A mass cytometry receptor occupancy study of natalizumab therapy in multiple sclerosis
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Background: Natalizumab is a therapeutic antibody that effectively reduces disease activity in relapsing remitting multiple sclerosis (RRMS) by binding α4 integrin on leukocytes and preventing leukocyte migration into the central nervous system (CNS). Natalizumab is administered intravenously at a standard dose of 300 mg every 4 weeks. Approximately half of treated patients report subjective wearing-off symptoms at the end of the dosing interval. This phenomenon is sparsely investigated, and it is not known whether it has a biological cause or is associated with poor therapeutic efficacy. Accumulating evidence suggests that extending the dosing interval to up to 8 weeks maintains therapeutic efficacy in many patients while reducing the risk of progressive multifocal leukoencephalopathy (PML), a rare but potentially lethal complication of therapy. This has prompted efforts to personalize dosing intervals. Natalizumab receptor occupancy (RO) correlates with therapeutic response and has been suggested as a biomarker to navigate individual dosing. RO is traditionally measured by flow cytometry, but spectral overlap limits the number of markers that can be measured simultaneously. This restricts RO assays to the analysis of major cell types, although rare cell populations are of potential therapeutic relevance. Mass cytometry is a cutting-edge technology that allows simultaneous analysis of more than 40 parameters on single cells, facilitating measurement of RO in a broader array of cell types together with more biomarkers of interest than possible by conventional flow cytometry. Although RO assays are widely used in flow cytometry, no RO assay utilizing mass cytometry has been published prior to this study. Objective: We aimed to develop a method for reliable RO measurement with high-parameter mass cytometry, and to study natalizumab RO and clinical characteristics in RRMS patients treated with natalizumab. Methods: We developed a novel method to measure RO with mass cytometry, allowing simultaneous in-depth immune monitoring and reliable measurement of natalizumab RO on multiple peripheral blood leukocyte subtypes. This was achieved by adapting antibody-binding beads from flow cytometry to standardize the varying detection sensitivity in mass cytometry, generating accurate and reproducible RO results. We applied the natalizumab RO assay in a cross-sectional study of 40 RRMS patients treated with natalizumab at the Department of Neurology, Haukeland University Hospital. Clinical and radiological signs of disease activity were recorded, and fatigue, cognitive function and wearing-off symptoms were evaluated. We followed the patients prospectively for one year. Results: In the cross-sectional study, we found that patients who reported wearing-off symptoms regularly (at the end of every 4-week dosing interval) had lower natalizumab RO in several leukocyte subtypes. Body mass index (BMI) was higher in patients who regularly had wearing-off symptoms, and high BMI was associated with low RO. After 1-year follow-up none of the patients displayed clinical or radiological signs of disease activity, but patients reporting wearing-off symptoms regularly had more severe fatigue and cognitive dysfunction. Conclusions: Low natalizumab RO may contribute to the wearing-off phenomenon and high BMI may be the underlying cause. Patients with wearing-off symptoms showed no increased short-term risk of RRMS disease activity, but they may be more vulnerable to therapeutic failure if dosing intervals are extended than patients with higher RO levels. This work provides new tools for future exploration of natalizumab and other therapeutic antibodies in the era of personalized medicine.
Has partsPaper I: Bringeland GH, Bade L, Blaser N, Budzinski L, Schulz AR, Mei HE, Myhr KM, Vedeler CA, Gavasso S. Optimization of Receptor Occupancy Assays in Mass Cytometry: Standardization Across Channels with QSC Beads. Cytometry A. 2019 Mar;95(3):314-322. The article is available at: http://hdl.handle.net/1956/22313
Paper II: Bringeland GH, Blaser N, Myhr KM, Vedeler CA, Gavasso S. Wearing-off at the end of natalizumab dosing intervals is associated with low receptor occupancy. Neurol Neuroimmunol Neuroinflamm. 2020 Feb 4;7(3). The article is available in the main thesis. The article is also available at: https://doi.org/10.1212/NXI.0000000000000678
Paper III: Bringeland GH, Myhr KM, Vedeler CA, Gavasso S. Wearing-off at the end of natalizumab dosing interval and risk of MS disease activity: a prospective 1-year follow-up study. 2020 Aug;415:116880. The manuscript is available in the main thesis. The published article is available at: https://doi.org/10.1016/j.jns.2020.116880