Lymphocyte Subsets Show Different Response Patterns to In Vivo Bound Natalizumab—A Flow Cytometric Study on Patients with Multiple Sclerosis

Abstract

Natalizumab is an effective monoclonal antibody therapy for the treatment of relapsing- remitting multiple sclerosis (RRMS) and interferes with immune cell migration into the central nervous system by blocking the α4 subunit of very-late activation antigen-4 (VLA-4). Although well tolerated and very effective, some patients still suffer from relapses in spite of natalizumab therapy or from unwanted side effects like progressive multifocal leukoencephalopathy (PML). In search of a routine-qualified biomarker on the effectiveness of natalizumab therapy we applied flow cytometry and analyzed natalizumab binding to α4 and α4 integrin surface levels on T-cells, B-cells, natural killer (NK) cells, and NKT cells from 26 RRMS patients under up to 72 weeks of therapy. Four-weekly infusions of natalizumab resulted in a significant and sustained increase of lymphocyte-bound natalizumab (p<0.001) which was paralleled by a significant decrease in detectability of the α4 integrin subunit on all lymphocyte subsets (p<0.001). We observed pronounced natalizumab accumulations on T and B cells at single measurements in all patients who reported clinical disease activity (n = 4). The natalizumab binding capacity of in vitro saturated lymphocytes collected during therapy was strongly diminished compared to treatment-naive cells indicating a therapy-induced reduction of α4. Summing up, this pilot study shows that flow cytometry is a useful method to monitor natalizumab binding to lymphocytes from RRMS patients under therapy. Investigating natalizumab binding provides an opportunity to evaluate the molecular level of effectiveness of natalizumab therapy in individual patients. In combination with natalizumab saturation experiments, it possibly even provides a means of studying the feasability of patient-tailored infusion intervals. A routine-qualified biomarker on the basis of individual natalizumab saturation on lymphocyte subsets might be an effective tool to improve treatment safety.

A routine-qualified flow cytometric method for the identification of multiple sclerosis patients with a reduced therapeutic effectiveness of natalizumab

“…It is important to have a biomaker which is informative if the treatment can be effective. This study, and previous studies from our group, have shown that the natalizumab saturation level of immune cells can be such a biomarker [11,12] . We demonstrated the importance of regular 4-weekly infusion intervals by showing that patients experience strong fluctuations in their cellular natalizumab saturation levels in case of prolonged infusion intervals of 6 -8 weeks [12] .…”

“…However, other studies indicated that natalizumab saturation levels of immune cells are a more robust parameter than CD49d expression levels for drawing conclusions about the success of natalizumab treatment [11,12] . In the current study, we examined whether analyzing the natalizumab saturation level on T lymphocytes by flow cytometry represents a sensitive method to early identify patients with a reduced therapeutic success and increased probability of infusion-related side effectiveness due to NAB.…”

Immunological biomarkers identifying natalizumab-treated multiple sclerosis patients at risk of progressive multifocal leukoencephalopathy

“…A sustained decrease of adhesion molecules involved in the leukocyte trafficking, alpha-4 and beta-1 integrin surface level in particular, was observed in T cells, B cells, natural killer cells, natural killer T cells, and dendritic cells (Wipfler et al, 2011;de Andrés et al, 2012;Harrer et al, 2012), but only CD11a expression after 2 years of natalizumab treatment, when PML incidence reaches its peak level, has been proposed as a PML biomarker (Jilek et al, 2014).…”

Routine-taugliche durchflusszytometrische Methode zur Identifikation von Multiple Sklerose PatientInnen mit einer nicht ausreichenden Therapieeffizienz unter einer Natalizumab-Therapie

“…Andere Studien zeigten jedoch, dass der NatalizumabSättigungsgrad ein stabilerer Parameter ist als die CD49d-Expression, um Rückschlüsse auf den Therapieerfolg von Natalizumab ziehen zu können [11,12]. Der NatalizumabSättigungsgrad berechnet sich aus dem Vergleich von in vivo gebundenen Natalizumab und dem in vitro mit Natalizumab gesättigten Immunzellen.…”