Fig 2: HB0043 effectively antagonized the production of IL-6 induced by IL-17A and IL-36 in NHDF cells. (A–C) The ability of HB0043 to block IL-36R signaling and IL-17A signaling was measured and compared to that of HB0034 and HB0017, using IL-17A and IL-36R blockade cell-based assay. The secretion of IL-6 was monitored, which was induced by 0.5 ng/ml human IL-17A and 15 ng/ml IL-36α (A), 1 ng/ml IL-36β (B) or 2 ng/ml IL-36γ (C) in NHDF cells overnight. Data are expressed as means ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.01, ****P < 0.0001, as determined by one-way ANOVA.

Fig 3: Direct, isolated CAD activation leads to senescence in HaCaT cells.(A) Treatment protocol to induce senescence in HaCaT-ICAD-mAID-GFP and HaCaT-ICAD-mAID-GFP cGAS-deficient cells. Stimulation with auxin (10 µM, 24 h) was conducted for 24 h every second day for 2 weeks, with 2 days recovery after the third stimulation. Cells were harvested 2 days after the last auxin treatment. (B) Representative microscopy pictures of HaCaT-ICAD-mAID-GFP cells after 2 weeks of auxin treatment. Cells were stained with β-galactosidase staining solution. Top panel shows a bright-field picture and bottom panel shows SA-β-Gal+ cells. Scale bar: 10 µm. Flat and enlarged cells (senescent phenotype) are marked by stars. SA-β-Gal+ and Edu+ cells were quantified by microscopy. Each symbol represents one separate experiment. (C) Lamin B1 protein expression was analyzed by western blot. GAPDH was used as loading control. (D) Gene expression was determined by PCR following the auxin treatment protocol in (A). Expression of P21, CXCL10, and IFNβ are shown. Expression is given as fold induction by auxin treatment. Each symbol represents one separate experiment. (E) Supernatants from the cells in (D) were analyzed by ELISA for IL-6. Each symbol represents one separate experiment. (F–H) BJ human fibroblasts were incubated for 7 days with conditioned medium from HaCaT-ICAD-mAID-GFP cells subjected to the protocol in (A) or exposed to solvent (DMSO). Normal medium (NM) was used as a control. (F) Percentage of proliferating (Edu+) cells was quantified by microscopy from at least 500 cells per group. Each symbol represents one separate experiment. (G) γH2AX+ cells were quantified by microscopy from at least 500 cells per group. Each symbol represents one separate experiment. (H) Expression of lamin B1 was determined by immunofluorescence staining and analysis by confocal microscopy (CTCF, corrected total cell fluorescence of lamin B1-stain). Each symbol represents one separate experiment (B, D–G) or one cell (H). Data are the means/SD. An unpaired parametric t test (with Welch’s correction) was used to calculate statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available online for this figure.

Fig 4: Induction of senescence by oncogenic RAS, and type-I interferon depends on CAD.(A–D) wt and CAD-deficient MEFs were transduced with empty retrovirus or pLNCX2-ER:H-RASG12V-virus and stimulated each day with tamoxifen to activate H-RASG12V. (A) Cells were stained with β-galactosidase and Edu staining solution and percentages of SA-β-Gal+ and Edu+ cells were quantified by microscopy after 10 days in culture. (B) Expression of senescence-associated genes 10 days after retroviral transduction. Gene expression was analyzed by RT-PCR and normalized to GAPDH. Relative expression compared to wt cells transduced with empty vector is shown. (C) Cells were lysed and western blot for H3K9me3 and Lamin B1 was performed. GAPDH was used as loading control. (D) Supernatants from cells cultured for 10 days post-transduction were analyzed by ELISA for IL-6. (E–H) wt and CAD-deficient MEFs were treated daily with 10 ng/ml of IFN-β for 10 days. (E) Cells were stained with β-galactosidase and Edu staining solution and percentages of SA-β-Gal+ and Edu+ cells were quantified by microscopy after 10 days in culture. (F) Expression of senescence-associated genes was analyzed after 10 days in culture by RT-PCR. The expression relative to untreated wt cells is shown. (G) Cells were lysed and western blot for Lamin B1 was performed. GAPDH was used as loading control. (H) Supernatants from cells cultured for 10 days were analyzed by ELISA for IL-6. Data are the mean/SD of 3 independent experiments. Each symbol represents one experiment. Unpaired parametric t test (with Welch’s correction) was used to calculate statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure.

Fig 5: HB0043 retained the affinities and blockade activity to both targets. (A) Schematics of the anti-IL-36R×IL-17A BsAb (HB0043) structure. HB0043 was constructed by linking a scFv targeting IL-36R to the C-terminal domain of the anti-IL-17A IgG backbone. (B) The kinetics profile of HB0043 and HB0017 (the parental anti-IL-17A mAb) binding to human IL-17A, and human IL-36R binding to HB0043 and HB0034 (the parental anti-IL-36R mAb) measured by SPR assay. (C) The ability of HB0043 to neutralize IL-17A signaling was measured and compared to that of HB0017, using an IL-17A blockade cell-based assay. The secretion of IL-6 was monitored, which was induced by human IL-17A (0.3 nM) and TNF-α (0.6 nM) in HT-1080 cell. (D) The inhibition of IL-6 releases from NCI/ADR-RES cells was used as an evaluation of IL-36R signaling inhibition. NCI/ADR-RES cells were stimulated with human IL36α (13.5 nM) in the presence of HB0043 (32 pM to 245 nM) and HB0034 (4 pM to 336 nM).