Journal: Nature Communications

Article Title: B7-H3-mediated cis-inhibition of EGFR by a tumor-selective bispecific antibody enhances anti-tumor efficacy and minimizes toxicities

doi: 10.1038/s41467-026-69703-7

Figure Lengend Snippet: a Screening workflow schematic. *7B6 binding affinity was below the detection limit. b In vitro efficacy and safety validation. Efficacy: IC 50 ratio of monovalent EGFR mAb over B7-H3/EGFR bsAb in PC9-B7-H3 cells. Safety: IC 50 ratio of B7-H3/EGFR bsAb in A431 skin cells vs PC9-B7-H3. The IC 50 values for B7-H3/EGFR bsAb are the means from three biological replicates. c Growth inhibition assays of NCI-H358 and A431 cell lines ( n = 3 biological replicates). d Competitive EGFR binding assay. NCI-H358 cells pre-incubated with fluorescently labeled Zalutumumab (Zalu) were treated with 20G5/Zalu bsAb or Zalu ( n = 3 biological replicates). e EGF receptor occupancy assay. Cells treated with 20G5/Zalu or Zalu were stained with AF647-Zalu to quantify unoccupied EGFR ( n = 3 biological replicates). f EGFR internalization. Cells were treated with 5 nM IBI334, Zalu, or Null/Zalu and surface EGFR levels were detected using a non-competing anti-EGFR antibody ( n = 3 biological replicates). g Western blot of EGFR pathway-related proteins in NCI-H358 cells treated with 20G5/Zalu, Zalu, Null/Zalu or combo (20G5/null + Null/Zalu) ( n = 2 biological replicates). h Time-course analysis of EGFR signaling inhibition by Western blot (left, n = 2 biological replicates), and quantitated band intensities (left). i , j Ligand blockade assays. Flow cytometry (left) measuring EGF ( i ) or TGF-α ( j ) binding after treatment ( n = 3 biological replicates). EGFR signaling pathway was analyzed by Western blot (right, n = 2 biological replicates). k – m Cis-inhibition experiment. Cells were seeded at low-density (3 × 10⁵ cells/10 cm²) to prevent cell-cell contact. l Western blots of WT SK-MES-1 vs B7-H3-overexpressing (B7-H3 OE ) SK-MES1 treated with indicated antibodies for 4 h ( n = 2 biological replicates). (m) Western blot of SK-MES-1 B7-H3 OE cells ( n = 2 biological replicates). n Cis- vs trans-inhibition. Fluorescence microscopy of pEGFR (red) in SK-MES-1 (white arrows) and GFP + SK-MES-1-B7-H3 OE (green) co-cultures with/without 1 nM 20G5/Zalu ( n = 3 biological replicates). Schematic (left) and representative images (right) are shown. For EGFR internalization ( f ), statistical significance was assessed by two-way ANOVA (Tukey’s post hoc test). Data bars are mean ± SD. Source data are provided as a Source Data file.

Article Snippet: For IHC, multiplexed immunofluorescence (mIF), and western blotting (WB), the following antibodies were utilized: EGFR (CST, 4267), pEGFR (CST, 3777), B7-H3 (CST, 14508), AKT (CST, 4691), pAKT (CST, 4060), ERK (CST, 4695), pERK (CST, 4370), S6 (CST, 2217S), pS6 (CST, 2211S), GAPDH (CST, 2218), Pan CK (Gene Tech, GM351514 ), and Ki67 (CST, 9027S).

Techniques: Binding Assay, In Vitro, Biomarker Discovery, Inhibition, Incubation, Labeling, Staining, Western Blot, Flow Cytometry, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: EGFR/TBK1-dependent mitochondrial quality control contributes to acquired resistance to temozolomide

doi: 10.64898/2025.12.19.695123

Figure Lengend Snippet: A. U251 cells were treated by TMZ. Cells were harvested, fixed and stained by pTBK1 antibody coupled to fluorescent secondary antibody. pTBK1 specific fluorescence was detected by flow cytometry and expressed as a ratio to D0 fluorescence (*: p<0.05 vs D0). B. EGFR phosphorylation was measured by flow cytometry by the use of phospho-specific antibodies (pEGFR Y845 or pEGFR Y1068) on U251 or U251 Rho0 cells. Fluorescence was normalized to the fluorescence measured at D0 (*: p<0.05, **: p<0.01). C. EGFR Y845 as measured as in B. in the presence or absence of NAC 5mM (*: p<0.05, **: p<0.01). D. Phosphorylation of EGFR (Y845 and Y1068) and TBK1 was measured as in B in the presence or absence of PP1 (Src inhibitor). E. Src and TBK1 phosphorylation was measured as in B in U251 and U251 EGFR-cells treated by TMZ for the time indicated on the graph (*: p<0.05, **: p<0.01, ***: p<0.001). F. U251 and U251 EGFR-cells were treated by TMZ 50µM twice a week and cells were counted by flow cytometry at each time point. Cell number was normalized by the cell count at D0. G. The sensitivity of U251 cells to erlotinib was evaluated by MTT during TMZ treatment and the IC50 was calculated by AAT Bioquest “IC50 Calculator” tool ( https://www.aatbio.com/tools/ic50-calculator ).

Article Snippet: TOM20 #612278, BD (Le Pont de Claix, France); TFAM #8076, Cell Signaling Technology (Danvers, MA, USA); EGFR #4267, Cell Signaling Technology; pEGFR Y845 #6963, Cell Signaling Technology; pEGFR Y1068 #3777, Cell Signaling Technology; TBK1 #3504, Cell Signaling Technology; pTBK1 #5483, Cell Signaling Technology; pSrc #sc-81521, Santa Cruz Biotechnology (Dallas, TX, France); VDAC #ab14734 Abcam, (Cambridge, UK); IP3R #sc-28614, Santa Cruz Biotechnology; LC3 #83506, Cell Signaling Technology or #L7543, Sigma Aldrich.

Techniques: Staining, Fluorescence, Flow Cytometry, Phospho-proteomics, Cell Counting

Journal: bioRxiv

Article Title: EGFR/TBK1-dependent mitochondrial quality control contributes to acquired resistance to temozolomide

doi: 10.64898/2025.12.19.695123

Figure Lengend Snippet: A. U251 cells were treated by TMZ. Cells were harvested, fixed and stained by pTBK1 antibody coupled to fluorescent secondary antibody. pTBK1 specific fluorescence was detected by flow cytometry and expressed as a ratio to D0 fluorescence (*: p<0.05 vs D0). B. EGFR phosphorylation was measured by flow cytometry by the use of phospho-specific antibodies (pEGFR Y845 or pEGFR Y1068) on U251 or U251 Rho0 cells. Fluorescence was normalized to the fluorescence measured at D0 (*: p<0.05, **: p<0.01). C. EGFR Y845 as measured as in B. in the presence or absence of NAC 5mM (*: p<0.05, **: p<0.01). D. Phosphorylation of EGFR (Y845 and Y1068) and TBK1 was measured as in B in the presence or absence of PP1 (Src inhibitor). E. Src and TBK1 phosphorylation was measured as in B in U251 and U251 EGFR-cells treated by TMZ for the time indicated on the graph (*: p<0.05, **: p<0.01, ***: p<0.001). F. U251 and U251 EGFR-cells were treated by TMZ 50µM twice a week and cells were counted by flow cytometry at each time point. Cell number was normalized by the cell count at D0. G. The sensitivity of U251 cells to erlotinib was evaluated by MTT during TMZ treatment and the IC50 was calculated by AAT Bioquest “IC50 Calculator” tool ( https://www.aatbio.com/tools/ic50-calculator ).

Article Snippet: TOM20 #612278, BD (Le Pont de Claix, France); TFAM #8076, Cell Signaling Technology (Danvers, MA, USA); EGFR #4267, Cell Signaling Technology; pEGFR Y845 #6963, Cell Signaling Technology; pEGFR Y1068 #3777, Cell Signaling Technology; TBK1 #3504, Cell Signaling Technology; pTBK1 #5483, Cell Signaling Technology; pSrc #sc-81521, Santa Cruz Biotechnology (Dallas, TX, France); VDAC #ab14734 Abcam, (Cambridge, UK); IP3R #sc-28614, Santa Cruz Biotechnology; LC3 #83506, Cell Signaling Technology or #L7543, Sigma Aldrich.

Techniques: Staining, Fluorescence, Flow Cytometry, Phospho-proteomics, Cell Counting