Contributions
Abstract: EP1416
Type: ePoster
Abstract Category: Clinical aspects of MS - 8 Clinical assessment tools
Natalizumab is a highly efficacious treatment option for patients with relapsing-remitting multiple sclerosis (MS), but carries a risk of JC polyomavirus driven progressive multifocal leukoencephalopathy (PML). Recent data has suggested that patients on extended interval dosing schedules have a reduced rate of PML consequent to lower natalizumab exposure. However, there is significant variation in steady state drug levels in natalizumab patients, suggesting a need for a therapeutic dose monitoring assay tha t can identify the patientswith the highest free trough levels of natalizumab that are candidates for dose extension. We have previously described a specific mimetope peptide for the capture and quantification of free and active natalizumab in serum. The peptide binds to the antigen binding site of natalizumab and competes for binding with the target α4 chain of integrin as shown by flow cytometry. We compared a mimetope peptide based ELISA assay with an anti-idiotype based assay using a commercially available anti-natalizumab antibody. Both assays were benchmarked against a cell based assay in which samples were applied to VLA -4 positive cell lines. The peptide based ELISA was consistently closer in value to the cell based assay while the anti-idiotype assay almost always reported a higher value. While both the peptide and anti-idiotype cross compete in ELISA and block natalizumab binding in the cell based assay, we hypothesized that the anti-idiotype antibody could bind to natalizumab that was incapable of binding target. We gently heat denatured natalizumab and observed a loss of binding in the cell based assay that was also observed in the peptide ELISA, whereas the anti-idiotype reported the full natalizumab concentration regardless of the time of heat treatment. Thus we postulate that there is significant but variable amounts of natalizumab that is no longer able to bind target at the time of trough sampling in treated patients and that the surrogate ligand peptide mimetope ELISA is a more accuratemeasure of active drug. A second generation peptide was developed by amino acid replacement scanning of the original mimetope. When used in the ELISA assay, this affinity matured peptide had roughly 10 times greater sensitivity, resulting in an assay with an estimated limit of detection of less than 500ng/ml. Full assay validation is ongoing.
Disclosure: Bradley Messmer: nothing to disclose
Abstract: EP1416
Type: ePoster
Abstract Category: Clinical aspects of MS - 8 Clinical assessment tools
Natalizumab is a highly efficacious treatment option for patients with relapsing-remitting multiple sclerosis (MS), but carries a risk of JC polyomavirus driven progressive multifocal leukoencephalopathy (PML). Recent data has suggested that patients on extended interval dosing schedules have a reduced rate of PML consequent to lower natalizumab exposure. However, there is significant variation in steady state drug levels in natalizumab patients, suggesting a need for a therapeutic dose monitoring assay tha t can identify the patientswith the highest free trough levels of natalizumab that are candidates for dose extension. We have previously described a specific mimetope peptide for the capture and quantification of free and active natalizumab in serum. The peptide binds to the antigen binding site of natalizumab and competes for binding with the target α4 chain of integrin as shown by flow cytometry. We compared a mimetope peptide based ELISA assay with an anti-idiotype based assay using a commercially available anti-natalizumab antibody. Both assays were benchmarked against a cell based assay in which samples were applied to VLA -4 positive cell lines. The peptide based ELISA was consistently closer in value to the cell based assay while the anti-idiotype assay almost always reported a higher value. While both the peptide and anti-idiotype cross compete in ELISA and block natalizumab binding in the cell based assay, we hypothesized that the anti-idiotype antibody could bind to natalizumab that was incapable of binding target. We gently heat denatured natalizumab and observed a loss of binding in the cell based assay that was also observed in the peptide ELISA, whereas the anti-idiotype reported the full natalizumab concentration regardless of the time of heat treatment. Thus we postulate that there is significant but variable amounts of natalizumab that is no longer able to bind target at the time of trough sampling in treated patients and that the surrogate ligand peptide mimetope ELISA is a more accuratemeasure of active drug. A second generation peptide was developed by amino acid replacement scanning of the original mimetope. When used in the ELISA assay, this affinity matured peptide had roughly 10 times greater sensitivity, resulting in an assay with an estimated limit of detection of less than 500ng/ml. Full assay validation is ongoing.
Disclosure: Bradley Messmer: nothing to disclose