There is considerable interest in the pharmaceutical industry toward development of antibody-based biotherapeutics because they can selectively bind diverse receptors and often possess desirable pharmacology. Here, we studied product characteristics of 89 marketed antibody-based biotherapeutics that were approved from 1986 to mid-2020 by gathering publicly available information. Our analyses revealed major trends in their emergence as the best-selling class of pharmaceuticals. Early on, most therapeutic monoclonal antibodies were developed to treat cancer, with CD20 being the most common target. Thanks to industrialization of antibody manufacturing technologies, their use has now blossomed to include 15 different therapeutic areas and nearly 60 targets, and the field is still growing! Drug manufacturers are solidifying their choices regarding types of antibodies and their molecular formats. IgG1 kappa continues to be the most common molecular format among marketed antibody-based biotherapeutics. Most antibody-based biotherapeutics approved since 2015 are either humanized or fully human, but the data we collected do not show a direct correlation between humanness and reported incidence of anti-drug antibodies. Furthermore, there have also been improvements in terms of drug product stability and high concentration liquid formulations suitable for subcutaneous route of administration, which are being approved more often in recent years. These improvements, however, have not been uniformly adopted across all therapeutic areas, suggesting that multiple options for drug product development are being used to serve diverse therapeutic purposes. Insights gained from this analysis may help us devise better end-to-end antibody-based biotherapeutic drug discovery and development strategies.

The aim of the present pilot study was the identification of micro-RNA changes over time during the development and progression of type 2 diabetes (T2D) in Zucker diabetic fatty rats (ZDF rats). T2D is a complex metabolic disorder that is characterized, inter alia, by progressive failure of pancreatic β cells to produce insulin, but also by functional or morphological modifications of others organ, such as liver, adipose tissue and the cardiovascular system. Micro-RNAs are a novel class of biomarkers that have the potential to represent biomarkers of disease progression. In this study, the onset and progression of diabetes was followed in ZDF rats from six weeks until 17 weeks of age. After an initial phase of hyperinsulinemia, the animals developed T2D and lost the capacity to produce sufficient insulin. Circulating miRNAs were measured from plasma samples at four time points: pre-diabetes (six weeks of age), hyperinsulinemia (eight weeks), β cell failure (11 weeks) and late-stage diabetes (17 weeks) using TaqMan miRNA arrays. Bioinformatic analysis revealed distinct changes of circulating miRNAs over time. Several miRNAs were found to be increased over the course of the disease progression, such as miR-122, miR-133, miR-210 and miR-375. The most significantly decreased miRNAs were miR-140, miR-151-3p, miR-185, miR-203, miR-434-3p and miR-450a. Some of the miRNAs have also been identified in type 2 diabetic patients recently and, therefore, may have the potential to be useful biomarkers for the disease progression of T2D and/or the treatment response for anti-diabetic medications.

Excess liver iron content is common and is linked to the risk of hepatic and extrahepatic diseases. We aimed to identify genetic variants influencing liver iron content and use genetics to understand its link to other traits and diseases. First, we performed a genome-wide association study (GWAS) in 8,289 individuals from UK Biobank, whose liver iron level had been quantified by magnetic resonance imaging, before validating our findings in an independent cohort (n = 1,513 from IMI DIRECT). Second, we used Mendelian randomisation to test the causal effects of 25 predominantly metabolic traits on liver iron content. Third, we tested phenome-wide associations between liver iron variants and 770 traits and disease outcomes. We identified 3 independent genetic variants (rs1800562 [C282Y] and rs1799945 [H63D] in HFE and rs855791 [V736A] in TMPRSS6) associated with liver iron content that reached the GWAS significance threshold (p <5 × 10-8). The 2 HFE variants account for ∼85% of all cases of hereditary haemochromatosis. Mendelian randomisation analysis provided evidence that higher central obesity plays a causal role in increased liver iron content. Phenome-wide association analysis demonstrated shared aetiopathogenic mechanisms for elevated liver iron, high blood pressure, cirrhosis, malignancies, neuropsychiatric and rheumatological conditions, while also highlighting inverse associations with anaemias, lipidaemias and ischaemic heart disease. Our study provides genetic evidence that mechanisms underlying higher liver iron content are likely systemic rather than organ specific, that higher central obesity is causally associated with higher liver iron, and that liver iron shares common aetiology with multiple metabolic and non-metabolic diseases. Excess liver iron content is common and is associated with liver diseases and metabolic diseases including diabetes, high blood pressure, and heart disease. We identified 3 genetic variants that are linked to an increased risk of developing higher liver iron content. We show that the same genetic variants are linked to higher risk of many diseases, but they may also be associated with some health advantages. Finally, we use genetic variants associated with waist-to-hip ratio as a tool to show that central obesity is causally associated with increased liver iron content.

Activation of the AMP-activated protein kinase (AMPK) is an attractive approach for the treatment of type 2 diabetes. AMPK activation reduces glucose levels in animal models of type 2 diabetes by increasing glucose uptake in skeletal muscles and reducing hepatic glucose production. Furthermore, AMPK activation ameliorates hepatic steatosis in animal models. For the clinical development of AMPK activators it is essential to have a reliable target engagement marker for appropriate dose finding and to support proof of clinical principle. While the activation of AMPK by quantification of the phosphorylation of AMPK at Thr¹⁷² in target tissues can be assessed pre-clinically, this is not feasible in clinical studies. Therefore, we attempted to identify and translate a peripheral target engagement biomarker downstream of AMPK activation for clinical use in blood samples. For pharmacological activation of AMPK, two AMPK activators were synthesized (compound 1 and 2). A compound with structural similarities but no pharmacological effect on AMPK phosphorylation was synthesized as negative control (compound 3). Whole blood from healthy volunteers was incubated with an AMPK activator for up to 6 hours and mRNA sequencing was performed. Additionally, human PBMCs were isolated to evaluate Thr¹⁷²-phosphorylation of AMPK in Western blots. In order to enable identification of translatable biomarker candidates, blood samples from HanWistar rats treated for two weeks with an AMPK activator were also subjected to mRNA sequencing. Furthermore, concentration-response curves for four biomarker candidates were recorded in human blood samples using Nanostring nCounter technology. Finally, ZDF rats were treated with increasing doses of compound 2 for five weeks to investigate the glucose-lowering efficacy. To investigate changes of mRNA expression of two selected biomarker candidates in this ZDF rat study, qRT-PCR was performed. Pharmacological activation of AMPK in human PBMCs revealed an increase in Thr¹⁷²-phosphorylation of AMPK, confirming target engagement in these blood cells. RNA sequencing of human blood samples identified 608 deregulated genes after AMPK activation. Additionally, AMPK activation led to deregulation of 367 genes in whole blood from HanWistar rats which mapped to the respective human genes. 22 genes out of the intersection of genes deregulated in both species are proposed as potential translatable target engagement biomarker candidates. The most prominent genes were transmembrane glycoprotein NMB (GPNMB, osteoactivin), calcium-binding protein A9 (S100A9), peptidoglycan recognition protein (PGLYRP1) and Ras homolog gene family, member B (RHOB). Specificity for AMPK was shown by testing inactive compound 3 in HanWistar rats. The exposure-effect relationship for GPNMB was investigated in a subchronic study in diabetic ZDF rats. GPNMB showed a dose-dependent up-regulation both acutely and after subchronic dosing. GPNMB up-regulation correlated with an increased Thr¹⁷²-phosphorylation of AMPK in liver and quadriceps muscle in rats. GPNMB has been identified as a translatable target engagement biomarker for use in clinical studies.