Background: Glioblastoma is “cold”. Consequently, immune checkpoint blockade therapy has failed to improve patients‘ survival, which is negatively correlated with patients’ peripheral MDSC counts. AHR is known to mediate immune-suppressive functions of certain tryptophan metabolites such as kynurenine; yet, there lack of reports on how AHR agonists affect glioma immunity. Methods/Objectives: We hypothesized that ITE could synergize with PD1 antibody as AHR is one major node of immune-suppressive pathways, and tested it using an immune-competent mouse glioma model. Results: The combination of ITE+PD1 antibody glioma MDSC was significantly reduced, along with increased infiltration of the CD4−CD8+ and CD4+CD8+ T cells, leading to extended mouse survival. ITE treatment alone significantly reduces the infiltration of CD11b+Ly6G+Ly6Clo cells, namely PMN-MDSCs, and neutrophils, while the combination with PD1 antibody significantly reduces all MDSCs plus neutrophils. The presence of ITE inhibits the expression of IL11 and the in vitro induction of MDSCs from mouse PBMCs by IL11. The identification of the ITE-AHR-IL11-MDSC pathway provides more mechanistic insights into AHR’s effects. The fact that ITE, which is otherwise immune-suppressive, can activate immunity in glioma indicates that searching for drugs targeting AHR should go beyond antagonists.

Aryl hydrocarbon receptor (AHR) was a master regulator of anti-tumor cell migration in various cell types. Whether and how AHR regulates glioma cell migration is largely unknown. We found that small molecule 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous AHR ligand, can significantly block glioma cell migration and invasion in vitro , ex vivo and in vivo . Knocking down AHR by siRNA abolished ITE’s migration-inhibiting effects. ITE increased the number of filopodia-like protrusion formation, but reduced protrusion attachment to the extracellular matrix, and inhibited the rear retraction of migrating glioma cells. Moreover, both mesenchymal and amoeboid migrating cells were observed in the DMSO control group while none of the cells display amoeboid migration in the ITE treated group. MYH9 was significantly reduced by ITE treatment in human glioma cells. Over-expression of MYH9 abrogated ITE’s migration-inhibiting effects, with the expression level of MYH9 correlated with cell migration ability. Since MYH9 is a component of non-muscle myosin IIA (NMIIA), which is essential for cell migration in 3D confined space, and not a discovered target of AHR, the fact that ITE affects MYH9 via AHR opens a new research and development avenue.

Nanobodies (VHHs, ~12-15 kDa) are single-domain antibody fragments valued for diagnostic applications, yet their engineering is frequently limited by poor solubility and aggregation. This study used the aggregation-prone anti-VirB4 nanobody a_b5 as a model to establish a two-stage solubility optimization strategy: rational single-point mutagenesis, then AI-driven de novo design. Consensus sequence analysis and CamSol prediction converged on glycine 54; G54A and G54D mutations reduced the Z-average hydrodynamic diameter by ~31% and ~32%, respectively, but particle sizes remained in the hundreds of nanometers, revealing an inherent ceiling for single-point mutagenesis. To surpass this limit, a modular AI pipeline integrating AlphaFold3 hotspot analysis, RFdiffusion backbone generation, dual-pathway ProteinMPNN sequence design (Vanilla and Soluble), AlphaFold3 ipTM screening, and 100-ns molecular dynamics was constructed. Among 780 designed sequences across 10 nanobody-antigen pairs, B5S achieved a CamSol solubility score of 3.007 (480% increase over wild-type 0.518). DLS confirmed a Z-average diameter of 6.084 nm with 43.4% monomer peak; B5S was solubly expressed in E. coli while the Vanilla counterpart B5V precipitated. Direct ELISA confirmed dose-dependent binding of B5S to VirB4 with apparent K_D ~856.2 nmol/L. These results demonstrate that AI-driven holistic redesign can simultaneously optimize binding interface quality and macromolecular solubility.