Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay
2018
The administration of
enzyme replacement therapies(ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed
neutralizing antibodies(NAbs), is crucial in understanding the effects of these antibodies on the drug's pharmacological profile. This protocol describes a cell-
based flowcytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and
titerassays to detect, identify, and establish the relative level of
neutralizing antibody
titerin subject samples. In this method, samples are first mixed with the
fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (
Jurkat cells)] that express a cell-surface cation-independent
mannose 6-phosphate receptor(CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the
fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the
fluorophore-conjugated ERT internalized by the
Jurkat cellsis measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative
drug-naivematrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then
serially dilutedto generate an
antibody titer. Semi-quantitative
antibody titersmay be correlated with measurements of drug safety and efficacy.
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