Opportunities For Drug Development Research Articles

Phosphorylation regulation of cardiac proteins in Babesia microti infected mice in an effort to restore heart function

BackgroundBabesia is a common protozoan parasite that infects red blood cells. In mice infected with Babesia microti, the red blood cells were lysed, resulting in decreased oxygen-carrying capacity. To compensate for low blood oxygen levels, stress on the heart was greatly increased. Babesiosis induces a variety of pathologies; meanwhile, heart tissues initiate self-repair responses to babesiosis-induced tissue damage to restore heart function.MethodsTo discover the molecular mechanisms of the damage and self-repair in the heart after B. microti infection in mice, we investigated the changes in protein expression and phosphorylation modification levels in heart tissues at 0, 5, 8, 11, and 19 days post-infection using data-independent acquisition (DIA) quantitative proteomics.ResultsThe numbers of global proteins we identified were 1934, 1966, 1984, 1989, and 1955 and of phosphopeptides were 5118, 5133, 5130, 5133, and 5140 at 0, 5, 8, 11, and 19 days, respectively, in heart cells after infection with B. microti. The results showed that after B. microti infection the differentially expressed proteins in mice mainly include fibrinogen α (Fgα), fibrinogen β (Fgβ), Serpina1b, Serpina1c, cathepsin Z, cytochrome c oxidases (COXs), RPS11, and RPS20. The proteins with phosphorylation changes mainly include 20-kDa light chain of myosin II (MLC20), myosin light chain kinase (MLCK), mitogen-activated protein kinase 14 (MAPK14), and Akt1. These proteins were mainly involved in coagulation processes, cell apoptosis, oxidative phosphorylation, and ribosomes.ConclusionsThe coagulation cascade-related proteins, apoptosis-related proteins, oxidative phosphorylation-related proteins, and other types of proteins are all involved in the damage and self-repair process in the heart after B. microti infection. These results offer a wealth of new targets for further exploration into the causes of heart disease induced by Babesia infection and are of great significance for novel drug development and new opportunities for targeted therapies.Graphical

Dual-cell culture system with identical culture environment for comparison of anti-cancer drug toxicity

Tumor-on-a-chip technology that enables the dynamic culture of cell lines or clinical specimens on a microfluidic device offers new opportunities for clinical diagnosis, drug development, and toxicology test. Circulation is one of the key conditions to simulate tumor and other human-specific physiological environments. The peristaltic pump is an excellent tool to achieve the circulation culture condition. However, the complexity of the experimental setup, the cost of equipment, and experimental variations between culture units limit its widespread applications. Herein, we report a microfluidic-based dual culture system (dual-CS) that could simultaneously support two identical culture units for two independent drug tests using a single peristaltic pump. After continuously perfusing for 5 days, the difference in the flow rates between the two units remained<1%. To demonstrate the application, the dual-CS was used to investigate the toxicities of anti-cancer drugs toward A549 lung cancer epithelial cells. The cisplatin shows a dose-dependent effect on A549 cells, while pembrolizumab is non-toxic to the cells. The combination of cisplatin and pembrolizumab shows a synergistic effect on A549 cells. The dual-CS that simultaneously recapitulates an identical physiological environment for two culture units not only minimize the equipment cost but also reduces experimental variations between culture units.

Systematic illumination of druggable genes in cancer genomes.

SUMMARYBy combining 6 druggable genome resources, we identify 6,083 genes as potential druggable genes (PDGs). We characterize their expression, recurrent genomic alterations, cancer dependencies, and therapeutic potentials by integrating genome, functionome, and druggome profiles across cancers. 81.5% of PDGs are reliably expressed in major adult cancers, 46.9% show selective expression patterns, and 39.1% exhibit at least one recurrent genomic alteration. We annotate a total of 784 PDGs as dependent genes for cancer cell growth. We further quantify 16 cancer-related features and estimate a PDG cancer drug target score (PCDT score). PDGs with higher PCDT scores are significantly enriched for genes encoding kinases and histone modification enzymes. Importantly, we find that a considerable portion of high PCDT score PDGs are understudied genes, providing unexplored opportunities for drug development in oncology. By integrating the druggable genome and the cancer genome, our study thus generates a comprehensive blueprint of potential druggable genes across cancers.

Kirenol: A Potential Natural Lead Molecule for a New Drug Design, Development, and Therapy for Inflammation.

Kirenol, a potential natural diterpenoid molecule, is mainly found in Sigesbeckia species. Kirenol has received a lot of interest in recent years due to its wide range of pharmacological actions. In particular, it has a significant ability to interact with a wide range of molecular targets associated with inflammation. In this review, we summarise the efficacy and safety of kirenol in reducing inflammation, as well as its potential mechanisms of action and opportunities in future drug development. Based on the preclinical studies reported earlier, kirenol has a good therapeutic potential against inflammation involved in multiple sclerosis, inflammatory bowel disorders, diabetic wounds, arthritis, cardiovascular disease, bone damage, and joint disorders. We also address the physicochemical and drug-like features of kirenol, as well as the structurally modified kirenol-derived molecules. The inhibition of pro-inflammatory cytokines, reduction in the nuclear factor kappa-B (NF-κB), attenuation of antioxidant enzymes, stimulation of heme-oxygenase-1 (HO-1) expression, and nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation are among the molecular mechanisms contributing to kirenol’s anti-inflammatory actions. Furthermore, this review also highlights the challenges and opportunities to improve the drug delivery of kirenol for treating inflammation. According to the findings of this review, kirenol is an active molecule against inflammation in numerous preclinical models, indicating a path to using it for new drug discovery and development in the treatment of a wide range of inflammations.

Tilianin: A Potential Natural Lead Molecule for New Drug Design and Development for the Treatment of Cardiovascular Disorders.

Cardiovascular disorders (CVDs) are the leading risk factor for death worldwide, and research into the processes and treatment regimens has received a lot of attention. Tilianin is a flavonoid glycoside that can be found in a wide range of medicinal plants and is most commonly obtained from Dracocephalum moldavica. Due to its extensive range of biological actions, it has become a well-known molecule in recent years. In particular, numerous studies have shown that tilianin has cardioprotective properties against CVDs. Hence, this review summarises tilianin’s preclinical research in CVDs, as well as its mechanism of action and opportunities in future drug development. The physicochemical and drug-likeness properties, as well as the toxicity profile, were also highlighted. Tilianin can be a natural lead molecule in the therapy of CVDs such as coronary heart disease, angina pectoris, hypertension, and myocardial ischemia, according to scientific evidence. Free radical scavenging, inflammation control, mitochondrial function regulation, and related signalling pathways are all thought to play a role in tilianin’s cardioprotective actions. Finally, we discuss tilianin-derived compounds, as well as the limitations and opportunities of using tilianin as a lead molecule in drug development for CVDs. Overall, the scientific evidence presented in this review supports that tilianin and its derivatives could be used as a lead molecule in CVD drug development initiatives.

Non-alcoholic fatty liver disease - opportunities for personalized treatment and drug development

ABSTRACT Introduction Non-alcoholic fatty liver disease (NAFLD) constitutes a highly prevalent liver disorder whose rise in prevalence is closely connected to the growing rates of obesity, dyslipidemia, and type 2 diabetes. Importantly, kinetics and likelihood of NAFLD onset and its progression to non-alcoholic steatohepatitis (NASH) and fibrosis differs considerably between individuals. In recent years, the understanding of NAFLD pathogenesis has increased substantially and a multitude of factors, genetic predispositions, molecular signatures or NAFLD-related liver injury and comorbidities have been identified. Areas Covered This article summarizes inter-individual differences in NAFLD, including genetic variations, epigenetic and metabolic alterations and differences in the microbiome. We also discuss how these features might be leveraged for treatment personalization. Expert opinion The complexity and heterogeneity of NAFLD provides considerable challenges for drug developers and has resulted in numerous costly project failures. We expect that increased knowledge and appreciation of patient-specific factors will facilitate better patient stratification and identification of those individuals that benefit most from a given therapeutic strategy. Furthermore, we anticipate that pathophysiologically relevant in vivo and ex vivo disease models as well as large-scale chemogenomic projects hold promise to drastically improve NAFLD drug development to complement lifestyle and surgical interventions with pharmacological approaches.

A narrative review for the Hippo-YAP pathway in cancer survival and immunity: the Yin-Yang dynamics.

The Hippo-YAP pathway is fast becoming a key instrument in regulating cancers. Binary oppositions, also known as the Yin-Yang dynamics in Chinese, have emerged in its effects. Some oppositions are attributable to in vitro and in vivo experimental conditions, some are due to differences between cancers or species, some are derived from its inherent duality endowed by upstream and downstream signaling, and some are yet unresolved mysteries. However, as bewildering they are, few have been noticed and defined so far. In this review, we first look back on the Hippo pathway which was initially identified more than a decade ago. We then focus on tumor biology, especially some latest popular mechanisms that regulate tumor cell survival, such as ferroptosis, autophagy, and apoptosis. The third chapter is concerned with the findings of the relationship between the Hippo pathway and tumor immunity on the microenvironment in which tumor cells progress. In each of these main sections the contradictory points of the Hippo pathway are elucidated and thoroughly examined. On one hand, the Yin-Yang dynamics of the Hippo pathway brings about considerable challenges for current research; on the other hand, this work will generate fresh insight into it and offer opportunities for subsequent drug development for cancer and regenerative medicine.

CSIG-31. MULTI-OMICS TO EDGE INTO PRECISION MEDICINE FOR DIPG

Abstract DIPG is an incurable pediatric brain tumor with 80% of patients harboring H3F3A (H3.3) mutation that substitutes methionine for lysine at position 27 (K27M), resulting in global depletion of H3.3K27 me3 (trimethylation). These histone mutations modify the epigenome and alter oncogenic transcription, causing oncogenic insults to progenitor cells in early neurodevelopment (1). To determine the reprogramming pathways in the cell context of H3.3K27M tumors, we conducted LC-MS based proteomic and phosphoproteomic analysis on seven patient-derived DIPG cell lines. Three normal neuronal stem cell lines were included as non-tumor brain cells for comparison. Pathway analysis identified 29 pathways that are significantly altered in DIPG compared to normal brain cells at both the protein abundance and phosphosite level. Notably, AKT and MAPK associated PI3K signaling, VEGF signaling, mTOR signaling, and HIF1a signaling were differentially active in H3.3K27M tumors compared to healthy control cell lines. We saw significantly higher activity of multiple kinases involved in axon guidance and cytoskeletal remodeling in DIPG, such as PTK2B, DYRK2, TTBK2 and MARK2. This is the first time to report an increased abundance and kinase activity of PYK2 protein (coded by PTK2B), a close homologue of FAK and its associated signaling in DIPG. PYK2 has been proposed to act in concert with Src to link Gi- or Gq-coupled receptors with the mitogen-activated protein (MAP) kinase signaling pathway (2). Because of the shared signaling across kinase pathways, targeting activated PYK2 in DIPG may complement inhibitors of other dysregulated signaling networks in DIPG such as MAPK2, VEGFR, PI3K and Src. Our data also found that IL13RA2 was upregulated in DIPG. We conclude that for H3 K27M DIPG tumors, campaigns to target PYK2, MAPK2, VEGFR, PI3K, Src and IL13Ra2 using small molecules that traverse the blood brain barrier loom as promising opportunities for drug development.

U.S. FDA Drug Approvals for Gynecological Malignancies: A Decade in Review.

Over the last decade, there has been tremendous progress in the treatment of patients with gynecologic cancers with a changing therapy landscape. This summary provides an overview of U.S. Food and Drug Administration (FDA) approvals for gynecologic cancers from 2010 to 2020, totaling 17 new indications. For each of the approved indications, endpoints, trial design, results, and regulatory considerations are outlined. Among these 17 indications, six received accelerated approval (AA) and 11 received regular approval (RA). As of September 2021, of the six AA, three have subsequently demonstrated clinical benefit resulting in conversion to RA and the remaining three have ongoing clinical trials that have not yet reported results. Approval decisions for these 17 indications were supported by primary efficacy endpoints of progression-free survival (n = 10), objective response rate (n = 6), and overall survival (n = 1) and showed a favorable benefit-risk profile. Among the 17 indications, 15 received priority review and three applications participated in one or more novel Oncology Center of Excellence initiatives, including Real Time Oncology Review, Assessment Aid, and Project Orbis. Current FDA thinking on drug development opportunities and regulatory initiatives currently under way will be discussed.

Pharmaco-metabolomics opportunities in drug development and clinical research.

Pharmaco-metabolomics uses metabolic phenotypes for the prediction of inter-individual variations in drug response and helps in understanding the mechanisms of drug action. The field has made significant progress over the last 14 years with numerous studies providing clinical evidence for personalised medicine. However, discovered pharmaco-metabolomic biomarkers are not yet translated into clinics due to a lack of large-scale validation. Integration of targeted and untargeted metabolomics workflows into pharmacokinetic analysis and drug development can advance the field from bench to bedside. Also, Indian pharmaceutical research and its bioanalytical infrastructure are in a position to take on these opportunities by addressing challenges such as appropriate training and regulatory compliance.

Two steps, one ligand: How PPARγ binds small-molecule agonists

In this issue of Structure,Shang and Kojetin (2021) present insights into the binding mechanism of artificial agonists to the PPARγ nuclear receptor. These data support a two-step model with induced fit and conformational selection aspects. This mechanism may exist in related receptors, providing new opportunities for drug development.

DHODH Hot Spots: An Underexplored Source to Guide Drug Development Efforts.

Dihydroorotate dehydrogenase (DHODH) has long been recognized as an important drug target for proliferative and parasitic diseases, including compounds that exhibit trypanocidal action and broad-spectrum antiviral activity. Despite numerous and successful efforts in structural and functional characterization of DHODHs, as well as in the development of inhibitors, DHODH hot spots remain largely unmapped and underexplored. This review describes the tools that are currently available for the identification and characterization of hot spots in protein structures and how freely available webservers can be exploited to predict DHODH hot spots. Moreover, it provides for the first time a review of the antiviral properties of DHODH inhibitors. X-ray structures from human (HsDHODH) and Trypanosoma cruzi DHODH (TcDHODH) had their hot spots predicted by both FTMap and Fragment Hotspot Maps web servers. FTMap showed that hot spot occupancy in HsDHODH is correlated with the ligand efficiency (LE) of its known inhibitors, and Fragment Hotspot Maps pointed out the contribution of selected moieties to the overall LE. The conformational flexibility of the active site loop in TcDHODH was found to have a major impact on the druggability of the orotate binding site. In addition, both FTMap and Fragment Hotspot Maps servers predict a novel pocket in TcDHODH dimer interface (S6 site). This review reports how hot spots can be exploited during hit-to-lead steps, docking studies or even to improve inhibitor binding profile and by doing so using DHODH as a model, points to new drug development opportunities.

3D assessment of intervertebral disc degeneration in zebrafish identifies changes in bone density that prime disc disease

Back pain is a common condition with a high social impact and represents a global health burden. Intervertebral disc disease (IVDD) is one of the major causes of back pain; no therapeutics are currently available to reverse this disease. The impact of bone mineral density (BMD) on IVDD has been controversial, with some studies suggesting osteoporosis as causative for IVDD and others suggesting it as protective for IVDD. Functional studies to evaluate the influence of genetic components of BMD in IVDD could highlight opportunities for drug development and repurposing. By taking a holistic 3D approach, we established an aging zebrafish model for spontaneous IVDD. Increased BMD in aging, detected by automated computational analysis, is caused by bone deformities at the endplates. However, aged zebrafish spines showed changes in bone morphology, microstructure, mineral heterogeneity, and increased fragility that resembled osteoporosis. Elements of the discs recapitulated IVDD symptoms found in humans: the intervertebral ligament (equivalent to the annulus fibrosus) showed disorganized collagen fibers and herniation, while the disc center (nucleus pulposus equivalent) showed dehydration and cellular abnormalities. We manipulated BMD in young zebrafish by mutating sp7 and cathepsin K, leading to low and high BMD, respectively. Remarkably, we detected IVDD in both groups, demonstrating that low BMD does not protect against IVDD, and we found a strong correlation between high BMD and IVDD. Deep learning was applied to high-resolution synchrotron µCT image data to analyze osteocyte 3D lacunar distribution and morphology, revealing a role of sp7 in controlling the osteocyte lacunar 3D profile. Our findings suggest potential avenues through which bone quality can be targeted to identify beneficial therapeutics for IVDD.

Molecular Imaging and the PD-L1 Pathway: From Bench to Clinic.

Programmed death-1 (PD-1) and programmed death ligand 1 (PD-L1) inhibitors target the important molecular interplay between PD-1 and PD-L1, a key pathway contributing to immune evasion in the tumor microenvironment (TME). Long-term clinical benefit has been observed in patients receiving PD-(L)1 inhibitors, alone and in combination with other treatments, across multiple tumor types. PD-L1 expression has been associated with response to immune checkpoint inhibitors, and treatment strategies are often guided by immunohistochemistry-based diagnostic tests assessing expression of PD-L1. However, challenges related to the implementation, interpretation, and clinical utility of PD-L1 diagnostic tests have led to an increasing number of preclinical and clinical studies exploring interrogation of the TME by real-time imaging of PD-(L)1 expression by positron emission tomography (PET). PET imaging utilizes radiolabeled molecules to non-invasively assess PD-(L)1 expression spatially and temporally. Several PD-(L)1 PET tracers have been tested in preclinical and clinical studies, with clinical trials in progress to assess their use in a number of cancer types. This review will showcase the development of PD-(L)1 PET tracers from preclinical studies through to clinical use, and will explore the opportunities in drug development and possible future clinical implementation.

Repurposing existing therapeutics, its importance in oncology drug development: Kinases as a potential target.

Repurposing the large arsenal of existing non‐cancer drugs is an attractive proposition to expand the clinical pipelines for cancer therapeutics. The earlier successes in repurposing resulted primarily from serendipitous findings, but more recently, drug or target‐centric systematic identification of repurposing opportunities continues to rise. Kinases are one of the most sought‐after anti‐cancer drug targets over the last three decades. There are many non‐cancer approved drugs that can inhibit kinases as “off‐targets” as well as many existing kinase inhibitors that can target new additional kinases in cancer. Identifying cancer‐associated kinase inhibitors through mining commercial drug databases or new kinase targets for existing inhibitors through comprehensive kinome profiling can offer more effective trial‐ready options to rapidly advance drugs for clinical validation. In this review, we argue that drug repurposing is an important approach in modern drug development for cancer therapeutics. We have summarized the advantages of repurposing, the rationale behind this approach together with key barriers and opportunities in cancer drug development. We have also included examples of non‐cancer drugs that inhibit kinases or are associated with kinase signalling as a basis for their anti‐cancer action.

Abstract 2966: Novel ex vivo model for ERα positive breast cancer

Abstract Three-dimensional ex vivo cultures from patient-derived tumor tissue offer new opportunities for drug development and personalized treatment of cancer. However, one of the main challenges in establishing a tumor type faithful ex vivo tissue culture system is in optimization of biochemical and physical microenvironment to preserve desired phenotypic features. A collaboration with the Helsinki University Hospital enables us to receive live patient-derived breast cancer tissue samples from most breast cancer surgeries performed in the Southern Finland, typically samples arriving twice a week. Together with a group of materials scientists from Aalto Technical University we have explored seven different 3D culture matrices and defined their biomechanical properties and stiffness values with rheological measurements. Four investigated matrices preserved the luminal CK8+ cell phenotype of the luminal cancers they derived from, whereas rest of the matrices that included commonly used Matrigel and collagen scaffolds promoted a switch in the cell identity from luminal CK8+ to basal CK14+ phenotype. Furthermore, the genetic expression profiles examined with NGS corresponded to observed phenotypic switch. We noticed significant species-specific differences between mouse mammary and human breast samples in their morphology, phenotypic responses and ERα+ preservation to microenvironmental matrix stiffness. Furthermore, a combination of scaffold stiffness and composition of biochemical matrix constituency is required for long-term maintenance of ERα+ luminal tumour phenotype. Therefore, both physical properties and composition of the microenvironment need to be optimized for maintenance of breast tumor type specific phenotype and genetic profiles of ex vivo tumor explant cultures. Citation Format: Pauliina M. Munne, Lahja Martikainen, Iiris Räty, Kia Bertula, Nonappa Nonappa, Aino Peura, Minna Mutka, Marjut Leidenius, Tuomo Meretoja, Panu Kovanen, Johanna Mattson, Paivi Heikkila, Heikki Joensuu, Olli Ikkala, Juha T. Klefstrom. Novel ex vivo model for ERα positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2966.

Abstract 318: Multi-omics to edge into precision medicine for DIPG

Abstract Effective targeted cancer therapies are limited due to lack of our understanding of disease pathogenesis at the molecular level. Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brain tumor with 80% of DIPGs harboring H3F3A (H3.3) mutation that lead to a substitution of methionine for lysine at position 27 (K27M). The tumors bearing H3.3K27M mutation arise throughout the midline structures with global depletion of H3.3K27 me3 (trimethylation). These histone mutations modify the epigenome and alter oncogenic transcription, causing oncogenic insults to progenitor cells in early neurodevelopment (PMID: 30453529). To determine the reprogramming pathways in the cell context of H3.3K27M tumors, we conducted liquid chromatography-mass spectrometry-based proteomic and phosphoproteomic analysis on seven patient-derived DIPG cell lines that can be stably passaged in serum-free neural stem cell media and displayed distinct morphologies, growth rates and chromosome abnormalities (PMID: 21368213). Three normal neuronal stem cell lines were included as non-tumor brain cells for comparison. Pathway analysis identified 29 pathways that are significantly altered in DIPG compared to normal brain cells at both the protein abundance and phosphosite level. Notably, AKT and MAPK associated PI3K signaling, VEGF signaling, mTOR signaling, and HIF1a signaling were differentially active in H3.3K27M tumors compared to healthy control cell lines. We saw significantly higher activity of multiple kinases involved in axon guidance and cytoskeletal remodeling in DIPG, such as PTK2B, DYRK2, TTBK2 and MARK2. This is the first time to report an increased abundance and kinase activity of Pyk2 protein (coded by PTK2B), a close homologue of FAK and its associated signaling in DIPG. Overexpression and autophosphorylation of Pyk2 are required to stimulate glioma cell migration (PMIDs: 15967096, 18648907). Pyk2 has also been proposed to act in concert with Src to link Gi- or Gq-coupled receptors with the mitogen-activated protein (MAP) kinase signaling pathway (PMID:12960403). Because of the shared signaling across kinase pathways, targeting activated Pyk2 in DIPG may complement inhibitors of other dysregulated signaling networks in DIPG such as MAPK2, VEGFR, PI3K and Src. Our data also found that IL13RA2 was upregulated in DIPG; others have shown that increased expression of IL13RA2 is associated with poor prognosis in GBM (PMIDs: 32913543, 30366424). We conclude that for H3 K27M DIPG tumors, campaigns to target Pyk2, MAPK2, VEGFR, PI3K, Src and IL13Ra2 using small molecules that traverse the blood brain barrier loom as promising opportunities for drug development. Citation Format: Nanyun Tang, Kristin Leskoske, Krystine Garcia-Mansfield, Ritin Sharma, Hannah Tolson, Patrick Pirrotte, Michael E. Berens. Multi-omics to edge into precision medicine for DIPG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 318.

From Genome to Drugs: New Approaches in Antimicrobial Discovery.

Decades of successful use of antibiotics is currently challenged by the emergence of increasingly resistant bacterial strains. Novel drugs are urgently required but, in a scenario where private investment in the development of new antimicrobials is declining, efforts to combat drug-resistant infections become a worldwide public health problem. Reasons behind unsuccessful new antimicrobial development projects range from inadequate selection of the molecular targets to a lack of innovation. In this context, increasingly available omics data for multiple pathogens has created new drug discovery and development opportunities to fight infectious diseases. Identification of an appropriate molecular target is currently accepted as a critical step of the drug discovery process. Here, we review how diverse layers of multi-omics data in conjunction with structural/functional analysis and systems biology can be used to prioritize the best candidate proteins. Once the target is selected, virtual screening can be used as a robust methodology to explore molecular scaffolds that could act as inhibitors, guiding the development of new drug lead compounds. This review focuses on how the advent of omics and the development and application of bioinformatics strategies conduct a “big-data era” that improves target selection and lead compound identification in a cost-effective and shortened timeline.

P063 The immunological landscape of intestinal fibrosis in Crohn’s Disease

Abstract Background Patients with Crohn’s disease (CD) often develop strictures, necessitating surgical intervention. The immune pathways expressed in the fibrotic regions, especially in the deeper intestinal layers, are poorly characterized hence hampering therapeutic development for anti-fibrotic agents. We performed a detailed analysis of the immune cell populations in both inflammation and fibrosis in the mucosa and deeper intestinal layers in CD patients. Methods Patients with stricturing CD (n=25) or colorectal cancer (CRC, n=10), undergoing ileocolonic resection were included. The resection specimen of CD patients was macroscopically subdivided into fibrotic, inflammatory but not fibrotic, and unaffected regions by an IBD-experienced histopathologist. The mucosal layer was furthermore separated from the deeper intestinal layers. Immune cells were isolated, followed by flow cytometry. Quantitative real time PCR (qRT-PCR) was performed for the eosinophil chemoattractant CCL11 coding for eotaxin-1 and normalized against the housekeeping genes RPL13a and PPIA. Wilcoxon matched-pairs sign rank test was performed on paired samples while Dunn’s multiple testing was performed for unpaired comparisons. Results Flow cytometric analysis in CD patients showed mainly B cell enrichment in fibrotic tissue compared to the unaffected tissue, both in the mucosa (30.2% vs 20.9% of CD45+ cells, p=0.001) as in deeper layers (34.4% vs 24.1% of CD45+ cells, p=0.01) (figure 1). Mature dendritic cells (mDC), alternatively-activated macrophages and eosinophils were also enriched but in the deeper layers of the fibrotic segment compared to the mucosa (4.3% vs 3.4%, 1.07% vs 0.04% and 2.3% vs 1.5% of CD45+ cells; p = 0.02, 0.008 and 0.02) (figure 2). Moreover, increased eosinophilic presence was also shown in the unaffected CD region compared to the fibrotic area, both in deeper layers (4.0% vs 2.3% of CD45+ cells; p=0.03) as the mucosa (2.4% vs 1.5% of CD45+ cells; p=0.01) as well as in the inflamed deeper layers compared to the superficial layers (3.3% vs 2.1% of CD45+ cells, p=0.04). These data were corroborated by increased CCL11 mRNA expression, in the deeper layers of fibrotic tissue compared to the mucosa (52.3% vs 31.0%, p=0.03). Conclusion Our results suggest a role for innate immune cells in CD complicated with fibrosis. Especially in the deeper layers, eosinophils, mature dendritic cells and alternatively-activated macrophages are implicated. The eosinophilic enrichment in the deeper layers may partly be attributed to an increased eosinophil recruitment by CCL11. Our results offer opportunities for anti-fibrotic drug development in CD.

A phase 1b study of nivolumab in patients with autoimmune disorders and advanced malignancies (AIM-NIVO).

TPS2676 Background: Nivolumab is an anti-PD1 monoclonal antibody approved for treatment of an increasing number of solid tumors and hematological malignancies. However, patients (pts) with history of autoimmune disorders are excluded from the majority of clinical trials testing immune-checkpoint inhibitors (ICI) such as anti-PD1/anti-PD-L1 antibodies. Consequently, the risks of flare ups, worsening of pre-existing autoimmune disorders or risk of de-novo immune related adverse events (irAEs) in pts with dysfunctional immune systems and tumor types who otherwise stand to benefit from ICI therapy are largely unknown, posing a challenge for oncologists. We are conducting a phase Ib study to test the hypothesis that nivolumab can be safely administered to pts with varying severity of Dermatomyositis, Systemic Sclerosis, Rheumatoid Arthritis, Systemic Lupus Erythematosus, Inflammatory Bowel Disease, Multiple Sclerosis and other autoimmune disorders (AIM-Nivo). Methods: AIM-Nivo is an open-label, multi-center ongoing phase Ib study with nivolumab 480mg IV every 28 days in pts with autoimmune diseases and advanced malignancies (NCT03816345). The study has autoimmune disease-specific cohorts overseen by a multidisciplinary group of experts. The primary objective is to assess the overall safety and toxicity profile of nivolumab in pts with autoimmune disorders and advanced malignancies. Secondary objectives are to evaluate the antitumor efficacy; the impact of nivolumab on the autoimmune disease severity indices; and to explore potential biomarkers of response, resistance, or toxicity for each of the autoimmune disease-specific cohorts. Key overall inclusion criteria include age ≥18 years, histologically confirmed advanced or metastatic malignancies in which ICI are approved or have shown clinical activity. Key overall exclusion criteria include prior therapy with anti-PD-1/PD-L1 antibodies. Specific eligibility criteria are defined for each disease-specific cohort. For each autoimmune disorder, severity level of the disease as defined by disease-specific severity indices will be assessed, and up to a total of 12 pts will be included in each disease cohort at each severity level (max 36 pts per cohort). Primary endpoints are dose-limiting toxicities, adverse events (AEs) and serious AEs. Continuous monitoring of toxicity will be conducted. Key secondary endpoints are best objective response per RECIST1.1; progression free and overall survival; and cohort specific tumor tissue, blood, and non-tumor tissue-based biomarkers. The AIM-Nivo trial opened in May 2019 and is enrolling pts through the National Cancer Institute Experimental Therapeutics Clinical Trials Network (ETCTN), Early Drug Development Opportunity Program (EDDOP), and Create Access to Targeted Cancer Therapy for Underserved Populations (CATCH-UP) sites. Clinical trial information: NCT03816345.