Journal: Journal of Animal Science and Biotechnology

Article Title: Baicalin alleviates mastitis in dairy cows by targeting IL-17RA to inhibit IL-17 signaling pathway activation

doi: 10.1186/s40104-026-01401-2

Figure Lengend Snippet: Schematic overview of baicalin's mechanism in alleviating mastitis by regulating the IL-17RA-mediated IL-17 signaling pathway. A Oral administration of baicalin to mice and dairy cows. B In vivo release of LPS by E. coli . C LPS activated the TLR4/MyD88/NF-κB pathway in epithelial cells. D NF-κB was activated within the cell nucleus. E E. coli infection increases IL-17A content in mammary glands. F IL-17A binds to IL-17RA on the cell membrane surface, thereby activating the IL-17 signaling pathway. G Activation of the MAPK signaling pathway and ERK signaling pathway. H Production of IL-6, TNFα and IL-1β. I TNFα activated TNF signaling pathway. J TJ structure damage. K Baicalin inhibits IL-17RA signal transduction, thereby preventing activation of the IL-17 signaling pathway

Article Snippet: An in vitro mastitis model was established by treating cells with 5 μg/mL LPS (Sigma, USA) for 12 h. Baicalin (purity ≥ 95%) was purchased from Macklin (Shanghai, China), with a concentration of 20 μmol/L for 24 h. Furthermore, the TNFα inhibitor SPD304 and recombinant IL-17A (rIL-17A) were purchased from MedChemExpress (MCE, USA) and were used at concentrations of 5 μmol/L (for 2 h) and 100 ng/mL (for 12 h), respectively.

Techniques: In Vivo, Infection, Membrane, Activation Assay, Transduction

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Expression and purification of CPBT0853. Panel ( A ) is a schematic representation of the generation of the bispecific antibody. Following phage display selection, and VHHs’ identification, the first produced version of bispecific antibody, named CPBT0853, was in the IgG1 format. The antibody was then reformatted into an IgG4 version (CPBT1174), and humanization was subsequently performed by substituting specific amino acids to reduce immunogenicity (CPBT1269). Panel ( B ) shows the SDS-PAGE analysis under non-reducing conditions, illustrating the expression profile of CPBT0853 over seven days following transient transfection in ExpiCHO-S cells. An arrow indicates the expected protein band. Panel ( C ) presents a representative chromatogram from the affinity purification step using MabCaptureC resin. Panel ( D ) displays the size-exclusion chromatography (SEC) profile of the final purified CPBT0853, confirming the high homogeneity of the sample.

Article Snippet: The binding of human, cynomolgus monkey ( Macaca fascicularis ), and mouse IL-6 and IL-17A (ACROBiosystems), human complex IL-17A/F (R&D System, Minneapolis, MN, USA) and human IL-17E and IL-17F (ACROBiosystems) to the anti-IL-6/anti-IL17A bispecific antibody CPBT0853 was analyzed using an Octet RED96 instrument (Sartorius AG).

Techniques: Expressing, Purification, Selection, Produced, Immunopeptidomics, SDS Page, Transfection, Affinity Purification, Size-exclusion Chromatography

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Purity analysis of CPBT0853. ( A ) Representative chromatogram obtained from capillary electrophoresis, showing the purity profile of the CPBT853 sample. ( B ) The corresponding electropherogram, providing a visual representation of the separations of the final product differentially treated, as indicated. Each subsequent line in the electropherogram ( B ) corresponds to the following peak marked in the red box ( A ).

Article Snippet: The binding of human, cynomolgus monkey ( Macaca fascicularis ), and mouse IL-6 and IL-17A (ACROBiosystems), human complex IL-17A/F (R&D System, Minneapolis, MN, USA) and human IL-17E and IL-17F (ACROBiosystems) to the anti-IL-6/anti-IL17A bispecific antibody CPBT0853 was analyzed using an Octet RED96 instrument (Sartorius AG).

Techniques: Electrophoresis

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Biophysical characterization of CPBT0853 using the Uncle platform. ( A ) Thermal stability and aggregation analysis of CPBT853 assessed using fluorescence and static light scattering (SLS) to determine melting temperature (T m ) and aggregation onset temperature (T agg ), respectively. ( B ) Dynamic light scattering (DLS) measurements performed at 25 °C and 95 °C, demonstrating the hydrodynamic radius and polydispersity index (PDI) of the sample under native and heat-stressed conditions in three technical repetitions.

Article Snippet: The binding of human, cynomolgus monkey ( Macaca fascicularis ), and mouse IL-6 and IL-17A (ACROBiosystems), human complex IL-17A/F (R&D System, Minneapolis, MN, USA) and human IL-17E and IL-17F (ACROBiosystems) to the anti-IL-6/anti-IL17A bispecific antibody CPBT0853 was analyzed using an Octet RED96 instrument (Sartorius AG).

Techniques: Fluorescence

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Binding affinity analysis of the CPBT0853 bispecific antibody. Representative bio-layer interferometry (BLI) binding curves for CPBT0853 interacting with human IL-17A ( A ), human IL-6 ( B ), human IL-17A/F heterodimer ( C ), cynomolgus monkey IL-17A ( D ), and cynomolgus monkey IL-6 ( E ) are shown. The absence of binding is demonstrated by the lack of interaction between CPBT0853 and mouse IL-6 ( F ).

Article Snippet: The binding of human, cynomolgus monkey ( Macaca fascicularis ), and mouse IL-6 and IL-17A (ACROBiosystems), human complex IL-17A/F (R&D System, Minneapolis, MN, USA) and human IL-17E and IL-17F (ACROBiosystems) to the anti-IL-6/anti-IL17A bispecific antibody CPBT0853 was analyzed using an Octet RED96 instrument (Sartorius AG).

Techniques: Binding Assay

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Blocking activity of the bispecific antibody CPBT0853 measured by bio-layer interferometry (BLI). Bar graphs represent quantitative data derived from BLI sensorgrams showing the effect of increasing concentrations of CPBT0853 on the binding of IL-17A ( A ) or IL-6 ( B ) to their respective receptors. The measured response [nm], reflecting the amount of receptor bound to the immobilized cytokine, decreases with increasing antibody concentration, indicating dose-dependent inhibition of cytokine–receptor interactions. Data represent mean ± SD from three independent experiments.

Article Snippet: The binding of human, cynomolgus monkey ( Macaca fascicularis ), and mouse IL-6 and IL-17A (ACROBiosystems), human complex IL-17A/F (R&D System, Minneapolis, MN, USA) and human IL-17E and IL-17F (ACROBiosystems) to the anti-IL-6/anti-IL17A bispecific antibody CPBT0853 was analyzed using an Octet RED96 instrument (Sartorius AG).

Techniques: Blocking Assay, Activity Assay, Derivative Assay, Binding Assay, Concentration Assay, Inhibition

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Neutralization potency of CPBT0853 targeting IL-17A ( A ) and IL-6, IL-17A. Representative graphs show IC 50 analysis performed using a reporter cell assay. For comparison, parental VHHs and monospecific referential antibodies were used. The cellular response, measured as cytokine-induced activation, progressively declined with increasing concentrations of the tested antibodies, indicating effective inhibition of signaling through IL-6 ( A ), IL-17A ( B ), and the IL-17A/F heterodimer ( C ). Data were analyzed using nonlinear regression with a three-parameter dose–response model (log [inhibitor] vs. response), fitted using GraphPad Prism software. The curves shown represent a representative biological replicate performed with two technical replicates. The IC 50 values summarized in were calculated from three independent biological replicates.

Article Snippet: The binding of human, cynomolgus monkey ( Macaca fascicularis ), and mouse IL-6 and IL-17A (ACROBiosystems), human complex IL-17A/F (R&D System, Minneapolis, MN, USA) and human IL-17E and IL-17F (ACROBiosystems) to the anti-IL-6/anti-IL17A bispecific antibody CPBT0853 was analyzed using an Octet RED96 instrument (Sartorius AG).

Techniques: Neutralization, Comparison, Activation Assay, Inhibition, Software

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Representative IC 50 curves showing inhibition of STATs phosphorylation in PBMCs stimulated with IL-6 and treated with increasing concentrations of the bispecific antibody CPBT0853. Phosphorylation of STAT1 ( A ) and STAT3 ( B ) was measured by flow cytometry. Data were normalized to cytokine-only controls and expressed as % pSTAT + lymphocytes. IC 50 values were calculated using a nonlinear regression with a three-parameter dose–response model (log [inhibitor] vs. response) in GraphPad Prism software. Each curve represents technical duplicates from PBMCs of one representative donor out of three tested.

Article Snippet: The binding of human, cynomolgus monkey ( Macaca fascicularis ), and mouse IL-6 and IL-17A (ACROBiosystems), human complex IL-17A/F (R&D System, Minneapolis, MN, USA) and human IL-17E and IL-17F (ACROBiosystems) to the anti-IL-6/anti-IL17A bispecific antibody CPBT0853 was analyzed using an Octet RED96 instrument (Sartorius AG).

Techniques: Inhibition, Phospho-proteomics, Flow Cytometry, Software

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Inhibition of cytokine production (IL-6 and IL-8) in HFLS cells by CPBT0853. Levels of IL-6 ( A ) and IL-8 ( B ) secreted into the culture medium after stimulation of the cells with IL-17A. Additionally, cells were treated with bispecific and monospecific antibodies, as indicated. Data are shown as the mean ± SD from a minimum of four independent experiments. The bispecific antibody CPBT0853 effectively reduced the IL-6 and IL-8 levels, with higher efficiency, as compared to the monospecific antibodies, indicating enhanced anti-inflammatory activity. Additionally, the humanized IgG4 form of CPBT0853 (CPBT1269) was evaluated for comparison, showing activity in cytokine inhibition similar to that of the parental/original IgG1 form.

Article Snippet: The binding of human, cynomolgus monkey ( Macaca fascicularis ), and mouse IL-6 and IL-17A (ACROBiosystems), human complex IL-17A/F (R&D System, Minneapolis, MN, USA) and human IL-17E and IL-17F (ACROBiosystems) to the anti-IL-6/anti-IL17A bispecific antibody CPBT0853 was analyzed using an Octet RED96 instrument (Sartorius AG).

Techniques: Inhibition, Activity Assay, Comparison