Contributions
Abstract: EP1710
Type: ePoster
Abstract Category: Therapy - disease modifying - 28 Long-term treatment monitoring
Background: Natalizumab is one of the therapeutic monoclonal antibodies developed for prevention of inflammatory attacks in multiple sclerosis (MS). It blocks integrins on leukocytes from adhering to the blood brain barrier, thereby hindering migrating into the central nervous system. All treated patients receive the same dose, but the clinical response is variable and saturation of natalizumab on integrins differs substantially between patients. Natalizumab saturation has been proposed as a biomarker to monitor and patient-tailor treatment, and different methods to measure this on a single-cell level have been proposed in previous flow cytometry studies. We have compared three of these methods, using a new technology for single-cell analysis: mass cytometry by time-of-flight (CyTOF), where antibodies are labelled with metal isotopes instead of fluorochromes, allowing analysis of over 50 parameters in a single cell.
Methods: Peripheral blood leukocytes from MS patients receiving natalizumab therapy (n=9) were labelled with antibodies against natalizumab, integrin and cell subtype markes prior to CyTOF analysis. Natalizumab saturation calculated by three previously described methods were compared: 1) natalizumab level compared to a reference sample in vitro saturated with natalizumab, 2) ratio natalizumab/integrin and 3) quantitation of relative numbers of natalizumab and integrin molecules on single cells using beads with known amounts of bound antibodies against natalizumab and integrin as a reference.
Results: Numbers of bound natalizumab was positively correlated to numbers of integrin on single cells. After a natalizumab infusion, numbers of both natalizumab and integrin molecules decreased, but the ratio natalizumab/integrin increased. Measuring natalizumab levels alone, therefore gave a false impression of decreasing natalizumab levels after infusion.
Conclusions: The method that best reflects saturation of natalizumab on target cells is a combination of previously described techniques: using beads for quantitation of natalizumab and integrin to calculate a ratio natalizumab/integrin. This approach will be of interest for monitoring other therapeutic antibodies.
Disclosure: Gerd Haga Bringeland: Nothing to disclose.
Christian Vedeler: Nothing to disclose.
Kjell Morten Myhr: Nothing to disclose.
Sonia Gavasso: Nothing to disclose.
Abstract: EP1710
Type: ePoster
Abstract Category: Therapy - disease modifying - 28 Long-term treatment monitoring
Background: Natalizumab is one of the therapeutic monoclonal antibodies developed for prevention of inflammatory attacks in multiple sclerosis (MS). It blocks integrins on leukocytes from adhering to the blood brain barrier, thereby hindering migrating into the central nervous system. All treated patients receive the same dose, but the clinical response is variable and saturation of natalizumab on integrins differs substantially between patients. Natalizumab saturation has been proposed as a biomarker to monitor and patient-tailor treatment, and different methods to measure this on a single-cell level have been proposed in previous flow cytometry studies. We have compared three of these methods, using a new technology for single-cell analysis: mass cytometry by time-of-flight (CyTOF), where antibodies are labelled with metal isotopes instead of fluorochromes, allowing analysis of over 50 parameters in a single cell.
Methods: Peripheral blood leukocytes from MS patients receiving natalizumab therapy (n=9) were labelled with antibodies against natalizumab, integrin and cell subtype markes prior to CyTOF analysis. Natalizumab saturation calculated by three previously described methods were compared: 1) natalizumab level compared to a reference sample in vitro saturated with natalizumab, 2) ratio natalizumab/integrin and 3) quantitation of relative numbers of natalizumab and integrin molecules on single cells using beads with known amounts of bound antibodies against natalizumab and integrin as a reference.
Results: Numbers of bound natalizumab was positively correlated to numbers of integrin on single cells. After a natalizumab infusion, numbers of both natalizumab and integrin molecules decreased, but the ratio natalizumab/integrin increased. Measuring natalizumab levels alone, therefore gave a false impression of decreasing natalizumab levels after infusion.
Conclusions: The method that best reflects saturation of natalizumab on target cells is a combination of previously described techniques: using beads for quantitation of natalizumab and integrin to calculate a ratio natalizumab/integrin. This approach will be of interest for monitoring other therapeutic antibodies.
Disclosure: Gerd Haga Bringeland: Nothing to disclose.
Christian Vedeler: Nothing to disclose.
Kjell Morten Myhr: Nothing to disclose.
Sonia Gavasso: Nothing to disclose.