Journal: Frontiers in Immunology

Article Title: Novel nanobody-161 binds tumor necrosis factor receptor 2 (TNFR2) to exert an anti-tumor effect but does not block TNFα-binding

doi: 10.3389/fimmu.2025.1694313

Figure Lengend Snippet: Characterization of Nanobody-161 by binding assays. (A) Binding of Nanobody-161 to human TNFR2 determined by ELISA (EC50 = 0.403 nM). Positive control antibody, OPI-SBT002e, and negative control, Y9, are shown. (B) Binding of Nanobody-161 to cynomolgus TNFR2 by ELISA (EC50 = 0.198 nM). Positive control antibody, OPI-SBT002e, and negative control, Y9, are shown. (C) Binding of Nanobody-161 to murine TNFR2 by ELISA. Positive control antibody, Y9, and negative control, OPI-SBT002e, are shown. (D) Binding of Nanobody-161 to human TNFR2-overexpressing HEK293 cells (huTNFR2-HEK293) by FACS (EC50 = 0.850 nM). Isotype control refers to native untransfected HEK293 cells. (E) Binding of Nanobody-161 to human TNFR2 overexpressing Jurkat cells (huTNFR2-Jurkat) by FACS (EC50 = 0.642 nM). Isotype control refers to native untransfected Jurkat cells. (F) Competitive inhibition assay with both Nanobody-161 and TNFα to show binding to huTNFR2-HEK293 cells. (G) Binding of Nanobody-161 to huTNFR2-HEK293 cells by FACS. Positive control: IgG1 which was known to bind HEK293 cells; isotype control: IgG1. (H) Binding of Nanobody-161 to Jurkat cells overexpressing human TNFR1 by FACS. Positive control: Anti-TNFR1 antibody; isotype control: IgG1. All experiments were repeated three times. Data are presented as mean ± SD or SEM. TNFR2, Tumor necrosis factor receptor 2; ELISA, Enzyme linked immunosorbent assay; MFI, mean fluorescence intensity; FACS, Fluorescence-activated cell sorting; SD, standard deviation; SEM, standard error of the mean.

Article Snippet: Human, monkey and mouse TNFR2 recombinant proteins were purchased from Sino Biological (10417-H03H, 90102-C08H and 50128-M08H).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Control, Inhibition, Fluorescence, FACS, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Novel nanobody-161 binds tumor necrosis factor receptor 2 (TNFR2) to exert an anti-tumor effect but does not block TNFα-binding

doi: 10.3389/fimmu.2025.1694313

Figure Lengend Snippet: Crystal structures of Nanobody-161 in complex with TNFR2. (A) Ribbon diagrams of the asymmetric unit of TNFR2 and Nanobody-161 complex in space group P212121. (B) Ribbon representation of Nanobody-161 showing the composition of 9 β-sheets from A to l CRD1: yellow; CRD2: green; CRD3: red. (C) Surface analysis of key residues involved in TNFR2 and Nanobody-161 interaction. TNFR2 epitope shown in light blue; Nanobody-161 paratope shown in pink. (D) Interface analysis showing TNFR2/Nanobody-161 interactions, including salt bridge, hydrogen bonds and hydrophobic interactions. TNFR2 CRD1: green; CRD2: blue; CRD3: orange; CRD4: cyan. Nanobody-161 CDR1: yellow; CDR2: green; CDR3: red. (E) Superimposition of Nanobody-161/TNFR2 and TNFα/TNFR2 complexes. Structure of the TNFα/TNFR2 complex was derived from the (PDB ID: 3ALQ). Left panel: TNFα: green; TNFR2: cyan; Nanobody-161: red. Overlapping regions are shown in yellow. Right panel: key interface residues in TNFR2. Colored residues participate in TNFα or Nanobody-161 binding. The residue shown in red participated in both interactions. TNFR2, Tumor necrosis factor receptor 2; PDB, protein data bank.

Article Snippet: Human, monkey and mouse TNFR2 recombinant proteins were purchased from Sino Biological (10417-H03H, 90102-C08H and 50128-M08H).

Techniques: Derivative Assay, Binding Assay, Residue

Journal: Frontiers in Immunology

Article Title: Novel nanobody-161 binds tumor necrosis factor receptor 2 (TNFR2) to exert an anti-tumor effect but does not block TNFα-binding

doi: 10.3389/fimmu.2025.1694313

Figure Lengend Snippet: Nanobody-161 effects on Tregs, CD8+ T cells and huTNFR2-expressing cells. (A) Effects of Nanobody-161 on TNFα-induced proliferation of Tregs in PBMCs. Isotype control: IgG1 targeting an irrevalent target except TNFR2. (B) Effects of Nanobody-161 on CD8+ T cell activation and IFN-γ secretion in vitro . (C, D) Effects of Nanobody-161 on ADCC in huTNFR2-HEK293 and huTNFR2-Jurkat cells. (E, F) Effects of Nanobody-161 on proliferation of huTNFR2-Jurkat and human tumor Jeko-1 cells. TNFR2, Tumor necrosis factor receptor 2; PBMCs, peripheral blood mononuclear cells; ADCC, antibody-dependent cell-mediated cytotoxicity; Tregs, T cells. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human, monkey and mouse TNFR2 recombinant proteins were purchased from Sino Biological (10417-H03H, 90102-C08H and 50128-M08H).

Techniques: Expressing, Control, Activation Assay, In Vitro

Journal: Frontiers in Immunology

Article Title: Novel nanobody-161 binds tumor necrosis factor receptor 2 (TNFR2) to exert an anti-tumor effect but does not block TNFα-binding

doi: 10.3389/fimmu.2025.1694313

Figure Lengend Snippet: Site-directed mutagenesis of key Nanobody-161 residues interacting with TNFR2. Binding and dissociation curves for Nanobody-161 WT and mutants R29A, F30G, R53A, S101A, Q102A, L103G, Y105G, F107G and R108A binding to histidine-tagged TNFR2-.TNFR2, Tumor necrosis factor receptor 2; WT, wild type.

Article Snippet: Human, monkey and mouse TNFR2 recombinant proteins were purchased from Sino Biological (10417-H03H, 90102-C08H and 50128-M08H).

Techniques: Mutagenesis, Binding Assay

Journal: Frontiers in Immunology

Article Title: Novel nanobody-161 binds tumor necrosis factor receptor 2 (TNFR2) to exert an anti-tumor effect but does not block TNFα-binding

doi: 10.3389/fimmu.2025.1694313

Figure Lengend Snippet: Effects of Nanobody-161 on apoptosis in huTNFR2-Jurkat cells treated with TNFα. (A–C) Dose-response curves for TNFα-mediated cell death of hTNFR2-Jurkat cells at different time points. (D–F) Dose-response curves for Nanobody-161 and cell death in huTNFR2-Jurkat cells pre-incubated with 15pM, 150pM and 1500pM TNFα for 5 min. (G) Dose-response curve for Nanobody-161 and cell death in huTNFR2-Jurkat cells pre-incubated with 58 pM TNFα for 30 min. (H) Dose-response curve for Nanobody-161 and cell death in huTNFR2-Jurkat cells pre-incubated with 150 pM TNFα for 30 min. TNFR2, Tumor necrosis factor receptor 2.

Article Snippet: Human, monkey and mouse TNFR2 recombinant proteins were purchased from Sino Biological (10417-H03H, 90102-C08H and 50128-M08H).

Techniques: Incubation

Journal: Frontiers in Immunology

Article Title: Novel nanobody-161 binds tumor necrosis factor receptor 2 (TNFR2) to exert an anti-tumor effect but does not block TNFα-binding

doi: 10.3389/fimmu.2025.1694313

Figure Lengend Snippet: The efficacy of Nanobody-161 in transgenic mouse model. (A) Effects of 0.5, 2.5 and 7.5 mpk Nanobody-161 on tumor volume over time in a mouse human TNFR2-expressing model of colon adenocarcinoma generated with MC38 cells. (B) Effects of 0.5, 2.5 and 7.5 mpk Nanobody-161 on body weight over time in a mouse model of colon adenocarcinoma generated with MC38 cells. (C) Effects of 0.5, 2.5 and 7.5 mpk Nanobody-161 on tumor weight on Day 35 (tumor weight was plotted as 0.01 g). (D) Effects of 0.5, 2.5 and 7.5 mpk Nanobody-161 on spleen weight on Day 35. No detectable spleen enlargement was found. (E) Photos of tumors dissected on Day 35. (F, G) Levels of serum ALT and AST on Day 19 (4th dose) and Day 24 (5th dose). TNFR2, Tumor necrosis factor receptor 2; CR, complete regression; ALT, alanine aminotransferase; AST, aspartate aminotransferase. * P < 0.05, ** P < 0.01.

Article Snippet: Human, monkey and mouse TNFR2 recombinant proteins were purchased from Sino Biological (10417-H03H, 90102-C08H and 50128-M08H).

Techniques: Transgenic Assay, Expressing, Generated

Journal: The Journal of Clinical Investigation

Article Title: Interruption of KLF5 acetylation promotes PTEN -deficient prostate cancer progression by reprogramming cancer-associated fibroblasts

doi: 10.1172/JCI175949

Figure Lengend Snippet: ( A and B ) Expression levels of Fgf9 mRNA and protein were higher in isolated CAFs when cocultured with 2 prostate cancer cell lines PC-3 and DU 145 with the KLF5 KR mutant, as detected by real-time qPCR ( A ) and ELISA ( B ). ( C ) Heatmap showing expression of activators and suppressors of FGF9 as reviewed from 617 publications. Red and green indicate the genes upregulated and downregulated by the Klf5 KR mutant. ( D ) The top ligands that signal fibroblasts from Krt4 + luminal cells were calculated by NicheNet, and their expression levels in Krt4 + luminal cells are shown as violin plots. ( E ) Plots of Tnf expression as detected by RNA-Seq. W/W , PB Cre Pten –/– Klf5 WT/WT ; KR/W , PB Cre Pten –/– Klf5 WT/KR ; KR/KR , PB Cre Pten –/– Klf5 KR/KR . ( F ) IHC staining for Tnf-α in mouse prostate tumors of the indicated genotypes. Scale bar: 50 μm. ( G ) The expression levels of TNF-α mRNA and protein were higher in DU 145 cells expressing the KLF5 KR mutant, as indicated by real-time qPCR (left) and ELISA (right). DU 145 cells were cultured under the indicated conditions. CAFs from Pten -deficient mouse prostate tumors were used to produce CM and cocultured with DU 145 cells. ( H ) TNF-α induced Fgf9 expression levels in CAFs, as indicated by real-time qPCR (left) and ELISA (right). ( I ) Blockage of TNF-α signaling by the neutralizing antibodies against TNF-α (5 ng/mL), TNFR1 (20 μg/mL), or TNFR2 (5 ng/mL) suppressed Fgf9 expression that was induced in CAFs by expression of the KLF5 KR mutant in DU 145 cells. Data are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed Student’s t test ( A , B , E , and G – I ) and 2-way ANOVA ( F ).

Article Snippet: Neutralizing antibodies against human TNF-α (SinoBiology, catalog no. 10602-R10N1), mouse tumor necrosis factor receptor 1 (TNFR1) (R&D System, catalog no. MAB430-100), and TNFR2 (SinoBiology, catalog no. 50128-RN204) were used for blocking TNF-α signaling in the cocultures.

Techniques: Expressing, Isolation, Mutagenesis, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Immunohistochemistry, Cell Culture