IMMUNOGENICITY TESTING USING FLOW CYTOMETRY - Flow Cytometry in Drug Discovery and Development - page 215

Patient 01
Patient 02
Patient 03

700

600

500

Assay
cut point

400

300

200

100

0

Baseline 4 weeks 12 weeks 24 weeks

FIGURE 10.7 Neutralizing activity in three patients following administration of a biother-
apeutic, measured by intensity of cell binding, and the assay cut point is indicated as a solid
horizontal bar.

24 week period; patient 01 develops a persistent neutralizing response, patient 02
shows no response, and patient 03 exhibits a transient response. Assignment of sample
score is a function of the quantitative readout from the instrument relative to the
assigned cut point. As mentioned previously in immunogenicity testing, the final
sample result is determined by a decision tree of ADA detection, specificity
confirmation, and neutralizing antibody characterization. Assessing the correlation
of a measured immune response following the administration of a biotherapeutic to
any clinical sequelae is perhaps the most meaningful aspect of immunogenicity
testing. Development of neutralizing antibodies to the biotherapeutic can be of
severe significance to the patient. These antibodies can abrogate the efficiency of
the therapeutic that may or may not be overcome by increasing the dosage. In cases
where patients no longer benefit from life-saving treatment due to the presence of
neutralizing antibodies, tolerance induction regimens are recommended [28].

10.5 SUMMARY

Flow cytometry is used successfully in immunogenicity testing to detect neutralizing
activity in antibody positive samples. We have described examples of assay devel-
opment using a biotherapeutic that binds a cell surface receptor and a biotherapeutic
enzyme that is actively internalized. Although NAb assays do not typically take
advantage of the multicolor capability of the flow cytometer, they do however utilize
the simultaneous cell gating and cell viability functions that are easily incorporated
into these types of assays. As with any assay development, application of factorial
design can shorten assay time lines and offer early indications of conditions that can
be used to develop robust flow cytometry assays. The technical considerations for
NAb flow cytometry assays include the potential for assay interferences from
biological matrices, circulating biotherapeutic and soluble target in the sample, in