Contributions
Abstract: EP1454
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Background: Anti-B-cell therapies, such as fully human anti-CD20 monoclonal antibody (Ab) ofatumumab, currently studied in Phase III clinical trials in remitting multiple sclerosis, have strong preclinical and clinical evidence of efficacy. As depletion of B cells may serve as a pharmacodynamic biomarker, we explored two methods in parallel, reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS), to compare their sensitivity for their quantification.
Objective: To compare the detection threshold of B-cell depletion in blood, as measured by the current standard method (FACS) with that of RT-PCR. Second, to evaluate whether RT-PCR is suitable to quantify anti-CD20 induced B-cell depletion in peripheral lymphoid organs.
Methods: C57BL/6 female mice (n=10; 7 weeks old) were treated with anti-CD20 mAb (20 µg or 200 µg) to induce B-cell depletion. Blood, spleen, and inguinal lymph nodes (LN) were collected and samples were split for FACS and RT-PCR analysis. Single lymphocyte suspensions were stained with anti-CD19-APC/Cy7, anti-CD3-APC, and SYTOX Blue for analysis using a Fortessa flow cytometer and FlowJo software. Total RNA was extracted by RNeasy Protect Animal Blood kit (blood) or RNeasy mini/micro (cell suspensions). A duplex TaqMan assay with either CD19-FAM or CD20-FAM probes, and endogenous control probe GAPDH-VIC was run in triplicates after reverse transcription.
Results: At 7 days after anti-CD20 treatment, a strong depletion of total B cells was observed in blood, regardless of the dose. Both FACS and RT-PCR methods showed that B-cell depletion was dose-dependent. Relative quantification of CD19 mRNA and CD20 mRNA showed nearly identical values. B-cell depletion was quantifiable in LN and spleen by RT-PCR. In spleen, B-cell depletion measured by RT-PCR displayed a larger spread, probably indicating higher sensitivity of detection that will be further explored by absolute quantification using digital RT-PCR.
Conclusions: Quantification of total B cells under anti-CD20 mAb treatment can be performed by both FACS and RT-PCR analysis, as shown here in experimental mice. RT-PCR allows measurement of B-cell depletion in blood and solid tissue. It requires significantly less specimen material, and simpler sample logistics. Moreover, RT-PCR can measure CD20 directly that is not possible by FACS. RT-PCR may be of potential advantage for application in a clinical setting.
Disclosure: This study was funded by Novartis Pharma AG, Basel, Switzerland.
All authors are employees of Novartis Pharma AG.
Abstract: EP1454
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Background: Anti-B-cell therapies, such as fully human anti-CD20 monoclonal antibody (Ab) ofatumumab, currently studied in Phase III clinical trials in remitting multiple sclerosis, have strong preclinical and clinical evidence of efficacy. As depletion of B cells may serve as a pharmacodynamic biomarker, we explored two methods in parallel, reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS), to compare their sensitivity for their quantification.
Objective: To compare the detection threshold of B-cell depletion in blood, as measured by the current standard method (FACS) with that of RT-PCR. Second, to evaluate whether RT-PCR is suitable to quantify anti-CD20 induced B-cell depletion in peripheral lymphoid organs.
Methods: C57BL/6 female mice (n=10; 7 weeks old) were treated with anti-CD20 mAb (20 µg or 200 µg) to induce B-cell depletion. Blood, spleen, and inguinal lymph nodes (LN) were collected and samples were split for FACS and RT-PCR analysis. Single lymphocyte suspensions were stained with anti-CD19-APC/Cy7, anti-CD3-APC, and SYTOX Blue for analysis using a Fortessa flow cytometer and FlowJo software. Total RNA was extracted by RNeasy Protect Animal Blood kit (blood) or RNeasy mini/micro (cell suspensions). A duplex TaqMan assay with either CD19-FAM or CD20-FAM probes, and endogenous control probe GAPDH-VIC was run in triplicates after reverse transcription.
Results: At 7 days after anti-CD20 treatment, a strong depletion of total B cells was observed in blood, regardless of the dose. Both FACS and RT-PCR methods showed that B-cell depletion was dose-dependent. Relative quantification of CD19 mRNA and CD20 mRNA showed nearly identical values. B-cell depletion was quantifiable in LN and spleen by RT-PCR. In spleen, B-cell depletion measured by RT-PCR displayed a larger spread, probably indicating higher sensitivity of detection that will be further explored by absolute quantification using digital RT-PCR.
Conclusions: Quantification of total B cells under anti-CD20 mAb treatment can be performed by both FACS and RT-PCR analysis, as shown here in experimental mice. RT-PCR allows measurement of B-cell depletion in blood and solid tissue. It requires significantly less specimen material, and simpler sample logistics. Moreover, RT-PCR can measure CD20 directly that is not possible by FACS. RT-PCR may be of potential advantage for application in a clinical setting.
Disclosure: This study was funded by Novartis Pharma AG, Basel, Switzerland.
All authors are employees of Novartis Pharma AG.