ADC-111 is an Antigen-1-targeting antibody–drug conjugate with approximately eight payloads conjugated to one monoclonal antibody. The primary mechanism of action of ADC-111 is payload-induced cytotoxicity after antigen–antibody binding-mediated internalization. A cell-based assay using T24 cells engineered with Antigen-1 expression was developed and phase-appropriately validated to measure the potency of ADC-111 drug substance and drug product. This specific and stability-indicating assay detected potency loss resulting from degradation in the complementarity-determining region of the antibody, and correlated closely with the results from the Antigen-1 binding enzyme-linked immunosorbent assay, demonstrating that the ADC-111 cell-based assay is antigen-binding dependent. The potency measured by the cell-based assay also showed good correlation with varying drug–antibody ratio values, but ELISA did not. Notably, the ready-to-use format of the cell-based assay had performance comparable to that of the continuous culture using the same procedure, allowing both formats to be used interchangeably. In addition, the same parental cell line T24 was engineered with other tumor antigens, and the corresponding cell-based assay also showed good specificity, demonstrating the proof-of-concept of using a single parental cell line for multiple ADCs to standardize and accelerate cell-based assay development.
With-No-Lysine Kinase 1 (WNK1) is an atypical serine/threonine kinase involved in ion homeostasis, blood pressure regulation, migration and proliferation. Its intrinsically disordered regions (IDRs) support dynamic phosphorylation patterns. Here, we compiled 895 human phosphoproteomic profiling datasets and 352 differential phosphoproteomic datasets to systematically characterise the phosphorylation landscape associated with WNK1. Among reported WNK1 phosphosites, S2027, S2029, and S2032, located within an IDR, were the most frequently detected. Using a one-sided Fisher’s Exact Test followed by three sequential bias-correction filters, we identified 387 phosphosites in other proteins (PsOPs) showing positive co-differential association and 34 showing negative co-differential association with these three WNK1 sites. Computational approaches and database integration identified two candidate upstream kinases (EIF2AK1 and MAPK3), eight binary interactors, and 26 candidate substrate proteins. GO enrichment revealed site-specific functional categories: S2027-associated proteins were enriched in Ras/small GTPase signaling, chromatin organization, and cell-cycle-related processes; S2029-associated proteins in chromosome organization and transcriptional regulation; and S2032-associated proteins mainly in GTPase-related molecular functions. These findings provide a phosphosite-centric resource and computationally supported candidate associations that generate testable hypotheses for further experimental investigation of WNK1-associated signaling.
Fabry disease is a rare genetic multisystemic lysosomal disease characterized by progressive cellular accumulation of globotriaosylceramide (GL-3) in various organs with specific cutaneous (angiokeratoma), neurological (pain), renal (proteinuria, chronic kidney failure), cardiovascular (cardiomyopathy, arrhythmia), cochleovestibular and cerebrovascular manifestations (transient ischemic attacks, strokes). The phenotypic expression depends on the age of onset and, in females, on the level of X-inactivation. Enzyme replacement therapy is currently the standard therapy for the disease. JSC GENERIUM recently developed a potential biosimilar of Fabrazyme®, GNR-062, and investigated its physicochemical and biological properties, pharmacokinetics and immunogenicity to assess its bioequivalence to Fabrazyme®. Direct evaluation of the N-glycosylation profile by HPLC revealed differences mainly related to the distribution of complex sialylated glycans. The overall sialylation level was comparable between the two products. The same conclusion follows from the results of direct analysis of the sialic acid content. Despite a small difference of a few percent in biphosphorylated glycan content, the overall measured M6P values were comparable. Thus, the internalization of agalsidase β into the inner space of lysosomes by binding to key receptors appear to be the same for both products. In a single-blind, randomized comparative study of the pharmacokinetics and safety of a single intravenous infusion of GNR-062 (JSC GENERIUM, Russia) and Fabrazyme® (Genzyme Europe B.V., The Netherlands) in a crossover design in healthy volunteers, the ratio of geometric means (90% CI) of AUCinf and Cmax for GNR-062 to agalsidase β was 0.9736 (0.9413–1.0070) and 0.9867 (0.9546–1.0199), respectively. These studies demonstrated pharmacokinetic bioequivalence of GNR-062 to agalsidase β. Immunogenicity findings are preliminary, as the study was not powered for formal immunogenicity equivalence, and the single-dose design in healthy volunteers does not reflect repeated long-term administration in patients with Fabry disease. In conclusion, GNR-062 demonstrated acceptable short-term tolerability in healthy volunteers and warrants further investigation as a potential therapeutic option for patients with Fabry disease.
Lisianthus (Eustoma grandiflorum) is an ornamental species of high commercial value due to its diverse flower colors and prolonged vase life. However, lisianthus propagation by seed is limited by its low germination rates and high genetic variability, compromising plant uniformity and production timelines. This study evaluated the effect of meta-Topolin (mT) and of 6-benzylaminopurine (BAP), aromatic cytokinins, on the in vitro micropropagation of lisianthus, aiming to develop an efficient protocol for mass propagation. In vitro established shoots were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of mT and BAP (0, 0.5, 1.0, or 1.5 mg L−1). The parameters assessed were shoot proliferation, shoot length, leaf number, root formation, and root length. Significant differences were observed among treatments. The highest proliferation rate (59.20 shoots per explant) was achieved with 0.5 mg L−1 mT, representing a 37-fold increase over the control. With the addition of BAP to the culture medium, the results were inferior. Higher mT concentrations promoted rooting and improved acclimatization success, with the highest survival rate (95%) recorded for plants from the 1.5 mg L−1 mT treatment. Low mT concentrations enhanced shoot multiplication but limited root formation, affecting nursery survival. Results demonstrated that meta-Topolin induces a higher shoot number during lisianthus micropropagation, and at higher concentrations, it enables in vitro rooting of the shoots generated during the proliferation stage. This provides a practical alternative for large-scale commercial propagation of this ornamental species.
Background/Objectives: Pharmaceutical residues in human urine have raised serious concerns about the safe reuse of urine in agriculture. The current study investigates and compares the concentrations of pharmaceuticals in urine samples collected from male patients undergoing active antiretroviral regimens and self-reported “healthy individuals” at a government hospital in Pretoria, South Africa, and determines how the residues changed during three months of ambient storage, which is the usual recommendation before use in agriculture. Methods: The urine samples were collected in a plastic bottle to prevent fecal contamination, and 15 targeted compounds were analyzed using solid-phase extraction and liquid chromatography–mass spectrometry. The linearity ranged from 0.9910 to 0.9999, 75.5% to 99.1% for recovery, and intra-day and inter-day precision were below 6%. The urine from each group was collected into one pooled sample and then analyzed both pre- and post-storage. Results: Thirteen pharmaceuticals were detected in urine from patients on regular medication before storage; in “healthy individuals”, detections rose from eight to twelve after storage. Concentrations in the composite samples after storage were higher for some of the compounds but lower for others, including caffeine, nevirapine, prednisolone and 17α-ethinylestradiol. Metformin was highest overall at 1186.25 µg/L and remained at 806.68 µg/L after storage. The highest concentration for all the pharmaceuticals was reported for metformin, with a value of 1186.25 µg/L, occurring in the group of patients on a regular regimen. There could be a possibility that the rise can be attributed to the hydrolysis of conjugated metabolites during storage; however, no analysis of conjugates was carried out. Conclusions: This study shows the need for appropriate treatment of human urine prior to its reuse as fertilizer and echoes the importance of further research into human exposure pathways and effective pharmaceutical removal strategies.
