Fig 2: Cytokine array analysis revealed high TIM3 expression on M2-like macrophage-derived CM. (A) Schematic representation of CM preparation from THP-1 cells. Complete media containing 5% FBS or 100% CM from 8505c and KTC-2 cells were used for the treatment of THP-1 cells for 48 h. The medium was discarded and then incubated with fresh medium. After 48 h, 100% CM from THP-1 cells were collected and analyzed by the Proteome Profiler Human XL Cytokine Array Kit. (B) Cytokine array profiling of CM derived from THP-1 cells. Antibodies for detection of each cytokine were spotted in duplicate. Reference spots and negative controls had six and two spots each, respectively. (C) Densitometric analysis revealed that TIM3 was consistently high in both CM THP-1 (8505c CM) and CM THP-1 (KTC-2). (D) TIM3 expression was verified by flow cytometry analysis. THP-1 cells treated with 8505c-derived CM for 18 h (D) and 24 h (E) compared to THP-1 control (incubated with complete media containing 5% FBS). Representative histograms (D,E) and quantification (F) are shown. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.005. Created with BioRender.

Fig 3: Model for M6903 partial competition with Gal-9 binding. Crystal structure of the Fab of M6903 bound to TIM-3 Ig-V domain (right side of figure) with a depiction of Gal-9 (represented as green cartoons) binding to the glycans on the TIM-3 Ig-V domain and the TIM-3 mucin domain (represented as a blue oval). Glycans are depicted as cyan squares and red circles bound by Gal-9. Coloring as in Fig 2A with Asn-99, as well as three additional predicted sites of glycosylation in the mucin domain colored magenta. Non-glycan mediated interaction to the IgV domain of TIM-3 is also shown schematically, although the exact binding site is unknown.38 Partial competition is represented by a red explosion symbol. The N- or C-terminal CRD of Gal-9 may bind to any of the glycans, and this may explain competition.

Fig 4: Decreased CEBPA expression leads to a decrease in Galectin-9 expression and an increase in the efficacy of cytotherapy. (A) Results of RT-PCR showed that the decrease of CEBPA leads to a significant decrease in the mRNA expression of Galectin-9 in U87. (B) Results of western blot showed that the decrease of CEBPA leads to a significant decrease in protein expression of Galectin-9 in U87. (C) Results of flow cytometry showed that the proportion of Galectin-9 and TIM-3 positive cells in patients with 1p/19q codel LGGs was significantly lower than that in patients with 1p/19q non-codel LGGs. There was no significant difference in the proportion of CD3 positive cells between the two groups. (D) Results of in vivo fluorescence imaging showed that the decrease of CEBPA can enhance the killing effect of T cells on tumor cells. The significance of the difference between the two groups was verified by Student’s t-test. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: M6903 inhibited TIM-3 binding to Gal-9, PtdSer, and CEACAM1. a) Binding of huTIM-3-Fc biotin (0.5 µg/mL) to plate-bound Gal-9 (2 µg/mL) was evaluated via ELISA after pre-incubation with serial dilutions of M6903 or isotype control (nM). b) Staurosporine (2 µg/mL, 18 hrs) induced apoptosis in Jurkat cells, leading to exposure of surface PtdSer. Apoptotic Jurkat cells were then incubated with rhTIM-3-Fc AF647 pre-incubated with various concentrations of M6903 or isotype control (µg/mL), and the binding of rhTIM-3-Fc AF647 on the Jurkat cells was measured by flow cytometry (MFI). c) Binding of recombinant His-tagged CEACAM1 to plate-bound huTIM-3-Fc (0.5 µg/well) was evaluated via ELISA after pre-incubation with serial dilutions of M6903 or isotype control (µg/mL). a-c) Non-linear best fit lines were generated for all plots using a Sigmoid dose-response equation and mean and standard deviation (SD) are presented.