cells for protein overexpression  (Thermo Fisher)

Journal: FEBS Open Bio

Article Title: Development and characterization of a novel human CD137 agonistic antibody with anti‐tumor activity and a good safety profile in non‐human primates

doi: 10.1002/2211-5463.13494

Figure Lengend Snippet: Binding, specificity, and blocking profiles of PE0116. (A) The binding activity of PE0116, Urelumab, and Utomilumab toward CHOK1‐hCD137 and CHOK1‐cynoCD137 cells was analyzed as concentration‐mean fluorescence intensity (MFI) curve. (B) Specificity of PE0116 to CD137 was conducted by ELISA after capturing 1 μg·mL −1 humanOX40, GITR, and CD137 as the antigen. All assays were performed in duplicate, and all error bars indicate the SD. (C) Cell‐based blocking assay was performed to evaluate PE0116, Urelumab, and Utomilumab using CHOK1‐hCD137 stable cell line and CD137L‐hFc‐Biotin with streptavidin‐Alexa Fluor 488 conjugate.

Article Snippet: Flow cytometry assay was performed to evaluate the ability of anti‐hCD137 to block the binding of CD137 and CD137L based on CHOK1 cell overexpressing hCD137 and CD137L‐hFc‐Biotin (15693‐H01H‐B; Sino Biological, Beijing, China) protein.

Techniques: Binding Assay, Blocking Assay, Activity Assay, Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Stable Transfection

Journal: FEBS Open Bio

Article Title: Development and characterization of a novel human CD137 agonistic antibody with anti‐tumor activity and a good safety profile in non‐human primates

doi: 10.1002/2211-5463.13494

Figure Lengend Snippet: Characterization of PE0116 in vitro functionality. (A) Luminescence value changed by HEK293‐CD137‐NF‐κB reporter cells treated with serially diluted PE0116, Urelumab, or Utomilumab (cross‐linking or not cross‐linking, antibodies were cross‐linked by anti‐Fc F(ab') 2 fragment, the molar ratio was 1 : 1.5). (B) Levels of IFN‐γ released by activated CD3 + T cells from three donors after 72 h of incubation with serially diluted PE0116, Urelumab, or Utomilumab in the presence of OKT3‐scFv displayed on the CHOK1 cell surface (cross‐linking or not cross‐linking). The EC50 value of Utomilumab non‐crosslink was blank in (B) (upper right panel), representing that the dates could not be analyzed by curve fitting. **** P < 0.0001 (hIgG4 vs. PE0116 at 6.67 n m in donor#2 or at 0.67 n m in donor#3 with cross‐linking) and *** P < 0.001 (hIgG4 vs. PE0116 at 66.7 n m in donor#2 without cross‐linking), as determined by two‐way ANOVA. All assays were performed in duplicate, and all error bars indicate the SEM.

Article Snippet: Flow cytometry assay was performed to evaluate the ability of anti‐hCD137 to block the binding of CD137 and CD137L based on CHOK1 cell overexpressing hCD137 and CD137L‐hFc‐Biotin (15693‐H01H‐B; Sino Biological, Beijing, China) protein.

Techniques: In Vitro, Incubation