Journal: Frontiers in Immunology
Article Title: An antigen-specific immunotherapeutic, AKS-107, deletes insulin-specific B cells and prevents murine autoimmune diabetes
doi: 10.3389/fimmu.2024.1367514
Figure Lengend Snippet: AKS-107 does not bind or activate the human or mouse insulin metabolic receptor. (A) Insulin receptor binding: human IM-9 cells that naturally express the human insulin metabolic receptor were treated with different concentrations of AKS-107 or rhINS in the presence of 0.5 µg/mL PE-rhINS and fixed with paraformaldehyde prior to flow cytometric analysis. PE-rhINS binding was calculated as MFI/sample in which 100% binding were cells bound with PE-rhINS in the absence of AKS-107 or cold rhINS. IC 50 values were calculated using the non-linear 4-parameter log[Inhibitor] vs. response-Variable slope (four parameters) algorithm in GraphPad Prism 10.1 software (data represent one of two experiments that had similar results). (B) Insulin receptor phosphorylation: The U2OS cells genetically engineered to express the human insulin receptor (PathHunter ® Insulin Bioassay, Eurofins/DiscoverX) were incubated with different concentrations of rh-INS (positive control), AKS-130 (active insulin-Fc IgG control), AKS-107, and human IgG mAb (negative control) and insulin receptor activation was assessed via activated kinase conversion of a chemiluminescence substrate (PathHunter Detection Reagent) (data represent one of two experiments that had similar results). IC 50 values were calculated using GraphPad Prism 10.1 software. The lack of insulin receptor activation by AKS-107 was determined in vivo in BALB/c mice after a single i.p. (C) or s.c. (D) injection of AKS-107 or AKS-130 via measurement of FBG levels over 7 days, with assessment of serum levels of AKS-107 via ELISA (n = 3 mice per group).
Article Snippet: The insulin receptor activation assay consisted of U2OS cells genetically engineered to express the human insulin receptor (PathHunter ® Insulin Bioassay kit, Eurofins/DiscoverX, Fremont, CA) that were incubated with different concentrations of rhINS (positive control), AKS-130 (active insulin-Fc analog positive control), AKS-107, or human IgG mAb (Fc negative control) and kinase activity was assessed by the addition of chemiluminescence substrate (PathHunter Detection reagent cocktail) using the PerkinElmer EnvisionTM instrument (see ).
Techniques: Binding Assay, Software, Phospho-proteomics, Bioassay, Incubation, Positive Control, Control, Negative Control, Activation Assay, In Vivo, Injection, Enzyme-linked Immunosorbent Assay
![AKS-107 does not bind or activate the human or mouse insulin metabolic receptor. (A) Insulin receptor binding: human IM-9 cells that naturally express the human insulin metabolic receptor were treated with different concentrations of AKS-107 or rhINS in the presence of 0.5 µg/mL PE-rhINS and fixed with paraformaldehyde prior to flow cytometric analysis. PE-rhINS binding was calculated as MFI/sample in which 100% binding were cells bound with PE-rhINS in the absence of AKS-107 or cold rhINS. IC 50 values were calculated using the non-linear 4-parameter log[Inhibitor] vs. response-Variable slope (four parameters) algorithm in GraphPad Prism 10.1 software (data represent one of two experiments that had similar results). (B) Insulin receptor phosphorylation: The U2OS cells genetically engineered to express the human insulin receptor <t>(PathHunter</t> ® Insulin Bioassay, Eurofins/DiscoverX) were incubated with different concentrations of rh-INS (positive control), AKS-130 (active insulin-Fc IgG control), AKS-107, and human IgG mAb (negative control) and insulin receptor activation was assessed via activated kinase conversion of a chemiluminescence substrate (PathHunter Detection Reagent) (data represent one of two experiments that had similar results). IC 50 values were calculated using GraphPad Prism 10.1 software. The lack of insulin receptor activation by AKS-107 was determined in vivo in BALB/c mice after a single i.p. (C) or s.c. (D) injection of AKS-107 or AKS-130 via measurement of FBG levels over 7 days, with assessment of serum levels of AKS-107 via ELISA (n = 3 mice per group).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4819/pmc10954819/pmc10954819__fimmu-15-1367514-g003.jpg)