Journal: bioRxiv
Article Title: De novo masking domains that gate TNF-family ligand assembly and activity
doi: 10.64898/2026.04.20.719557
Figure Lengend Snippet: A single masked TNFα or 4-1BBL module was fused to a one-armed, affinity-attenuated trastuzumab variant to generate an antibody format that undergoes activation-dependent homotrimerization. Homotrimerization was first validated using the masked TNFα construct (b–d), and functional consequences were then evaluated using an analogous masked 4-1BBL fusion in assays with HER2-positive SK-BR-3 cells and Jurkat-Lucia h4-1BB reporter cells (e, f). Protein concentrations are reported per molecular species (monomeric or trimeric antibody, as indicated). Where indicated, trimeric species were re-isolated by SEC before downstream assays. a, Proposed mechanism for delivery of membrane-type TNFLs. Before activation, the construct is predominantly monomeric with reduced apparent target engagement and masked TNFL function. Protease-triggered activation promotes assembly into a trimeric state with increased functional valency and the capacity to drive receptor crosslinking, thereby enhancing signaling. b, Design of antibody formats used to test the concept: OATmab_mTNFα, OATmab, and Tmab. All constructs are based on an affinity-attenuated trastuzumab variant and contain a C-terminal peptide tag on the light chain for site-selective drug conjugation . In OATmab_mTNFα, TNFα (orange) within the masked TNFα module was fused to the C-terminus of one heavy chain. One-armed formats were assembled using knob-into-hole Fc heterodimerization mutations. c, Oligomeric states of OATmab and OATmab_mTNFα assessed by analytical SEC before (black) and after (gray) TEV activation. The arrow indicates the TEV-dependent shift in the main peak consistent with homotrimer formation. d, Binding of monomeric and trimeric antibody species to HER2 (left) and TNFR2 (right) measured by ELISA. Data are shown as mean ± s.d. from n = 3 technical replicates. e, Binding of monomeric and trimeric antibody species to HER2-positive SK-BR-3 cells measured by cell-based ELISA. Data are shown as mean ± s.d. from n = 3 technical replicates. f, Functional activity of monomeric and trimeric antibody species measured in Jurkat-Lucia h4-1BB reporter cells cultured alone (dashed lines) or co-cultured with SK-BR-3 cells (solid lines). Data are shown as mean ± s.d. from n = 3 technical replicates.
Article Snippet: The biological activity of antibody-fused masked 4-1BBL was evaluated using Jurkat-Lucia h4-1BB reporter cells in the presence or absence of HER2-positive SK-BR-3 cells (ATCC).
Techniques: Variant Assay, Activation Assay, Construct, Functional Assay, Isolation, Membrane, Drug discovery, Conjugation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, In-Cell ELISA, Activity Assay, Cell Culture
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![a Schematic overview of the SILAC pulse-chase experimental setup. H4-Ctrl and H4-aSyn cells were initially cultured in SILAC media containing light or heavy isotopes. Twenty-four hours after transfection with mock, FICD-WT, or FICD-E234G, the pulse-chase was initiated by switching the SILAC media to the opposite isotope condition (light-to-heavy or heavy-to-light). Isotope incorporation was monitored at multiple chase time points (2 h, 8 h, and 24 h) by LC-MS/MS to quantify the overall protein turnover. Depicted is the pulse-chase experimental workflow initiated with light SILAC medium. b Hierarchical clustering of half-lives from 938 commonly identified proteins. Z-score: normalized half-lives scale from lower (blue) to higher (red) levels. c Subcellular compartment analysis. Boxplots show distribution of protein half-lives grouped by GO cellular compartment annotation in H4-Ctrl (left) and H4-aSyn (right) cells. The x-axis shows the log2-transformed protein half-lives in log 2 (T1/2) [h]. Each box spans the interquartile range (IQR: 25 th -75 th percentile), with the center line indicating the median. Whiskers extend to data points within 1.5× IQR from the lower and upper quartiles, outliers are represented by dots. FICD-E234G overexpression significantly reduces global protein turnover compared with FICD-WT overexpression in both H4-Ctrl and H4-aSyn cells (n = 4). Statistical analysis: two-tailed Wilcoxon-matched-pairs signed rank test. p* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_95/10__64898_slash_2026__03__30__715195/10__64898_slash_2026__03__30__715195___F5.large.jpg)
