Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: Generation of targeted immunocytokine mimetics (ICMs) in a bispecific bivalent camelid-derived sdAb-based architecture after yeast surface screening (A) Trimeric (rh) TNF (blue) triggers trimeric TNFR1 (light green) downstream signaling. Bispecific bivalent ICMs are able to elicit trimeric TNFR1 downstream signaling by binding of HER2-targeting sdAbs (purple) to HER2 (light purple) and engaging trimeric TNFR1 (light green) with camelid-derived TNFR1-targeting paratopes (green). Structural visualization was generated with PyMOL software version 2.3.0, based on PDB entries 7K7A and 1TNF and structural modeling as described in the section. (B) Enrichment after three sorting rounds of each library against (rh) TNFR1 ECD. A two-dimensional sorting strategy was applied to detect full-length VHH display simultaneous to antigen binding at a concentration of 1 μM. Plots show 5 × 10 4 events of the corresponding sorting output and the percentage of gated cells to visualize enrichment.

Article Snippet: Three camelids were immunized with (rh) TNFR1 ECD (Sino Biological, 10872-H08H).

Techniques: Derivative Assay, Binding Assay, Generated, Software, Concentration Assay

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: TNFR1-and HER2-specific bispecific bivalent ICMs (2 + 2) trigger reporter cell activity (A) HEK-Blue TNF reporter cells were incubated with two fixed concentrations of 10 nM and 1 nM of bsAbs, as well as 0.1 nM and 0.01 nM of (rh) TNF as a positive control and 0.1 nM of (rh) IL-18 as a negative control. After 24 h of incubation, secreted embryonic alkaline phosphatase activity was measured via OD 640 . Reporter activity was normalized to (rh) TNF signal. (B) Dose-titration of agonistic bsAbs in HEK-Blue TNF reporter cells. Mean values ±SEM of three independent experiments are shown. ∗∗∗∗ p < 0.0001,∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 calculated by utilizing one-way ANOVA analyses and Bonferroni test.

Article Snippet: Three camelids were immunized with (rh) TNFR1 ECD (Sino Biological, 10872-H08H).

Techniques: Activity Assay, Incubation, Positive Control, Negative Control, Titration

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: AlphaFold3 models of TNFR1-ICM11 complexes (A) Native TNFR1–TNF reference structure for epitope orientation (adapted from PDB: 1TNR ). The following contact residues defines TNF binding site: Lys18, Ser49, His52, Trp93, Glu95, Arg132, Lys143, Lys144, Glu42, Ser43, His55, Cys56, Cys59, Ser60, Lys61, Arg63, Lys64, Glu65, and Met66. (B–D) AF3 models for 1:1, 2×, and 3× TNFR1-ICM11 assemblies. The AF3 confidences are indicated in the figure: ipTM provides confidence in the interface quality, and pTM summarizes the overall complex topology. The resulting contact residues of the 1:1 complex are: Arg63, Glu65, His91, Tyr92, Trp93, Glu95, Asn96, Gln99, Phe101, Lys118, Arg132, Glu133, Glu135, Glu147, and Lys150. Shared TNFR1 hotspot residues shared by both TNF and ICM11: Arg63, Glu65, Trp93, Glu95, Arg132. Interface/contact residues were computed via Molecular Operating Environment (MOE) software.

Article Snippet: Three camelids were immunized with (rh) TNFR1 ECD (Sino Biological, 10872-H08H).

Techniques: Binding Assay, Software

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: Engineering of TNFR1 paratope valencies enables augmented cell death induction of MCF-7 cells (A) Schematic depiction of valency-engineered TNFR1xHER2 targeting ICMs. A TNFR1-targeting VHH is fused N -terminally to the hinge region of an immune effector-silenced IgG1 Fc. A HER2-targeting VHH is fused C- terminally by employing a 15 amino acid linker (3xGly 4 Ser), resulting in the initially generated (2 + 2) format. To increase valencies of TNFR1, multivalent constructs were designed by linking TNFR1-targeting VHHs in tandem for tetravalent (4 + 2) or in tridem for hexavalent (6 + 2) targeting of TNFR1 and bivalent binding to HER2. All building blocks were separated by a 15 amino acid linker (3xGly 4 Ser). (B) TNFR1xHER2 multivalent formats were scrutinized for their killing capacities on HER2 expressing MCF-7 cells in comparison to their corresponding TNFR1xHEL negative control ICMs and (rh) TNF. Killing was monitored by green fluorescence signal with SYTOX Green Dead Cell Stain after 96 h of incubation. Killing signal was normalized to (rh) TNF signal. (C) Kinetics of MCF-7 cell death induction by (rh) TNF, bivalent TNFR1xHER2 ICM11 (2 + 2) as well as tetravalent TNFR1xHER2 ICM11 (4 + 2) and hexavalent TNFR1xHER2 ICM11 (6 + 2). Mean values ±SEM of four independent experiments are shown.

Article Snippet: Three camelids were immunized with (rh) TNFR1 ECD (Sino Biological, 10872-H08H).

Techniques: Generated, Construct, Binding Assay, Expressing, Comparison, Negative Control, Fluorescence, Staining, Incubation

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: HER2-targeted TNFR1-agonists induce TNF-like caspase-1, caspase-3, and caspase-8 activation decoupled from NF-κB signaling in MCF-7 cells (A–C) Caspase-1/3/8 activation of HER2-expressing MCF-7 cells by ICM11 derivatives compared to (rh)TNF. MCF-7 cells were stimulated with increasing compound concentrations for 72 h. Caspase activities were detected intracellularly with FAM-FLICA(R) Caspase 1 Assay Kit (Biomol), CaspaTag Caspase-3 In situ Assay Kit (Merck Millipore), and CaspaTag Caspase-8 In Situ Assay Kit (Merck Millipore). Caspase-1/3/8 activation was normalized to (rh) TNF. (D) NF-κB activation in MCF-7 cells triggered by ICM11 derivatives and (rh) TNF. MCF-7 cells were stimulated with increasing concentrations of ICMs and (rh) TNF for 40 min. NF-κB was stained intracellularly with AF488-labeled anti-NF-κB staining antibody (BD) after lysis, fixation, and permeabilization of cells. (E) Remaining relative cell death of MCF-7 cells after treatment with ICM11 derivatives or (rh)TNF in the presence or absence of caspase inhibitors. MCF-7 cells were incubated with caspase-1 inhibitor (InvivoGen), caspase-3 inhibitor (R&D Systems), caspase-8 inhibitor (InvivoGen), or pan-caspase inhibitor (InvivoGen) at 50 μM and a fixed (rh) TNF or ICM concentration of 5 nM for 72 h. Killing was monitored by green fluorescence signal with SYTOX Green Dead Cell Stain and normalized to (rh) TNF signal. Mean values ±SEM of four independent experiments for each figure are shown. ∗∗∗∗ p < 0.0001,∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 calculated by utilizing two-way ANOVA multiple analyses and Bonferroni test.

Article Snippet: Three camelids were immunized with (rh) TNFR1 ECD (Sino Biological, 10872-H08H).

Techniques: Activation Assay, Expressing, In Situ, Staining, Labeling, Lysis, Incubation, Concentration Assay, Fluorescence

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: HER2-specific TNFR1-engaging ICMs elicit diminished pro-inflammatory cytokine release in PBMCs compared to (rh) TNF Pro-inflammatory cytokine release of human PBMCs triggered by (rh) TNF in comparison to TNFR1xHER2 ICMs. Human PBMCs were stimulated with 100 nM, 10 nM, 1 nM, and 0.1 nM of (rh) TNF or ICMs for 24 h. Cytokine release was quantified with MSD proinflammatory panel 1 (human) Kit (MSD). Mean values ±SEM of four independent experiments are shown. ∗∗∗∗ p < 0.0001,∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 calculated by utilizing two-way ANOVA multiple analyses and Bonferroni test.

Article Snippet: Three camelids were immunized with (rh) TNFR1 ECD (Sino Biological, 10872-H08H).

Techniques: Comparison

Journal: bioRxiv

Article Title: Fructose utilization by GM-CSF-differentiated macrophages aggravates autoimmune inflammation via MG-derived AGE–RAGE signaling

doi: 10.64898/2026.01.26.701899

Figure Lengend Snippet: A. GM-CSF human macrophages were stimulated with LPS (50 ng/mL) for 18 h in glucose-, fructose- (10 mM), or sugar-free media. IL-1β, IL-6, and IL-23 levels were quantified by ELISA. For IL-1β detection, 4 mM ATP was added during the final 2 h (n = 6). B. IL1B mRNA expression was analyzed by RT-qPCR under identical conditions (n = 5). C. Immunoblotting of cell lysates and supernatants from (A) was performed to assess pro- and mature (m) forms of IL-1β and caspase-1 (Casp-1). Band intensities were quantified by densitometry and normalized to β-actin (n = 5). D–E. Glycolytic activity was assessed by Seahorse extracellular flux analysis of ECAR following sequential injection of glucose, fructose (10 mM), or no sugar, followed by oligomycin (2 µM) and 2-DG (50 mM). Data represents five technical replicates (n = 3). F. HIF-1α protein and mRNA levels were measured in LPS-stimulated GM-CSF macrophages under indicated sugar conditions (n = 3–4). G–J. SKG mice were injected with curdlan to induce arthritis and administered fructose in drinking water starting 3 days prior to immunization. Glucose levels in serum and synovial fluid (SF) were assessed at day 42 (n = 5–8). H–I. mRNA levels of Hif1a and HIF-1α target genes in synovial cells were analyzed by RT-qPCR (n = 5). J. Intracellular HIF-1α expression was measured by flow cytometry in CD45⁺CD11b⁺F4/80⁺ synovial cells (n = 9–10). Graphs represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by Mann–Whitney U test (A–C, E–J) or unpaired t-test (D, F).

Article Snippet: Cells were plated in 96-well U-bottom plates and stimulated with anti-CD3/CD28-coated beads for 7 days under the following cytokine conditions: recombinant human (rh) IL-1β (30 ng/mL), rhIL-6 (30 ng/mL), rhIL-23 (10 ng/mL), and rhTGF-β (10 ng/mL) (all from R&D Systems, Minneapolis, MN) for Th17 polarization, rhIL-2 (100 IU/mL; PeproTech) and rhIL-12 (10 ng/mL; R&D Systems) for Th1 polarization, and rhIL-2 (100 IU/mL) alone for non-polarizing control.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Injection, Flow Cytometry, MANN-WHITNEY