This method can help answer key questions in the immunogenicity and bioanalytical fields about the impact of neutralizing antibodies on drug safety, efficacy, and pharmacokinetic and pharmacodynamic profiles. The main advantage of this technique is that it provides a sensitive and biologically relevant cell-based method for measuring drug-specific neutralizing antibodies in a semi-quantitative manner. In this technique, fluorophore-labeled enzymes enter Jurkat cells through the binding of a mannose-6-phosphate residue to the Cation-Independent Mannose-6-Phosphate Receptor or CI-M6PR.

Neutralizing antibodies that prevent the enzyme receptor interaction induce a decrease in fluorescence as measured by flow cytometry. To begin, seed 7.5 times 10 to the fourth Jurkat cells in 100 microliters of cell growth medium per well in a 96-well round bottom cell culture plate for a 14 to 20-hour incubation at 37 degrees Celsius and 5%carbon dioxide with 95%humidity. Dilute the assay samples in serum-free medium first at a one to 2.5 ratio.

Then add 60 microliters of each sample to the appropriate wells of a 96-well white round bottom non-binding polypropylene plate. This will be the sample incubation plate. For samples that have screened positive and need to be confirmed, vortex a vial of Enzyme Replacement Therapy or ERT beads and add the appropriate number of beads to a 15 milliliter conical tube for another vortexing.

Then place the tube in a magnetic tube rack for two minutes and carefully remove the supernatant. After the fourth wash, add 100 microliters of beads to the appropriate wells of a new white 96-well round bottom non-binding polypropylene plate. When all of the beads have been plated, place the plate on a 96-well side-skirted magnet and allow the beads to form a pellet before carefully removing the clear supernatant.

Next, add 140 microliters of each sample into the appropriate wells and seal the plate with plastic film for shaking for at least 60 minutes on a rotary shaker at approximately 800 RPM at room temperature. Then place the confirmatory plate on the 96-well side-skirted magnet to allow the beads to form another pellet and add 60 microliters of sample to the appropriate wells of a 96-well white round bottom non-binding polypropylene sample incubation plate. For samples that have been screened and are confirmed positive, a titer series may be performed.

To prepare a titer series, serially dilute the sample in the pooled matrix a sufficient number of times to cross the predetermined titer cut point and add 60 microliters of each diluted sample to the appropriate wells of the sample incubation plate and 60 microliters of the appropriate concentrations of fluorophore-labeled ERT to the appropriate wells of a new 96-well white round bottom non-binding polypropylene plate according to the text protocol. Then pipette the fluorophore ERT sample solution several times to mix and wrap the plates in foil for a 14 to 20-hour incubation at two to eight degrees Celsius. The next morning, warm the sample plates in a 37 degree Celsius bead bath for 10 to 15 minutes and check the plated cells on an inverted microscope to confirm that the cells are healthy and evenly distributed across the wells.

When the plates have warmed, add 100 microliters of sample and fluorophore-labeled ERT mixture to the cell plate according to the experimental plate map and return the cells to the cell culture incubator for another three hours and 15 minutes. At the end of the incubation, pellet the cells by centrifugation and confirm the presence of a pellet at the bottom of each well. Holding the plate at 30 to 45 degree angle, carefully remove the supernatant in each well without disturbing the pellets and wash the cells three times in 200 microliters of DPBS per well per centrifugation.

After the last wash, add 100 microliters of live dead stain to each well for a 15-minute incubation at room temperature in the dark. At the end of the incubation, pellet the cells by centrifugation and wash the cells with 200 microliters of DPBS per well. Resuspend the washed pellets in 100 microliters of two to eight degree Celsius 1%paraformaldehyde per well and seal the plate for gentle pulse vortexing.

Then wrap the plate in foil for a 10-minute fixation at two to eight degrees Celsius. To analyze the cell viability by flow cytometry, mix the cells with 50 microliters of DPBS per well to achieve a single cell suspension and read the cells on a flow cytometer according to standard flow cytometry protocols. Fluorophore conjugated ERT uptake can then be assessed according to the Median Fluorescence Intensity or MFI of the live single cells.

Before being tested in the assay, an experimental screening cut point is calculated as a threshold for determining the positive and negative samples. In this example, quality controls spiked with high or low amounts of neutralizing antibodies are shown. Positive samples are then tested in a confirmatory assay using drug conjugated magnetic beads to deplete the drug-specific antibody from the samples.

Samples with a recovery ratio greater than the confirmatory cut point are considered to be positive. Samples that screened and confirmed positive are serially diluted and tested in the titer assay to determine the relative titer of neutralizing antibodies in each sample. Titer is the interpolated value between two dilutions that cross the titer cut point.

Shown here, the live single cell MFIs assessed fluorophore conjugated ERT uptake. For example, in this experiment, the matrix spiked with the positive control antibody inhibited fluorophore conjugated ERT uptake resulting in a low median fluorescence intensity. In contrast, cells incubated with the matrix in the absence of the positive control antibody exhibited a higher median fluorescence intensity demonstrating uptake of the fluorophore conjugated ERT.

While attempting this procedure, it is important to remember to maintain strict incubation windows to obtain reliable and consistent results. It is also important to optimize the assay for each enzyme replacement therapy. After its development, this technique paved the way for immunogenicity and bioanalytical scientists in drug development to explore the impact of cell uptake neutralizing antibodies on ERT safety and efficacy.

Don't forget that working with human samples can be extremely hazardous and that all of the samples should be treated as if they're potentially infectious while performing this procedure.

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