Noninvasive Early Identification of Therapeutic Benefit from Immune Checkpoint Inhibition.

Although treatment of non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs) can produce remarkably durable responses, most patients develop early disease progression. Furthermore, initial response assessment by conventional imaging is often unable to identify which patients will achieve durable clinical benefit (DCB). Here, we demonstrate that pre-treatment circulating tumor DNA (ctDNA) and peripheral CD8 T cell levels are independently associated with DCB. We further show that ctDNA dynamics after a single infusion can aid in identification of patients who will achieve DCB. Integrating these determinants, we developed and validated an entirely noninvasive multiparameter assay (DIREct-On, Durable Immunotherapy Response Estimation by immune profiling and ctDNA-On-treatment) that robustly predicts which patients will achieve DCB with higher accuracy than any individual feature. Taken together, these results demonstrate that integrated ctDNA and circulating immune cell profiling can provide accurate, noninvasive, and early forecasting of ultimate outcomes for NSCLC patients receiving ICIs.

O-GlcNAcylation promotes tumor immune evasion by inhibiting PD-L1 lysosomal degradation.

Programmed-death ligand 1 (PD-L1) and its receptor programmed cell death 1 (PD-1) mediate T cell-dependent immunity against tumors. The abundance of cell surface PD-L1 is a key determinant of the efficacy of immune checkpoint blockade therapy targeting PD-L1. However, the regulation of cell surface PD-L1 is still poorly understood. Here, we show that lysosomal degradation of PD-L1 is regulated by O-linked N-acetylglucosamine (O-GlcNAc) during the intracellular trafficking pathway. O-GlcNAc modifies the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a key component of the endosomal sorting machinery, and subsequently inhibits its interaction with intracellular PD-L1, leading to impaired lysosomal degradation of PD-L1. O-GlcNAc inhibition activates T cell-mediated antitumor immunity in vitro and in immune-competent mice in a manner dependent on HGS glycosylation. Combination of O-GlcNAc inhibition with PD-L1 antibody synergistically promotes antitumor immune response. We also designed a competitive peptide inhibitor of HGS glycosylation that decreases PD-L1 expression and enhances T cell-mediated immunity against tumor cells. Collectively, our study reveals a link between O-GlcNAc and tumor immune evasion, and suggests strategies for improving PD-L1-mediated immune checkpoint blockade therapy.

Curvature-sensing peptide inhibits tumour-derived exosomes for enhanced cancer immunotherapy.

Tumour-derived exosomes (T-EXOs) impede immune checkpoint blockade therapies, motivating pharmacological efforts to inhibit them. Inspired by how antiviral curvature-sensing peptides disrupt membrane-enveloped virus particles in the exosome size range, we devised a broadly useful strategy that repurposes an engineered antiviral peptide to disrupt membrane-enveloped T-EXOs for synergistic cancer immunotherapy. The membrane-targeting peptide inhibits T-EXOs from various cancer types and exhibits pH-enhanced membrane disruption relevant to the tumour microenvironment. The combination of T-EXO-disrupting peptide and programmed cell death protein-1 antibody-based immune checkpoint blockade therapy improves treatment outcomes in tumour-bearing mice. Peptide-mediated disruption of T-EXOs not only reduces levels of circulating exosomal programmed death-ligand 1, but also restores CD8+ T cell effector function, prevents premetastatic niche formation and reshapes the tumour microenvironment in vivo. Our findings demonstrate that peptide-induced T-EXO depletion can enhance cancer immunotherapy and support the potential of peptide engineering for exosome-targeting applications.

Polyphenol Supplementation Enhances the Efficacy of PD-1/PD-L1 Inhibitors Against Cancer: A Meta-Analysis of Animal Studies.

BACKGROUND: This study performed a meta-analysis to evaluate the combined effects of polyphenols and anti-programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) inhibitors. METHODS: Relevant studies were collected from electronic databases. Standardized mean differences (SMDs) or hazard ratio (HR) was calculated by Stata 15.0 software. RESULTS: Sixteen preclinical studies were included. The overall meta-analysis showed that, compared to anti-PD-1/PD-L1 alone, polyphenol combined therapy significantly reduced the tumor volume (SMD = -3.28), weight (SMD = -2.18), number (SMD = -2.17), and prolonged the survival (HR = 0.45) of mice (all P < 0.001). Pooled analysis of mechanism studies indicated polyphenol combined therapy could increase the number of cytotoxic CD8+ T cells (SMD = 3.88; P < 0.001), IFN-γ+ CD8+ T cells (SMD = 2.38; P < 0.001), decrease the number of myeloid-derived suppressor cells (SMD = -2.52; P = 0.044) and Treg cells (SMD = -4.00; P = 0.004) and suppress PD-L1 expression in tumors (SMD = -13.41; P < 0.001). Subgroup analyses demonstrated curcuminoids, flavonoids, and stilbene changed the tumor volume, the percentage of CD8+ T cells, IFN-γ+CD8+ T cells, and PD-L1 expression. CONCLUSION: Polyphenol supplementation may be a promising combined strategy for patients with poor response to anti-PD-1/PD-L1 monotherapy.

Siglecs-7/9 function as inhibitory immune checkpoints in vivo and can be targeted to enhance therapeutic antitumor immunity.

Given the role of myeloid cells in T cell activation and in the antitumor response, targeting checkpoint molecules expressed on this population represents a promising strategy to augment antitumor immunity. However, myeloid checkpoints that can be effectively used as immunotherapy targets are still lacking. Here, we demonstrate the therapeutic potential of targeting the myeloid receptors Siglec-7 and Siglec-9 in vivo. By using a humanized immunocompetent murine model, we demonstrate that human Siglec-7 and Siglec-9, in addition to the murine homolog Siglec-E, inhibit the endogenous antitumor immune response, as well as the response to tumor-targeting and immune checkpoint inhibiting antibodies in vivo. The impact of these Siglecs on tumor progression is highly dependent on the anatomical distribution of the tumor and, as a consequence, the local tumor microenvironment, as tumors with a more immune-suppressive tumor microenvironment are less sensitive to Siglec perturbation. Finally, to assess the potential of these two receptors as targets for immunotherapy, we developed Fc engineered blocking antibodies to Siglec-7 and Siglec-9 and demonstrate that Siglec-7 and Siglec-9 blockade can significantly reduce tumor burden in vivo, demonstrating the therapeutic potential of targeting these two receptors.

GSK-3 Inhibitor Elraglusib Enhances Tumor-Infiltrating Immune Cell Activation in Tumor Biopsies and Synergizes with Anti-PD-L1 in a Murine Model of Colorectal Cancer.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that has been implicated in numerous oncogenic processes. GSK-3 inhibitor elraglusib (9-ING-41) has shown promising preclinical and clinical antitumor activity across multiple tumor types. Despite promising early-phase clinical trial results, there have been limited efforts to characterize the potential immunomodulatory properties of elraglusib. We report that elraglusib promotes immune cell-mediated tumor cell killing of microsatellite stable colorectal cancer (CRC) cells. Mechanistically, elraglusib sensitized CRC cells to immune-mediated cytotoxicity and enhanced immune cell effector function. Using western blots, we found that elraglusib decreased CRC cell expression of NF-κB p65 and several survival proteins. Using microarrays, we discovered that elraglusib upregulated the expression of proapoptotic and antiproliferative genes and downregulated the expression of cell proliferation, cell cycle progression, metastasis, TGFß signaling, and anti-apoptotic genes in CRC cells. Elraglusib reduced CRC cell production of immunosuppressive molecules such as VEGF, GDF-15, and sPD-L1. Elraglusib increased immune cell IFN-γ secretion, which upregulated CRC cell gasdermin B expression to potentially enhance pyroptosis. Elraglusib enhanced immune effector function resulting in augmented granzyme B, IFN-γ, TNF-α, and TRAIL production. Using a syngeneic, immunocompetent murine model of microsatellite stable CRC, we evaluated elraglusib as a single agent or combined with immune checkpoint blockade (anti-PD-1/L1) and observed improved survival in the elraglusib and anti-PD-L1 group. Murine responders had increased tumor-infiltrating T cells, augmented granzyme B expression, and fewer regulatory T cells. Murine responders had reduced immunosuppressive (VEGF, VEGFR2) and elevated immunostimulatory (GM-CSF, IL-12p70) cytokine plasma concentrations. To determine the clinical significance, we then utilized elraglusib-treated patient plasma samples and found that reduced VEGF and BAFF and elevated IL-1 beta, CCL22, and CCL4 concentrations correlated with improved survival. Using paired tumor biopsies, we found that tumor-infiltrating immune cells had a reduced expression of inhibitory immune checkpoints (VISTA, PD-1, PD-L2) and an elevated expression of T-cell activation markers (CTLA-4, OX40L) after elraglusib treatment. These results address a significant gap in knowledge concerning the immunomodulatory mechanisms of GSK-3 inhibitor elraglusib, provide a rationale for the clinical evaluation of elraglusib in combination with immune checkpoint blockade, and are expected to have an impact on additional tumor types, besides CRC.

Current Understanding on Why Ovarian Cancer Is Resistant to Immune Checkpoint Inhibitors.

The standard treatment of ovarian cancer (OC) patients, including debulking surgery and first-line chemotherapy, is unsatisfactory because of recurrent episodes in the majority (~70%) of patients with advanced OC. Clinical trials have shown only a modest (10-15%) response of OC individuals to treatment based on immune checkpoint inhibitors (ICIs). The resistance of OC to therapy is caused by various factors, including OC heterogeneity, low density of tumor-infiltrating lymphocytes (TILs), non-cellular and cellular interactions in the tumor microenvironment (TME), as well as a network of microRNA regulating immune checkpoint pathways. Moreover, ICIs are the most efficient in tumors that are marked by high microsatellite instability and high tumor mutation burden, which is rare among OC patients. The great challenge in ICI implementation is connected with distinguishing hyper-, pseudo-, and real progression of the disease. The understanding of the immunological, molecular, and genetic mechanisms of OC resistance is crucial to selecting the group of OC individuals in whom personalized treatment would be beneficial. In this review, we summarize current knowledge about the selected factors inducing OC resistance and discuss the future directions of ICI-based immunotherapy development for OC patients.

Increased serum cholesterol and long-chain fatty acid levels are associated with the efficacy of nivolumab in patients with non-small cell lung cancer.

BACKGROUND: Lipids have immunomodulatory functions and the potential to affect cancer immunity. METHODS: The associations of pretreatment serum cholesterol and long-chain fatty acids with the objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were evaluated in 148 patients with non-small cell lung cancer who received nivolumab. RESULTS: When each lipid was separately evaluated, increased low-density lipoprotein (LDL)-cholesterol (P < 0.001), high-density lipoprotein (HDL)-cholesterol (P = 0.014), total cholesterol (P = 0.007), lauric acid (P = 0.015), myristic acid (P = 0.022), myristoleic acid (P = 0.035), stearic acid (P = 0.028), linoleic acid (P = 0.005), arachidic acid (P = 0.027), eicosadienoic acid (P = 0.017), dihomo-γ-linolenic acid (P = 0.036), and behenic acid levels (P = 0.032) were associated with longer PFS independent of programmed death ligand 1 (PD-L1) expression. Meanwhile, increased LDL-cholesterol (P < 0.001), HDL-cholesterol (P = 0.009), total cholesterol (P = 0.036), linoleic acid (P = 0.014), and lignoceric acid levels (P = 0.028) were associated with longer OS independent of PD-L1 expression. When multiple lipids were evaluated simultaneously, LDL-cholesterol (P = 0.003), HDL-cholesterol (P = 0.036), and lauric acid (P = 0.036) were independently predictive of PFS, and LDL-cholesterol (P = 0.008) and HDL-cholesterol (P = 0.031) were predictive of OS. ORR was not associated with any serum lipid. CONCLUSIONS: Based on the association of prolonged survival in patients with increased serum cholesterol and long-chain fatty acid levels, serum lipid levels may be useful for predicting the efficacy of immune checkpoint inhibitor therapy.

EPHA7 mutation as a predictive biomarker for immune checkpoint inhibitors in multiple cancers.

BACKGROUND: A critical and challenging process in immunotherapy is to identify cancer patients who could benefit from immune checkpoint inhibitors (ICIs). Exploration of predictive biomarkers could help to maximize the clinical benefits. Eph receptors have been shown to play essential roles in tumor immunity. However, the association between EPH gene mutation and ICI response is lacking. METHODS: Clinical data and whole-exome sequencing (WES) data from published studies were collected and consolidated as a discovery cohort to analyze the association between EPH gene mutation and efficacy of ICI therapy. Another independent cohort from Memorial Sloan Kettering Cancer Center (MSKCC) was adopted to validate our findings. The Cancer Genome Atlas (TCGA) cohort was used to perform anti-tumor immunity and pathway enrichment analysis. RESULTS: Among fourteen EPH genes, EPHA7-mutant (EPHA7-MUT) was enriched in patients responding to ICI therapy (FDR adjusted P < 0.05). In the discovery cohort (n = 386), significant differences were detected between EPHA7-MUT and EPHA7-wildtype (EPHA7-WT) patients regarding objective response rate (ORR, 52.6% vs 29.1%, FDR adjusted P = 0.0357) and durable clinical benefit (DCB, 70.3% vs 42.7%, FDR adjusted P = 0.0200). In the validation cohort (n = 1144), significant overall survival advantage was observed in EPHA7-MUT patients (HR = 0.62 [95% confidence interval, 0.39 to 0.97], multivariable adjusted P = 0.0367), which was independent of tumor mutational burden (TMB) and copy number alteration (CNA). Notably, EPHA7-MUT patients without ICI therapy had significantly worse overall survival in TCGA cohort (HR = 1.33 [95% confidence interval, 1.06 to 1.67], multivariable adjusted P = 0.0139). Further gene set enrichment analysis revealed enhanced anti-tumor immunity in EPHA7-MUT tumor. CONCLUSIONS: EPHA7-MUT successfully predicted better clinical outcomes in ICI-treated patients across multiple cancer types, indicating that EPHA7-MUT could serve as a potential predictive biomarker for immune checkpoint inhibitors.

Local and Targeted Delivery of Immune Checkpoint Blockade Therapeutics.

Immune checkpoint blockade (ICB) therapy elicits antitumor response by inhibiting immune suppressor components, including programmed cell death protein 1 and its ligand (PD-1/PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). Despite improved therapeutic efficacy, the clinical response rate is still unsatisfactory as revealed by the fact that only a minority of patients experience durable benefits. Additionally, "off-target" effects after systemic administration remain challenging for ICB treatment. To this end, the local and targeted delivery of ICB agents instead could be a potential solution to maximize the therapeutic outcomes while minimizing the side effects.In this Account, our recent studies directed at the development of different strategies for the local and targeted delivery of ICB agents are discussed. For example, transdermal microneedle patches loaded with anti-programmed death-1 antibody (aPD1) and anti-CTLA4 were developed to facilitate sustained release of ICB agents at the diseased sites. Triggered release could also be achieved by various stimuli within the tumor microenvironment, including low pH and abnormally expressed enzymes. Recently, the combination of an anti-programmed death-ligand 1 antibody (aPD-L1) loaded hollow-structured microneedle patch with cold atmospheric plasma (CAP) therapy was also reported. Microneedles provided microchannels to facilitate the transdermal transport of CAP and further induce immunogenic tumor cell death, which could be synergized by the local release of aPD-L1. In addition, in situ formed injectable or sprayable hydrogels were tailored to deliver immunomodulatory antibodies to the surgical bed to inhibit tumor recurrence after primary tumor resection. In paralell, inspired by the unique targeting ability of platelets toward the inflammatory sites, we engineered natural platelets decorated with aPD-L1 for targeted delivery after tumor resection to inhibit tumor recurrence. We further constructed a cell-cell combination delivery platform based on conjugates of platelets and hematopoietic stem cells (HSCs) for leukemia treatment. With the homing ability of HSCs to the bone marrow, the HSC-platelet-aPD1 assembly could effectively deliver aPD1 in an acute myeloid leukemia mouse model. Besides living cells, we also leveraged HEK293T-derived vesicles with PD1 receptors on their surfaces to disrupt the PD-1/PD-L1 immune inhibitory pathway. Moreover, the inner space of the vesicles allowed the packaging of an indoleamine 2,3-dioxygenase inhibitor, further reinforcing the therapeutic efficacy. A similar approach has also been demonstrated by genetically engineering platelets overexpressing PD1 receptor for postsurgical treatment. We hope the local and targeted ICB agent delivery methods introduced in this collection would further inspire the development of advanced drug delivery strategies to improve the efficiency of cancer treatment while alleviating side effects.

Potential predictive value of change in inflammatory cytokines levels subsequent to initiation of immune checkpoint inhibitor in patients with advanced non-small cell lung cancer.

For a definite indication for immunotherapy, finding appropriate biomarkers that are predictive of treatment responses is necessary. Inflammatory cytokines which play critical roles in immunity against infectious sources or cancer cells are suggested to activate immune cells after initiation of immune checkpoint inhibitors (ICI). Through activation of immune cells such as T cells, natural killer cells, macrophages, or tumor infiltrating dendritic cells, inflammatory cytokines usually increase after programmed death (PD)-1/PD-L1 axis blockade. There have been several studies evaluating the predictive value of early changes in inflammatory cytokines in non-small cell lung cancer (NSCLC) patients undergoing immunotherapy. In this mini-review, we went through recent articles on potential blood level values of inflammatory cytokines in NSCLC patients receiving ICI and their early change around commencement of ICIs in predicting response to treatment and disease progression. The studies evaluated cytokines including interleukin (IL)-2, 6, 8, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α for predictability for responses to ICI. A combination cytokine panel can help predict the response and prognosis of patients with NSCLC who are receiving ICI treatment. Furthermore, a more individualized ICI treatment will be available if responses and change in tumor burden can be predicted. However, most of the studies on cytokines in NSCLC patients receiving ICIs had a small number of patients, and the heterogeneous measurement time points. Nevertheless, cytokines such as IL-8 and IFN- γ have considerable potential predictive value for immunotherapy response, which is worthy of further studies. To utilize blood cytokines levels as biomarkers for immunotherapy, a larger study with uniform measurement protocol is necessary.

Targeting cryptic-orthosteric site of PD-L1 for inhibitor identification using structure-guided approach.

Approved mAbs that block the protein-protein interaction (PPI) interface of the PD-1/PD-L1 immune checkpoint axis have led to significant improvements in cancer treatment. Despite having drawbacks of mAbs only few a compounds are reported till date against this axis. Inhibiting PPIs using small molecules has emerged as a significant therapeutic opportunity, demanding for the identification of drug-like molecules at an accelerated pace under the hit-to-lead campaigns. Due to the PD-L1's cross-talk with PD-1/CD80 and its overexpression on cancer cells, as well as the availability of its crystal structures with small molecules, it is an enticing therapeutic target for structure-assisted small molecule design. Furthermore, the selection of chemical databases enriched with focused designing for PPI interfaces is crucial. Therefore, in this study we have utilized the Asinex signature library for structure-assisted virtual screening to find the potential PD-L1 inhibitors by targeting the cryptic PD-L1 interface, followed by induced fit docking for pose refinements in the pocket. The obtained hits were then subjected to interaction fingerprinting and ligand-based drug-likeness investigations in order to evaluate and analyze their drug-like qualities (ADME). Twelve compounds qualified for molecular dynamics simulations, followed by thermodynamic calculations for evaluation of their stability and energetics inside the pocket. Two novel compounds with different chemical moieties have been identified that are consistent throughout the simulation, mimicking the interactions and binding energies with BMS-1166. These compounds appear as potential therapeutic candidates to be explored experimentally, thereby paving the way for the development of novel leads as immunomodulators.

Oncolytic Adenovirus Armed with BiTE, Cytokine, and Checkpoint Inhibitor Enables CAR T Cells to Control the Growth of Heterogeneous Tumors.

No single cancer immunotherapy will likely defeat all evasion mechanisms of solid tumors, including plasticity of tumor antigen expression and active immune suppression by the tumor environment. In this study, we increase the breadth, potency, and duration of anti-tumor activity of chimeric antigen receptor (CAR) T cells using an oncolytic virus (OV) that produces cytokine, checkpoint blockade, and a bispecific tumor-targeted T cell engager (BiTE) molecule. First, we constructed a BiTE molecule specific for CD44 variant 6 (CD44v6), since CD44v6 is widely expressed on tumor but not normal tissue, and a CD44v6 antibody has been safely administered to cancer patients. We then incorporated this BiTE sequence into an oncolytic-helper binary adenovirus (CAdDuo) encoding an immunostimulatory cytokine (interleukin [IL]-12) and an immune checkpoint blocker (PD-L1Ab) to form CAdTrio. CD44v6 BiTE from CAdTrio enabled HER2-specific CAR T cells to kill multiple CD44v6+ cancer cell lines and to produce more rapid and sustained disease control of orthotopic HER2+ and HER2-/- CD44v6+ tumors than any component alone. Thus, the combination of CAdTrio with HER2.CAR T cells ensures dual targeting of two tumor antigens by engagement of distinct classes of receptor (CAR and native T cell receptor [TCR]), and significantly improves tumor control and survival.

Organ Specificity and Heterogeneity of Cancer-Associated Fibroblasts in Colorectal Cancer.

Fibroblasts constitute a ubiquitous mesenchymal cell type and produce the extracellular matrix (ECM) of connective tissue, thereby providing the structural basis of various organs. Fibroblasts display differential transcriptional patterns unique to the organ of their origin and they can be activated by common stimuli such as transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF) signaling. Cancer-associated fibroblasts (CAFs) reside in the cancer tissue and contribute to cancer progression by influencing cancer cell growth, invasion, angiogenesis and tumor immunity. CAFs impact on the tumor microenvironment by remodeling the ECM and secreting soluble factors such as chemokines and growth factors. Differential expression patterns of molecular markers suggest heterogeneous features of CAFs in terms of their function, pathogenic role and cellular origin. Recent studies elucidated the bimodal action of CAFs on cancer progression and suggest a subgroup of CAFs with tumor-suppressive effects. This review attempts to describe cellular features of colorectal CAFs with an emphasis on their heterogeneity and functional diversity.

The Future of Cancer Diagnosis, Treatment and Surveillance: A Systemic Review on Immunotherapy and Immuno-PET Radiotracers.

Immunotherapy is an effective therapeutic option for several cancers. In the last years, the introduction of checkpoint inhibitors (ICIs) has shifted the therapeutic landscape in oncology and improved patient prognosis in a variety of neoplastic diseases. However, to date, the selection of the best patients eligible for these therapies, as well as the response assessment is still challenging. Patients are mainly stratified using an immunohistochemical analysis of the expression of antigens on biopsy specimens, such as PD-L1 and PD-1, on tumor cells, on peritumoral immune cells and/or in the tumor microenvironment (TME). Recently, the use and development of imaging biomarkers able to assess in-vivo cancer-related processes are becoming more important. Today, positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) is used routinely to evaluate tumor metabolism, and also to predict and monitor response to immunotherapy. Although highly sensitive, FDG-PET in general is rather unspecific. Novel radiopharmaceuticals (immuno-PET radiotracers), able to identify specific immune system targets, are under investigation in pre-clinical and clinical settings to better highlight all the mechanisms involved in immunotherapy. In this review, we will provide an overview of the main new immuno-PET radiotracers in development. We will also review the main players (immune cells, tumor cells and molecular targets) involved in immunotherapy. Furthermore, we report current applications and the evidence of using [18F]FDG PET in immunotherapy, including the use of artificial intelligence (AI).

Case-Control Study of the Immune Status of Humans Infected With Zoonotic Gorilla Simian Foamy Viruses.

BACKGROUND: Zoonotic simian foamy viruses (SFVs) establish persistent infections in humans, for whom the long-term consequences for health are poorly described. In this study, we aimed to characterize blood-cell phenotypes and plasma biomarkers associated with gorilla SFV infection in humans. METHODS: We used a case-control design to compare 15 Cameroonian hunters infected with gorilla SFV (cases) to 15 controls matched for age and ethnicity. A flow cytometry-based phenotypic study and quantification of plasma immune biomarkers were carried out on blood samples from all participants. Wilcoxon signed-rank tests were used to compare cases and controls. RESULTS: Cases had a significantly higher percentage of CD8 T lymphocytes than controls (median, 17.6% vs 13.7%; P = .03) but similar levels of B, natural killer, and CD4 T lymphocytes. Cases also had a lower proportion of recent CD4 thymic emigrants (10.9% vs 18.6%, P = .05), a higher proportion of programmed death receptor 1 (PD-1) expressing memory CD4 T lymphocytes (31.7% vs 24.7%, P = .01), and higher plasma levels of the soluble CD163 scavenger receptor (0.84 vs .59 µg/mL, P = .003) than controls. CONCLUSIONS: We show, for the first time, that chronic infection with SFV is associated with T lymphocyte differentiation and monocyte activation.

Characterization of PD-L1 binding sites by a combined FMO/GRID-DRY approach.

The programmed cell death protein 1 (PD-1) and its ligand, PD-L1, constitute an important co-inhibitory immune checkpoint leading to downregulation of immune system. Tumor cells developed a strategy to trigger PD-1/PD-L1 pathway reducing the T cell anticancer activity. Anti-PD-L1 small drugs, generally with improved pharmacokinetic and technological profiles than monoclonal antibodies, became an attractive research topic. Nevertheless, still few works have been published on the chemical features of possible binding sites. In this work, we applied a novel computational protocol based on the combination of the ab initio Fragment Molecular Orbital (FMO) method and a newly developed GRID-DRY approach in order to characterize the PD-L1 binding sites, starting from PD-1/PD-L1 and PD-L1/BMS-ligands (Bristol-Mayers Squibb ligands) complexes. The FMO method allows the calculation of the pair-residues as well as the ligand-residues interactions with ab initio accuracy, whereas the GRID-DRY approach is an effective tool to investigate hydrophobic interactions, not easily detectable by ab initio methods. The present GRID-DRY protocol is able to determine the energy contributions of each ligand atoms to each hydrophobic interaction, both qualitatively and quantitatively. We were also able to identify the three specific hot regions involved in PD-1/PD-L1 protein-protein interaction and in PD-L1/BMS-ligand interactions, in agreement with preceding theoretical/experimental results, and to suggest a specific pharmacophore for PD-L1 inhibitors.

Modeling Immune Checkpoint Inhibitor Efficacy in Syngeneic Mouse Tumors in an Ex Vivo Immuno-Oncology Dynamic Environment.

The immune checkpoint blockade represents a revolution in cancer therapy, with the potential to increase survival for many patients for whom current treatments are not effective. However, response rates to current immune checkpoint inhibitors vary widely between patients and different types of cancer, and the mechanisms underlying these varied responses are poorly understood. Insights into the antitumor activities of checkpoint inhibitors are often obtained using syngeneic mouse models, which provide an in vivo preclinical basis for predicting efficacy in human clinical trials. Efforts to establish in vitro syngeneic mouse equivalents, which could increase throughput and permit real-time evaluation of lymphocyte infiltration and tumor killing, have been hampered by difficulties in recapitulating the tumor microenvironment in laboratory systems. Here, we describe a multiplex in vitro system that overcomes many of the deficiencies seen in current static histocultures, which we applied to the evaluation of checkpoint blockade in tumors derived from syngeneic mouse models. Our system enables both precision-controlled perfusion across biopsied tumor fragments and the introduction of checkpoint-inhibited tumor-infiltrating lymphocytes in a single experiment. Through real-time high-resolution confocal imaging and analytics, we demonstrated excellent correlations between in vivo syngeneic mouse and in vitro tumor biopsy responses to checkpoint inhibitors, suggesting the use of this platform for higher throughput evaluation of checkpoint efficacy as a tool for drug development.

Notch signaling genes and CD8+ T-cell dynamics: Their contribution to immune-checkpoint inhibitor therapy in oral squamous cell carcinoma: A retrospective study.

BACKGROUND: Aberrant Notch signaling pathway has been related with the tumorigenesis in head and neck region, involving oral cavity. Here, we report the correlation between mutations in the Notch signaling pathway and CD8+ T-cell infiltration via PD-L1, which lead to enhanced antitumor immunity and may target for immune-checkpoint inhibitors (ICIs) therapy. METHODS: This retrospective study analyzed the results of immunohistochemical staining for PD-L1 and CD8+ T-cell infiltration in 10 patients and whole-exome sequencing (WES) was conducted on five of these patients to identify frequently mutated genes. RESULTS: Four of 10 patients were positive for PD-L1 and CD8+ T. By analyzing WES in three of these four patients, we notably identified the mutations of NOTCH1, FBXW7, and noncoding RNA intronic mutation in NOTCH2NLR in two of these three patients. This study may enable better selection of ICI therapy with CD8+ T-cell infiltration via PD-L1 expression for oral squamous cell carcinoma patients with mutations in Notch signaling pathway.

Antigen-loaded Monocyte Administration and Flt3 Ligand Augment the Antitumor Efficacy of Immune Checkpoint Blockade in a Murine Melanoma Model.

Undifferentiated monocytes can be loaded with tumor antigens (Ag) and administered intravenously to induce antitumor cytotoxic T lymphocyte (CTL) responses. This vaccination strategy exploits an endogenous Ag cross-presentation pathway, where Ag-loaded monocytes (monocyte vaccines) transfer their Ag to resident splenic dendritic cells (DC), which then stimulate robust CD8 + CTL responses. In this study, we investigated whether monocyte vaccination in combination with CDX-301, a DC-expanding cytokine Fms-like tyrosine kinase 3 ligand (Flt3L), could improve the antitumor efficacy of anti-programmed cell death (anti-PD-1) immune checkpoint blockade. We found that Flt3L expanded splenic DC over 40-fold in vivo and doubled the number of circulating Ag-specific T cells when administered before monocyte vaccination in C57BL/6 mice. In addition, OVA-monocyte vaccination combined with either anti-PD-1, anti-programmed cell death ligand 1 (anti-PD-L1), or anti-cytotoxic T lymphocyte antigen-4 (anti-CTLA-4) suppressed subcutaneous B16/F10-OVA tumor growth to a greater extent than checkpoint blockade alone. When administered together, OVA-monocyte vaccination improved the antitumor efficacy of Flt3L and anti-PD-1 in terms of circulating Ag-specific CD8 + T cell frequency and inhibition of subcutaneous B16/F10-OVA tumor growth. To our knowledge, this is the first demonstration that a cancer vaccine strategy and Flt3L can improve the antitumor efficacy of anti-PD-1. The findings presented here warrant further study of how monocyte vaccines can improve Flt3L and immune checkpoint blockade as they enter clinical trials.