Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: Characterization of the three anti-IL-33 antibodies. (A) Sequence alignment of etokimab, itepekimab, and tozorakimab. CDRs and FRs were defined according to the IMGT system. (B) SPR analysis measuring the binding affinity of the three antibodies and ST2 to IL-33. Serially diluted IL-33 (starting concentration 11.11 nM) was injected over immobilized ligands for a 180 s association and 300 s dissociation. (C) Inhibition of IL-33-induced signaling by the antibodies. HEK-Blue IL-33 reporter cells were stimulated with IL-33 at 2 ng/mL (triangles) or 30 ng/mL (circles) in the presence of a series of each antibody. After 22 hours, SEAP activity in the supernatant was quantified by measuring absorbance at 620 nm. Itepekimab, etokimab, and tozorakimab are shown in blue, green, and purple, respectively. Data points represent mean ± SD ( n = 3). Dose–response curves were fitted, and IC 50 /IC 90 values were calculated using GraphPad Prism which were summarized in the right panel table. (D) Blocking of IL-33–induced signaling on human peripheral blood mononuclear cell (PBMCs). Antibody and 1 nM IL-33 were added to human PBMCs for 19 hours. IFN-γ release was measured by means of ELISA. All data are represented as means ± SDs.

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques: Sequencing, Binding Assay, Concentration Assay, Injection, Inhibition, Activity Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: Structural overview of antibody – IL-33 complexes. (A) Structures of IL-33 (medium orchid) in complex with etokimab Fab (salmon), itepekimab Fab (olive drab), and tozorakimab Fab (dark turquoise), shown from left to right. The rightmost panel shows a superposition of all three Fab – IL-33 complexes, illustrating their relative positions and orientations. (B) Structure of the IL-33–ST2 complex (dark slate blue; PDB: 4KC3). The interface of D1/D2 domains and IL-33 named Site 1, interface of D3 domain and IL-33 named Site 2. (C) Structural alignment of the three Fab – IL-33 complexes with the ST2–IL-33 complex, showing the spatial relationship between each antibody and ST2. Colors are consistent with panels (A) and (B).

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques:

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: Interactions of etokimab and itepekimab with IL-33. (A, B) Epitopes on IL-33 (medium orchid) and paratopes on (A) etokimab (heavy chain, salmon; light chain, orange) and (B) itepekimab (heavy chain, olive drab; light chain, coral) are colored in dark gray, binding residues within are highlighted. (C, D) Detailed interaction networks for (C) Etokimab – IL-33 and (D) Itepekimab – IL-33 complexes. Hydrogen bonds and salt bridges are indicated by black dashed and solid lines, respectively; a red solid line denotes an interaction involving both. Interaction residues within are highlighted in related color. (E) Structural footprint of etokimab (salmon), itepekimab (olive drab), tozorakimab (dark turquoise), and ST2 (dark slate blue) on IL-33 (dark gray). Shared binding residues are listed in the lower panel.

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques: Binding Assay

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: Molecular basis of tozorakimab recognition of IL-33. (A) Binding interface between tozorakimab and IL-33. The epitope on IL-33 is shown in dark gray, with key contacting residues highlighted. The paratope is composed of the heavy chain (dark turquoise) and light chain (yellow-green). (B) Structural footprint comparison between tozorakimab and ST2 on IL-33. The tozorakimab footprint is shown in dark turquoise, while the ST2 Site 1 interface is colored dark slate blue. Shared binding residues are colored and highlighted according to their chain origin in tozorakimab. (C) Detailed molecular interactions in the tozorakimab – IL-33 complex. Hydrogen bonds and salt bridges are indicated by black dashed and solid lines, respectively; a red solid line denotes an interaction involving both. Relevant residues are colored consistently with panels A and B. (D) Electrostatic surface potential analysis of IL-33 (left) and tozorakimab (right). Surfaces are colored from red (negative charge) to blue (positive charge). The binding footprints of tozorakimab (dark turquoise) and IL-33 (medium orchid) are outlined. Dashed circles indicate complementary charged patches that facilitate binding.

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques: Binding Assay, Comparison

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: Characterization of tozorakimab and related mutants. (A) Binding affinity of tozorakimab and mutants to IL33 was assessed by indirect ELISA. Wells were coated with IL33 (2 μg/mL). An anti-human IgG-HRP antibody (promega, cat. no. W4031 ) was used for detection. (B) Inhibition of IL-33-induced signaling by the tozorakimab and mutants. HEK-Blue IL-33 reporter cells were stimulated with IL-33 at 0.5 nM in the presence of a series of each antibody. After 22 hours, SEAP activity in the supernatant was quantified by measuring absorbance at 620 nm. Data points represent mean ± SD ( n = 3). Dose – response curves were fitted and EC 50 or IC 50 values were calculated using GraphPad prism ( n = 3); results are summarized in the right panel table.

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques: Binding Assay, Indirect ELISA, Inhibition, Activity Assay

Journal: mAbs

Article Title: Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy

doi: 10.1080/19420862.2026.2639673

Figure Lengend Snippet: SPR competition assays of three antibodies and ST2. IL-33 was immobilized on a CM5 sensor chip. Injections were performed in two steps: first, protein 1 was injected for 150 s, followed by a mixture of protein 1 and protein 2 for 150 s. From left to right: competition profiles for itepekimab + ST2, etokimab + ST2, and tozorakimab + ST2, ST2 = 200 nM，Abs = 100 nM.

Article Snippet: HEK-BlueTM IL-33 cells (InvivoGen, catalog no. hkb-hil33) were utilized for bioactivity assessment of IL-33 through monitoring NF-κB and AP-1 pathway activation.

Techniques: Injection

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 was upregulated and positively correlated with NET formation in renal transplant patients. ( A ) Paired analysis of serum IL-33 levels, ( B ) paired analysis of serum MPO-DNA levels, ( C ) paired analysis of serum citH3 levels in renal transplant patients preoperatively and postoperatively ( n = 20). ( D ) Correlation analysis for postoperative serum IL-33 level and serum MPO-DNA level (Spearman r = 0.6892, P = 0.0008). ( E ) Correlation analysis of postoperative serum IL-33 level and serum citH3 level (Spearman r = 0.6486, P = 0.002). * P < 0.05, ** P < 0.01

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques:

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 and NETs were elevated following renal I/R in mice. ( A - B ) Serum IL-33 levels ( A ) and renal tissue homogenate IL-33 levels ( B ) after renal I/R ( n = 6). ( C - D ) Representative blots ( C ) and statistical analysis ( D ) of IL-33 protein expression levels in renal tissues after I/R ( n = 6). ( E - F ) Representative images ( E ) and statistical analysis ( F ) of IL-33 immunohistochemistry in renal tissues after I/R ( n = 6). Scale bar was 50 μm. ( G - H ) IL-33 expression and distribution among the groups (blue staining indicate DAPI for nuclei), IL-33 (red) and CD31 (green) ( n = 6). Scale bar was 50 μm. (I-J) Serum MPO-DNA (I) and citH3 levels ( J ) following renal I/R ( n = 6). ( K - L ) Representative blots ( K ) and statistical analysis ( L ) of citH3 protein expression levels in renal tissues after I/R ( n = 6). ( M - N ) NET formation in in the indicated groups (blue indicate DAPI staining), MPO (red) and citH3 (green) ( n = 6). Scale bar was 20 μm. ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 promoted NET generation during renal IRI. ( A ) A diagram showing WT mice receiving renal IRI or sham surgery following intraperitoneal injection of rIL-33 (10 µg per mouse) or vehicle (PBS). ( B - C ) Serum MPO-DNA ( B ) and citH3 levels ( C ) of mice in each group ( n = 6). ( D - E ) Representative blots ( D ) and statistical analyses ( E ) of citH3 protein expression levels in renal tissues in the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups through DAPI staining (blue), MPO (red) and citH3 (green) ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated groups ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues in the indicated groups ( n = 6). ( L - N ) Renal tissues from mice in the indicated groups stained with HE and KIM-1 ( L ), followed by score of tubular injury ( M ) and quantitative analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. * P < 0.05, ** P < 0.01

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Injection, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 exacerbated renal IRI by inducing NET formation. ( A ) A diagram showing the time node of intraperitoneal injection of GSK484 (4 mg/kg) and rIL-33 (10 µg per mouse) in WT mice before renal I/R. (B-C) Serum MPO-DNA ( B ) and citH3 levels ( C ) in the indicated groups ( n = 6). ( D - E ) Representative blots ( D ) and statistical analysis ( E ) of citH3 expression levels in renal tissues of the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups as determined using the DAPI (blue), MPO (red) and citH3 (green) staining ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated groups ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues from the indicated groups ( n = 6). ( L - N ) Renal tissues of mice in the indicated groups stained with HE and KIM-1 ( L ), followed by score of tubular injury ( M ) and quantitative analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. ** P < 0.01, *** P < 0.001

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Injection, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: Blocking IL-33 improved renal IRI by reducing NET formation. ( A ) The diagram of WT mice receiving renal IRI or sham surgery after intraperitoneal injection of anti-IL-33 monoclonal antibody (Anti-IL-33) (10 µg per ounce) or vehicle (IgG2b Isotype Control). ( B - C ) Serum MPO-DNA ( B ) and citH3 levels ( C ) of mice in the indicated groups ( n = 6). (D-E) Representative blots ( D ) and statistical analysis ( E ) of citH3 protein expression levels in renal tissues in the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups following DAPI (blue), MPO (red) and citH3 (green) staining ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated group ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues in the indicated groups ( n = 6). ( L - N ) Renal tissues of mice in the indicated groups subjected to HE and KIM-1 staining ( L ), followed by score of tubular injury ( M ) and statistical analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. ** P < 0.01, *** P < 0.001

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Blocking Assay, Injection, Control, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 stimulated NET generation in vitro. ( A - B ) Neutrophils incubated with PBS, PMA (100nM) and various concentrations of rIL-33 (20, 60, 100 ng/mL) for 4 h. rIL-33 stimulation increased MPO-DNA ( A ) and citH3 levels ( B ) in the neutrophil culture medium in a dose-dependent manner relative to the PBS group ( n = 3). ( C - D ) Relative to the PBS group, rIL-33 activated neutrophils to increase citH3 protein in a dose-dependent manner ( n = 3). ( E ) Compared with the PBS group, rIL-33 increased the expression of ST2 mRNA on neutrophils ( n = 3). ( F ) Representative scanning electron microscopy graphs of neutrophils treated with PBS or rIL-33 (100ng/mL) for 4 h. Scale bar was 10 μm. ( G ) The NET formation as indicated by DAPI (blue), MPO (red) and citH3 (green) staining, was comparable between rIL-33 (100ng/mL) and PMA group. Scale bar was 50 μm. ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: In Vitro, Incubation, Expressing, Electron Microscopy, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 induced NET formation via ST2/PI3K/Akt and ST2/PAD4 signaling pathways. Mouse bone marrow-derived neutrophils were treated with PBS or rIL-33 (100ng/mL) for 4 h, followed by RNA sequencing ( n = 3). ( A - B ) Volcano plot ( A ) and heatmap ( B ) showing differential gene expression between PBS and rIL-33 groups. ( C ) GO enrichment analysis of DEGs. ( D ) KEGG pathway enrichment analysis of DEGs. Bone marrow-derived neutrophils from WT and ST2 KO mice were treated with PBS or rIL-33 (100ng/mL) for 4 h. The production of MPO-DNA ( E ) and citH3 ( F ) in the cell culture medium of ST2 KO neutrophils treated with IL-33 was markedly decreased relative to the IL-33-treated WT neutrophils ( n = 3). ( G ) Confocal microscopy was conducted to examine NET formation (co-localization of DAPI, MPO and citH3) in each group. ( H - I ) The expression of PI3K, p-PI3K, Akt, p-Akt and citH3 in WT and ST2 KO neutrophils stimulated by rIL-33 as determined by Western blot ( n = 3). ( J - K ) WT neutrophils were treated with 10µM LY294002 (PI3K inhibitor) or 10µM MK2206 (Akt inhibitor) for 24 h, and 100ng/mL rIL-33 was added on the 20th h to stimulate WT neutrophils for 4 h. The expression of PI3K, p-PI3K, Akt, p-Akt and citH3 in neutrophils was determined by Western blot ( n = 3). ( L ) Gene expression of Padi1, Padi2, Padi4 and Padi6 in neutrophils stimulated with rIL-33 ( n = 3). The gene expression data were expressed as log 2 (FPKM + 1). ( M - N ) PAD4 protein expression levels and quantitative analysis in WT and ST2 KO neutrophils treated with rIL-33 were quantified by Western blots ( n = 3). ( O - P ) WT neutrophils were pretreated with GSK484 (PAD4 inhibitor) for 30 min after treatment with 100ng/mL rIL-33 for 4 h. The protein expression of citH3 in neutrophils was determined by Western blot ( n = 3). ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Protein-Protein interactions, Derivative Assay, RNA Sequencing, Gene Expression, Cell Culture, Confocal Microscopy, Expressing, Western Blot

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: Schematic diagram of the mechanism by which IL-33 exacerbated renal IRI by enhancing NET formation during renal I/R

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: