Development of a New Cell-Based AP-1 Gene Reporter Potency Assay for Anti-Anthrax Toxin Therapeutics

Area of Science:

• Microbiology
• Immunology
• Toxicology

Background:

• Bacillus anthracis toxin is a key virulence factor, composed of protective antigen (PA) and enzymatic moieties (lethal factor [LF] and edema factor [EF]).
• Anti-PA antibodies are crucial for anthrax prophylaxis and treatment, but existing potency assays do not fully capture the toxin's pathological effects.
• Current assays measure inhibition of cell death or cAMP levels, which may not accurately reflect in vivo toxin activity.

Purpose of the Study:

• To develop a novel cell-based gene reporter potency assay for anti-PA antibodies.
• To establish an assay that reflects a key pathogenic effect of anthrax toxin in human cells.
• To create a reliable bioassay for lethal toxin (LT) activity and therapeutic antibody characterization.

Main Methods:

• Development of a cell-based assay using Hepa1c1c7 cells transduced with an AP-1 reporter lentiviral construct.
• Quantification of anti-PA antibody potency based on the inhibition of LT-induced c-Jun protein degradation.
• Qualification of the assay for specificity, accuracy, repeatability, intermediate precision, and linearity.

Main Results:

• Successful development of a cell-based gene reporter assay for anti-PA antibodies.
• The assay accurately measures LT-induced c-Jun degradation, a relevant pathogenic mechanism.
• The assay demonstrated qualification for key performance characteristics including specificity and accuracy.

Conclusions:

• The developed assay serves as a valuable bioassay for LT activity.
• This assay is applicable for the characterization and quality control of anti-PA therapeutic antibodies.
• The assay has potential applications for other products targeting the AP-1 signaling pathway.