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Monoclonal antibody-based immune checkpoint inhibitors, which have brought breakthrough effects in cancer treatments, are expected to assist in the treatment of viral diseases. However, antibody therapies may cause immune-related side effects, such as inflammation and pneumonia, due to cytokine storms. Small-molecule PD-1/PD-L1 inhibitors are an alternative to monoclonal antibody-based therapeutics. We have identified a novel small-molecule PD-1/PD-L1 inhibitor having a functional group (disulfide group), namely compound 2 (molecular weight: 456.6), from our library of sulfur-containing protein–protein interaction inhibitor compounds. Compound 2 selectively bound to PD-L1 over PD-1, with the dissociation rate constant (K D ) of 77.60 ± 4.44 nM (obtained by affinity analysis) and showed promising T cell activation recovery. A molecular docking simulation study between 2 and PD-L1 suggested that 2 binds to PD-L1 in a binding mode different from those of other small-molecule PD-L1/PD-1 inhibitors. Notably, oral administration of 2 to mice pre-infected with influenza A virus (A/NWS/33, H1N1 subtype) caused a significant increase in the neutralizing antibody titers, as well as recovery from influenza-induced pneumonia. Overall, 2 provides insight for the development of therapeutic drugs against early viral infections, with both virus titer-reducing and antibody titer-boosting effects. Moreover, 2 is widely used as a rubber peptizing agent in the production process of tires and other rubber products. Our findings may provide useful information for investigating its influence on living organisms. The online version contains supplementary material available at 10.1038/s41598-025-17982-3. Subject terms: Drug discovery and development, Pharmacology, Screening, Structure-based drug design

Chimeric Antigen Receptor (CAR) T cell therapy is a pivotal treatment for hematological malignancies. However, CAR T cell products exhibit batch-to-batch variability in cell number, quality, and in vivo efficacy due to donor-to-donor heterogeneity, and pre/post-manufacturing processes, and the manufacturing of such products necessitates careful testing, both post-manufacturing and pre-infusion. Here, we introduce the Cell Trajectory Modulation (CTM) assay, a microfluidic, label-free approach for the rapid evaluation of the functional attributes of CAR T cells based on biophysical features (i.e., size, deformability). CTM assay correlates with phenotypic metrics, including CD4:CD8 ratio, memory subtypes, and cytotoxic activity. Validated across multiple donors and culture platforms, the CTM assay requires fewer than 10,000 cells and delivers results within 10 minutes. Compared to labeled flow cytometry processing, the CTM assay offers real-time data to guide adaptive manufacturing workflows. Thus, the CTM assay offers an improvement over existing phenotypic assessments, marking a step forward in advancing CAR T cell therapy manufacturing. CAR T cell manufacturing faces significant challenges that impact cell quality and in vivo efficacy. This necessitates reliable cellular characterization methods. Here the authors present a real-time, label-free, microfluidic method that profiles cellular biophysical properties and correlates them to activation state and CAR T potency, facilitating the rapid phenotypic cell assessment during production.

Hematologic adverse events are common dose-limiting toxicities in drug development. Classical animal models for preclinical safety assessment of immunotherapies are often limited due to insufficient cross-reactivity with non-human homologous proteins, immune system differences, and ethical considerations. Therefore, we evaluate a human bone marrow (BM) microphysiological system (MPS) for its ability to predict expected hematopoietic liabilities of immunotherapeutics. The BM-MPS consists of a closed microfluidic circuit containing a ceramic scaffold covered with human mesenchymal stromal cells and populated with human BM-derived CD34+ cells in chemically defined growth factor-enriched media. The model supports on-chip differentiation of erythroid, myeloid and NK cells from CD34+ cells over 31 days. The hematopoietic lineage balance and output is responsive to pro-inflammatory factors and cytokines. Treatment with a transferrin receptor-targeting IgG1 antibody results in inhibition of on-chip erythropoiesis. The immunocompetence of the chip is established by the addition of peripheral blood T cells in a fully autologous setup. Treatment with T cell bispecific antibodies induces T cell activation and target cell killing consistent with expected on-target off-tumor toxicities. In conclusion, this study provides a proof-of-concept that this BM-MPS is applicable for in vitro hematopoietic safety profiling of immunotherapeutics. Subject terms: Biologics, Haematopoiesis, Lab-on-a-chip, Drug safety