cell responses, while virus-specific T cells in chronic hepatitis B (CHB) are rare and exhibit immune exhaustion that includes programmed-death-1 (PD-1) expression on virus-specific T cells. Thus, an immunotherapy able to expand and activate virus-specific T cells may have therapeutic benefit for CHB patients. Like HBV-infected patients, woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells with substantial individual variation in these parameters. We used woodchucks infected with WHV to assess the safety and efficacy of anti-PD-L1 monoclonal antibody therapy (αPD-L1) in a variety of WHV infection states. Experimentally-infected animals lacked PD-1 or PD-L1 upregulation compared to uninfected controls, and accordingly, αPD-L1 treatment in lab-infected animals had limited antiviral effects. In contrast, animals with naturally acquired WHV infections displayed elevated PD-1 and PD-L1. In these same animals, combination therapy with αPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared to ETV treatment alone, but with efficacy restricted to a minority of animals. Pre-treatment WHV surface antigen (sAg) level was identified as a statistically significant predictor of treatment response, while PD-1 expression on peripheral CD8+ T cells, T cell production of interferon gamma (IFN-γ) upon in vitro antigen stimulation (WHV ELISPOT), and circulating levels of liver enzymes were not. To further assess the safety of this strategy, αPD-L1 was tested in acute WHV infection to model the risk of liver damage when the extent of hepatic infection and antiviral immune responses were expected to be the greatest. No significant increase in serum markers of hepatic injury was observed over those in infected, untreated control animals. These data support a positive benefit/risk assessment for blockade of the PD-1:PD-L1 pathway in CHB patients and may help to identify patient groups most likely to benefit from treatment. Furthermore, the efficacy of αPD-L1 in only a minority of animals, as observed here, suggests that additional agents may be needed to achieve a more robust and consistent response leading to full sAg loss and durable responses through anti-sAg antibody seroconversion.

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

Entecavir (ETV) is a deoxyguanosine analog competitive inhibitor of hepatitis B virus (HBV) polymerase that exhibits delayed chain termination of HBV DNA. A high barrier to entecavir-resistance (ETVr) is observed clinically, likely due to its potency and a requirement for multiple resistance changes to overcome suppression. Changes in the HBV polymerase reverse-transcriptase (RT) domain involve lamivudine-resistance (LVDr) substitutions in the conserved YMDD motif (M204V/I +/- L180M), plus an additional ETV-specific change at residues T184, S202 or M250. These substitutions surround the putative dNTP binding site or primer grip regions of the HBV RT. To determine the mechanistic basis for ETVr, wildtype, lamivudine-resistant (M204V, L180M) and ETVr HBVs were studied using in vitro RT enzyme and cell culture assays, as well as molecular modeling. Resistance substitutions significantly reduced ETV incorporation and chain termination in HBV DNA and increased the ETV-TP inhibition constant (K(i)) for HBV RT. Resistant HBVs exhibited impaired replication in culture and reduced enzyme activity (k(cat)) in vitro. Molecular modeling of the HBV RT suggested that ETVr residue T184 was adjacent to and stabilized S202 within the LVDr YMDD loop. ETVr arose through steric changes at T184 or S202 or by disruption of hydrogen-bonding between the two, both of which repositioned the loop and reduced the ETV-triphosphate (ETV-TP) binding pocket. In contrast to T184 and S202 changes, ETVr at primer grip residue M250 was observed during RNA-directed DNA synthesis only. Experimentally, M250 changes also impacted the dNTP-binding site. Modeling suggested a novel mechanism for M250 resistance, whereby repositioning of the primer-template component of the dNTP-binding site shifted the ETV-TP binding pocket. No structural data are available to confirm the HBV RT modeling, however, results were consistent with phenotypic analysis of comprehensive substitutions of each ETVr position. Altogether, ETVr occurred through exclusion of ETV-TP from the dNTP-binding site, through different, novel mechanisms that involved lamivudine-resistance, ETV-specific substitutions, and the primer-template.

PurposeCheckMate 915 (NCT03068455) compared adjuvant nivolumab monotherapy versus combination nivolumab+ipilimumab in patients with resected stage III/IV melanoma. This exploratory analysis was performed to identify biomarkers that correlate with benefit from adjuvant immunotherapy.Patients and methods1,844 patients received nivolumab 480 mg every 4 weeks or nivolumab 240 mg every 2 weeks with ipilimumab 1 mg/kg every 6 weeks. Tumor and peripheral biomarkers were evaluated, including tumor-informed circulating tumor DNA (ctDNA) at postresection baseline and on-treatment, for their association with recurrence-free survival and distant metastases-free survival.ResultsBiomarker analyses were conducted in 60–96% of the intention-to-treat population. ctDNA positivity at baseline (seen in 16.2% of patients) and on-treatment was associated with higher risk of recurrence than ctDNA negativity (HR, 1.97; 95% CI, 1.57 to 2.46), with a high specificity (87%) and modest sensitivity (39%). ctDNA status, tumor mutational burden (TMB) status (TMB < or ≥350 mutations/tumor) and interferon gamma-RNA signature score (< or ≥median) evaluated together, as well as ctDNA status with tumor CD8 or cell programmed death ligand 1 expression, were more predictive of survival than ctDNA alone. Tumor bulk RNA-seq expression patterns identified gene expression at baseline associated with recurrence.ConclusionsThis study represents the largest assessment of ctDNA and other baseline tumor and peripheral biomarkers for predicting recurrence-free survival in patients with resected melanoma receiving adjuvant immunotherapy. ctDNA alone and in combination with more established biomarkers predicted recurrence-free and distant metastasis-free survival and has potential utility for assessing and monitoring the risk of recurrence in patients with resected melanoma treated with immunotherapy in the adjuvant setting.Trial registration numberNCT03068455.

BackgroundImmunotherapy (IO) has changed the treatment paradigm for patients with metastatic melanoma. IO combination therapy such as anti-PD1 nivolumab (NIVO), anti-CTLA4 ipilimumab (IPI) and anti-LAG3 relatlimab (RELA) have replaced chemotherapy as first line treatment. Although candidate biomarkers such as interferon gamma gene expression, BRAF mutation, PD-L1 tumor expression, high tumor mutational burden and CD8 T-cell infiltration have been explored for their predictive value, no definitive biomarkers for IO response have been identified. Our study explores novel exploratory biomarkers that can help us understand which metastatic melanoma patients will have an increased benefit from either anti-CTLA4 or anti-CTLA4/PD1 treatment.MethodsScRNAseq profiling of peripheral blood mononuclear cells (PBMCs) was performed on a subset of CheckMate-069 clinical trial (NCT01927419) samples, which included 52 patients with metastatic melanoma treated with either IPI (n=26) or NIVO + IPI (n=26), using the 5 prime 10x Genomics platform (total n=52, samples=104). PBMCs from patients were profiled at Baseline (C1D1) and after 2 cycles of therapy (C3D1) and separated as responders (R, n=20) or non-responders (NR, n=32). We deployed Seurat R package for cell phenotype determination including Tregs and interrogated drug response in R vs. NR, mechanisms of drug resistance/action, pharmacodynamic (PD) markers, and biomarker of response.ResultsScRNAseq analysis revealed that peripheral regulatory T-cells (Tregs) did not decrease post IO regardless of treatment type (IPI or NIVO+IPI) or response. In fact, the proportion of Treg cells increased post IO regardless of treatment or response, despite an observed decrease in their cell of origin proportion (CD4+ T-cells) (figure 1A). Although Tregs do not decrease, their ‘suppressive’ phenotype was down-modulated preferentially in responders as indicated by the selective downregulation of EZH2 and FOXP3 expression in responders (figure 1B). A lower Treg cell proportion at Baseline and higher CD8-central-memory cell proportion at C3D1 served as a predictive response biomarker of IPI and NIVO+IPI respectively. The ratio of CD8+Teffector memory to Treg also predicts outcome benefit to anti-CTLA4 and may serve as a potential predictive biomarker (figure 2).ConclusionsOur analysis serves to elucidate a missing piece of anti-CTLA4 therapy mechanism of action and identifies potential new biomarkers that can be applied to inform stratification for combination therapy trials. Although Tregs were not depleted, EZH2 was selectively upregulated in T-regs of non-responders upon IPI treatment indicating that a combination therapy of EZH2i+anti-CLTA4 may increase response rates. Additionally, CD8+effector memory/Treg cells ratio may serve as a new biomarker predictive of anti-CTLA4 benefit.AcknowledgementsWe acknowledge the contribution of Megan Wind-Rotolo and Evisa Gjini to the design of this work prior to their departure from the companyTrial RegistrationNCT01927419Study of Nivolumab (BMS-936558) Plus Ipilimumab Compared With Ipilimumab Alone in the Treatment of Previously Untreated, Unresectable, or Metastatic Melanoma (CheckMate 069)Ethics ApprovalAll subjects consented to this research.ConsentWritten informed consent was obtained by BMS from the patient for this exploratory research and publication of the resultsAbstract 624 Figure 1(A) Tregs cell proportion are not depleted upon either IPI or Nivo+IPI treatment regardless of response (B) Treg suppressive features are downregulated in responders and upregulated in non-responders. Colors denote different response groupsAbstract 624 Figure 2A higher CD8 effector memory cell to T-reg ratio at baseline predicts better outcome to Ipi

Integrated experimental and modeling methods using both surface plasmon resonance–based kinetic measures and cell‐binding data have been used to account for the kinetics of individual receptors, their apparent binding affinity, and more important their cross‐linking behavior. See PDF] Model state variables and parameters Symbol Description Suggested units k a Free antibody association rate with free antigen nM−1 day−1 k r Dissociation rate of monovalent antibody–antigen complexes day−1 k xa Cross‐linking rate for antibody–antigen complexes μm2 molecule−1 day−1 N cell Number of cells in the assay well (often around 1 × 105 for avidity experiments) number N nmole A conversion factor from nanomole to molecules (6.022 × 1014) molecule nanomole−1 r cell Assumed cell radius μm V w Volume of the assay well L X A Amount of free antibody nanomole X B Amount of bound antibody nanomole X C1 Amount of single antigen and antibody complex on the cell surface nanomole X C2 Amount of double antigen and antibody complex on the cell surface nanomole X RT Total amount of antigen on the cell surface nanomole B Density of total antibody bound on the cell surface molecule μm−2 σ Confinement length nanometer k a,lgnd Solution phase association rate of receptor‐ligand complexes nanomolar−1 day−1 k xa,lgnd Two‐dimensional association rate of receptor‐ligand complexes μm2 molecule−1 day−1 V convert Volumetric conversion factor (1 × 10−18) L nm−1 μm−2 f v Vascular fraction for the tumor dimensionless f ICF Fraction of the nonvascular tumor volume that is intracellular dimensionless f ECM,A Fraction of the interstitial tumor volume that is not accessible to antibody due to extracellular matrix dimensionless k in,DAg Rate constant for antigen molecules to diffuse into the synapse day−1 k out,DAg Rate for antigen diffusing out of the synapse day−1 Q prod,contact Production rate of antigen for cells in contact molecule day−1 k sdeg Surface antigen degradation rate day−1 k xr,lgnd Dissociation rate of receptor–ligand complexes day−1 S syn Total surface area directly involved in cell–cell contact in the tumor (synapse), same on antigen‐expressing or ligand‐expressing cells μm2 S Agnonsyn Surface area on antigen‐expressing cells in the tumor that are engaged in cell–cell contacts, but this portion of the surface is not directly involved in the synapse μm2 V t Total tumor volume L X At Amount of free antibody in the tumor nanomole X Agsyn Amount of free antigen in cellular synapses nanomole X Agnonsyn Amount of free antigen on contacting cells but outside of synapses nanomole X C1syn Amount of single‐antigen antibody complexes in cellular synapses nanomole X C2syn Amount of double‐antigen antibody complexes in cellular synapses nanomole X lgndsyn Amount of free ligand in cellular synapses nanomole X CAglgnd Amount of antigen–ligand complexes in cellular synapses nanomole Illustration of models. (a) Cartoon of antibody‐binding minimodel depicting monovalent antibody, first bond formation, and cross‐linking. Note that different regions and characteristics of the curve are differentially sensitive and best able to report different underlying biophysical parameters, such as the cross‐linking, monovalent association rates, and total receptor site densities. (c) Illustration of a bigger model incorporating bivalent antibody–antigen interactions and competition for ligand. To better assess these issues, experimental checks coupled with simulation, such as observing dissociation kinetics of bound antibody preattached to cells with a pulse‐chase incubation, can serve as additional verification for estimated cell‐binding kinetic parameters.

BackgroundSpatial patterns of CD8+ T cells in the tumor microenvironment are associated with clinical outcomes in patients with advanced solid tumors. However, attempts to quantify spatial topology are hindered by challenges in manual scoring, heterogeneous immune-cell infiltrates, and interpathologist variability. Artificial intelligence (AI)–powered analysis can quantify CD8 topology in a biologically meaningful, reproducible, and scalable way. Using an AI-driven algorithm, we retrospectively assessed CD8 topology as a biomarker of response to immunotherapy in patients with advanced melanoma.MethodsWe trained a random forest classifier to predict CD8 topology using parenchymal and stromal CD8+ immune-cell measurements derived from a deep-learning platform (PathAI, Boston, MA). For model validation, pathologists manually classified CD8 immunohistochemistry (C8/144B, Agilent, Santa Clara, CA) in melanoma samples into inflamed (CD8+ cells in tumor parenchyma), excluded (CD8+ cells restricted to stroma), and desert (deficient in CD8+ cells) patterns. We explored the association with overall survival (OS) in a subset of patients with previously untreated metastatic melanoma who received nivolumab + ipilimumab (NIVO+IPI, n=102) or NIVO alone (n=107) in the CheckMate 067 phase 3 trial. Retrospective analysis of baseline AI-defined CD8 topology was performed alone and combined with manually scored programmed death ligand 1 (PD-L1) expression on tumor cells.ResultsClassifier model predictions were concordant with manual scoring (determined by a consensus of pathologists) and non-inferior to the agreement between 2 pathologists, via Cohen’s kappa coefficient k=0.79 and k=0.65, respectively. No statistically meaningful differences in outcomes were observed between CD8-excluded and CD8-inflamed phenotypes within the PD-L1 ≥1% population. However, patients with PD-L1 <1%/CD8-excluded tumors exhibited longer median OS compared with those with PD-L1 <1%/CD8-inflamed (table 1). 38% (40/104) of PD-L1 <1% tumors were CD8-excluded. Within PD-L1 <1%, patients with an excluded phenotype also exhibited lower frequency of severe adverse events (grade ≥3) than patients with inflamed phenotype following treatment: NIVO+IPI, 75% (n=20) vs 91% (n=11); NIVO, 61% (n=18) vs 80% (n=15). Compared with PD-L1 status, the composite biomarker (AI-classified CD8-excluded plus PD-L1 ≥1%) identified a larger group of patients who had greater survival benefit with NIVO+IPI or NIVO alone (table 2).Abstract 387 Table 1Immunotherapy outcomes by CD8+ topology in PD-L1<1% melanomaIn a subset of patients with melanoma and tumor cell PD-L1 expression <1% in the CheckMate 067 clinical trial, those with a CD8-excluded phenotype demonstrated longer overall survival compared with those with a CD8-inflamed phenotype when treated with NIVO±IPI.Abstract 387 Table 2Composite biomarker outcomes in Checkmate 067In patients with melanoma in the CheckMate 067 clinical trial, the composite biomarker (AI-classified CD8-excluded phenotype plus PD-L1 expression ≥1%) identified more biomarker-positive patients and demonstrated increased overall survival benefit vs PD-L1 status alone for patients treated with NIVO±IPI. Hazard ratios represent patients with a PD-L1 expression of ≥1% compared with PD-L1 <1% or patients with a PD-L1 expression of ≥1% and CD8-excluded phenotype compared with PD-L1 expression <1% and not CD8-excluded.ConclusionsThis study explores the utility of combining AI-powered CD8 topology classifications with PD-L1 expression as a composite biomarker associated with immunotherapy response. In patients with PD-L1 <1% melanoma, median OS with NIVO+IPI was significantly longer in patients with CD8-excluded tumors than with an inflamed phenotype. Further studies are underway to identify mechanisms underlying responses to NIVO+IPI.AcknowledgementsWe would like to thank the team at PathAI for development of the AI classifier, and Dako, an Agilent Technologies, Inc. company, for collaborative development of the PD-L1 IHC 28-8 pharmDx assay. Editorial support was provided by Emily Motola, PharmD, and Matthew Weddig of Spark Medica Inc.Trial RegistrationClinicaltrialsgov number NCT01844505Ethics ApprovalThe study protocol and all amendments were approved by local institutional review boards, and the protocol was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines, as defined by the International Conference on Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. All patients provided written informed consent before enrollment.

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

BackgroundCombination checkpoint blockade with anti-PD-1 and anti-CTLA-4 antibodies has shown promising efficacy in melanoma. However, the underlying mechanism in humans remains unclear. A better understanding of the cellular and molecular mechanisms of αPD-1 and αCTLA-4 individually, and in combination, will guide the development of safer and more effective combination immunotherapy strategies.MethodsWe performed multimodal (scRNA + TCR + epitope) analysis across time in 32 stage IV melanoma patients treated with anti-PD-1, anti-CTLA-4, or anti-PD-1 + anti-CTLA-4 (combination) therapy. In order to understand the effect of checkpoint blockade on T cells at a single-clone resolution, we developed a novel algorithm Cyclone to track temporal clonal dynamics and their underlying cell states.ResultsAnti-CTLA-4 induced more durable immune responses than anti-PD-1, whereas combination therapy mobilized greater and more sustained immune responses. Using Cyclone, we identified 6 clonotypic trajectories with distinct temporal patterns. These analyses revealed that checkpoint blockade induced waves of immune responses composed of distinct clonotypes that peaked at different timepoints. Combination therapy generated clonal effector and exhausted CD8 T cells (TEX) responses that peaked at 6–9 weeks after treatment. Focused analyses of TEX in additional cohorts identified that anti-CTLA-4 induced robust expansion and proliferation of progenitor TEX, which synergized with anti-PD-1 to generate a large and durable reinvigoration of TEX. Immune profiling of a cohort of patients that first received anti-CTLA-4, followed by anti-PD-1 revealed that these enhanced progenitor responses were largely due to anti-CTLA-4 therapy. The induction of progenitor TEX by anti-CTLA-4 were independently validated using samples collected from the Checkmate 238 clinical trial of adjuvant nivolumab versus ipilimumab in resectable melanoma.ConclusionsFirst, durable immune responses represent waves of immune responses generated by different T cell clones. Second, progenitor TEX induced by CTLA-4 blockade may contribute to durable immune responses through self-renewal and replenishing of the TEX pool.Ethics ApprovalPatients were consented for blood collection under the University of Pennsylvania Abramson Cancer Center’s melanoma research program tissue collection protocol UPCC 08607, in accordance with the Institutional Review Board. For specimens from Checkmate 238, PBMC were obtained following informed consent under an IRB-approved protocol at NYU.

Purpose A same-day PET imaging agent capable of measuring PD-L1 status in tumors is an important tool for optimizing PD-1 and PD-L1 treatments. Herein we describe the discovery and evaluation of a novel, fluorine-18 labeled macrocyclic peptide-based PET ligand for imaging PD-L1. Methods [ 18 F]BMS-986229 was synthesized via copper mediated click-chemistry to yield a PD-L1 PET ligand with picomolar affinity and was tested as an in-vivo tool for assessing PD-L1 expression. Results Autoradiography showed an 8:1 binding ratio in L2987 (PD-L1 (+)) vs. HT-29 (PD-L1 (-)) tumor tissues, with >90% specific binding. Specific radioligand binding (>90%) was observed in human non-small-cell lung cancer (NSCLC) and cynomolgus monkey spleen tissues. Images of PD-L1 (+) tissues in primates were characterized by high signal-to-noise, with low background signal in non-expressing tissues. PET imaging enabled clear visualization of PD-L1 expression in a murine model in vivo, with 5-fold higher uptake in L2987 (PD-L1 (+)) than in control HT-29 (PD-L1 (-)) tumors. Moreover, this imaging agent was used to measure target engagement of PD-L1 inhibitors (peptide or mAb), in PD-L1 (+) tumors as high as 97%. Conclusion A novel 18 F-labeled macrocyclic peptide radioligand was developed for PET imaging of PD-L1 expressing tissues that demonstrated several advantages within a nonhuman primate model when compared directly to adnectin- or mAb-based ligands. Clinical studies are currently evaluating [ 18 F]BMS-986229 to measure PD-L1 expression in tumors.