Although there have been compelling advances in the cancer immunotherapy space recently in the form of chimeric antigen receptor (CAR) modified T-cells and checkpoint inhibitors, advanced tools to ...explore the therapeutic mechanisms of their combination are not adequately developed or widely available. To address this growing need, we developed a robust quantitative fluorescent immunohistochemistry platform using multiplex AQUA (Automated Quantitative Analysis) technology to evaluate checkpoint inhibitor expression, enumerate CAR T cells and determine the interaction between tumor cells and immune cells via novel co-localization algorithms. We explored utility of this method both in preclinical- and clinical model systems. In an immunodeficient mouse model of B-cell lymphoma, we evaluated homing of CAR T cells to malignant B-cells in primary lymphoid organs. We determined the phenotype and functional status of the CAR T cells via multiplex analyses of CD4, CD8, PD1 and FOXP3 expression. Additionally, to enable combination immunotherapies in Diffuse Large B-Cell Lymphoma (DLBCL) setting, we explored prevalence of adaptive immune resistance mechanisms in the form of PD1 and PD-L1 expression in immune- and tumor cell compartments via landmarks created by cytoplasmic and nuclear stains in both primary and secondary biopsies from DLBCL patients (n = 63). To support patient selection for CAR T trials, we quantified expression and prevalence of relevant tumor antigens that could not be scored reproducibly by traditional methods to yield objective cut points. We anticipate utilization of these quantitative multiplexed IHC methods for optimal selection of patients into upcoming novel combination immunotherapy trials
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Abstract Immune phenotypes (IP), defined by the tumor-infiltrating lymphocyte (TIL) distribution within the tumor microenvironment (TME), is prognostic and predictive of treatment response. Here, ...machine learning (ML) models that characterize the TME were deployed in non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) to exhaustively label TILs directly from hematoxylin and eosin (H&E)-stained whole slide images (WSI), compute IP and quantify TIL distribution, tasks which are manually untenable. ML-powered TME models (PathAI, Boston, MA; commercially available as PathExplore™) for NSCLC and HNSCC that quantify tissue regions (e.g. tumor, epithelium, and stroma) and cells (e.g. lymphocytes) in H&E-stained WSI were deployed on 126 NSCLC and 103 HNSCC commercial samples. Ground truth CD3 and CD8 scores were obtained from paired immunohistochemically-stained WSI (HALO, Indica Labs, Albuquerque, NM). Inflamed, desert, or excluded IPs (based on data-driven cutoffs) were inferred from the mean TIL density in epithelium and stroma (slide-level IP, sIP), and from the fraction of “hot” patches in all 100um x 100um patches tiling the tumor and stroma areas (patch-level IP, pIP). TIL spatial distribution was measured by the Morisita-Horn index that computes the patch-wise overlap between TILs and cancer cells (MHI, ranging from 0 to 1), and epithelial-stromal interface distance index (EDI) indicating the hot stromal patch bias toward the epithelium (-EDI) or stroma (+EDI). ML-predicted TIL densities highly correlated with ground truth CD3+ and CD8+ cell densities (NSCLC/HNSCC CD3, CD8: tumor Pearson r= 0.88/0.85, 0.74/0.74; epithelium r= 0.76/0.71, 0.88/0.62; stroma r= 0.74/0.87, 0.61/0.76). High s- and pIP agreement was seen (NSCLC 93% and HNSCC 83%); 6/26 discordant cases were driven by TIL hotspots with high density (116%-310% of the cutoff) but few (median = 26%) hot patches in the epithelium. MHI was higher for the inflamed vs excluded IP (p < 0.0001 for NSCLC and HNSCC) and intra-group variability was high (NSCLC/HNSCC inflamed: 0.66±0.11/0.31±0.14, excluded: 0.51±0.12/0.26±0.17, desert: 0.48±0.09/0.28±0.17; mean±std). EDI was lowest and negative in inflamed IP but near zero in desert and excluded IP (NSCLC/HNSCC inflamed: -43±4um/-184±19um, excluded: 1.6±4um/-41±13um, desert: 18±4um/-14±13; mean±sem). Excluded and desert IPs had roughly equal + and - EDI cases (NSCLC +/-: 57/46; HNSCC +/-: 39/29). ML-powered IP prediction using TIL distribution enables accurate and rapid profiling of the TME using routine histopathology. pIPs were concordant with sIPs and highlight TIL heterogeneity. Spatial markers (MHI and EDI) reveal differences between IP classes and intra-group heterogeneity relevant for drug discovery and patient stratification. Investigating prognostic associations of these markers is a promising direction for future studies. Citation Format: Nhat Le, Bahar Rahsepar, Jennifer Hipp, Jake Conway, Ylaine Gerardin, Emma Krause, Ciyue Shen, Raymond Biju, Michael Nercessian, Nicholas Indorf, Sandrine Degryse, Miles Markey, Victoria Mountain, Pranjal Vaidya, William Wijaya, Aditee Shrotre, Patrick Caplazi, David Inzunza, Joann Palma, Erik Huntzicker, Catherine Tribouley, Diana Chen, Raluca Prediou, Francine Chen, Kevin Kolahi. ML quantification of tumor-Infiltrating lymphocytes distinguishes immune-phenotypes and reveals phenotypic heterogeneity abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 905.
Glucocorticoids are used widely in the treatment of inflammatory diseases, but use is accompanied by a significant burden of adverse effects. It has been hypothesized that gene- and cell-specific ...regulation of the glucocorticoid receptor by small molecule ligands could be translated into a therapeutic with an improved risk-benefit profile. MK-5932 is a highly selective glucocorticoid receptor modulator that is anti-inflammatory in vivo with an improved profile on glucose metabolism: Bungard et al. (2011). Bioorg. Med. Chem. 19, 7374–7386. Here we describe the full biological profile of MK-5932. Cytokine production following lipopolysaccharide (LPS) challenge was blocked by MK-5932 in both rat and human whole blood. Oral administration reduced inflammatory cytokine levels in the serum of rats challenged with LPS. MK-5932 was anti-inflammatory in a rat contact dermatitis model, but was differentiated from 6-methylprednisolone by a lack of elevation of fasting insulin or glucose levels after 7 days of dosing, even at high exposure levels. In fact, animals in the vehicle group were consistently hyperglycemic at the end of the study, and MK-5932 normalized glucose levels in a dose-dependent manner. MK-5932 was also anti-inflammatory in the rat collagen-induced arthritis and adjuvant-induced arthritis models. In healthy dogs, oral administration of MK-5932 exerted acute pharmacodynamic effects with potency comparable to prednisone, but with important differences on neutrophil counts, again suggestive of a dissociated profile. Important gaps in our understanding of mechanism of action remain, but MK-5932 will be a useful tool in dissecting the mechanisms of glucose dysregulation by therapeutic glucocortiocids.
The adenovirus major late promoter (MLP) is induced to very high levels after the onset of the viral DNA replication. Previous studies have identified sequence elements located downstream of the MLP ...startsite (DE1, between +85 and +98; DE2, between +100 and +120) implicated, together with the upstream promoter element, in this late-phase-specific transcriptional activation. One protein (DEF, now renamed OEF-A), induced during the late phase of viral infection, has been identified and shown to bind to the DE1 element (Jansen-Durretal.,1989, J. Virol. 63, 5124–5132). Here we report about a distinct late-phase-specific protein (DEF-B) and its interactions with DEF-A. DNA-bindlng studies reveal that DEF-B interacts with the 5′ part of DE2 (DE2b), whereas DEF-A, besides its interaction with DE1, also binds to the 3′ portion of DE2 (DE2a), but with a lower affinity than for DE1. Furthermore, when added together, DEF-A and DEF-B cooperatively assemble onto the DE2 element as a heteromeric complex which is substantially more stable than the complexes formed by each protein alone. Using an In vivo transcriptional assay of the MLP, we show that DEF-A and DEF-B both have intrinsic transactivating properties.
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