Journal: Nature Communications
Article Title: Targeting site-specific N-glycosylated B7H3 induces potent antitumor immunity
doi: 10.1038/s41467-025-58740-3
Figure Lengend Snippet: a Tumor growth of 4T1-hB7H3 cells in female 8 week-old BALB/c mice following treatment with Ab-82 antibody ( n = 9 mice per group). The treatment protocol is summarized by the arrows. Tumor volume (left) and tumor weight upon autopsy on day 23 (right) were calculated. b , c Quantitative IHC analysis of mouse CD4, mouse CD8, mouse GZMB expression ( b ), and human B7H3 expression ( c ) ( n = 9 mice per group). d Working flow chart for humanization of Ab-82 by using genetic engineering technology. e Binding affinity (K D ) analysis of Hu-Ab-82 by Biacore T200. f Tumor growth of A549 cells in female 7 week-old huPBMC-NOG-MHC I/II-2 KO Mice following treatment with Hu-Ab-82 antibody ( n = 8 tumors per group, 4 Mice bearing established tumors on both flanks). The treatment protocol is summarized by the arrows. Tumor volume (left) and tumor weight upon autopsy on day 26 (right) were calculated. g , h Quantitative IHC analysis of human CD8, human GZMB expression ( g ), and human B7H3 expression ( h ) ( n = 8 tumors per group, 4 Mice bearing established tumors on both flanks). i The indicated cells were treated by Ab-82 at different concentrations for 48 h, followed by western blotting to detect the total B7H3 protein level with anti-human B7H3. j Cell surface B7H3 was bound by Ab-82 for 30 min or not, followed by flow cytometry with commercial anti-B7H3 (Cat. 17-2769-42, eBioscience) ( n = 3 biological independent samples). k A549 cells were treated by Ab-82 at different concentrations for 24 h, followed by flow cytometry with commercial anti-B7H3 (Cat. 17-2769-42, eBioscience) ( n = 3 biological independent samples). l Cell surface B7H3 was labeled by Ab-82 and internalized for 5 min, followed by flow cytometry to measure B7H3 level ( n = 3 biological independent samples). m Cell surface B7H3 was labeled by Ab-82 and internalized at different time points in the presence of 50uM primaquine, followed by flow cytometry to measure B7H3 level ( n = 3 biological independent samples). n Cells were treated by 20 μg/ml pHrodo red-labeled Ab-82 for 24 h, followed by immunofluorescence analysis with LSM880.BF, bright field. o Cells were treated by 20 μg/ml pHrodo red-labeled Ab-82 for different time, followed by flow cytometry to measure Ab-82 internalization level ( n = 3 biological independent samples). p The indicated cells were treated by 20 μg/ml pHrodo red-labeled Ab-82 for different time, followed by flow cytometry to measure Ab-82 internalization level ( n = 3 biological independent samples). NS, not significance. The p -value in ( a – c ) and ( f – h ) and ( o ) was determined by one-way ANOVA with Dunnett’s multiple comparisons test. The p -value in ( p ) was determined by one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent mean ± SD. The data in ( a – h ) are representative of two independent experiments. The data in ( i – p ) are representative of three independent experiments.
Article Snippet: Cells were treated with primaquine (Cat. T0850, TargetMol) incubated at 37 °C to allow internalization of antibody-labeled B7H3.
Techniques: Expressing, Binding Assay, Western Blot, Flow Cytometry, Labeling, Immunofluorescence
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