Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2.

The effect of targeted therapeutics on anticancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Because ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T-cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Moreover, we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways, and increased activity of transcriptional regulons driven by Atf5, Mafg, and Zbtb7a. This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest, skewing immature MDSC development toward monocytic lineage cells. In vivo, we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally, in the tumor immune infiltrate. Thus, our data reveal transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off-target effects of dabrafenib. SIGNIFICANCE: An important, but poorly understood, aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities.

Dabrafenib alters MDSC differentiation and function by activation of GCN2.

The effect of targeted therapeutics on anti-cancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Since ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Moreover, we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways, and increased activity of transcriptional regulons driven by Atf5 , Mafg , and Zbtb7a . This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest, skewing immature MDSC development towards monocytic lineage cells. In vivo , we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally, in the tumor immune infiltrate. Thus, our data reveals transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off target effects of dabrafenib.

Development of an In Vitro Test Method to Replace an Animal-Based Potency Test for Pertactin Antigen in Multivalent Vaccines.

There is increasing interest to replace animal-based potency assays used routinely to test vaccines, since they are highly variable, are costly, and present ethical concerns. The development of relevant in vitro assays is part of the solution. Using pertactin (PRN) antigen as an example in DTaP-IPV (diphtheria, tetanus, acellular pertussis, and inactivated poliovirus) vaccines, a PRN antigenicity ELISA was developed using two monoclonal antibodies with a high affinity to unique PRN epitopes, relevance to human immune responses, and evidence of functionality. The ELISA measured consistent PRN antigenicity between the vaccine lots and was validated to demonstrate its accuracy, precision, linearity, and specificity. Notably, the PRN antigenicity ELISA was more sensitive than the mouse-based potency test and could more effectively differentiate between degraded and intact vaccine lots compared to the in vivo test. From these studies, the PRN antigenicity ELISA is proposed as an in vitro replacement for the in vivo potency test for PRN in DTaP-IPV-based formulations. Important considerations in this study included comprehensive antibody characterization, testing of multiple vaccine lots, method validation, and comparison to animal-based potency. Together, these factors form part of an overall strategy that ensures reliable and relevant in vitro assays are developed to replace animal tests.

Epitope Profiling of Diphtheria Toxoid Provides Enhanced Monitoring for Consistency Testing during Manufacturing Process Changes.

In the vaccine industry, multiple physicochemical, immunological, in vitro and in vivo analytical methods are applied throughout the manufacturing process to characterize and monitor the quality of vaccines. Presented here is the Single Epitope Antigenicity Test (SEAT), an innovative, quantitative epitope profiling method which provides an extended immunochemical analysis for diphtheria toxoid (DTxd) to be used for consistency testing during manufacturing process changes. The method uses BioLayer Interferometry (BLI) and a panel of monoclonal antibodies (mAbs) to independently assess nine individual antigenic sites of DTxd. The panel includes mAbs which are functional, bind distinct sites on DTxd and are able to distinguish intact DTxd from that which has been exposed to heat treatment. The SEAT method was qualified for precision, accuracy, and linearity, and was used to define a preliminary comparability range for DTxd made using the current manufacturing process. DTxd lots manufactured using alternate processes were assessed in the context of this range to determine the impact on DTxd antigenicity. Epitope profiling by SEAT provides quantitative information on the integrity of multiple important antigenic regions of DTxd, and therefore represents a valuable tool in a comprehensive analytical test package which can be used to support manufacturing process changes for vaccines.

Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

Challenges establishing a multi-purpose fecal microbiota transplantation stool donor program in Toronto, Canada.

Background: The success of fecal microbiota transplantation (FMT) programs depends on maintaining suitable stool donors. We describe challenges recruiting and retaining universal donors in the first 2 years of an FMT clinical and research program in Toronto and identify opportunities for improvement. Methods: A four-stage screening process is used to identify suitable FMT donors in the Microbiota Therapeutics Outcomes Program. Donor screening follows Health Canada recommendations and excludes persons with history or risk for diseases associated with dysbiosis. Donors are rescreened microbiologically approximately every 1-3 months and answer ongoing health, exposure, and dietary questionnaires. Results: In the first 2 years of our program, 5 of 322 (1.6%) prospective stool donors passed initial screening, and only 2 (0.6%) were retained. Most prospective donors were excluded on telephone screening, at which point high BMI, medication use, and family history of relevant illness were common exclusions. No candidate was excluded because of a concerning physical examination. Microbiologic reasons for donor exclusion included carriage of Blastocystis hominis (n = 2), Helicobacter pylori (n = 2), extended spectrum beta-lactamase producing organisms (n = 1), Shiga-toxin producing Escherichia coli (n = 1), and sapovirus (n = 1). Universal donors were lost temporarily because of travel, antibiotic exposures, and transient carriage of antibiotic-resistant organisms. Conclusions: Recruiting and retaining suitable donors for FMT is challenging because of rigorous exclusions and labour-intensive screening processes. We present considerations for efficiency in donor screening, including targeting recruitment populations, expanded website self-screening, eliminating physical examinations, and streamlining post-travel risk assessment.

Historique: Le succès des programmes de transplantation de microbiote fécal (TMF) dépend de la rétention de donneurs fécaux appropriés. Les auteurs décrivent les difficultés à recruter et à conserver des donneurs universels dans les deux premières années d'un programme clinique et de recherche de TMF à Toronto ainsi qu'à déterminer les possibilités d'amélioration. Méthodologie: Un processus de sélection en quatre étapes permet de déterminer les donneurs de TMF appropriés au sein du programme de résultats thérapeutiques du microbiote. La sélection des donneurs suit les recommandations de Santé Canada et exclut les personnes ayant des antécédents ou un risque de maladies associés à la dysbiose. Les donneurs reprennent une sélection microbiologique environ tous les un à trois mois et répondent à des questionnaires sur la santé, l'exposition et le régime alimentaire. Résultats: Au cours de deux premières années du programme, cinq des 322 donneurs prospectifs de matière fécale (1,6 %) ont réussi la sélection initiale, et seulement deux (0,6 %) ont été retenus. La plupart des donneurs prospectifs ont été exclus à la sélection téléphonique; un IMC élevé, la prise de médicaments et des antécédents familiaux de maladie pertinente étaient des exclusions courantes. Aucun candidat n'a été exclu à cause d'un examen physique inquiétant. Les raisons microbiologiques d'exclure les donneurs incluaient le portage de Blastocystis hominis (n = 2), d'Helicobacter pylori (n = 2), d'organismes producteurs de bêta-lactamase à large spectre (n = 1), d'Escherichia coli producteur de la toxine de Shiga (n = 1) et du sapovirus (n = 1). Des donneurs temporaires étaient perdus temporairement à cause de voyages, d'exposition à des antibiotiques et de portage transitoire d'organismes antibiorésistants. Conclusions: Il est difficile de recruter et de retenir des donneurs appropriés de TMF en raison des exclusions rigoureuses et des processus de dépistage fastidieux. Les auteurs présentent des considérations d'efficacité pour le dépistage des donneurs, y compris le ciblage de populations à recruter, l'autodépistage élargi dans les sites Web, l'élimination des examens physiques et la rationalisation de l'évaluation du risque après le voyage.