Description
Applications
CAR-T Cell-Mediated Cytotoxicity
Raji (HT-HiBiT) TCK Bioassays are amenable for use with CAR-T effector cells. Raji Cells (HT-HiBiT) were incubated with serially diluted human CD19 CAR-T cells at the indicated E:T (effector-to-target) ratios. After a 4-, 24-, 48- or 72-hour induction, Bio-Glo-NB™ TCK Reagent was added.
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
Raji (HT-HiBiT) TCK Bioassays reflect the ADCC MOA of biologic drugs and demonstrate specificity for cell surface antigens. Raji Cells (HT-HiBiT) were incubated with Human PBMC, ADCC-qualified effector cells in the presence of serial titrations of antibodies as indicated. After a 5-hour induction, Bio-Glo-NB™ TCK HiBiT Luciferase Reagent was added.
T Cell-Dependent Cell-Mediated Cytotoxicity (TDCC)
Raji (HT-HiBiT) TCK Bioassays reflect the TDCC MOA of biologic drugs and demonstrate specificity for cell surface antigens. Raji Cells (HT-HiBiT) were incubated with human T Cells (CD8+) in the presence of serial titrations of biologic drugs as indicated. After a 24-hour induction, Bio-Glo-NB™ TCK Reagent was added.
Antibody-Dependent Cellular Phagocytosis (ADCP)
Raji (HiBiT) TCK Bioassays reflect the ADCP MoA of biologic drugs. Raji Cells (HiBiT) were incubated with Human Macrophages (ADCP-qualified) in the presence of serial titrations of rituximab. After a 24-hour induction, Bio-Glo-NB™ Lytic Reagent was added. Loss of signal reflects HiBiT degradation following phagocytosis of the Raji target cell.
After substrate was added, luminescence was quantified using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism software. HT = HaloTag, the fusion partner for the HiBiT tag.
Data sourced from the Human Protein Atlas transcriptomic data of human cell lines. Version 23.0, updated on 23-06-19 [ The Human Protein Atlas ].
When Raji (HT-HiBiT) target cells are cocultured with appropriate effector cells (e.g., CAR-T cells, PBMCs or cytotoxic T cells), they can be specifically used to monitor the activity of biologics directed against receptors expressed on the Raji Cells (HT-HiBiT), such as CD19, CD20 or CD22. When Raji Cells (HT-HiBiT) are used in assays with biologics against TAAs they do not express, target cells are not lysed and no increase in luminescent signal is observed. Representative data for different MoA are above.
Protein Knockouts to Determine Antigen Specificity
A.
B.
C.
Raji (HT-HiBiT) TCK Bioassays reflect T cell engager (TCE) activity in wild-type and surface antigen knockout cells. Parental and knockout cells were incubated with Human T Cells (CD8+) in the presence of serial titrations of biologic drugs for 24 hours. Panel A. Raji cell lines expressing CD19 show a dose-dependent increase in percent-specific lysis values for blinatumumab (CD3 × CD19 TCE), whereas CD19 KO cell lines do not. Panel B. Raji cell lines expressing CD20 show a dose-dependent increase in percent-specific lysis values for mosunetuzumab (CD20 × CD3 TCE), whereas CD20 KO cell lines do not. Panel C. Raji cells express BCMA and show a dose-dependent increase in percent-specific lysis values for a CD3 × BCMA TCE. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. Luminescence was measured using the GloMax® Discover System following addition of the Bio-Glo-NB™ TCK Reagent.
