Obinutuzumab-Induced B Cell Depletion Reduces Spinal Cord Pathology in a CD20 Double Transgenic Mouse Model of Multiple Sclerosis

Abstract

B cell-depleting therapies have recently proven to be clinically highly successful in the treatment of multiple sclerosis (MS). This study aimed to determine the effects of the novel type II anti-human CD20 (huCD20) monoclonal antibody (mAb) obinutuzumab (OBZ) on spinal cord degeneration in a B cell-dependent mouse model of MS. Double transgenic huCD20xHIGR3 (CD20dbtg) mice, which express human CD20, were immunised with the myelin fusion protein MP4 to induce experimental autoimmune encephalomyelitis (EAE). Both light and electron microscopy were used to assess myelination and axonal pathology in mice treated with OBZ during chronic EAE. Furthermore, the effects of the already established murine anti-CD20 antibody 18B12 were assessed in C57BL/6 wild-type (wt) mice. In both models (18B12/wt and OBZ/CD20dbtg) anti-CD20 treatment significantly diminished the extent of spinal cord pathology. While 18B12 treatment mainly reduced the extent of axonal pathology, a significant decrease in demyelination and increase in remyelination were additionally observed in OBZ-treated mice. Hence, the data suggest that OBZ could have neuroprotective effects on the CNS, setting the drug apart from the currently available type I anti-CD20 antibodies.

Keywords: B cells; EAE; anti-CD20; mAb; multiple sclerosis; neurodegeneration; obinutuzumab.

Figure 1

Disease course of mice treated with anti-CD20 or respective isotype control antibodies. (A) Wild-type (wt) cohort: Treated on day 30 after the peak of experimental autoimmune encephalomyelitis (EAE) with 18B12 or muIgG2a, respectively (n = 13). (B) CD20dbtg cohort 1: Treated on day 30 after the peak of EAE with OBZ or huIgG1, respectively (n = 12). (C) CD20dbtg cohort 2: Treated on day 50 after the peak of EAE with OBZ or huIgG1, respectively (n = 12). Treatment periods are marked in grey. Values are displayed as mean values ± standard error of the mean (SEM).

Figure 2

Spinal cord histopathology in wt mice treated from day 30 after the EAE peak with 18B12 or muIgG2a, respectively. (A) Representative light microscopic images of the spinal cord. The lesion area can be seen to the right of the dashed line. The scale bar represents 75 µm. (B) Representative electron micrographs of the spinal cord. The scale bar represents 5 µm. An exemplary axolytic axon is marked with a star. Images of untreated non-immunised mice have been added in both A and B for reference. (C) Quantification of the treatment effect on lesion area, g-ratio, demyelination, remyelination, axolysis and number of axons. Values are displayed as mean values ± standard error of the mean (SEM). *: p < 0.05; **: p < 0.01. Statistical significance was determined using the Mann–Whitney U test.

Figure 3

Spinal cord histopathology in CD20dbtg mice treated from day 30 after the EAE peak with OBZ or huIgG1, respectively. (A) Representative light microscopic images of the spinal cord. The lesion area can be seen to the right of the dashed line. The scale bar represents 75 µm. (B) Representative electron micrographs of the spinal cord. The scale bar represents 5 µm. A diamond marks an exemplary demyelinating axon and a star an axolytic axon, respectively. Images of untreated non-immunised mice have been added in both (A) and (B) for reference. (C) Quantification of the treatment effect on lesion area, g-ratio, demyelination, remyelination, axolysis and number of axons. Values are displayed as mean values ± standard error of the mean (SEM). *: p < 0.05; **: p < 0.01. Statistical significance was determined using the Mann–Whitney U test.

Figure 4

Spinal cord histopathology in CD20dbtg mice treated from day 50 after the EAE peak with OBZ or huIgG1, respectively. (A) Representative light microscopic images of the spinal cord. The scale bar represents 75 µm. (B) Representative electron micrographs of the spinal cord. The scale bar represents 5 µm. An exemplary demyelinating axon is marked with a diamond, three remyelinating axons with arrows and an axolytic axon with a star, respectively. Images of untreated non-immunised mice have been added in both (A) and (B) for reference. (C) Quantification of the treatment effect on the lesion area, g-ratio, demyelination, remyelination, axolysis and number of axons. Values are displayed as mean values ± standard error of the mean (SEM). *: p < 0.05; **: p < 0.01. Statistical significance was determined using the Mann–Whitney U test.

Figure 5

Serum NfL levels measured in each group (n = 6–7 mice per group). The dashed line indicates average values in untreated non-immunised mice (n = 3–4). Values are displayed as mean values ± standard error of the mean (SEM).

Figure 6

Correlation between the percentage of axolysis and serum neurofilament light levels in all mice from cohorts wt, CD20dbtg1 and CD20dbtg2 (n = 37). The regression line and 95% confidence intervals are shown.

Figure 7

Exemplary images of axons in electron micrographs pertaining to the respective categories and the symbols used to mark them in the images in the results section.