Understanding Immunogenicity Testing
Understanding Immunogenicity Testing
Immunogenicity testing consists of the measurement of antibodies generated against a drug treatment and helps to determine its safety and efficacy. These tests are mandatory for therapeutic proteins in clinical studies. On a case-by-case basis and from a scientific rationale, immunogenicity could also be analyzed in preclinical studies and for peptide and oligonucleotide therapeutics.
Immunogenicity assessed with a multi-tiered-based approach
- The first step is a screening assay to detect antibodies that bind to the drug. The assay is usually done in a bridging format using a ligand binding assay (usually ELISA or MSD) with a labeled drug for capture and detection. A cut point is set to give 5% false positive samples to minimize the risk of getting false negative results and ensure patient safety. A sensitivity of at least 100 ng/mL is recommended and the assay should be drug-tolerant and without interference from the target.
- A confirmation assay is used to reduce the number of false positive responses generated in the screening tier and ensure that all samples are correctly classified as positive or negative. This is done by competition with an excess of unlabeled drugs, usually with the same assay setup as in the screening assay. It is recommended to have a cut-off point that gives a 1% false rate in this step.
- The magnitude of anti-drug antibodies (ADAs) in the samples that are confirmed positive are characterized in a titer assay. The same assay format as in the screening step is used in the titer assay. The titer is the highest dilution that gives results above a cut-off point.
- Characterization of the neutralizing capacity is done for ADAs in samples that are confirmed positive. Neutralizing ADAs inhibits the interaction between the drug and its target and could thus neutralize the therapeutic effect. The neutralization assay should reflect the mode of action of the drug and could be a cell-based assay or a competitive ligand binding assay.
- In some cases, further characterization is needed, such as the assessment of cross-reactivity to endogenous antigens, domain specificity for multi-domain products, or isotyping of the ADAs.
There are recent suggestions from the bioanalytical communities to streamline the immunogenicity testing procedure (read more; Anti-Drug Antibodies: A Challenge for Biopharmaceutical Development).
References
GUIDANCE DOCUMENT from FDA: Immunogenicity Testing of Therapeutic Protein Products — Developing and Validating Assays for Anti-Drug Antibody Detection, 2019. https://www.fda.gov/media/119788/download
GUIDANCE DOCUMENT from EMA: Guideline on Immunogenicity assessment of therapeutic proteins, 2017. https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-immunogenicity-assessment-therapeutic-proteins-revision-1_en.pdf
